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Polysaccharide-based nucleic acid nanoformulations

Koen Raemdoncka, Thomas F. Martensa,b, Kevin Braeckmansa,b, Jo Demeestera, Stefaan C. De

Smedta,*

a Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences,

Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium

b Center for Nano-and Biophotonics, Faculty of Pharmaceutical Sciences, Ghent University,

Harelbekestraat 72, B-9000 Ghent, Belgium

*Corresponding author during submission: Dr. Koen Raemdonck

koen.raemdonck@ugent.be

Tel: +32 9 2648095

Fax: +32 9 2648189

Faculty of Pharmaceutical Sciences, Ghent University

Harelbekestraat 72, B-9000 Ghent, Belgium

Corresponding author after submission: Prof. Dr. Stefaan C. De Smedt

stefaan.desmedt@ugent.be

Tel: +32 9 2648076

Fax: +32 9 2648189

Faculty of Pharmaceutical Sciences, Ghent University

Harelbekestraat 72, B-9000 Ghent, Belgium

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Abstract

Therapeutic application of nucleic acids requires their encapsulation in nanosized carriers that enable

safe and efficient intracellular delivery. Before the desired site of action is reached, drug-loaded

nanoparticles (nanomedicines) encounter numerous extra-and intracellular barriers. Judicious

nanocarrier design is highly needed to stimulate nucleic acid delivery across these barriers and

maximize the therapeutic benefit. Natural polysaccharides are widely used for biomedical and

pharmaceutical applications due to their inherent biocompatibility. At present, there is a growing

interest in applying these biopolymers for the development of nanomedicines. This review highlights

various polysaccharides and their derivatives, currently employed in the design of nucleic acid

nanocarriers. In particular, recent progress made in polysaccharide-assisted nucleic acid delivery is

summarized and the specific benefits that polysaccharides might offer to improve the delivery

process are critically discussed.

Keywords

siRNA

plasmid DNA

Dextran

Chitosan

Hyaluronic acid

Cyclodextrin

Nanomedicine

Gene therapy

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Table of Contents

1. Introduction

1.1. Nucleic acid nanotherapeutics

1.2. Polysaccharides in nucleic acid delivery

2. Dextran-based nucleic acid nanotherapeutics

2.1. Introduction to dextran

2.2. Dextran-based matrices

2.3. Dextran as an additive in nucleic acid nanotherapeutics

2.3.1. Polyelectrolyte complexes with dextran sulphate

2.3.2. Polyelectrolyte complexes with modified dextran

2.3.3. Dextran coated nucleic acid nanoparticles

3. Chitosan-based nucleic acid nanotherapeutics

3.1. Introduction to chitosan

3.2. Chitosan-based matrices

3.3. Chitosan as a polycation in nucleic acid nanotherapeutics

3.3.1. Polyelectrolyte complexes with native chitosan

3.3.2. Polyelectrolyte complexes with modified chitosan

3.3.3. Hybrid chitosan-based polyelectrolyte complexes

3.4. Nanoparticles coated with chitosan

4. Hyaluronic acid-based nucleic acid nanotherapeutics

4.1. Introduction to hyaluronic acid

4.2. Hyaluronic acid-based matrices

4.3. Hyaluronic acid as an additive in nucleic acid nanotherapeutics

4.3.1. Hyaluronic acid polyelectrolyte complexes

4.3.2. Hyaluronic acid-based conjugates

4.3.3. Hyaluronic acid core-shell particles

4.4. Influence of hyaluronic acid molecular weight

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5. Cyclodextrin-based nucleic acid nanotherapeutics

5.1. Introduction to cyclodextrins

5.2. Cyclodextrin polymer nanoparticles

5.2.1. Polymers with cyclodextrin backbone

5.2.2. Cyclodextrin modifications of pre-existing polymers

6. Other polysaccharides in nucleic acid nanotherapeutics

6.1. -Glucans

6.2. Alginate

6.3. Arabinogalactan, pullulan and pectin

7. Conclusions and future perspectives

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1. Introduction

1.1.Nucleic acid nanotherapeutics

Macromolecular single and double-stranded nucleic acids, such as antisense oligonucleotides (AsON),

small interfering RNA (siRNA), and plasmid DNA (pDNA) show great therapeutic promise in the

treatment of a wide variety of pathologies including cancer and inflammatory, neurological,

cardiovascular or metabolic disorders [1,2]. This is clearly reflected by the many clinical trials

launched in the past two decades evaluating the safety and efficacy of nucleic acid therapeutics for

these different pathologies [1,3]. With gene therapy, the underlying genetic cause of a pathology

could be treated instead of standard symptom management. Where a dominant disease-causing

gene is overexpressed, sequence-specific gene silencing therapy via AsON or siRNA can block the

expression of the encoded protein on the post-transcriptional level. On the other hand, if a genetic

mutation causes deficient production of a vital protein, gene supplementation via pDNA could

potentially restore functional protein expression [4].

To attain a therapeutic phenotype, the aforementioned nucleic acid drugs require delivery into the

target cell, albeit at a distinct intracellular location. AsON and siRNA function at the post-

transcriptional level through sequence-specific binding with their target messenger RNA (mRNA) in

the cytosol, opposite to pDNA that has to reach the cell nucleus to access the transcriptional

machinery [2]. Several physical delivery methods, such as electroporation, hydrodynamic injection

and ultrasound-assisted delivery, have shown to be successful in transferring naked nucleic acids to

target cells. However, these methods are generally quite impractical or limited to local delivery in

easily accessible tissues such as skin and muscle [4,5]. Another well-known strategy to assist nucleic

acid therapeutics in reaching their intracellular site of action, is formulating them in nanosized

carriers. Although these can be exploited for systemic delivery, drug loaded nanoparticles (NPs) still

encounter numerous obstacles en route to their intracellular target upon in vivo administration

(Figure 1) [2,6]. In general, NPs should protect the nucleic acid payload from enzymatic degradation,

improve its biodistribution toward the diseased tissue and allow delivery into the desired

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intracellular compartment of the target cells [2]. Typically, nucleic acid nanocarriers can be divided in

viral and nonviral vectors. Viruses are by far the most potent gene therapy vectors since they have

naturally evolved to acquire optimal nucleic acid delivery capabilities, also explaining their prominent

use in clinical trials [3,7]. Nonetheless, viral vectors have a limited payload capacity and their use in

viral gene therapy entails inherent safety risks [6,7]. Therefore, gene therapy research has

increasingly focused on optimizing the delivery of nucleic acids with synthetic nonviral delivery

agents such as (cationic) liposomal formulations and polymers [2]. Despite a lower transfection

efficiency compared to viral vectors, their low immunogenicity, versatility and ease of manufacturing

make nonviral nanomedicines promising alternatives for gene therapy [7].

The size, geometrical shape and surface chemistry of nanomedicines predominantly dictate their in

vivo behavior at the nano-bio interface [8,9]. Following intravenous delivery, NPs should be large

enough to avoid fast renal clearance via glomerular filtration, but still be able to cross the capillary

endothelium (extravasation) in order to reach the target tissue interstitium. For instance, passive

tumor targeting of drug-loaded NPs can be assisted by the so-called enhanced permeability and

retention (EPR) effect. Defective angiogenesis in rapidly growing tumors often entails the formation

of leaky blood vessels through which NPs can easily extravasate. The impaired lymphatic drainage

further contributes to the accumulation of the nanocarriers in the tumor extracellular matrix [10].

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Figure 1. Overview of the main biological barriers for nucleic acid therapy following intravenous

injection of nucleic acid nanoparticles (NPs). These should be large enough to circumvent renal

clearance, but small enough to be able to extravasate and accumulate in the target tissue. Moreover,

the nucleic acid NP should evade uptake by the mononuclear phagocyte system (MPS) and has to

protect the encapsulated nucleic acid against degradation. Once the carrier has reached the target

cell, it needs to deliver the therapeutic RNA or DNA to the cytoplasm. This generally involves cellular

uptake through endocytosis, followed by escape from the endosome and carrier disassembly in the

cytosol. When nuclear entry is required, the nuclear-envelope embedded nuclear pore complex

poses an additional barrier. ECM: extracellular matrix, NPC: nuclear pore complex. Adapted with

permission from ref [6]. Copyright Elsevier.

Passive tissue accumulation is also correlated with blood circulation time. Thus NPs are envisioned

that remain sufficiently stable in the bloodstream and are able to evade fast removal from the

systemic circulation by the mononuclear phagocyte system (MPS). Decorating the NP surface with

hydrophilic polymers, such as poly(ethylene glycol) (PEG), is an established strategy to improve

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colloidal stability in the extracellular environment and to reduce recognition by specialized MPS

phagocytes [2].

In addition, ample control over the NP fate at the subcellular level following cellular uptake has

become increasingly important, aiming to amplify the percentage of the endocytosed nucleic acid

drugs that is effectively delivered into the cell cytoplasm or the nucleoplasm [11]. Cellular

internalization of nanomedicines can be mediated by adsorptive or receptor-mediated endocytosis.

While the former involves non-specific hydrophobic and/or ionic interactions with the cell-surface

proteoglycans, the latter requires modification of the NP surface with targeting ligands that can

specifically recognize and bind membrane-anchored receptor proteins. It is generally assumed that

nucleic acid nanotherapeutics are internalized via an endocytic uptake mechanism [2,11].

Mammalian cells have been shown to display distinct endocytic entry portals and there is emerging

evidence that the transfection efficiency is strongly linked to the uptake mechanism which on its turn

is influenced by the nanocarriers physicochemical properties. Following internalization, NPs typically

will accumulate in endolysosomes containing various acid hydrolases. To avoid nucleic acid

degradation, the nanocarrier should preferably escape from the endosomes into the cell cytoplasm

and release the encapsulated or complexed nucleic acid. Furthermore, when delivery into the cell

nucleus is required (e.g. for transgene expression), the nuclear envelope poses another important

barrier. Plasmid DNA or nucleic acid NPs generally are too bulky for translocation across the nuclear

envelope-embedded nuclear pore complex (NPC), that only allows passive diffusion of molecules

with a hydrodynamic diameter below 10 nm [11]. For this reason, the transgene expression obtained

via non-viral gene delivery systems is often dependent on cell division, during which the nuclear

envelope is disassembled [12].

Rational nanocarrier design and a fundamental insight into the underlying extra-and intracellular

barriers are needed to potentiate nucleic acid delivery across these barriers and maximize the

therapeutic benefit. Both natural and synthetic materials are currently investigated as building blocks

in biomedical nanotechnology. This review focuses on polysaccharides and their derivatives in

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nanocarrier design and details recent important contributions toward improved AsON, siRNA and

pDNA delivery. In particular, this review will highlight specific advantages ascribed to both

established and less well-known polysaccharides used as influential constituents in nucleic acid

nanocarriers.

1.2.Polysaccharides in nucleic acid delivery

Polysaccharides are defined as polymeric carbohydrate structures composed of repeating

monosaccharide units adjoined by glycosidic bonds. They form an important class of naturally

occurring biopolymers that can be obtained from various natural and abundant resources, such as

algae (e.g. alginate), plants (e.g. pectins, cellulose, cyclodextrins), microorganisms (e.g. dextran,

pullulan) and animals (e.g. chitosan, hyaluronan) [13]. Polysaccharides are often quite heterogeneous

in structure and chemical composition, distinguishing neutral or charged, linear or branched and low

or high molecular weight (Mw) polymers with varying hydrophilicity (Figure 2). In addition, the

chemical microenvironment in which these polymers are dissolved may greatly influence their

physicochemical identity and hence also their interaction with therapeutic nucleic acids. This

diversity in polysaccharide structure therefore also entails the formation of nucleic acid NPs with

varying biophysical properties [7], affecting the efficiency with which the various existing extra-and

intracellular barriers in nucleic acid delivery can be overcome [1,2,6]. In general, owing to their

natural origin, polysaccharides are often described as biodegradable, biocompatible and non-

immunogenic, which are attractive properties for pharmaceutical and biomedical applications. For

this reason, polysaccharides are also often employed in hybrid nanostructures to reduce the toxicity

of (synthetic) materials (e.g. poly(ethylene imine) or PEI). Various protocols exist for the production

of nucleic acid loaded polysaccharide NPs, for which the reader is referred to other published reviews

on the topic [13,14].

The in vivo biodistribution of nanocarriers can be markedly influenced by the incorporation of

specific polysaccharides. Many polysaccharides, e.g. chitosan, alginate and hyaluronic acid (HA), are

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excellent bioadhesive materials warranting their application in tissue engineering and mucosal drug

delivery [14]. Alternatively, the presence of a dense layer of polysaccharides in a brush-like

configuration on the nanocarrier surface (e.g. dextran and chitosan) may also lengthen their

circulation time in the bloodstream by reduced MPS recognition, as a result of steric shielding and

mitigated complement activation [14-16]. In addition, the ability to recognize particular

carbohydrate-binding cell-surface receptors confers the use of polysaccharides (e.g. hyaluronic acid)

or oligosaccharide motifs (e.g. galactosan side chains in pectins) as targeting moieties for nucleic acid

delivery to specific cell types [17,18]. Instead of evading MPS phagocytes, treatment of inflammatory

disorders often requires specific targeting to phagocytic cell types (macrophages, dendritic cells),

which is the key advantage of -glucan containing NPs that recognize the ‘dendritic-cell-associated C-

type lectin-1’ or dectin-1 receptor frequently expressed on antigen presenting cells (APCs).

Another advantage linked to the polysaccharide structure is the ease of chemical modification due to

the availability of various functional groups (e.g. hydroxyls, amines, carboxylic acids) on the glycosidic

units. Most often, modifications are introduced with the aim to overcome specific hurdles such as

insufficient nucleic acid binding, fast MPS clearance and/or endosomal escape. Frequently

researchers evaluated modification with PEI to overcome endosomal sequestration. It is believed

that the endosomal buffering by the amine groups in PEI result in osmotic rupture of the endosomes

and release of the endosomal content into the cytosol (often referred to as the proton sponge effect)

[2]. Cyclodextrins are of particular interest toward further derivatization because of their intrinsic

ability to form stable inclusion complexes with hydrophobic ‘guest’ molecules (see 5.1.). All of the

virtues listed above conjointly make polysaccharides promising and attractive biomaterials in many

drug delivery applications.

2. Dextran-based nucleic acid nanotherapeutics

2.1. Introduction to dextran

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Dextran is a highly water-soluble polysaccharide of bacterial origin, produced by Lactobacillus,

Leuconostoc and Streptococcus species. It is composed predominantly of -1,6-linked glucopyranose

units with low degree of 1,3-branching (Figure 2) [19]. Native dextran is characterized by a high Mw

and polydispersity, both of which can be tailored by controlled hydrolysis and subsequent

fractionation [19].

Figure 2. Chemical structures of polysaccharides frequently incorporated in nanocarriers for nucleic

acid delivery. Dextran consists of repeating -1,6-linked D-glucose units with low degree of -1,3-

branching. Chitosan is a positively charged polysaccharide with repeating D-glucosamine and N-

acetyl-D-glucosamine units that are linked via -(1,4) glycosidic bonds. Hyaluronan is an anionic

polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide building

blocks, linked via alternating -1,4 and -1,3-glycosidic bonds. Alginate is composed of alternating

blocks of β-(1,4)-D-mannuronic acid and α-(1,4)-L-guluronic acid. Pullulan consists of -1,6-linked

maltotriose units. Schizophyllan has a main chain of 1,3--D-linked glucose units with 1,6--D-glucosyl

side groups every third glucose. For details on the more complex chemical structure of pectin and

arabinogalactan, the reader is referred to references as mentioned in section 6.3.

Dextran chains may also be produced using dextran sucrase for the transfer of D-glucopyranose from

sucrose to acceptor molecules [20]. Importantly, low Mw dextran in particular already entails a long

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history of clinical applications in humans, e.g. as plasma volume expander, to enhance peripheral

blood flow or as rheological excipient in artificial tears [19,21]. The combined advantages of its

hydrophilic character, biocompatibility, biodegradability and ease of chemical derivatization all relate

to its suitability as drug delivery biopolymer [22].

2.2.Dextran-based matrices

Different types of dextran-based NPs have been described in the literature with the aim to enhance

intracellular nucleic acid delivery. The group of Stefaan De Smedt aimed to design controlled-release

carriers for siRNA delivery based on dextran [23,24]. Cationic dextran hydrogel NPs (dextran

nanogels, dex-NGs) were successfully prepared by an inverse emulsion photopolymerization of

dextran hydroxyethyl methacrylate (dex-HEMA) in mineral oil. To incorporate cationic charges in the

nanoscopic hydrogel network, cationic methacrylate monomers were copolymerized with dex-HEMA.

An important feature of these hydrogel particles is their biodegradability under physiological

conditions, owing to the hydrolysable carbonate ester linking the HEMA moieties to the dextran

backbone [25]. Preformed dex-NGs could encapsulate siRNA based on electrostatic interaction with a

maximal loading exceeding 50 pmol of siRNA per µg of lyophilized dex-NGs [25]. Although an

excellent reporter gene knockdown was obtained in hepatoma cells, confocal fluorescence

microscopy revealed that a substantial fraction of the endocytosed siRNA loaded dex-NGs (siDEX-

NGs) accumulates in acidified organelles, likely endolysosomes. This prompted the authors to

investigate the impact of photochemical internalization (PCI) on the obtained RNAi effect [26]. PCI is

an established endosomolytic method that involves the use of amphiphilic photosensitizers (PS) that

accumulate in the membranes of endocytic vesicles [27]. In this study, mesotetraphenylporphine

carrying two sulfonate groups on adjacent phenyl rings (TPPS2a) was used. Upon illumination with a

specific light source, excitation of the PS compound induces the formation of reactive oxygen species

(ROS), primarily singlet oxygen (Figure 3A). This highly reactive intermediate can damage cellular

components, but the effect is mainly confined to the local production site owing to its short range of

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action and short lifetime [27]. This localized effect will therefore selectively disrupt the endosomal

membranes, releasing the entrapped macromolecules or NPs into the cytosol. Since its discovery in

1999 [28], PCI has been successfully applied to stimulate the cytosolic delivery of several types of

macromolecules (peptides, proteins, nucleic acids), incorporated in non-viral carrier systems [29]. It

was found that application of PCI, even several days post-transfection, was able to significantly

improve and lengthen the gene knockdown obtained with siDEX-NGs (Figure 3B and 3C). These data

indicate that PCI is able to liberate a fraction of the siRNA or siDEX-NGs that remain trapped in

intracellular organelles. Applying vesicular compartments as drug depots can thus be regarded as a

potential strategy to prolong the therapeutic response when endosomal escape is effect-limiting

[26]. Nevertheless, dex-NGs loaded with anti-tumor necrosis factor (anti-TNF- siRNA also

mediated high gene silencing and minor toxicity or off-target transcriptional changes in LPS activated

macrophages, without assistance of any endosomolytic tool [30]. In this report, siDEX-NGs

outperformed other polymeric nanocarriers such as trimethylated chitosan (TMC), G4/G7 poly(amido

amine) (PAMAM) dendrimers and poly(DL-lactic-co-glycolic acid) (PLGA) NPs [30].

Naeye et al. extended this work by decorating siDEX-NGs with a hydrophilic PEG shell to develop a

drug delivery platform capable of intravenous siRNA administration [31]. Although PEGylating the

nanogels did not prevent the partial dissociation of the encapsulated siRNA in human plasma,

fluorescence single particle tracking (fSPT) measurements revealed that PEGylation was required to

prevent their aggregation. Because intravenous application not only implies the contact with plasma

constituents but also with millions of blood cells, the interactions between blood cells and

(PEGylated) siDEX-NGs were investigated [32].

None of the nanogel formulations caused significant erythrocyte lysis, but positively charged

nanogels were shown to induce platelet aggregation. Flow cytometry data confirmed that nanogels

hardly bind to erythrocytes while a clear charge dependent interaction with platelets and leukocytes

was observed, with positively charged nanogel again demonstrating significant cellular binding [32].

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Figure 3. Influence of photochemical internalization (PCI) on in vitro siRNA delivery via biodegradable

cationic dextran nanogels (dex-NGs). (A) Schematic graph clarifying the endosomolytic mechanism of

PCI with the amphiphilic photosensitizer TPPS2a. (B) Confocal micrographs visualize the improved

delivery of green fluorescently labeled siRNA (siGLO) into Huh-7 hepatoma cells. (C) The application

of PCI at a later time-point after transfection with siDEX-NGs enables prolonged gene silencing in

contrast to a cationic lipoplex formulation (Lipofectamine™ RNAiMAX). Reprinted and adapted with

permission from ref [26]. Copyright Elsevier.

Tripathi et al. reported on a different type of cationic modification of dextran NPs. To this end,

dextran was first crosslinked with 1,4-butanediol diglycidyl ether. Partial oxidation of dextran

hydroxyl groups to form aldehydes enabled grafting of branched PEI (bPEI). The resulting dextran-g-

bPEI nanocomposites were able to electrostatically bind pDNA and displayed endosomal buffering,

likely contributing to endosomal escape. The gene expression profile was evaluated in vitro and in

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vivo and showed maximal transgene expression in the spleen of Balb/c mice, possible due to capture

by splenic MPS phagocytes [33].

In an attempt to bypass chemotherapeutic failure due to multidrug resistance (MDR) in

osteosarcoma, Susa and coworkers formulated siRNA targeting the ATP binding cassette transporter

B1 (ABCB1, MDR1) gene in lipid-modified dextran-based polymeric NPs [34]. Overexpression of

MDR1 may be the predominant factor that confers resistance to a broad spectrum of

chemotherapeutics in many types of cancer [35]. Dextran was chosen as the primary NP building

block by virtue of its biocompatibility and biodegradability. The dextran particles were modified with

stearyl moieties and equipped with a PEG coat. The authors show that RNAi-mediated MDR1

downregulation by dextran carriers in drug-resistant osteosarcoma cell lines reversed the MDR

phenotype and re-sensitized these cell lines to the cytotoxic effect of doxorubicin.

The group of Jean Fréchet reported on a tunable and modular particulate system, constructed from

acetal-modified dextran (Ac-DEX) and designed for protein antigen and vaccine adjuvant delivery to

antigen presenting cells (APCs) [36,37]. The hydrophobic acetal groups make the modified Ac-DEX

soluble in organic solvents but insoluble in water, allowing the feasible production of Ac-DEX based

micro-and nanoparticles for protein antigen encapsulation via standard emulsification methods [36].

These acid-degradable particles were further optimized for gene delivery in both phagocytic and non-

phagocytic cells by incorporating small amounts of a degradable cationic polymer (poly--amino ester

or PBAE) in Ac-DEX particles [38]. For the intracellular delivery of siRNA via this polymeric platform,

spermine-modified dextran was applied for the preparation of Ac-DEX, instead of including PBAEs

during particle production (Figure 4). The authors argue that the cationic nature of spermine enables

a better complexation of the negatively charged siRNA and improved interaction with the target cell

membrane. Spermine is a tetravalent organic amine that is present in mammalian cells in millimolar

concentrations and is considered non-cytotoxic [39]. Following endocytic uptake, acid-catalyzed

hydrolysis of the particles will occur in the endolysosomal compartments releasing the siRNA. The

siRNA is thought to reach the cell cytoplasm as a result of endosomal bursting by virtue of an amine-

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induced proton sponge effect. This is further potentiated by intraluminal osmotic pressure build-up

through endosomal accumulation of spermine-Ac-DEX degradation products [40].

Figure 4. Dextran modified with hydrophobic acetal and cationic spermine moieties (spermine-Ac-

DEX) can be used to form NPs for siRNA encapsulation and intracellular delivery. The chemical

structure of spermine-Ac-DEX and a representative scanning electron micrograph of the resulting

siRNA-loaded NPs are shown. Reprinted with permission from ref [40]. Copyright American

Chemical Society.

2.3.Dextran as an additive in nucleic acid nanotherapeutics

2.3.1. Polyelectrolyte complexes with dextran sulphate

Alternatively, the negatively charged dextran sulphate (DS) can be used to prepare nanosized

polyelectrolyte complexes (PECs) together with cationic natural or synthetic (bio)polymers, such as

chitosan (see below) [41] or PEI [42,43]. PECs result from direct interaction of oppositely charged

polyelectrolytes in solution and within PEC NPs polycations are the driving force for complexation of

negatively charged nucleic acids. PEC structure and stability are primarily influenced by the polymer

characteristics (Mw, flexibility, charge density) and the chemical environment (pH, ionic strength,

temperature) [14]. The rationale behind the use of (polysaccharide) polyanions as an additive in PEC

drug formulations is to create more stable nanocomplexes, to minimize polycation-induced toxicity

and/or to stimulate cellular interactions via carbohydrate-binding receptors [14,42,44-46]. PECs have

often been used for protein and small molecule delivery [14,41]. However, Cho et al. reported on

PECs composed of DS and the positively charged poly-L-arginine (PLR) polypeptide to encapsulate

siRNA targeting the epidermal growth factor receptor (EGFR) [44]. Given the small and rigid nature of

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an siRNA duplex, this may result in less stable and larger complexes with polycations (polyplexes)

compared to pDNA polyplexes. The incorporation of polyanions, such as DS or HA, has shown to

result in more compact siRNA polyplexes [45]. In vitro gene silencing and in vivo tumor growth

inhibition in head and neck cancer cells was demonstrated by Cho et al. with their optimized PEC

formulation [44]. Intratumoral injection was employed in the in vivo xenograft mouse model instead

of intravenous injection, since the authors recognize that improved PEC stability in the extracellular

environment is critical before systemic administration can be considered [44]. In another report,

pDNA delivery in human corneal cells was achieved by using hybrid NPs built from cationized gelatin,

modified with the low Mw oligoamine spermine, and the polysaccharide polyanions DS or

chondroitin sulphate. The particles were further stabilized by ionic crosslinking of the cationized

gelatin with pentasodium tripolyphosphate (TPP), thus obtaining particles with a more hydrogel-like

structure. It was found that the hybrid particles modestly improved the in vitro toxicity profile when

compared with the particles prepared in the absence of polyanions, without interfering with their

transfection efficiency. Again, the in vivo colloidal stability and gene delivery performance in relevant

animal models remain to be elucidated for this gene delivery system [46].

2.3.2. Polyelectrolyte complexes with modified dextran

Many examples can be found in the literature describing drug delivery assisted by chemically

modified dextran conjugates [20]. In the context of nucleic acid delivery, cationic modifications of the

dextran backbone with natural or synthetic oligo- or polyamines are preferred to allow electrostatic

interaction and formation of stable polyplexes with therapeutic nucleic acids. Many research groups

investigated conjugates of dextran and PEI in an attempt to reduce the conspicuous toxicity of the

latter polymer and improve the stability of its polyplexes in the presence of serum through a

PEGylation-like shielding effect. Early reports mainly focused on high Mw linear or branched PEI,

however with limited success in confining PEI-mediated toxicity while maintaining gene transfer

activity or polyplex stability [47-49]. The cytotoxicity of PEI is dependent on the polymer architecture

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and increases with increasing Mw [50,51]. It has been demonstrated that reduced toxicity and

consistent biological activity can coincide when assembling small non-toxic PEI units into larger

biodegradable polymeric structures which is a prerequisite for efficient nucleic acid complexation

into NPs [52,53]. For this reason, other groups investigated the grafting of low Mw

oligoethyleneimine (OEI) moieties onto (biodegradable) dextran chains. In general, this approach

resulted in a more satisfying outcome with regard to combining decreased cytotoxicity with

maintained or even increased NP integrity and intracellular gene transfer ability [54-57].

Instead of employing small synthetic cationic units, several reports describe the use of natural

occurring oligoamines in the design of cationic dextran conjugates. It is hypothesized that the use of

biogenic amines would increase the biocompatibility of the resulting polycation. Azzam et al.

synthesized a library of over 300 different polycations by grafting spermine oligoamines to a dextran

or arabinogalactan backbone. Only the dextran-spermine polycations of defined Mw were able to

efficiently transfect different cell lines in vitro [58]. The obtained dextran-spermine conjugates were

further modified with PEG [59] or hydrophobic oleate residues [60] to gain colloidal stability in the

extracellular environment and improve intracellular gene delivery, respectively. It was demonstrated

that the micellar form of oleate-modified dextran-spermine achieved better transfection efficiencies

in vitro at high serum concentrations (50%) in contrast to unmodified dextran-spermine [61]. In a

recent comparable approach, Yang et al. reported on the grafting of another biogenic oligoamine, i.e.

agmatine, to low Mw dextran chains. Further hydrophobic modification of the dextran hydroxyls was

performed with lauric acid, thereby resulting in a polymeric construct composed exclusively from

biocompatible endogenous molecules. The authors showed that both agmatine and fatty acid

conjugation contributed to the high reporter gene expression levels obtained in COS-7 and HEK 293

cells [62]. Agmatine is produced by decarboxylation of L-arginine via the mitochondrial enzyme

arginine decarboxylase and is widely distributed in mammalian tissues. Its involvement in diverse

physiological processes and its low in vivo toxicity both translate in a variety of potential clinical

applications of exogenous agmatine [63]. Thus, agmatine-modified dextran cannot merely be

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regarded as an inert excipient since it should be taken into consideration that its degradation

products could entail unforeseen pharmacological effects. In another report, Thomas et al. described

the modification of dextran with protamine, which is a naturally occurring cationic oligopeptide

isolated from salmon sperm [64]. Protamines are highly specialized sperm chromosomal proteins

responsible for the compaction of DNA. They carry arginine rich motifs and peptide sequences that

may act as a nuclear localization signal (NLS), both contributing to a more favorable intracellular

pDNA delivery [65,66]. Dextran-protamine conjugates mediated high transfection efficiency in vitro

without significant cytotoxicity and appeared to be haemocompatible, providing opportunities

toward in vivo application [64]. Several other examples of cationized dextrans have been described in

the literature which will not be discussed in detail here [67-69].

2.3.3. Dextran coated nucleic acid nanoparticles

Polysaccharide coating of NPs has been considered as a biomimetic alternative to PEG in order to

provide them with so-called ‘stealth’ properties and improve their in vivo performance [16]. For

many types of polysaccharides (e.g. dextran, chitosan, pullulan, hyaluronic acid) such surface

coatings have been successfully demonstrated via various methodologies. Native dextran, charged

dextran (dextran sulphate or diethyl amino ethyl (DEAE)-dextran) and hydrophobically modified

dextran were evaluated to coat polymeric particles as well as liposomal vesicles. The deposition of a

dextran coat could modulate protein adsorption, improve colloidal stability and mitigate complement

activation [14-16]. In a recent report, layer-by-layer (LbL) polyelectrolyte coated quantum dots (QDs),

with a terminal polysaccharide outer layer (dextran sulphate (DS) or hyaluronic acid (HA)) were

evaluated for their in vivo biodistribution in mice. Both DS and HA terminated particles exhibited

significantly prolonged circulation times (elimination half-lives of 3.2 h and 8.4 h, respectively) when

compared with uncoated QDs. However, the particles capped with a DS outer layer also showed

increased liver accumulation, possibly due to the interaction with receptors (HARE receptor, also see

20

4.1.) on the liver sinusoidal endothelial cells (LSEC) [70] and/or phagocytic uptake by liver

macrophages (Kupffer cells) triggered by the high negative charge of DS [70,71].

To date, only few reports are available in the literature describing dextran coated NPs in the context

of nucleic acid delivery. Early reports describe dextran-g-poly(L-lysine) coated poly(lactic acid) (PLA)

NPs and DEAE-dextran modified poly(alkylcyanoacrylate) NPs for pDNA and AsON delivery

respectively [72-74]. More recently, Delgado et al. published on the design of solid lipid nanoparticles

(SLNs), modified with dextran and protamine, for gene delivery [66,75]. An aqueous solution of

dextran was incubated with protamine and pDNA prior to mixing with preformed SLNs. In the

resulting gene delivery vector, dextran-protamine-DNA complexes are electrostatically adsorbed on

the SLN surface. The particles displayed significant transgene expression in vivo following ocular

administration in rats [66] and intravenous injection in mice [75] and gene transfer was more

successful when protamine and dextran were incorporated in the formulation. As mentioned above

(2.3.2), the arginine-rich polycationic protamine aids in DNA complexation and may enhance DNA

entry into the nucleus [65,66,75]. The presence of dextran on the particle surface is important

toward improved in vivo biocompatibility. In addition, dextran seemed to influence the cellular

uptake mechanism, leading to improved transfection efficiency by promoting clathrin-mediated

endocytosis [66,75].

3. Chitosan-based nucleic acid nanotherapeutics

3.1.Introduction to chitosan

Chitosan is obtained by partial deacetylation of chitin, which is the natural main structural

component of the crustaceans exoskeleton and the cell wall of fungi [76,77]. It is a linear and

positively charged polysaccharide with repeating D-glucosamine and N-acetyl-D-glucosamine units

that are linked via -(1,4) glycosidic bonds (Figure 2) [77]. Because of its cationic nature, chitosan is a

very popular candidate among natural polysaccharides for nucleic acid complexation and delivery

[13]. Additional advantages described for chitosan, in line with the other polysaccharides mentioned

21

in this review, are its low cytotoxicity, low immunogenicity and its biodegradability [13,78-80]. A

more specific beneficial feature that can be ascribed to chitosan and chitosan derivatives are their

mucoadhesive and permeation-enhancing properties, explaining the frequent use in mucosal drug

delivery and tissue engineering [81-84]. Irrespective of these advantages, the low water-solubility of

chitosan at physiological pH is an important limitation for its clinical use. Deacetylation of chitosan

exposes D-glucosamine primary amines with a pKa ~6.5, explaining why chitosan is only soluble in an

acidic aqueous environment [85]. Therefore, also for chitosan an important amount of effort has

been put forward to chemically modify this polysaccharide, e.g. by quaternization of the D-

glucosamine moieties [86], in order to make it more amenable for drug delivery (see below). The

nucleic acid delivery performance of native chitosan and its derivatives is highly dependent on a wide

variety of formulation-related factors, e.g. chitosan Mw, degree of deacetylation, type of salt and NP

preparation technique. The influence of these parameters has recently been reviewed in detail by

Mao et al. [78]. For a more in depth view on chitosan-based nucleic acid delivery systems, we also

would like to refer the reader to a review by Buschmann et al., published in this same theme-issue of

Advanced Drug Delivery Reviews. Given the enormous wealth of literature reports describing

chitosan nanotherapeutics, this review will mainly focus on the most recent advances made with this

type of polysaccharide for various nanoarchitectures and various (chemical) modifications. Several

preparation techniques can be harnessed for the production of nanosized particles from chitosan

[84], among which covalent or ionic chitosan crosslinking (chitosan hydrogel NPs) and self-assembly

with nucleic acids (chitosan polyplexes) are most often reported in the context of nucleic acid

delivery.

3.2.Chitosan-based matrices

Therapeutic nucleic acids can be encapsulated in chitosan hydrogel nanoparticles (nanogels) in which

chitosan polymeric chains are interconnected via covalent or ionic crosslinkages. Although physical

crosslinking methods generally lead to 3D polymer networks of inferior mechanical strength, ionic

22

crosslinking is the preferred method as it avoids the use of cytotoxic crosslinking agents (e.g.

glutaraldehyde) and can occur under relatively mild conditions [13,14]. It involves the formation of

ionic inter-and intramolecular linkages between chitosan protonated amines with the aid of small

multivalent ionic molecules. As mentioned before, TPP has been most widely used as polyanion

crosslinker for chitosan [13,77,84]. Chitosan nanogels can be formed by merely mixing an alkaline

TPP phase with an acidic chitosan solution, paying proper attention to the chitosan:TPP ratio to yield

stable solidified NPs [77]. Chitosan nanogels may consist solely of chitosan or can be prepared in the

presence of other hydrophilic macromolecular compounds. In this way, ionic gelation has been

frequently applied to further stabilize chitosan-based polyelectrolyte complexes with various

polyanions and/or bioactive nucleic acids. The resulting chitosan NPs are thus held together by

cooperative electrostatic interactions with both nucleic acids and TPP, giving rise to more compact

nanostructures [77]. Moreover, it has also been demonstrated that the condensed crosslinked

polymer matrix allows a more sustained gene expression, ascribed to a more extended release time

of the pDNA [87,88].

The group of Maria Alonso pioneered TPP induced ionic gelation of chitosan to form nanoscopic drug

delivery carriers [77]. Csaba et al. formulated pDNA and short dsDNA oligonucleotides into

chitosan/TPP crosslinked NPs prepared with chitosan of varying Mw. Low Mw chitosan (10 kDa)

provided more compact nanocarriers (~100 nm) opposed to high Mw chitosan (125 kDa) on account

of the lower viscosity of the former polymer dispersion, in line with other reports in the literature

[89]. Importantly, also the efficiency of transfection seemed highly dependent on the chitosan Mw,

with low Mw chitosan/TPP NPs showing superior gene transfer in vitro. In addition, low Mw

chitosan/TPP particles displayed a marked transgene expression following intratracheal

administration in mice, albeit comparable with the corresponding low Mw chitosan polyplexes in the

absence of TPP [87]. This study provided the basis for further optimization of chitosan/TPP NPs for

siRNA and pDNA delivery. To further enhance the transfection efficiency, colloidal stability and the

toxicity profile of the original chitosan/TPP formulation, PEG moieties were grafted to the chitosan

23

backbone and HA was included. To prepare the hybrid NPs, a preformed HA/TPP mixture was added

to a chitosan-g-PEG solution under magnetic stirring. The resulting HA/chitosan-g-PEG/TPP nanogels

demonstrated efficient transgene expression and moderate gene silencing in HEK 293T cells, albeit

that gene suppression values were similar to the commercial lipofectamine 2000 formulation [90].

Katas and Alpar directly compared the physicochemical properties and in vitro RNAi activity of

chitosan-siRNA NPs prepared via three different methods: complex coacervation, ionic gelation in the

presence of siRNA and adsorption of siRNA onto the surface of preformed chitosan nanogels. They

found that siRNA entrapment in chitosan/TPP nanogels showed better reporter gene suppression

compared to the other preparation methods, likely due to the improved siRNA loading [91], thereby

corroborating earlier results from Maria Alonso’s group obtained with oligonucleotides [77].

In a combined effort to improve both nanocarrier-mediated cellular delivery and transgene

expression efficiency at the level of the pDNA itself, Gaspar et al. formulated compact supercoiled

(sc) pDNA topoisoforms in chitosan NPs by ionotropic gelation [92]. It is known that sc pDNA

outperforms its relaxed open circular (oc) and linearized counterparts with regard to transfection

efficiency [93], thus warranting further optimization of pDNA topology in gene transfer applications.

The authors succeeded in purifying sc pDNA via high throughput arginine affinity chromatography

and subsequently encapsulated the recovered pDNA into TPP induced chitosan nanogels.

Importantly, the mild reaction conditions for the preparation of the chitosan NPs only induced minor

topoisoform conversion, in contrast to what is reported in the literature for other NP manufacturing

techniques [92].

3.3.Chitosan as a polycation in nucleic acid nanotherapeutics

3.3.1. Polyelectrolyte complexes with native chitosan

The preparation of chitosan PECs is based on the process of complex coacervation in which

oppositely charged polyelectrolytes are mixed together. As highlighted earlier, chitosan has been

studied elaborately for nucleic acid complexation and condensation into polyplex NPs, where the

24

negatively charged therapeutic nucleic acid functions as counter polyion in the complex coacervate.

The most straightforward chitosan polyplex formulation is obtained by merely mixing native chitosan

with the nucleic acid therapeutics, preferably at acidic pH [94-96]. Klausner et al. applied ultrapure

chitosan oligomers for ocular gene delivery. Low Mw chitosan with a high degree of deacetylation

achieved in vivo demonstrated reporter transgene expression levels superior to PEI after intrastromal

injections in rat corneas [97].

However, the chitosan polymer chains should have a sufficient charge density at physiological pH to

ensure good complexation and stability of the resulting polyplex in the extracellular environment,

which is often markedly hampered by the partial protonation of the glucosamine groups [81,98].

Intracellular nucleic acid delivery is further restricted by limited endosomal escape, likely due to its

weak buffering capacity, making native chitosan less efficient in comparison with other cationic

polymers such as PEI [80,99]. To impart improved nucleic acid delivery properties to chitosan, many

alterations to its native structure have been proposed. The repeating glucosidic units in chitosan

contain two hydroxyl groups and one primary amine that are regarded as potential reactive sites for

chemical modifications, depending on the desired biomedical application [81].

3.3.2. Polyelectrolyte complexes with modified chitosan

To increase the aqueous solubility of chitosan and the colloidal stability of chitosan PECs at

physiological pH, quaternization of the primary amine was investigated by many groups. In the

context of nucleic acid therapy N,N,N-trimethyl chitosan chloride (TMC) is most often reported [100].

To further optimize the delivery properties of TMC, Verheul et al. reported on the preparation of

nanoparticles based on thiolated TMC [101]. The introduction of thiol groups enables the formation

of intra- and intermolecular disulfide bonds that can provide additional NP stability in high salt

conditions or in the presence of competing anions [101,102]. The presence of free thiol groups also

enables further functionalization with e.g. thiolated PEG or HA [102] and may lead to enhanced

muco-adhesive properties by formation of disulfide bonds with extracellular mucin glycoproteins.

25

Moreover, the latter interaction could stimulate cellular internalization and therefore also gene

transfer, which was demonstrated by Lee et al. in various cell lines in vitro and in vivo in mice [88].

To further address the issue of forming siRNA polyplexes with inferior colloidal stability relative to

pDNA, the concept of polymeric siRNAs was recently proposed as an alternative strategy towards

more stable siRNA polyplexes [103]. It is expected that increasing the Mw and total number of

anionic charges by virtue of siRNA polymerization would result in more stable and condensed

nanosized complexes with polycations due to increased multivalent electrostatic interactions. Lee

and coworkers demonstrated the polymerization of thiolated siRNA (poly-siRNA) via reducible

disulfide linkages that can be selectively cleaved again in the reductive intracellular environment

following cytosolic delivery in the target cell [104]. However, when poly-siRNA was incubated with

cationic thiolated glycol chitosan (TGC) in HEPES buffer (pH 8), unstable complexes were formed as a

result of the weak charge interactions. Nevertheless, the electrostatic complexation enabled

additional intra-and intermolecular disulfide bonding within the TGC polymer chains, leading to

better condensed nanostructures (Figure 5). The TGC polyplexes displayed improved resistance

against polyanion destabilization and showed superior tumor accumulation over PEI polyplexes

following systemic injection in tumor-bearing mice. Moreover, significant vascular endothelial growth

factor (VEGF) silencing and concomitant reduction in tumor growth was observed for the TGC

polyplexes [104].

Recognizing the key role of intracellular vector unpackaging and nuclear entry in determining the

efficacy of transgene expression, Zhao et al. focused on the modification of chitosan with short

phosphorylatable peptides to overcome these impediments [105]. As mentioned before,

uncomplexed pDNA and nucleic acid NPs are too large to reach the nucleoplasm, given that the

nuclear-envelope embedded NPC only allow passive diffusion of molecules with a hydrodynamic

diameter below 10 nm [11].

26

Figure 5. (A) Production of polymeric siRNA (poly-siRNA) via disulfide groups. (B) Thiolated glycol

chitosan (TGC) can form polyplex structures with poly-siRNA via electrostatic interaction. Subsequent

crosslinking of TGC thiol groups leads to more condensed NPs. The intracellular release of

monomeric siRNA is mediated by glutathione (GSH) stimulated reduction of the disulfide bonds.

Reprinted with permission from ref [104]. Copyright Wiley-VCH.

NLS containing proteins are recognized by nuclear transport receptors like importins and transportins

that actively enhance protein translocation across the NPC. Anchoring NLS peptides to pDNA and

even cationic polymers is believed to aid in nuclear delivery of transgenes, albeit with limited success

[106,107]. Therefore, a peptide was selected carrying a N-terminal Simian virus 40 large T antigen

(SV40 T-ag) derived NLS sequence and a serine-containing motif that could be selectively

phosphorylated by kinases present in nuclear cell lysates. The C-terminal end of the selected peptide

consisted of two additional arginines to increase the isoelectric point and enable the formation of

stably condensed DNA polyplexes at physiological pH. Following nuclear translocation, it was

hypothesized that the introduction of anionic phosphate groups by virtue of nuclear kinase activity

on the oligopeptide, would entail specific intranuclear polyplex dissociation. The peptide modified

chitosan polyplexes showed modest improvement in gene transfer efficiency when compared to

lipofection [105]. In this report the authors mainly focused on intracellular polyplex stability and the

nuclear import of polyplex and/or transgene, but unfortunately did not consider the importance of

the endosomal barrier in nucleic acid delivery. This issue is addressed by many others, given the fact

27

that the often observed low transfection efficiency of chitosan is thought to be related to its less

efficient escape from endosomes [78]. It is indeed known that the endosomal buffering capacity of

chitosan is significantly lower opposed to PEI [80,99]. However, in this theme issue Buschmann et al.

also describe that when comparing PEI with chitosan based on molar ionisable amines instead of

mass, fully deacetylated chitosan actually may have a larger endolysosomal buffering capacity than

PEI. In addition, some reports claim several other mechanisms by which chitosan could overcome the

endosomal barrier, e.g. its direct charge-dependent membrane destabilizing effect or indirect as a

result of endosomal enzymatic degradation. In the latter case, accumulation of chitosan degradation

products would evoke an increase in osmotic pressure eventually leading to perturbation of the

endolysosomal membrane [78].

Nevertheless, to bestow chitosan with improved endosomal destabilizing properties, both polymers

and small molecules have been grafted to the chitosan backbone, mainly with the aim to obtain a

cationic polymer capable of buffering the lumen of endolysosomes. In line with chemical

modifications carried out on other polysaccharides already described in this review, several authors

reported on the modification of chitosan or chitosan derivatives with PEI [108-113]. However, this

may again raise concerns regarding the cytotoxicity of the resulting conjugates. As an alternative,

Ghosn et al. used a 1-step carbodiimide reaction to modify the glucosamine functional groups of

chitosan with imidazol-4-acetic acid (IAA), thus introducing secondary and tertiary amines and

concurrently also endosomal buffering capacity [114]. A similar approach was used by Pego and

coworkers [99,115]. In vitro transgene expression and gene silencing using chitosan-IAA polyplexes

with pDNA and siRNA respectively was significantly improved over unmodified chitosan [116]. To

prevent aggregation of the polyplexes in vivo, PEG chains with a Mw of 5000 Da were anchored to

the surface of the polyplexes via a succinimidyl ester. PEGylated chitosan-IAA polyplexes mediated

substantial gene silencing in lung and/or liver following intranasal and intravenous administration

[114]. Chang et al. modified chitosan with pending histidine amino acids of which the imidazole ring

confers endosomal buffering capacity [117], a modification also recently exploited for dextran [67].

28

Likewise, urocanic acid that also bears an imidazol ring, was coupled to chitosan to increase its

transfection efficiency [118].

In an attempt to mimic the cellular translocation mediated by cell penetrating peptides (CPPs),

guanidinylated chitosan (GCS) was synthesized for gene and siRNA delivery [119,120]. CPPs have

demonstrated the ability to deliver peptides and nucleic acids in to cells. The most extensively

studied CPPs are characterized by their high content of positively charged arginines and lysines,

which are believed to play a major role in the cellular binding and intracellular delivery of

biomacromolecules. Although there is no consensus yet on their specific mechanism of cellular

internalization, it is believed that CPPs can assist bulky macromolecular payloads and even NPs in

crossing the cellular barriers [121]. The guanidinium group of arginine amino acid residues seems

imperative for cellular translocation, thereby providing a rationale for GCS evaluation as nucleic acid

nanocarrier [122]. It was demonstrated by Zhai et al. that GCS could improve DNA condensation at

neutral pH and improved cellular uptake when compared to native chitosan, both likely mediated by

the strong basic character of the guanidinium group that replaces the glucosamine primary amine

[119]. Unfortunately, no data on the cellular uptake mechanism and/or intracellular trafficking of the

guanidinylated polyplexes was provided. Correspondingly, GCS was used to complex siRNA in a

follow-up study investigating the potential of the GCS carrier toward pulmonary gene silencing via

intratracheal administration. To further improve pulmonary targeting, the 2-adrenergic receptor (2-

AR) agonist salbutamol was ligated to the GCS polymer. Evidence suggests that 2-AR agonists could

be used as a targeting ligand to ameliorate in vivo receptor-mediated delivery of nanotherapeutics in

the lung [123]. The salbutamol functionalized GCS particles achieved increased gene silencing over

non-targeted GCS polyplexes in the lungs of EGFP transgenic mice [120].

Several other examples of modified or native chitosan, equipped with appending cellular targeting

moieties, can be highlighted [110]. For instance, mannosylated chitosan-g-PEI polyplexes could be

targeted to APCs bearing mannose lectin receptors, as demonstrated in vitro in a macrophage cell

line [124]. Galactosylated chitosan was synthesized toward targeted gene transfer in hepatocytes via

29

the asialoglycoprotein receptor [125,126]. In another report, folate was applied as a targeting ligand

in the context of pulmonary tumor targeting [127]. Recently, CD7-specific single-chain antibodies

were conjugated to chitosan for targeted siRNA delivery to T-cells [128]. The group of Anil Sood

succeeded in developing a cyclic Arg-Gly-Asp (RGD) peptide anchored chitosan NP for siRNA delivery

(Figure 6) [129]. The peptide was coupled to the chitosan backbone by thiolation of glucosamine

residues using the crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The

RGD peptide specifically binds to v3 integrin receptors often overexpressed in tumors. In addition,

the v3 integrin receptor is also selectively expressed by endothelial cells of the tumor vasculature,

making it an attractive binding site for anti-angiogenic therapy [130]. The NPs showed distinct tumor

(vascular) targeting in vivo in distinct orthotopic murine models of ovarian carcinoma. Tumor

selective delivery of siRNA, targeting genes involved in ovarian cancer progression, led to marked

inhibition of tumor growth [129].

The group of Ana Pego further built on their imidazol-modified chitosan to design a nanocarrier

capable of targeting the peripheral nervous system (PNS) [131]. In addition to imidazol grafting,

remaining glucosamine moieties were reacted with 2-iminothiolane. Thiolation was pursued to

enable grafting of the targeting ligand via a bifunctional PEG spacer. The non-toxic carboxylic

terminal fragment of tetanus toxine (HC fragment) was chosen as targeting moiety because of its

ability to mediate neuron cell-specific internalization. Although specific targeting was demonstrated

in a neuroblastoma cell line and a primary dorsal root ganglia (DRG) neuron culture model, the

percentage of transfected primary neurons was markedly reduced when compared to the HC

functionalized PEI-based vector[131,132]. It remains to be clarified if the targeted chitosan-based

formulation will be able to target the PNS in vivo and if sufficient expression levels can be achieved to

attain a therapeutic benefit in peripheral neuropathies [131].

30

Figure 6. In vivo gene silencing of periostin (POSTN), involved in cell survival and invasion, via siRNA

loaded chitosan nanoparticles (CH-NP) or integrin-targeted RGD-CH-NP. (A) The cyclic RGD peptide is

coupled to the chitosan backbone via disulfide coupling chemistry. (B and C) A single intravenous

injection of POSTN siRNA/RGD-CH-NP (0.15 mg/kg) resulted in significantly improved POSTN silencing

compared with the non-targeted formulation in an SKOV3ip1 (v3 integrin positive) murine model of

ovarian cancer, as assessed by both western blotting (B) and immunohistochemistry (C). Reprinted

and modified with permission from ref [129]. Copyright American Association for Cancer Research.

31

3.3.3. Hybrid chitosan-based polyelectrolyte complexes

Chitosan PECs have also been constructed containing other polyanions (e.g. polysaccharides and

polypeptides) in addition to the nucleic acids [13,133-135]. For instance, Liao et al. demonstrated

enhanced gene silencing with ternary CS/siRNA/poly--glutamic acid complexes [134]. In another

example, hybrid NPs of chitosan and HA could instigate gene transfer into primary chondrocytes for

the treatment of osteoarthritis. The presence of HA in the particles is believed to enhance receptor-

mediated uptake via interaction with the CD44 cell-surface receptor (see 4.1), that is highly

expressed on osteoarthritic chondrocytes [136]. As briefly mentioned in 2.3.1., the polyanions may

also bring about the added advantage of improved colloidal stability as a result of multivalent ionic

interactions, which is of particular interest for siRNA containing formulations [137].

3.4.Nanoparticles coated with chitosan

In addition to the nanocarriers described above, in which chitosan mainly governs NP formation and

nucleic acid complexation, chitosan may function as a coating material for surface modification of

various types of NPs. For instance, many groups reported on the modification of biodegradable and

biocompatible poly(D,L-lactic-co-glycolic acid) (PLGA) NPs with chitosan [138]. Because of its cationic

nature, chitosan easily binds with the negatively charged PLGA giving rise to core-shell NPs endowed

with a positive zeta-potential. Mostly, chitosan is applied during the emulsion evaporation method

used to prepare PLGA particles. It is thus postulated that the chitosan polymer chains can be found

both within the PLGA polymer matrix as on its surface. This surface modification can strongly

enhance the particles affinity for negatively charged cellular membranes and trigger subsequent

endocytic uptake. Moreover, because of chitosan affinity for mucosal surfaces and its permeation

enhancing properties, NPs equipped with a chitosan coat are attractive for mucosal delivery

[14,16,77]. The group of Claus-Michael Lehr developed chitosan-modified PLGA NPs to deliver

therapeutic oligonucleotides, adsorbed onto the surface of preformed chitosan-coated NPs, to lung

cancer cell lines [139,140]. Yuan et al. evaluated chitosan-PLGA nanoparticles for siRNA delivery into

32

HEK 293T cells. Since the siRNA was pre-complexed with chitosan prior to emulsification, this

formulation strategy will likely encapsulate a fraction of the siRNA in the PLGA core [141]. Tahara and

coworkers optimized an emulsion solvent diffusion method without sonication to prepare chitosan-

coated PLGA NPs. The encapsulation efficiency of siRNA in the PLGA matrix was amplified by its pre-

complexation with cationic lipids (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) and its

addition to the PLGA containing organic phase before emulsification in a chitosan supplemented

aqueous stabilizer solution [142].

Next to PLGA, other types of solid hydrophobic NPs have been used as template for chitosan coating.

Poly(isohexylcyanoacrylate) (PIHCA) with chitosan shell were used to complex siRNA targeting the

Ras homologous A (RhoA) protein that has been shown to promote cell proliferation and metastasis

[143]. Repeated intravenous injections every 3 days during a 30 day period of the anti-RhoA siRNA

formulation in nude mice inhibited xenografted breast cancer proliferation >90% at an siRNA dose of

0.15 mg/kg. Of equal importance, this treatment regimen was shown to be without in vivo toxicity

when compared with the untreated anesthetized animals as judged by body weight progression,

histological assessment of vital organs and quantification of biochemical markers for hepatic, renal

and pancreatic function [143].

More recently, de Martimprey and coworkers applied chitosan-coated poly(isobutylcyanoacrylate)

(PIBCA) NPs for in vivo siRNA delivery via intratumoral injection to treat papillary thyroid carcinoma

[144]. To enable intravenous delivery of this nanoformulation and passive EPR-mediated tumor

targeting, the authors succeeded in downsizing the PIBCA and PIHCA particles below 100 nm by

adding pluronic as a surfactant during NP synthesis. Multiple intravenous injections with a

cumulative siRNA dose of 5 mg/kg led to almost complete blockage of tumor growth in the same

murine tumor model [145]. The NPs were synthesized via redox radical emulsion polymerization as

opposed to earlier reports by the same group in which an anionic emulsion polymerization was

employed [146]. The former method has the advantage that the polysaccharide chains adopt a

33

distinct configuration on the NP surface that can bypass complement activation thus contributing to

lower recognition by the MPS [147].

Following the development of chitosan-coated liposomes (frequently termed chitosomes) for

mucosal delivery of macromolecules (e.g. oral delivery of insulin), some reports also addressed the

usefulness of these hybrid vesicular NPs for nucleic acid delivery [148,149]. For instance, the

pRc/CMV-HBs(S) plasmid, encoding the S region of the hepatitis B antigen, was encapsulated in

neutral liposomes consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), egg

phosphatidylcholine (egg PC) and cholesterol [150]. Glycol chitosan was adsorbed onto the surface of

the liposomes and subsequently administered intranasally to mice to evaluate their suitability for

non-invasive immunization via the nasal mucosa. The glycol chitosan coated liposomes exhibited

humoral, mucosal and cellular immune responses that were superior to immunization with naked

pDNA [150].

4. Hyaluronic acid-based nucleic acid nanotherapeutics

4.1.Introduction to hyaluronic acid

Hyaluronic acid (HA) is a nonsulfated glycosaminoglycan (GAG), composed of alternating disaccharide

units of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA), linked by alternating β-1,4

glycosidic bonds and β-1,3 glucuronidic bonds (Figure 2) [151]. Despite its plain appearance, its

functions and interactions are extremely diverse depending on its size. In mammalian organisms,

native HA is usually found as an unbranched, high Mw (several million Daltons) linear polymer with

exceptional physicochemical properties. In this high Mw form, HA is one of the main constituents of

the extracellular matrix (ECM) and is known to have a mechanical and structural role in the synovial

fluid, the vitreous humour of the eye and in connective tissues of e. g. the umbilical cord and dermis

[152]. Aside from these extracellular functions conveyed by the physicochemical properties of HA,

so-called HA binding hyaladherins extend the functionality of HA to the modulation of cellular fate by

receptor-mediated intracellular signaling. For example, HA has been suggested to influence cell

34

signaling to the cyclin D1 pathway [153], regulate the immune response [154] and influence both cell

proliferation and migration [155,156]. Some hyaladherins play important roles in the degradation

and uptake pathways of HA, mostly cell receptors in contact with the extracellular environment. To

date, already several of these HA-specific cell receptors have been identified, such as cluster

determinant 44 (CD44), receptor for hyaluronate-mediated motility (RHAMM), HA receptor for

endocytosis (HARE) and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), all with specific

functions. More detailed information about these various HA binding proteins and their respective

functions can be found in an extensive review recently published by Jiang et al.[157].

Nowadays, HA is easily extracted from rooster combs or produced by microbial fermentation [158].

In the medical field, non-immunogenic, highly viscous HA solutions are commercially available as a

surgical aid in ophthalmology and as viscosupplementation for synovial fluids in patients with

osteoarthritis [152]. The ubiquitous distribution of HA throughout the body and it’s intrinsic

properties such as hydrophilicity, biocompatibility, biodegradability and non-immunogenicity all

endorse the use of HA for biomedical needs. Aside from these advantages, several other

characteristics have encouraged the use of HA in drug delivery. The muco-adhesive properties of HA

were exploited to increase the residence time of small molecule therapeutics on ocular mucosa or in

topical wound healing [159,160], whereas the hydrophilic nature of HA was employed to reduce

unspecific interactions with proteins in the blood stream and prolong in vivo circulation time [161].

Nearly ten years ago, it was documented that the hydrophilic nature of HA could also serve as a

cryoprotectant in the preparation of unilamellar liposomal formulations [162]. When using liposomes

for drug delivery, the unilamellar structure is of utmost importance. Upon rehydration after

lyophilization, the emulsions usually revert back to multilamellar liposomes, yet it was found that the

covalent attachment of HA on the unilamellar liposomes could preserve their structure after

rehydration. Finally, one of the most promising advantages of HA is based on the previously

mentioned interactions with HA-specific cell receptors such as CD44, rationalizing HA as a targeting

strategy to CD44-(over) expressing tissues, such as tumors and diseased livers [163-165]. This

35

targeting strategy was elegantly documented in vitro by Surace et al. in HA receptor-expressing cells

compared to control cells (Figure 7) [166]. Taken together, it is widely acknowledged that HA can

provide several advantages to drug delivery, and in the next section we would like to highlight the

use of HA specifically for nucleic acid delivery vectors.

Figure 7: In vitro targeting properties of HA towards the CD44 receptor, as demonstrated by Surace

et al. Comparative transfection efficiency in MCF-7 (A) and MDA-MB231 (B) cells treated with

DE:DOPE lipoplexes (black) and lipoplexes containing 0.10 mg/mL of HA-DOPE conjugate (white) in

the presence of increasing amounts of anti-CD44 antibody Hermes-1 and anti-ErbB2 antibody used as

control. It is noticed that lipoplexes containing the HA-DOPE conjugate show an increased

transfection efficiency in HA receptor-containing MDA-MB231 cells compared to MCF-7 cells. This

increase in transfection efficiency is downregulated by increasing amounts of anti-CD44 antibody,

indicating a CD44 receptor-specific uptake in MDA-MB231 cells. Reproduced with permission from

ref [166].

36

4.2.Hyaluronic acid-based matrices

Since HA is advantageous in terms of biodegradability and non-immunogenicity, and given its use in

drug delivery [152,158], it would make sense to propose this biopolymer as a nucleic acid delivery

vector. Unfortunately, due to the negative charge of both HA and nucleic acids, nucleic acid NPs

based solely on HA are not that common. Regardless, some groups have reported on the ability of HA

matrices to incorporate therapeutic nucleic acids. HA hydrogels and microspheres were evaluated

for the sustained release of pDNA encoding for platelet derived growth factor (PDGF) [167] and β-

galactosidase [168], respectively. The HA matrices were formed by an adipic dihydrazide (ADH)

crosslinking reaction, where pDNA and high Mw HA are mixed together, after which ADH and 1-

ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are added to ensure crosslinking. Another group

employed a film of ADH-crosslinked HA as a physical barrier to prevent post-operative peritoneal

adhesions, where the film was also able to release previously incorporated pDNA encoding for

hyaluronan synthase 2 in the peritoneal cavity over a prolonged period of time [169]. In all three

studies, the sustained release of the pDNA was confirmed in vitro by degradation of the matrices in a

hyaluronidase solution. The released pDNA was shown to be intact and bioactive by in vitro

transfection assays using commercially available transfection agents. Consequently, aside from the in

vivo data by Yun et al. [168], it must be emphasized that in these studies only the bioactivity of the

released pDNA was assessed. This way, the macroscopic HA matrices are only evaluated in terms of

their ability to protect therapeutic DNA, not to ensure its uptake in the target cells, one of the most

difficult barriers to overcome in nucleic acid delivery. To do so, some groups have proposed the use

of HA hydrogels not only for their biocompatible features, but also for their known interactions with

HA-specific cell receptors. In contrast with the larger hydrogels discussed previously, Lee et al.

reported using an emulsion method and ultrasonication to fabricate nano-sized biodegradable thiol-

crosslinked HA nanogels, with anti-GFP-siRNA physically entrapped during the emulsification process

[170]. The disulfide-linkages in the hydrogels were incorporated to ensure cargo release in the

reductive intracellular environment, as was demonstrated by the release of intact siRNA in a GSH-

37

concentration dependent fashion. Compared to cationic polymers such as PEI and PLL, the HA

hydrogels showed much lower cytotoxicity and higher gene silencing capabilities in the presence of

serum. Additionally, the purpose of HA to guarantee specific uptake of the hydrogels in HA receptor-

positive cells was verified by a competitive binding assay in HCT-116 cells, where the co-incubation of

HA/siRNA-hydrogels with free HA drastically decreased the gene silencing effect, indicative of the

specific uptake of the hydrogels by HA receptor-mediated endocytosis [170] .

4.3.Hyaluronic acid as an additive in nucleic acid nanotherapeutics

4.3.1. Hyaluronic acid polyelectrolyte complexes

As mentioned before, HA on its own is not considered a good nucleic acid vector due to its negative

charge. Therefore, HA is mostly used as an additive to an existing nucleic acid carrier to endow the

latter with added advantages. As it is known that most nucleic acid vectors have a positive charge,

anionic HA can be electrostatically complexed with these cationic polymers and the nucleic acids to

form PECs by supramolecular self-assembly. By mixing low Mw HA with biodegradable PLR, PECs

capable of complexing siRNA were easily formed [171]. The authors proposed to incorporate HA in

the siRNA-vector to grant additional HA receptor-specific targeting capabilities. Besides an increased

colloidal stability in physiological conditions, the proposed targeting advantage was confirmed by an

in vitro uptake profile and in vivo gene silencing effect which was positively correlated with CD44

receptor density. Crosslinked hyaluronic acid-chitosan oligomer (HA-CSO) PECs were synthesized by

de la Fuente et al. to improve the gene delivery potential in the cornea by prolonging residence time

of gene delivery NPs on the ocular mucosa due to HA’s muco-adhesive nature [172]. The particles in

this study were prepared by an ionotropic gelation technique, where CSOs were mixed with a

solution containing HA, TPP and the desired pDNA. Furthermore, the authors propose that HA might

influence the intracellular trafficking of the gene carriers towards the perinuclear region of the cell,

given previously published data by Tammi et al. [173] and Evanko et al. [174]. This premise was

further studied by Contreras-Ruiz et al. by visualizing the uptake of the same crosslinked HA-CSO

38

hydrogels using live cell fluorescence microscopy [175]. It was shown that the NPs were effectively

taken up in an HA receptor-specific process. Further experiments with chemical inhibitors of

endocytic processes demonstrated that the particles were not taken up by clathrin-mediated

endocytosis, but rather by caveolin-mediated endocytosis, which was confirmed by colocalization

studies of NPs with caveolin [175]. As it has previously been reported that caveolin-dependent

endocytosis bypasses the lysosomal degradation pathway and delivers its cargo near the Golgi

apparatus or in the perinuclear region [176], the authors postulate the idea of HA as a targeting

ligand not only for HA-specific cell receptors, but also for intracellular compartments (Figure 8).

4.3.2. Hyaluronic acid-based conjugates

For a more stable and permanent availability of HA on the nucleic acid vector, some groups prefer to

covalently attach an HA-molecule to the building blocks of the NP before the self-assembly. Several

HA-conjugates have already been described in the literature. For example, Takei et al. conjugated

low Mw HA to PLL by a reductive amination reaction using sodium cyanoborohydride (NaBH3CN) as a

reductant [177]. The resulting copolymer was able to efficiently complex pDNA due to the cationic

PLL. The HA bestowed the carrier with in vivo HARE receptor-specific targeting properties, as

demonstrated by the enrichment of particles at liver sinusoidal endothelial cells (LSECs) after

intravenous injection in animal models. Usually though, the carboxyl-groups on the HA are activated

with EDC in acid buffer to form a highly reactive O-acylisourea, which can easily react in a basic

medium with a free amine to form a covalent amide-linkage. These free amine groups are frequently

encountered on most known non-viral polymeric nucleic acid vectors, such as PEI. By conjugating PEI

on a HA backbone via amide-bonds, Jiang et al. envisioned a non-viral nucleic acid carrier with the

target-specific and biocompatible advantages of HA, whilst retaining the ability to escape the

endosomes via the buffering effect of the amine-containing PEI [178].

39

Figure 8: Internalization pathway for hyaluronic acid (HA) – chitosan oligomer (CSO) NPs, as proposed

by Contreras-Ruiz et al.. The HA on the particles interacts with CD44 receptors in the plasma

membrane, triggering the caveolae-mediated internalization of the NPs. From the caveosome, NPs

are sorted to the endoplasmic reticulum (ER) and then to the nucleus, bypassing the lysosomal

degradation pathway. CD44 receptors and caveolin are then recycled and carried back to the

membrane through the Golgi network. The authors therefore envision HA as a targeting ligand not

only for the CD44 receptor, but also for the caveolae-mediated endocytic pathway. Reproduced with

permission from ref [175].

40

These conjugates were able to efficiently complex siRNA by means of electrostatic interactions with

the amine groups of PEI. The authors demonstrated the HA receptor-specific binding in vitro with

preferred uptake and enhanced silencing effects in LYVE-1-expressing cells (B16F1 cells) compared to

HEK 293 cells, which do not express HA-specific receptors.

To determine how the chemical conjugation of PEI to HA would affect its receptor-specificity, the

authors investigated in a follow-up study the receptor-mediated uptake of QD-modified HA in

different mol %, and showed an increased accumulation of QD-HA conjugates in the liver up to 35

mol % modification, and a non-targeted systemic distribution with higher mol % modifications

[178,179]. Less mol % modification with PEI resulted in the inability of the conjugates to form

complexes with siRNA due to the shortage of positive groups, which was demonstrated in their first

study [178]. To complement the in vitro targeting studies of the HA coated NPs, Jiang et al. further

investigated the particles in terms of in vivo targeting. After injection in xenografted C57BL/6 mice,

the authors note that the HA coated particles preferably accumulate in tissues with HA receptor-

expressing cells, such as the liver, kidneys and tumors, in agreement with in vivo data on the QD-HA

conjugates [179]. The siVEGF-PEI/HA particles were significantly better at suppressing tumor growth

compared to uncoated siVEGF/PEI complexes [180]. Aside from the main advantage of HA receptor

specificity, the authors also noted that the HA PEI conjugates showed less cytotoxicity in vitro

compared to PEI alone, yet still more cytotoxic than HA alone.

To further optimize the nucleic acid carrier, the group proposed to use low Mw PEI crosslinked with

cystamine bisacrylamide (CBA). This disulfide-linked PEI is known to be less cytotoxic because of its

biodegradability in reductive environments, and Park et al. examined if an HA/SS-PEI conjugate

would still show the same targeting abilities. These optimized particles were employed in in vivo

studies for tumor therapy with anti-VEGF-siRNA and to treat liver cirrhosis with anti-transforming

growth factor (TGF)-β siRNA targeted to the liver [181,182]. In a first study, the authors

demonstrated an enhanced therapeutic effect of the siVEGF/SS-PEI/HA-nanoparticles after

intratumoral injection compared to siVEGF/SS-PEI-particles, indicating the preservation of tumor-

41

specific targeting (Figure 9) [182]. In a follow-up study, a therapeutic effect was noticed after

intravenous injection, which they attribute in part to the maintained targeting effects towards

cirrhotic livers, yet also to the decreased unspecific interactions with blood proteins and longer

circulation time on the other.

Figure 9: Anti-tumoral therapeutic effect of anti-VEGF-siRNA (siVEGF)/(PEI-SS)-b-HA complex in

female balb/c mice where CT-26 colon cancer cells were injected for tumor inoculation and growth.

(A) Tumor volume change with increasing time after intratumoral injection of a control of 5% glucose

solution, siVEGF/PEI-SS, non-specific Luc siRNA (siLuc)/(PEI-SS)-b-HA, and siVEGF/(PEI-SS)-b-HA

complexes. The treatments were performed three times after 8, 11, and 14 days. The results

represent mean ± SD (n = 3). (B) Photo-images of dissected tumor tissues after 20 days. Both in (A)

and (B), the increased therapeutic effect of HA-containing particles is clearly seen. Reproduced with

permission from [182]. Copyright Elsevier.

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Figure 10. In vivo biodistribution of Cy5.5-labeled particles consisting of 5β-cholanic acid

(CA)/hyaluronic acid (HA)-conjugates. 2, 6 and 10 denote the degree of substitution (DS), defined as

the number of CA molecules per 100 sugar residues of HA. (A) In vivo non-invasive fluorescence

imaging of HA-NPs in tumor-bearing mice. Time-dependent whole body images of athymic nude mice

bearing SCC7 tumors after intravenous injection of HA-NPs. Arrows indicate the sites of tumors. (B)

Ex vivo fluorescence images of normal organs and tumors collected at two days post-injection of HA-

NPs. (C) Quantification of the ex vivo tumor targeting characteristics of HA-NPs in tumor-bearing

mice. Error bars represent standard deviation (n = 3). Reproduced with permission from [183].

Copyright Elsevier.

This enhanced biodistribution of HA-coated particles was correspondingly visualized in a study by

Choi et al., where HA-nanoparticles were intravenously injected in tumor-bearing mice and analyzed

at different time-points (Figure 10) [183]. In contrast to the HA/polycation-conjugates used by Park et

al., the HA particles in the study of Choi and coworkers had a hydrophobic segment incorporated,

such as 5β-cholanic acid, to further ensure the spontaneous self-assembly of core/shell-structured

nanoparticles. This has already been used in a drug delivery setting, where the authors hoped the

HA-shell would confer colloidal stability and mobility in the vitreous humour, the extracellular matrix

43

in the center of the eye [184]. Here, hydrophobic small molecule drugs could easily be loaded in the

hydrophobic core, but in the context of nucleic acid delivery the loading of hydrophilic nucleic acids

could pose a problem. Nevertheless, there have been reports of hydrophobation of nucleic acids with

CTAB [185], enabling its complexation in the hydrophobic core, although the possible toxic effects of

CTAB [146] was not further investigated in this study. A more elegant approach was proposed by

Shen et al., who added a cationic spermine segment to the HA block copolymers for the

complexation of siRNA [186]. In their study, the authors wanted to elucidate the uptake mechanism

of HA coated particles, and found that they were mostly endocytosed by caveolae-dependent

endocytosis. These findings nicely concur with the proposed hypothesis of Contreras-Ruiz and

colleagues to employ HA as a targeting ligand towards the caveosome [175]. Nevertheless, a

different study by Zaki et al. reported on the clathrin-dependent uptake mechanisms of both

uncoated and HA-coated nucleic acid NPs [187]. The differences between the findings of Zaki et al. on

the one hand [187], and Shen et al. [186] and Contreras-Ruiz et al. [175] on the other, could be

explained in part by the different cell types used or the size of the particles. Indeed, despite the fact

that the findings of Contreras-Ruiz et al. [175] agree with those of Shen et al. [186], the latter authors

attributed the caveolae-dependence to the size of the particles rather than claiming HA can be used

as a targeting ligand to an intracellular compartment. It has been reported that particle size can

influence the uptake pathway where larger particles around 200 – 500 nm in size would preferably

be endocytosed in a caveolae-dependent manner [188]. In short, the intracellular targeting of NPs

can’t solely be attributed to the presence of HA and regarding the previously mentioned studies,

some other remarks should also be taken in consideration. When interpreting data from chemical

endocytosis inhibitor experiments, care should always be taken. Aside from the risk of eliciting

cytotoxicity, the effects of these inhibitors are usually poorly characterized, non-specific and cell

type-dependent [11,189], all of which might influence the findings of the previously mentioned

studies.

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4.3.3. Hyaluronic acid core-shell particles

The conflicting results between caveolae-dependent endocytosis [175,186] and clathrin-dependent

endocytosis [187] could also be attributed to a different particle structure. Instead of incorporating

HA in a PEC or a covalently attached micelle-coat, Zaki et al. employed an electrostatic “post-coating”

strategy, where HA is added to a preformed crosslinked chitosan/TPP core to ensure HA availability

by means of an outer HA-shell. Indeed, this electrostatic complexation could prove beneficial, as it

had been suggested by Kim et al. that chemical modification could disturb the functionalities of

native HA [171]. Moreover, because the negative charge density of HA is not high enough to displace

the electrostatic bonds between the nucleic acids and the polymer, HA would be unable to displace

the nucleic acids or even disturb supramolecular self-assembled PECs [190]. Electrostatic coating of

preformed cationic PEI/pDNA polyplexes was documented by Hornof et al. [191], who justified a low

Mw HA coating by facilitating uptake in human corneal epithelial (HCE) cells via CD44-mediated

endocytosis. Wang et al. employed the same strategy to improve stability in physiological conditions

and specifically target HA receptor-expressing cells [192]. Again, HA was added to preformed

PEI/pDNA binary polyplexes, resulting in negatively charged ternary complexes which displayed

increased stability in physiological salt media compared to the uncoated polyplexes. Furthermore,

shielding of the polyplexes with HA resulted in a significant increase in transfection efficiency in HA

receptor-expressing HepG2 cells, while a decreased transfection efficiency was observed in HEK 293T

cells. Interestingly, the significant rise in transfection efficiency in the HepG2 cells could not be

related to an increased uptake. The lack of correlation between uptake and transfection efficiency

had already been reported by a previous study of Ruponen et al. [190], where it is suggested that

downstream intracellular processing mechanisms might contribute more to the final transfection

efficiency than uptake alone. Wang et al. attributed the increase in transfection efficiency to the

relaxation of DNA/polycation interactions which would allow the gene transcription machinery to

more easily reach the complexed DNA [192], a hypothesis previously postulated by Ito et al. [193]. In

45

their study, the authors describe the formation of ternary HA coated PEI/pDNA complexes and

specifically investigate the influence of HA on transcription of complexed DNA. They observe with in

vitro assays that HA increases both DNA transcription as well as DNA relaxation and conclude that HA

might display high mobility group (HMG)-like properties, which enhance transcription by granting the

transcription machinery easier access to the genes. Similarly, Xu et al. noticed an increase in

transfection efficiency independent of uptake when evaluating ternary HA/CLPEI/pDNA complexes in

NIH/3T3 cells [194]. These polyplexes consist of pDNA complexed by low Mw PEI-fragments

crosslinked with disulfide linkages (CLPEI), which are electrostatically coated with HA for increased

stability in the extracellular environment. The authors hypothesized that increase in transfection

efficiency was because of the HA which helps DNA unpacking triggered by the intracellular CLPEI

degradation, by further loosening the interactions between complexed DNA and low Mw polycations.

Likewise, it was observed by de la Fuente et al. that the combination of HA and low Mw CSOs in their

hydrogels resulted in very high transfection efficiencies, which was also explained by the proposed

role of HA as a transcription activator and facilitator of DNA unpacking [172].

Instead of polymers, other commonly used NA vectors are cationic liposomes [2]. The group of Dan

Peer has focused on the use of HA by covalently attaching it to the surface of preformed liposomes.

They have previously shown that liposomes with mitomycin D preferentially accumulate at CD44-

overexpressing tumor tissue when coated with high Mw HA [161]. They noticed that the effect of HA

is two-fold: (1) HA acts as a targeting ligand in vitro to cells overexpressing hyaluronan receptors; (2)

HA provides the particles with a longer circulation time in vivo by eluding unspecific interactions with

blood components. What is more, the authors discovered a new advantage of HA, being its use as a

cryoprotectant [162]. In a later study, the liposomes coated with HA are used for the in vivo delivery

to leukocytes of siRNA against cyclin D1 [195]. The targeting to the leukocytes was not done by using

the HA as a ligand, rather HA was used as an easily modifiable intermediate to graft targeting

antibodies. Furthermore, HA was used for liposomal stability during rehydration after lyophilization

and to prolong the circulation time in vivo.

46

Some groups argue that a post-coating as described before, where HA is covalently attached to

preformed liposomes, does not grant enough control over loading density which is deemed highly

necessary for proper targeting [166]. By conjugating high Mw HA molecules on DOPE lipids via an

EDC-facilitated amide-bond, liposomes with an outer HA shell could be made with sufficient control

of HA loading. These liposomes were evaluated in terms of delivery of both pDNA [166] and siRNA

[196]. Surace et al. prepared liposomes consisting of cationic lipid [2-(2,3-didodecyloxypropyl)-

hydroxyethyl] ammonium bromide (DE) and DOPE, complexed with pCMV-Luc plasmids [166]. These

lipoplexes were then evaluated in CD44-expressing MDA-MB-231 cells, as well as in MCF-7 control

cells with very low CD44 expression (Figure 7). Taetz et al. used DOTAP:DOPE liposomes to validate

HA receptor-targeted gene silencing of the HA coating in CD44-expressing A549 cells and in non-

hyaladherin-expressing Calu-3 cells [196]. Both studies demonstrated a very low toxicity in all cell

lines, attributed to the negative surface charge. Also, the targeting properties of the NPs were

confirmed by an increased transfection efficiency in CD44-expressing cells, compared to a lower

transfection in the control cell lines.

In conclusion, the use and advantages of HA in the field of nucleic acid delivery are being actively

investigated in combination with many known nucleic acid delivery vectors and in many different

particle structures. Therefore, it should be noted that every method has its own merits and pitfalls,

yet a clear consensus on NP structure has not been found yet.

4.4.Influence of hyaluronic acid molecular weight

In the wealth of literature concerning the use of HA as an aid in drug or nucleic acid delivery, a lot of

contradictory findings have been documented. As previously stated, this could be explained in part

by a different NP structure or the different cell types used. However, the Mw of the HA molecules

should also be taken into account, because it has been documented that the functions of free HA in

the body is mostly determined by its Mw [197]. For example, high Mw HA chains are non-

immunogenic whereas low Mw HA oligomers are suspected to elicit immune-responses. It has also

47

been documented that HA oligomers have a tumor-suppressive ability [156], whereas HA of a

different Mw are thought to have a general stimulating effect of tumor growth [155]. These effects

are presumably modulated by HA-binding receptors overexpressed in the tumors, indicating the

interaction of HA with hyaladherins is also dependent on Mw of the HA molecule [18]. Yet, as a

targeting ligand towards the CD44 receptor, HA has been used in its low Mw form [171,191], its high

Mw form [166,196] and a middle Mw form [170]. Some groups acknowledge the alleged effect of

Mw on HA functions and have investigated accordingly. Hornof et al. have tried to optimize an

electrostatically shielded particle with different Mw’s of HA (<10 kDa, 10 – 30 kDa, 30 – 50 kDa)

[191]. The authors find that a low Mw coating (<10 kDa) is most adept at providing the carriers both

with colloidal stability in physiological media as with a targeting function. However, the HA fractions

used in this study only cover a very small part of the entire range of Mw, mostly in the low Mw range.

A more comprehensive study by Mizrahy et al. compared 5 different fractions, ranging from low Mw

6.4 kDa HA to high Mw 1500 kDa HA, for the effects on innate immune response and CD44 targeting

[198]. Contrary to the previous findings, they document that the binding affinity towards the CD44

receptor is increased with increasing HA Mw. In conclusion, because it is generally accepted that the

different functions of HA in the body are solely regulated by its Mw, this factor should not be

overlooked when employing HA in a drug or nucleic acid delivery setting.

5. Cyclodextrin-based nucleic acid nanotherapeutics

5.1.Introduction to cyclodextrins

Cyclodextrins (CDs) can be defined as naturally occurring cyclic oligosaccharides, consisting of -1,4

linked D-glucopyranose units (Figure 2), that are produced by enzymatic conversion of starch. CDs

are characterized by a typical amphiphilic topology, with a hydrophilic exterior and an inner

hydrophobic cavity, which endows CDs with the capacity of forming molecular inclusion complexes

with hydrophobic ‘guests’ [199]. Depending on the number of glucose building blocks, one can

distinguish between a hexameric (-CDheptameric (-CD and octameric (-CD) form. In a

48

pharmaceutical context, CDs have been frequently applied for the formulation of poorly water-

soluble drugs, to improve drug stability or to enhance drug permeability across biological membranes

[200]. However, a wealth of scientific reports available in the literature describes the implementation

of native or chemically modified CDs in nanocarrier design to improve the formulation of

biomacromolecules or enhance their delivery across the extra-and intracellular barriers. More than a

decade ago, a body of evidence already indicated the potential applications of CDs in therapeutic

formulations of peptides, proteins and oligonucleotides [201,202]. It was shown that the

complexation with CDs entailed improved cellular delivery, resistance to endonucleases and reduced

immunogenicity [199,200,202]. Currently, CD containing delivery systems, mostly composed of CD

containing polymers, are of interest for gene and siRNA delivery. Besides cationic CD polymers, also

CD-based polyrotaxanes, CD-based dendrimers and monodisperse CD derivatives have been used for

pDNA and/or siRNA delivery, which will not be discussed in further detail here. Recent

comprehensive reviews specifically on CD-mediated drug delivery are available [199,200,203].

5.2.Cyclodextrin polymer nanoparticles

The concept of using CD-containing polymers (CDPs) as drug carriers already exists for several

decades and they have been explored in the field of gene delivery since 1999 [200,204]. The general

added benefit of incorporating a CD moiety in a pharmaceutical nanocarrier is two-fold [199]: (1) CDs

have the ability to act as membrane absorption enhancers and (2) the unique feature to form CD

molecular inclusion complexes enables multifunctional surface modification of nanocarriers to

improve in vivo stability and cellular targeting, the latter being of particular interest in relation to NA

NPs. CDPs in gene therapy can be structurally divided in polymers with CDs in their backbone or pre-

existing polymers to which CD derivates are grafted. A general feature of these polymers is that they

are positively charged to allow electrostatic complexation with nucleic acid therapeutics.

49

5.2.1. Polymers with cyclodextrin backbone

The group of Mark E. Davis was the first to synthesize CDPs with polycationic signature for gene

delivery [200,205-209] and significant advances have been made since then. The cationic CDPs were

able to condense pDNA (~5 kbp) into nanosized polyplexes (CDPlexes) that achieved equal in vitro

transfection efficiencies compared to PEI and Lipofectamine™ [204]. Further optimization of the

polymer structure and its transfection activity was attained by capping the polymer ends with

imidazol groups [210]. It is hypothesized that this modification bestows the CDPlexes with endosomal

buffering capacity that would allow enhanced endosomal escape of the transgene complex on

account of the proton-sponge effect [211]. Moreover, the authors claim that the imidazol-modified

CDPlexes show enhanced intracellular unpackaging which could also contribute to the observed

improvement in transgene expression [210]. To optimize CDPs for systemic administration, it was

demonstrated by Pun and Davis that CDPlexes built from linear cationic CDPs and pDNA could be

furnished with a PEG hydrophilic shell. This was achieved through the addition of adamantane

modified PEG chains (Ad-PEG) to preformed CDPlexes, resulting in the formation of Ad-CD inclusion

complexes at their surface. PEGylation of polyplexes prevents their aggregation in the systemic

circulation and minimizes non-specific interactions [2]. In addition, targeting of specific cell types can

be made possible by coupling receptor ligands to the distal end of the Ad-PEG chains. CDPlexes

coated with galactosylated Ad-PEG were specifically internalized by hepatoma cells through

receptor-mediated endocytosis via the asialoglycoprotein receptor [212]. Likewise, the use of

transferrin (Tf) as a targeting ligand enables efficient internalization of CDPlexes in malignant cells

overexpressing the transferrin receptor [213]. Davis and coworkers also pioneered the translation of

this concept towards siRNA delivery (Figure 11) [214-216]. Ad-PEG-Tf modified CDP complexes with

siRNA (siCDPlexes) targeting the ribonucleotide reductase subunit M2 (RRM2) showed significant

tumor growth inhibition in murine models [217-220] and dose-escalating studies in non-human

primates demonstrated that multiple systemic NP administrations, with doses up to 9 mg siRNA/kg,

were well tolerated. The delivery concept was termed RONDEL™ (“RNAi/Oligonucleotide

50

Nanoparticle Delivery”, Calando Pharmaceuticals) and was applied in the first in-human phase I

clinical trial with a targeted siRNA containing NP, administered to patients with solid cancer

refractory to standard-of-care therapies [221]. Targeted NPs were detected in post-treatment tumor

biopsy sections and significant RRM2 downregulation was observed both on the mRNA and protein

level. Moreover, in one patient sample 5’-RLM-RACE (RNA Ligand Mediated – Rapid Amplification of

Complementary DNA Ends) PCR demonstrated the presence of an RNAi-specific RRM2 mRNA

cleavage fragment, indicating that the in vivo delivered anti-RRM2 siRNA can effectively activate the

RNAi pathway [221].

Figure 11. (A) Chemical structure of -cyclodextrin containing polycation, end-capped with imidazol

groups (CDP-im), for in vivo delivery of siRNA and pDNA. (B) Schematic representation of the ionic

self-assembly of siRNA and CDP-im into nanosized particles and their functionalization with

poly(ethylene glycol) (PEG) chains and targeting ligands (e.g. transferrin, Tf) via the formation of

inclusion complexes with adamantane (Ad) (RONDEL™ delivery technology). Adapted from ref

[214,221].

51

Likewise, this delivery platform was exploited by Brahmamdam et al. in a murine cecal ligation and

puncture (CLP) model of sepsis. Immune suppression through apoptotic loss of competent immune

effector cells significantly contributes to morbidity and mortality in this pathology. In vivo delivery of

RONDEL™ formulated siRNA to splenic CD4+ T and B lymphocytes, targeting the cell death proteins

Bim and PUMA, could significantly diminish the apoptotic depletion of these immune cells and

reversed sepsis-induced immune suppression [222]. Regardless of PEGylation however, it was

demonstrated in mice, monkeys and humans that the siRNA/CDP NPs are rapidly cleared from the

circulation [215,220,223]. The different components of the RONDEL™ delivery technology self-

assemble into particles with a size ~70 nm, large enough to avoid renal filtration. Additionally, the

presence of a PEG corona should suffice to prevent rapid uptake by the MPS and dissociation in the

systemic circulation. In a recent report, Zuckerman et al. explained this seemingly paradoxical

clearance phenomenon by a more detailed look into the renal filtration barrier [220]. Apparently,

sub-100 nm polyplexes may extravasate across the glomerular fenestrated endothelium and

disassemble at the glomerular basement membrane (GBM), through interaction with the abundant

negatively charged proteoglycans (e.g. heparan sulfate). The released siRNA can subsequently be

filtered into the urinary space. Interestingly, the authors hypothesize that this clearance mechanism

maybe of general importance for cationic siRNA polyplexes that are small enough to cross the

glomerular endothelium [220].

Focusing on the intracellular delivery potential and with the aim to enable photo-induced

spatiotemporal control on the RNAi effect, Bøe et al. investigated the influence of PCI on the gene

silencing performance of siRNA CDPlexes, previously developed by the group of Mark Davis [224].

The authors chose to work with the unmodified CPDs, lacking the endosomolytic imidazol groups, to

maximize light-directed control over the intracellular siRNA delivery. With photochemical treatment

a gene knockdown of ~90% was obtained at the mRNA level in two different cell lines, while the RNAi

effect was nearly absent when PCI was not applied. Given that PCI treatment is under investigation

52

for in vivo anti-tumor applications [225], the authors foresee that combining unmodified siRNA

CDPlexes with PCI could be an attractive strategy for systemic light-directed tumor targeting [224].

CDPs have also been modified with OEI units to enhance pDNA delivery. Linear high Mw CDPs were

constructed by Srinivasachari and Reineke through Cu(I)–catalyzed azide-alkyne 1,3 dipolar

cycloaddition (“click reaction”), linking a diazo--cyclodextrin monomer with a series of dialkyne-

oligoethyleneimine monomers. The resulting CDPs achieved good reporter gene transfection

efficiency in HeLa cells [226]. In another report, OEI units (OEI600) were crosslinked with 2-

hydroxypropyl (, or )-cyclodextrin following their activation with carbonyl diimidazol (CDI). The

resulting polymers demonstrated a lower toxicity and comparable or higher transfection efficiency in

SKOV-3 human ovarian carcinoma cells or SK-BR-3 human breast cancer cells compared to bPEI (25

kDa) [227,228]. An interesting feature for these polymers is their biodegradability by virtue of the

carbamate linkages, ensuring lower polymer toxicity [199]. The -hydroxypropyl-CD-OEI600 polymer

was further modified with an oligopeptide (MC-10) targeting the human epidermal growth factor

receptor 2 (HER2), that is frequently overexpressed on breast and ovarian cancer. Targeted polyplex

formulations with the interferon- (INF- gene could transfect SKOV-3 cells in vitro and in vivo,

resulting in an enhanced anti-tumor effect in SKOV-3 tumor-bearing mice, compared with the non-

targeted formulation [229]. Likewise, a folate-targeted polyplex formulation, based on -CD-OEI

grafted with folic acid, could mediate gene expression in melanoma-bearing mice with a comparable

efficiency as adenoviral transduction [230].

5.2.2. Cyclodextrin modifications of pre-existing polymers

Instead of a polymeric CD backbone, several research groups have described the attachment of CD

moieties to pre-existing (cationic) polymers. This chemical engineering strategy allows to better

modulate their gene transfer potential and/or inherent toxicity. In addition, this again provides a

platform to include additional functionalities often required for in vivo use [199]. Among this class of

CDPs, PEI modifications obviously take a prominent position, with PEI being the gold-standard in non-

53

viral gene delivery. However, as mentioned before, the use of PEI as a transfection reagent is

severely hindered by its high cytotoxicity. It was the group of Mark Davis that first investigated the

effect of CD grafting on commercially available linear and branched PEI in depth [231]. Transgene

expression in PC3 cells was reduced as a function of increased CD attachment for both PEIs, while it

mitigated their in vitro toxicity. The inclusion of Ad-PEG in the formulation proved to be essential to

maintain NP colloidal stability at physiological salt concentration. Intriguingly, the PEGylated CD-

grafted linear PEI achieved better in vitro gene transfection than linear PEI alone and could induce

liver gene expression following systemic administration in mice [231]. An elegant example of the

versatility of employing CDs in NP design was given by Zhang et al., who reported on the

supramolecular inclusion-driven self-assembly of NPs with a core-shell architecture [232]. The

nanoassemblies are composed of -CD grafted PEI and poly(-benzyl-L-aspartate) (PBLA), with PBLA

microdomains forming the hydrophobic core that is surrounded by a positively charged PEI

hydrophilic shell. The self-assembly into NPs is driven by the formation of inclusion complexes

between the hydrophobic benzyl groups and the pending CD moieties. The amphiphilic character of

the resulting NPs could be harnessed for the combined drug delivery of small hydrophobic drugs,

encapsulated in the hydrophobic center, and hydrophilic macromolecular drugs (e.g. proteins and

nucleic acids), complexed to the hydrophilic surface. This dual drug delivery concept was

demonstrated with dexamethasone and luciferase encoding pDNA in an osteoblast cell line [232].

Besides PEI, the grafting of CD units has also proved valuable to enhance the gene delivery potential

of other cationic polymers, such as poly-L-lysine (PLL) [233] and chitosan [234]. The group of Maria

Alonso proposed hybrid polysaccharide nanocarriers, containing both chitosan and cyclodextrin

building blocks, for gene delivery to the pulmonary epithelium. The particles were constructed by

TPP induced ionic gelation of native chitosan in the presence of anionic -cyclodextrin derivatives and

pDNA. The hybrid particles, prepared with low Mw chitosan (~10 kDa), succeeded in enhanced gene

expression in differentiated Calu-3 cells when compared to the conventional chitosan formulation

[234].

54

As a final example, the CD derivatization of neutral instead of cationic polymers has been put forward

by Kulkarni et al. in the context of siRNA and pDNA delivery [235,236]. They reported the

development of a degradable cationic polymer construct based on the self-assembly of

monosubstituted cationic -CD (amino--CD+) derivatives with a poly(vinyl alcohol) (PVA) polymer

backbone bearing PEG2000 and cholesteryl or adamantane modified grafts. The multivalent host-guest

interactions of the amino--CD+ with the pendant Chol/Ad-PVA-PEG enables electrostatic

complexation with nucleic acids into stabilized nanosized particles. The hydrophobic ‘guest’ anchors

are coupled to the PVA-PEG polymer via an acid-labile acetal linkage that can be hydrolyzed in

endolysosomal compartments following internalization by the target cells, mediating complex

disassembly. Polyplexes formed with siRNA or pDNA achieved comparable or higher biological

activity in vitro compared to 25 kDa bPEI or Lipofectamine 2000 while being far less toxic [235,236].

6. Other polysaccharides in nucleic acid nanotherapeutics

The polysaccharides discussed above are notable to many research groups involved in gene therapy.

Nonetheless, other less well-know polysaccharides may also be beneficial toward nucleic acid

delivery. This section aims to cite some remarkable contributions to gene transfer and/or gene

silencing made by -glucan, alginate, arabinogalactan, pullulan and pectin-based nanotherapeutics.

6.1.-Glucans

-Glucans are a heterogenous group of carbohydrates built from repeating D-glucose units, linked

together via -glycosidic bonds, and are primarily found in the cell wall of fungi [237]. As mentioned

before for other polysaccharides, many research groups also pursued cationic modification of the

original -glucan polysaccharide, e.g cellulose [238,239] and shizophyllan [240,241], to enable

electrostatic complexation of therapeutic nucleic acids. However, -1,3-glucans such as curdlan,

lentinan and shizophyllan, also have the intrinsic ability to form macromolecular complexes with

55

homopolynucleotides based on hydrogen bonding [242], a feature that was recently adopted by

Takedatsu et al. for the delivery of antisense oligonucleotides to macrophages [243]. These authors

employed schizophyllan (SP), that consists of a -1,3-D-glucan main chain with one -1,6-D-glucosyl

side chain every three glucose units (Figure 2). SP exists as a single chain at alkaline conditions and

adopts a triple helix structure at neutral pH. When neutralization of an alkaline solution of SP occurs

in the presence of homopolynucleotides, a hybrid triple helix can be formed in which two SP main

chain glucoses interact with one polynucleotide base [242,243]. Although SP can only hybridize with

homopolymeric sequences, the authors could form complexes of SP with functional AsON by

endowing the latter with a poly(dA) tail. -1,3-D-glucans are selectively bound by the pattern

recognition receptor (PRR) dectin-1 present on the surface of APCs and subsequently internalized by

phagocytosis. Complexation of AsON with SP would therefore enable their selective delivery to

macrophages, which are attractive target cells due to their role in promoting pathogenic

inflammation in several immune-related diseases such as atherosclerosis and inflammatory bowel

disease (IBD). Takedatsu and coworkers demonstrated the delivery of AsONs targeting the

macrophage-migration inhibitory factor (MIF) via SP complexation to CD11b+ macrophages, leading

to effective MIF suppression. Moreover, intraperitoneal administration of the antisense MIF/SP

complexes could attenuate intestinal inflammation in a murine dextran sodium sulphate (DSS)-

induced colitis model [243].

Besides -glucan polymer chains, also microparticulate -glucan shells have been exploited for nucleic

acid delivery. Aouadi et al. impressively demonstrated oral siRNA delivery to macrophages of the gut-

associated lymphatic tissue (GALT) to be able to downregulate pathogenic inflammatory responses.

Phagocytosis was mediated by formulating the siRNA in micrometer-sized -1,3-D-glucan shells

(GeRPs) obtained by solvent extraction of baker’s yeast. The GeRP core consisted of yeast tRNA,

Endo-Porter [244] (EP), PEI and siRNA in a layer-by-layer format to assist with cytoplasmic siRNA

release triggered by phagosomal acidic pH. Orally delivered GeRPs were able to systemically silence

TNF- in macrophages recovered from several MPS related tissues, since GALT associated

56

macrophages may disseminate from the gut. Moreover, silencing of the mitogen-activated protein

kinase kinase kinase kinase 4 (Map4k4) via oral gavage of GeRPs could protect mice from a lethal

dosis of lipopolysaccharide (LPS) owing to inhibited production of TNF- and IL-1.

To simplify this complex five-component GeRP formulation, in a follow-up study only the EP

amphipathic peptide was used to complex the siRNA in the GeRP core [245]. EP was identified as the

critical component in this formulation, enabling siRNA entrapment based on electrostatic interaction

and potentiating phagosomal destabilization. Moreover, omitting PEI from the original GeRP

formulation is believed to be beneficial to constrain in vitro and in vivo toxicity [245]. It has to be

noted that the specific interaction of (particulate) -1,3-glucans with membrane-bound and cytosolic

pattern recognition receptors (PRRs), such as dectin-1 and NLPR-3 inflammasome, may trigger a pro-

inflammatory response [246,247]. Although this may promote their usefulness as adjuvants in

(genetic) vaccination strategies [237,248], in part it also contradicts their application as delivery

agents of anti-inflammatory therapeutics. Nonetheless, in the above-mentioned reports no effects

were observed in control experiments with neither uncomplexed shizophyllan [243] nor empty -1,3-

D-glucan shells [249].

6.2.Alginate

Alginate is an anionic naturally occurring polysaccharide that can be obtained from brown algae and

bacteria such as Azotobacter and Pseudomonas [250]. Alginate is defined as a linear block copolymer

composed of regions with consecutive -1,4-D-mannuronic acid residues (M-blocks), -L-guluronic

acid residues (G-blocks) and alternating M and G residues (MG-blocks) (Figure 2). The copolymer

composition and its Mw may vary significantly with source and species. Because of its

biocompatibility, low toxicity and muco-adhesiveness, also alginate is an attractive polymer for

biomedical applications [14]. In addition, in analogy with chitosan, alginate can form 3D hydrophilic

networks (hydrogels) via ionic gelation induced by divalent cations (e.g. Ca2+), an attractive feature

toward controlled drug release applications. Gel formation predominantly involves the association

57

between G-blocks on adjacent polymer chains in the presence of divalent cations as ionic crosslinker.

Alginate-based macro-and microscopic hydrogels have mainly been investigated for controlled small

molecule and protein delivery in the context of wound healing and tissue regeneration [250]. A

limited number of reports also describes alginate-based nanostructures as nucleic acid carrier.

In accordance with reports on e.g. HA and DS, alginate has also been exploited in pursuit of

alleviating PEI mediated cytotoxicity [251]. Patnaik et al. showed successful gene and siRNA

transfection in various cell lines in vitro with PEI-alginate nanocomposites composed of different Mw

PEI (25 kDa or 750 kDa) [252,253]. For transfection purposes, the obtained PEI-alginate

nanocomposites were complexed with pDNA or siRNA. To PEGylate the complexes, PEG4000-

bis(phosphate) was incorporated into the amalgam wherein 10% of the PEI amine groups ionically

interact with the anionic phosphate end-groups on the homobifunctional PEG. At an optimal alginic

acid content, the nanocomposites outperformed native PEI while being almost non-toxic [252,253]. It

should however be questioned in future work to what extent these electrostatic complexes will hold

together in biological media before proceeding toward in vivo studies. Jiang et al. endowed

preformed PEI/DNA polyplexes with an alginate coat and demonstrated that this enhanced reporter

gene expression in vivo in comparison to the uncoated complexes [254,255]. More recently, He et al.

also reported on PEI-alginate conjugates by grafting 2 kDa PEI to an alginate backbone with the

corresponding aim to optimize the balance between efficiency and toxicity [256].

Making use of alginate’s ability to form a crosslinked gel-like structure, Shardool and Mansoor

encapsulated pDNA encoding the anti-inflammatory murine IL-10 into alginate NPs with a 60%

loading efficiency. The negatively charged surface of the particles was electrostatically modified with

a tuftsin targeting peptide sequence (TKPR) via a hexameric L-arginine motif preceeding a tetrameric

L-glycine spacer. Tuftsin is known to stimulate receptor-mediated phagocytic uptake and serves to

direct the DNA loaded alginate NPs to macrophages. Transfecting IL-10 in murine J774A.1

macrophages with the tuftsin modified particles could significantly block subsequent LPS stimulated

TNF- expression.

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6.3.Arabinogalactan, pullulan and pectin

In addition to the dextran-spermine conjugates [61], described under 2.3.2., the group of Y.

Barenholz also reported on the grafting of spermine to an arabinogalactan backbone [58,257].

Opposite to dextran-spermine conjugates, the arabinogalactan-spermine derivatives however failed

to induce substantial transgene expression. The authors attempt to explain this disparity by pointing

at the difference in polysaccharide structure. While dextran is a linear -glucan, arabinogalactan is a

highly branched natural polysaccharide, composed mainly of galactose and arabinose residues.[14]

The branching structure may be responsible for the low level of spermine conjugation observed in

arabinogalactan compared to dextran. Importantly, this will also entail a decreased percentage of

secondary amines in arabinogalactan, interfering with the polymers ability to induce a proton sponge

effect following endocytic uptake [257].

Pullulan is known as a neutral and non-toxic exopolysaccharide of fungal origin composed of -1,6-

linked maltotriose units. It is produced from starch by the fungus Aureobasidum pullulans. Pullulan

has a unique linkage pattern that contributes to its adhesiveness and ability to form fibers and

biodegradable films. These characteristic physicochemical properties explain its use in food and

cosmetic industry as well as various biomedical applications [258]. Because of its non-ionic nature,

several research groups turned to cationic modifications of pullulan to enable the formation of

electrostatic complexes with pDNA for in vitro and in vivo gene delivery [259-261]. Interestingly it has

been shown that pullulan, equal to arabinogalactan, has intrinsic liver targeting properties, resulting

from its interaction with the asialoglycoprotein receptor present on liver parenchymal hepatocytes

[262]. However, it has to be noted that extensive chemical modification of the native polysaccharide

may greatly influence its affinity for the liver [261].

Another polysaccharide rarely reported for gene delivery is pectin, an important constituent of the

cell wall of fruits and vegetables, best known for its use as gelling and thickening agent. It is a very

complex and heterogenous polysaccharide, both in polymer length as in chemical composition.

Pectin extracts predominantly consist of homogalacturonan regions (HG) composed of repeating α-

59

1,4-D-galacturonic acid units. HG regions, which are often designated as ‘smooth regions’ are

attached to so-called ‘hairy regions’, that are built from alternating 1,2-linked -D-galactose and -L-

rhamnose residues. The hairy regions are highly branched with neutral arabinan, galactan and

arabinogalactan side chains. Although occurring less frequently, pectin extracts can also contain

other highly branched structures, adding to its complexity [263]. It is hypothesized that the

galactose-rich side chains of pectin stimulate binding to galactose-binding lectins (galectins),

expressed on the cell surface of the cancer cells, making pectin a potential carrier polymer in cancer

therapy [17,264]. However, although the galacturonic acid groups in pectin are partially methylated,

unmodified galacturonic acid moieties confer its anionic nature which is not beneficial for nucleic

acid complexation. Therefore, Katav and coworkers modified citrus pectin with primary, tertiary or

quaternary amine groups and formulated polyplexes with GFP encoding pDNA. Reporter gene

expression in HEK 293 cells was highest for the quaternized pectin although further improvements on

endosomal escape and intracellular disassembly may be required [264]. Alternatively, ionotropic

gelation of pectin in the presence of divalent cations (Ca2+, Mg2+, Mn2+) and pDNA may lead to the

formation of DNA loaded micro-and nanogel particles thereby avoiding potential cytotoxicity induced

by cationic modifications, albeit with limited success in transfection efficiency [265]. Very recently,

Zhou et al. reported on the derivatization of hyperbranched amylopectin with various oligoamines.

The newly constructed amylopectin derivatives exhibited lowered erythrocyte lysis and cytotoxicity

when compared with branched PEI, but did not outperform the latter polymer with regard to

transfection efficiency [266].

7. Conclusions and future perspectives

Polysaccharides are promising candidates in nanocarrier development for nucleic acid delivery, due

to several intrinsic advantages (table 1). Most natural polysaccharides discussed in this review share

the important benefit of biocompatibility and biodegradability. However, to be successful in nucleic

acid delivery, a first requirement for polysaccharide-based nanocarriers is to ensure efficient nucleic

60

acid encapsulation and protection. Because of its polycationic nature, chitosan is frequently applied

for the assembly of polyelectrolyte complexes (PECs) with negatively charged nucleic acids.

Conversely, other polysaccharides require complexation with polycations or chemical modification

with oligo-or polyamines to enable nucleic acid complexation. Fortunately, polysaccharides possess

many functional groups that allow straightforward derivatization. Although reworking the structure

of a native polysaccharide can both be beneficial to nucleic acid complexation and intracellular

nucleic acid delivery, the inherent biocompatibility can no longer be guaranteed. It is therefore

imperative to perform an in depth evaluation of the potential toxicity of modified polysaccharides

and resulting NPs. In view of that, even though the potential of nanocarriers to improve delivery of

nucleic acid therapeutics cannot be denied, their transition from bench to bedside will strongly rely

on the emerging field of nanotoxicology to assess the parameters governing the toxic effects inflicted

by nanomaterials. Polysaccharides and their derivatives have been used to optimize nucleic acid

nanocarrier design toward in vivo applications. Prolonging the blood circulation time of NPs, by

endowing them with stealth properties, could promote passive tumor targeting via the enhanced

permeability and retention (EPR) effect. Many polysaccharides also possess excellent bioadhesive

properties, justifying their use in mucosal drug delivery strategies (e.g mucosal DNA vaccination). In

addition, several cell types express carbohydrate-binding receptors recognizing specific

oligosaccharide motifs, enabling active targeting at the cellular level depending on the type of

polysaccharide used. However, to clearly state a therapeutic benefit, additional in vivo data in

validated and representative animal disease models are urgently required. As reviewed above, many

types of polysaccharides with variable chemical composition, molecular structure and polymer

length, have been employed in nucleic acid nanocarrier design. In concert with a range of different

methodologies available for NP production, this entails a plethora of existing polysaccharide-based

nanotherapeutics, albeit often with unpredictable biological and physicochemical properties. Besides

thorough physicochemical characterization, future research should therefore aim for a clearer view

on the (un-)specific interactions at the nano-bio interface, both on the extra-and intracellular level.

61

More detailed insight in the underlying mechanisms of the nucleic acid delivery process will strongly

contribute to the design of tomorrow’s safe and effective nucleic acid delivery systems.

62

Table 1. Predominant beneficial properties contributing to improved nucleic acid delivery typically

ascribed to the most commonly used polysaccharides in biomedical and pharmaceutical applications

Polysaccharide advantage dextran cyclodextrin chitosan hyaluronan

Low cytotoxicity

Non-immunogenic§

Nucleic acid complexation

Ionotropic gelation

Ease of chemical modification

Membrane permeation enhancer

muco-adhesive

‘stealth’ properties*

Receptor targeting

§ also dependent on other parameters, such as molecular weight (e.g. hyaluronic acid) and nano-

architecture (e.g. dextran and chitosan)

*stealth properties include steric shielding, decreased unspecific binding and reduced complement

activation

Acknowledgments

The Research Foundation Flanders (FWO) and the Ghent University Special Research Fund are

gratefully acknowledged for their financial support.

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Graphical abstract