mechanistic studies on transcutaneous subunit vaccine deliverythe investigations described in this...

Mechanisti

c studies on transcutaneous subunit vaccine deliverySuzanne Bal

Mechanistic studies on transcutaneous subunit vaccine delivery

Microneedles, nanoparticles and adjuvants

Suzanne Bal

Mechanistic studies on

transcutaneous subunit vaccine

delivery Microneedles, nanoparticles and adjuvants

The investigations described in this thesis were performed at the Division of Drug Delivery

Technology of the Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden,

The Netherlands. The studies were financially supported by TIPharma (D5-106-1; Vaccine

delivery: alternatives for conventional multiple injection vaccines).

ISBN: 978-90-816587-1-3

© 2011 Suzanne Bal

Cover design by Jeroen Ophof

Printed by Wöhrmann Print Service (Zutphen, The Netherlands)

Mechanistic studies on

transcutaneous subunit vaccine

delivery Microneedles, nanoparticles and adjuvants

Proefschrift

ter verkrijging van

de graad van Doctor aan de Universiteit Leiden,

op gezag van Rector Magnificus prof. mr. P.F. van der Heijden,

volgens besluit van het College voor Promoties

te verdedigen op dinsdag 15 februari 2011

klokke 15.00 uur

door

Suzanne Marleen Bal

geboren te Den Haag

in 1983

Promotiecommissie

Promotoren: Prof. Dr. J.A. Bouwstra

Prof. Dr. W. Jiskoot

Overige leden: Prof. Dr. M. Danhof

Prof. Dr. J.B. Haanen

Prof. Dr. C.J. Boog

Prof. Dr. Y. Perrie (Aston University)

Table of contents

Chapter 1 Introduction, aim and outline of this thesis 1

Chapter 2 Advances in transcutaneous vaccine delivery: do all ways lead to Rome? 9

Part I: Safety and efficacy of microneedle pre-treatment on human volunteers

Chapter 3 In vivo assessment of safety of microneedle arrays in human skin 55

Chapter 4 Influence of microneedle shape on the transport of a fluorescent dye

into human skin in vivo

75

Part II: TMC-based formulations for intradermal and transcutaneous vaccination

Chapter 5 Efficient induction of immune responses through intradermal

vaccination with N-trimethyl chitosan containing antigen formulations

95

Chapter 6 Microneedle-based transcutaneous immunisation in mice

with N-trimethyl chitosan adjuvanted diphtheria toxoid

formulations

121

Chapter 7 Small is beautiful: N-trimethyl chitosan-ovalbumin conjugates for

microneedle-based transcutaneous immunisation

143

Chapter 8 Adjuvanted, antigen loaded N-trimethyl chitosan nanoparticles for

nasal and intradermal vaccination: adjuvant- and site-dependent

immunogenicity in mice

159

Part III: Cationic liposomes to co-deliver antigen and adjuvant

Chapter 9 Co-encapsulation of antigen and adjuvant in cationic liposomes affects

the quality of the immune response in mice after intradermal

vaccination

177

Chapter 10 Adjuvant effect of cationic liposomes and CpG depends on

administration route: intranodal, intradermal, transcutaneous and nasal

immunisation in mice

197

Chapter 11 Summary and perspectives 223

Chapter 12 Nederlandse samenvatting 237

Chapter 13 Curriculum Vitae 247

Chapter 14 List of publications 251

Chapter 15 Nawoord 257

Chapter 1

Introduction, aim and outline of this

thesis

Chapter 1

2

Introduction, aim and outline

3

Introduction

In recent years the search for alternatives for the classical manner of vaccination by

injection into the muscle or subcutaneous tissue has increased tremendously. Improved

safety, patient compliance and better efficacy are the most important reasons to develop

novel vaccine delivery techniques [1, 2]. One of the alternative vaccination sites is the skin.

Previously regarded as an unconquerable barrier, the skin was first described as an

attractive immunisation site by Glenn et al., who showed that it was possible to induce an

immune response by topical application of cholera toxin (CT) or heat-labile enterotoxin (LT)

on intact mice [3] and human skin [4]. Their research has led to the development of a

vaccine patch against traveller’s diarrhoea currently being tested in a phase III study [5]

and one against influenza in a phase II study. These studies have boosted the research on

novel techniques to apply a vaccine via the skin. An elegant example is the application of

microneedle arrays, needles that pierce the stratum corneum while being short enough to

avoid pain sensation. The first microneedle arrays introduced comprised solid

microneedles that can be used to pre-treat the skin [6]. More recently a variety of types,

including hollow, solid, coated and dissolvable microneedles, have been developed [7].

The research on efficient vaccination focuses not only on the delivery method and

administration route, but also on the composition of vaccines. Safety issues with the

traditional life-attenuated and inactivated vaccines have advanced the development of

subunit vaccines, which are based on a purified protein, peptide or gene fragment of the

pathogen and are less reactogenic than traditional vaccines. The main drawback of such

subunits is their poor immunogenicity, which necessitates the addition of an adjuvant in

order to yield a good immune response. An adjuvant is an additive that enhances the

immunogenicity of an antigen. The adjuvant field is rapidly evolving. For a long time

colloidal aluminium salts (alum) were the only approved adjuvants, but more recently

squalene emulsions (MF59) and monophosphoryl lipid A have been licensed for usage in

Europe. The more thorough understanding of the innate immune system has stimulated

the research on developing new adjuvants. Another promising approach to increase the

immunogenicity of subunit antigens is their formulation into (nano)particles [8, 9].

Particulates facilitate the uptake by the professional antigen presenting cells, such as

dendritic cells, due to their similarity in size to pathogens. Furthermore, they can protect

the antigen from enzymatic breakdown, allow sustained antigen release over time and

offer the possibility of co-encapsulation of adjuvants. Knowledge on the effects of antigen

formulation for transcutaneous vaccination, however, is sparse.

Chapter 1

4

Aim

The principal aim of the studies described in this thesis is to design subunit vaccine

formulations that can be combined with microneedles for transcutaneous immunisation. In

order to achieve this, understanding of the requirements of both the microneedles and the

formulations needs to be acquired. Therefore several sub-aims needed to be formulated.

• The safety and efficacy of different microneedle arrays.

• The immunogenicity of the different vaccine formulations when used for

vaccination via the skin. For this purpose the formulations are injected

intradermally, to avoid the complicating factor of transport into the skin.

• The effectiveness of the formulations when applied transcutaneously with

microneedle arrays.

Thesis outline

In chapter 2 the literature regarding transcutaneous immunisation is reviewed, with a

strong focus on the immunological characteristics that makes the skin an excellent

vaccination site and a critical view on the many different devices developed to deliver

vaccines across the stratum corneum barrier. Adjuvants and particulate delivery systems

currently used in (pre)clinical transcutaneous immunisation studies are also discussed.

The research described in the thesis is divided into three parts: the microneedle arrays

used for transcutaneous vaccination (part I); the development and efficacy of several

generations of N-trimethyl chitosan (TMC) based formulations (part II); and the usage of

adjuvanted liposomal formulations for vaccination purposes (part III).


The ability of solid microneedle arrays differing in shape and length (200-550 µm) to

disrupt the skin barrier is evaluated in chapter 3. The microneedles are applied with an

electrical applicator onto the skin of human volunteers and the following parameters are

studied: pain sensation, skin redness and blood flow as a measure of skin irritation and

transepidermal water loss to indicate barrier disruption. These measurements are

repeated in time to assess the closure time of the conduits.

This is followed by the visualisation of the microneedle conduits by confocal laser scanning

microscopy in chapter 4. Two different solid microneedle arrays and the commercially

available Dermastamp®, all three containing 300 µm long microneedles are applied onto

the skin of human volunteers before or after the application of a fluorescent dye,

fluorescein. The transport of fluorescein into the skin through the formed conduits is both

qualitatively and quantitatively determined.


5


This part focuses on preparing and testing different formulations based on the positively

charged polymer N-trimethyl chitosan (TMC). This polymer has been successfully used

preclinically as an adjuvant for mucosal vaccine delivery. In chapter 5 nanoparticles are

prepared by ionic cross-linking of TMC with tripolyphosphate using either ovalbumin (OVA)

or diphtheria toxoid (DT) as an antigen. These nanoparticles are physicochemically

characterised and tested for their ability to enhance antigen uptake by dendritic cells (DCs)

in vitro, DC maturation and T cell activation. The immunogenicity of the formulations is

tested in Balb/c mice after intradermal injection. Antibody titres are measured to study the

humoral immune response.

In chapter 6 mice are immunised with DT-loaded TMC nanoparticles, a mixture of TMC and

DT and non-adjuvanted DT by applying the formulations on microneedle pre-treated skin.

The antibody titres induced by vaccination via transcutaneous immunisation are compared

to those after administration via the intradermal route. To obtain information on the

efficiency of transport through the conduits, the localisation of TMC, as a solution and in

nanoparticulate form, is visualised ex vivo.

Several parameters that can affect transcutaneous immunisation are optimised in chapter

7 by prolonging the application time of the formulations and using a smaller antigen-

adjuvant entity, a TMC-OVA conjugate. To study the combined effect of diffusion through

the conduits into the skin, transport to the draining lymph nodes and antigen uptake by

DCs, the formulations are also applied by intradermal or intranodal injection. Besides the

antibody titres, the amount of OVA+ DCs in the draining lymph nodes is quantified.

A second generation of OVA-loaded TMC nanoparticles is developed in chapter 8. A

selection of adjuvants including Toll-like receptor ligands lipopolysaccharide (LPS),

PAM3CSK4 (PAM), CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and

the GM1 ganglioside receptor ligand, cholera toxin B subunit are co-incorporated with the

antigen into TMC nanoparticles. The immunogenicity of the formulations is assessed by

determining the antibody response after nasal and intradermal vaccination.


The use of cationic liposomes, another type of nanoparticles, for intradermal vaccination is

described in chapter 9. Two different Toll-like receptor ligands, PAM and CpG are

encapsulated in OVA-containing liposomes. The ability of these ligands to interact with

their receptors is studied in Toll like receptor (2 and 9) transfected HEK cells and their DC

stimulating properties are investigated. Both humoral and cellular immune responses after

intradermal immunisation are measured.

The formulation requirements for different administration routes are addressed in chapter

10. Liposomes containing OVA and CpG, as well as a mixture of soluble OVA and CpG, are

Chapter 1

6

administered via the transcutaneous, nasal, intradermal and intranodal route and the

serum IgG and IgG subclass titres are measured. To further understand the working

mechanism of the liposomes, the uptake of antigen and adjuvant by DCs, both in vitro and

in vivo in the draining lymph nodes, is determined.


7

References

1. Giudice EL and Campbell JD, Needle-free vaccine delivery. Adv Drug Deliv Rev, 2006. 58(1): p. 68-89.

2. O'Hagan DT and Rappuoli R, Novel approaches to vaccine delivery. Pharm Res, 2004. 21(9): p. 1519-

30.

3. Glenn GM, Rao M, Matyas GR, and Alving CR, Skin immunization made possible by cholera toxin.

Nature, 1998. 391(6670): p. 851.

4. Glenn GM, Taylor DN, Li X, Frankel S, Montemarano A, and Alving CR, Transcutaneous immunization:

a human vaccine delivery strategy using a patch. Nat Med, 2000. 6(12): p. 1403-6.

5. Frech SA, DuPont HL, Bourgeois AL, McKenzie R, Belkind-Gerson J, Figueroa JF, Okhuysen PC,

Guerrero NH, Martinez-Sandoval FG, Melendez-Romero JHM, Jiang ZD, Asturias EJ, Halpern J, Torres

OR, Hoffman AS, Villar CP, Kassem RN, Flyer DC, Andersen BH, Kazempour K, Breisch SA, and Glenn

GM, Use of a patch containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: A

phase II, randomised, double-blind, placebo-controlled field trial. Lancet, 2008. 371(9629): p. 2019-

2025.

6. Henry S, McAllister DV, Allen MG, and Prausnitz MR, Microfabricated microneedles: A novel

approach to transdermal drug delivery. J Pharm Sci, 1998. 87(8): p. 922-5.

7. Donnelly RF, Raj Singh TR, and Woolfson AD, Microneedle-based drug delivery systems:

Microfabrication, drug delivery, and safety. Drug Deliv, 2010. 17(4): p. 187-207.

8. Singh M, Chakrapani A, and O'Hagan D, Nanoparticles and microparticles as vaccine-delivery

systems. Expert Rev Vaccines, 2007. 6(5): p. 797-808.

9. Rice-Ficht AC, Arenas-Gamboa AM, Kahl-McDonagh MM, and Ficht TA, Polymeric particles in vaccine

delivery. Curr Opin Microbiol, 2010. 13(1): p. 106-12.

Chapter 2

Advances in transcutaneous vaccine

delivery: do all ways lead to Rome? Suzanne M. Bal*, Zhi Ding*, Elly van Riet, Wim Jiskoot, Joke A. Bouwstra

* Authors contributed equally

Journal of Controlled Release 2010, 148(3): 266-282

Chapter 2

10

Abstract

Transcutaneous immunisation (TCI) is a promising alternative to vaccine delivery via the

subcutaneous and intramuscular routes, due to the unique immunological characteristics

of the skin. The increasing knowledge of the skin immune system and the novel delivery

methods that have become available have boosted research on new vaccination strategies.

However, TCI has not yet been exploited to its full potential, because the barrier function

of the stratum corneum, the top layer of the skin, is difficult to overcome. In this review we

first discuss the immune system of the skin, focusing on the role the different types of skin

residing dendritic cells play in the immune response. Subsequently, adjuvants and the large

variety of devices, in particular microneedles, developed to deliver vaccines into the skin

are summarised. Clearly, many ways have been explored to achieve efficient

transcutaneous vaccination with varying success. The perspectives of the most promising

concepts will be discussed.

Advances in transcutaneous vaccine delivery: do all ways lead to Rome?

11

Introduction

Over the last two centuries, vaccination has been one of the most successful medical

interventions in reduction of infectious diseases [1, 2]. A good vaccine is safe, administered

in a minimally invasive manner and, most importantly, capable of eliciting a strong,

protective immune response. Currently available vaccines can be classified into three

categories: live-attenuated, inactivated and subunit vaccines. From a safety perspective

subunit vaccines are preferred over live-attenuated and inactivated pathogens. However,

purified antigens generally are poorly immunogenic and therefore require to be

formulated with adjuvants [3, 4].

Nearly all subunit vaccines are administered by intramuscular (IM) or subcutaneous (SC)

injection, but alternative routes of administration are widely explored in the search for

more effective and safer vaccines. Injection requires syringes, needles, and trained

personnel. Moreover, injection can be painful and cause stress, especially in children. For

pediatric vaccination programs, poor compliance is one of the reasons for incomplete

vaccination coverage [5]. Finally, muscle and SC tissue contain less antigen presenting cells

(APCs) than skin tissue, adding to the belief that they are not ideal sites for vaccination.

The disadvantages of injectable vaccines have boosted the research on nasal [6],

transcutaneous [7], oral [8] and pulmonary delivery of vaccines [9].

The transcutaneous route is particularly attractive because the skin is highly accessible and

has unique immunological characteristics. It has been known for a long time that an

effective immune response can be induced via the skin and many different approaches

have been tried. One successful example of transcutaneous vaccination is scarification in

the case of smallpox immunisation in humans [10]. The presence of professional antigen

presenting cells (APCs) in the epidermis and dermis mediates the immune response

following cutaneous immunisation [11]. Another primary reason for considering the

transcutaneous route is the potential for safe immune stimulation, as it avoids the direct

contact between potent (sometimes even slightly toxic) adjuvants with the general

circulation [12]. However, the uppermost layer of the skin, the stratum corneum, acts as a

barrier for diffusion and thereby forms a major obstacle to transcutaneous immunisation

(TCI), e.g. vaccination through intact or pre-treated skin. Currently, the main challenges for

cutaneous immunisation are to enhance the transport of antigens across the skin barrier

and to improve the immunogenicity of topically applied subunit vaccines.

This review will focus on approaches for improving TCI. It starts with a description of the

barrier and immunological functions of the skin. As TCI is an emerging field, many

techniques have been employed to elicit an efficient immune response. We will summarize

these techniques and make a distinction between approaches for enhancing

transcutaneous antigen delivery and for improving the immunogenicity of subunit vaccine

Chapter 2

12

formulations (addition of adjuvants). For clarity, different terms related to immunisation

via the skin are defined in Table 1.

Table 1. Skin immunisation.

Term Interpretation

Cutaneous immunisation Both intradermal and transcutaneous immunisation

Intradermal immunisation Antigen delivery into the dermis via a syringe and hollow needle

Transcutaneous immunisation Antigen delivery into the epidermis and/or dermis through

intact or pre-treated skin

Immunological function of the skin

Skin structure

The skin is the largest organ of the human body. It represents the outermost physical

barrier between the body and the surrounding environment. It protects us against external

mechanical impacts, ultraviolet radiation, dehydration, and microorganisms. The skin

consists of three main layers: epidermis, dermis, and subcutaneous fat tissue (figure 1).

The epidermis is the outermost layer of the skin. The human epidermis varies in thickness

from 50 to 150 μm. The barrier function of the skin is located in the upper 15-20 µm, the

stratum corneum. This layer consists of rigid, desmosome-linked epithelial cells, known as

corneocytes, embedded in a highly organized lamellar structure formed by intercellular

lipids. The unique arrangement of this layer results in a practically impermeable barrier

which reduces the passage of molecules, especially those larger than 500 Da [13].

Underneath the stratum corneum resides the viable epidermis. The main cell type in the

viable epidermis is the keratinocyte. However, melanocytes, Merkel cells and Langerhans

cells (LCs, figure 2), although less abundantly present, also play important roles in the

functioning of the viable epidermis. Underneath the viable epidermis the dermis is located.

The important cell classes in the dermis are fibroblasts, mast cells, and dermal DCs (dDCs).

The dermis also contains blood vessels, lymph vessels, nerves and an abundant level of

collagen fibres. This skin layer is the major site of cellular and fluid exchanges between the

skin and the blood and lymphatic networks [14]. Beneath the dermis lays the

subcutaneous fat tissue, an assembly of adipocytes linked by collagen fibres. It forms a

thermal barrier, but also stores energy and functions as a mechanical cushion for the body

[15]. Appendages such as sweat glands, pilosebaceous units, and hair follicles are

structures penetrating the skin and originate either from the dermis or the subcutaneous

fat tissue. These appendages form important discontinuities in the skin structure [14].


13

Besides the barrier function, the skin also has important immunological functions with an

imperative role for the skin residing APCs, such as LCs and dDCs, which communicate with

keratinocytes, mast cells and subsets of T lymphocytes. Although considerable amounts of

microbes are covering our skin, homeostasis is maintained and we stay remarkably

healthy. When microbes break the skin barrier, the immune system faces a number of

questions: whether or not to respond, and how to respond. This decision can be a matter

of life and death exemplified by for instance leprosy [17]. The skin is involved in both

innate and adaptive immunity. The adaptive response can generate memory responses

and therefore generally becomes more effective with each successive encounter with the

same antigen, whereas the innate immune mechanism provides an immediate, but short-

lasting defence against infections. The immune system of man and mouse differ in several

aspects; unless stated otherwise, in this review the human immune system is discussed.

Figure 2. Electron microscopy image of human

skin, showing keratinocytes (white arrows) and a

LC (black arrow).

Figure 1. Structure of the skin. The skin consists of

three main layers: epidermis, dermis, and

subcutaneous fat tissue. The barrier function of

the skin is located in the uppermost layer, the

stratum corneum. Image adapted from Watt [16].

Chapter 2

14

Innate immunity

The innate immune system fights infections in an unspecific manner using fever, the

complement system, phagocytic and natural killer cells, naturally occurring antibodies and

anti-microbial peptides (figure 3).

Keratinocytes, accounting for about 90% of the total epidermal cell population, play an

important role in innate immunity in the skin. In case of danger, e.g. skin barrier

disruption, keratinocytes produce a wide range of cytokines, chemokines and antimicrobial

peptides [18]. In this way they are able to kill invading pathogens and recruit immune cells.

Examples are the cytokines interleukin-1α (IL-1α), IL-1β, granulocyte-macrophage colony-

stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α), which interact with DCs

and help to maintain an appropriate balance between reactivity and tolerance of the

immune system [19, 20]. For example, migration and maturation of LCs are initiated by

pro-inflammatory cytokine IL-1β and keratinocyte-derived TNF-α [21, 22]. Another

example is the expression of CCL20 by keratinocytes that attracts LCs [23, 24]. In addition

keratinocytes have been reported to function as non-professional APCs, via surface

expression of major histocompatibility complex (MHC) class II molecules [25].

Besides keratinocytes, also neutrophils, macrophages, mast cells and natural killer cells

secrete cytokines that influence DC maturation [26-28]. DCs are the most important APC in

the skin and play a vital role in both the innate and adaptive immune response. Skin

residing DCs, LCs and dDCs, together with macrophages recruited from circulating blood,

exert their sentinel role by sampling and processing potential pathogens invading the skin.

Immature DCs are activated by numerous agents derived from microbes and cells of the

innate and adaptive immune system. These responses are initiated by binding of the

agents to pathogen-recognition receptors (PRRs). Although PRRs are expressed on many

cell types, research on PRR activation mainly focuses on DCs, because of their important

role in controlling immune responses [29]. Among agents that trigger these receptors

pathogen-associated molecular patterns (PAMPs) are most relevant in the context of this

review. PAMPs usually represent exogenous signals, such as the conservative motifs of

microbial products [30]. The function of DCs in the initiation and regulation of the adaptive

immune response will be discussed later in this review.

Pattern-recognition receptors

The innate immune response is mediated by the PRRs, of which Toll-like receptors (TLRs)

have been a central focus for immunologists and vaccinologists after they were discovered

by Medzitov and Janeway in 1997 [31]. TLRs are important PRRs involved in host defence

against a variety of pathogens in general and also in the skin. So far, ten TLR members have

been identified in humans and three more in mice, each thought to selectively recognize


15

diverse bacterial or viral stimuli or endogenous signals [32]. TLRs can be divided into

subfamilies, according to the ligands they recognise and to their cellular localisation. The

subfamily of TLR1, 2, 4 and 6 recognises lipids, whereas TLR3, 7, 8, and 9 recognise nucleic

acids [30]. Generally, TLRs detecting bacterial products other than nucleic acids (TLR1, 2, 4,

5, 6 and 11) are expressed on the cell surface, whereas those detecting nucleic acids (TLR

3, 7, 8, and 9) are located intracellularly, typically on late endosomes or lysosomes [33].

In the skin, most studies focus on TLR expression on LCs and dDCs, which is dissimilar and

also differs from other subtypes of DCs at mucosal surfaces or in the blood circulation.

Epidermal LCs freshly isolated from the human skin express TLR1-3, 6 and 10 but not TLR4

and 5 [34, 35]. dDCs do express TLR4 and 5, in addition to TLR2, 6, 8 and 10 necessary for

recognition of bacterial PAMPs [36]. Besides DCs, keratinocytes also express TLR1-6, 9 and

10 [37-40]. Furthermore, Yu et al. recently showed that cultured human melanocytes

express TLR2-4, 7 and 9 [41], which attributes a possible role for these cells in the immune

response. The TLR distribution on immune active skin cells (human and mouse) are

presented in Table 2. Some of the data are still under debate because of different isolation

methods for generating the specific types of cells. This DC heterogeneity and the

differences in the epithelial microenvironment may influence the immune modulation

function of certain adjuvants and thereby the choice of adjuvants for TCI.

When activated, TLRs recruit adapter molecules within the cytoplasm of cells to propagate

a signal, which ultimately leads to the induction or suppression of genes that orchestrate

the inflammatory response. It is generally accepted that the detection of pathogens by

TLRs initiates the mobilization of the host defence against most, if not all, infectious

agents. However, recent results highlight the role of other PRRs that cooperate with TLRs

or compensate for TLR specialization [49]. In the absence of TLR activities, most viruses and

intracellular bacteria are recognized by alternative intracellular receptor families, including

nucleotide oligomerisation domain (NOD)-like receptors (NLRs) [50-52], retinoic acid

inducible gene based (RIG)-I-like receptors (RLRs) and C-type lectin-like receptors (CLRs)

[53-55]. In general, activation and maturation of DCs are the consequence of signal

Cell type Human Mouse

Keratinocytes 1-6, 9, 10 2, 4, 7, 9

LCs 1, 2, 3, 6, 10 2, 3, 4, 7, 9

dDCs 2, 4, 5, 6, 8, 10 9

Myeloid DCs 1-4 1-4, 7, 9

Plasmacytoid DCs 7, 9 7, 9

Macrophages/Monocytes 1, 2, 4, 5, 8 3, 4, 7, 9

Mast cells 3, 9 2, 3, 4, 7, 8, 9

Table 2. TLR distribution in

immune active skin cells

[34-40, 42-48].

Chapter 2

16

transduction within the PRR network, resulting in appropriate immunity against invading

pathogens.

Adaptive immunity

Adaptive immunity provides pathogen-specific, long-lasting protection to the host. Similar

to those at other immunological sites, skin DCs are an important link between innate and

adaptive immunity (figure 3) [29]. Upon activation, the DCs will maturate and migrate to

the lymph nodes, where they present epitopes via MHC I and II to respectively CD8+ and

CD4+ T cells [29]. Adaptive immunity starts with activation and polarization of lymphocytes

via DC-T cell interaction, followed by proliferation of T and B lymphocytes in the secondary

lymphoid organs (figure 3). T and B cells develop from a common lymphoid progenitor in

the bone marrow. T cells differentiate further into either CD4+ (helper) or CD8

+ (cytotoxic)

T cells. Antigen recognition by B and T lymphocytes differs from that by cells of the innate

immune system in that the latter recognize conservative motifs using PRRs, whereas each

B- or T-cell receptor specifically recognizes a unique epitope. Below we will briefly describe

the general function of T and B cells in the immune response, followed by the specific role

of skin DCs in induction of the adaptive immune response.

Figure 3. Schematic representation of the cells involved in the general innate and adaptive

immune response. Upon infection with a pathogen the cells of the innate immune response

offer immediate, but short-lasting help. This leads to DC activation, which forms the bridge

between the innate and adaptive immunity. The cells of the adaptive immune response

provide pathogen-specific, long-lasting protection.


17

Effector cells of the adaptive immune response

T cell activation depends not solely on specific recognition by the T cell receptor of antigen

presented by APCs; the interaction of co-stimulatory molecules (CD80 and CD86 on APC

with CD28 on T cells), the secretion of stimulatory cytokines (IL-2) and a polarization signal

(e.g. IL-4 and interferon-γ (IFN-γ)) are also necessary [43, 56]. TLR recognition by APCs

contributes to this activation process. As mentioned above there are two different types of

effector T lymphocytes, but the CD4+ T cells (also called T helper, or Th cells) are further

classified in different subsets. The best studied subtypes are the Th1 and Th2 cells. Most

bacterial and viral products, including nearly all TLR ligands drive the differentiation

towards a Th1 functional phenotype [57, 58]. Th1 cells secrete IL-2 and IFN-γ cytokines,

support the production of IgG2a antibodies in mice (IgG1 in humans) and stimulate cell-

mediated immunity against intracellular pathogens [59]. In the presence of parasitic

pathogens and allergens, naïve CD4+ T cells differentiate into Th2 cells. Th2 type cytokines,

including IL-4, IL-5 and IL-13, mediate humoral immunity and support the production of the

IgG1 (in mice) and IgE subclasses. The discovery of Th17 that are induced by extracellular

bacteria and were also implicated to have a role in (auto)immune disorders, regulatory T

cells (Treg), follicular helper T cells (Tfh) [60-62] and more recently also Th9 and Th22 [63-

66], the latter being described to be important in skin homeostasis and pathology [65, 66],

further complicates the CD4+ paradigm (figure 3). The dominant type of immune response

induced is determined by many factors, including the route of antigen delivery, antigen

dose, duration of antigen presentation, number, or frequency of immunisations and

inclusion of adjuvants. The main function of CD8+ T cells is to kill tumour cells or cells

infected by viruses or intracellular bacteria. Naïve CD8+ T cells become cytotoxic T cells

(CTL) when they are activated by DCs presenting antigens in the context of MHC I in the

lymph nodes. Upon activation they migrate back to the sites of infection to clear infected

or tumour cells. The activation of a CD8+

T cell response is the main mechanism of vaccines

developed for cancer therapy. Th1 CD4+ T cells seem to be required to help CD8

+ T cells

fight certain pathogens. Cross-talk between both types of effector T cells is mediated by

CD40-CD40L interactions [67].

The humoral immune response is mediated by B lymphocytes. These cells recognize free

(soluble) antigen in the blood or lymph using their membrane-bound IgM or IgD, which act

as B cell receptors. In most cases, B cell activation, e.g. clonal proliferation and terminal

differentiation into plasma cells, requires not only recognition of antigens, but also

cytokines produced by activated CD4+ T cells. Special antigens, such as repeating

carbohydrate epitopes from many bacteria, may also directly stimulate B cells by cross-

linking the IgM antigen receptors, thereby activating them in a T cell-independent manner

[68]. B cells can take up antigens and present them by MHC II to CD4+ T cells. Interactions

between B cells and CD4+ T cells mutually stimulate each other. Activated Th2 cells express

Chapter 2

18

CD40L on their surface which can interact with CD40 on B cells. In this way, the activation

of more effector T cells and the production of antibodies are sustained [69]. These

antibodies assist in the destruction of microbes by binding to them, thereby making them

easier targets for phagocytes and facilitating activation of the complement system.

The basis of vaccination lays in the existence of memory B and CD4+ and/or CD8

+ T cells.

These cells enable faster and stronger responses to pathogen-derived antigens

encountered before [70]. They are long-lived and, upon contact with a familiar antigen,

start dividing quickly and induce secretion of large amounts of antibodies and/or cellular

responses. This process is nicely illustrated by the enhanced immune response obtained

after booster vaccinations.

Interestingly, recent findings implicate an important role for skin resident T cells in

memory responses. Not only were they found to outnumber the T cells in the blood [71],

but in addition memory T cells were found to survive long-term in the skin and are crucial

in the control of an infection upon a secondary challenge [72, 73]. Most interestingly for

vaccination purposes, it was found that after antigen presentation to naïve T cells by DC in

the lymph nodes, skin homing effector memory T cells were not only migrating to the site

of infection, but distributed to all parts of the skin. After the pathogen was cleared, these

cells remained resident locally in the skin. Moreover, during primary infection, proliferating

T cells in the skin draining lymph nodes were also found to be distributed to lymph nodes

draining other tissues, and subsequently these cells were found to reside in those

peripheral tissues, including gut and lung [74]. How infection or immunisation via the skin

can lead to local as well as systemic memory responses was recently reviewed by Clark [75]

and implicates that immunisation through the skin can generate widespread systemic

immunity through populations of tissue resident effector memory T cells.

Skin DCs as a bridge between innate and adaptive immunity

The DCs link the innate to the adaptive immune response. They not only sample the

environment, but afterwards they process antigens and undergo a maturation and

differentiation process. In the skin, differentiation of LCs and dDCs during maturation

includes increased expression of MHC and co-stimulatory molecules, increased production

of cytokines such as IL-1β, IL-6, IL-12, and chemokines such as CXCL1, 2, 3, 8 and CCL3-5, as

well as the enhanced migration of these cells from the skin to the draining lymph nodes

[76, 77]. In the lymph nodes, skin-derived DCs present the processed antigens of the

pathogen, together with the activation stimuli, to naïve resting T-lymphocytes surrounding

them [78, 79]. This occurs in an antigen-specific fashion and results in the T cell expansion

into extremely potent immune stimulatory cells, controlling the development of adaptive

immunity [80].


19

Several distinct types of DCs are present in human skin and this is an emerging field of

research [81, 82]. The most evident distinction is between the LCs and the dDCs, two types

of myeloid DCs. LCs are epidermal DCs that account for only 1% of the total epidermal cell

population, but cover nearly 20% of the skin surface area [83]. Human LCs can be

distinguished from other subsets of DCs by their expression of langerin/CD207, a C-type

lectin that induces the formation of a unique intracytoplasmic organelle, the Birbeck

granule [84]. Furthermore LCs express E-cadherin and high levels of CD1a, responsible for

the presentation of lipid antigens to T cells [85]. Two subsets of DCs in the dermis have

been distinguished until now: CD14+ dDCs and CD1a

+ dDCs [86]. CD14

+ dDCs are most

easily characterized by expression of DC-SIGN (DC-specific intercellular adhesion molecule-

3 (ICAM-3)- grabbing non-integrin), also known as CD209, in addition to CD1c and CD11b

[87, 88]. Dermal CD1a+ DCs were shown to express an intermediate phenotype between

CD14+

DCs and LCs (figure 4, [36]). dDCs are present in higher numbers than LCs in the skin.

These cells are continuously produced from the hematopoietic stem cells and distributed

in an immature state as antigen-capturing cells.

Recently a new subset of skin DCs has been found in mice, i.e. the langerin+ CD103

+ dDC

[89-91]. This subtype differs from LCs and the classical dDCs by a low expression of CD11b

and high expression of CD103 [92, 93]. Furthermore, LCs were found to express epithelial-

cell adhesion molecule (EpCAM) [91, 94], an adhesion molecule that distinguishes them

from both types of mouse dDCs. There is some speculation that the CD1a+ subset of dDCs

in human skin might correspond to the langerin+ CD103

+ dDC found in mice, but that still

remains to be investigated [81].

Figure 4. Dendritic cells

present in the epidermis and

dermis of human skin. These

cells differ in respect to the

expression of cell markers

and the interaction with

cells of the adaptive immune

response. While LCs are

more involved in the

interaction with CD4+ (to

preferentially induce Th2)

and CD8+ T cells, CD14

+ dDCs

have the ability to induce B

cells to switch isotype and

become plasma cells by

direct contact and via the

induction of follicular helper

T cells (CD4+). CD1a

+ dDCs

express an intermediate

phenotype [88, 95-98].

Chapter 2

20

The different DC subsets in human skin each have distinct functions in the adaptive

immune response (figure 4). Both dDCs and LCs isolated from human skin were shown to

activate naïve CD4+ T cells, but LCs induced the secretion of Th2 type cytokines, which

CD14+ DCs did not [95]. The CD1a

+ dDCs provoked some secretion of Th2 type cytokines,

but less compared to the LCs. CD14+ dDCs promote the differentiation of naïve B cells into

IgM-secreting plasma cells through the secretion of IL-6 and IL-12 [99, 100]. This effect was

not observed with LCs, which not only failed to induce high levels of IgM production, but

isotype switching of naïve B cells and the production of IgG was also only induced by CD14+

dDCs [95]. These results, together with studies performed in mice showing that langerin-

DCs preferentially migrated to the outer paracortex of the lymph nodes, just beneath the B

cell follicles [101, 102], indicate that CD14+ dDCs are important for the induction of

humoral immune responses.

Human LCs were shown to have a function in the CD8+

T cell response [95]. Both isolated

and in vitro cultured LCs were shown to induce proliferation of naïve CD8+ T cells to a

higher extent than CD14+ dDCs [95, 103]. Also in mice a role was ascribed to LCs in the

cross-presentation of antigens to CD8+ T cells, as it was shown that upon stimulation

langerin+ DCs migrate into the T cell-rich inner paracortex [102]. However, in this study the

relative contribution of LCs and CD103+ langerin

+ was not explored. This would be very

interesting as a recent study suggests that in mice the CD103+ langerin

+ dDCs are

responsible for cross-presentation in vivo [92]. Coming back to the immune response in

humans, the function of the CD1a+ dDC remains not fully understood. Currently, this topic

is of great interest and important for the design of novel vaccines targeting specific DC

subsets [104].

Even though these studies clearly indicate the crucial role of skin resident DCs in the

immune response, soluble antigen can also directly diffuse to the draining lymph nodes

through the lymphatic system [105]. Here the antigen can be taken up by a large

population of lymph node resident DCs. This process is much faster, thereby inducing two

distinct waves of antigen delivery to lymph nodes [101], which can induce different

immune responses [106].

Transcutaneous immunisation

As mentioned before, for subunit vaccines the co-application of adjuvants with the

antigen(s) is required for induction of a strong immune response. This approach also holds

for TCI, but for successful TCI transport of the antigen and adjuvant across the skin poses

an additional challenge. Here we will briefly discuss the adjuvants used for TCI, followed by

a more in depth overview of the physical methods utilized to overcome the stratum

barrier.


21

Adjuvants used in TCI

Due to the advances in understanding innate immunity, the range of adjuvant candidates is

enlarging dramatically. In many established as well as experimental vaccine formulations,

ligands for PRRs, cytokines or messenger molecules involved in the signal transduction of

PRRs are incorporated (as reviewed by Wilson-Welder et al. [4]). The most commonly used

adjuvants are colloidal aluminium hydroxide and aluminium phosphate, commonly

referred to as alum [107, 108]. Other adjuvants recently approved for human use are

monophosphoryl lipid A (MPL) and MF59, an oil-in water emulsion containing squalene,

which has been accepted in Europe. These three adjuvants have not been used in TCI,

probably due to their relatively large size, which limits transport across the skin barrier.

Furthermore the depot effect of alum is undesirable for TCI. There are many other

experimental adjuvants commonly used; here we will focus on those used for TCI. Since

these adjuvants are still in pre-clinical development, the discussion below concerns animal

(mouse) studies.

Bacterial exotoxins

Bacterial ADP-ribosylating exotoxins possess a high degree of adjuvanticity and are

therefore the adjuvants that are most often used pre-clinically for TCI. Among them,

cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) are the ones most intensively

studied [109]. CT and LT bind to the GM1-ganglioside receptor (B subunit) and have ADP-

ribosyl transferase activity (A subunit) [110-114]. CT and LT do not only function as

adjuvants, but in addition provoke the formation of anti-CT and -LT antibodies. In the first

TCI study, Glenn et al. showed that application of CT on intact mouse skin resulted in anti-

CT antibodies [115]. As this was an excellent result, this study was followed by many others

showing that CT enhances the immune response against other antigens [115-120]. LT was

shown to possess similar adjuvanticity [118, 121-125]. These studies are summarized in

Table 3A. CT and LT do not only improve the total immune response, but affect the quality

of the immune response as well, although this is still under debate. While there are studies

indicating that mainly a Th1 bias with enhanced IgG2a levels is induced [126-130], others

point to a Th2 bias [119, 120, 123, 131] or a mixed response [121, 122]. Besides antibody

responses, it was shown that CT can induce a cytotoxic T cell response [120] and that the

CTA and CTB subunit are responsible for different cytokine expression from restimulated

lymphocytes isolated from the spleen of immunized mice [132]. Of course the antigen,

mouse model and dose can also have a profound influence on the elicited immune

response. Additional studies are needed to further elucidate how CT and LT affect the

immune response. It remains an important question how bulky antigens as well as

adjuvants can penetrate the stratum corneum barrier when applied on intact skin. Beignon

Chapter 2

22

et al. showed that CT could penetrate hydrated mouse skin in vivo, and was found

preferably around the hair follicles [133]. However, there are also studies in which the skin

is pre-treated by abrasion, which could play a significant role.

TLR ligands

As described above, TLRs are important signalling molecules which cells use to sense

danger. It is therefore a logical approach to use either purified or synthetic TLR ligands as

adjuvants for vaccination purposes. One example is CpG. Prokaryotic DNA contains

unmethylated CpG dinucleotides within nucleic acid motifs that are recognized by TLR9 of

vertebrates [134]. By signalling through TLR9, CpG induces the secretion of pro-

inflammatory cytokines such as IL-12, TNF-α and IFN-γ, resulting in a Th1 biased response

[135, 136]. Scharton-Kersten et al. first showed that CpG functions as an adjuvant when

applied with DT on intact skin, as elevated anti-DT IgG titres were observed [121]. The

same was observed for TCI with CpG co-administered with the model antigen ovalbumin

(OVA) or DNA vaccine encoding influenza M protein [131, 137]. In the skin CpG induces LC

and DC maturation and migration of these APCs to the lymph nodes [138, 139]. CpG is

capable of modulating pre-existing immune response causing a switch from a Th2 biased

response to a Th1 biased response [131, 140, 141]. Topical application of a HIV peptide

together with a mixture of CT and CpG induced a strong HIV-specific CTL response resulting

in protection against a mucosal challenge [142]. CpG has been used as an adjuvant in

clinical immunisation studies using different vaccination routes [143-145], however not yet

in clinical TCI studies. Other TLR ligands have also been used pre-clinically in TCI studies

and are summarised in Table 3A. The many different strains and type of vaccines used in

influenza immunisation studies are listed in Table 3B.

Table 3B. Type of influenza antigen used in studies mentioned in Table 3A.

Reference Type of influenza vaccine used

[118, 131, 147]

[137]

[127] [129]

HA:307-319 peptide

M protein DNA vaccine

H3N2 subunit vaccine (A/Panama/2007/99 RESVIR-17)

Inactivated H1N1 virus (A/PR/8/34)

Physical methods to overcome the skin barrier

Disruption of the skin barrier increases the transcutaneous permeation of antigen and

makes it more readily available for sampling by APCs (figure 5). Moreover, it is known that

skin barrier disruption can activate the immune system, inducing the secretion of pro-

inflammatory cytokines by keratinocytes and resulting in DC activation [152, 153]. This

makes it attractive to develop physical methods to overcome the skin barrier.


23

Table 3A. Adjuvants used in pre-clinical TCI studies.

Adjuvant Dose (µg) Antigen Dose (µg) Type of immune

response Reference

CT 100

100

25-100

25-50

100

100

100

50-100

50

DT

DT

TT*

Influenza

Influenza

VP1 (FMD**)

G2Na/G5

(RSV***)

OVA

PCLUS3-P18IIIB

(HIV****)

100

100

25-50

100

30-100

100

150

25-300

50

IgG, mixed

IgG1/IgG2a, CD4+

IgG, IgG2a

IgG, CD4+

IgG, IgG1, CD4+

IgG, mixed

IgG1/IgG2a

IgG1

IgG1

IgG, IgG2a, CTL, CD4+

CTL

[115, 117,

121, 146]

[126, 127,

130]

[115, 122]

[118, 131,

137]

[127, 129]

[133]

[119]

[120, 132]

[142]

LT (and

derivatives)

20-100

1-100

100

50

50

DT

TT

β-gal

BSA*****

Influenza

10-100

5-100

100

100

100

IgG, IgG2a, CD4+

IgG, IgG1, CD4+

IgG

IgG

CD4+

[121, 128]

[121-123,

147]

[118]

[125]

[118, 147]

CpG 10-100

12.5-100

100

50

50-500

DT

Influenza

TT

HIV

OVA

5-100

100

20

50

50-100

IgG, IgG2a, CD4+

IgG2a, CD4+

IgG, IgG2a

CTL

IgG, CTL, CD4+

[121, 140]

[131, 137]

[123]

[142]

[148, 149]

TLR7

Imiquimod Resiquimod

50000

100

OVA

OVA

150

100

CTL

CTL

[150]

[151]

* TT: tetanus toxoid

** FMD: feet and mouth disease

*** RSV: respiratory syncytial virus

**** HIV: human immunodeficiency virus

***** BSA: bovine serum albumin

Chapter 2

24

One important factor to consider is what/where to target. As mentioned above LCs and

dDCs in the skin are located in different skin layers and have a dissimilar, but not yet

completely understood, function in the skin immune system. Therefore, to assume that

any means of barrier disruption will lead to the desired immune response is not true.

Development of vaccine delivery devices should go in close collaboration with

immunological studies into the exact function of the skin residing immune cells. The clinical

safety of the devices described here, though of major importance, has received little

attention so far. As the scope of this review is on the efficacy rather than the safety and

the latter was recently reviewed by Donnelly et al. [154], we will not discuss this subject.

The most widely used method to date to overcome the skin barrier for cutaneous

immunisation is intradermal (ID) injection, invented by Mendel and Mantoux in the early

1900s [155] (figue 6A). With ID injection it is possible to deliver antigens into the dermis

precisely and reproducibly. Clinical trials with hepatitis B, influenza, and therapeutic cancer

vaccines have shown that ID vaccination is safe and effective. In many cases, stronger

immune responses with a lower antigen dose compared to SC or IM injection were

observed [156, 157]. However, traditional ID injection requires well-trained healthcare

workers; therefore new devices for ID injection are being developed. One example is the

Becton Dickinson (BD) microinjection system, SoluviaTM

(figure 6B). This is a prefilled

syringe with a single 1.5 mm-long, 30G intradermal needle designed to deliver 100-200 µl

fluid. It is now commercially available for a trivalent seasonal influenza vaccine (Sanofi-

Pasteur) [158, 159]. These studies underline the effectiveness of the skin as a site of

immunisation, but ID injection still employs long needles and causes pain. For vaccination

of healthy people TCI in a minimal-invasive manner would be more desirable.

Figure 5. Schematic illustration of several physical approaches and devices developed for TCI.


25

Microneedle arrays

One approach towards painless TCI is to dramatically reduce the size of needles so that

they are barely perceptible. The concept of the microneedle array for drug delivery

purposes essentially dates back to a patent, filed in 1976, by Gerstel and Place [160].

However, it was not until the 1990s that the technique became viable, as by then

fabrication techniques became available to produce these microneedle arrays in a

potentially cost-effective manner.

The term microneedles in the definition used here refers to needles shorter than 1 mm.

Theoretically, microneedles only need to pierce the 15-20 μm thick stratum corneum

before reaching the viable epidermis. However, the skin is an elastic, heterogeneous tissue

and slightly stretched in vivo. The mechanical and structural properties of the skin vary

significantly with age, skin type, hydration level, body location and among individuals [161,

162]. To ensure effective and reproducible piercing regardless of these factors,

microneedles need to be much longer than 20 µm [163], although the use of an applicator

may reduce the required microneedle length. Other parameters, such as microneedle

diameter, insertion depth, microneedle tip geometry and microneedle density also

influence skin perforation and antigen delivery [163-165]. For instance, very thin

microneedles are fragile, which results in an increased risk for fracture in the skin. To

overcome this risk, increased microneedle density helps to spread the surface forces

between each microneedle, thereby decreasing the risk for fracture in the skin [166]. On

the other hand, increased microneedle density can give rise to the ‘bed of nails’ effect and

not improves antigen delivery [163].

Numerous methods have been developed to fabricate a wide range of microneedles as

recently reviewed by Donnely et al. [154]. Microneedle technology is under active research

and various strategies were developed using microneedle arrays in transdermal drug

delivery, including TCI (Table 4A) [167, 168]. Below we will discuss the most important

strategies pursued so far.

Solid microneedles

A straightforward method is to perforate the skin with solid microneedle arrays and apply

antigens to the skin surface for subsequent diffusion into the skin. Henry et al.

demonstrated four orders of magnitude increase in permeability for calcein and BSA

through human epidermis in vitro after penetration with a microneedle array of 150 μm

needle length (figure 6E) [169]. Banks et al. reported that the flux across microneedle array

pre-treated skin was augmented by increasing the charge of the drug [170]. In our group,

Verbaan et al. showed that 200 nm particles can diffuse through conduits formed by a

solid microneedle array (300 μm long, 4×4 array, figure 6C) [171]. This microneedle array

was applied at a speed of 3 m/s by an electric applicator. In the absence of such an

Chapter 2

26

applicator no conduits were formed. For its application in TCI, Ding et al. have

demonstrated that pre-treatment of the skin using the same type of microneedle arrays

leads to a major improvement (1000-fold increase in antibody titres) in the

immunogenicity of topically applied DT in mice [127]. The immune response was further

boosted by co-application of CT. Given the fact that with microneedle pre-treatment only a

fraction of the vaccine formulation applied is transported to the APCs in the skin, the dose

of antigens can be further refined. We also showed that the immune responses induced

can be additionally improved and modulated by selective addition of adjuvants [126],

which may lower the antigen dose required. In general, microneedle array pre-treatment is

considered a simple approach for TCI with great potential, but parameters such as dose

and application time should be optimized. Recent studies indicate that the smaller the

entity, the easier the transport through the conduits, thereby limiting the potential of for

instance liposomes and nanoparticles as antigen carriers in TCI [130, 172]. More groups are

currently focusing on using solid microneedles for skin pre-treatment [173, 174], and these

systems could be used in future TCI studies. 3M has developed the Microstructured

Transdermal System (MTS) using solid microneedles, either coated or uncoated [175]. In

collaboration with VaxInnate these microneedles will be used for the delivery of an

influenza vaccine.

Coated microneedle arrays

Arrays of vaccine coated microneedles have been developed as an alternative to

microneedle pre-treatment. Coated microneedle arrays may not be very attractive for

transdermal drug delivery as only a limited amount of active compounds can be coated

onto the needles. However, this amount might be sufficient for antigens to generate a

protective immune response [167]. The concept of coated microneedle arrays is that they

are inserted into the skin and then removed, thus depositing their payload to a maximum

depth determined by the length of the microneedle and the application manner. Matriano

et al. showed, using 1 μg OVA on pre-coated microneedle arrays, a 100-fold increase in

immune response compared to IM injection of the same dose [176]. They used an array

with 300 μm long titanium microneedles, applied to the skin by an impact insertion

applicator. Later, Widera et al. from the same group carried out an extensive study on the

influence of OVA-coated microneedle properties on the immune response. The immune

response was found to be dose dependent, however, practically independent of depth of

delivery, density of microneedles, or area of application. Notably, OVA delivered with short

microneedles (225 μm) in a high density array (725 microneedles/cm2) induced a similar

immune response as compared to longer microneedles (600 μm) at a lower density (140

microneedles/cm2) [163]. This led to the development of the Macroflux system

® which is

now in a phase I clinical study for TCI with an influenza vaccine (figure 6D).


27

Coatings are usually applied by dipping microneedles in a vaccine formulation. A

systematic study performed by Gill and Prausnitz demonstrated that excipients reducing

surface tension of the coating solution improve coating uniformity, while excipients

increasing solution viscosity increase coating thickness. The amount of antigen coated can

be adjusted by the concentration of the coating solution. Coatings could be localized just

to the needle shafts and formulated to dissolve within 20 s in porcine cadaver skin [177-

180]. Another method is to use gas-jet coating, to achieve more uniform coating of densely

packed microprojections (figure 6F) [181]. Two groups focusing on coated microneedles

are the groups of Prausnitz and Kendall. While the former group uses rather long (up to

700 µm) and sparsely packed microneedles the latter uses very short (30-90 µm) and

densely packed microneedles, also called NanopatchTM

[181-183]. Initially a large

difference was reported in the amount of vaccine deposited in the skin, only 15% for the

short densely packed compared to 90% for the long sparsely packed microneedles [178,

181]. However, by applying the short microneedles with a speed of 2.5 m/s their pay-load

could be doubled, even though the majority of the vaccine still remained on the

NanopatchTM

[183].

Both types of coated microneedles have been employed successfully for TCI, in

immunisation studies with OVA, H3N2 influenza antigen, Fluvax® 2008, inactivated

influenza virus and Hepatitis C DNA vaccine, with doses ranging between 0.4 and 10 µg

[178-185]. Humoral and cellular antibody responses comparable to those induced after IM

or gene gun immunisation were observed.

From a formulation point of view it could be an advantage that dried antigen formulated

on the surface of the microneedles may improve the long-term stability [186]. However,

coating of antigens has also been reported to reduce the immunogenicity of the vaccine,

needing trehalose to partially retain the activity [185, 187].

Hollow microneedle arrays

By solid microneedle array pre-treatment, antigen delivery is based on passive diffusion

along the conduits created by the microneedles. Although this is a relatively easy approach

from a technical point of view, not all of the dose applied will be available to activate

immune cells in the skin due to limited transport through the conduits. Using hollow

microneedle arrays to inject the vaccine to a well defined depth in the skin, one can

precisely steer the flow rate using a syringe or a pump and provide a more controlled

vaccine delivery. The main technical demands are avoiding leakage and clogging of the

microneedles during injection [188]. Clogging can be prevented by using a bevelled tip

[164]. However, given the short needle length allowed, it will increase the chance for

leakage. Therefore an optimum in the flow rate, needle length and localization of the

opening are demanded. Furthermore, insertion of the microneedles using a drilling or

Chapter 2

28

vibrating motion may avoid the tissue compaction [165, 189]. Martanto et al. investigated

the influence of different parameters on the infusion flow rate and found the location of

the tip opening and retraction of the microneedle before injection to be of major

importance [164, 165].

The first hollow microneedle array, 150 μm long, made of silicon, was presented by

McAllister et al. in the late 1990s [190]. Recently, the potential of hollow microneedles for

vaccination purposes has received attention as it can both be used for TCI and ID

vaccination depending on the microneedle length [7, 191]. Van Damme et al. delivered α-

RIX influenza vaccine (3.3 μg of HA per strain) using a hollow microneedle array (450 μm

long, 4×1, MicronJet® developed by Nanopass, figure 6H & 6I) and elicited immune

responses similar to those induced by 15 μg HA per strain administered IM in human

volunteers [156].

Dissolvable microneedle arrays

Usage of dissolvable or biodegradable materials containing the vaccine components is an

elegant way to deliver a vaccine without the possibility of microneedles breaking off in the

skin. Miyano et al. were the first to report about maltose based microneedles [198],

followed by Ito et al., who used dextrin microneedles for the delivery of insulin and

erythropoietin [199, 200]. More data emerged recently, following this trend [201-205].

Before TCI studies using these systems, successful delivery of large molecules such as the

above mentioned insulin and erythropoietin and also BSA [201, 203, 205] and IgG [204]

was reported. Recently, Sullivan et al. showed that immunisation with polymeric

dissolvable microneedles containing inactivated influenza virus resulted in a strong

antibody and cellular response and provided protection against an influenza challenge

[206].The main challenge is to develop a fabrication technique which allows antigen to be

incorporated into the matrix of the microneedle materials in a mild procedure without

causing antigen breakdown and compromising material strength. The high temperatures

necessary to mould polymers led to significant drug loss [205]. Sullivan et al. proposed a

photo-polymerization method to use UV light to form microneedles without compromising

the activity of β-galactosidase [207].

Two companies, TheraJect and BioSerenTach (figure 6J) are currently developing

dissolvable microneedle systems for vaccine delivery. The TheraJect VaxMat®, made of a

sugar matrix containing vaccine components, are fabricated in various lengths from 100

μm to 1,000 μm and assembled with an adhesive patch. Upon piercing, the microneedles

dissolve and antigen diffuses into the epidermis and dermis within a few minutes [208].

Given water-proof packaging, fast-dissolving microneedle arrays provide a one-step

solution for TCI.


29

Figure 6. Examples of approaches and devices used for ID immunisation (A, B) and TCI (C-O). (A)

classical ID immunisation; (B) SoluviaTM

(BD) [192]; (C) Applicator with a solid microneedle array as

used in [171]; (D) solid microneedles of the Macroflux® [176]; (E) array of silicon microneedles

[169]; (F) Coated microneedles [183]; (G) coated and hollow microneedle arrays (3M); (H) silicon

hollow microneedle [127]; (I) hollow microneedle array, MicronJet® (NanoPass) [156]; (J)

dissolvable microneedle array from BioSerenTach [193]; (K &L) blunt-tipped microneedle array,

OnVax® (BD) and its electron microscopy image [194]; (M) smart vaccine patch from Intercell [195];

(N) PassPortTM

patch (Altea) [196]; (O) powder jet systems, adapted from [197].

Chapter 2

30

Other microneedle arrays

BD’s OnVax®

device employs blunt-tipped microneedles being 50–200 μm in length over a 1

cm2 area (figure 6K and L). These “microenhancer arrays” were used to gently scrape the

skin containing a vaccine solution in order to expose the epidermis to the vaccine without

pain sensation [194]. Using a hepatitis B DNA vaccine (100 μg dose), stronger and less

variable immune responses were achieved compared to IM and ID injection with the same

dose. Moreover, 100% of seroconversion was achieved after only two immunisations,

whereas only 40-50% conversion was obtained by the conventional techniques. This

enables “wipe and go” vaccination with easy self-administration [194]. However, although

DNA vaccines can be produced in larger quantity with lower costs compared to subunit

vaccines, the amount of vaccine delivered using this device is very low.

The EasyVaxTM

device has been designed to insert coated microneedle arrays into the skin

followed by electrical pulses to deliver DNA into the cells (figure 6M). Following this

procedure, TCI with a smallpox DNA vaccine induced neutralizing antibody titres greater

than those elicited by the traditional live virus vaccine administered by scarification [209].

Even though the animal studies with the EasyVaxTM

are promising, the main drawback of

this approach is the complexity of the device.

The use of an applicator

Verbaan et al. showed that 300 μm long microneedles were not able to pierce the skin

when applied manually. It was found that the elasticity of the skin results in folding of the

skin surrounding the microneedles [210]. Consequently, an electric applicator, providing an

injection speed of 3 m/s, enabled the 300 μm long microneedle arrays, and even the 245

μm long ones to pierce the skin effectively and reproducibly (figure 6C) [171]. Crichton et

al. showed that, by varying the application speed of coated microneedles, the amount of

microneedles piercing the skin and the delivered dose can be increased [183]. More

importantly, they showed that the antigen can be targeted either to the epidermis or to

both the epidermis and the dermis, so one can decide whether to deliver the majority of

the vaccine only to the LCs or also to the dDCs. These studies highlighted the necessity of

an applicator. It is conceivable that a higher velocity is needed to counteract the elasticity

and ensure efficient penetration of the skin. A mechanical applicator device is superior to

manual application as it can provide an adjustable yet consistent projection speed, with

minimal inter-individual variability. Applicators available on the market or under

development are either integrated with the microneedle patch or supplied as a separate

device, for single or repeated use, respectively [154]. It is possible to pierce manually using

longer microneedles, but with a less precise penetration depth.

Some trends can be noticed from studies performed during the last ten years in this field:


31

i) instead of piercing on dermatomed skin in vitro, since recently very relevant

experimental evaluations are being performed also in vivo;

ii) an impact applicator or insertion device is often used. It provides defined

projection speed (faster than applied manually) of the microneedle arrays, thereby

enhancing the uniformity of skin piercing and allowing shorter needle lengths;

iii) coated microneedle arrays (as well as dissolvable ones) may provide TCI with

simple patch design, resulting in competitive products on the market;

iv) hollow microneedle arrays have gained more attention for their potential of precise

dose control, while the device design needs to be improved with respect to

leakage-free injection and simplicity;

v) antigen doses used in TCI fall in broad range depending on the animal model and

the delivery methods. Studies on dose-dependency and dose refinement should be

included in future TCI studies.

Other approaches

Besides microneedles, a large number of approaches have emerged to overcome the skin

barrier. These methods have been reviewed extensively elsewhere [167, 211, 212] and we

will shortly discuss the most promising techniques for TCI (Table 4B).

One way to overcome the stratum corneum barrier is to remove it by tape-stripping,

abrasion or thermo-ablation. Glenn et al. were a pioneer in this field, showing that mild

abrasion results in the removal of approximately 29% of the stratum corneum, which

greatly enhances the passive diffusion of an antigen. Stratum corneum disruption prior to

applying a vaccine patch (containing 50 μg LT) resulted in IgG titres comparable to those

obtained after active toxin infection and those induced by oral cholera vaccine [213, 214].

Later on they developed the Skin Preparation System (SPS) which was successful in phase I

and II studies against traveller’s diarrhoea [149, 215, 216] and has currently entered phase

III development (figure 6M). This would be the first vaccine delivered with a patch on the

market. The mechanism of action of these patches partly depends on occlusion of the skin

they cover, which increases the hydration of the skin. Increased hydration progressively

increases its permeability, due to swelling of the corneocytes, pooling of fluid in the

intercellular spaces and dramatic microscopic changes in its structure at very high

hydration levels [217]. The PassPortTM

patch system (figure 6N), developed by Altea,

creates 80 micropores within a 1 cm2 area using thermo-ablation [218]. An applicator is

employed to release a single pulse of energy. TCI using this system by application of a

prime and two booster vaccinations with 3 μg doses of recombinant H5 influenza

hemagglutinin adjuvanted with 25 μg CpG with 4 week intervals induced robust serum

antibody responses in mice and provided protection against a lethal challenge with a highly

pathogenic avian H5N1 influenza virus [218].

Chapter 2

32

Besides heat, ultrasound and electrical pulses have also been used to disturb the stratum

corneum. These techniques have not yet been used extensively, due to complicated

devices which still need to be optimized. However, preliminary studies show that both

methods are able to induce an immune response, although with relatively high antigen and

adjuvant doses [219, 220].

Finally, vaccines can be delivered by powder or liquid jet injections. A lot of studies have

been performed using epidermal powder immunisation, showing protective immune

responses against influenza, hepatitis B and DT with doses ranging from 0.2 – 5 µg [140,

221-223]. This device is now acquired by Pfizer (PMEDTM

, figure 6O) to target dry powder

DNA vaccines to mainly the epidermis of the skin [224-229]. The high impact with which

very small sugar or gold coated particles enter the skin will disrupt cells, thereby inducing

LC activation and migration from the skin in a similar fashion as after microneedle

application [222]. This disruption causes mild side effects, such as application site burning,

which usually resolves within hours [225, 229]. Liquid jet injections, very popular until the

1985 hepatitis outbreak [230], have now regained interest with safer design, e.g.

disposable cartridges prefilled with vaccines [231].

Table 4. New technologies targeting vaccine delivery into the skin.

A: Microneedle-related approaches

Technology Vaccine (development phase) Company or Ref

Hollow needles (ID)

• Soluvia™

• Nanoject

• other systems

Hollow microneedles (TCI)

• MicronJet®

Trivalent inactivated seasonal influenza

vaccine (phase III)

not available

recombinant protective anthrax vaccine

(pre-clinical)

Trivalent subunit (HA) seasonal influenza

vaccine (phase I)

BD/Sanofi-Pasteur,

[159]

Debiotech

BD, [232]

NanoPass, [156]

Solid microneedles

• MTS*

• OnVax®

• other systems

OVA, M2E-flagellin influenza subunit

vaccine (pre-clinical)

Hepatitis B DNA vaccine (pre-clinical)

DT, influenza subunit vaccine (pre-

clinical)

3M & VaxInnate

[175]

BD, [194]

[126, 127, 130, 172]


33

Coated microneedles

• Macroflux®

• MTS

• other systems

Influenza vaccine (phase I)

OVA (pre-clinical)

not available

OVA, hepatitis C DNA vaccine,

inactivated influenza virus (pre-clinical)

Zosano (Alza)

[163, 176]

3M

[178-183]

Dissolvable microneedles

• VaxMat®

• other systems

not available

Inactivated influenza vaccine

TheraJect

BioSerenTach, [193,

206]

Microneedles with

electroporation

• EasyVax®

Smallpox DNA vaccine (pre-clinical)

[209]

B: Other physical and chemical approaches

Technology Vaccine/(development phase) Company or Ref

Skin abrasion

• SPS**

• CSSS***

Trivalent inactivated seasonal influenza

(phase II)

LT for travelers’ diarrhea (phase III)

Virosomal influenza subunit vaccine

(clinical phase II)

Recombinant protective anthrax antigen

(pre-clinical)

Inactivated influenza/tetanus vaccine or

subunit influenza vaccine(phase I)

DT (pre-clinical)

Melanoma or HIV epitopes (phase I)

Vaccinia Ankara (pre-clinical)

Iomai/Intercell

[233, 234]

[235]

[236]

[237, 238]

[121]

[239]

[240]

Low frequency (20 kHz)

ultrasound

TT (pre-clinical) [219]

Electroporation

• Elgen® / CELLECTRA

®

• other systems

HIV & influenza DNA vaccines

OVA peptide (pre-clinical), pGL3

luciferase DNA

Inovio, [241, 242]

[220, 243]

Chapter 2

34

Thermo-ablation

• PassPortTM

system

(recombinant) influenza protein (pre-

clinical)

Altea, [218]

Jet immunisation

• PMEDTM

(powder)

• Biojector® 2000

(liquid)

• PharmaJet® (liquid)

• Mini-ject

HIV DNA vaccine (pre-clinical)

HSV**** type 2 DNA vaccine (phase I)

DNA melanoma gp100 (phase I)

Influenza DNA vaccine (phase I)

Hepatitis B DNA vaccine (phase II)

Influenza DNA vaccine (phase I)

Malaria DNA vaccine (phase I)

HIV DNA vaccine (phase I)

Inactivated hepatitis A vaccine(phase I)

Rotavirus, Dengue DNA (pre-clinical)

Inactivated polio vaccine (phase I)

Measles-mumps-rubella

Yellow fever

not available

[226]

[228]

[227]

[225]

[224]

[229]

[244, 245]

Bioject, [246]

[109]

[247, 248]

PharmaJet Inc.

Valeritas

* MTS: microstructured transdermal system

** SPS: Skin preparation system

*** CSSS: cyanoacrylate skin surface stripping

**** HSV: herpes simplex virus

The stages of development of the approaches mentioned are summarized in Table 4. The

long list of strategies/devices, developed to overcome the skin barrier and enable painless

TCI, reflects a very competitive and fast developing field. Combining techniques might be

necessary to target the preferred APCs. For instance, tape-stripping and microneedle

arrays with very short needle lengths will expose mainly LCs to the antigens following TCI,

whereas ligands binding to specific receptors may be utilized to home an antigen to a

single skin DC subset.


35

Design of novel formulations

Formulation of antigens in particulate carriers is a popular strategy to improve vaccine

delivery, also via the transcutaneous route [249, 250]. The usage of nanoparticles as

antigen carriers has several advantages. They can retain the antigen at the delivery site for

a prolonged period [4] and improve the uptake of antigens by APCs, because of their

similar size and structure to microorganisms, the natural pathogens which are actively

sampled by the APCs [251]. Another advantage is the possibility to encapsulate both

antigen and adjuvant in the same particle, which is suggested to enhance the

immunogenicity [252]. However, the usage of nanoparticles for TCI so far is limited. The

focus has mainly been on lipid vesicles, i.e. closed spherical structures consisting of bilayers

of hydrated amphiphilic lipids or other amphiphilic compounds. Especially cationic

liposomes have been extensively explored as carriers for protein and DNA vaccines as they

can carry both membrane-associated and water soluble antigens [253, 254]. In particular,

elastic vesicles, which have a flexible bilayer, have been used as they are supposed to

penetrate the stratum corneum more easily as compared to conventional liposomes.

Transfersomes®

Transfersomes® are ultra-deformable liposomes, generated by incorporation of a

surfactant in the lipid bilayer [255, 256]. Transfersomes® are applied non-occlusively as it

has been suggested that the hydration gradient in the stratum corneum will drive the

intact vesicles into the viable epidermis [257]. However, this claim has not yet been

substantiated [258]. Nevertheless, several groups have independently reported that

Transfersomes® substantially increase the transport of small molecules across the stratum

corneum [255, 259-261].

The use of Transfersomes® to formulate antigens in TCI has also been reported in a few

studies. When using antigens such as human serum albumin, gap junction protein and TT,

potent humoral immune responses were induced in murine models with antibody levels

comparable to those obtained through SC injection [262-264]. Transfersomes® prepared

with soybean phosphatidylcholine (PC), Span 80 and ethanol, were loaded with hepatitis B

surface antigen (HBsAg). Comparable IgG titres and much higher secretory IgA titters

against HBsAg were induced when elastic liposomes loaded with 10 μg HBsAg were applied

onto intact mouse skin as compared to those obtained by IM injection of the same dose of

alum-adsorbed HBsAg [265]. However, in these studies no washing step was included after

topical antigen application on the back of the animals to remove the remaining

formulations. This raises the question if the immune responses were purely induced by TCI

or if oral delivery also contributed, e.g. through grooming of the rodents. In contrast in our

group elastic cationic liposomes made of PC, Span 80 and DOTAP (1,2-dioleoyl-3-

Chapter 2

36

trimethylammonium-propane chloride salt) did not improve the immune response when

loaded with DT and applied topically for 1 hour on intact mouse skin while the mice were

kept under anaesthesia [172].

Other elastic vesicles

Van den Bergh et al. introduced a series of surfactant-based elastic vesicles, consisting of a

bilayer-forming surfactant (sucrose-laurate ester), a surfactant (octaoxyethylene-laurate

ester) and a charge inducer (sodium bistridecyl sulfo succinate) [266, 267]. Enhanced

transdermal diffusion through intact skin of low-molecular weight drugs incorporated in

elastic vesicles has been observed and vesicular structures were visualized in deep layers

of the stratum corneum [268-270]. However, in the TCI studies using elastic vesicle-

incorporated DT on intact mouse skin, no enhanced immune response compared to a DT

solution was induced [172]. Other generations of elastic vesicles have also been evaluated

in TCI, e.g. with high percentage of ethanol being introduced into the vesicles, the

ethosomes; or constructed from non-ionic surfactant and cholesterol, the niosomes. TCI of

HBsAg-loaded ethosomes (composed of soybean PC and ethanol) has been reported to

induce immune response comparable to IM injection of HBsAg-alum [271]. BSA-loaded

niosomes, composed of Span 60, Span 85, cholesterol and stearylamine, were coated with

a modified polysaccharide O-palmitoyl mannan for targeted delivery to the LCs. This

niosomal formulation elicited significantly higher serum IgG titres as compared with alum-

adsorbed BSA and plain uncoated niosomes in TCI, but still lower than those obtained after

IM injection of an equivalent dose of BSA-alum [272].

Non-elastic nanoparticles

Besides elastic vesicles a modest number of groups have investigated the use of polymeric

nanoparticles for TCI, so far with limited success. Not surprisingly, Mattheolabakis et al.

found no advantage of antigen encapsulation in negatively charged polylactic acid (PLA)

nanoparticles when applied on intact skin [273]. Much smaller virus-like particles (40 nm),

when adjuvanted with CpG were able to induce humoral and cellular immune responses

[274]. To overcome the skin barrier we applied DT-loaded N-trimethyl chitosan (TMC)

nanoparticles on microneedle pre-treated skin [130]. Applying these nanoparticles for one

hour did not enhance the immune response compared to a DT solution. However, using a

longer application time, the nanoparticles were more efficient in potentiating the immune

response than a DT solution showing that TMC nanoparticle diffusion might be an

important limiting factor for the potency in TCI (unpublished results). Conjugating the

antigen to the polymer, thereby creating a smaller unit, could further increase the

potential of TMC [275-277]. In related in vitro studies it was shown that TMC itself acts as

an adjuvant and stimulates DC maturation [278, 279].


37

Other formulation issues

The delivery of nanoparticles in TCI needs to be further optimized and studies on the effect

of size, charge and intrinsic adjuvant properties of particulate carrier systems are needed.

Another important issue in the development of formulations for TCI is antigen stability. As

previously mentioned, coating of vaccines onto microneedles and the formulation into

biodegradable microneedles can affect the stability and effectiveness of the vaccine.

Similarly, encapsulation of antigens in nanoparticles may compromise their stability and

antigenicity. Moreover, when particulate antigen carriers are used, the colloidal stability of

the formulation should be addressed.

Limitations of animal models

The physiological differences between lab animals and humans should be taken into

consideration when transferring techniques of skin barrier disruption between species. For

example, skin of humans and mice is similarly densely populated with immune active cells,

especially LCs [280, 281]. However, human skin is much thicker and less hairy than mouse

skin. The epidermis of human skin is approximately 150 µm thick, compared to only 10 µm

in mice [282, 283]. Correspondingly, human LCs are located deeper in the skin [280]. This

fact is often not considered in the development of (micro)needle devices. Needle-lengths

vary considerably, up to 1 mm in length. The total skin thickness in mice is about 500 µm

[283], so if longer needles are used in mice studies they may reach the subcutaneous tissue

in addition to the dermis. Another difference between human skin and mouse skin is that

the latter is more hairy and consequently has more hair follicles. It has been shown that

hair follicles can be used for drug delivery [284] and nanoparticles were shown to

accumulate in the hair follicles and be taken up by surrounding APCs [240, 273] . TCI via

the hair follicles is also possible in human skin, as recently a clinical phase I study showed

induction of CD8+ T cells after immunisation with an inactivated influenza vaccine [237].

Finally, an important limitation of animal models is the restricted application time of

vaccines. Usually animals need to be anesthetized to prevent them from grooming and oral

vaccine delivery. Several of the particulate formulations mentioned above were only

applied for 1 hour [130, 172]. In humans patches can easily be left on the skin for up to 24

hours. These factors need to be taken into account when designing vaccination studies and

interpreting the data.

Concluding remarks

The skin is an important immunological site and, although it poses a complex barrier, has

the potential to be an ideal non-invasive vaccination site. TCI provides effective, easy-to-

use and painless vaccination with fewer side effects and safer handling than the

Chapter 2

38

conventional injections. The main challenges are to ensure accurate delivery of antigens

into the epidermal and/or dermal skin tissue and to formulate antigens with adjuvants

and/or carrier systems for selective activation of the proper PRRs existing in the skin DC

subsets. Many different approaches have been developed of which several ways may lead

to successful TCI (table 4). The most promising systems combine barrier disruption (most

probably with microneedles) with the addition of an adjuvant to the vaccine formulation.

For particulate formulations to be successful, this barrier disruption is of crucial

importance. However the most efficient way still needs to be found, which will require

joint efforts from immunologists, vaccinologists, pharmaceutical scientists and (fine)

mechanical engineers. Only then TCI can be further improved and essentially revolutionize

the current vaccination practice. The ideal strategy is to combine skin barrier disruption

approaches with use of adjuvants. With the advance in understanding the functional

specialization of skin DC subsets, immune modulation by targeted delivery of antigen and

adjuvant predominantly to one of these skin DC subsets is theoretically possible yet

challenging.

Acknowledgements

This research was performed under the framework of TI Pharma project number D5-106-1;

Vaccine delivery: alternatives for conventional multiple injection vaccines. The authors

thank Aat Mulder for providing the electron microscopy picture of the skin.


39

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280. Mulholland WJ, Arbuthnott EA, Bellhouse BJ, Cornhill JF, Austyn JM, Kendall MA, Cui Z, and Tirlapur

UK, Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin

model adopted for epidermal powdered immunization. J Invest Dermatol, 2006. 126(7): p. 1541-8.

281. Friedmann PS, Disappearance of epidermal Langerhans cells during PUVA therapy. Br J Dermatol,

1981. 105(2): p. 219-21.

282. Falstie-Jensen N, Spaun E, Brochner-Mortensen J, and Falstie-Jensen S, The influence of epidermal

thickness on transcutaneous oxygen pressure measurements in normal persons. Scand J Clin Lab

Invest, 1988. 48(6): p. 519-23.

283. Azzi L, El-Alfy M, Martel C, and Labrie F, Gender differences in mouse skin morphology and specific

effects of sex steroids and dehydroepiandrosterone. J Investig Dermatol, 2004. 124(1): p. 22-27.

284. Lauer AC, Lieb LM, Ramachandran C, Flynn GL, and Weiner ND, Transfollicular drug delivery. Pharm

Res, 1995. 12(2): p. 179-86.

PART I: SAFETY AND EFFICACY OF MICRONEEDLE PRE-TREATMENT ON HUMAN VOLUNTEERS

Chapter 3

In vivo assessment of safety of

microneedles in human skin Suzanne M. Bal, Julia Caussin, Stan Pavel, Joke A. Bouwstra

European Journal of Pharmaceutical Sciences 2008, 35(3): 193-202

Chapter 3

56

Abstract

Microneedle arrays are promising devices for the delivery of drugs and vaccines into or

through the skin. However, little is known about the safety of the microneedles. In this

study we obtained insight in the ability of microneedles to disrupt the skin barrier, which

was evaluated by transepidermal water loss (TEWL). We also determined the safety in

terms of skin irritation (skin redness and blood flow) and pain sensation. We applied

microneedle arrays varying in length and shape on the ventral forearms of 18 human

volunteers. An effect of needle length was observed, as TEWL and redness values after

treatment with solid microneedle arrays of 400 μm were significantly increased compared

to 200 μm. The blood flow showed a similar trend. Needle design also had an effect.

Assembled microneedle arrays induced higher TEWL values than the solid microneedle

arrays, while resulting in less skin irritation. However, for all microneedles the irritation

was minimal and lasted less than 2 hours. In conclusion, the microneedle arrays used in

this study are able to overcome the barrier function of the skin in human volunteers, are

painless and cause only minimal irritation. This opens the opportunity for dermal and

transdermal delivery of drugs and vaccines.

Safety of microneedle arrays

57

Introduction

Even though the skin is an attractive site for drug delivery, the stratum corneum, the upper

part of the epidermis, poses a barrier to the transport of most compounds. In recent years

a large number of methods have been developed to increase the permeation across this

skin barrier. Among these methods are chemical enhancement such as the use of

penetration enhancers and novel formulations and physical enhancement, such as

iontophoresis and electroporation [1-5]. Recently microneedles have gained much

attention, as they can create little holes in the stratum corneum. Microneedles can be

fabricated from a large number of different materials, such as silicon, glass, metal and

polymers, and differ in length and in shape [6-8]. The microneedles are excellent

candidates for transdermal and dermal delivery. One of the most attractive applications of

the microneedle arrays is to use them for transcutaneous vaccination. Microneedle studies

are often focused on the fabrication of microneedle arrays. Studies on the enhanced

delivery across the skin [9-15] and the increase in immune response generated [16-18] are

in progress. An important question that needs to be resolved is whether these

microneedles induce skin irritation [15, 17, 19].

Skin irritation is a reversible inflammatory reaction that can lead to erythema and oedema

[20, 21]. Many chemical substances act as skin irritants and the mechanism of this process

is not completely understood, but the production of cytokines by epidermal cells is

deemed important. Keratinocytes, which comprise 95% of the epidermal cells, are the

major source of cytokines. Activated Langerhans cells also secrete cytokines, but to a lesser

extent [22]. In response to barrier disruption, keratinocytes produce a variety of cytokines

of which interleukin-1α (IL-1α) is the most important one. Preformed and active IL-1α is

already present in resting keratinocytes and after it has been released, it stimulates further

release of more IL-1α and other cytokines such as IL-8, IL-6, granulocyte-macrophage

colony-stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α) [23-25]. This

cytokine cascade leads to dermal vasodilatation and cellular infiltration in the epidermis,

which directs the restoration of the skin barrier function [26, 27]. Physical barrier

disruption by tape stripping or UV radiation is also known to result in release of IL-1α and

the resulting inflammation reaction [25, 28, 29]. It may therefore be possible that

microneedles also induce an inflammatory reaction.

There are many non-invasive biophysical techniques to assess skin irritation and barrier

disruption, such as transepidermal water loss (TEWL), skin colour, laser Doppler flowmetry,

capacitance, reflectance spectroscopy, ultrasound and visual scoring [30-35]. In this study

the safety and barrier disruption caused by microneedle arrays was investigated in healthy

subjects. Erythema was evaluated by skin colour assessment and by laser Doppler imaging

(LDI). LDI is an optical technique that measures the movement of red blood cells. Light

Chapter 3

58

from a laser beam is directed onto the skin. Moving red blood cells scatter the laser light in

a different way than static tissue resulting in a frequency shift. This shift is photodetected

and processed to provide a blood flow value [36, 37]. The barrier function was investigated

by measuring the TEWL [38]. After treatment with different types of microneedle arrays

the TEWL, LDI, redness and painscore were assessed on regular intervals during 2 hours.

The length of the microneedles as well as the shape of the tip of the microneedles varied.

Materials and methods

Volunteers

Eighteen non-smoking healthy volunteers (9 men and 9 women), aged between 21 and 30

years (mean ± SD, 25 ± 3), with no pre-existing skin conditions participated in the study.

They were asked not to apply any cosmetic formulations on the ventral forearm during

seven days before the study and to refrain from coffee and tea on the day of the study.

The study was approved by the Medical Ethical Committee from the Leiden University

Medical Centre.

Microneedles

Two different types of microneedle arrays were used. Solid metal microneedle arrays

(figure 1a and b) with a length of 200, 300 or 400 μm (200S, 300S and 400S) were obtained

from Transferium (Almelo, The Netherlands). These needles are made from stainless steel

wire with a diameter of 200 μm and are die-cut to a tangential shape. The needles were

placed in a 4 by 4 pattern in a polyetheretherketone mould (diameter 9 mm) with a pitch

of 1.25 mm. Assembled hollow metal microneedle arrays (figure 1c and d) with a length of

300 and 550 μm (300A, 550A) were obtained from Philips (Philips Research Europe,

Eindhoven, The Netherlands). These needles were manufactured from commercially

available 30G hypodermic needles and have a diameter of 300 μm [14]. These needles

were positioned in a 4x4 pattern in a polyetheretherketone mould with a pitch of 1.25

mm, similarly to the solid microneedle arrays.

To precisely tailor the insertion speed of the microneedle array into the skin to 3 m/s a

custom made electrical applicator was used (Fine Mechanical Department, Leiden

University). An array of microneedles was positioned at the end of the applicator and held

in place by a metal holder. A Perspex cover protects this metal holder. The device contains

a coil through which on demand current passes, which results in a magnetic driving force

that launches a metal rod out of the coil, moving the attached microneedle array.


59

Experimental procedure

The study was conducted at 23˚C in a temperature controlled room. The subjects

acclimatised in this room for 30 minutes prior to the start of the study. Three circular areas

were marked on the left ventral forearm and five on the right ventral forearm of each

subject. The circular areas were located at approximately the same position on each

forearm. However, to ensure that these areas were not located on a vein, which would

interfere with the blood flow measurements, the subcutaneous blood flow was imaged

before treatment with the microneedle arrays with a laser Doppler imager (LDI)

(MoorLDLS, Moor Instruments, Devon, UK). The distance between the LDI measuring head

and the skin was set to 15 cm and the images were analysed by calculating the mean blood

flow over an area of 0.64 cm2, corresponding to the size of the mould of the microneedle

array. The values are expressed as perfusion units (PU).

In order to compare the effect of increasing microneedle length, 200S, 300S and 400S

microneedle arrays were applied on the left ventral forearm of 18 volunteers in a

randomised manner. This experiment was always performed in the morning, between 10

AM and 12 PM. On the right ventral forearm of 15 volunteers five microneedle treatments

were carried out to compare single application of the 300S microneedles to the following

treatments: i) twofold application of the 300S microneedles, ii) application of 300A and iii)

application with 550A, which served as a positive control and iv) application of an empty

mould which served as a negative control. All positions were randomised in comparison to

the 300S to correct for differences between the application sites. This experiment was

always performed in the afternoon, between 1 PM and 3 PM.

Figure 1. The microneedle arrays used in this study are i) solid metal microneedles in a 4x4 array (300S, a) and a higher magnification of a single microneedle (b) and ii) assembled hollow metal microneedles in a 4x4 array (300A, c) and a higher magnification of a single microneedle (d).

Chapter 3

60

Before applying the microneedle arrays, baseline values were recorded for the barrier

function (TEWL), the subcutaneous blood flow and the skin colour. The TEWL was

measured with a Tewameter TM210 (Courage+Khazaka, Köln, Germany). After placing the

probe on the skin, the TEWL values were recorded for a period of 1 min after which an

average reading during this time interval was calculated. The values are expressed in g h-1

m-2. The skin colour was measured using a Minolta CR-300 chromameter (Minolta Ltd,

Milton Keynes, UK). The chromameter was calibrated against a colour standard before

measuring each subject, according to the method defined by the manufacturer. The probe

of the apparatus was placed gently onto the skin and the colour was measured on the a*

scale, the red-green Commission Internationale de l'Éclairage (CIE) axis [39]. The treated

areas were also visually inspected for skin damage. The measurements were performed

directly after application (0 min) and repeated after 15, 30, 45, 60, 90 and 120 minutes.

The subjects were also asked to rate the pain of application on a 1-10 scale directly after

the treatment.

Statistical analysis

Statistical analysis was performed with Prism 4 for Windows (GraphPad, San Diego, U.S.A).

Data of TEWL, redness and LDI are presented as mean ± SEM (n = 18 for left ventral

forearm and n = 15 for right ventral forearm). Because the data for the pain scoring did not

show a normal distribution, a box-and-whiskers plot was used to present these data. A

repeated measurement analysis of variance (ANOVA) was combined with a Bonferroni

multiple comparison post test.

Results

Barrier function

The TEWL values after treatment with the 200S, 300S and 400S are provided in figure 2a.

Prior to treatment, TEWL values were around 9.5 g h-1 m-2. The 200S treatment did not

result in increased TEWL values and 15 minutes after piercing the TEWL values only

decreased and reached values that were below the initial baseline values. After piercing

with the 300S, TEWL values increased immediately and declined after 15 minutes reaching

baseline values after 30 minutes. The pattern of the TEWL values obtained after treatment

with the 400S was similar to that obtained with the 300S, but the effect lasted 15 minutes

longer. Treatment with the microneedle arrays showed a trend that longer microneedles

result in a higher increase in TEWL values. Only a significant difference in response was

observed between the 400S and 200S (table 1A). In figure 2b the increase in TEWL after

treatment with microneedles of different shapes, positive control (550A), negative control


61

(550A) and twofold application is provided. In this study all treatments were compared to

the treatment with the 300S microneedle arrays. For almost all microneedle arrays the

TEWL values increased and reached a peak directly after application. After the first time

point at 0 min the TEWL decreased very slowly, but did not return to the baseline value

within the time frame of the experiment. The 300A was the only treatment that reached

its maximum TEWL values not directly after piercing, but 15 minutes later. TEWL did not

increase after treatment with the control. As shown in table 1B, treatment with the 300S

did not increase the TEWL to a significantly higher level than after the control treatment.

The highest TEWL values (maximum of 11.8 g h-1 m-2) were obtained with the 550A

(p<0.001 in comparison to the 300S). Furthermore, the 300A resulted in a significant higher

increase in TEWL than the solid microneedle array of the same length (p<0.001) and

piercing twice with the 300S microneedle array increased the TEWL significantly compared

to a single 300S microneedle treatment (p<0.001).

Pain

In figure 3a and b box-and-whisker plots of the pain scores as reported by the volunteers

are shown. The pain scores of all treatments are similar and very low. No significant

differences in pain caused by microneedles of different length or shape were found. The

median value of all microneedle arrays was 1, except for the 550A were the median was 2.

This array also had the highest maximum pain score of 6. Even though the scores after

microneedle treatment and control did not differ significantly, the latter did have the

smallest interquartile range.

A) TEWL 400S vs 300S 400S vs 200S 300S vs 200S

Mean difference 0.548 1.04 0.495 95 % CI 0.149 to 0.947 0.644 to 1.44 0.0962 to 0.894 p value p< 0.01 p< 0.001 p> 0.05 B) TEWL 300S vs 300A 300S vs 2x 300S 300S vs control 300S vs 550A

Mean difference -0.884 -0.844 0.458 -1.95 95 % CI -1.33 to -0.435 -1.29 to -0.395 0.00961 to 0.907 -2.40 to -1.51 p value p< 0.001 p< 0.001 p< 0.05 p< 0.001

Table 1. Pairwise comparison of TEWL values (g h-1 m-2) between microneedle arrays of different length (A) and type (B).

Chapter 3

62

Figure 3. Box-and-whisker plots of the pain scores after treatment with different microneedle arrays. (A) Solid metal microneedle arrays of 200, 300 and 400 μm needle length. n = 18. (B) Solid metal microneedle arrays of 300 μm in comparison to different types of microneedle arrays. n = 15.

Figure 2. TEWL values before and after applying different microneedle arrays (t=0). (A) Solid metal microneedle arrays of 200, 300 and 400 μm needle length. (B) Solid metal microneedle arrays of 300 μm in comparison to different types of microneedle arrays. Data is presented as average values ± SEM of 18 (A) or 15 (B) volunteers.


63

Skin Irritation

As a determinant of the degree of irritation the redness of the skin and the blood flow was

examined. Figure 4a shows the change in redness (Δa) for the solid microneedle arrays of

different length. After application of each microneedle array an increase in Δa was

observed. After 15 min the Δa values were maximal and reached values of 1.8 absorption

units (AU) for the 300S and 400S and of 1.4 AU for the 200S could be detected. From this

time on the Δa values decreased and reached baseline values for the 200S after 60 minutes

and for the 300S and 400S after 90 minutes. As shown in table 2A treatment with the 400S

resulted in significant higher Δa values than treatment with the 200S (P<0.001). In figure

4b the Δa after treatment with the 300S was compared to different types of microneedle

arrays. Treatment with the empty mould resulted in maximum values directly after

application and almost immediately afterwards the baseline values were reached. For all

microneedle arrays, the Δa values were maximal 15 minutes after application, and

remained elevated for at least 90 minutes. Treatment with the 550A and the 300S resulted

in Δa values that were still higher after 2 hours than before treatment. As shown in table

2B, treatment with the 300S resulted in an increase that was significantly higher than after

the control treatment (P<0.001). After treatment with the 550A, similar Δa levels were

reached as with the 300S, while significantly lower values compared to the 300S were

found after treatment with the 300A (P<0.01), even though after treatment with the 300A

very small spots of blood redness were observed in the skin. Piercing with the 550A also

resulted in small blood spots in the skin. Single and twofold piercing with the 300S

microneedle array did not result in significant differences in Δa values.

Monitoring changes in subcutaneous blood flow using the LDI was another way to assess

skin irritation. In figure 5 examples of pictures and perfusion images of skin reactions after

5 different applications of microneedle arrays are shown. The figure shows scans of the

same skin area before treatment and at different time points after treatment. The change

in blood flow compared to the baseline values after application of the 200S, 300S and 400S

was derived from the blood flow images and is shown in figure 6a. Immediately after

treatment the blood flow increased, but reduced to baseline values within 45 minutes.

However, no significant differences in blood flow were found after treatment with 400S,

300S and 200S microneedles (table 3A). As shown in figure 6b pressing an empty mould

against the skin resulted in a slight increase the subcutaneous blood flow, as an increase of

25 PU could be observed, but after 30 minutes the baseline value was reached again.

Applying the microneedle arrays resulted in an immediate increase in blood flow followed

by a rapid decrease. The 300S resulted in a significantly higher increase in blood flow than

after treatment with the control (p<0.001). The blood flow returned to baseline values

within 60 minutes for all microneedle arrays except the 550A, which values remained

Chapter 3

64

elevated for at least 2 hours. Treatment with the solid microneedle arrays resulted in a

trend of a more pronounced blood flow increase than after applying the assembled

microneedles (table 3B). Twofold and single piercing of 300S microneedle arrays did not

result in significant differences in blood flow.

A) Δa 400S vs 300S 400S vs 200S 300S vs 200S

Mean difference 0.181 0.537 0.356 95 % CI -0.112 to 0.481 0.237 to 0.837 0.0559 to 0.656 p value p> 0.05 p< 0.001 p< 0.05 B) Δa 300S vs 300A 300S vs 2x 300S 300S vs control 300S vs 550A

Mean difference 0.804 0.408 2.01 0.119 95 % CI 0.244 to 1.36 -0.152 to 0.967 1.45 to 2.57 -0.441 to 0.679 p value p < 0.01 p > 0.05 p < 0.001 p > 0.05

Figure 4. The change in redness (Δa) at different time points after the application of microneedle arrays (t=0) in comparison to the redness before application. (A) Solid metal microneedle arrays of 200, 300 and 400 μm needle length. (B) Solid metal microneedle arrays of 300 μm in comparison to different types of microneedle arrays. values. Data is presented as average values ± SEM of 18 (A) or 15 (B) volunteers.

Table 2. Pairwise comparison of induced redness between microneedle arrays of different length (A) and type (B).


65

Δblood flow 400S vs 300S 400S vs 200S 300S vs 200S

Mean difference 19.25 18.82 -0.432 95 % CI -0.177 to 38.68 -0.608 to 38.25 -19.86 to 19.00 p value p> 0.05 p> 0.05 p> 0.05 Δblood flow 300S vs 300A 300S vs 2x 300S 300S vs control 300S vs 550A

Mean difference 15.79 3.823 40.57 -12.6 95 % CI -9.646 to 41.22 -21.61 to 29.26 15.14 to 66.01 -38.04 to 12.83 p value p > 0.05 p > 0.05 p < 0.001 p > 0.05

Table 3. Pairwise comparison of the increase in blood flow (PU) between microneedle arrays of different length (A) and type (B).

Figure 5. Laser Doppler pictures and perfusion images of a forearm of a volunteer. The figure shows scans of the same skin area before treatment and at different time intervals after treatment.

Chapter 3

66

Discussion

The aim of this study was to obtain insight in the ability of microneedles to disrupt the

barrier of the skin and to determine the safety of microneedle treatment in terms of skin

irritation and pain sensation. For this purpose we used microneedles varying in

microneedle length, diameter and shape. In one study we investigated the effect of

increasing microneedle length and in another study single application of the 300S

microneedles was compared to treatment with microneedles of different shape and to

twofold application.

First, the influence of the microneedles arrays on the barrier function was assessed. For

microneedle arrays with the same shape, only treatment with 400S resulted in a significant

difference in TEWL in comparison to 200S. Treatment with the 300S was also compared to

treatment with microneedle arrays with a differently shaped tip. We found a significant

difference between the 300S and the 300A and 550A, indicating that needle shape is an

important parameter for barrier disruption. The 300S did not increase the TEWL

significantly compared to the control treatment. However, in in vitro studies we did show

that these needles could pierce human skin by visualising the conduits [40]. The 300S

microneedle arrays were used in the study focusing on needle length and in the study

Figure 6. The change in blood flow at different time points after the application of microneedle arrays (t=0) in comparison to the blood flow before application. (A) Solid metal microneedle arrays of 200, 300 and 400 μm needle length. (B) Solid metal microneedle arrays of 300 μm in comparison to different types of microneedle arrays. Data is presented as average values ± SEM of 18 (A) or 15 (B) volunteers.


67

focusing on needle shape. Slight differences in TEWL were observed in both studies. In the

microneedle length study, the elevated TEWL values lasted 30 minutes, while in the

microneedle shape study the TEWL values remained elevated and increased again after 90

minutes. It is possible that this was caused by circadian variations. The microneedle shape

study was performed between 1 and 3 PM. Le Fur et al. showed that TEWL values on the

forearm reach a peak at 8 am and at 4 pm and a minimum at noontime [41, 42].

An important reason to develop microneedles for dermal vaccination is to decrease the

pain and discomfort that the current delivery of vaccines by injection causes. Several

recent studies indicate that approximately 20% of the children suffered serious distress

from vaccinations [43]. For this reason we also assessed the pain that treatment with our

microneedle arrays might induce. We demonstrated that treatment with microneedle

arrays varying in microneedle length, diameter and shape did not cause pain to most of the

volunteers. This is in agreement with results from Kaushik et al., who showed that the pain

sensation caused by microneedle arrays containing 400 microneedles with a length of 150

μm did not differ significantly from a smooth surface [19]. The pain score of the

microneedle arrays do have a larger interquartile range than the control. However, pain

scoring is a subjective matter and two volunteers did perceive all microneedle arrays as

uncomfortable.

To assess the safety of the microneedles, the irritation that these needles might induce

was measured both with a chromameter and a LDI. Both methods measure erythema,

which is one of the fundamental markers of inflammation [23]. However, a chromameter

measures only the superficial redness, while a LDI measures the blood flow much deeper in

the skin. The exact penetration depth of the laser depends on pigmentation, but on

average the image is reflecting the blood flow until a skin depth of 1 mm [44]. Because the

vasodilatation response caused by the inflammation reaction in the dermis is faster than

the redness response on the surface of the skin, the blood flow reached its maximum value

directly after treatment with the microneedles, while the maximum Δa was measured 15

minutes after microneedle treatment. The results of both methods correlate excellently.

For microneedle arrays of the same needle type, an increase in length results in an

increase in Δa. Although treatment with microneedles of varying length did not result in

significant blood flow differences, a similar trend was observed. When focusing on the

microneedles of different shapes, treatment with the 300S induced clearly more irritation

than the control treatment and the 300A microneedles, while between the 300S and 550A

no significant differences in skin irritation were observed. Taking the Δa and blood flow

data together, the assembled microneedle arrays result in less skin irritation than the solid

ones. The effects observed are in agreement with data from Sivamani et al., who observed

a higher maximum blood flow after microneedle application of methyl nicotinate

compared with topical application [45].

Chapter 3

68

Which microneedle arrays are most suitable to use for transdermal delivery and dermal

vaccination purposes? The assembled microneedle arrays have the advantage that they

disrupt the stratum corneum barrier to a higher extent, while they induce slightly less

irritation. The most likely explanation for the difference in irritation and TEWL between the

two needle types is the sharpness of the tips. The solid metal microneedles are 200 μm

thick at the base and the tapered shaft of the needles has a length of approximately 280

μm. The slope of the angle is therefore 45°. The assembled hollow metal microneedles are

made from 30G needles, which are 300 μm thick at the base, but the tapered shaft of the

needles has a length of approximately 1.2 mm. The angle is therefore more acute, resulting

in a very sharp tip. For this reason, they can make deeper incisions into the skin, as is

suggested by the presence of small blood spots on the skin surface after application of

these microneedles. On the other hand, piercing with the solid microneedle arrays appears

to form a larger cut and therefore causes slightly more skin damage and irritation. This

could mean that the assembled microneedles increase penetration of drugs, without

unwanted side effects caused by irritation. However, previous in vitro transport studies

across human skin performed by Verbaan et al. showed that pre-treatment with the solid

microneedle arrays resulted in significant higher fluxes of cascade blue than pre-treatment

with the assembled microneedle arrays [40]. Chilcott et al. also postulated that there is no

correlation between increased TEWL levels and increased transdermal transport [46].

Further transport studies have to be performed to confirm the significant difference

between solid and assembled microneedles in vivo. In case of dermal vaccination, the

irritation caused by the solid microneedle arrays could be an advantage. It was shown that

mechanical barrier disruption induces cytokine release and in that way initiates an

inflammatory reaction [28, 29]. The Langerhans cells that are recruited to the site of

irritation can take up antigens and consequently initiate an immune response. In this way

the irritation caused by the microneedle arrays could function as an enhancer. Langerhans

cells are located in the lower epidermis [47], that is approximately 150 μm thick [48].

Verbaan et al. postulated that microneedle arrays do not pierce the skin with their full

length, because they have to overcome the bulk elastic tissue compression of the skin [14].

It is therefore advisable to use microneedles that are longer than 150 μm. From this study

can be concluded that the minimal length should be 300 µm, because shorter needles did

not pierce the skin.

To further evaluate the irritation data, we performed a pilot study in which we compared

the TEWL, redness and blood flow values to those directly after tape stripping. We chose

for tape stripping as this has been used for many years and is reported to be non-invasive

[32]. After 10 tape strips the TEWL reached values of 15 g h-1 m-2 and remained at that

value for at least 2 hours. This was higher than after treatment with the 550S. This larger

increase in barrier disruption was accompanied by a higher degree of irritation. The Δa and


69

increase in blood flow after 10 tape strips were 4 AU and 160 AU respectively, which is

higher than the Δa of 2 AU and the increase in blood flow of 140 AU reached after

application of the 550S. The effect of tape stripping on the blood flow was short lasting,

similar to the effect of the microneedle arrays. Only after removing 30 tape strips the

blood flow appeared to remain elevated for 2 hours. The Δa after removing 10, 20 or 30

tape strips lasted longer than after application of the microneedle arrays, probably

because with tape stripping the stratum corneum is removed and more superficial damage

is done. Previously, Li et al. studied the effects of iontophoresis on TEWL and skin redness

[49]. They found comparable redness values (Δa of 4 AU) to the values we obtained after

microneedle application, but the redness persisted for a longer time. In this study also

increased TEWL values were observed, but it is difficult to compare these values to the

values obtained in our study, as the skin was hydrated with buffer solution for the duration

of the iontophoresis and this also causes an increase in TEWL.

In conclusion, this study has shown that application of solid and assembled metal

microneedle arrays with a length of up to 550 μm can be used to overcome the barrier

function of the skin. Furthermore, human volunteers perceived their application as

painless. Finally, it causes only minimal irritation in comparison to for instance tape

stripping, which is accepted to be non-invasive. The shape and the length of the

microneedle arrays have an influence on the degree of irritation, but for all microneedle

arrays the irritation is short lasting.

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70

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penetration enhancers in transdermal drug delivery. Curr Med Chem, 2000. 7(6): p. 593-608. 2. Kalia YN, Naik A, Garrison J, and Guy RH, Iontophoretic drug delivery. Adv Drug Deliver Rev, 2004.

56(5): p. 619-658. 3. Akimoto T and Nagase Y, Novel transdermal drug penetration enhancer: synthesis and enhancing

effect of alkyldisiloxane compounds containing glucopyranosyl group. J Control Release, 2003. 88(2): p. 243-252.

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with a multifactorial approach: comparison of eight non-invasive measuring techniques on five

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variations of the skin barrier function in humans: Transepidermal water loss, stratum corneum

hydration, skin surface pH, and skin temperature. J Invest Dermatol, 1998. 110(1): p. 20-23. 43. Jacobson RM, Swan A, Adegbenro A, Ludington SL, Wollan PC, and Poland GA, Making vaccines more

acceptable - methods to prevent and minimize pain and other common adverse events associated

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of methyl nicotinate: stratum corneum penetration. Skin Res Technol, 2005. 11(2): p. 152-156. 46. Chilcott RP, Dalton CH, Emmanuel AJ, Allen CE, and Bradley ST, Transepidermal water loss does not

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Chapter 4

Influence of microneedle shape on the

transport of a fluorescent dye into

human skin in vivo Suzanne M. Bal, Annelieke C. Kruithof, Raphaël Zwier, Ekkehart Dietz, Joke

A. Bouwstra, Jürgen Lademann, Martina C. Meinke

Journal of Controlled Release 2010, 147(2):218-224

Chapter 4

76

Abstract

Microneedles can enhance the penetration of vaccines into the skin for transcutaneous

vaccination. In this study for the first time the influence of microneedle geometry on the

transport through the formed conduits was visualised on human volunteers by confocal

laser scanning microscopy. Three differently shaped 300 µm long microneedle arrays were

selected and fluorescein was applied either before or after piercing. Based on the intensity

a distinction was made between regions with high and low intensity fluorescence (HIF and

LIF). The areas of both intensities were quantified over time. In most cases HIF areas were

only present in the stratum corneum, while LIF areas were also present in the viable

epidermis. The areas were larger if fluorescein was applied after piercing compared to

before piercing. After 15 minutes almost no HIF was present anymore at the skin surface.

The microneedle geometry, but not the manner of application affected the shape and

depth of the conduits. In conclusion we showed that the different microneedle arrays are

able to form conduits in the skin, but the geometry of the microneedles influences the

penetration of the fluorescent dye. This is the first step towards a more rational design of

microneedle arrays for transcutaneous vaccination.

Transport of a fluorescent dye into human skin

77

Introduction

Microneedles are used in the field of transcutaneous drug delivery for more than a decade

[1]. They were developed as either a replacement for the traditional needle and syringe or

to facilitate the transport across the skin barrier. Microneedles are shorter than traditional

needles, generally less than 1 mm long, but long enough to breach the stratum corneum

barrier, the upper 20 µm of the skin. Because of the elasticity of the skin, the microneedles

need to have a certain length to achieve insertion into the skin [2]. This length depends on

the manner of insertion, manual or with an applicator. Verbaan et al. showed that, when

applied with a certain velocity, microneedles with a length of 300 µm can successfully and

reproducibly pierce the skin [3]. Microneedles of this length are generally not perceived as

painful [4, 5]. The application of microneedles creates small conduits in the skin. These

conduits are large enough to allow penetration of high molecular weight compounds

across the stratum corneum, which can not be transported into the skin passively [6].

However, compared to piercing with a conventional needle, there is only minimal microbial

infiltration through the conduits and microorganisms do not reach the dermis [7]. More

recently microneedles have also been used for vaccination [8-12].

Several types of microneedles have been developed. The microneedles can either be

hollow or solid. Hollow microneedles can be used to inject a vaccine into the skin. This

increases the bioavailability of the vaccine compared to solid microneedles, because the

majority of the dose ends up in the skin. One disadvantage is the possibility of leakage to

the skin surface that is expected to increase when shorter microneedles are used. Hollow

microneedles have been successfully used for vaccination purposes. Recently, van Damme

et al. published a clinical trial showing that a 5 times lower dose of influenza vaccine

administered via the MicronJet (NanoPass Technologies), consisting of 4 hollow

microneedles with a length of 450 µm, induced similar titres compared to the full dose

administered intramuscularly [13]. Solid microneedle arrays can either be coated with the

vaccine or used to pre-treat the skin after which a patch containing the vaccine is applied.

Matriano et al showed that by coating solid microneedles with ovalbumin a 50-fold higher

immune response was induced compared to intramuscular administration of the same

dose in guinea pigs [11]. The most straightforward manner is to use solid microneedles to

pierce the skin resulting in small conduits. Ding et al. showed a significant increase in

antibody response in mice after diphtheria toxoid application on microneedle pre-treated

skin in comparison to untreated skin. The application of cholera toxin as an adjuvant could

amplify the titres to comparable levels as after subcutaneous immunisation [8].

Even though microneedles are a very promising tool for transcutaneous immunisation,

little is known about the optimal type (hollow or solid), geometry, length and shape of the

arrays. Most studies focus on the immune response generated, while the dimensions of

Chapter 4

78

the formed conduits and the transport across these conduits are less well studied. A more

thoroughly investigation of these parameters may allow for a rational design of

microneedle arrays for transcutaneous vaccination.

Recently we showed that by using confocal laser scanning microscopy (CLSM) it is possible

to visualise the conduits made by microneedle treatment in human volunteers [14]. To our

knowledge this was the first time that microneedle conduits were visualised in an in vivo

study on human volunteers. The transport of a fluorescent dye into the skin could be

visualised over time. To examine the influence of the shape of the microneedles, in the

present study we compared three different microneedle arrays. Two types of arrays were

developed in our lab and have already proven to be painless [4] and to be able to induce

enhanced immune responses [8, 15]. They were compared to the commercially available

Dermastamp®. All three types of arrays consist of solid stainless steel microneedles with a

length of 300 µm, but with a variable microneedle shape. We explored the behaviour of

the conduits formed by these microneedles in 6 healthy subjects over time by CLSM. As a

model drug fluorescein was used. To investigate if the application method had an influence

on the formed conduits, the fluorescent dye was applied before and after piercing.

Materials and Methods

Volunteers

Six healthy volunteers (5 women and 1 man), aged between 20 and 58 years (mean 33

years) with no pre-existing skin conditions participated in the study. The study was

approved by the Ethics Committee from the University Hospital Charité (Berlin, Germany)

in accordance with the Rules of Helsinki.

Materials

Three different types of microneedle arrays were used. In figure 1 light microscopy images

and schematic drawings of the microneedles are shown.

1) Assembled metal microneedle arrays (figure 1A and D) with a length of 300 µm (300A)

were manufactured from commercially available 30G hypodermic needles [16]. These

microneedles were positioned in a 4x4 pattern in a polymer mould (diameter 5 mm)

with a pitch of 1.25 mm. The microneedles are 300 µm in diameter at the base and

have a very sharp tip with an angle of 15°.

2) Stainless steel microneedles prepared by electrical discharge machining (300ED). The

microneedles have a square base of 250 x 250 µm and a length of 300 µm and are also

positioned in a 4x4 pattern with a pitch of 1.25 mm. The shape of the tip is defined by a

diagonal plane which runs from the top of one side of the square pillar to the opposed


79

bottom, in this way forming an angle of approximately 40° relative to the bottom

surface (figure 1B and E).

To apply these types of microneedles in a controlled manner an electrical applicator was

used, as described before [16]. With this applicator the microneedles are applied onto the

skin with a speed of 3 m/s.

3) These microneedles were compared to the commercially available Dermastamp®

(Dermaroller S.a.r.l., Wolfenbuettel, Germany). The Dermastamp®

consists of 6

microneedles (one in the center, radially surrounded by 5 others). These microneedles

also have a length of 300 µm, a diameter of 120 µm and a pitch distance of 2 mm

(figure 1C and F). They were applied manually three times on the same area by turning

the stamp approximately 45°.

To visualise the conduits a 0.2% solution of sodium fluorescein was used (Alcon Pharma

GmbH, Freiburg, Germany).

Confocal laser scanning microscopy

The fluorescent dye in the conduits was visualised with in vivo confocal laser scanning

microscopy (CLSM) (Stratum® System, OptiScan, Melbourne, Australia) as previously

described [17]. A hand-held device containing the optical system and the focus tuning is

connected to the basic station containing an Argon laser (488 nm). The measuring area is

235 x 235 µm2 and the penetration depth of the Argon laser in human skin is about 200 µm

[18]. The intensity of the laser varied between 450 and 520 µW.

Figure 1. Images of the

different microneedle

arrays used in this study.

A: 300A microneedle

array, assembled of 30G

needles. B: 300ED

microneedle array made

of stainless steel. C:

Dermastamp® consisting

of 6 microneedles. In

figure D, E and F higher

magnification images of

single microneedles are

shown.

Chapter 4

80

Experimental procedure

The microneedles and the formulations were applied on the ventral forearms of the

volunteers. The skin was disinfected before the formulations and the microneedle systems

were applied. On each volunteer all three microneedle systems were tested in triplicate in

a randomised order. The fluorescein solution (50 µl / 0.8 cm2) was applied in two different

manners. Either the skin was first treated with the microneedles after which the dye was

applied for 1 minute, or the dye was applied before microneedle treatment. In the latter

case the dye was removed immediately after microneedle treatment. Afterwards the

conduit was visualised with CLSM. At 5, 10 and 15 minutes after application images were

taken at different depths. At least 5 images were taken to monitor the dye at the surface,

the lateral and vertical distribution and the maximal penetration depth. Between the

measurements the laser was set out of focus to avoid bleaching. As a control a drop of

fluorescein was applied to untreated skin. This control experiment showed that at the

concentration used in this study no bleaching of the fluorescein occurred.

Data analysis

The images were analysed with respect to fluorescence pixel intensity and area using

Image J (National institute of health, USA). The pixel intensity was categorised into three

different classes: the class with the highest pixel intensity was set between 230 and 255 AU

and the signal was referred to as high intensity fluorescence (HIF); the class with pixel

intensity between 230 and 14 AU is referred to as low intensity fluorescence (LIF) and the

class with pixel intensity values below 14 AU is regarded as background. The

autofluorescence of the skin was always below 14 AU. These thresholds were selected

based on analysing 31 random images of different depths taken from two volunteers. The

thresholds were selected in such a way that at least 90% of the test pixels were inside the

described classes. The fluorescent signal from other skin structures such as furrows or hair

follicles was removed manually. The area of either HIF or LIF was calculated by the number

of pixels in the specified intensity areas. The following parameters were analysed: the area

of HIF at the surface; the maximum area of LIF in the skin and its corresponding depth; and

the total depth of the conduit determined by the maximum depth where LIF can be

detected. The parameters are further explained in figure 2.


The data analysis was performed considering that the measurements within one volunteer

are not independent. Therefore, the statistical analysis of the univariate and the

multivariate effect of the factors microneedle system, time, and application method has to


81

be based on random effect models. The generalised estimation equation (GEE) -method of

SPSS (SPSS Inc., Chicago, Illinois) was used for statistical analysis.

Results

Adverse effects

Application of the microneedle arrays was not perceived as painful. After application with

the 300A microneedles, occasionally small blood spots could be observed visually where

they had pierced the skin. In those cases with the CLSM often erythrocytes could be found

in the conduits. With the other types of microneedles no bleeding was detected. When

small blood spots were detected, the fluorescent images were not included in the analysis.

Dimensions of the conduits formed by the different microneedle arrays

In summary, 95 conduits were investigated and 285 images were taken at different time

points and at different depths. In figure 3 representative images obtained after the pre-

treatment with the different microneedle arrays and subsequent dye application are

shown. The differences in shape of the conduits formed by the three microneedle arrays

can be observed. The images show the conduits 5 minutes after application of the

fluorescent dye at different depths in the skin. The shape of the conduits was dependent

on the type of microneedle array used. The 300A microneedles form half-moon shaped

conduits and the dye areas are much larger than those visualised after treatment with the

other two arrays. The conduits formed after application of the two other arrays are more

Figure 2. Schematic

representation of diffusion

pattern of the fluorescent

dye after microneedle

treatment. The grey levels

represent the difference in

fluorescent intensity.

Chapter 4

82

round-shaped and similar in size. From the images a difference in fluorescence intensity in

depth can be observed. In the upper layers of the skin the bright HIF is clearly visible, while

at the deeper skin layers a more diffuse low intensity signal is present. In the images taken

at the surface of the skin also the shape of the corneocytes is visible. The fluorescent dye

preferably diffuses through the lipid regions surrounding the corneocytes, outlining the

cells. The shape of the conduits was not influenced by the method of application: applying

the dye before or after piercing induced similarly shaped conduits (data not shown).

To be able to study the shape of the conduits and the diffusion of the fluorescent dye

through the conduits over time, images were made at 5, 10 and 15 minutes after dye

application. Figure 4 shows representative images made after application of the 300A

microneedle array at different depths over time. The images obtained after treatment with

the other two microneedle arrays showed comparable patterns. From these images it

appears that the largest area of HIF was present at the skin surface at all time points.

However, this was not always the case. In around 40 % of the cases (an example is

provided in figure 5) the largest area of HIF was observed somewhat deeper in the skin,

but within the upper 20 µm of the skin, the stratum corneum. This percentage differs

between the arrays: for the Dermastamp® this occurred in 48% of the cases and for the

300A only in 30%. The area of HIF at the surface decreased over time and generally after

15 minutes almost no HIF could be detected at the skin surface or deeper in the conduit. In

addition, figure 4 shows that the area of LIF also reduced over time. Furthermore, the

depth where still fluorescence could be detected decreased over time. While after 5 and

10 minutes the LIF is still observed at a depth of 100 µm in the skin, after 15 minutes this

can only be seen until a depth of 60 µm.


83

Figure 3. Representative

images of the conduits

formed by the different

microneedle arrays after

pre-treatment with the

microneedles followed by

application of the

fluorescent dye. Images

were taken 5 minutes

after the application of the

dye at different depths.

Dimensions of the images

are 235 by 235 µm2.

Figure 4. Representative

images of a microneedle

conduit formed by the

300A microneedles at

different depths over

time. The fluorescent dye

present in the conduits

can be observed.

Dimensions of the images

are 235 by 235 µm2.

Chapter 4

84

Area of HIF at the skin surface after different microneedle applications

From the images the area containing HIF present at the skin surface could be calculated. At

5 min in almost all conduits (96%) HIF appeared. After 10 min in 72% of the conduits HIF

could be observed and after 15 min still 55% of the conduits showed HIF, but the area of

this fluorescence strongly decreased over time (p<0.001). As mentioned before, the shape

of the conduits was not influenced by the manner of applying the microneedle arrays.

However, the area containing HIF present both at the surface and in the deeper layers of

the skin was affected by the application method. In figure 6 the area containing HIF at the

skin surface after both methods of microneedle application is plotted over time. The

fluorescence in the furrows was not taken into account. For all arrays there is a distinct

difference between piercing before or after application of the fluorescent dye (figure

6/table 1). The size of the areas was twice as large when the dye was applied after

microneedle treatment (p<0.05). This was the case at all time points.

The differences in needle shape are reflected by the obtained areas of HIF. After piercing

with the 300ED microneedles and the Dermastamp® similarly sized areas of HIF were

formed at the surface which decreased over time in a comparable manner. For both

methods of application, treatment with the 300A microneedles resulted in an area of HIF

at the surface that was twice the size compared to those observed with the two other

types of microneedle arrays (p<0.001). It has to be mentioned that for applying the dye

Figure 5. Selected images of a microneedle

conduit formed by the Dermastamp®

showing the area of HIF below the surface

in the skin. The fluorescent dye present in

the conduits can be observed. Dimensions

of the images are 235 by 235 µm2.


85

before piercing with the 300ED microneedles the data are only obtained from 3

volunteers. Because of the square shape of these 300ED microneedles, they tended to

push the dye away when applied and this made it very difficult to find the conduits.

Therefore it was decided to skip this application for the remaining volunteers. As a

consequence for the statistical analysis comparing the two application methods only the

300A and the Dermastamp® were included. For both the 300A and the Dermastamp

® a

significantly larger area of HIF was present at the surface of the skin if the microneedles

were used to pre-treat the skin (p<0.05).

Factor Depth of conduit Maximum area of HIF Maximum area of LIF

Microneedle system *** *** ***

Before and after ‡ n.s. * n.s.

Time *** *** ***

Interaction

microneedle system –

time ** n.s. *

Interaction before

and after – time ‡ n.s. * n.s.

* p<0.05

** p<0.01

*** p<0.001

n.s . not significant

‡ Only the 300A and the Dermastamp® are taken into account.

Figure 6. The area of HIF measured at the skin surface. A and B: area of HIF present at different

time points on the surface of the skin when the microneedles were used to pierce either

before (A) or after (B) dye application. Mean ± SEM of 6 volunteers, except for the data of

piercing with the 300ED microneedles after fluorescein application which is of 3 volunteers.

Table 1. Data analysis by the GEE-method of the influence of the different microneedle arrays,

application methods and time on the analysed parameters. If not mentioned otherwise only

the results of piercing before dye application.

Chapter 4

86

Area of LIF in the skin

Deeper into the skin still some HIF could be observed, but mostly regions of LIF were

found. In figure 7 the maximum area of LIF from the conduit (Fig 7A) and the

corresponding depth (Fig 7B) at which these areas are located are shown as a function of

time. Only the data of applying the fluorescein after microneedle treatment are shown.

However, the data of applying the fluorescent dye before microneedle application showed

the same trend.

Comparing the different microneedle arrays, the maximum area of LIF (figure 7A) showed

the same trend as that of the HIF at the surface (figure 6B). The largest area of LIF can be

observed after pre-treatment with the 300A microneedles (p<0.001), with a size

approximately twice that induced by the other two arrays. The maximum areas of LIF after

application of the 300ED and the Dermastamp® did not differ significantly from each other.

The decrease in the size of the area of LIF over time also shows a similar profile as that of

the HIF at the surface. The maximum area of LIF after application of the Dermastamp® was

found deeper in the skin compared to the other two arrays (p<0.01). After 5 minutes the

maximum area of LIF was found at the deepest location in the skin. Over time not only the

size of the area, but also the depth at which the maximum LIF was located decreased

(p<0.001).

For all arrays also the maximum depth of the conduit was determined by the depth at

which the last image was taken where LIF could still be detected. In figure 8 the data are

shown of fluorescein dye application before and after microneedle treatment for the

different time points. No significant differences in conduit depth between both application

methods were found. After microneedle pre-treatment the deepest conduits were

Figure 7. Maximum area of LIF when the dye is applied after microneedle treatment at different

time points (A) and the corresponding depth (B) at which these areas are located. Mean ± SEM

of 6 volunteers.


87

observed with the Dermastamp® (p<0.001), which could be observed until a depth of 170 ±

13 µm into the skin. For the 300A and 300ED microneedles, LIF could be observed until 120

± 10 µm. The depth of the conduits formed by applying the fluorescein before application

did not differ between the 300A and the Dermastamp®. The depth of the conduit

decreased over time, but after 15 minutes for all three microneedle arrays the LIF could

still be detected at a depth of 60 µm, depending on the microneedles used.

Discussion

Microneedle based vaccination has received a lot of attention in the past years and offers

great promise as a replacement for the traditionally used hypodermic needle and syringe.

Advances in the mechanical field have allowed scientists to develop a great variety of

microneedles, differing not only in material, but also in shape and length [6]. By means of

vaccination studies information has been gained about their functionality [8-13, 19, 20],

but data on the influence of microneedle shape on the conduit characteristics and the

transport though the conduits is scarce.

In this study CLSM was used to visualise the conduits made by three solid, stainless steel

microneedle arrays differing in microneedle shape in vivo in humans. CLSM provides the

opportunity to gather information about the geometric parameters of the microneedle

conduits by collecting images at different depths. Moreover, the behaviour of the diffusion

of the fluorescent dye along the conduits over time can be studied to determine the

dynamics of the transport through the conduits. Most studies performed so far are in vitro

studies using different visualisation techniques. Coulman et al. visualised the microneedle

Figure 8. Total depth of the conduits formed by the three different microneedle arrays. In figure A

the data of piercing before fluorescein application and in figure B that of piercing after fluorescein

application is shown. Mean ± SEM of 6 volunteers, except for the data of piercing with the 300ED

microneedles after fluorescein application which is of 3 volunteers.

Chapter 4

88

conduits in human epidermal membranes made by 280 µm long silicon microneedles. They

used scanning electron microscopy (SEM) to show the conduit area at the skin surface and

the deposition of fluorescently labelled nanoparticles in this conduit area [21]. Badran et

al. used light microscopy and SEM to visualise the conduits made by the Dermaroller® in

vitro. They showed that

the size of the conduits was related to the length of the

microneedles [22]. Even though with these techniques the shape of the conduits at the

surface can be visualised, they do not provide information about the depth of the conduits

and the transport into the skin. With CLSM it is possible to obtain insight into the transport

process both in vitro and in vivo without the necessity to process the skin, as the tissue is

optically sectioned. Verbaan et al. used CLSM to visualise the transport of FITC labelled

polystyrene nanoparticles up to a depth of 250 µm into the skin in vitro after application of

the 300A microneedles [3]. In another study Verma et al. also used CLSM to visualise in

vitro the conduits made by Dermarollers® differing in microneedle length and geometry

[23]. Both parameters influenced the penetration depth of DiI (1,1’-Dioctadecyl-3,3,3’,3’-

tetramethylindocarbo-cyanine perchlorate). All these studies provide insight into the

microneedle conduit dimensions, but the need for an in vivo evaluation of the microneedle

conduits remains.

In the present study the transport of a fluorescent dye through the conduits could be

visualised in vivo. We showed that the shape of the microneedles influences the

dimensions of the conduits. Three different microneedle arrays were used. The 300A

microneedle array has already successfully been used for drug delivery and vaccination

purposes [3, 8, 15, 16] and the 300ED microneedles were recently developed in our lab.

The Dermastamp®, a variation on the Dermaroller

®, was developed mainly for cosmetic

usage, but recently also their usage for drug delivery purposes was studied [22, 23]. It has

however not yet been used in vaccination studies. Even though the length of the

microneedles of all three arrays was 300 µm, the 300A microneedles, with a very sharp tip

and applied with an electrical applicator, formed the largest conduits. The base areas of

the 300A and 300ED have a comparable size, but the tips of the 300ED microneedles have

a different shape and are less sharp than the 300A microneedles. Therefore the effective

area of contact between the microneedle and the skin is larger, requiring more force to

penetrate the skin to a similar depth as the 300A microneedles [24]. The 300A and 300ED

(4x4 arrays, 16 microneedles) are applied with an applicator, to ensure reproducible

piercing. The Dermastamp® consists of 6 very sharp microneedles, of a much smaller

diameter compared to the 300A (see figure 1), therefore less force was necessary for

insertion into the skin [24]. This difference in microneedle diameter and number of

microneedles may explain that although the deepest conduits were found after application

of the Dermastamp®, only after application of the 300A microneedles occasionally minor

bleeding was observed. The smaller diameter of the Dermastamp® microneedles reduces


89

the chance of reaching a blood vessel. In a previous study the 300A microneedles were

used to study skin irritation and reduction in skin barrier [4]. In that study the 300A

microneedles did not induce bleeding. Even though the application speed of the applicator

was the same in both studies, in the previous study the microneedles were placed in a

larger back plate. The smaller back plate used in the current study might increase the

pressure applied to the skin and thereby the microneedles may pierce deeper into the skin.

Most probably, by adjusting the speed of microneedle insertion, bleeding can be

prevented. Even though the penetration depth of the fluorescein differed between the

different microneedle applications, for all three microneedle arrays fluorescence could be

detected in the epidermis, indicating successful breaching of the stratum corneum. A

general limitation of the CLSM is the low sensitivity at depths larger than 150-200 µm,

thereby failing to detect fluorescence intensity at this depth. The depths achieved suggest

that the epidermis and to some extent the dermis is reached, which would be very useful

for drug and vaccine delivery.

In previous studies TEWL measurements showed that the conduits remained open for a

few hours [4, 25] under non occlusive conditions. This means that after microneedle pre-

treatment drugs can be delivered through the conduits for a longer period of time. The

present study shows that once the fluorescein has entered the conduits, it rapidly diffuses

both in the lateral and vertical direction. Within 15 minutes a strong reduction in both high

and LIF was observed indicating spreading of the dye. Control experiments were

performed to determine whether bleaching plays a role in the reduced intensities over

time. At the concentrations and conditions used in this study no bleaching of the

fluorescein was observed (data not shown). Therefore the reduction in fluorescent

intensity implies that vaccines might also be easily and rapidly transported to dendritic

cells. Immunisation studies with microneedles demonstrated that an enhanced immune

response can be induced by microneedle pre-treatment [8, 15]. Even though we found

differences in the dye areas present in the skin between the two application methods, the

maximum depth where still LIF could be detected did not differ. This means that the

amount of dye that enters the conduits will end up at a similar depth in the skin. An

explanation for the higher areas of both high and LIF found for piercing before dye

application might be the different penetration times. When the fluorescent dye is applied

prior to piercing, it was immediately removed after piercing. This means that the dye could

only enter the conduits if it is being taken along with the microneedles during piercing. The

penetration time for dye application after piercing was 1 minute, during which time

fluorescein could migrate into the open conduit. Longer application times could reduce

these differences.

It would be interesting to repeat the visualisation studies with nanoparticles, as these are

often used in vaccination studies. Transport of nanoparticles through intact skin is

Chapter 4

90

practically impossible. Studies have indicated that if nanoparticles are applied to intact skin

they will primarily form a depot in the stratum corneum [26, 27] or remain in the hair

follicles [28]. The conduits formed in this study should allow the penetration of

nanoparticles into the skin, but as reviewed by Milewski et al. both the formulation and

the pore lifetime are important parameters for successful transport into the skin [29]. By

combining nanoparticles with microneedle arrays, they could reach the Langerhans cells

and dendritic cells present in the epidermis and the dermis, respectively.

In conclusion, all three microneedle arrays are able to form conduits in vivo in human skin.

The shape of the microneedles and the application speed both influence the shape and

depth of the conduits that are formed. Sharp microneedles, such as present in the 300A

and the Dermastamp®, are good candidates to use for transcutaneous vaccination.

Whereas these studies show that microneedle sharpness and diameter affect the

distribution of a fluorescent dye into the skin, other parameters, such as microneedle

length, material and type, may also have a profound influence. The comparison of these

parameters is necessary to draw conclusions about the ideal microneedle device.

Acknowledgements


Vaccine delivery: alternatives for conventional multiple injection vaccines.

The authors like to thank Horst Liebl for supplying us with the Dermastamp®.


91

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PART II: TMC-BASED FORMULATIONS FOR INTRADERMAL AND TRANSCUTANEOUS

VACCINATION

Chapter 5

Efficient induction of immune responses

through intradermal vaccination with

TMC containing antigen formulations Suzanne M. Bal, Bram Slütter, Elly van Riet, Annelieke C. Kruithof, Zhi Ding,

Gideon F.A. Kersten, Wim Jiskoot, Joke A. Bouwstra

Journal of Controlled Release 2010, 142(3):374-383

Chapter 5

96

Abstract

The function of N-trimethyl chitosan (TMC) in immunisation via the skin is unknown.

Therefore we investigated the immunogenicity of both antigen-containing TMC

nanoparticles and TMC/antigen solutions after intradermal injection. Nanoparticles were

prepared with a size around 200 nm and a positive zetapotential. In vitro, TMC

nanoparticles increased the uptake of OVA by dendritic cells (DCs) and both nanoparticles

and TMC/OVA mixtures were able to induce upregulation of MHC-II, CD83 and CD86.

These activated DCs could induce a Th2 biased T cell proliferation. A solution of plain OVA

did not induce DC maturation or T cell proliferation. In vivo, mice were injected thrice with

TMC based formulations containing either OVA or diphtheria toxoid (DT), a more relevant

antigen. All TMC containing formulations were able to increase the IgG titres compared to

unadjuvanted antigen and induced a Th2 biased immune response. When using DT-

containing TMC formulations, IgG titres and neutralising antibody titres could match up to

those obtained after subcutaneous injection of DT-Alum. In conclusion, both soluble

TMC/antigen mixtures and TMC nanoparticles are able to induce DC maturation and

enhance immune responses after intradermal injection. This demonstrates that TMC

functions as an immune potentiator for antigens delivered via the skin.

Intradermal vaccination with N-trimethyl chitosan formulations

97

Introduction

Currently, most vaccines in development are subunit vaccines. In comparison to live

attenuated vaccines and whole inactivated organisms subunit vaccines are safer, because

of their higher purity. However, the high purity reduces the immunogenicity of subunit

vaccines, which poses a challenge in designing formulations [1]. Subunit vaccines often

require adjuvants to achieve a more effective immune response. Adjuvants are substances

or devices that enhance the delivery or immunogenicity of an antigen. Improved delivery

can prolong the localisation time of the antigen at the site of action and increased

immunogenicity implies better activation of antigen presenting cells (APCs), especially

dendritic cells (DCs) [2]. Since it has become clear that APCs have such a central role in the

first steps of the immune response, interest into the choice of the administration route has

increased. In the muscle, the most common vaccination site, only few APCs are present [3].

Higher numbers are found in the skin and the mucosal membranes, where invading

pathogens are usually encountered.

In this study we investigate the skin as immunisation site and the use of N-trimethyl

chitosan (TMC) as an adjuvant. In the skin two types of APCs, Langerhans cells (LCs) and

DCs are present in the epidermis and the dermis, respectively [4, 5]. The main function of

these cells is to take up an antigen, process it and present it to T cells. The presence of LCs

and DCs in the skin is one of the great values of vaccination via the skin, which may be a

dose-sparing alternative to conventional immunisation routes. For instance, low antigen

doses administered intradermally (i.e. injected into the dermis) have been reported to

elicit a similar or better immune response compared to higher doses given intramuscularly

[6-8]. The only intradermal vaccines currently on the market are against BCG and rabies,

and recently Intanza(R)

was approved as an intradermal influenza vaccine. An excellent

review on recent clinical studies of intradermal immunisation was published by Nicolas et

al. [9].

To deliver the antigen to the APCs, it is important to have an interaction of the antigen

with a particle [10-13]. Particles do not only increase the exposure time of the antigen to

DCs, but can also improve the uptake and maturation of DCs, because their size is more

comparable to that of viruses or bacteria. The ideal size of a particle has not yet been

determined and may depend on the immunisation route, but some evidence exists that

skin APCs preferentially take up small nanoparticles [14]. Nanoparticles can be prepared

from natural polymers such as starch [15] and chitosan [16] or synthetic polymers such as

D-poly L-lactate (PLA) and poly (DL-lactic-co-glycolic acid) (PLGA) [17]. Chitosan is present

in small amounts in some micro-organisms and fungi, and can be derived by deacetylation

of the naturally occurring polysaccharide chitin [16]. Its main drawback is the insolubility at

a physiological pH. Chitosan can be made more water-soluble by chemical modification.

Chapter 5

98

One possible modification is the introduction of three methyl groups on the NH2 group of

chitosan resulting in TMC [18, 19]. This soluble, positively charged derivate has been

studied as an adjuvant in vaccine delivery applications for various routes of administration,

such as oral [20, 21], nasal [22-25] and pulmonary [26]. However, in all these studies the

adjuvant effect of TMC was mainly ascribed to its mucoadhesive properties. In vaccination

via the skin this does not play a role and to our knowledge TMC has never been used in

transcutaneous (i.e. through application onto the skin) or intradermal vaccination. It has

only been shown to enhance the transdermal delivery of low-molecular-weight drugs [27].

Recently, studies performed in our lab showed that TMC nanoparticles can induce

maturation of DCs in vitro [20]. This indicates that TMC has adjuvant properties besides its

function as a mucoadhesive. Our interest is to investigate TMC as an adjuvant in

transcutaneous or intradermal vaccination. In transcutaneous vaccination, the efficiency of

the delivery system not only depends on the interactions of the delivery system with the

DCs, but is also largely dependent on the transport of the vaccine across the skin barrier.

The outermost layer of the skin, the stratum corneum, acts as a formidable barrier for the

transport of compounds. One of the methods to overcome this barrier is by using

microneedles to pierce small conduits in the skin [28]. Although transcutaneous

vaccination is our final goal, in this study we will focus on the efficiency of TMC as an

adjuvant. Therefore we will avoid the complicating factor of the transport issue across the

skin barrier mentioned above. We will explore the immune potentiation of TMC to

determine whether the characteristics of the TMC polymer itself or the particulate nature

play a prominent role in the adjuvant effect. It is known that TMC nanoparticles can

function as an adjuvant using other routes of administration, but whether the TMC

polymer itself also functions as an immune potentiator is unclear. For this purpose TMC

nanoparticles and mixtures of a TMC and an antigen were compared concerning in vitro DC

maturation, the in vitro T cell proliferation and finally the immune potentiation in

intradermal vaccination. Formulations made of TMC with different degrees of

quarternisation (DQ) and loaded with ovalbumin (OVA) or diphtheria toxoid (DT) were

used.


Materials

Chitosan (MW 120 kDa) with a degree of deacetylation of 92% was obtained from Primex

(Alversham, Norway). Pentasodium tripolyphosphate (TPP), N-(2-hydroxyethyl) piperazine-

N’-(2-ethanesulphonic acid) (HEPES) and Tween 20 were obtained from Sigma Aldrich

(Zwijndrecht, The Netherlands). Ovalbumin grade VII was obtained from Calbiochem

(Merck KGaA, Darmstadt, Germany) FITC and Alexa647 labelled ovalbumin (OVAFITC and


99

OVAAF647 respectively) were purchased from Invitrogen (Breda, The Netherlands).

Diphtheria toxin (DTa 79/1) and DT (batch 98/40, protein content 12.6 mg/ml by BCA

assay, 1 μg equal to approximately 0.3 Lf) were a kind gift from the Dutch Vaccine Institute

(NVI, Bilthoven, The Netherlands). Horseradish peroxidase (HRP) conjugated goat anti-

mouse IgG (γ chain specific), IgG1 (γ1 chain specific) and IgG2a (γ2a chain specific) were

purchased from Southern Biotech (Birmingham, USA). Chromogen 3, 3', 5, 5'-

tetramethylbenzidine (TMB) and the substrate buffer were purchased from Invitrogen.

Nimatek® (100 mg/ml Ketamine, Eurovet Animal Health B.V., Bladel, The Netherlands),

Oculentum Simplex (Farmachemie , Haarlem, The Netherlands), Rompun® (20 mg/ml

Xylazine, Bayer B.V., Mijdrecht, The Netherlands) and the injection fluid (0.9% NaCl) were

obtained from a local pharmacy. All other chemicals were of analytical grade.

Animals

Female BALB/c mice (H2d), 8-weeks old at the start of the vaccination study were

purchased from Charles River (Maastricht, The Netherlands), and maintained under

standardised conditions in the animal facility of the Leiden/Amsterdam Centre for Drug

Research, Leiden University. The study was carried out under the guidelines compiled by

the Animal Ethic Committee of the Netherlands.

TMC synthesis

TMC with a variable DQ was synthesised by methylation of chitosan by using iodomethane

in the presence of a strong base (NaOH) as described previously [29]. In short, 2 g of

chitosan and 4.8 g sodium iodide were dissolved in 80 mL 1-methyl-2-pyrrolidone and after

stirring for 20 minutes at 60°C 12 mL 15% NaOH and 12 mL iodomethane were added. The

mixture was refluxed for 60 minutes after which the TMC was precipitated with ethanol

and diethyl ether. To synthesise TMC with an increasing DQ an additional amount of NaOH

(5-14 mL) and 5 mL of iodomethane were added before precipitation. The obtained

polymer was purified by dialysis against 1% NaCl for 4 days followed by dialysis against

water for 2 days at 4°C. Finally the product was freeze-dried. The purified TMC was

analysed by 1H-nuclear magnetic resonance (NMR) spectroscopy. For this measurement

the TMC was dissolved in D2O and the spectrum was recorded at 80°C with a DMX 400

MHz NMR spectrometer (Brucker, Switzerland). The degree of quarternisation was

calculated according to the following equation:

DQ = [[(CH3)3] / [H] x 1/9] x 100 (1)

Chapter 5

100

[(CH3)3] is the integral of the trimethyl amino group at 3.3 ppm and [H] is the integral of

the hydrogen peaks of the carbon 1 atom of TMC between 4.7 and 5.7 ppm [19].

Preparation and characterisation of TMC formulations

Both TMC nanoparticles and mixtures of TMC and antigen were prepared. For nanoparticle

preparation TMC of two different DQ was used, namely 15 and 30% (TMC15 and TMC30).

TMC nanoparticles were prepared by ionic complexation with TPP as was previously

described [20]. Shortly, for a 10 mL batch of nanoparticles an aqueous solution of TPP (1

mg/mL) was added drop wise to 5 mL of TMC solution (10 mg) in 5 mM HEPES pH 7.0 while

stirring until the solution became slightly opalescent. OVA and DT loaded nanoparticles

were prepared by dissolving the antigen (1 mg) in the TMC solution before adding the TPP

solution. The amount of TPP added depended on the antigen and on the DQ of the TMC

used (varying between 1.3 and 2.0 mL). After 1 hour of stirring, the nanoparticle

suspension was centrifuged for 15 minutes at 10000 g on a glycerol bed and the pellet was

resuspended in a 5 mM HEPES buffer adjusted to pH 7.0.

Mixtures of TMC and OVA or DT were prepared by mixing a solution of TMC and the

antigen in a 2.5:1 ratio. The size of the nanoparticles and mixtures was determined by

dynamic light scattering (DLS) and the zetapotential was determined by laser Doppler

velocimetry using Zetasizer® Nano ZS (Malvern Instruments, UK). The amount of TMC and

TPP in the nanoparticles was determined with a ninhydrin assay and a phosphate

determination, respectively [30, 31].

Nanoparticle visualisation

To characterise the morphology of the nanoparticles, they were visualised with scanning

electron microscopy. 50 µl of 0.1% w/v nanoparticle suspension was air dried overnight on

an adhesive sample holder. Afterwards the samples were gold/palladium sputtered using a

sputter coater device K650X (Emitech, Hailsham, UK) and analysed with a JEOL JSM-6700F

scanning electron microscope (Jeol, Tokyo, Japan).

Loading efficiency of TMC nanoparticles

The amount of encapsulated OVA or DT in the nanoparticles was determined by measuring

the amount of protein remaining in the supernatant with a micro-BCA protein assay

(Pierce, Rockford, IL, USA) after centrifugation (15 minutes, 10000 g). The same was done

with the TMC/antigen mixtures to determine the adsorption of antigen to the TMC

polymer. For the OVAFITC loaded nanoparticles the amount of OVA was determined by

measuring the amount of OVAFITC in the supernatant with a FS920 fluorimeter (ex 488 nm,

em 520 nm) (Edinburgh Instruments, Campus Livingston, UK). To avoid differences in


101

fluorescence due to pH changes, 25 µl of 5 M NaOH was added to all samples. For both

methods a non-loaded nanoparticle suspension was used as a blank to correct for

interference by TMC. The loading efficiency was determined with the following equation:

Loading efficiency = ((Total amount of protein – Free protein)/Total amount of protein) x 100% (2)

In vitro stability of TMC nanoparticles

To measure the colloidal stability of the nanoparticles, after centrifugation they were

resuspended in 1 ml distilled water and diluted to a final TMC concentration of 1.6 mg/mL

in phosphate buffered saline pH 7.4 (PBS: NaCl: 8 g/l, KCl: 0.4 g/l, KH2PO4: 0.4 g/l, Na2HPO4:

2.86 g/l). The nanoparticles were stored at 37°C and their size was measured after 1, 2, 4,

24 and 48 hours.

In vitro release of ovalbumin from TMC nanoparticles

Nanoparticles containing 250 µg OVAFITC were prepared and after centrifugation (15

minutes 10000 g) resuspended in 1 ml distilled water and diluted to a final TMC

concentration of 1 mg/ml in PBS pH 7.4 containing 0.1% Tween 20 (sink conditions). The

nanoparticles suspension was stirred continuously in the dark at 37°C for 9 days and every

day a 300 µl sample was taken. The samples were centrifuged for 15 minutes at 15000 g

and the amount of OVAFITC in the supernatant was determined after the addition of 25 µl

of 5 M NaOH.

Generation of human monocyte derived dendritic cells

Monocytes were isolated from whole blood or buffy coat (obtained from blood bank,

Sanquin, The Netherlands) by Ficoll and Percoll density centrifugation [32]. These

monocytes were purified from platelets by monocyte adherence to 24 well plates (Corning,

Schiphol, The Netherlands) followed by washing. To differentiate into immature DCs, the

monocytes were cultured for 6 days at a density of 0.5 x 106 cells/well in RPMI 1640,

supplemented with 10% FCS, 1% glutamine, 1% v/v Penicillin/Streptomycin, granulocyte

macrophage-colony stimulating factor (GM-CSF) 250 U/ml and IL-4 100 U/ml (Invitrogen)

at 370C and 5% CO2. Medium was refreshed after 3 days.

Dendritic cell association

To assess the effect of nanoparticle encapsulation on antigen uptake, DCs were incubated

at 370C in serum free RPMI 1640 (with 500 U/ml GM-CSF) containing OVAFITC (2 µg/mL) in

solution, mixed with TMC15 or TMC30 or encapsulated in TMC nanoparticles. For the in

vitro studies formulations containing OVA were used; both because of the availability of

Chapter 5

102

fluorescently labelled antigen and straightforward comparison to previous studies

performed in our lab [20]. The DCs were incubated with the formulations for 4 hours. After

1, 2 or 4 hours the cells were washed three times with PBS containing 1% w/v bovine

serum albumin and 2% v/v FCS and the association of OVAFITC with DCs was quantified

using flow cytometry (FACS Canto II, Becton Dickinson, Breda, The Netherlands). Living

cells were gated based on forward and side scatter, OVAFITC association was expressed as

the mean fluorescence intensity (MFI) in the FL-1 channel. To verify whether the

formulations were not only associated with the DCs, but also actively taken up, the same

study was repeated at 40C. Histogram overlays were created with WinMDI 2.9.

Upregulation of DC maturation markers

DCs were incubated for 48 hours in RPMI 1640 containing 500 U/mL GMCSF and 10% FCS

with the same formulations as for the DC association study. Since no difference in DC

association between TMC15 and TMC30 nanoparticles was observed, only TMC15 was

used in this study. LPS (100 ng/mL) was used as a positive control. After 48 hours the

supernatant was removed and stored at -200C until ELISA analysis of IL-6 and IL-12

secretion (PeliKine-compact kit, Sanquin, Amsterdam, The Netherlands). The cells were

washed three times with PBS containing 1% w/v bovine serum albumin and 2% v/v FCS and

incubated for 30 minutes with 20x diluted anti-HLADRFITC, anti-CD83PE and anti-CD86APC

(BD, Breda, The Netherlands) in the dark at 40C. Cells were washed and the expression of

MHC-II, CD83 and CD86 on the cells was quantified using flow cytometry. Living cells were

gated based on forward and side scatter, the amount of MHCII, CD83 and CD86 positive

cells were expressed as the MFI in the FL-1, FL-2 and FL-4 channel relative to the LPS

control.

Confocal microscopy

The uptake of antigen by DCs was visualised with confocal laser scanning microscopy

(CLSM). DCs were plated at a density of 1 x 105 cells to a poly-L-lysine coated petridish with

glass bottom and allowed to adhere for 30 minutes. Afterwards the cells were incubated

with the formulations containing OVAAF647 for 1 hour. After 45 minutes 0.1 mM

LysoTracker Green (Invitrogen) was added. Images were processed using a Bio-Rad

Radiance 2100 confocal laser scanning system equipped with a Nikon Eclipse TE2000-U

inverted microscope and a 40x air objective. The images were captured using an argon

laser at 488 nm with a 515/30 nm emission filter and a red diode at 633 nm with a 660

long pass emission filter. Image acquisition was controlled using the Laser Sharp 2000

software (Bio-Rad, Hercules, USA).


103

T cell activation

To assess the tendency of the formulations to induce a Th1 or Th2 response, CD4+ T cells

were purified from PBMC using MACS beads (Miltenyi Biotec, Bergisch-Gladbach,

Germany) according to the manufacturer’s protocol. After 48 hrs of incubation of the DCs

with the formulations, 5 x 103 cells were cocultured with 2 x 10

4 T cells in RPMI 1640

containing 10% FCS and 20 pg/mL Staphylococcus enterotoxin B (SEB; Sigma-Aldrich) in 96-

well flat-bottom culture plates. After 5 days, the T cells were transferred to a 24 well plate

and IL-2 (10 U/mL) was added. The cells were cultured for 9 more days and on day 14 the T

cells were restimulated for 6 hours with 100 µg/mL phorbol myristate acetate (PMA) and

0.5 mg/mL ionomycin (Sigma-Aldrich). After 3 hours 5 mg/mL Brefeldin A (Sigma-Aldrich)

was added to be able to detect the intracellular production of IL-4 and IFN-γ with flow

cytometry. Live cells were gated based on forward and side scatter, the amount of IL-4 and

IFN-γ positive cells were expressed relative to the LPS control. As Th1 and Th2 controls a

mixture of LPS (100 ng/mL) with Heat-killed Listerla monocytogenes (HKLM; 10

8 U/mL) or

with a soluble extract of schistosome eggs (25 µg/mL) were used respectively.

Intradermal immunisation

The immunogenicity of intradermally administered OVA and DT loaded TMC15

nanoparticles and a mixture of antigen and TMC15 was assessed in mice. The in vivo

studies were performed with both antigens as DT is a relevant antigen for immunisation

and a model to evaluate the protection after vaccination is available [33]. The mice were

vaccinated thrice with three weeks intervals. Groups of 8 mice were injected intradermally

with a Hamilton syringe equipped with a 30-Gauge needle [34]. A total volume of 30 µL

containing 5 µg (1.5 Lf) OVA or DT dissolved in PBS, encapsulated in TMC15 nanoparticles

or mixed with an equivalent amount of TMC was injected into the abdominal skin under

anaesthesia (by intraperitoneal injection of 150 mg/kg Ketamine and 10 mg/kg Xylazine).

As a control 100 µL containing 5 µg of antigen in PBS or in case of DT adsorbed to

aluminium phosphate (Adju-Phos®; Brenntag Biosector, Denmark) was injected

subcutaneously. The DT adsorbed to aluminium phosphate (DT-Alum) was prepared as

previously described and the adsorption was between 70 and 80% [35]. One day before

each immunisation blood samples were collected from the tail vein. Three weeks after the

last vaccination the mice were sacrificed. Just before euthanasia total blood was collected

from the femoral artery. Blood samples were collected in MiniCollect® tubes (Greiner Bio-

one, Alphen a/d Rijn, The Netherlands) till clot formation and centrifuged 10 minutes at

10,000 g to obtain cell-free sera. The sera were stored at -80 ºC until further use.

Chapter 5

104

Detection of serum IgG, IgG1 and IgG2a

OVA and DT specific antibodies (IgG, IgG1 & IgG2a) were determined by sandwich ELISA as

described previously [36]. Briefly, 96 well plates (Microlon®, Greiner Bio-one, Alphen a/d

Rijn, The Netherlands) were coated overnight at 4°C with 100 ng OVA or 140 ng DT in

coating buffer (0.05 M sodium carbonate/ bicarbonate, pH 9.6) per well. Afterwards the

plates were blocked by incubation with 1% (w/v) BSA in PBS containing 0.05% Tween 20

for 1 hour at 37°C. Two-fold serial dilutions of sera from individual mice were applied to

the plates and incubated for 2 hours at 37°C. Plates were incubated with HRP-conjugated

goat antibodies against either mouse IgG, IgG1 or IgG2a (Invitrogen) for 1.5 hour at 37°C

and antibodies were detected by TMB and measuring optical density at 450 nm. Antibody

titres were expressed as the reciprocal of the sample dilution that corresponds to half of

the maximum absorbance at 450 nm of a complete s-shaped absorbance-log dilution

curve.

Vero cell test

The levels of diphtheria toxin-neutralising antibodies in mouse sera were assessed by a

Vero cell test as previously described [33]. First, serum complement was inactivated by

heating at 56ºC for 45 minutes. Following, twofold serial dilutions of individual sera were

prepared in complete medium 199 (CM199, Gibco, Breda, The Netherlands) and applied to

96 well plates (CELLSTAR®, Greiner Bio-One). Then, 2.5 x 10

-5 Lf of diphtheria toxin was

added to each well and the plates were incubated for 2 hours at 37ºC for neutralisation.

Subsequently, medium containing 1.25 x 104 Vero cells was added to each well. As a

control antitoxin and untreated cells were included. The plates were incubated at 37ºC in

5% CO2 for 6 days and afterwards the presence of living cells was verified by light

microscopy. The antibody titres were obtained from the serum dilution factor that still

resulted in living cells.


Statistical analysis was performed with Prism 5 for Windows (Graphpad, San Diego, USA).

Data are presented as mean ± S.D. for all results. Statistical significance was determined

either by a one way or a two way analysis of variance (ANOVA) with a Bonferroni post-test,

depending on the experiment set-up. The results of the Vero cell test were analysed by a

Kruskal-Wallis test with a Dunn's multiple comparison post-test.


105

Results

Synthesis of TMC with varying degrees of quarternisation

By synthesising TMC according to the one step method described by Hamman and Kotzé

[29] we were able to obtain TMC with a reproducible DQ around 15%. By increasing the

amount of base (NaOH) in the additional step, the trimethylation could be increased in a

controlled manner (figure 1). From this figure it can be observed that initially the chitosan

was mostly dimethylated and by adding more NaOH trimethylation was induced. As

postulated by Domard et al., the NaOH increases the pH by reacting with the hydroiodic

acid formed during the reaction [37]. For that reason, a deficit of sodium hydroxide might

limit the trimetylation. By gradually increasing the amount of sodium hydroxide, the DQ

could be increased in a controlled manner up to 70%. Recent studies performed by Verheul

et al. showed that this method of TMC synthesis induces only a slight decrease in

molecular weight due to chain scission [38].

Characterisation of TMC15 and TMC30 formulations

After having optimized the TMC synthesis, nanoparticles were prepared with TMC15 and

TMC30 as this is in the DQ range where previously no toxicity was observed [22, 38, 39].

DLS studies revealed that with both TMC15 and TMC30 nanoparticles of a size between

200 and 300 nm could be made, depending on the antigen that was encapsulated. The

presence of the nanoparticles was confirmed by SEM images (figure 2C). The nanoparticles

were irregularly in shape, which is consistent with previous studies [39]. It was possible to

vary the size of the particles to a minor extent by varying the amount of TPP added (data

not shown). For TMC nanoparticles containing OVA the ratio (w/w) TMC:TPP to obtain

stable nanoparticles was 6.7:1 for TMC15 and 5:1 for TMC30. If more TPP was added,

aggregation of the particles could be observed. The same trend was observed if DT was

used as an antigen (table 1). Nanoparticles of both types of TMC were positively charged.

The zetapotential of the TMC30 nanoparticles was slightly higher than that of the TMC15

Figure 1. Effect of the amount of

NaOH used during the second

step of TMC synthesis on the

percentage of trimethylation of

TMC. Mean ± SD of three

independent batches are shown.

The batches with 7 and 14 ml of

NaOH in the second step were

prepared only once.

Chapter 5

106

nanoparticles. Since this effect was present regardless of the antigen, it can probably be

ascribed to the higher DQ of TMC30.

The colloidal stability of the nanoparticles in PBS was measured during a period of 2 days.

We observed a difference between TMC15 and TMC30. In PBS, the size of the TMC15

nanoparticles started to increase and after 24 hours the nanoparticles had already doubled

(figure 2A). In contrast, the size of the TMC30 nanoparticles was stable in PBS for at least 2

days (figure 2B).

Nanoparticles

Antigen Size

[nm]

PDI Zetapotential

[mV]

Protein

loading [%]

TMC

[%]

TPP

[%]

TMC15 - 219 ± 1 0.13 ± 0.02 16.2 ± 0.8 - 75 ± 1 47 ± 3

TMC15 OVA 276 ± 6 0.21 ± 0.04 10.6 ± 0.3 69 ± 1 75 ± 3 57 ± 5

TMC15 DT 211 ± 4 0.15 ± 0.01 12.9 ± 0.8 70 ± 3 56 ± 1 41 ± 2

TMC30 - 248 ± 9 0.20 ± 0.05 17.4 ± 0.9 - 71 ± 1 43 ± 6

TMC30 OVA 344 ± 16 0.26 ± 0.03 13.5 ± 1.3 78 ± 5 66 ± 2 52 ±4

TMC30 DT 228 ± 2 0.14 ± 0.01 13.6 ± 0.2 78 ± 7 35 ± 2 27 ± 2

The percentage of protein, TMC and TPP shown in the table indicate the amount present in the

nanoparticles after purification in comparison to the amount added during preparation.

The amount of protein associated with the nanoparticles or TMC solution was assessed.

For TMC15 around 70% of the added antigen (OVA or DT) could be loaded into the

nanoparticles, while for TMC30 this was almost 80% (table 1). The difference in antigen

loading between TMC15 and TMC30 nanoparticles might be explained by the slightly lower

DQ of TMC15, leading to a lower protein association. No difference in loading was

observed between OVA and DT. However, a distinct difference between the amount of

TMC and TPP present in the DT loaded nanoparticles compared to the OVA loaded

nanoparticles was found. The interaction of the antigen with the TMC seems to be

different for DT and OVA. The difference in association was also observed for the

TMC/antigen mixtures. In TMC/OVA mixtures only 5% of the antigen was associated with

the TMC polymer, while in TMC/DT mixtures this was almost 50% (data not shown). This

difference is probably due to dissimilarity in zetapotential between OVA and DT at pH 7

(OVA -11.2 ± 3.3 mV and DT -20.7 ± 2.2 mV).

The release profile of OVAFITC from the TMC30 and TMC15 particles was similar; showing

an initial burst release (figure 2D). The burst release of the TMC15 nanoparticles was

slightly higher, probably also due to the lower DQ. After the initial release an equilibrium

was reached, showing no further release over the next nine days. This release profile is in

agreement with results obtained by Amidi et al. who attributed the burst release to

Table 1. Physicochemical characteristics of TMC15 and TMC30 nanoparticles.


107

antigen that is loosely bound to the particle surface [39]. This means that most of the

antigen is encapsulated in the nanoparticles.

DC association and uptake of TMC-ovalbumin formulations

Antigen association to DCs was measured by stimulating immature DCs with OVAFITC

formulations. As shown by flow cytometry the association of OVAFITC to DCs increased with

time compared to unstimulated DCs. Both types of nanoparticles increased the association

tremendously compared to applying a solution of OVAFITC (figure 3A). A 2.5 fold increase

could already be observed after one hour and after four hours the association had

increased 7.5 fold compared to a solution of OVAFITC. Interestingly, a TMC/OVAFITC mixture

did not increase the association of OVAFITC to DCs. No difference was observed between

TMC15 and TMC30 regarding their effect on OVAFITC uptake.

Control studies performed at 4°C to inhibit active uptake and CLSM visualisation studies

both showed that the positively charged nanoparticles adhered to the DCs. Figure 3B

Figure 2. Characteristics of OVA loaded TMC15 and TMC30 nanoparticles. A/B: Size of the TMC15

nanoparticles (A) and TMC30 (B) nanoparticles during 2 days incubation at 37°C. C: SEM images of

TMC15 nanoparticles show that the particles have an average size between 200 and 300 nm and

are irregularly shaped. D: Release of OVA over time from the nanoparticles in PBS at 37°C. Values

are the average ± SD of three independent batches.

Chapter 5

108

shows that OVAFITC from a solution was only taken up at 37°C, while DCs treated OVAFITC-

loaded TMC-nanoparticles also had increased MFI values at 4°C. However, association does

not completely explain the increased MFI values, because the uptake of OVAFITC

encapsulated in TMC nanoparticles was slightly higher at 37°C than at 4°C at. Confocal

microscopy studies with TMC nanoparticles containing OVAAF647 confirmed these results

(figure 4). Free OVA or in a mixture of OVA and TMC in solution was taken up by DCs and

ended up in the lysosomes, as shown by co-localisation of OVAFITC and lysosome staining.

Overlays made with transmission microscopy showed that if OVAAF647 was encapsulated in

nanoparticles it was mainly present on the surface of DCs. Only a small part of the OVAAF647

was co-localised with the lysosomes. Similar results were observed for TMC15 and TMC30

(data not shown). The increased association may be favourable for antigen uptake,

because the cells are in contact with the antigen for a longer period.

Effect of TMC15 formulations on DC maturation

It was already known that nanoparticles can improve the uptake of an antigen [10-13].

However, uptake alone is not enough to induce an immune response. Therefore the ability

of the formulations to induce DC maturation was explored. Even though the

characterisation and DC uptake studies were performed with both TMC15 and TMC30, for

practical reasons in all further studies only TMC15 is used. Since the characterisation and

uptake studies showed only minor differences between TMC15 and TMC30, it seems that

the difference in DQ in the studied range did not have an influence on the function of the

TMC.

Flow cytometry measurements showed that even though a solution of OVA was taken up

by DCs, it did not induce upregulation of the expression of the maturation markers CD83,

Figure 3. A: Association

of OVAFITC with DCs after

application of different

formulations for 1, 2 and

4 hrs at 37°C. Results

expressed as mean MFI

± SEM relative to free

OVAFITC at t=1 hr (n=6).

B: Representatives of

the difference between

DC association of OVAFITC

after 4 hrs at 4 and 37°C.


109

CD86 and MHCII (figure 5A). LPS, on the other hand showed a distinct increase of the

expression of these three markers. If the antigen was either encapsulated in TMC15

nanoparticles or mixed with a TMC15 solution, the levels of CD83 and CD86 increased

significantly compared to those after OVA application. A trend of increased MHCII levels

could also be observed.

To obtain additional insight into the maturation status of the DCs, the secretion of IL-6 and

IL-12 was measured. Both OVA loaded TMC nanoparticles and a mixture of OVA and TMC

induced secretion of IL-6 and IL-12 (figure 5B). TMC nanoparticles induced a much higher

cytokine secretion than a TMC solution. Even more IL-12 was produced than after LPS

stimulation. This confirms that TMC functions as an immune potentiator. To verify that the

DC maturation was not caused by LPS contamination in our samples, they were applied to

TLR-4 transfected HEK cells. The LPS content was found to be below the detection limit

(<0.1 ng/ml, data not shown).

Figure 4. CLSM images showing the presence of OVAAF647 in DCs 1 hr after the application of

different formulations. The lysosomes are stained with LysoTracker Green to show possible co-

localisation of the OVA and the lysosomes.

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110

Induction of T cell stimulation by TMC formulations

Since the TMC-OVA interaction is based on electrostatic interactions, it is expected that the

TMC nanoparticles will be degraded once they reach the lysosomes, releasing the OVA to

be processed and presented to T cells. By using the effector DCs obtained after 48 hrs of

maturation to stimulate naïve T cells, we studied the T cell response in vitro. After 14 days

we determined the subsets of effector T cells by analysing the intracellular production of

IFN-γ (Th1 biased response) and IL-4 (Th2 biased response). Figure 6 shows that LPS

induced a mixture of Th1 and Th2 effector T cells, in agreement with previous studies [40].

The other formulations were compared to LPS. In comparison to LPS, the OVA loaded

TMC15 nanoparticles and the mixture of OVA and TMC15 induced more cells to secrete IL-

4 than IFN-γ. The TMC-containing formulations were strong inducers of the proliferation of

Th2 effector T cells, since the Th1/Th2 ratios were 0.26 for TMC15 nanoparticles and 0.57

for the TMC15/OVA mixture. This was in the same range as an extract of schistosome eggs,

a known stimulus of Th2 development, which gave a Th1/Th2 ratio of 0.28 (see figure 6).

Figure 5. Effect of different formulations on the maturation of DCs. A: MHC-II, CD83 and CD86

expression; B: production of IL-12 and IL-6. Values are expressed as mean MFI ± SEM relative

to LPS of seven experiments (A) and in figure B a representative example is shown.


111

Antibody levels after intradermal immunisation

No severe side reactions to the intradermal or subcutaneous injections were observed. For

the both the TMC- and alum- containing formulations sometimes a white firmness could

be observed at the injection site, most likely due to depot formation. The IgG titres were

measured in the sera of the mice before each immunisation and three weeks after the last

immunisation. The results obtained with OVA and DT are very comparable. Intradermal or

subcutaneous injection with plain antigen gave the lowest titres. TMC15-TPP-OVA

nanoparticles were applied subcutaneously and intradermally. Both intradermal and

subcutaneous injection of the nanoparticles induced significantly higher antibody titres

compared to the plain OVA. No differences between intradermal and subcutaneous could

be observed. Besides TMC nanoparticles, also a TMC/OVA mixture was applied.

Intradermal application of either TMC nanoparticles or a TMC/OVA mixture induced 5x,

14x and 8x higher anti-OVA IgG titres compared to plain OVA after respectively the prime,

first and second boost immunisations (figure 7A).

For DT, after the first immunisation, only 6 out of 8 mice which were injected intradermally

with plain DT, showed detectable anti-DT IgG levels (figure 7C). Also here application of the

TMC based formulations enhanced the immune response, even more pronounced than for

OVA. Intradermal injection of TMC15 nanoparticles containing DT or a mixture of DT and

TMC15 induced 200 fold higher titres (p<0.001) after the first immunisation compared to

intradermal DT injection. The difference in response was less distinct after the first and

second boost, but still respectively 60x and 4x higher IgG levels were obtained (p<0.001).

The IgG titres of intradermal TMC15-TPP-DT nanoparticles and a TMC15/DT mixture were

comparable to those after subcutaneous DT-alum injection. For both these groups the

Figure 6. Ability of DCs stimulated with different formulations to induce the activation of Th1

(IFN-γ) or Th2 (IL-4) effector T cells. Relative contributions compared to LPS are shown. Th1 (LPS

+ HKLM) and Th2 (LPS + an extract of schistosome eggs) controls were included. Results from

one representative experiment out of two performed.

Chapter 5

112

titres already reached their maximum values after the first boost. In figure 7B and D the

IgG1 and IgG2a titres after the second boost are shown. The ratio of IgG1:IgG2a gives an

indication if the immune response is Th1 or Th2 biased. For all groups of both antigens the

IgG2a titres were lower than the IgG1 titres and in general developed only after the first

boost (data not shown). The IgG1/IgG2a ratio did not differ significantly between the

groups and in all cases Th2 biased responses were achieved. The IgG2a titres were slightly

higher after DT compared to OVA immunisation and only here significant differences

between intradermal application of the TMC formulations and plain OVA were observed

(figure 7D). For OVA only an effect of the TMC was present if the nanoparticles were

applied subcutaneously (figure 7B).

Figure 7. Antibody titres after subcutaneous (SC) and intradermal (ID) vaccination with DT or

OVA. Total anti DT (A) and OVA (C) IgG titres after the first and two subsequent boost

vaccinations; IgG1 and IgG2a levels against DT (B) and OVA (D) after the second boost. Mean

titres ± SD of 8 mice are shown. **/*** p<0.01/0.001 compared to ID OVA or DT. ‡ p<0.001

compared to SC OVA.


113

The neutralising antibody titres from the Vero cell test confirmed the IgG antibody titre

results (figure 8). The TMC based formulations applied intradermally increased the

production of neutralising antibodies compared to unadjuvanted DT. This result was most

clearly visible after the first boost (figure 8A) when no neutralising antibodies could be

detected for intradermal DT, but the TMC based formulations already induced substantial

antibody titres. Their neutralising capacity was the same compared to subcutaneous DT-

alum, but a trend of faster kinetics could be observed.

Discussion

Vaccination via the skin offers great promise as an alternative for intramuscular or

subcutaneous vaccination, but to induce an efficient immune response to subunit vaccines,

also for this delivery route adjuvants are necessary. Different types of adjuvants have

already been used, which evoke or increase the transcutaneous immune response [41-45].

Another adjuvant TMC has already shown great promise in mucosal vaccination, which is

thought to be largely due to its mucoadhesive properties. In this study we explored the

properties of TMC as an adjuvant in immunisation via the skin. To focus on the adjuvant

effect and not on the transport through the skin, which plays a prominent role in

transcutaneous vaccination, the formulations were administered by intradermal injection.

The antibody titres after intradermal immunisation show the potential of TMC as an

inducer of an immune response after intradermal immunisation. Total IgG, IgG1 and IgG2a

titres after immunisation with either OVA or DT loaded TMC15 nanoparticles and

Figure 8. Diphtheria toxin-neutralising antibody titres after subcutaneous (SC) and intradermal

(ID) vaccinations after the first (A) and second boost (B). Serum samples were collected and the

titres determined by a Vero cell test. Data are expressed as the highest dilution that was still

capable of protecting the Vero cells from the challenge of diphtheria toxin.

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TMC15/OVA and TMC15/DT mixtures were significantly higher than after immunisation

with an antigen solution. From the Vero cell test it can be concluded that TMC15, both in a

solution or as a nanoparticle increases the total neutralising antibody titres and their

kinetics compared to plain DT. Interestingly, for both antigens a TMC15 solution works as

well as TMC15 nanoparticles. This is in contradiction to the results obtained by Amidi et al.,

who observed a significant increase in IgG titres after intranasal immunisation with

influenza-loaded TMC nanoparticles compared to an antigen/TMC solution [25]. Boonyo et

al. also showed that a solution of TMC20 and OVA applied intranasally did not induce

significantly higher IgG titres compared to OVA alone [23]. In this study we show that by

mixing DT and TMC, almost 50% of the DT was associated with the TMC. For OVA this was

only 5%.. However, antigen adsorption does appear to have no influence on the obtained

antibody titres. The promising results obtained with these mixtures indicate that although

nanoparticles are very important in for instance intranasal vaccination, this seems not to

be the case in intradermal vaccination. Given that by intradermal injection the antigen is

immediately at its site of action, nanoparticles might not be necessary. In intranasal

delivery the transport across the epithelium also plays a role and nanoparticles are known

to enhance the transport across M-cells [46]. Since transport also plays an important role

in transcutaneous vaccination, it is interesting to investigate if nanoparticles are an

advantage for this delivery route.

To understand the in vivo results, in vitro DC and T cell studies were carried out. TMC

nanoparticles, but not a TMC solution, were able to increase the uptake of OVA by

immature DCs. This effect could be ascribed to the association of the TMC nanoparticles

with the DCs. An increased association was not observed for a TMC solution. The

interactions between the TMC and the OVA are stronger in the nanoparticle formulation

compared to the mixture. The positive charge of the TMC is responsible for the association

to the DCs and therefore the nanoparticles are able to enhance the association of OVA,

while a TMC solution is not. The maturation of DCs was shown not to be dependent on the

particulate nature of the TMC. Both a TMC15 solution and the TMC15 nanoparticles were

able to upregulate the levels of maturation markers and to induce increased IL-12 and IL-6

levels. Furthermore, all TMC based formulations were able to induce Th2 biased T cell

proliferation.

If the in vitro and in vivo results are compared, it is clear that both the DC model and the

mouse model show that TMC15 is an immune potentiator. It is not possible to draw the

same conclusion from the T cell model. In vitro, the TMC15 based formulations are strong

inducers of Th2 biased proliferation, but the same is not observed in vivo. This can be due

both to the antigens and the mice model that was used. Since immunisation with plain

OVA or DT already induces a clear Th2 biased immune response, no further effect of

TMC15 on the Th1/Th2 ratio could be observed. Both IgG1 and IgG2a levels were


115

increased. The same was observed by Amidi et al after nasal immunisation with influenza

antigen loaded TMC nanoparticles [25]. Future immunisation studies with antigens that

favour a Th1 immune response or by using for instance C57BL/6 mice can potentially show

the effect of TMC on the Th1/Th2 ratio.

Both in vitro and in vivo it was shown that a TMC solution as well as TMC nanoparticles can

act as an adjuvant. The mechanism behind this immune modulation remains unclear, but

there are different possibilities. It is generally thought that positively charged compounds

can act as a ‘danger-signal’ for DCs and function as a signal 0 adjuvant [2]. In vitro DC

maturation studies with positively charged poly-L-lysine coated polystyrene microparticles

showed enhanced DC uptake and an increased amount of CD83 positive cells [13].

However, negatively charged modified poly(γ-glutamic acid) nanoparticles [47] and

negatively charged liposomes [48] also induced increased expression of maturation

markers. Studies with chitosan, the precursor of TMC, have shown contradictory results as

well. It was shown that chitosan increased DC maturation [49], but also studies in which

chitosan has no effect on DCs have been published [50, 51].

If TMC as an adjuvant is compared to Alum, the in vivo data show that similar IgG and

neutralising antibody titres were obtained after intradermal immunisation with the TMC

formulations as after subcutaneous DT-Alum injection. Zaharoff et al. used a solution of

chitosan as an adjuvant in subcutaneous immunisation and found it to be superior to

aluminium hydroxide [52]. Its adjuvant effect was attributed to the ability of chitosan to

retain the antigen at the site of injection. It is possible that TMC has the same function.

Further studies should focus on the adjuvant mechanism of TMC and compare it to other

known adjuvants.

In conclusion, intradermal immunisation with TMC nanoparticles as well as with TMC

solutions can elicit strong IgG and neutralising antibody titres against two different

antigens. This demonstrates that the skin is an excellent vaccination site and that TMC

based formulations have great potential. In the next step we will focus on the transport of

the antigen across the skin barrier. Trancutaneous vaccination studies with the TMC

formulations and microneedles, which were already used in previous vaccination studies

[36, 53], will be performed.

Acknowledgements



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Chapter 6

Microneedle-based transcutaneous

immunisation in mice with TMC

adjuvanted diphtheria toxoid

formulations Suzanne M. Bal, Zhi Ding, Gideon F.A. Kersten, Wim Jiskoot, Joke A.

Bouwstra

Pharmaceutical Research 2010, 27(9):1837-1847

Chapter 6

122

Abstract

The purpose of this study was to gain insight into the delivery and immunogenicity of N-

trimethyl chitosan (TMC) adjuvanted diphtheria toxoid (DT) formulations applied

transcutaneously with microneedles. Mice were vaccinated with DT-loaded TMC

nanoparticles, a solution of TMC and DT (TMC/DT) or DT alone. The formulations were

applied onto the skin before or after microneedle treatment with two different 300 µm

long microneedle arrays and also injected intradermally (ID). As a positive control alum

adjuvanted DT (DT-alum) was injected subcutaneously (SC). Ex vivo confocal microscopy

studies were performed with rhodamine-labelled TMC.

Independent of the microneedle array used and the sequence of microneedle treatment

and vaccine application, transcutaneous immunisation with the TMC/DT mixture elicited 8-

fold higher IgG titres compared to the TMC nanoparticles or DT solution. The toxin

neutralising antibody titres from this group were similar to those elicited by SC DT-alum.

After ID immunisation, both TMC-containing formulations induced enhanced titres

compared to a DT solution. Confocal microscopy studies revealed that transport of the

TMC nanoparticles across the microneedle conduits was limited compared to a TMC

solution. In conclusion, TMC has an adjuvant function in transcutaneous immunisation with

microneedles, but only if applied in a solution.

Transcutaneous immunisation with N-trimethyl chitosan formulations

123

Introduction

Transcutaneous immunisation (i.e. immunisation through vaccine application onto the

skin) has the potential to be an excellent non-invasive vaccination route [1]. This is

desirable as injection of a vaccine with a needle and a syringe is not only painful [2], but it

also bears a risk of transmission of infection with, e.g., hepatitis B or C, or human

immunodeficiency virus [3]. Furthermore, the skin is densely populated with antigen

presenting cells (APCs) [4]. In the epidermis the Langerhans cells (LCs) are present and in

the dermis the dermal dendritic cells (DCs) [5, 6]. The main function of these professional

APCs is to sample their environment, process antigens and present specific epitopes to T

cells. Studies using intradermal immunisation (i.e. injection of the antigen into the dermis)

have shown that this delivery route can result in similar or even enhanced immune

responses compared to intramuscular immunisation [7, 8].

During recent years particle based immunisation has gained more emphasis [9]. The

advantage of nanoparticles is that they can function as a depot [10] and are more

efficiently taken up by DCs than plain antigens [11]. Therefore, nanoparticles may function

as an adjuvant. Nanoparticles can be prepared from polymers, such as poly (DL-lactic-co-

glycolic acid) (PLGA) or N-trimethyl chitosan (TMC). TMC is a derivate of chitosan that

bears a permanent positive charge and is therefore water soluble over a wide pH range.

TMC nanoparticles have mainly been used in mucosal immunisation [12-14], but recently

we showed that TMC can also function as an immune potentiator in intradermal

immunisation [15]. Interestingly, we observed that the adjuvant effect could be ascribed

primarily to the TMC polymer itself rather than to its formulation in nanoparticles. After

intradermal injection of diphtheria toxoid (DT) loaded TMC nanoparticles or a solution of

TMC and DT (TMC/DT mixture), mice developed 4 fold higher IgG titres compared to those

induced by plain DT. These results indicate that in intradermal vaccination antigen-loaded

TMC nanoparticles are not superior to soluble TMC/antigen mixtures, in contrast with e.g.

intranasal vaccination [16, 17]. This might be attributed to the fact that with intradermal

injection antigen and adjuvant are immediately delivered to an APC-rich environment,

thereby making nanoparticles unnecessary.

Transcutaneous vaccination differs from intradermal vaccination in that the antigen first

has to be transported into the skin. Only then it can be taken up by skin resident APCs and

induce APC maturation. The natural function of the skin is to protect the body from the

environment [18]. This function is exerted by the upper part of the epidermis, the stratum

corneum. Even though this part is only 15 µm thick in human skin, it proves to be an

excellent barrier. One way to breach this barrier is by using microneedles. The idea of using

microneedles for transdermal drug delivery dates back to 1971 [19], but only in the 1990s

the first microneedles were developed [20]. Since then their usage has increased and many

Chapter 6

124

different microneedles have become available. Some devices are currently being tested in

clinical trials [21] and several others are in pre-clinical development [22-24]. The use of

microneedles for vaccine delivery can be based on different principles: hollow

microneedles can be used for injection of liquids; solid microneedles can either be coated

with the antigen of interest or used for perforation of the skin prior to vaccine application.

The main advantage of microneedles is that they are long enough to penetrate the stratum

corneum, but short enough to avoid pain and major discomfort [25, 26]. During the past

few years we have been studying solid microneedle arrays to pre-treat the skin, followed

by vaccine application. In previous studies from our group it was shown that microneedle

pre-treatment significantly increased antibody titres in transcutaneous vaccination studies

with DT [27, 28].

In this study we will focus on the transport of DT-loaded TMC nanoparticles and TMC/DT

mixtures into the skin, by applying them as liquid formulations in combination with two

types of solid microneedles. Immunisation studies in mice were employed to compare the

antibody responses elicited by microneedle-based delivery to intradermal delivery. To

visualise the transport of soluble and particulate TMC into the skin, the adjuvant was

fluorescently labelled and confocal microscopy studies were performed.


Materials

Chitosan (MW 120 kDa) with a degree of deacetylation of 92% was obtained from Primex

(Alversham, Norway). Cholera toxin (CT), pentasodium tripolyphosphate (TPP), N-(2-

hydroxyethyl) piperazine-N’-(2-ethanesulphonic acid) (HEPES) and rhodamine B

isothiocyanate were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands).

Diphtheria toxin (DTa 79/1) and DT (batch 98/40, protein content 12.6 mg/ml by BCA

assay, 1 μg equal to approximately 0.3 Lf) were a kind gift from the Netherlands Vaccine

Institute (NVI, Bilthoven, The Netherlands). Horseradish peroxidase (HRP) conjugated goat

anti-mouse IgG (γ chain specific), IgG1 (γ1 chain specific) and IgG2a (γ2a chain specific)

were purchased from Southern Biotech (Birmingham, USA). Chromogen 3, 3', 5, 5'-

tetramethylbenzidine (TMB) and the substrate buffer were purchased from Invitrogen

(Breda, The Netherlands). Nimatek® (100 mg/ml Ketamine, Eurovet Animal Health B.V.,

Bladel, The Netherlands), Oculentum Simplex (Farmachemie, Haarlem, The Netherlands),

Rompun® (20 mg/ml Xylazine, Bayer B.V., Mijdrecht, The Netherlands) and the injection

fluid (0.9% NaCl) were obtained from a local pharmacy. All other chemicals were of

analytical grade.


125

Animals

Female BALB/c mice (H2d), 8-weeks old at the start of the vaccination study and male

hairless (skh-1) mice, 7-9 weeks old were purchased from Charles River (Maastricht, The

Netherlands) and maintained under standardised conditions in the animal facility of the

Leiden/Amsterdam Centre for Drug Research, Leiden University. The study was carried out

under the guidelines compiled by the Animal Ethic Committee of the Netherlands.

Vaccine formulations

TMC with a degree of quarternisation of 15% was synthesised from chitosan in a one step

methylation reaction as described previously [15]. The LPS content of TMC was found to be

below the detection limit (<0.1 ng/ml for 1 mg/ml TMC) when tested on TLR-4 transfected

HEK cells (data not shown). For confocal microscopy studies the TMC was labelled at the

amine group with rhodamine B isothiocyanate. TMC was dissolved in a 0.1 M carbonate

buffer pH 9 and rhodamine B isothiocyanate was added in a TMC:rhodamine ratio of 15:1.

After subsequent dialysis in 1% NaCl and water until no rhodamine could be detected in

the dialysis solution (measured by fluorescence), the TMC-rhodamine solution was freeze

dried.

TMC nanoparticles were prepared by ionic complexation with TPP. A TMC:TPP (w/w) ratio

of 6.7:1 was used as described before [15]. Briefly, for the preparation of DT loaded

nanoparticles, 1 mg of DT was added to a 5 mM HEPES pH 7 solution containing 10 mg

TMC. After addition of TPP and 1 hour of stirring, the nanoparticle suspension was

centrifuged for 15 minutes at 10000 g on a glycerol bed. The pellet was resuspended in 10

mM phosphate buffer adjusted to pH 7.4. The size of the nanoparticles was determined by

dynamic light scattering (DLS) and the zetapotential was determined by laser Doppler

velocimetry using a Zetasizer® Nano ZS (Malvern Instruments, UK). The amount of DT in the

particles was measured with a micro-BCA protein assay (Pierce, Rockford, IL, USA). TMC/DT

mixtures were prepared by mixing them in a 2.5:1 (w/w) ratio. To further potentiate the

immune response in some cases CT was added to the formulations in a DT:CT ratio of 1:1

(w/w) just before usage. Finally, DT adsorbed to aluminium phosphate (Adju-Phos®;

Brenntag Biosector, Denmark) (DT-alum) was prepared in a DT:alum ratio of 1:30 as

previously described and the adsorption was between 70 and 80% [29].

Microneedles

Two types of microneedle arrays were used. Assembled metal microneedle arrays with a

length of 300 µm (300A) were manufactured from commercially available 30G hypodermic

needles [30]. 30G needles have a diameter of 300 µm at the base and a tapered shaft of

approximately 1.2 mm, thereby forming an angle of approximately 15 degrees. These

Chapter 6

126

microneedles were positioned in a 4x4 pattern in a polymer mould (diameter 5 mm) with a

pitch of 1.25 mm. The second type of array consists entirely of stainless steel and the

microneedles were prepared by electrical discharge machining (300ED). Similar as the

300A, the 300ED microneedles are 300 µm long and are positioned in a 4x4 pattern with a

pitch of 1.25 mm. They differ from the 300A microneedles in shape as can be observed in

figure 1. They have a square base of 250 x 250 µm and the tip of the microneedles is less

sharp than that of the 300A microneedles. The shape of the tip is defined by a diagonal

plane which runs from the top of one side of the square pillar to the opposed bottom, in

this way forming an angle of approximately 40 degrees relative to the bottom surface. An

electrical applicator was used to apply the microneedles with a speed of 3 m/s to ensure

reproducible piercing of the skin [25].

Immunisation studies

The immunogenicity of the DT-loaded TMC nanoparticles and TMC/DT mixtures was

assessed in an immunisation study in mice using the two types of microneedle arrays. The

microneedle arrays were applied on the abdominal skin under anaesthesia (by

intraperitoneal injection of 150 mg/kg Ketamine and 10 mg/kg Xylazine). The microneedles

were applied in two ways: either before or after application of the formulations. In both

cases 70 µl of the formulations containing 100 µg DT were applied on the skin for 1 hour

(±2 cm2 area restricted by a metal ring). After the application the skin was washed with

lukewarm water to remove the remaining amount of formulation. Groups of 8 mice were

vaccinated thrice with a three weeks interval. To circumvent the skin barrier, the

formulations (5 µg DT/30 µL formulation) were also injected intradermally with a Hamilton

syringe equipped with a 30-Gauge needle as described before [15]. As a positive control

DT-alum (5 µg DT and 150 µg alum/100 µL) was injected subcutaneously (SC). In some

Figure 1. The two types of microneedles

used in this study. A: Array of 300A

microneedles, manufactured from

commercially available 30G needles. B:

Array of 300ED microneedles, made of

stainless steel. Both arrays contain sixteen

microneedles with a length of 300 µm. In

figure C and D higher magnification

images of a single 300A microneedle (C)

and a 300ED microneedle (D) are shown.


127

groups CT was used as an additional adjuvant: 10 µg per mouse for intradermal, 100 µg for

the microneedle groups. One day before each immunisation blood samples were collected

from the tail vein. Three weeks after the last vaccination the mice were sacrificed. Just

before euthanasia total blood was collected from the femoral artery. Blood samples were

collected in MiniCollect® tubes (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) till clot

formation and centrifuged 10 minutes at 10,000 g to obtain cell-free sera. The sera were

stored at -80°C until further use.


DT specific antibodies (IgG, IgG1 & IgG2a) in the sera were determined by sandwich ELISA

as described previously [27]. Briefly, plates were coated overnight with 140 ng DT. After

blocking, two-fold serial dilutions of sera from individual mice were applied to the plates.

HRP-conjugated antibodies against IgG were added and detected by TMB. Antibody titres

were expressed as the reciprocal of the sample dilution that corresponds to half of the

maximum absorbance at 450 nm of a complete s-shaped absorbance-log dilution curve.

Vero cell test

The levels of diphtheria toxin-neutralising antibodies in mouse sera were assessed by a

Vero cell test as described previously [31]. Control samples included were reference anti-

serum and untreated cells. The plates were incubated at 37°C in 5% CO2 for 6 days and

afterwards the presence of living cells was verified by light microscopy. The neutralising

antibody titres were obtained from the serum dilution factor that still resulted in living

cells.

Analysis of in vivo transport into the skin by confocal microscopy

To visualise the transport into the skin of TMC nanoparticles compared to a TMC solution,

hairless (skh-1) mice were treated with empty rhodamine-labelled TMC nanoparticles or a

TMC solution. In this case the 300A microneedles were used and the mice were pre-

treated with the microneedles before occlusive application of the formulations. An equal

concentration of rhodamine-labelled TMC was used in both formulations, as determined

by fluorescence spectroscopy (FS920 fluorimeter, Edinburgh Instruments, Campus

Livingston, UK). After 1 hour of application the formulations were removed with a cotton

bud. To visualise the distribution of the nanoparticles and solution in the skin, the

formulations were also injected intradermally. After euthanasia of the mice, the treated

skin area was removed, immediately mounted on a sample holder and visualised with a

confocal laser scanning microscope. Images were taken every 10 µm, over a total depth of

300 µm. Images were processed using a Bio-Rad Radiance 2100 confocal laser scanning

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128

system equipped with a Nikon Eclipse TE2000-U inverted microscope and either a 4X plan

fluor or a 10X plan air objective (Nikon, Japan). The images were captured using a helium

neon laser at 543 nm, with a 570 long pass emission filter. Image acquisition was

controlled using the Laser Sharp 2000 software (Bio-Rad, Hercules, USA). The amount of

TMC in the conduits was estimated from the images using Image J (National institute of

health, USA). The distribution area of TMC was calculated by the number of pixels in the

specified area containing a level of fluorescence above the threshold value. Threshold

settings were 20 AU (lower threshold) and 255 (upper threshold). A fluorescent intensity

below 20 AU was regarded as background fluorescence.



Data are presented as mean ± SD for the immunisation studies and as mean ± SEM for the

confocal results. Statistical significance was determined by a two way analysis of variance

(ANOVA) with a Bonferroni post-test. The results of the Vero cell test were analysed by a

Kruskal-Wallis test with a Dunn's multiple comparison post-test.

Results

Physicochemical characteristics of the formulations

DT-loaded TMC nanoparticles were prepared with a mean size of 211 ± 4 nm and a PDI of

0.15 ± 0.01. They were positively charged (zetapotential 12.9 ± 0.8 mV in 10 mM sodium

phosphate pH 7.4) and the loading efficiency of DT in the nanoparticles was about 70%. In

the TMC/DT mixtures ca. 50% of the DT was adsorbed to the TMC, which is likely due to

the fact that TMC and DT carry opposite charges at pH 7.4 [15]. As reported previously, the

release of the antigen from the nanoparticles in PBS was characterised by an immediate

burst without any further release over the next 9 days [15].

Combining microneedles and nanoparticles for transcutaneous vaccination

In figure 2A the anti-DT IgG titres after application of the 300A microneedles are shown. By

applying a solution of DT on microneedle pre-treated skin an immune response was

initiated, with IgG titres being 100 fold higher compared to application of DT on intact skin

[27]. Still, these titres were significantly lower compared to those obtained after SC

application of DT-alum, the positive control. Formulating DT into TMC nanoparticles did

not further increase the immune response. In contrast, when a mixture of TMC and DT

solutions was applied on microneedle pre-treated skin, the IgG titres after the second

boost were 8 fold higher compared to application of a solution of DT (p<0.001) and


129

comparable to those elicited by SC DT-alum. Similar results were obtained for the

neutralising antibody titres (figure 2B): after the second boost with a TMC/DT mixture a

clear trend of enhanced titres compared to a solution of DT were observed, whereas

nanoparticles did not enhance the titres. The titres after application of the TMC/DT

mixture were not significantly different from those elicited by SC DT-alum (p=0.26).

It was thought that by applying the formulation before microneedle treatment, the

microneedles might carry the formulation with them into the skin (figure 2A). This

application method indeed induced higher IgG titres after the first boost (p<0.01), but the

IgG and neutralising antibody titres after the second boost did not differ significantly from

those obtained after the original sequence of application, i.e. microneedle treatment prior

to applying the nanoparticles. This indicates that both methods of microneedle application

result in similar immunogenicity. Because the dose is more controlled when the

formulations are applied after microneedle pre-treatment, it was decided to continue with

this application method in the following studies.

To investigate the effect of the shape of the microneedle array, two different arrays were

used: the 300A and the 300ED. Even though the microneedle arrays differ in shape and

sharpness (figure 1), similar IgG and neutralising antibodies were observed after pre-

treatment using either of the two arrays (figure 3).

To further potentiate the immunogenicity of the formulations CT was added to the

nanoparticles. Figure 3A shows that addition of CT, as compared to nanoparticles alone,

Figure 2. IgG (A) and neutralising antibody (B) titres obtained after piercing with 300A

microneedles followed by application of a DT solution, a TMC/DT mixture or DT-loaded TMC

nanoparticles (TMC NP) as compared to SC DT-alum. A: IgG titres after prime and two booster

vaccinations. Mean and SD of 8 mice. B: Neutralising antibody titres after second boost. Data

are expressed as the highest dilution that was still capable of protecting the Vero cells against

challenge with diphtheria toxin. ** p< 0.01, *** p<0.001.

Chapter 6

130

significantly enhanced the IgG titres after the prime and two subsequent booster

vaccinations (p<0.001). The titres obtained after the second boost were comparable to

those after SC DT-alum immunisation. The results of the neutralising antibody assay

confirmed these results (figure 3B). Furthermore, CT not only had an effect on the total IgG

titres, but also affected the IgG1/IgG2a ratio. After immunisation with DT and TMC mainly

IgG1 titres were induced (figure 3C), which is indicative of a Th2 biased response [32, 33].

The addition of CT to the nanoparticle formulations increased the IgG2a titres significantly

(p<0.01), pointing to a more Th1 skewed response.

Intradermal immunisation with TMC-based formulations

In transcutaneous immunisation, the transport of topically applied vaccine into the skin

could be an important barrier to delivery of the vaccine to the APCs in the skin. To

eliminate this transport factor, the formulations were injected intradermally. In figure 4

the antibody titres are shown after intradermal injection of mice with a DT solution, a

TMC/DT mixture and DT-loaded TMC nanoparticles with and without CT. In line with a

previous study [15], the TMC nanoparticles resulted in significantly higher IgG titres

compared to those elicited after intradermal injection of a DT solution (figure 4A) and

higher neutralising antibody titres were also observed (figure 4B). DT-loaded TMC

nanoparticles and TMC/DT mixture induced comparable antibody titres and the levels

were not significantly different from those obtained after SC immunisation with DT-alum

Figure 3. Effects of microneedle array type and co-administration of CT on the immunogenicity

of the DT-loaded TMC nanoparticles (TMC NP) after microneedle pre-treatment. A: IgG titres

after prime and 2 booster vaccinations. B: Neutralising antibody titres after 2nd

boost. Data are

expressed as the highest dilution that was still capable of protecting the Vero cells against

challenge with diphtheria toxin. C: IgG1 and IgG2a titres after 2nd

boost. A/C: Mean and SD of 8

mice. B: Individual values and geometric mean of 8 mice are shown. * p<0.05, ** p< 0.01, ***

p<0.001.


131

(figure 4A,B). When CT was added to the nanoparticle formulation it accelerated and

potentiated the immune response (figure 4A). Higher IgG titres were obtained compared

to the SC DT-alum control after the prime and first boost (p<0.01). Furthermore, after the

second boost, the addition of CT induced significantly higher neutralising antibody titres

(p<0.05) compared to nanoparticles without CT (figure 4B). After intradermal

immunisation with all formulations the main IgG subtype produced was IgG1, but the DT-

loaded TMC nanoparticles enhanced the production of IgG2a antibodies (p<0.01). For the

CT-containing formulation the IgG2a response was most pronounced (p<0.001).

Visualisation of TMC transport into skin

Figure 5 shows representative images of the transport of fluorescently labelled TMC

nanoparticles and a TMC solution into the microneedle conduits. With confocal microscopy

easily all conduits could be visualised (figure 5A), indicating that piercing with the

microneedle arrays was successful. In the conduits fluorescence was present in the deeper

layers of the skin, until a depth of approximately 150 µm (figure 5B). This image also

illustrates the shape of the conduits. Higher magnification images of single conduits were

also made (figure 6). The images show that at the skin surface, adjacent to the conduits,

fluorescence was observed in the furrows. Deeper in the skin, the dye was solely present in

the conduits. The fraction of the TMC-rhodamine that will be transported into the skin

through the conduits is small. To compare the transport into the skin of both formulations

the area containing measurable dye fluorescence and the fluorescent intensity were

calculated from the images. In figure 7 the area containing TMC-rhodamine is plotted

Figure 4. IgG (A) and neutralising antibody (B) titres after intradermal (ID) vaccination with the

different formulations. A: IgG titres after prime and 2 booster vaccinations. B: Neutralising

antibody titres after 2nd

boost. Data are expressed as the highest dilution that was still capable

of protecting the Vero cells against challenge with diphtheria toxin. C: IgG1 and IgG2a titres

after 2nd

boost. A/C: Mean and SD of 8 mice. B: Individual values and geometric mean of 8 mice

are shown. • significantly higher compared to ID DT. ‡ significantly higher compared to SC DT-

alum. * p<0.05, ** p< 0.01.

Chapter 6

132

against the skin depth. The maximum area of TMC-rhodamine (solution and nanoparticles)

was not found at the surface, but at a depth of 20-30 µm in the skin. When TMC-

rhodamine was applied in solution, it was distributed over a larger area in the skin

compared to application of TMC-rhodamine nanoparticles. The distribution areas differ

significantly at a depth of 30 to 70 µm (p<0.05), indicating a broader distribution of the

TMC-rhodamine solution. No difference in the penetration depth was observed between

the TMC-rhodamine solution and TMC-rhodamine nanoparticles.

Figure 6: X,y images (parallel to the skin surface) of a single conduit at different depths. On the

top layer images after application of a TMC-rhodamine solution are shown and on the bottom

layer those obtained after application of TMC-rhodamine nanoparticles.

Figure 5. Representative images of

microneedle conduits in mouse skin.

A: x,y image (parallel to skin surface)

showing 8 conduits at the skin

surface. B: x,z image (perpendicular

to the skin surface) showing

penetration of TMC-rhodamine until

a depth of approximately 150 µm.


133

Images were also taken after intradermal injection of rhodamine-labelled TMC by (figure

8). In this case the fluorescence could be observed over a depth of 200 µm. It is evident

that the TMC is distributed over a much larger area when applied as a solution (figure 8A)

than in nanoparticulate form (figure 8B).

Discussion

A combination of microneedles with adjuvants implements the two main requirements for

effective minimally invasive transcutaneous immunisation: increased transport across the

stratum corneum and induction of a protective immune response. Both the microneedles

and the TMC-based formulations have already proven their effectiveness in previous in

vivo studies [15, 27, 28]. Moreover, because of the positive charge of the TMC, it easily

forms complexes with the negatively charged antigen DT and can induce maturation of DCs

in vitro [15] The potent adjuvant effect of TMC in the skin is clearly shown after

intradermal injection. Both the TMC/DT mixture and the TMC nanoparticles induced an

equally strong immune response, eliciting similar titres as after SC DT-alum administration.

Besides intradermal immunisation, microneedle-based application of a TMC/DT mixture

also increased the antibody titres compared to the application of a DT solution. However,

topically applied DT-loaded TMC nanoparticles were not able to enhance the immune

response. The method of microneedle application or the type of microneedle array used

could not improve the immunogenicity of the TMC nanoparticles. Similarly as observed

with the TMC nanoparticles, positively charged liposomes also failed to enhance the

immune response against DT after microneedle pre-treatment [34]. Even though

nanoparticles are mentioned as a promising tool for transcutaneous vaccination [9], their

usage so far is limited. Studies focussed on the transport of different types of nanoparticles

Figure 7. Determination of the

fluorescence in the skin after

microneedle pre-treatment and

application of either TMC-rhodamine

nanoparticles or a TMC-rhodamine

solution. Area containing fluorescence

plotted against the skin depth. Mean

and SEM of 3 mice.

Chapter 6

134

across intact skin have shown that in most cases the nanoparticles remain in the stratum

corneum or the hair follicles [35-37]. The few groups that claim successful penetration of

nanoparticles into the skin either lack an adequate explanation of the discrepancies found

for the transport of nanoparticles differing in size and charge [38] or could not

demonstrate whether the nanoparticles themselves or only the released dye penetrates

the skin [39]. Lipid based vesicles, which are thought to penetrate the skin more easily,

were shown to remain in the stratum corneum [40-42]. From these studies it can be

concluded that for successful delivery of nanoparticles into the skin, the stratum corneum

barrier needs to be breached. Our group reported for the first time the in vitro

visualisation of transport of commercially available polymeric nanoparticles with a size of

200 nm into human skin after pre-treatment with the 300A microneedles [30]. The

nanoparticles could be traced until a depth of approximately 250 µm. These results were

confirmed by Coulman et al. who pre-treated the skin with 280 µm long microneedles and

showed that nanoparticles with a size of 138 nm were able to penetrate into the epidermis

[43]. To further investigate the transport of TMC nanoparticles and a TMC solution,

visualisation studies were performed. We visualised ex vivo the transport of positively

charged TMC nanoparticles into the skin. We showed that these nanoparticles could be

transported through the microneedle conduits, though to a lower extent compared to the

polymer in solution. The conduit area containing TMC-rhodamine was larger for the

solution than for the nanoparticles, indicating a broader distribution of the TMC solution. A

possible reason for this is blockage of the conduits due to the electrostatic interactions

between the positively charged nanoparticles and the negatively charged skin. Also, the

presence of proteins may cause aggregation of the nanoparticles, making the transport of

the nanoparticles through the conduits to the APCs an important limiting factor. It should

be noted that by prolonging the application time of the formulations (>1 hour), the

transport might be boosted.

The addition of an additional adjuvant (CT) to the nanoparticles increased the IgG and

neutralising antibody titres after microneedle pre-treatment, reaching similar IgG levels as

after SC immunisation with DT-alum. It is worthwhile to mention that next to enhanced

total antibody levels, addition of CT also induced substantially higher IgG2a titres and,

hence, affected the IgG1/IgG2a ratio, both after intradermal and after transcutaneous

application. This is in agreement with other transcutaneous immunisation studies

performed with DT [28], the cross-reacting material (CRM197) of diphtheria toxin [44] and

inactivated influenza virus [45]. Elevated IgG2a titres could be beneficial in case of

vaccination against viruses or intracellular bacteria, where a more Th1 biased response is

required.


135

For DT, high IgG1 rather than IgG2a titres seem to correlate with protection [28, 46]. In our

studies vaccination with DT mainly results in the production of IgG1 antibodies, indicating a

Th2 biased response as is usually reported for toxins [47]. After transcutaneous

immunisation TMC strengthened this Th2 bias, in agreement with our in vitro studies on

human monocyte derived DCs and T cells [14, 15]. Contrarily, after intradermal

administration both TMC formulations enhanced significantly the production of IgG1, but

even more that of IgG2a. This could be explained by the fact that after intradermal

injection the formulations will reach the dermis, while with the microneedles also the

epidermis is targeted. Previous studies in mice have shown that by immunising via the

epidermis mainly a Th2 biased response is elicited [48-51]. It is thought that in these cases

the immune response is initiated mainly by epidermal LCs that take up the antigen and

migrate to the lymph nodes [51]. In addition, Klechevsky et al. showed that human LCs

upon stimulation with CD40L efficiently induced the secretion of Th2 type cytokines by T

cells [52]. In the dermis of mice two types of DCs are present, the classical dermal DCs and

a recently discovered langerin+ DCs [53-55]. This subtype differs from LCs and the classical

dermal DCs by a low expression of CD11b and high expression of CD103. The exact role of

these dermal langerin+ CD11b

- CD103

+ DCs in the humoral immune response is not clear

yet, but the presence of different DC subtypes in the epidermis and dermis could explain

the different immune response generated after transcutaneous and intradermal

immunisation.

Conclusion

For successful transcutaneous immunisation both the transport into the skin and the

activation of the APCs are important. This can be achieved by the combination of

microneedles and an adjuvant. TMC offers great promise as an adjuvant for

transcutaneous immunisation, but not when formulated in nanoparticles. Nanoparticles

which are much smaller might be more suitable to use in combination with microneedle

arrays. Moreover, as this study showed that a mixture of TMC and DT is able to increase

Figure 8. X,y images (parallel to the

skin surface) showing the

distribution pattern in the dermis

after intradermal injection of

either a TMC-rhodamine solution

(A) or TMC-rhodamine

nanoparticles (B).

Chapter 6

136

the immunogenicity, conjugation between the polymer and the antigen could be a better

option to further potentiate the immune response [56], also via the transcutaneous route.

Acknowledgement


Vaccine delivery: alternatives for conventional multiple injection vaccines. The authors

thank Bram Slütter for his helpful suggestions.


137

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Chapter 7

Small is beautiful: N-trimethyl chitosan-

ovalbumin conjugates for microneedle-

based transcutaneous immunisation Suzanne M. Bal, Bram Slütter, Wim Jiskoot, Joke A. Bouwstra

Submitted for publication

Chapter 7

144

Abstract

For microneedle-based transcutaneous immunisation the formulation can greatly impact

the transport of the antigen and adjuvant into the skin and subsequently to the lymph

nodes. Therefore we immunised mice with ovalbumin (OVA) formulated in three different

ways with N-trimethyl chitosan (TMC). TMC + OVA mixtures, TMC-OVA conjugates and

TMC/OVA nanoparticles were applied transcutaneously, intradermally and intranodally, to

explore the effect of the formulations’ physical form on the number of OVA+

dendritic cells

(DCs) in the lymph node and the resultant immunogenicity (serum IgG titres).

Transcutaneously, the TMC-OVA conjugate induced the highest IgG levels and resulted in

more OVA+ DCs in the lymph nodes after 24 h than the other TMC formulations.

Intradermally, all TMC-adjuvanted OVA formulations increased IgG titres compared to

plain OVA. These formulations accumulated in the skin, prolonging OVA delivery to the

lymph nodes. The prolonged delivery of TMC-adjuvanted OVA to lymph node resident DCs

was also observed after intranodal immunisation, but in this case the higher uptake did not

correspond with elevated antibody titres compared to plain OVA.

In conclusion, TMC-OVA conjugates are not more immunogenic, but are better taken up by

DCs than TMC + OVA mixtures and penetrate the skin more efficiently than TMC/OVA

nanoparticles.

Transcutaneous immunisation with conjugates of N-trimethyl chitosan and ovalbumin

145

Introduction

Transcutaneous immunisation as an alternative for the conventional intramuscular or

subcutaneous vaccination routes has received a lot of attention in the past years [1].

Although the skin is an attractive vaccination site due to the presence of high amount of

antigen presenting cells, the stratum corneum prevents efficient diffusion of vaccines into

the skin. This barrier can be breached by using for instance microneedles [2, 3]. Different

types of microneedles are available, each with their own advantages and disadvantages

[2]. In our lab we have used solid microneedles to effectively pierce both human and

mouse skin [4-6]. The microneedles have proven to be successful for transcutaneous

immunisation [7-10]. Solid microneedles are more easily fabricated compared to hollow

and biodegradable microneedles and lack the inconvenience of possible leakage associated

with hollow microneedles. They were introduced by Henry et al. in 1998, who showed a 4

orders of magnitude increase in the transport of calcein through microneedle pre-treated

skin in vitro [11]. Ding et al. showed that microneedle pre-treatment resulted in 1000-fold

increase in antibody titres against diphtheria toxoid compared to application on intact skin

[10]. However, still much higher doses are necessary to provoke comparable titres as after

subcutaneous injection. This is probably a result of the low amount of antigen that reaches

the dendritic cells (DCs) in the skin.

To improve the uptake of antigen that does reach the DCs, antigens are often formulated

into nanoparticles. This is a logical strategy as all pathogens are particulates. Nanoparticles

are better taken up by DCs and can function as an antigen depot [12-14]. Additionally, co-

localisation of antigen and adjuvant in a nanoparticle results in simultaneously delivery to

the same DC, which is thought to be pivotal for induction of potent immune responses [15,

16]. In general, nanoparticles have proven to be successful [17, 18], but recent studies

using liposomes or N-trimethyl chitosan (TMC) nanoparticles for intradermal and

transcutaneous immunisation have questioned their benefit for these delivery routes [7, 8,

19]. TMC however is an interesting polymer since it has intrinsic adjuvant properties and

induces DC maturation [19, 20]. Moreover, it has successfully been used for vaccination via

various administration routes [7, 19-23]. In transcutaneous vaccination with microneedle

arrays the diffusion of TMC nanoparticles, after application for 1 h on microneedle pre-

treated skin, was shown to be significantly impaired compared to that of a TMC solution

[7]. The delivery of nanoparticles into the skin can be optimised by prolonging the

application time or by using smaller vaccine entities. The extended application of the

formulations will allow more accumulation in and diffusion through the skin. By using

conjugates between TMC and the model antigen ovabumin (OVA), we hypothesize that the

diffusion through the conduits is improved, whereas the co-localisation of antigen and

adjuvant is retained [24]. We have previously described the synthesis of this conjugate

Chapter 7

146

[24], which links the antigen and the adjuvant by a disulfide bond, ensuring release once

the conjugate is taken up by DCs [25, 26]. These conjugates enhanced DC uptake and

maturation and were equally or more immunogenic compared to TMC nanoparticles after

intramuscular vaccination [24] or nasal immunisation [27], respectively.

In this study we immunised mice with OVA-loaded TMC nanoparticles, a TMC-OVA

conjugate, a mixture of a TMC and OVA solution and plain OVA by applying the

formulations for 2 h on microneedle pre-treated skin. The immunogenicity of the

formulations was assessed by measuring the antibody titres. The efficiency of the delivery

of the antigen through the conduits to the lymph nodes was assessed by determining the

amount of OVA positive (OVA+) DCs in the draining lymph nodes. To discriminate between

the different transport aspects: diffusion through the conduits into the skin; transport from

the skin to the lymph nodes and DC uptake in the lymph nodes, the formulations were also

administered by intradermal or intranodal injection.

Materials and Methods

Materials

TMC with a degree of quaternisation of 15% was synthesised from 92% deacetylated

chitosan (MW 120 kDa, Primex, Siglufjordur, Iceland) as described previously [19].

Endotoxin free OVA grade VII was obtained from Merck (Darmstadt, Germany).

Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (γ chain specific), IgG1 (γ1

chain specific) and IgG2a (γ2a chain specific) were purchased from Southern Biotech

(Birmingham, USA). Invitrogen (Breda, The Netherlands) supplied AlexaFluor647 labelled

OVA (OVAAF647), chromogen 3,3',5,5'-tetramethylbenzidine (TMB) and the substrate buffer

and all cell culture reagents. Anti CD11c-PE/Cy5 was acquired from Becton Dickinson

(Breda, The Netherlands). Nimatek® (100 mg/ml Ketamine, Eurovet Animal Health B.V.,

Bladel, The Netherlands), Oculentum Simplex (Farmachemie, Haarlem, The Netherlands)

and Rompun® (20 mg/ml Xylazine, Bayer B.V., Mijdrecht, The Netherlands) were obtained

from a local pharmacy. Phosphate buffered saline (PBS) pH 7.4 was obtained from Braun

(Oss, The Netherlands). N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), dithiothreitol

(DTT), pentasodium tripolyphosphate (TPP), N-(2-hydroxyethyl) piperazine-N’-(2-

ethanesulphonic acid) (HEPES) and all other chemicals were purchased at Sigma-Aldrich

(Zwijndrecht, The Netherlands), unless stated otherwise.

Animals

Female BALB/c mice, 8 weeks old at the start of the vaccination study were purchased

from Charles River (Maastricht, The Netherlands) and maintained under standardised


147

conditions in the animal facility of the Leiden/Amsterdam Center for Drug Research, Leiden

University. The study was carried out under the guidelines compiled by the Animal Ethic

Committee of the Netherlands.

Vaccine formulations

TMC nanoparticles were prepared by ionic complexation with TPP and OVA as described

before [19]. Briefly, OVA followed by TPP were added to a 0.2% (w/v) of TMC in 5 mM

HEPES (pH 7.4) in a 10:1.0:1.7 TMC:OVA:TPP ratio under continuous stirring. The

nanoparticles suspension was centrifuged for 15 min at 10,000 g on a glycerol bed and

resuspended in 5 mM HEPES (pH 7.4). A mixture of TMC and OVA was prepared by adding

solutions of both components together in a 2.5:1 (w/w) ratio (TMC + OVA). TMC-OVA

conjugates were synthesised and purified as described previously [24]. Briefly, 10 mg TMC

and 5 mg OVA were separately exposed to a 10 fold molar excess of SPDP for 1 h at room

temperature, resulting in approximately 2 functionalized groups per TMC and per OVA

molecule. Functionalised TMC was treated with DTT for 30 min at room temperature to

obtain thiolated TMC. Thiolated TMC and functionalised OVA were mixed a 1:1 molar ratio

to allow disulfide bond formation overnight. The size of the nanoparticles and the

conjugate was measured with dynamic light scattering and the zetapotential was

determined by laser Doppler velocimetry using a Zetasizer(R)

Nano ZS (Malvern,

Instruments, United Kingdom). Formulations containing OVAAF647 with similar size and

zetapotential were produced by substituting OVA by its fluorescent counterpart.

Microneedles

Assembled metal microneedle arrays with a length of 300 µm (300A) were manufactured

from commercially available 30G hypodermic needles [4]. These microneedles were

positioned in a 4x4 pattern in a polymer mould (diameter 5 mm) with a pitch of 1.25 mm.

An electrical applicator was used to apply the microneedles with a speed of 3 m/s to

ensure reproducible piercing of the skin.

Immunisation study

Groups of 8 mice (transcutaneous immunisation) or 5 mice (intradermal and intranodal

immunisation) were vaccinated with the above mentioned formulations. All immunisations

were applied under anaesthesia by intraperitoneal injection of 150 mg/kg ketamine and 10

mg/kg xylazine. Transcutaneous immunisation with the microneedles was performed as

described previously [10]. However, whereas in previous studies the formulations were

applied occlusively for 1 h, in the present study in most cases the formulation application

was extended to 2 h (~2 cm2 area restricted by a metal ring) before they were washed off.

Chapter 7

148

A dose of 100 µg was applied in a volume of 70 µl. To circumvent the skin barrier, the

formulations (2 µg/30 µl) were also injected intradermally with a 30G needle as described

before [19]. To circumvent transport to the lymph nodes, the formulations (0.2 µg/10 µl)

were also injected directly into the inguinal lymph node as described by Johansen et al

[28]. After 3 weeks blood samples were drawn from the tail vein and the mice received a

similar booster vaccination. After 6 weeks total blood was collected from the femur artery

and the mice were sacrificed. Blood samples were collected in MiniCollect® tubes (Greiner

Bio-one, Alphen a/d Rijn, The Netherlands) till clot formation and centrifuged for 10

minutes at 10,000 g to obtain cell-free sera. The sera were stored at −80°C until further

use.

Antigen uptake by DCs in the lymph nodes

Mice were vaccinated as described above, but with OVAAF647-containing formulations. After

4 or 24 h mice were sacrificed and inguinal lymph nodes were collected. Single cell

suspensions were obtained in RPMI with 10% foetal calf serum, 50 µM β-mercaptoethanol,

2 mM glutamine, 1 mM sodium pyruvate and 500 U/L penicillin/streptomycin, by grinding

the lymph nodes through 70 µm cell strainers. Lymphocytes were washed with PBS

containing 1% w/v bovine serum albumin and stained with CD11c-PE-Cy7. Cells were

analysed with flow cytometry using a FACSCanto II (Becton Dickinson). DC population was

determined based on the expression of CD11c and the number of OVA+ cells in this

population was quantified.


OVA specific antibodies (IgG, IgG1 & IgG2a) in the sera were determined by sandwich ELISA

as described previously [19]. Briefly, plates (NUNC, Roskilde, Denmark) were coated

overnight with 100 ng OVA. After blocking, two-fold serial dilutions of sera from individual

mice were applied into the wells. HRP-conjugated antibodies against IgG, IgG1 and IgG2a

were added and detected by TMB. Absorbance was determined at 450 nm with an EL808

micro plate reader (Bio-Tek Instruments, Bad Friedrichshall, Germany). Antibody titres





Statistical significance was determined by a two-way analysis of variance (ANOVA) with a

Bonferroni post-test.


149

Results

In a pilot study, the effect of prolonging the application time of a vaccine formulation on

microneedle pre-treated skin was studied in the absence of TMC (figure 1). A two-fold

increase of the application time resulted in a nine-fold and thirty-fold amplification of IgG

titres after the prime and boost immunisation, respectively (p<0.001). Even for a relatively

small soluble antigen like OVA (9 nm) the delivery through the much larger conduits is a

slow process. This emphasises the importance of studying the delivery parameters for

microneedle-based transcutaneous vaccination and provides rationale for extending the

application time in the studies with the TMC formulations. Based on these results it was

decided to use 2 h application for transcutaneous immunisation.

Immunisation studies with TMC-based formulations

We immunised mice with OVA, a mixture of TMC + OVA, TMC nanoparticles and TMC-OVA

conjugates via the transcutaneous, intradermal and intranodal route. The formulations

used for vaccination had a similar TMC:OVA ratio and were of a broad size range (table 1).

Transcutaneously, the TMC-OVA conjugate, the smallest co-localised entity in this study,

outperformed the other formulations (figure 2A). After the prime vaccination the

conjugate induced significantly higher IgG titres than the other three formulations

(p<0.001). Also after the boost the TMC-OVA conjugate proved to be significantly better

than OVA alone (p<0.01), although also the TMC nanoparticles significantly elevated the

IgG titres compared to plain OVA (p<0.05). A physical mixture of TMC + OVA elicited IgG

levels that were not significantly higher than plain OVA (p=0.25).

Figure 1. OVA specific IgG titres after

transcutaneous immunisation. After

microneedle pre-treatment an OVA

solution was applied for 1 or 2 h on the

skin. Mean + SEM of 8 mice. *** p<0.001

Chapter 7

150

Formulation TMC:OVA (w/w) Size [nm]

OVA n.a. 8 ± 1

TMC + OVA 2.5:1 n.a.*

TMC-OVA conjugate 2:1 28 ± 1

TMC/OVA nanoparticles 2.5:1 276 ± 6

*= n.a. not applicable

When the formulations were administered by intradermal injection, circumventing

transport along the conduits, all TMC-based formulations were equally potent in enhancing

the IgG titres compared to non-adjuvanted OVA (figure 2B). Both after prime and boost

vaccination the titres were elevated (p<0.001) by using TMC in the formulations. After

intranodal vaccination, where the antigen is directly injected at the site where the immune

response is initiated, all four formulations induced similarly high IgG titres (figure 2C).

Besides the total IgG titres, the IgG1 and IgG2a titres after the boost were measured as

well. Almost exclusively IgG1 was produced after immunisation with all formulations via

the three different delivery routes, indicative of a Th2 biased response as reported before

[7, 29, 30]. Only after intradermal immunisation with the TMC nanoparticles four out of

five mice also had measurable IgG2a titres (values around 100, data not shown).

DC uptake in the lymph nodes

To elucidate the influence of transport on the observed antibody titres, the number of DCs

in the lymph nodes that had taken up OVA was quantified after application of fluorescently

labelled OVA via the different immunisation routes. Transcutaneous application of the

Figure 2. OVA specific IgG titres after a prime and a booster vaccination by the

transcutaneous (2 h application, A), intradermal (B) and intranodal route (C). Data are

expressed as the mean ± SEM of 8 (A) or 5 mice (B/C). * p<0.05, ** p<0.01, *** p<0.001.

Table 1. Characteristics of formulations.


151

formulations resulted in a very low number of OVA+ DCs in the lymph nodes (figure 3A and

D); after 4 h no OVA+ DCs could be detected, but after 24 h application of OVA or the TMC-

OVA conjugate did result in OVA uptake by DCs. For the other formulations the levels were

barely higher than the background fluorescence.

Intradermally, after 4 h the highest amount of OVA could be found in the lymph nodes if a

solution of OVA was administered, whereas the TMC-based formulations reduced the

direct lymph node drainage in a size dependent manner (figure 3B). After 24 h TMC + OVA

mixture and the TMC nanoparticles were able to elevate the OVA uptake, whereas the

TMC-OVA conjugate did not have a significant effect (figure 3E). This extended delivery of

OVA could be ascribed to a depot effect as both 4 and 24 h after intradermal injection of

all OVAAF647-containing TMC formulations a depot was visible at the injection site (figure 4).

This depot was not present if a solution of OVA was used. It is known that for instance

liposomal formulations can form a depot in the skin [31] and here we show that this is also

the case for TMC. Since TMC is a positively charged polymer it likely will interact with

(negatively charged) cells and collagen matrix present in the dermis. Also the linear

structure of the polymer could promote entanglement in the collagen matrix.

Intranodal injection resulted in the largest population of OVA+ DCs; up to 40% of the DCs in

the lymph nodes had taken up OVA (figure 3C and F). The IgG titres correspond well with

the DC uptake in the lymph nodes, as after 4 h the different formulations induced

comparable OVA uptake. For plain OVA the uptake was a fast process: whereas after 4 h

40% of the DCs were OVA+, after 24 h the amount of OVA

+ DCs had already decreased by

10-fold. The TMC-based formulations were able to prolong the exposure to OVA after 24 h

(p=0.08), just as was the case after intradermal immunisation.

Figure 3. Quantification of the amount of OVA+ DCs in the lymph nodes 4 (A-C) and 24 h (D-F)

after A/D: transcutaneous vaccination with microneedle pre-treatment, B/E: intradermal

immunisation and C/F: intranodal vaccination. Data are expressed as mean ± SEM of at least

3 mice. * significantly different compared to OVA (p<0.05).

Figure 4. Picture of injection site 24 h after

intradermal injection of TMC nanoparticles.

Chapter 7

152

Discussion

Vaccines are administered via a variety of routes, but knowledge on the different

requirements of vaccine formulations for the various delivery routes is sparse [32]. In this

study we compared four different formulations for transcutaneous, intradermal and

intranodal vaccination. Non-adjuvanted OVA and three TMC-containing OVA formulations

were selected that differ with respect to size (OVA conjugated with TMC, or encapsulated

in larger TMC nanoparticles) and co-localisation of adjuvant and antigen (nanoparticles and

conjugate versus TMC + OVA in solution). In this study we did not only compared the

formulations with respect to the antibody response they induced, but also quantified the

antigen uptake in the lymph nodes. This was the first time that the antigen uptake in the

lymph nodes after transcutaneous immunisation with microneedles was quantified.

Guebre-Xabier et al. could detect OVA+ DCs in the draining lymph nodes 24 h after

application of OVA together with heath labile enterotoxin (LT) on abraded mice skin [33].

In a similar manner Belyakov et al. measured the uptake of LT applied on abraded skin and

could measure LT+ DCs after 48 h [34]. However, the effect of formulating antigens into

nanoparticles or conjugates on the DC uptake in the lymph nodes after transcutaneous

immunisation has not been measured before. It provides an elegant method of comparing

the efficiency of different vaccine formulations. Furthermore, it makes it possible to

compare different immunisation routes.


153

Interestingly all three delivery routes showed a different effect of the formulations. For

vaccination via the transcutaneous route the importance of the size of the

adjuvant/antigen combination as well as the co-localisation of antigen and adjuvant was

evident. The smaller conjugate was superior in inducing a fast serum IgG response and in

enhancing the OVA uptake in the lymph nodes compared to the TMC nanoparticles and a

TMC + OVA mixture. Even though plain OVA was also taken up by DCs in the lymph nodes,

the lack of co-stimulatory components in this formulation resulted in a less effective

antibody response.

The DC uptake studies in the lymph nodes make it evident that despite the much higher

dose applied transcutaneously compared to intradermal and intranodal injection, the

amount of OVA that is taken up by DCs in the lymph nodes is significantly lower. This

matches the previously published data comparing the OVA+ DCs after transcutaneous

application, intradermal and subcutaneous injection of OVA [33]. They showed that 1 h of

transcutaneous application of a much higher antigen dose resulted in lower numbers of

OVA+ DCs compared to intradermal administration. We observed similar results after

quantifying the OVA uptake from OVA/CpG liposomes administered via the transcutaneous

and intradermal route (unpublished data).

Prolongation of the application time resulted in significantly elevated IgG levels after

administration of OVA. Moreover, whereas in a previous study applying the TMC

nanoparticles for 1 h on microneedle pre-treated skin did not result in elevated IgG titres

[7], in the current study after application for 2 h significantly higher titres compared to an

OVA solution were obtained. For a mixture of TMC + OVA the opposite was observed: even

though adding TMC to an antigen (diphtheria toxoid) improved the antibody titres in a

previous after 1 h of application [7], this effect was not observed after 2 h of application. A

logical next step would be to further prolong the application time. This has not been done

so far, as it is difficult to anesthetise the animals for a longer period. However, if the

microneedle approach would be applied to humans instead of mice, patches could easily

be worn for up to 24 h or even longer. This is expected to lead to lower doses required for

successful transcutaneous immunisation.

Whereas for transcutaneous vaccination the entity size is the most important parameter,

for intradermal vaccination this apparently plays a minor role. Transport to the lymph

nodes from the dermis is a relatively fast process, as the lymphatic vessels are present just

below the epidermis and have a diameter of 10-80 µm [35]. Other factors, such as the

depot formation are of considerable importance. It is known that the retention of the

antigen and its slow release from the depot can stimulate the immune response, by the

attraction of antigen presenting cells [31, 36]. However, if the antigen passage to the

lymph nodes is impaired because of electrostatic interactions with the extracellular matrix

in the skin, this can have a detrimental effect on the immune response. This was illustrated

Chapter 7

154

by the TMC-OVA conjugate, where the antigen and adjuvant are covalently linked. The

disulfide bond linkage will only be degraded once the conjugate is taken up into the DCs

reducing cytoplasm, thereby prohibiting direct drainage of OVA from the injection site to

the lymph nodes. The necessary uptake by and subsequent migration of DCs to the lymph

nodes means that conjugated OVA will reach the lymph nodes over a longer time span.

After subcutaneous injection of cationic liposomes that also formed a depot, the maximum

amount of antigen could be detected in the lymph nodes 5 days past injection [37]. A

kinetic study of lymph node trafficking might reveal that the OVA from the conjugate will

reach the lymph nodes at a later time point, as is expected from the antibody titres where

the three TMC-containing formulations were equally potent. It remains to be studied

whether the adjuvant effect of TMC can be further improved by shielding its positive

charge, for instance by PEGylation to reduce the depot formation [38].

Intranodally, it was surprising that the controlled antigen release in the lymph nodes with

the TMC-containing formulations did not correlate with elevated IgG titres. It was expected

that the in vivo DC uptake after intranodal injection would correlate with the in vitro DC

uptake, where the TMC-OVA conjugate and the nanoparticles increased the OVA uptake

after 4 h and an TMC + OVA solution did not [19, 24]. Apparently in the present study the

in vitro model is not representative for the in vivo situation. Bachmann et al. stated that in

the lymphoid tissue there are abundant co-stimulation signals present and as long as there

is a sufficient load of antigen, no adjuvant is necessary [39]. This may explain why no

beneficial effect of the TMC was observed. Moreover, damage to the surrounding tissue as

a result of the intranodal injection could provide a danger signal similar to that of an

adjuvant [40].

Conclusion

The optimal vaccine formulation differs for each administration site as optimum delivery

parameters for one route of administration can not simply be extrapolated to other routes.

Focusing on transcutaneous vaccination, the size of the vaccine entity and co-localisation

of antigen and adjuvant are crucial parameters when designing formulations to effectively

enhance the immune response. Conjugates of an antigen and an adjuvant offer in this

respect better perspectives than (nano)particles.

Acknowledgement




155

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Chapter 8

Adjuvanted, antigen loaded TMC

nanoparticles for nasal and intradermal

vaccination: adjuvant- and site-

dependent immunogenicity in mice Suzanne M. Bal*, Bram Slütter*, Rolf J. Verheul, Joke A. Bouwstra, Wim

Jiskoot


Submitted for publication

Chapter 8

160

Abstract

N-trimethyl chitosan (TMC) nanoparticles have been shown to increase the

immunogenicity of subunit antigens after nasal and intradermal administration. This work

describes a second generation of TMC nanoparticles containing ovalbumin as a model

antigen (TMC/OVA nanoparticles) and an adjuvant (TMC/adjuvant/OVA nanoparticles). The

selection of adjuvants included Toll-like receptor (TLR) ligands lipopolysaccharide (LPS),

PAM3CSK4 (PAM), CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and

the GM1 ganglioside receptor ligand, cholera toxin B (CTB) subunit. The

TMC/adjuvant/OVA nanoparticles were characterised physico-chemically and their

immunogenicity was assessed by determining the serum IgG, IgG1, IgG2a titres and

secretory IgA levels in nasal washes after intradermal and nasal vaccination in mice.

After nasal vaccination, TMC/OVA nanoparticles containing LPS or MDP elicited higher IgG,

IgG1 and sIgA levels than non adjuvanted TMC/OVA particles, whereas nanoparticles

containing CTB, PAM or CpG did not. All nasally applied formulations induced only marginal

IgG2a titres. After intradermal vaccination, the TMC/CpG/OVA and TMC/LPS/OVA

nanoparticles provoked higher IgG titres than plain TMC/OVA particles. Additionally, the

TMC/CpG/OVA nanoparticles were able to induce significant IgG2a levels. None of the

intradermally applied vaccines induced measurable sIgA levels.

Altogether, our results show that co-encapsulation of an adjuvant with the antigen in TMC

nanoparticles can significantly increase the immunogenicity of the antigen. However, the

strength and quality of the response depends on the adjuvant as well as the route of

administration.

Adjuvant selection for nasal and intradermal vaccination

161

Introduction

Most human vaccines are administered via injection into muscle or subcutaneous tissue.

Notwithstanding the success of this approach, during the last decades it has also become

apparent that muscle and subcutaneous tissue may not be the most ideal sites to induce

an immune response. The skin and the mucosal linings for instance contain more immune

cells capable of initiating an immune response [1, 2], which is most likely a consequence of

the fact that pathogens generally invade the human body via these tissues. Various

examples have shown that intradermal vaccination is more effective than intramuscular

administration as the same level of protection is reached by injection of a smaller dose [3-

5]. Moreover, applying the vaccine via the route through which the pathogen would

normally invade could induce a type of immune response that provides better protection

[6]. Nasal vaccination often induces the production of secretory IgA (sIgA) antibodies that

can neutralise pathogens colonising the mucosal linings [7], whereas intramuscular

administration does not induce sIgA.

Currently there are several vaccines on the market that use a different administration

route (e.g., oral, intradermal and nasal) and they are well perceived by the vaccinee [8].

However, many of these vaccines are of live-attenuated nature, which makes them

unsuitable for administration to young children, elderly or immune-compromised patients.

Replacement of these vaccines by subunit vaccines would be a great improvement for

safety reasons and would make them suitable for administration to these groups.

However, such vaccines are difficult to develop as plain subunit antigens are poorly

immunogenic. To enhance their immunogenicity, subunit antigens can be formulated into

particulate vaccine delivery systems. This improves the uptake by antigen presenting cells

(APCs) and when adjuvants are included it can also enhance the activation of these APCs

[9]. Especially approaches that combine antigen and adjuvant into a particle have been

shown to result in a strong immune response [10, 11]. We have recently shown that N-

trimethyl chitosan (TMC) nanoparticles loaded with ovalbumin (OVA) as a model subunit

antigen increased the immune response after nasal [12] as well intradermal administration

[13]. Inclusion of an adjuvant may further improve the immunogenicity of TMC

nanoparticles.

The aim of the present study was to co-encapsulate various adjuvants in OVA-loaded TMC

(TMC/OVA) nanoparticles and to evaluate if these additional danger signals can further

enhance the efficacy of the TMC/OVA nanoparticles when administered nasally or

intradermally in mice. We selected 5 potential adjuvants based on their physicochemical

properties and their reported adjuvant effect after intradermal and nasal administration:

lipopolysaccharide (LPS) [14, 15], CpG [16, 17], PAM3CSK4 [17, 18], muramyldipeptide

(MDP) [19, 20] and the non-toxic beta subunit of cholera toxin (CTB) [21, 22]. These

Chapter 8

162

adjuvants were co-complexed with OVA into TMC nanoparticles, rather than co-

administered, as co-localization of antigen and adjuvant into one entity has been reported

to be very beneficial for the resulting immune response [10, 11, 23, 24]. The size and

zetapotential were measured to ensure that all particles had a similar physical form. The

adjuvanted nanoparticles were administered nasally and intradermally to mice to assess

the extent of the immune response (OVA specific IgG titres) and the type of immune

response (IgG1/IgG2a, secretory IgA (sIgA)) that was elicited.


Materials

TMC with a degree of quaternisation of 15% was synthesised from 92% deacetylated

chitosan (MW 120 kDa, Primex, Siglufjordur, Iceland) as described previously [25].

Endotoxin free OVA grade VII was obtained from Merck (Darmstadt, Germany).

Lipopolysaccharide (LPS) from E.Coli 0111:B4, Pam3Cys-Ser-(Lys)4 (PAM), and CpG

oligonucleotide 1826 were obtained from Invivogen (Toulouse, France). Horseradish

peroxidase (HRP) conjugated goat anti-mouse IgA, IgG (γ chain specific), IgG1 (γ1 chain

specific) and IgG2a (γ2a chain specific) were purchased from Southern Biotech

(Birmingham, USA). Invitrogen (Breda, The Netherlands) supplied chromogen 3, 3', 5, 5'-

tetramethylbenzidine (TMB) and the substrate buffer and all cell culture reagents.

Nimatek® (100 mg/ml Ketamine, Eurovet Animal Health B.V., Bladel, The Netherlands),

Oculentum Simplex (TEVA, Haarlem, The Netherlands) and Rompun® (20 mg/ml Xylazine,

Bayer B.V., Mijdrecht, The Netherlands) were obtained from a local pharmacy. Phosphate

buffered saline (PBS) pH 7.4 was obtained from Braun (Oss, The Netherlands). Cholera

toxin B subunit (CTB), muramyl dipeptide (MDP) and all other salts/chemicals were

purchased at Sigma-Aldrich (Zwijndrecht, The Netherlands), unless stated otherwise.

Animals

Female BALB/c mice, 8 weeks old at the start of the vaccination study were purchased

from Charles River (Maastricht, The Netherlands) and maintained under standardised

conditions in the animal facility of the Leiden/Amsterdam Center for Drug Research, Leiden

University. The study was carried out under the guidelines compiled by the Animal Ethic

Committee of the Netherlands.

Plain TMC/OVA nanoparticles

TMC/OVA nanoparticles were prepared as described before [26]. Briefly, 1 mg OVA was

dissolved in 10 ml 0.1% (w/v) TMC in 5 mM Hepes pH 7.4. Under continuous stirring 1.7 ml


163

0.1% (w/v) TPP was added to obtain an opalescent dispersion. Nanoparticles were

collected by centrifugation (10 min, 12000 g) and resuspended in water. For size and

zetapotential measurements using a Nanosizer ZS apparatus (Malvern Instruments,

Malvern, UK), nanoparticles were diluted in 5 mM Hepes pH7.4 until slightly opalescent

dispersions was obtained. Supernatants were stored to determine the loading efficiency

with a BCA assay following the manufacturer’s guidelines (Pierce, Perbio Science, Etten-

Leur, The Netherlands).

Adjuvanted TMC/OVA nanoparticles

Adjuvanted nanoparticles were prepared in the same way as non-adjuvanted TMC

nanoparticles, as the adjuvant was co-dissolved with OVA in the TMC solution. TMC/CpG

nanoparticles were the only exception, and were prepared by replacing TPP with strongly

negatively charged CpG (serving as physical crosslinker and adjuvant), as described

previously [16]. To remove unencapsulated OVA or adjuvant, nanoparticles were collected

by centrifugation (10 min, 12000 g) and resuspended in water. To determine the loading

efficiencies of the adjuvants fluorescently labelled analogues were used and the amount of

adjuvant in the supernatant was determined by fluorescence spectroscopy (FS920

fluorimeter, Edinburgh Instruments, Campus Livingston, UK).

Based on the pre-determined loading efficiencies of each adjuvant (table 2), the initial

amount of adjuvant was chosen in such a way (table 1) that the different TMC

nanoparticles carried similar amounts of OVA and adjuvant in a 1:1 weight/weight ratio

were prepared.

Formulation TMC

[mg] TPP [mg]

OVA [mg]

Adjuvant

[mg]

TMC/OVA 10 1.8 1.0 -

TMC/CTB/OVA 10 2.0 1.0 0.83 TMC/LPS/OVA 10 2.0 1.0 1.7

TMC/PAM/OVA 10 2.0 1.0 5.0

TMC/MDP/OVA 10 2.0 1.0 1.3

TMC/CpG/OVA 10 - 1.0 0.5

Immunisation study

Groups of 8 mice (nasal) or 5 mice (intradermal) were vaccinated with the above

mentioned formulations. Nasally the mice received 10 µg antigen and 10 µg adjuvant in a

volume of 10 µl PBS (5 µl/nostril) and intradermally 2 µg of each in a volume of 30 µl PBS

Table 1. Initial amounts of components used for formulation of adjuvants into TMC/OVA

nanoparticles.

Chapter 8

164

was applied. Intradermal immunisations were carried out under anaesthesia by

intraperitoneal injection of 150 mg/kg ketamine and 10 mg/kg xylazine with a 30G needle

as described before [27]. After 3 weeks blood samples were drawn from the tail vein and

the mice received a similar booster vaccination. After 6 weeks total blood was collected

from the femur artery and the mice were sacrificed. Blood samples were collected in

MiniCollect® tubes (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) till clot formation

and centrifuged for 10 minutes at 10,000 g to obtain cell-free sera. The sera were stored at

−80°C until further use.

Detection of serum IgG, IgG1, IgG2a and secretory IgA

OVA specific antibodies (IgG, IgG1 & IgG2a) in the sera and sIgA in the nasal washes were

determined by sandwich ELISA as described previously [27]. Briefly, plates were coated

overnight with 100 ng OVA. After blocking, two-fold serial dilutions of sera from individual

mice were applied to the plates. HRP-conjugated antibodies against IgG, IgG1, IgG2a or IgA

were added and detected by TMB. Absorbance was determined at 450 nm with an EL808

micro plate reader (Bio-Tek Instruments, Bad Friedrichshall, Germany). Antibody titres



Statistics


Statistical significance was determined either by a one way or a two way analysis of

variance (ANOVA) with a Bonferroni post-test, depending on the experiment set-up.

Results

Characterisation of the nanoparticles

Inclusion of adjuvants into TMC nanoparticles did not alter the physical nature of the

particles substantially. All adjuvanted particles showed a similar average diameter

(between 300-400 nm) and all were modestly positively charged (+13-21 mV). The capacity

to encapsulate OVA was only marginally affected by the inclusion of any of the adjuvants

(table 2). The loading efficiency of the adjuvant, however, greatly differed depending on

the characteristics of the adjuvant. The strongly negatively charged species CpG and CTB

easily complexed with the nanoparticles, whereas the positively charged adjuvant, PAM,

associated much less efficiently with the positively charged TMC nanoparticles. The loading

efficiency of the amphiphilic adjuvants LPS (weakly negatively charged) and MDP (neutral)

was 35% and 42%, respectively. So, LPS and MDP were more efficiently encapsulated than


165

the positively charged PAM, but less efficiently than the hydrophilic, negatively charged

adjuvants CpG and CTB.

Formulation Size

[nm] PDI*

ZP** [mV]

LE***

OVA [%]

LE

Adjuvant

TMC/OVA 314 ± 31 0.12 18.2 ± 1.8 63 ± 6 -

TMC/CTB/OVA 323 ± 39 0.29 14.7 ± 2.4 56 ± 4 68-74

TMC/LPS/OVA 365 ± 46 0.33 13.3 ± 2.9 52 ± 0.1 32-37

TMC/PAM/OVA 375 ± 99 0.11 15.5 ± 0.2 59 ± 7 8.1-9.4

TMC/MDP/OVA 418 ± 89 0.15 13.6 ± 1.7 60 ± 1 41-43

TMC/CpG/OVA 304 ± 22 0.20 20.9 ± 2.0 52 ± 7 52-62

*PDI = polydispersity index, **ZP = zetapotential and ***LE = loading efficiency. n=3 +/- SEM ‡n=2

Total serum IgG response after nasal and intradermal vaccination

The differently adjuvanted TMC/OVA formulations were administered nasally and

intradermally to study their adjuvanticity and the site-dependency thereof. After nasal and

intradermal vaccination TMC/OVA nanoparticles induced higher IgG titres compared to

OVA alone (figure 1A, B). In some cases the inclusion of an adjuvant into the TMC/OVA

particle increased the immunogenicity even further.

Nasally, the LPS- and MDP-loaded TMC/OVA nanoparticles elicited higher IgG titres

compared to TMC/OVA nanoparticles (p<0.05; figure 1A). Encapsulation of CTB, PAM or

CpG into TMC/OVA nanoparticles did not significantly affect the total serum IgG response

compared to TMC/OVA nanoparticles.

After intradermal injection, TMC/LPS/OVA nanoparticles elicited higher IgG levels than

plain TMC/OVA nanoparticles after both a priming (p<0.05) and a booster dose (p<0.01). In

contrast to nasal administration, after a priming dose intradermal administration of

TMC/CpG/OVA nanoparticles significantly increased IgG titres compared to plain TMC/OVA

nanoparticles (p<0.05; figure 1B) and co-encapsulation of MDP had no effect.

Encapsulation of CTB and PAM into TMC/OVA nanoparticles did not lead to elevated IgG

titres compared to non-adjuvanted TMC/OVA nanoparticles.

Table 2. Characteristics of adjuvanted TMC nanoparticles.

Chapter 8

166

IgG subtyping of the immune response

Besides the IgG titres, the IgG1 and IgG2a antibody titres were measured to obtain insight

into the type of immune response elicited by the different formulations. For both

administration routes the main subtype produced after vaccination with OVA alone,

TMC/OVA nanoparticles and TMC/adjuvant/OVA particles was IgG1, which followed a

similar trend as the total IgG titres after the boost.

Nasally administered LPS- or MDP loaded TMC/OVA nanoparticles elicited higher IgG1

titres than plain TMC/OVA particles (figure 2A), whereas the other adjuvants did not show

significant effects on the IgG1 response. None of the formulation induced substantial

IgG2a levels.

After intradermal immunisation, TMC/OVA nanoparticles induced the production of

significantly more IgG1 compared to a solution of OVA, but no additional effect of the

encapsulation of adjuvants was observed. However, TMC nanoparticles containing CpG

significantly boosted the IgG2a production (p<0.001), causing a decrease in the IgG1/IgG2a

ratio compared to TMC/OVA nanoparticles (figure 2B).

Figure 1. OVA specific serum IgG titres after nasal (A) and intradermal (B) immunisation.

Data are presented as mean ± SEM of 8 (A) or 5 (B) mice. * p<0.05, ** p<0.01, *** p<0.001.


167

Figure 2. OVA specific serum IgG1 (white bars) and IgG2a (black bars) titres 3 weeks after a

booster dose nasally (A) and intradermally (B). Data are presented as mean ± SEM, ** p<0.01

***p<0.001.

Figure 3. Secretory IgA levels in nasal

washes of individual mice 3 weeks

after a nasal booster dose. Bar

represents mean.

Chapter 8

168

Production of sIgA

Secretory IgA is an important mediator of mucosal immunity and can therefore provide

protection against respiratory pathogens. Intradermal administration did not induce

detectable sIgA levels in the nasal washes (data not shown). In contrast, nasal vaccination

with TMC/OVA nanoparticles containing LPS, MDP or CpG did result in increased levels of

sIgA in some mice (figure 3). The nasal application of plain TMC nanoparticles or

nanoparticles adjuvanted with CTB or PAM did not trigger sIgA production.

Discussion

The field of adjuvants is rapidly evolving. Whereas alum has been the only approved

adjuvant for many years, recently squalene emulsions (MF-59) and monophosphoryl lipid A

(a LPS derivate) have been licensed for use in Europe. Increased knowledge on the

activation of the innate immune system has led to the identification of new adjuvants that

activate APCs specifically via Toll-like receptors (TLRs) [28] or NOD-like receptors (NLRs).

Moreover, detoxification of known adjuvants (MPL instead of LPS; CTB instead of cholera

toxin) and the development of new adjuvants exploiting the increased knowledge on

activation of the innate immune system (CpG, PAM3CSK4, MDP), will probably increase the

arsenal of adjuvants for commercial human vaccines in the future. This progress is crucial

for the development of subunit vaccines as the addition of an adjuvant seems inevitable in

order to yield a strong immune response. The large number of DCs in the dermis and nasal

epithelium potentially makes application of adjuvanted vaccines at these sites very

attractive, as it can directly result in activation of DCs. Nonetheless, a delivery system to

enhance the uptake of both the antigen and the adjuvant will be an important utensil, as

generally physical mixtures of adjuvant and antigen are inferior to systems where both

components are co-localised.

TMC nanoparticles are excellent antigen carriers as they associate with DCs and, because

of their intrinsic adjuvanticity, activate DCs [26, 27]. As a consequence, in direct

comparison with other vaccine delivery systems TMC nanoparticle have shown to be a

more effective carrier for mucosal or dermal administration than PLGA nanoparticles [12],

positively charged liposomes (unpublished data) and chitosan nanoparticles [26]. Co-

encapsulation of adjuvants has been reported to further increase the immunogenicity of

several carrier systems [10, 11, 23, 24]. Therefore we studied the co-encapsulation of

antigen an adjuvant in TMC nanoparticles. The OVA dose chosen was twofold lower than in

previous studies [12, 16, 27] to be able to better detect the effect of the encapsulated

adjuvant.

In alignment with previous studies [12, 16, 27], the beneficial effect of TMC nanoparticles

as a carrier system was clearly observed in this study. OVA-loaded TMC nanoparticles


169

enhanced the IgG and IgG1 titres compared to nasal or intradermal administration of OVA

alone.

The activity of the adjuvants appeared to be administration site specific. Nasally, co-

encapsulation of LPS and MDP in TMC/OVA nanoparticles elicited higher IgG titres than

TMC/OVA nanoparticles, whereas intradermally LPS and CpG were the most effective

adjuvants. It has been reported that the expression of TLRs and NLRs on DCs is dependent

on the micro-environment of the DC and the DC subset [29-32], which may explain the

differential effects of adjuvants when comparing the nasal and intradermal route. For

instance, the effect of the NOD2 ligand MDP could be explained in this manner. NOD2

plays an important role in Crohn’s disease [33, 34] and NOD2-deficient mice are more

susceptible to Listeria monocytogenes and Bacillus anthracis, two bacteria that cause

infection via a mucosal site [35, 36]. Moreover, Bogefors et al. recently reported that

different NLRs, including NOD2, are present in the nose [31]. This implicates an important

role for the NOD2 receptor in mucosal immunity, which concurs with the positive effect

found for MDP after nasal administration. Regarding intradermal vaccination, the

receptors for LPS (TLR4) and CpG (TLR9), the two adjuvants that showed a strong effect

after intradermal administration, are readily expressed on murine keratinocytes,

Langerhans cells and DCs [30, 37]. Previous murine vaccination studies via the skin have

shown the adjuvanticity of CpG [38, 39], which induced migration of Langerhans cells and

DCs from the skin to the lymph nodes [40, 41].

In a previous nasal vaccination study the TMC/CpG/OVA nanoparticles were equally potent

as non-adjuvanted TMC nanoparticles and particularly stimulated the IgG2a response [16].

However, since the applied dose in the present study was two times lower, this effect may

have been masked. These results together with the elevated sIgA levels for 3 out of 8 mice

indicate that whereas CpG can function as an adjuvant for nasal vaccination, the adjuvant

dose may be crucial.

For both the intradermal and nasal route, CTB was unable to further promote the antibody

titres compared to non-adjuvanted TMC/OVA nanoparticles. We have shown before that

CT is able to boost the immune response after intradermal administration [13] and for

both vaccination via the skin and nose CT is a well known adjuvant [42]. However, the

toxicity of CT is a concern for nasal and intradermal administration. Especially after a nasal

vaccine containing heat-labile enterotoxin (LT, a potent mucosal adjuvant with ADP-

ribosylating activity like CT) was withdrawn from the market [43], CT is not considered a

promising adjuvant for human nasal use anymore. CTB is a less toxic CT analogue [44] and

successful nasal administration of CTB as an adjuvant has been reported [20, 22, 45, 46].

However, in a few cases it has also been linked to the induction of tolerance [47-49], the

opposite of what is desired in the current vaccination study. Anjuère et al. compared the

ability of CT and CTB to provoke an immune response after transcutaneous immunisation

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[50]. They reported CTB to be poorly efficient in inducing anti-OVA IgG levels, whereas CT

provoked a strong humoral immune response. This shows that the adjuvant effect of CTB

depends on the antigen, the formulation and the adminstration route.

Besides the extent of the immune response, also the type of immune response is an

important parameter to consider, when selecting an adjuvant. TMC nanoparticles appear

to be a Th2-biasing carrier system, as described before [13, 16, 51], regardless of the

administration route. Encapsulation of most of the adjuvants did not significantly change

the Th1/Th2 ratio. LPS and PAM have been reported to augment the Th1 response after

intramuscular and intraperitoneal administration [52, 53], but do not appear to elicit this

effect after nasal or intradermal immunisation when co-encapsulated with the antigen in

TMC nanoparticles. Only intradermal administration of TMC/CpG/OVA nanoparticles was

able to counter the Th2 bias and increase the IgG2a levels (indicative of a Th1 response).

Nasally, this effect of CpG was not observed, whereas an earlier study using a CpG dose

that was twice as high, reported a clear Th1 biasing effect of TMC/CpG/OVA nanoparticles

[16], also indicating an important role for the adjuvant dose. Overall, the effect of an

adjuvant seems to be greatly dependent on the dose, the type of antigen, the way it is

formulated and –last but not least– the site of administration.

Conclusion

Inclusion of an adjuvant into antigen loaded TMC nanoparticles for nasal and intradermal

vaccine delivery can be good strategy to improve the immunogenicity of the antigen. The

success of this approach strongly depends on the selection of the adjuvant in conjunction

with the site of administration.

Acknowledgement

This work was performed within the framework of Top Institute Pharma project number

D5-106.


171

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46. Rask C, Fredriksson M, Lindblad M, Czerkinsky C, and Holmgren J, Mucosal and systemic antibody

responses after peroral or intranasal immunization: effects of conjugation to enterotoxin B subunits

and/or of co-administration with free toxin as adjuvant. APMIS, 2000. 108(3): p. 178-86.

47. Sun JB, Czerkinsky C, and Holmgren J, Mucosally induced immunological tolerance, regulatory T cells

and the adjuvant effect by cholera toxin B subunit. Scand J Immunol, 2010. 71(1): p. 1-11.

48. Aspord C and Thivolet C, Nasal administration of CTB-insulin induces active tolerance against

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591-7.

50. Anjuere F, George-Chandy A, Audant F, Rousseau D, Holmgren J, and Czerkinsky C, Transcutaneous

immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective

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PART III: CATIONIC LIPOSOMES TO CO-DELIVER ANTIGEN AND ADJUVANT

Chapter 9

Co-encapsulation of antigen and

adjuvant in cationic liposomes affects the

quality of the immune response in mice

after intradermal vaccination Suzanne M. Bal, Sander Hortensius, Zhi Ding, Wim Jiskoot, Joke A. Bouwstra

Vaccine 2010 (in press)

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Abstract

Enhanced immunogenicity of subunit antigens can be achieved by antigen encapsulation in

liposomes and the addition of immune potentiators. In this study we co-encapsulated

ovalbumin (OVA) and a Toll-like receptor (TLR) ligand (PAM3CSK4 (PAM) or CpG) in cationic

liposomes and investigated the effect of the formulations on dendritic cell (DC) maturation

in vitro and on the immune response in mice after intradermal immunisation. Co-

encapsulation of PAM did not affect the OVA content of the liposomes, but co-

encapsulation of CpG led to a decrease in OVA content by 25%. After liposomal

encapsulation, both ligands retained the ability to activate TLR-transfected HEK cells,

though PAM only induced activation at elevated concentrations. DC maturation induced by

liposome-based adjuvant formulations was superior compared to the free adjuvants.

Encapsulation of PAM and CpG in liposomes did not influence the total IgG titres compared

to the antigen/adjuvant solution, but OVA/CpG liposomes shifted the IgG1/IgG2a balance

more to the direction of IgG2a compared to non-encapsulated CpG. Moreover, only this

formulation resulted in IFN-γ production by restimulated splenocytes from immunised

mice. These data show that co-encapsulation of antigen and immune potentiator in

cationic liposomes can affect the type of immune response generated after intradermal

immunisation.

Quality of the immune response after intradermal vaccination with adjuvanted lipsomes

179

Introduction

Vaccines should be capable of eliciting a strong and protective immune response, but are

also required to be safe. Subunit antigens are regarded safer than live-attenuated and

inactivated pathogens, but lack strong immunogenicity. Optimising the formulation of

subunit vaccines could be instrumental in improving the immunogenicity and therefore in

the development of safe and effective vaccines [1]. Approaches to achieve a higher efficacy

include optimising the delivery to and interaction with dendritic cells (DCs) and the

addition of adjuvants to improve the activation of these DCs.

Lessons to improve the interaction with DCs can be learned from nature, as almost all

pathogens are particulates. Particles are better taken up by DCs and may provide an

additional benefit by offering prolonged antigen delivery due to slow antigen release [2].

Liposomes are elegant and flexible nanoparticulates that have been used for a long time as

drug delivery systems. Actually, when they were used for the first time in the

pharmaceutical field in 1974, it was for the delivery of vaccines [3]. Since then they have

been used successfully for the delivery of protein antigens [4-6] and DNA vaccines [7, 8]. By

changing the lipid composition of liposomes, their characteristics can be varied. The usage

of positively charged lipids, for instance, creates cationic liposomes. It has become clear

that cationic liposomes are one of the most effective liposomal delivery systems for

antigens to antigen presenting cells [9-12].

Liposomes themselves may improve the uptake of antigens by DCs, but generally lack

intrinsic adjuvanticity [11, 13]. By co-encapsulation of an adjuvant, the immunogenicity of

liposomes can be improved. Adjuvants have been classified by Schijns as substances that

activate the immune system [14]. Adjuvants i) interact with pattern recognition receptors

(PRRs) (Signal 0) [15, 16]; ii) are co-stimulatory molecules necessary for activating naïve T

cells (Signal 2) or iii) act as a ‘danger-signal’ [17]. Pathogens express specific pathogen-

associated molecular patterns (PAMPs) that are recognised by PRRs, of which the Toll-like

receptors (TLRs) are an important subclass. All cells, but mainly antigen presenting cells

such as DCs, have TLRs that recognise specific ligands. In humans 11 different TLRs have

been identified, the majority of them being specific for microbial products. Most TLRs are

present on the cell surface, but TLRs that recognise nucleic acids (TLR3, 7, 8 and 9) are

located intracellularly [18].

In this study we co-encapsulated a model antigen, ovalbumin (OVA) and two TLR ligands in

cationic liposomes. The selected TLR ligands are Pam3CSK4, a synthetic lipoprotein

consisting of a tri-palmitoyl-S-glyceryl cysteine lipopeptide with a pentapeptide SKKKK

(PAM), and unmethylated CpG oligonucleotide (CpG). PAM is recognised by TLR2 in

association with TLR1, both cell surface expressed receptors. CpG is a TLR9 ligand, which is

expressed intracellularly. By co-encapsulation in liposomes it is ensured that both the

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180

antigen and the adjuvant are co-delivered to the DCs, which is considered essential for

induction of a strong immune response [19-21]. To examine the effect of co-encapsulation,

a comparison was made to solutions of OVA mixed with the respective adjuvants. The

formulations were tested in vitro for their DC-stimulating properties and their

immunogenicity was studied in vivo by intradermal injection, an immunisation route which

has regained interest in recent years due to the dose-sparing potential compared to

intramuscular immunisation [22-25].


Materials

Soybean phosphatidylcholine (PC), 1,2-dioleoyl-3-trimethylammonium-propane chloride

salt (DOTAP) and 1,2-dioleoyl-sn-glycero-3-ghosphoethanolamine (DOPE) were kindly

provided by Lipoid GmbH (Ludwigshafen, Germany). Ovalbumin grade VII was obtained

from Calbiochem (Merck KGaA, Darmstadt, Germany). FITC-labelled ovalbumin (OVAFITC)

was purchased from Invitrogen (Breda, The Netherlands). PAM, rhodamine-labelled PAM,

CpG 2006 and 1826 and their FITC-labelled analogs were purchased from Invivogen

(Toulouse, France). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (γ chain

specific), IgG1 (γ1 chain specific) and IgG2a (γ2a chain specific) were purchased from

Southern Biotech (Birmingham, USA). Chromogen 3, 3', 5, 5'-tetramethylbenzidine (TMB)

and the substrate buffer were purchased from Invitrogen. All cell culture media, including

serum and trypsin were purchased from Gibco (Invitrogen). Nimatek® (100 mg/ml

Ketamine, Eurovet Animal Health B.V., Bladel, The Netherlands), Oculentum Simplex

(Farmachemie, Haarlem, The Netherlands), Rompun® (20 mg/ml Xylazine, Bayer B.V.,

Mijdrecht, The Netherlands) and the injection fluid (0.9% NaCl) were obtained from a local

pharmacy. Phosphate buffered saline (PBS) pH 7 was obtained from Braun (Oss, The

Netherlands). All other chemicals were of analytical grade.

Animals

Female BALB/c mice (H2d), 8-weeks old at the start of the vaccination study were

purchased from Charles River (Maastricht, The Netherlands), and maintained under

standardised conditions in the animal facility of the Leiden/Amsterdam Center for Drug

Research, Leiden University. The study was carried out under the guidelines compiled by

the Animal Ethic Committee of the Netherlands.


181

Liposome preparation and characterisation

Liposomes were prepared using the film rehydration method [26] followed by extrusion.

Soy-derived phosphatidyl choline (PC), dioleoyl trimethyl ammonium propane (DOTAP) and

dioleoyl phosphatidyl ethanolamine (DOPE), dissolved in chloroform, were mixed in a 9:1:1

molar ratio in a flask. A thin lipid film was formed at the bottom of this flask using a rotary

evaporator. The residual organic solvent was removed by nitrogen flow. The film was

rehydrated in a 10 mM phosphate buffer pH 7.4 (7.7 mM Na2HPO4 and 2.3 mM NaH2PO4)

containing 1 mg/ml OVA. The final concentration of lipids was 5% (w/v). The dispersion

was shaken in the presence of glass beads at 200 RPM for 2 hrs at room temperature. To

obtain monodisperse liposomes, the dispersion was extruded (LIPEXTM

extruder, Northern

Lipids Inc., Canada) 4 times through a carbonate filter with a pore size of 400 nm and 4

times through a filter with a pore size of 200 nm (Nucleopore Millipore, Amsterdam, The

Netherlands). For adjuvanted liposomes, after rehydration either PAM or CpG was added

to a final concentration of 2 mg/ml. The dispersions were freeze-dried and subsequently

rehydrated in the same buffer solution. Extrusion was performed as described above.

The size and zetapotential of the liposomes were determined by dynamic light scattering

and laser Doppler velocimetry, respectively, using a Zetasizer® Nano ZS (Malvern

Instruments, UK). The amount of OVA, PAM and CpG present in the liposomes was

determined by using their fluorescently labelled analogs (10% of used OVA, PAM or CpG

were labelled). The free antigen and adjuvant were separated from the liposomes by

filtration using a Vivaspin 2 centrifugal concentrator (PES membrane, MWCO 300 kDa,

Sartorius Stedim, Nieuwegein, The Netherlands) and quantified using a FS920 fluorimeter

(Edinburgh Instruments, Campus Livingston, UK). The stability of the OVA-loaded

liposomes and OVA release from the liposomes was determined in PBS pH 7.4. Liposomes

containing OVAFITC were diluted to a 0.5% lipid concentration and stored at 37°C under

constant stirring. Samples were taken at selected time intervals and the size of the

liposomes and antigen encapsulation were measured after filtration.

Activity of TLR ligands

HEK293 cells, stably transfected with human CD14/TLR2 or TLR9 and a NF-κB inducible IL-8

(TLR2) or luciferase (TLR9) plasmid [27, 28], were maintained in Dulbecco's Modified Eagle

Medium (DMEM), supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate

and 10 μg/ml ciprofloxacin. To the HEK293-CD14/TLR2 cells 5 μg/ml puromycin and to the

HEK293/TLR9 cells 700 µg/ml Geneticin (G418) was added as a selection marker. For

stimulation experiments, both cell types were seeded at a density of 4.0 × 104

cells/well in

96-well flat bottom plates and stimulated the next day. The cells were stimulated with the

formulations containing different concentrations of PAM (maximum 450 ng/ml) or CpG

Chapter 9

182

(maximum 10 µg/ml). Medium was used as a negative control. TLR2 stimulation was

measured by determining the IL-8 production in supernatants after 24 hr using a

commercial kit (Sanquin, Amsterdam, The Netherlands), following the manufacturer's

recommendations. The HEK-293/TLR9 cells were stimulated for 6 hrs with the

formulations. The luciferase expression was determined with a luciferase assay kit

(Promega, Leiden, The Netherlands) according to the manufacturer’s manual, using a

DLReady Berthold Centro XS luminometer (Berthold Detection Systems, Germany).

DC activation

Monocytes were isolated from human donor blood before each experiment by Ficoll and

Percoll density centrifugation and depletion of platelets was performed by surface

adherence of the monocytes in 24-well plates (Corning, Schiphol, The Netherlands) as

described previously[29]. The monocytes were cultured for 6 days at 37°C and 5% CO2

after seeding at a density of 0.5 x 106 cells/well in RPMI 1640, supplemented with 10% v/v

FCS, 2 mM glutamine, 1 mM sodium pyruvate and 500 U/L penicillin/streptomycin. To

differentiate monocytes into immature DCs 250 U/ml granulocyte macrophage-colony

stimulating factor (GM-CSF) and 100 U/ml IL-4 (Invitrogen) was added. Medium was

refreshed after 3 days.

DC were incubated for 48 hrs at 37°C in RPMI 1640 containing 500 U/ml GM-CSF with 2

μg/ml OVA, either free or encapsulated into liposomes with and without PAM or CpG.

Mixtures of OVA with PAM or OVA with CpG were used as controls and LPS (100 ng/ml,

Invivogen) was added as a positive control. Cells were washed 3 times with PBS containing

1% w/v bovine serum albumin and 2% v/v FCS and incubated for 30 min with a mixture of

20x diluted anti-HLADR-FITC, anti-CD83-PE and anti-CD86-APC (Becton Dickinson) in the

dark at 4°C. Cells were washed and the expression of MHCII, CD83 and CD86 was

quantified using flow cytometry (FACSCanto II, Becton Dickinson) relative to LPS, assuming

100% maturation for LPS-treated DC. Live cells were gated based on forward and side

scatter.

Intradermal immunisation

Groups of 8 mice were immunised with the OVA-loaded liposomes with and without PAM

or CpG by intradermal injection into the abdominal skin as described previously [29].

Besides the liposomes, solutions of OVA or OVA with PAM or CpG in PBS were injected and

subcutaneous injection of OVA served as a control. The mice were vaccinated twice with

three weeks intervals with a dose of 5 µg OVA and 10 µg PAM or CpG in a total volume of

30 µl. To maintain this ratio between antigen and adjuvant, liposomes used for the

immunisation study were not filtered to remove free antigen and adjuvant. Blood samples


183

were collected from the tail vein one day before each immunisation. Three weeks after the

last vaccination the mice were sacrificed. Just before euthanasia total blood was collected

from the femoral artery. Afterwards the spleens were removed. Blood samples were

collected in MiniCollect® tubes (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) till clot

formation and centrifuged 10 min at 10,000 g to obtain cell-free sera. The sera were stored

at −80 °C until further use.

Detection of IgG, IgG1 and IgG2a

OVA specific antibodies (IgG, IgG1 & IgG2a) in the sera were determined by sandwich ELISA

as described previously [29]. Briefly, plates were coated overnight with 100 ng OVA/well.

After blocking, two-fold serial dilutions of sera from individual mice were applied to the

plates. HRP-conjugated antibodies against IgG, IgG1 or IgG2a were added and detected by

TMB. Antibody titres were expressed as the reciprocal of the sample dilution that

corresponds to half of the maximum absorbance at 450 nm of a complete s-shaped

absorbance-log dilution curve.

T cell activation

The spleens from immunised mice were maintained in RPMI with 10% FCS, 50 µM β-

mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate and 500 U/L

penicillin/streptomycin. Cell suspensions were obtained using a cell strainer (70 µm,

Becton Dickinson). Cells were washed and cultured in 96-well flat bottom plates at a

density of 2.0×105

cells/well in triplicate and restimulated with 40 µg/ml OVA. ConA

(Sigma-Aldrich) 5 µg/ml was used as a positive control. After 3 days the supernatants were

collected and stored at -80 °C until further use. The amount of IFN-γ in the supernatant

was determined by ELISA using a commercial kit (Becton Dickinson) according to the

manufacturer’s instructions.

Statistics


Statistical significance was determined either by a one way or a two way analysis of

variance (ANOVA) with a Bonferroni post-test, depending on the experiment set-up.

Results

Liposome characteristics

With the film rehydration method OVA-containing liposomes with an average size of 130

nm and a positive zetapotential could be prepared in a reproducible manner (table 1).

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Ultrafiltration showed that nearly 100% OVA was associated with the liposomes. PAM

could be easily incorporated into the liposomes (~85%) and the incorporation did not

affect the (measured) liposome characteristics. The addition of CpG did influence the

liposome characteristics as the size augmented by two-fold. Furthermore, CpG reduced

OVA association with the liposomes, probably due to competition between the antigen

and the adjuvant as both compounds bear a negative charge.

Size [nm]

PDI ZP [mV]

LE OVA [%]

LE Adjuvant

[%]

OVA

liposomes 130 ± 10 0.19 ± 0.03 23 ± 1 98 ± 2 -

OVA/PAM

liposomes 128 ± 9 0.25 ± 0.01 20 ± 2 96 ± 3 85 ± 4

OVA/CpG

liposomes 263 ± 22 0.30 ± 0.09 18 ± 2 72 ± 5 61 ± 6

PDI = polydispersity index, ZP = zetapotential, LE = loading efficiency

The stability and release of the OVA liposomes was studied over time in PBS at 37°C.

Dilution in PBS had an initial effect on the size of the liposomes as their size decreased

from 130 nm to 90 nm, but remained stable during the following 8 days (figure 1). During

this period OVA was released from the liposomes. An initial burst release of 25% was

observed and after 5 hrs already 50% of the OVA was no longer associated with the

liposomes. During the following 8 days the remaining OVA was slowly released.

Preservation of TLR-activation of liposome encapsulated PAM and CpG

PAM and CpG are two TLR ligands. The effect of ligand encapsulation in OVA liposomes on

their interactions with the TLRs was studied on HEK293 cells transfected with either TLR2

(receptor for PAM) or TLR9 (receptor for CpG). Non-adjuvanted liposomes and a solution of

Table 1. Characteristics of liposomal formulations. All data are averages ± SD of at least 3

different batches.

Figure 1. Size and OVA release of OVA

liposomes over time in PBS (pH 7.4) at

37°C. Mean ± SEM of 3 individual

batches.


185

OVA did not induce TLR2 or TLR9 activation (data not shown). PAM in solution was a

stronger TLR2 activator compared to the liposome encapsulated PAM (figure 2A). A 15-fold

higher dose of PAM was necessary to obtain the same level of IL-8 production from the

HEK293-CD14/TLR2 cells. Both PAM in solution and OVA/PAM liposomes activated the cells

in a concentration dependent manner.

CpG activated TRL9-transfected HEK cells in a concentration dependent way as well.

Encapsulation of CpG in liposomes did not affect its ability to activate TLR9, as no

difference in activation between a solution of CpG and OVA/CpG liposomes was observed

(figure 2B).

Adjuvanted liposomes activate DCs

DCs express TLRs which upon stimulation with TLR ligands induces the expression of

maturation markers on the DC’s surface. Encapsulation of both adjuvants had a clear effect

on the DC activation. Application of 10 µg/ml of the OVA/PAM liposomes significantly

elevated the MHCII and CD83 expression (p<0.01) compared to untreated cells and this

activation proved to be concentration dependent (figure 3A and B). Moreover, a similar

pattern was observed for the CD86 levels. After application of a PAM solution also a trend

of elevated MHCII and CD83 levels was observed, but these levels were not significantly

higher compared to untreated DCs. PAM had a minor effect on the CD86 expression (figure

3C).

The effect of CpG encapsulation was more pronounced. Whereas a CpG solution did not

activate the DCs at all, encapsulation of CpG in liposomes induced increased MHCII, CD83

Figure 2. Preservation of TLR-activating capacity of encapsulated TLR ligands tested on

HEK293 cells transfected with TLR2 or TLR9. A: ability of OVA/PAM liposomes and a PAM

solution to activate TLR2-transfected HEK-293 cells; B: ability of OVA/CpG liposomes and a

CpG solution to activate TLR9-transfected HEK-293 cells. Data are mean ± SEM of 3 different

experiments.

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and CD86 expression (figure D-F). The level of expression obtained with the highest CpG

concentration was comparable to that induced by LPS, the positive control.

Intradermal vaccination with adjuvanted liposomes

To investigate whether the improved DC activation ability in vitro correlated with the

immunogenicity in mice, an immunisation study was performed. The liposomal

formulations and physical mixtures of OVA with CpG or PAM were applied intradermally.

Figure 3. Upregulation of DC maturation markers by OVA/PAM liposomes (A-C) and

OVA/CpG liposomes (D-F). M = medium and the concentrations are expressed in µg/ml. The

values are expressed as mean fluorescence intensity (MFI) ± SEM relative to LPS of triplicate

measurements in two separate experiments.


187

Both the OVA-specific total serum IgG titres (figure 4) and the antibody subclass (IgG1 and

IgG2a, figure 5) were measured.

The addition of either PAM or CpG into liposomes significantly increased the

immunogenicity of OVA-loaded liposomes (p<0.05), which did not enhance the immune

response to an OVA solution. Incorporation of the TLR ligands in OVA-containing liposomes

induced similar IgG titres as compared to the physical mixtures of OVA and the adjuvant.

However, the liposomes did influence the IgG1/IgG2a balance of the immune response

(figure 5). The main IgG subtype induced by non-adjuvanted OVA was IgG1. The addition of

PAM resulted in equally elevated IgG1 and IgG2a levels upon intradermal immunisation.

Encapsulation of OVA alone in liposomes and co-encapsulation of OVA and PAM resulted

in a tendency of altering the balance more towards IgG2a (figure 5B). Co-administration of

CpG with OVA significantly shifted the IgG1/IgG2a balance towards IgG2a (p<0.05). This

alteration was even more pronounced when OVA and CpG were co-encapsulated in

liposomes (p<0.001).

Besides the humoral immune response, the effect of the different formulations on the

cellular immunity was investigated by measuring the IFN-γ production by restimulated

splenocytes. Th1 cells produce IFN-γ which is reported to induce isotype switching and

IgG2a production [30, 31]. In agreement with the antibody subclass titres, only

formulations containing CpG, which resulted in the highest IgG2a titres, induced the

production of measurable IFN-γ levels and these levels were the highest for mice receiving

OVA/CpG liposomes (figure 6).

Figure 4. OVA specific IgG titres in serum after a prime and subsequent booster

immunisation. Mean ± SD of 8 mice. † significantly higher than ID OVA. ‡ significantly higher

than ID OVA liposomes.

ID Intradermal; SC subcutaneous

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Discussion

Liposomes are an attractive delivery system for vaccines as they protect the antigen from

degradation, opsonise the uptake of the encapsulated antigen by DCs and provide

controlled release of the antigen over time. Moreover, it is a versatile system that permits

the inclusion of various adjuvants. This is reflected by the fact that high encapsulation

efficiencies of both PAM and CpG were achieved, whereas both adjuvants have very

different physical chemical characteristics. This is an important feature, as in line with

Figure 5. OVA specific serum IgG1 (black bars) and IgG2a (white bars) titres after the second

boost. A: IgG1 and IgG2a titres after three immunisations. Mean ± SD of 8 mice. B:

Corresponding IgG1/IgG2a ratios of individual mice. Non-responders for IgG1 or IgG2a were

excluded. Bar represents geometric mean. * p<0.05 *** p<0.001.

Figure 6. IFN-γ production by

splenocytes after restimulation with

OVA. Mean + SEM of 5 mice are shown.

* p<0.05


189

other reports [11, 13], this study shows that cationic liposomes themselves are not that

immunogenic; OVA loaded liposomes did not enhance the antibody response compared to

free OVA. The inclusion of adjuvants into liposome-based formulations will therefore be

necessary to improve their application in vaccination strategies.

Here we showed that co-encapsulation of antigens and TLR ligands in liposomes can

enhance antigen delivery in vitro and combine this with potent stimulation of the innate

immune response as can be concluded from the vaccination study with PAM- or CpG-

containing liposomes. The anti-OVA serum IgG titres after the prime and booster

vaccinations with these adjuvanted liposomes were significantly higher than those

obtained with non-adjuvanted liposomes or plain OVA. Interestingly, the IgG titres elicited

in mice vaccinated with a physical mixture of OVA and PAM or CpG, were comparable with

those elicited by those that were immunised with PAM- or CpG-adjuvanted liposomes. This

is in accordance with previous studies by us and other groups, where no additional effect

of liposomes on the IgG titres was observed after vaccination via different routes [11, 13,

32]. It not only holds true for liposomes, but also for antigen-loaded N-trimethyl chitosan

nanoparticles [29]. This raises questions regarding the usefulness of nanoparticles for

intradermal immunisation. However, IgG titres not necessarily correlate with protection

and are therefore not the only parameter to express the extent or quality of an immune

response. A cellular response, which can be measured by the production of IgG2a

antibodies and IFN-γ production by T-cells, can sometimes be more predictive [33]. The

present study shows that liposomes did influence the quality of the immune response. A

trend of higher IgG2a levels compared to antigen and adjuvant solutions was observed for

all three liposomal formulations. Similar results were also reported by Brgles et al. after

subcutaneous immunisation; OVA-containing liposomes were able to modulate the

immune response towards a Th1/CD8+ cytotoxic T lymphocyte (CTL) direction, without

influencing the overall intensity of the immune response [13].

How liposomes modify the quality of the response remains to be clarified. The in vitro DC

study clearly demonstrates that CpG, and to a lesser extent also PAM, needs to be

encapsulated to activate the DCs. This is in accordance with a study by Fernandes et al.

who showed that the liposomal incorporation of two other triacylated lipopeptides

enhanced the proliferation of murine splenocytes [34], which could be attributed to

improved adjuvant uptake by the DCs [20, 21]. The prominent advantage of liposomal

encapsulation of CpG correlates excellent with the cellular localisation of the PAM and CpG

receptors. Whilst TLR2 is expressed on the cell surface, TLR9 is present in the endosomal

compartment. Conceivably, CpG profits more from liposomal delivery than PAM. For PAM

this is illustrated in vitro as liposome encapsulation decreases its ability to stimulate

HEK293-CD14/TLR2 cells, probably due to reduced interaction with the receptor. It is

known that liposomal incorporation can have a profound influence on the

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immunomodulatory properties of lipoproteins [35]. PAM’s functionality is dependent on

different structural components. The peptide segment linked to the carboxyl terminus of

the palmitoyl lipopeptide, the SKKKK sequence, was shown to elevate the adjuvant activity

compared to other peptide sequences [36]. Changes to the lipopeptide fatty acid chains,

the O-linked fatty acids in particular, appear to have a substantial effect on the signalling

through TLRs. The palmitoyl groups (C16) provide better adjuvant activity than longer and

shorter fatty acids [37, 38]. If the interaction of either of these moieties with the TLR2 is

disturbed, the adjuvanticity will be diminished. Liposomal encapsulation can also have a

positive effect on the adjuvanticity as it improves the solubility of PAM [39] and the DC

uptake of OVA, which may improve DC maturation. However, probably due to loss of

interaction with the TLR2, this did not enhance the immune response in vivo.

For CpG, improved DC uptake of OVA/CpG liposomes facilitates the interaction with the

endosomal TLR9 [18, 40], thereby inducing DC maturation. The in vivo situation is more

complicated. Even though the DCs will preferentially take up the liposomes, the speed and

duration of antigen and adjuvant exposure will differ between the solution and the

liposomal formulations. CpG and OVA in solution will probably reach the lymph nodes

faster than the liposomes, but only liposomes ensure uptake of CpG and OVA by the same

DC, which was reported to influence the type of immune response generated [21]. Indeed,

the enhanced DC uptake does result in a more Th1-biased response, which is most

pronounced for the CpG-containing liposomes. Similar results were reported by Gursel et

al., who showed that co-encapsulation of OVA and CpG in cationic liposomes induced

elevated IgG2a titres and IFN-γ secretion compared to free CpG after intraperitoneal

injection [41]. It has to be noted that liposome size also affects the Th1/Th2 bias; larger

liposomes tend to induce a Th1 shift [42, 43]. As OVA/CpG liposomes are larger this may

further shift the immune response towards Th1.

Finally the bias towards a more cellular response by the liposomes could also be attributed

to the presence of DOPE in the liposomes. DOPE, a neutral pH-sensitive lipid, is capable of

improving delivery of CpG into the cytosol following APC uptake [44]. Endosomal escape is

crucial for MHC I presentation of the antigen and the induction of CTL responses. It has

been reported that liposomes complexed with antigen and either CpG or poly(I:C), which

binds to TLR3 that is also expressed intracellularly, are capable of cross priming CD8+ T cells

[45]. Whether this is also the case after intradermal immunisation with our liposomes

requires further investigation, but the elevated IFN-γ production is a first indication that a

CTL response could be induced [46].

In conclusion, the advantage of co-encapsulation of antigen and adjuvant in cationic

liposomes is their potency to steer the immune bias. This depends on the type of adjuvant

used, as CpG, binding to the intracellular TLR9, induced the production of IgG2a antibodies


191

and a potent cellular immune response after intradermal immunisation, whereas PAM,

ligand of extracellular TLR2, did not.

Acknowledgement

This research was performed under the framework of TI Pharma project number D5-106

“vaccine delivery: alternatives for conventional multiple injection vaccines”. The authors

thank Bram Slütter for critically reading the manuscript.

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101.

Mechanistic studies on transcutaneous vaccine delivery : microneedles, nanoparticles and adjuvants Bal, Suzanne Marleen Chapter 10 under embargo until February 2nd 2015

Chapter 11

Summary and perspectives

Chapter 11

224


225

Summary

Microneedle-based transcutaneous immunisation is an appealing alternative to the

classical manner of injecting vaccines by intramuscular or subcutaneous route.

Importantly, as a consequence of the fact that the skin is in direct contact with the

environment and should protect the body against pathogens, it contains more antigen

presenting cells, such as dendritic cells (DCs) than the muscles or subcutaneous tissue and

thereby offers the possibility to induce a more effective immune response. However, the

perspective of the vaccinee, who generally prefer painless and safer vaccinations [1], is

perhaps even more important. The combination of microneedles and adjuvanted subunit

vaccines may offer effective vaccination whereas ensuring patient safety and vaccine

application in a painless manner.

The principal aim of this thesis was to design subunit vaccine formulations that can be

combined with microneedles for transcutaneous immunisation. The research focuses on

both vaccine efficacy and safety as it starts with evaluating the safety and effectiveness of

solid microneedles, followed by the development of adjuvanted formulations that can be

delivered via the conduits made by the microneedles.

This thesis starts with a general introduction to the field of transcutaneous immunisation

(chapter 2), giving an overview of the broad assortment of devices developed to deliver a

vaccine transcutaneously. After successful transport into the skin, the vaccine should be

taken up by the skin DCs. Pathogens are often particles. Formulating antigens into

nanoparticles resembles the pathogens in terms of size, thereby promoting DC uptake.

Two types of nanoparticles are used in the studies described in this thesis: N-trimethyl

chitosan (TMC) nanoparticles and cationic liposomes. Both have proven to be excellent

vaccine delivery systems [2, 3], but their potential for vaccination via the skin remains to

be elucidated.


In chapter 3 studies focusing on the safety of microneedle piercing in human volunteers,

an often overlooked parameter, are described in terms of skin irritation (skin redness and

blood flow) and pain sensation. Solid microneedles of different design and length (200-550

µm) were compared and, besides irritation, their ability to disrupt the stratum corneum

was evaluated by the transepidermal water loss (TEWL). Longer microneedle length (400

µm) resulted in increased TEWL, redness and blood flow compared to 200 μm long

microneedles. Needle design also had an effect. Of the two differently shaped

microneedles, the ones with the sharpest tip induced higher TEWL values, while resulting

Chapter 11

226

in less skin irritation. Most importantly, all microneedles resulted in minimal irritation

which lasted less than 2 hours.

In a subsequent clinical study (chapter 4) for the first time the influence of microneedle

geometry on the transport of fluorescein through the formed conduits was visualised with

confocal laser scanning microscopy. Based on the fluorescence intensity a distinction was

made between regions with high and low intensity fluorescence (HIF and LIF, respectively).

In most cases HIF areas were only present in the stratum corneum, while LIF areas were

also present in the viable epidermis. After 15 minutes almost no HIF was observed

anymore at the skin surface, whereas still LIF could be detected until a depth of 60 µm. All

microneedle arrays were able to form conduits in the skin, but the geometry of the

microneedles affected the shape and depth of the conduits. Microneedles with a sharp

needle tip were able to penetrate the skin to a higher extent than microneedles with a

blunter tip.


To prepare immunogenic subunit antigen formulations, the model antigen ovalbumin

(OVA) and a relevant antigen, diphtheria toxoid (DT), were formulated into nanoparticles

composed of TMC. As a comparison physical mixtures of TMC and the antigens were also

prepared. The studies described in Chapter 5 revealed that nanoparticles with a size

around 200 nm and a positive zetapotential could be prepared. A burst antigen release of

30% from these nanoparticles was observed in vitro, followed by no further antigen

release over a time span of 8 days. In an in vitro human DC model we showed that TMC

nanoparticles increased the uptake of OVA and that both nanoparticles and TMC/OVA

mixtures were able to induce upregulation of maturation markers (MHC-II, CD83 and

CD86) on these DCs. Co-cultures with T cells revealed production of cytokines of a Th2

biased profile. In vivo the humoral immune response was evaluated by measuring the total

serum IgG antibodies and antibody subclasses after intradermal vaccination in mice. For

both the OVA and DT vaccination studies, the TMC nanoparticles as well as the

TMC/antigen mixture were able to increase the IgG titres compared to non-adjuvanted

antigen and induced a Th2 biased immune response. Using DT-containing TMC

formulations, IgG titres and toxin-neutralising antibody titres could match up to those

obtained after subcutaneous injection of DT-alum.

The same formulations were used for transcutaneous immunisation using 300 µm long

microneedles in the studies reported in chapter 6. Two different microneedle arrays were

used and the formulations were applied before or after microneedle treatment.

Independent of the microneedle array used and the sequence of microneedle treatment

and vaccine application, transcutaneous immunisation with the physical mixture of TMC

and DT elicited 8-fold higher IgG titres compared to DT-loaded TMC nanoparticles or a DT


227

solution. Additional ex vivo confocal microscopy studies revealed that transport of the TMC

nanoparticles across the microneedle conduits was limited compared to a TMC solution.

To optimise the transport of vaccine formulation across the conduits formed by

microneedle pre-treatment the application time of the formulations was prolonged in

chapter 7. An extension from 1 to 2 hours of transcutaneous application of an OVA

solution resulted in a 30-fold increase of IgG titres. Besides the application time also the

effect of antigen-adjuvant entity size and co-localisation was found to be of crucial

importance. Superior IgG levels were induced by a TMC-OVA conjugate (28 nm) after the

prime vaccination and this coincided with higher numbers of OVA positive DCs found in the

lymph nodes. After the boost both the conjugate and the nanoparticles elevated the IgG

titres compared to an OVA solution. The same formulations were also applied via

intradermal and intranodal injection to study the aspect of delivery through the skin and to

the lymph nodes. Intradermally TMC, irrespective of its physical form, was essential for

increased antibody titres. These formulations formed a depot in the skin and prolonged

OVA delivery to the lymph nodes. The prolonged delivery to lymph node resident DCs was

also observed intranodally, but it did not correspond with elevated antibody titres. These

findings emphasise that each delivery route has different requisites for the ideal vaccine

formulation.

Chapter 8 describes a second generation of TMC nanoparticles containing a selection of

adjuvants including Toll-like receptor ligands lipopolysaccharide (LPS), PAM3CSK4 (PAM),

CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and the GM1

ganglioside receptor ligand, cholera toxin B subunit. The effectiveness of these adjuvant

loaded TMC particles was assessed after intradermal and nasal vaccination. After nasal

vaccination, TMC/OVA nanoparticles containing LPS or MDP elicited higher IgG, IgG1 and

sIgA levels than non adjuvanted TMC/OVA particles, whereas nanoparticles containing

CTB, PAM or CpG did not. All nasally applied formulations induced only marginal IgG2a

titres. After intradermal vaccination, the TMC/CpG/OVA and TMC/LPS/OVA nanoparticles

provoked higher IgG titres than plain TMC/OVA particles. Additionally, the TMC/CpG/OVA

nanoparticles were able to induce significant IgG2a levels. None of the intradermally

applied vaccines induced measurable sIgA levels. These results confirm the conclusions of

the previous chapter, i.e. the vaccine formulation, including the adjuvant, should be

tailored to the needs of the route of administration.


In this part cationic liposomes, which in contrast to TMC nanoparticles do not possess

intrinsic immunogenicity, were used to co-encapsulate an antigen (OVA) and an adjuvant

(PAM or CpG).

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228

In the studies described in chapter 9 we showed that, after liposomal encapsulation, both

adjuvants retained the ability to activate TLR-transfected HEK cells, though PAM in

liposomes only induced activation at elevated concentrations compared to a PAM solution.

In vitro DC maturation induced by the adjuvanted liposomes was superior compared to the

free adjuvants. For intradermal immunisation, encapsulation of PAM and CpG in liposomes

did not influence the total IgG titres compared to the antigen/adjuvant solution, but

OVA/CpG liposomes shifted the IgG1/IgG2a balance more to the direction of IgG2a

compared to non-encapsulated CpG. Moreover, only this formulation resulted in a cellular

immune response as measured by IFN-γ production by restimulated splenocytes from

immunised mice.

To obtain insight into the benefit of liposomes for various vaccination routes, we

immunised mice via the intranodal, intradermal, transcutaneous (with microneedle pre-

treatment) and nasal route with OVA/CpG liposomes and a mixture of OVA and CpG

(chapter 10). OVA/CpG liposomes increased the IgG and IgG1 titres compared to OVA after

intradermal and nasal administration. This effect could be attributed to the presence of

CpG, as co-administration of OVA and CpG in solution induced similar (intradermal) or even

higher (nasal) titres than OVA/CpG liposomes. After transcutaneous administration of an

OVA and CpG solution, also elevated IgG titres were observed. Intranodally all formulations

were equally potent. Although the serum IgG and IgG1 titres might suggest no added value

of liposomes, for all routes, co-encapsulation resulted in the production of relatively more

IgG2a than IgG1. Whereas after intradermal and intranodal vaccination with OVA/CpG

liposomes the number of OVA+ and CpG

+ DCs in the LNs increased, lower numbers were

detected after transcutaneous and nasal vaccination compared to administration of a

solution of OVA and CpG.

Conclusion

The approaches described in this thesis have generated new insights into the main

requirements for transcutaneous immunisation. Microneedles definitively have the

potential to be an excellent utensil for the delivery of vaccines into the skin. However, the

skin is a very elastic organ and the actual conduits formed by microneedle pre-treatment

will be considerably smaller than the diameter of the microneedles (chapter 4). These

conduits can remain open for up to 2 hours (chapter 3) under non-occlusive conditions,

but this time can be prolonged to 72 hours under occlusive conditions [4]. The conduit

dimensions and lifetime should be considered when developing formulations to apply on

microneedle pre-treated skin. As reported in chapter 7, extending the application time

from 1 to 2 hours can increase the antibody titres by 30-fold, thereby revealing the

beneficial effect of TMC nanoparticles for transcutaneous immunisation. Application times

can be further prolonged if applied to humans. Mice need to be anaesthetised to prevent


229

them from grooming, which restricts the application time. However, a smaller antigen-

adjuvant entity is preferable, as it will be transported more efficiently through the

microneedle conduits. In this respect the TMC-OVA conjugate proved to be the best

choice: a formulation which retains the co-delivery of antigen and adjuvant, while being as

small as possible. This issue together with other perspectives are further discussed in the

following section.

Perspectives

Ideal formulation for microneedle-based transcutaneous immunisation

The studies described in this thesis clearly define size of the adjuvant-antigen entity as the

most important parameter to consider when designing a formulation to be applied

transcutaneously after microneedle pre-treatment. This implies that the TMC-OVA

conjugate (28 nm) offers better perspectives than the TMC/OVA nanoparticles and the

OVA/CpG liposomes (200-300 nm). However, the preferred vaccine has additional

requirements besides the size of adjuvant-antigen entity. The desired type of immune

response and the adjuvant choice are also essential considerations. The CpG-containing

TMC nanoparticles and liposomes were the only formulations that could reverse the bias

of the induced immune response from Th2 to a more balanced Th1/Th2 response by

inducing the production of IgG2a antibodies. After intradermal vaccination with the

OVA/CpG liposomes a strong production of IFN-γ from restimulated splenocytes could be

measured, indicating induction of a cellular Th1 response (chapter 9). Not all vaccines

require the same immune response, but most of the currently approved adjuvants, such as

alum, induce a Th2, rather than a Th1 response [5]. The induction of a strong cellular and

Th1 response is indispensable for an appropriate response against viruses and intracellular

pathogens. Consequently, the quality of the immune response, which can be steered by

the vaccine formulation, depends on the disease against which the vaccine is developed. A

formulation containing TMC-antigen and TMC-CpG conjugates may induce a more Th1

biased response. Another option is to conjugate the antigen to monophosphoryl lipid A

(MPL), which was shown to promote Th1 responses [6]. Since intradermal immunisation

with LPS-containing TMC nanoparticles induced superior antibody titres compared to non-

adjuvanted TMC nanoparticles (chapter 8), conjugation of antigens to MPL (a derivative of

LPS) as recently described by Tang et al. [7] might result in a potent Th1 skewing

formulation for transcutaneous vaccination. These are just some options, and both the

conjugation of antigens to adjuvants and the effectiveness of the formed conjugates

remains to be studied. Co-localisation of antigen and adjuvant remains an important tool

to enhance the immunogenicity of a subunit vaccine, but should not be established by

Chapter 11

230

using a particulate delivery system. Antigen-adjuvant conjugates, due to their smaller size,

may be more suitable for transcutaneous vaccination.

Besides the entity size and the adjuvant choice, also TMC itself could be further optimised.

TMC can be synthesised from chitosan using the classical synthesis as described by Sieval

et al. [8], but more recently Verheul et al. developed an elegant and controlled method to

synthesise TMC without O-methylation and with a tailorable degree of quarternisation [9].

This type of chitosan can be synthesised with more precise and reproducible

characteristics, which may be an asset of approval for use in humans. A second important

characteristic of TMC is that, irrespective of the synthesis method, it carries a positive

charge. This might limit its transport across the microneedle conduits and was shown to

induce depot formation after intradermal injection (chapter 7). The formulation could

therefore benefit from for instance PEGylation to shield the positive charge. PEGylation of

positively charged nanoparticles was shown to improve antigen expression after DNA

vaccination via tattooing compared to unPEGylated nanoparticles [10]. It would be

interesting to study the effect of PEGylation of the TMC nanoparticles on the transport

through microneedle conduits and on the immune response generated. An important

notion is that the positive charge of TMC is at least partly responsible for the interaction

with DCs. The influence of PEGylation on the DC-stimulating properties of the TMC should

therefore be investigated.

Transcutaneous vaccination: what is the target?

The type of immune response that is induced not only depends on the adjuvants used, but

also relates to the type of DC that is targeted. An important consideration is whether

transcutaneous immunisation should target the epidermis, the dermis, or both. Until now

the main focus has been on the breaching of the stratum corneum barrier. Many effective

devices have been developed to achieve this goal as described in chapter 2. The time has

come to set our focus somewhat deeper in the skin. This, however, implicates that we

have to make a distinction between mice and man. Murine skin is much thinner than

human skin: the average epidermis thickness in mice is 10 µm, compared to 150 µm in

humans [11, 12]. This makes applying a vaccine solely to the epidermis in mice an

unfeasible challenge. Nevertheless, it has become clear that the epidermis and the dermis

contain different types of DCs, with distinct, but not completely understood,

immunological functions [13]. The separation between Langerhans cells in the epidermis

and dermal DCs in the dermis is oversimplified, as there are now at least two types of

dermal DCs described, both in human and murine skin. Next to the ‘classical’ dermal CD14+

DC, the human dermis contains a second type of DC that is CD1a+ and does not express DC-

SIGN [14]. In mice, a new subset of dermal DCs has been found, the langerin+ CD103

+

dermal DC [15]. Whether this subtype is the equivalent of the human CD1a+ dermal DC


231

remains to be investigated. It has been postulated that the CD14+ DCs are more linked to a

humoral immune response, whereas the Langerhans cells preferably induce cellular

responses [16]. However, further research is necessary to elucidate the role of the

different DC subtypes. To study the effect of reaching only one of these subtypes in a

mouse study, knock-out mouse models have to be developed or targeting ligands like

langerin or DC-SIGN should be included in the vaccine. At the same time the differences

and similarities of the skin immune system of mice and man needs to be thoroughly

compared.

Are needles needless for vaccination?

Non-invasive vaccination is often mentioned as the ideal method of applying a vaccine.

Whereas this is possible by mucosal vaccination, applying a formulation onto the skin

without disrupting the barrier will most probably not induce a potent immune response.

Although cholera toxin and heat-labile enterotoxin are exceptions that can induce an

immune response if topically applied [17], even for these very potent antigens barrier

disruption is preferred [18]. For instance, in the patch against travellers’ diarrhoea

currently in phase III, heat-labile toxin is applied with a Skin Prep System to mildly abrade

the skin [19, 20]. This system functions by pulling an abrasive strip over the skin in a force

controlled manner, enabling a 10-fold reduction of the dose compared to intact skin.

Microneedles can exert the same function as this skin abrasion system, i.e. disruption of

the skin barrier. Many different types of microneedles have been developed (solid, coated,

dissolvable and hollow), but it is not yet clear what the perfect type is and this might

depend on the selected antigen and adjuvant. Using solid microneedles for pre-treatment

is a straightforward manner, but as only a small fraction of the applied amount of antigen

will enter the skin, and even less will reach the lymph nodes (chapter 7 & 10), the dose

applied will be relatively high compared to other vaccination strategies. Prolongation of

the application time may make it possible to define the dose more precisely. By using

hollow microneedles the exact dose can be injected into the skin, but trained physicians

are needed to inject the vaccine. In particular, breakage and leakage are risks that should

not be underestimated. Another important parameter is the microneedle material. Hollow

and solid microneedles are often made of silicon or metal (stainless steel). Although silicon

allows the fabrication of microneedles of many different shapes, it is an expensive process

and requires clean room processing [21]. More importantly, it is not an FDA approved

material, making the regulatory path considerably longer compared to microneedles made

of stainless steel, which does have FDA approval. However, both materials are not

biodegradable, leading to concerns about what happens if the needles break off in the

skin. Alternatively, polymeric microneedles that have retained microneedle strength while

being biocompatible and biodegradable are becoming available.

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Coated or dissolvable microneedles are the most elegant devices to apply a vaccine into

the skin, but developments are still in its infancy. By coating a vaccine onto the

microneedles no additional patch or infusion solution is necessary, but only a small amount

can be delivered into the skin and often stabilisers (e.g. trehalose) have to be added to

preserve the immunogenicity of the vaccine. Dissolvable microneedles allow the possibility

of controlled release of the antigen depending on the encapsulation technique used [22].

Recently it was shown that mice could be protected against a lethal influenza challenge by

a single immunisation with polymer microneedles containing 6 µg of inactivated influenza

vaccine [23]. Although this is only the first study to use dissolvable microneedles for

vaccination purposes it shows that this type of microneedles offer great potential for the

future of microneedle-based immunisation.

Besides the different manners of applying microneedles, factors that need to be taken into

account are microneedle length, density and shape. These parameters vary greatly

between the currently available microneedle arrays and studies comparing different arrays

are sparse.

Perceived safety of vaccination – a role for microneedles?

In recent years vaccination has been a hot topic, both in the research community and in

the general population. New vaccines developed against for instance the human

papillomavirus (HPV) and the swine flu have raised questions in the society about the

safety of vaccination in general and of these vaccines in particular. Entering the term “HPV

vaccine” in a search engine results in a disturbing amount of sites advising people against

getting the vaccination. Although this cannot directly be correlated to the perception of

the general population, it is an indication that the current vaccination strategy is not ideal.

Besides people who refuse vaccination because of fear of needles or religious believes, the

number of people doubting the vaccine safety has increased rapidly and the world wide

usage of internet has boosted the influence of these ‘anti-vaccine’ groups [24]. Even

though serious side effects are rare, the occurrence is usually broadly reported and this

aids to the negative view on vaccination. It is therefore of crucial importance that health

care professionals cooperate with the media to ensure people of the benefit of

vaccinations, even if many diseases against which we are vaccinated are almost eradicated

[25]. One example to properly address the fear of people, is the “Six common

misconceptions about immunization” [26] published by the United States Center for

Disease Control and Prevention together with the World Health Organization, discussing

the most common objections to vaccination. This expression of distrust in vaccines shows

that scientists’ view on vaccination probably differs significantly from that of at least part

of the public. Scientists regard vaccines as valuable agents against many life-threatening

diseases, which has boosted research on for instance effective adjuvants and the


233

investigation of therapeutic vaccines against cancer and HIV. However, it remains to be

seen if these new vaccines will be accepted by the public, as concerns on the safety of even

the most commonly used aluminium salt adjuvants is rising [27]. The development of safe

and potent vaccines and delivery methods is essential in this respect.

Microneedles can function as an attractive alternative to the classical immunisation

method as it allows for vaccination in a minimally invasive, pain-free manner. In this light,

the study by Birchall et al. is of particular interest as it evaluates the opinion of the public

and healthcare professionals on microneedles [28]. Both groups had a positive view on

microneedle technology and believed that it would be a pain-free alternative for paediatric

vaccinations, people with needlephobia and in the treatment of chronic diseases. The

problems raised were of a practical kind. For instance, even though in the opinion of

healthcare professionals self-administration is an important benefit, 75% of the people in

the public focus groups rather had them applied by healthcare professionals. This

underlines the fact that people tend to be suspicious of new technologies as most people

preferred the hollow microneedles, which resemble most the current manner of

vaccination. The most important concern raised was the uncertainty of delivering the

appropriate dose. Both the public and professional groups mentioned the necessity of a

feed-back mechanism, such as a colour indicator to ensure proper usage. This is valuable

information for academic groups and industry working on the development of

microneedle-based vaccines and should be taken into account in the development process.

This type of studies show that including the potential consumers in a relatively early stage

may help to achieve public acceptance of the future product and to take away unnecessary

objections against vaccinations in general, and microneedle-based vaccinations in

particular. The development of microneedle-based vaccines may, provided that the

technical issues will be solved, contribute to an improved perception about vaccination by

the public and consequently, better vaccine coverage in the future.

Chapter 11

234

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Chapter 12

Nederlandse samenvatting

Chapter 12

238


239

Toedieningsroute van vaccins

In de afgelopen jaren is het onderzoek naar verandering van de traditionele wijze van

vaccinatie enorm toegenomen. Betere veiligheid, meer gemak voor de patiënten en betere

effectiviteit zijn de belangrijkste redenen om nieuwe manieren te ontwikkelen om een

vaccin toe te dienen. Een van deze alternatieven is vaccineren via de huid (transcutane

vaccinatie). Omdat de huid in direct contact staat met de omgeving heeft het als taak om

ons te beschermen tegen ziekteverwekkers. Om deze reden bevat de huid meer

immuuncellen dan bijvoorbeeld een spier, waar meestal een vaccin in geïnjecteerd wordt.

In de huid zitten veel dendritische cellen (DCs). Deze DCs spelen een belangrijke rol bij het

tot stand komen van een immuunrespons en hierdoor kan vaccinatie via de huid zorgen

voor een effectieve immuunrespons.

Uit de eerste transcutane vaccinatie studies eind jaren 90 bleek dat het mogelijk is om een

immuunrespons te induceren door toediening van cholera toxine of enterotoxine op

intacte muizen- of mensenhuid. Dit onderzoek heeft ertoe geleid dat vaccins in

pleistervorm tegen reizigersdiarree en griep nu getest worden in klinische studies

(respectievelijk fase III en II). Om een vaccin in voldoende mate de huid in te krijgen, moet

het door de hoornlaag (stratum corneum, de natuurlijke barrière van de huid) heen. Een

manier om dat te doen is door middel van micronaalden. Deze naalden zijn lang genoeg

om door het stratum corneum heen te prikken, maar kort genoeg om pijnsensatie te

vermijden. De micronaalden prikken tot in de opperhuid (epidermis), maar bereiken niet

de lederhuid (dermis) waar de zenuwuiteinden zitten. Er zijn verschillende soorten

micronaalden: holle en massieve micronaalden en meer recentelijk zijn ook oplosbare

micronaalden ontwikkeld.

Vaccinformulering

Het onderzoek naar efficiëntere manieren van vaccinatie richt zich niet alleen op de

toedieningsroute, maar ook op de samenstelling van vaccins. Veiligheidsproblemen met de

traditionele vaccins gemaakt uit verzwakte en geïnactiveerde pathogenen hebben geleid

tot de ontwikkeling van subunit vaccins, die één of meer gezuiverde antigenen bevatten

(het actieve gedeelte van het vaccin). Het belangrijkste nadeel van deze subunit vaccins is

dat ze minder immunogeen zijn, en daarom is het noodzakelijk om een hulpstof (adjuvans)

toe te voegen om een efficiënte immuunrespons te induceren. Gedurende een lange tijd

waren colloïdale aluminiumzouten (alum) de enige goedgekeurde hulpstoffen, maar

recentelijk mogen in Europa squaleen emulsies (MF59) en monophosphoryl lipid A ook

gebruikt worden. Momenteel wordt er veel onderzoek verricht naar het ontwikkelen van

nieuwe hulpstoffen. Een andere veelbelovende benadering om de immunogeniciteit van

subunit vaccins te verhogen is om ze te formuleren in (nano)deeltjes. Deze deeltjes

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worden beter opgenomen door DCs, vanwege hun vergelijkbare grootte met pathogenen.

Verder kunnen ze het antigeen beschermen tegen enzymatische afbraak, zorgen ze voor

vertraagde afgifte van het antigeen en kan een antigeen samen met een adjuvans in een

deeltje verpakt worden. Kennis over de ideale vaccinformulering voor transcutane

vaccinatie is echter schaars.

Focus van dit proefschrift

Het belangrijkste doel van de studies beschreven in dit proefschrift is het ontwikkelen van

een efficiënte manier van transcutane vaccinatie. Hiervoor worden subunit vaccins

geformuleerd in nanodeeltjes en deze deeltjes worden met behulp van massieve

micronaalden toegediend. Het onderzoek richt zich zowel op de werkzaamheid en de

veiligheid van het vaccin en begint met het evalueren van de veiligheid en effectiviteit van

het gebruik van massieve micronaalden, gevolgd door de ontwikkeling van nanodeeltjes

die zowel het antigeen als een adjuvans bevatten. Deze nanodeeltjes worden getest op

celsystemen en in muizen om de werking ervan te onderzoeken en ze te vergelijken met

mengsels van antigeen en adjuvans in buffer en met conjugaten van antigeen en adjuvans.

Samenvatting van dit proefschrift

Dit proefschrift begint met een algemene inleiding op het gebied van transcutane

vaccinatie (hoofdstuk 2), en geeft een overzicht van de vele mogelijkheden om een vaccin

transcutaan toe te dienen. Adjuvans en formuleringen (nanodeeltjes) die op dit moment

gebruikt worden in preklinische en klinische studies worden ook besproken.

Het onderzoek dat beschreven staat in dit proefschrift bestaat uit drie delen. In het eerste

deel wordt het gebruik van verschillende micronaalden met elkaar vergeleken. Het tweede

deel beschrijft het ontwikkelen en testen van verschillende formuleringen op basis van N-

trimethyl chitosan (TMC). In het derde deel wordt de ontwikkeling van liposoom

formuleringen beschreven die zowel een antigeen als een adjuvans bevatten.

Deel I: Het testen van de veiligheid en werkzaamheid van voorbehandeling met

micronaalden op proefpersonen

Hoofdstuk 3 richt zich op het onderzoeken van de veiligheid van de

micronaaldbehandeling op proefpersonen. De huidirritatie (roodheid van de huid en de

doorbloeding), pijnsensatie en het effect op het waterverlies door de huid werden

onderzocht. Massieve micronaalden die verschilden in vorm en lengte (200 tot 550

micrometer) werden vergeleken. Voor alle micronaalden was de ontstane irritatie

minimaal en verdween binnen 2 uur. Het gebruik van langere (400 micrometer)


241

micronaalden resulteerde in een verhoogd waterverlies en een toename in roodheid en in

doorbloeding in vergelijking tot 200 micrometer lange micronaalden. Naaldvorm had ook

een effect. De micronaalden met de scherpste punt zorgen voor meer waterverlies en

minder huidirritatie.

In een volgende klinische studie (hoofdstuk 4) werd de invloed van de vorm van de

micronaalden op de diffusie van fluoresceïne door de gevormde openingen in de huid

gevisualiseerd met confocale fluorescentiemicroscopie. Bij het analyseren van de beelden

werd onderscheid gemaakt tussen regio's met hoge en lage fluorescentie intensiteit. In de

meeste gevallen was een hoge fluorescentie intensiteit alleen aanwezig in het stratum

corneum, terwijl lage fluorescentie intensiteit ook aanwezig was in de epidermis. 15

minuten na het weghalen van de formulering werd bijna geen hoge intensiteit meer

waargenomen aan het huidoppervlak, terwijl fluorescentie met een lage intensiteit nog

steeds kon worden gedetecteerd tot een diepte van 60 micrometer. Met alle types

micronaalden waren we in staat humane huid in vivo te perforeren, maar de vorm van de

micronaalden bepaalde de hoeveelheid fluorescentie in de huid en tot welke diepte de

fluorescentie gemeten kon worden. Micronaalden met een scherpe punt zorgden voor een

hogere fluorescentie intensiteit in de huid dan micronaalden met een stompere punt.

Deel II: TMC formuleringen voor intradermale en transcutane vaccinatie

Om immunogene antigeen formuleringen te maken werden nanodeeltjes ontwikkeld

gemaakt van TMC. Een model antigeen, ovalbumine (OVA), en een relevant antigeen,

difterie toxoid (DT), werden in deze nanodeeltjes ingebouwd. Om het effect van

nanoparticles te onderzoeken werden ook mengsels van TMC en de antigenen gemaakt.

De studies in hoofdstuk 5 beschrijven de ontwikkeling van positief geladen nanodeeltjes

met een diameter van ongeveer 200 nm. Uit studies met een in vitro model van humane

DCs bleek dat TMC nanodeeltjes de opname van OVA bevorderen en dat zowel de

nanodeeltjes als een mengsel van TMC en OVA immuunstimulerende eigenschappen

hebben. Uit T cel studies bleek dat vooral de Th2 respons werd bevorderd (Th2 cellen

stimuleren B cellen tot het produceren van antistoffen). De afweerrespons in muizen werd

bepaald na het injecteren van de formuleringen in de huid (intradermale injectie). In zowel

de studies met OVA als die met DT verhoogden de TMC nanodeeltjes en de mengsels de

antistoftiters in vergelijking met antigeen zonder adjuvans. Het antistoffenprofiel was Th2

gekenmerkt (IgG1). Uit de studies met DT en TMC bleek dat deze formuleringen voor even

hoge totale en toxine-neutraliserende antistoftiters zorgden als een onderhuidse

(subcutane) injectie met DT-alum.

De formuleringen werden ook gebruikt voor transcutane immunisatie met behulp van 300

μm lange micronaalden, zoals beschreven staat in hoofdstuk 6. Twee verschillende types

micronaalden (zoals beschreven in hoofdstuk 3) werden gebruikt en de formuleringen

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werden op de huid aangebracht voor of na behandeling met de micronaalden. Transcutane

immunisatie met een mengsel van TMC en DT leidde tot 8 maal hogere antistoftiters ten

opzichte van DT-bevattende TMC nanodeeltjes, onafhankelijk van het gebruikte

micronaaldtype en manier van opbrengen. Uit ex vivo confocale microscopie studies bleek

dat TMC nanodeeltjes minder efficiënt de huid in getransporteerd werden dan een TMC

oplossing.

Om de opname van het vaccin in de huid te verhogen, werd de applicatieduur van de

vaccinformuleringen verlengd (hoofdstuk 7). Een verlenging van de applicatieduur van 1

tot 2 uur resulteerde, in het geval van een OVA oplossing, tot een 30-voudige toename van

antistoffen. Naast applicatieduur, bleek ook de grootte van de deeltjes in de

vaccinformulering en co-localisatie van het antigeen en het adjuvans van groot belang te

zijn. Een conjugaat van OVA-TMC (28 nm) resulteerde in hogere antistoftiters dan de TMC

nanodeeltjes. Na transcutane vaccinatie met deze formulering had een groter aantal DCs in

de lymfeknopen OVA opgenomen dan na vaccinatie met OVA bevattende nanodeeltjes. Na

een tweede vaccinatie verhoogden zowel het conjugaat als de nanodeeltjes de

antistoftiters in vergelijking met een OVA oplossing.

Om het aspect van transport door de huid en naar de lymfeknopen te onderzoeken

werden dezelfde formuleringen ook toegediend via injectie in de huid of direct in de

lymfeknoop (intranodaal). Intradermaal was de aanwezigheid van TMC in de formulering

(ongeacht in welke vorm) van essentieel belang voor het verkrijgen van verhoogde

antistoftiters. TMC formuleringen vormden een depot in de huid, wat leidde tot langdurige

OVA afgifte naar de lymfeknopen. Deze langdurige OVA afgifte werd ook waargenomen na

injectie in de lymfeknoop, maar dit leidde niet tot verhoogde antistoftiters. Deze

bevindingen benadrukken dat iedere toedieningsroute andere eisen aan de ideale

vaccinformulering stelt.

In hoofdstuk 8 wordt een tweede generatie van TMC nanodeeltjes beschreven waar een

adjuvans aan toegevoegd is. Als adjuvantia werden de Toll-like receptor liganden

lipopolysaccharide (LPS), PAM3CSK4 (PAM) en CpG DNA; de NOD-like receptor 2 ligand

muramyl dipeptide (MDP) en de GM1 Ganglioside receptor ligand cholera toxine B (CTB)

gebruikt. Het effect van deze adjuvantia op de afweerreactie werd onderzocht na

intradermale en nasale vaccinatie. Toevoeging van LPS of MDP aan de OVA-bevattende

TMC nanodeeltjes leidt na nasale vaccinatie tot verhoogde antistoftiters, terwijl CTB, PAM

en CpG geen effect hadden. De afweerreactie werd gekenmerkt door Th2 karakteristieke

antistoffen. Intradermaal had de toevoeging van LPS en CpG een positief effect op de

antistoftiters. Bovendien zorgde het toevoegen van CpG voor de productie van Th1

karakteristieke antistoffen (Th1 cellen zetten macrofagen en cytotoxische T cellen aan tot

het doden van geïnfecteerde cellen). Deze resultaten bevestigden de conclusies in het


243

vorige hoofdstuk, namelijk dat de gekozen toedieningsroute medebepalend is voor de

keuze van de optimale formulering van het vaccin.

Deel III: Positief geladen liposomen voor de gezamelijke toediening van antigeen en

adjuvans

In dit deel werden positief geladen liposomen (deeltjes met een waterige kern omsloten

door een hydrofobe laag die voornamelijk uit fosfolipiden bestaat) gebruikt. In

tegenstelling tot TMC nanodeeltjes zijn liposomen van zichzelf niet immuunstimulerend,

maar ze kunnen gebruikt worden om zowel een antigeen (OVA) als een adjuvans (PAM of

CpG) in in te sluiten.

Uit de studies beschreven in hoofdstuk 9 blijkt dat na insluiting in liposomen beide

adjuvantia nog steeds in staat waren om TLR-getransfecteerde HEK-cellen te activeren. De

hiervoor benodigde concentratie PAM in OVA/PAM liposomen was hoger dan in een

OVA/PAM mengsel. De in vitro DC activatie door de OVA/PAM liposomen en OVA/CpG

liposomen was superieur aan die geïnduceerd door de mengsels. Uit intradermale

immunisatiestudies bleek dat het insluiten van PAM en CpG in liposomen geen invloed had

op de totale antistoftiter. Echter toediening van OVA/CpG liposomen induceerde een Th1

gekenmerkte respons, terwijl een mengsel van OVA en CpG een Th2 respons veroorzaakte.

Bovendien resulteerde het gebruik van OVA/CpG liposomen in een cellulaire

immuunrespons, zoals bleek uit de IFN-γ productie door splenocyten van geïmmuniseerde

muizen.

Om inzicht te verkrijgen in het gebruik van liposomen voor diverse vaccinatie routes,

hebben we muizen intranodaal, intradermaal, transcutaan en nasaal gevaccineerd.

Hiervoor werden OVA/CpG liposomen gebruikt. De liposoom formuleringen werden

vergeleken met een mengsel van OVA en CpG en een OVA oplossing (hoofdstuk 10). Na

intradermale en nasale toediening verhoogden de OVA/CpG liposomen de antistoftiters in

vergelijking met een OVA oplossing. Dit effect werd veroorzaakt door CpG, aangezien

toediening van een mengsel van OVA en CpG leidde tot vergelijkbare titers als

(intradermaal) of zelfs hogere (nasaal) titers dan OVA/CpG liposomen. Na transcutane

toediening van een oplossing van OVA en CpG werden ook verhoogde antistoftiters

gemeten. Na intranodale injectie gaven alle formuleringen even hoge antistoftiters.

Hoewel de totale antistoftiters niet wijzen op een toegevoegde waarde van het insluiten

van het antigeen en adjuvans in liposomen, resulteerde het gebruik van lipsomen voor alle

toedieningsroutes in verhoogde productie van Th1 karakteristieke antistoffen. Ook werd

de opname van OVA en CpG door de DCs in de lymfeknopen onderzocht. Hoewel na

intradermale en intranodale injectie van OVA/CpG liposomen het aantal DCs dat OVA en

CpG had opgenomen verhoogd was ten opzichte van een mengsel van OVA en CpG,

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resulteerde de liposoom formulering in lagere aantallen OVA/CpG positieve DCs na

transcutane en nasale vaccinatie. De studies in hoofdstuk 9 en 10 tonen aan dat het

mogelijk is om door het gebruik van liposomen de kwaliteit van de immuunrespons te

verbeteren, maar dat de grotere afmetingen van deze liposomen ze minder geschikt

maken voor gebruik in de neus of op de huid.

Conclusies

De studies beschreven in dit proefschrift hebben geleid tot nieuwe inzichten in de

belangrijkste eisen voor efficiënte transcutane vaccinatie. Het gebruik van micronaalden

heeft voordelen voor transcutane vaccinatie. Echter, de huid is zeer elastisch waardoor de

openingen die gemaakt worden aanzienlijk kleiner zullen zijn dan de diameter van de

micronaalden. Bij het ontwikkelen van formuleringen voor transcutane vaccinatie met

micronaald-voorbehandeling moet rekening gehouden worden met de afmetingen van

deze openingen en de periode dat ze open blijven. Verlenging van de applicatieduur kan de

antistoftiters drastisch verhogen, waardoor de toegevoegde waarde van (bijvoorbeeld)

TMC nanodeeltjes voor transcutane vaccinatie zichtbaar wordt. Voor toepassing op

mensen kan de applicatieduur nog verder worden verlengd. Het is gebleken dat

formuleringen die zorgen voor gelijktijdige toediening van het antigeen en adjuvant het

meest effectief zijn. Ook de grootte van het vaccin speelt een belangrijke rol. Een relatief

klein conjugaat van antigeen en adjuvant is efficiënter dan de grotere TMC nanodeeltjes.

Op deze manier blijft de gelijktijdige toediening van het antigeen en adjuvant behouden,

maar als een zo klein mogelijke eenheid, zodat hij efficiënt kan worden vervoerd via de

openingen gemaakt door de micronaalden.

Chapter 13

Curriculum Vitae

Chapter 13

248

Curriculum Vitae

249

Suzanne Bal was born on August 9th 1983 in Den Haag. After graduating from the Dalton

Scholengemeenschap she started her study Bio-Pharmaceutical Sciences at the University

of Leiden. During her study she did two internships with the titles “Needle-array enhanced

iontophoretic delivery of cascade blue labeled dextrans” and “Dendritic cell activation and

cytokine secretion after application of Ag85B plasmid DNA adsorbed to PLGA-PEI

nanoparticles” at the department of Drug Delivery Technology at the Leiden/Amsterdam

Center for Drug Research (LACDR). In August 2006 she obtained her Master’s degree and in

September of the same year she started her PhD project at the same department under

the supervision of Prof. Dr. Joke Bouwstra and Prof. Dr. Wim Jiskoot. The research was

performed under the framework of TI Pharma project number D5-106 “vaccine delivery:

alternatives for conventional multiple injection vaccines” and resulted in this thesis. In

November 2010 she started as a postdoc at the division of Experimental Immunology and

Pulmonology at the Academical Medical Center of the University of Amsterdam.

Chapter 14

List of publications

Chapter 14

252

List of publications

253

Verheul RJ, Slütter B, Bal SM, Bouwstra JA, Jiskoot W, Hennink WE: Covalently stabilized

trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination

(submitted)

Bal SM*, Slütter B*, Ding Z, Jiskoot W, Bouwstra JA: Adjuvant effect of cationic liposomes

and CpG depends on administration route (submitted)

Bal SM*, Slütter B*, Verheul RJ, Bouwstra JA, Jiskoot W: Adjuvanted, antigen loaded N-

trimethyl chitosan nanoparticles for nasal and intradermal vaccination: adjuvant- and site-

dependent immunogenicity in mice (submitted)

Bal SM, Slütter B, Jiskoot W, Bouwstra JA: Small is beautiful: N-trimethyl chitosan-

ovalbumin conjugates for microneedle-based transcutaneous immunisation (submitted)

Bal SM, Hortensius S, Ding Z, Jiskoot W, Bouwstra JA: Co-encapsulation of antigen and Toll-

like receptor in cationic liposomes affects the quality of the immune response in mice after

intradermal vaccination (Vaccine 2010, In press)

Slütter B, Bal SM, Que I, Kaijzel E, Löwik C, Bouwstra JA, Jiskoot W: Antigen-adjuvant

nanoconjugates for nasal vaccination: an improvement over the use of nanoparticles? (Mol

Pharm 7(6): 2207-2215 2010)

Bal SM*, Ding Z*, van Riet E, Jiskoot W, Bouwstra JA: Advances in transcutaneous vaccine

delivery: Do all ways lead to Rome? (J Control Release 148(3): 266-282 2010)

Slütter B, Bal SM*, Keijzer C*, Mallants R, Hagenaars N, Que I, Kaijzel E, van Eden W,

Augustijns P, Löwik C, Bouwstra, J, Broere F, Jiskoot W: Nasal vaccination with N-trimethyl

chitosan and PLGA based nanoparticles: Nanoparticle characteristics determine quality and

strength of the antibody response in mice against the encapsulated antigen (Vaccine

28(38): 6282-6291 2010)

Bal SM, Kruithof AC, Zwier R, Dietz E, Bouwstra JA, Lademann J, Meinke MC: Influence of

microneedle shape on the transport of a fluorescent dye into human skin in vivo (J Control

Release 147(2): 218-224 2010)

Bal SM, Ding Z, Kersten GF, Jiskoot W, Bouwstra JA: Microneedle-based transcutaneous

immunisation in mice with N-trimethyl chitosan adjuvanted diphtheria toxoid formulations

(Pharm Res 17(9): 1837-1847 2010)

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Ding Z, Bal SM, Romeijn S, Kersten GF, Jiskoot W, Bouwstra JA: Transcutaneous

immunization studies in mice using diphtheria-toxoid loaded vesicle formulations and a

microneedle array (Pharm Res 28 (1): 145-58 2010)

Bal SM, Kruithof AC, Liebl H, Tomerius M, Bouwstra JA, Lademann J, Meinke M: In vivo

visualization of microneedle conduits in human skin using laser scanning microscopy (Laser

Phys Lett 7(3): 242-246 2010)

Bal SM, Slütter B, van Riet E, Kruithof AC, Ding Z, Kersten GF, Jiskoot W, Bouwstra JA:

Efficient induction of immune responses through intradermal vaccination with N-trimethyl

chitosan containing antigen formulations (J Control Release 142(3): 374-383 2010)

Bivas-Benita M, Lin MY, Bal SM, van Meijgaarden KE, Franken KLMC, Friggen AH, Junginger

HE, Borchard G, Kleijn MR, Ottenhof THM: Pulmonary delivery of DNA encoding

Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA-PEI nanoparticles

enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice

(Vaccine 27(3): 4010-4017 2009)

Bal SM, Caussin J, Pavel S, Bouwstra JA: In vivo assessment of microneedle arrays in human

skin (Eur J Pharm Sci 35(3): 193-202 2009)

Verbaan FJ, Bal SM, van den Berg DJ, Dijksman JA, van Hecke M, Verpoorten H, van den

Berg A, Luttge R, Bouwstra JA: Improved piercing of microneedle arrays in dermatomed

human skin by an impact insertion method (J Control Release 128(1): 80-88 2008)

Verbaan FJ, Bal SM, van den Berg DJ, Groenink HWW, Verpoorten H, Lüttge R, Bouwstra

JA: Assembled microneedle arrays enhance the transport of compounds varying over a

large range of molecular weight across human dermatomed skin (J Control Release 117(2):

238–245 2007)


Chapter 15

Nawoord

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Nawoord

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Nawoord

Het is niet te geloven, maar er is een einde gekomen aan mijn tijd in Leiden. Het was

zonder te overdrijven de mooiste periode uit m’n leven tot nu toe. En daar hebben veel

mensen aan bijgedragen. In totaal heb ik bijna 6 jaar bij DDT rondgelopen. Stages bij Ferry

en Maytal bevielen zo goed dat het voor mij de gewoonste zaak van de wereld was om

door te gaan als aio. Iedereen op het lab heeft op de een of andere manier wel bijgedragen

aan mijn project. De een meer op het wetenschappelijke vlak en de ander meer bij de

sociale gebeurtenissen. Maar als ‘onze’ vakgroep iets bewijst, is dat je beide nodig hebt om

goed onderzoek te kunnen doen. Natuurlijk wil je nooit iemand vergeten te noemen, en ik

hoop dan ook maar dat ik dat niet ga doen, maar een aantal mensen wil ik speciaal

noemen.

Iedereen van kamer 718, toch wel bij uitstek de gezelligste kamer, al was het alleen maar

omdat hier ook de borrels gehouden werden.

Als eerste wil ik Bram bedanken, mijn ‘aio-broer’. We zaten samen op het TMC project en

weet niet waar ik zonder jou geweest was: samen schelden op de FACS, 60 ELISA-platen op

een dag doen en de verplichte dingetjes zoals proberen een Nieuw-Zeelandse professor bij

te houden in de kroeg. Zonder jou had ik het een stuk zwaarder gehad en had ik veel

minder gelachen. Het is best raar dat je nu in Iowa zit en dus niet bij mijn promotie kan

zijn.

Ding, you were the senior PhD student in the room and ELISA king until we stole your title.

You were a very valuable partner in all the animal experiments and I really liked all the

intense immunology discussions. All the best in Tübingen with your wife.

En dan is er natuurlijk nog Daniel, die dit waarschijnlijk toch niet leest, omdat je bij je

‘echte’ vrienden bent ;) Maar toch, je was onmisbaar. Niet alleen voor de sfeer in de kamer

en tijdens borrels, maar ook omdat je altijd met net een andere blik naar alles kijkt. Ik ben

heel blij dat je mijn paranimf wil zijn.

Verder mag ik natuurlijk Olly niet vergeten. Ongeveer tegelijk bij DDT begonnen en helaas

was jij de eerste die onze kamer verliet. Natuurlijk kwamen er ook weer nieuwe mensen

bij, Ana and Koen, I have no doubt that you will keep the spirit of the room alive.

Daarnaast waren er nog Elly en Myrra aan wie ik heel veel heb gehad. Elly, zonder jou

hadden Bram en ik al het celwerk nooit kunnen doen. Je kwam in een leeg cellab en hebt

ervoor gezorgd dat alles opgezet werd. Helaas (voor ons) vertrok je naar Japan, wat een

groot gemis voor de vakgroep was. Nadat Elly vertrok, heeft Myrra veel van de cellab taken

overgenomen. Ook jij was altijd bereid om de helpende hand toe te steken en me bij te

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staan met je formuleringskennis. The vaccine team was further strengthened by

Christophe and also Benn joined us for a short time.

Varsha, jij was er altijd. Samen konden we heerlijk gebruik maken van de rust in het cellab

om alles te bespreken wat ons dwars zat. De rest begreep af en toe niets van ons, maar

uiteindelijk hebben we het nu maar goed voor elkaar, toch ;)

Mijn studenten mag ik ook niet vergeten. Iwan, Annelieke en Sander hebben me veel werk

uit handen genomen en bijgedragen aan drie hoofdstukken van dit proefschrift.

Aan het begin van mijn aio-periode was Julia echt de koningin van het lab (en niet alleen

qua lab-cleaning). Samen hebben we een in vivo studie gedaan en ons vermaakt in de VS.

En we denken nog weemoedig terug aan toen het lab zo opgeruimd was.

I really enjoyed being part of such an international group. Especially during my third year

there were many master students, PhD students and postdocs visiting the lab from all over

the world. Line, Abina, Francisco, Hugo, Nuch, Tomo, Christian, Dana, Diogenes, Maria

Chiara, Romano, Lies, Veerle, Zuhal, Julia and Melanie. Thank you all for the very nice

atmosphere in the lab and the many activities that were initiated. Of course I will not

forget our DDT football team. Even though I was the only girl in the team and not as

talented as some of you, you always made me feel a real part of the team.

Two visitors were extra special for me. Sun and Tue, we did so many fun outings of which

Texel was our first and my favourite. We had to endure every type of weather you can

imagine on one day and ended with a beautiful cycling trip on the beach. Perfect!

The core of DDT should also be mentioned: Vasco, Andrea, Robert R and Robert P, Bashak,

Miranda, Jeroen, Michelle, Aat, Lolu, Stefan and Gert. Many of you are also leaving DDT or

have already left. At least we all have good memories of our time as a group.

Connie verdient ook een eigen regel, want zonder haar zou alles een chaos zijn op het lab.

Als je een week op vakantie was, begon iedereen al te klagen dat dingen ineens niet meer

zo soepel gingen.

Dan zijn er nog wat mensen van buiten Leiden die genoemd mogen worden: Rolf, onze

TMC man uit Utrecht. Na vier jaar is het je nog niet gelukt om me ervan te overtuigen dat

jouw TMC beter is dan onze Leidse meuk, maar we hebben uiteindelijk toch een studie met

z’n drieën gedaan. Thomas van het VUmc, helaas was onze samenwerking maar van korte

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261

duur. De plannen waren mooi, maar er was te weinig tijd voor de uitvoering. Gideon,

bedankt voor het kritisch lezen van de DT manuscripten. Und vielen dank für Martina

Meinke und Jürgen Lademann. We managed to write two really nice papers together and I

really enjoyed the trips to Berlin.

Maar het leven is niet alleen werken en ik wil dan ook zeker mijn ouders bedanken voor

hun steun en interesse in alles wat ik aan het doen was en ben. Hoewel mijn leven erg is

veranderd, voelt het nog steeds erg fijn om thuis te komen. En voor mijn ‘kleine’ zusje,

succes met de grote stap die je gaat zetten. Een eigen huis, daarin loop je toch echt op me

voor!

En dan is er nog… Gino, mijn afleiding tijdens de lange dagen dat ik thuis aan het schrijven

was. Er zitten vast al snel haren van jou in mijn boekje. En tenslotte Stijn, die ik alleen

mocht noemen als ik vond dat hij echt bijgedragen had. Wat natuurlijk zo is, want zonder

jouw relativisme was ik de laatste periode nooit doorgekomen. Je hebt ervoor gezorgd dat

ik het avontuur in het verre Brabant aandurfde en ik hoop dat we samen nog veel

avonturen mogen beleven.

Suzanne

Stellingen

Behorende bij het proefschrift

Mechanistic studies on transcutaneous subunit vaccine delivery

Microneedles, nanoparticles and adjuvants

1. Microneedle pre-treatment enables painless delivery of compounds into the skin.

This thesis

2. Although nanoparticles are suitable to ensure concomitant delivery of antigen and adjuvant to

dendritic cells, smaller antigen-adjuvant conjugates are more suitable for transcutaneous

vaccination.

This thesis

3. Because of the intrinsic adjuvanticity of N-trimethyl chitosan (TMC), TMC-based nanoparticles

can induce a more potent immune response than liposomes.

This thesis

4. In pre-clinical studies on transport into the skin the limitations of the animal model form a

difficult barrier to overcome.

This thesis

5. “A different perception or interest in the administration routes seems to exist between

pharmaceutically and immunologically oriented scientists working in the field of vaccines.”

P. Johansen et al. J Control Release 2010 148 (1)56-62

6. “Although studies of delivery systems have often been separated from those of adjuvants, it is

now clear that the basis for adjuvant performance may lie with its particulate nature.”

A.C. Rice-Ficht et al. Curr Opin Microbiol 2010 13 (1) 106-12

7. “The prospect of elimination of several hundred million cases of dehydrating diarrhea in infants

and hundreds of thousands of deaths with a vaccine that is needle-free, stable at room

temperature, and easy to administer has led to a vigorous program to develop a patch and

pretreatment regimen suitable for such an application.”

G.M. Glenn et al. Infect Immun 2007 75(5) 2163-70

8. “During the first half of the 19th

century the cowpox vaccine against smallpox infection was

maintained by arm-to-arm transfer of the virus.”

P.D. Ellner Infection 1998 26(5) 263-9

9. “If you’re a pre-doc before getting a PhD and a post-doc afterwards, that means you’re only a

“doc” for an infinitesimal amount of time.”

Jorge Cham, PhD comics 8/20/2010

10. Vertraagde aflevering speelt zowel een rol bij het transport van vaccins als bij dat van mensen.

11. Voor buitenstaanders moet het toch een opluchting zijn als je na 4 jaar promoveren eindelijk

“afstudeert”.

12. Een bezoek aan een ontwikkelingsland doet je beseffen dat we in Nederland in plaats van te

klagen, blij moeten zijn dat we het zo verschrikkelijk goed hebben.

mechanistic studies on transcutaneous subunit vaccine deliverythe investigations described in this...

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