liposomes – successful carrier systems for targeted delivery of drugs

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Drug Delivery PEPTIDES, PROTEINS & LIPOSOMES

B U S I N E S S B R I E F I N G : P H A R M A T E C H 2 0 0 3

I n t r o d u c t i o n

Liposomes are colloidal, vesicular structures basedon (phospho)lipid bilayers. Their characteristicsdepend on the manufacturing protocol and choiceof bilayer components. They can be as small as20nm and as large as 10µm in diameter. Theliposomes can be unilamellar (meaning only onebilayer surrounds an aqueous core) or multilamellar(several bilayers oriented concentrically around anaqueous core). In addition, the choice of bilayercomponents determines the ‘rigidity’ (or ‘fluidity’)and the charge of the bilayer. For example,saturated phospholipids with long acyl chains suchas dipalmitoylphosphatidylcholine form a rigid,rather impermeable bilayer structure, while the unsaturated phosphatidylcholine species fromnatural sources (egg or soy bean phospha-tidylcholine) give much more permeable and lessstable bilayers. The introduction of positively ornegatively charged lipids provides the liposomeswith a surface charge.

Liposome surfaces can be readily modified. By attaching polyethylene glycol (PEG) units to the bilayer, the circulation time of liposomes in the bloodstream is increased dramatically.Alternatively, homing molecules can be attached toliposome bilayers to make these structures target-site-specific. Size, lamellarity, bilayer rigidity,charge and bilayer surface modifications: all theseparameters determine the fate of liposomes on theshelf and in vivo.

Over the years, the behaviour of liposomes has beeninvestigated in much detail,1,2 and algorithms can beused to help the pharmaceutical formulation scientistselect the proper liposome type.

L i p o s ome s – A pp l i c a t i o n s

Liposomes are used as carriers for drugs andantigens. The primary reason for this is that theycan serve several different purposes (see Table 1).2

Liposomes can direct a drug to a certain target.This aspect of liposome drug delivery will bediscussed later in more detail. Liposomes can alsoprolong the duration of drug exposure, acting as aslow-release reservoir. This has been demonstratedin a number of studies, for example with theantimalarial drug chloroquine or the radicalscavenger superoxide dismutase.3

Liposomes can protect a drug against degradation(for example metabolic degradation). Conversely,liposomes can protect the patient against side effectsof the encapsulated drug. For example, the doselimitation of the cytotoxic drug doxorubicin is its(irreversible) damage to heart muscles. Liposomeencapsulation greatly reduces exposure of the heartto doxorubicin and thereby its cardiotoxicity. Otherexamples are the reduction of haemolytic effects ofdrugs by liposome encapsulation and the protectionagainst local irritation on intradermal, subcutaneousor intramuscular injection of a tissue-irritating drug.4

As liposomes can solubilise lipophilic compounds,this solubilising potential can be used to injectpoorly water-soluble, lipophilic compoundsintravenously. If a fast pharmacological response isdesired, then ‘fragile’ liposomes with ‘fluid’ bilayersshould be selected.

Considering this list of applications and the existingliterature, it is clear that liposomes provide anextremely flexible drug carrier modality with manypotential applications and an impressive track record asa carrier system.

Professor Daan J A Crommelin isScientific Director of the UtrechtInstitute for PharmaceuticalSciences (UIPS). He is alsoProfessor at the Department ofPharmaceutics at Utrecht Universityand Adjunct Professor at theDepartment of Pharmaceutics andPharmaceutical Chemistry at theUniversity of Utah. His researchfocuses on advanced drug deliveryand drug targeting strategies.Professor Crommelin is also ChiefScientific Officer of OctoPlus, aLeiden-based company specialising inthe development of pharmaceuticalproduct formulations and advanceddrug delivery systems.

Dr Gert W Bos has a post-doctorateresearch and development functionat the faculty of PharmaceuticalSciences of Utrecht University (UIPS)and a business developmentposition at OctoPlus. He obtainedhis Master’s in Chemical Technologyin 1993 at the University of Twenteand subsequently earned his PhD in the field of Biomaterial Sciencefrom the same university.

Gert Storm was appointed asProfessor (Drug Targeting Chair) atthe University of Utrecht in 2000and, in 1999, as Adjunct Professor at the Department of Pharmaceutics,Royal School of Pharmacy,Copenhagen, Denmark. His researchinterests are in the fields ofbiopharmaceutics and drug targetingand he acts as a consultant to anumber of pharmaceutical companies.Professor Storm is a member of theeditorial (advisory) board of the J.Drug Targeting, J. Liposome Research,S.T.P. Pharma Sciences and Eur. J.Pharm. Sciences and is SpecialFeatures Editor of Pharm. Research.

1. G Gregoriadis (1993), Liposome Technology, Vol I, II, III (ed.), 2nd edition, CRC Press, Boca Raton, Florida, US.2. G Storm and D J A Crommelin, “Liposomes: quo vadis?”, Pharmaceutical Science & Technology Today, 1 (1998),

pp. 19–31.3. C Oussoren, G Storm, D J A Crommelin and J Senior (2000), “Liposomes for sustained drug release”, Sustained-release

injectable products (Eds J Senior and M Radomsky), Interpharm Press, Engelwood, Colorado, US, pp. 137–180.4. F Kadir, C Oussoren and D J A Crommelin (1999), “Liposomal formulations to reduce irritation of intramuscularly and

subcutaneously administered drugs”, Injectable drug development, techniques to reduce pain and irritation (Eds PK Gupta and G A Brazeau), Interpharm Press, Denver, Colorado, US, pp. 337–354.

a report by

P r o f e s s o r D a a n J A C r omme l i n , D r G e r t W B o s and

P r o f e s s o r G e r t S t o rm

Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University

L iposomes – Succes s fu l Carr ie r Sys tems for Targeted De l i ver y o f Drugs

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T a r g e t i n g w i t h L i p o s ome s

‘ C o n v e n t i o n a l ’ L i p o s o m e s

Liposomes can be used for site-specific delivery of

drugs. The remainder of this article will focus ontargeting to tissues upon parenteral administration –intravenously, intratumourally or in cavities such asthe intraperitoneal cavity.

To fully appreciate the potential, but also thelimitations of the drug-targeting concept withliposomes injected directly into the bloodstream, it isessential to understand those pathophysiological andanatomical conditions that control the fate of colloidalparticles in the body. In addition, it is important toappreciate the opportunities that surface modificationsoffer in directing liposomes to their target. Figure 1depicts schematically the four basic types of liposome‘surface make-up’. Non-surface-modified liposomeseither disintegrate in the bloodstream (‘fluid state’liposomes) or they circulate and are picked uppredominantly by macrophages (Kupffer cells) in theliver and also in the spleen. The rate and extent oftheir uptake depends on bilayer rigidity, liposome sizeand dose (small liposomes with rigid bilayers tend tocirculate for a number of hours). This predominantuptake by macrophages has been used to deliverantimicrobial agents effectively to these macrophageswhen they are infected with intracellular pathogens.Dramatic improvements in therapeutic potential havebeen reported.5,6 Another therapeutic goal that hasbeen identified is the possibility of deliveringimmuno-modulating agents (for example muramyldipeptide or tripeptide) to enhance the antitumouraland antiviral activity of macrophages.

‘ S t e a l t h ’ L i p o s o m e s

Two important observations were made andreported in the 1980s regarding the fate ofintravenously administered liposomes. First, it wasfound that the endothelial lining of ‘healthy’ bloodvessels forms an efficient barrier to liposomal escapefrom the blood circulation upon intravenousadministration. Only in sinusoidal tissue is escape

Figure 1: Schematic Representation of Four Major

Liposome Types

Conventional

Targeted

Stealth

Cationic

Conventional liposomes are either neutral or negatively charged. Sterically stabilised

(‘stealth’) liposomes carry polymer coatings to obtain prolonged circulation times.

Immunoliposomes (‘antibody-targeted’) may be either conventional or stealth. For

cationic liposomes, several ways to impose a positive charge are shown (mono, di

or multivalent interactions). Adapted from Storm and Crommelin (1998).2

5. D J A Crommelin and H Schreier (1994), “Liposomes”, Colloidal Drug Delivery Systems (Ed. J Kreuter), MarcelDekker Inc., pp. 73–190.

6. R M Schiffelers, G Storm and I A J M Bakken-Woudenberg, “Liposome-encapsulated amioglycosides in preclinical andclinical studies”, J. Antimicrob. Chemother., 48 (2001), pp. 333–344.

Table 1: Reasons to Use Liposomes as Drug Carriers

Solubilisation Liposomes may solubilise lipophilic drugs that would otherwise be difficult to administer intravenously.

Protection Liposome-encapsulated drugs are inaccessible to metabolising enzymes; conversely, body components (such as

erythrocytes or tissues at the injection site) are not directly exposed to the full dose of the drug.

Duration of action Liposomes can prolong drug action by slowly releasing the drug in the body.

Directing potential Targeting options change the distribution of the drug through the body.

Internalisation Liposomes are endocytosed or phagocytosed by cells, opening up opportunities to use ‘liposome-dependent drugs’. Lipid-

based structures (not necessarily liposomes) are also able to bring plasmid material into the cell through the same

mechanism (non-viral transfection systems).

Amplification Liposomes can be used as adjuvants in vaccine formulations.

Adapted from Storm and Crommelin (1998).2

Figure 2: Principle of Drug Targeting with Immunoliposomes

Drug containing immunoliposome

Targetcell

Non-targetcell No immunospecific binding

+

+ +

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possible for small liposomes. However, in a numberof solid tumours and at sites of inflammation, theendothelium is more permeable and allowsextravasation of small liposomes. This results inaccumulation of liposomes at the tumour site and atinflammatory sites (for example bacterial infectionsand arthritic joints), but, in general, forconventional liposomes, removal from thecirculation is too fast to benefit from this escapemechanism. Thus, long circulation times ofliposomes are required to take full advantage of this‘leaky endothelium’ effect. This brings us to thesecond important finding. Coating liposomes withPEG reduces the rate of uptake by macrophages(‘stealth’ effect) and leads to a prolonged presence ofliposomes in the circulation and consequentlyample time for these liposomes to escape from thecirculation through a leaky endothelium.

This stealth principle has been used to develop thesuccessful doxorubicin-loaded liposome productthat is currently marketed as Doxil® or Caelyx® fortreatment of solid tumours. Recently, impressivetherapeutic improvements were described by usingcorticosteroid-loaded liposomes in experimentalarthritic models.

By far the most attention regarding the applicationof long-circulating liposomes has been on their potential to escape from the blood

circulation. However, these long-circulatingliposomes may also act as a reservoir for prolongedrelease of a therapeutic agent. Woodle, et al.reported on a remarkably long pharmacologicalaction of vasopressin when formulated in long-circulating liposomes.7

L i p o s o m e s w i t h ‘ H o m i n g ’ D e v i c e s

An important consideration is how to make

2. Active targeting 2. Transfer of lipophiliccompounds

1. Passive targetingExtracellularcontents release

6. TAT–peptide-mediated translocation

5. Intracellularfusion

3. Liposomeinternalisation

4. Membranefusion

Nucleus

Figure 3: Potential Ways by which Targeted Immunoliposomes can Achieve Cytosolic Drug Delivery

Passive (1) and active (2) targeting, receptor-mediated endocytosis (3), fusion with the plasma membrane (4) or intracellularly (5) or TAT-mediated translocation.

DTA diINF-7

Anti-EGFR

PEG

Figure 4: Peptide-induced Release of ‘Stealth Liposome’-entrapped Diphtheria

Toxin (DTA) – A Liposome-dependent Drug

The peptide diINF-7 is a fusogen based on the N-terminal domain of influenza virus HA-2 and is activated upon a pH drop.

Anti-EGFR acts as a ‘homing’ device.

7. M C Woodle, G Storm, M S Newman, J L Jekot, L R Collins, F C Martin and F C Szoka, “Prolonged systemic deliveryof peptide drugs by long-circulating liposomes: illustation with vasopressin in the Brattleboro rat”, PharmaceuticalResearch, 9 (1992), pp. 260–265. 211



liposome uptake tissue or cell-specific. Figure 2shows liposomes with antibodies attached covalentlyto the surface of the liposomes. Most work onliposome targeting has focused on antibodies orantibody fragments attached to the surface,8 butother homing devices have been considered as well.For example, plasminogen-coated liposomes were

designed to reach fibrin clots specifically(plasminogen has an affinity for fibrin) in order todeliver fibrinolytics.

More recently, reports have appeared on arginine-glycine-aspartic acid (RGD) peptide-driventargeting of liposomes to endothelial cells in orderto block angiogenesis.9 Saccharide-directedtargeting has also been described, for example theuse of saccharide antennae (including galactose) todirect liposomes to hepatocytes.

Targeted liposomes should have ready access to thetarget site and should not be taken up bymacrophages before encountering their targettissue or cells. Therefore, nowadays, stealthtechnology is often combined with attachment of ahoming device to the terminal end of the PEGchain that is exposed to the aqueous medium.Specific attachment of the targeted liposomes totheir target has been successful; however, hard andconvincing experimental data on therapeuticadvantages is scarce.

P r o s p e c t s o f L i p o s ome D r u gT a r g e t i n g S t r a t e g i e s

There has been investigation into how liposometargeting can lead to tissue-specific therapeuticeffects. Upon interaction with the target cell, anumber of approaches have been proposed and someof these have proven to offer therapeutic advantagesin animal models. The more successful approaches sofar are discussed as follows.

S t r a t e g y O n e

( s e e F i g u r e s 3 a n d 4 )

The immunoliposomes are interacting with a cellsurface receptor that is endocytosed, leading toimmunoliposome internalisation upon immuno-liposome–cell interaction. For a successful action ofthe liposome-associated drug, escape from theendosome is often required as many drugs areinactivated when the endosome matures from theendosomal state into a lysosome. For endosomalescape, fusogenic peptides (often derived from virusessuch as the influenza virus) have been proposed, or,alternatively, pH-dependent liposomes are used thatdestabilise the endosomal membrane when the pHlevel drops (see Figure 3). The liposome structure thatis now required comprises several components withtheir specific functions: the liposome as carrier, theantibody as homing device, PEG as stealth coat, the

8. E Mastrobattista, G A Koning and G Storm, “Immunoliposomes for targeted delivery of antitumor drugs”, AdvancedDrug Delivery Reviews, 40 (1999), pp. 103–127.

9. E Mastrobattista, D J A Crommelin, J Wilschut and G Storm, “Targeted liposomes for delivery of protein-based drugs intothe cytoplasm of tumor cells”, J. Liposome. Res., in press (2002).

Figure 5: Schematic Presentation of a Selective Transfer Model Proposed for a

Lipophilic Prodrug of the Anticancer Agent FUdR-dP from Immunoliposomes

to the Plasma Membrane of Tumour Cells

Bystander effect

Lysosome

Endosome

Nucleus

After target cell binding, the immunoliposome-incorporated FUdR-dP is transferred to the plasma membrane of the tumour

(1). The prodrug is internalised (2) and hydrolysed intralysosomally (3). The active drug FUdR then diffuses into the

cytoplasm (4), from where it is either transferred into the nucleus (5, site of action) or extracellularly (6, ‘bystander effect’).

Figure 6: Concept of Antibody-directed Enzyme Prodrug Therapy with

Immunoliposomes

Antigen-binding fragment

Enzyme

Immuno-enzymosome

Tumour cell

Prodrug

Active drug

The immunoenzymosomes are first allowed to bind to target cells, then a prodrug is given, which is activated by the

immunoenzymosomes in close proximity to the target cell. Subsequently, the active drug can kill the cell.

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fusogen as endosomal escape tool and (last but notleast) the drug. This drug preferably belongs to thecategory ‘liposome-dependent drugs’, becausemaximal advantage can then be obtained from thedrug targeting approach. An example of a liposome-dependent drug is the A chain of diphtheria toxin.Without a carrier to deliver this compound into thecytosol, it is inactive. Only in the cytosol does it exertits extremely high toxicity by blocking ribosomeactivity efficiently (see Figure 4).

S t r a t e g y T w o ( s e e F i g u r e 5 )

An effective strategy developed by Scherphof, et al.8 is based on the selective binding ofimmunoliposomes that contain a lipophilic prodrug.This prodrug is transferred selectively from the cell-bound immunoliposomes into the target cell.Subsequently, the prodrug is converted in thelysosome into the active drug. From the lysosome, itleaks into the cytoplasm and maybe even to theoutside of the cell, causing a so-called ‘bystandereffect’ (see Figure 5).

S t r a t e g y T h r e e ( s e e F i g u r e 6 )

In antibody-dependent enzyme prodrug therapy, aprodrug is converted only at sites where its converting

enzyme is delivered. Site-specific delivery of theenzyme is performed by using a site-specific antibody.

To make target site enzyme delivery more efficient,enzymes can be attached to immuno-liposomes.Now, many enzyme molecules can be delivered tothe target site on one targeted immunoliposome (seeFigure 6).

Con c l u s i o n

Considering the complexity of these three targetingstrategies and the strong desire in thepharmaceutical world ‘to keep things simple’, well-established technologies such as liposome stealthtechnologies and other existing drug-carrying lipidcomplexes are preferred in the short run. Only incases where these ‘simple’ solutions fail and newstrategies for life-threatening diseases are on thedrawing board will the industry further developthose strategies that have been successful in anacademic setting.

Every component of such complex structures asdepicted in Figure 4 should be chosen carefully tofit into the delivery strategy, and challenges will beencountered regarding reproducibility, stability andupscaling methodologies. ■


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liposomes – successful carrier systems for targeted delivery of drugs

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