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Drug Delivery


Liposomes ­ Successful Carrier Systems for Targeted Deliver y of Drugs

a report by

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

Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University


Liposomes ­ Applications

Liposomes are colloidal, vesicular structures based on (phospho)lipid bilayers. Their characteristics depend on the manufacturing protocol and choice of bilayer components. They can be as small as 20nm and as large as 10µm in diameter. The liposomes can be unilamellar (meaning only one bilayer surrounds an aqueous core) or multilamellar (several bilayers oriented concentrically around an aqueous core). In addition, the choice of bilayer components determines the `rigidity' (or `fluidity') and the charge of the bilayer. For example, saturated phospholipids with long acyl chains such as dipalmitoylphosphatidylcholine form a rigid, rather impermeable bilayer structure, while the unsaturated phosphatidylcholine species from natural sources (egg or soy bean phosphatidylcholine) give much more permeable and less stable bilayers. The introduction of positively or negatively charged lipids provides the liposomes with 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 to liposome bilayers to make these structures targetsite-specific. Size, lamellarity, bilayer rigidity, charge and bilayer surface modifications: all these parameters determine the fate of liposomes on the shelf and in vivo. Over the years, the behaviour of liposomes has been investigated in much detail,1,2 and algorithms can be used to help the pharmaceutical formulation scientist select the proper liposome type.

Liposomes are used as carriers for drugs and antigens. The primary reason for this is that they can serve several different purposes (see Table 1).2 Liposomes can direct a drug to a certain target. This aspect of liposome drug delivery will be discussed later in more detail. Liposomes can also prolong the duration of drug exposure, acting as a slow-release reservoir. This has been demonstrated in a number of studies, for example with the antimalarial drug chloroquine or the radical scavenger superoxide dismutase.3 Liposomes can protect a drug against degradation (for example metabolic degradation). Conversely, liposomes can protect the patient against side effects of the encapsulated drug. For example, the dose limitation of the cytotoxic drug doxorubicin is its (irreversible) damage to heart muscles. Liposome encapsulation greatly reduces exposure of the heart to doxorubicin and thereby its cardiotoxicity. Other examples are the reduction of haemolytic effects of drugs by liposome encapsulation and the protection against local irritation on intradermal, subcutaneous or intramuscular injection of a tissue-irritating drug.4 As liposomes can solubilise lipophilic compounds, this solubilising potential can be used to inject poorly water-soluble, lipophilic compounds intravenously. If a fast pharmacological response is desired, then `fragile' liposomes with `fluid' bilayers should be selected. Considering this list of applications and the existing literature, it is clear that liposomes provide an extremely flexible drug carrier modality with many potential applications and an impressive track record as a carrier system.

Professor Daan J A Crommelin is Scientific Director of the Utrecht Institute for Pharmaceutical Sciences (UIPS). He is also Professor at the Department of Pharmaceutics at Utrecht University and Adjunct Professor at the Department of Pharmaceutics and Pharmaceutical Chemistry at the University of Utah. His research focuses on advanced drug delivery and drug targeting strategies. Professor Crommelin is also Chief Scientific Officer of OctoPlus, a Leiden-based company specialising in the development of pharmaceutical product formulations and advanced drug delivery systems. Dr Gert W Bos has a post-doctorate research and development function at the faculty of Pharmaceutical Sciences of Utrecht University (UIPS) and a business development position at OctoPlus. He obtained his Master's in Chemical Technology in 1993 at the University of Twente and subsequently earned his PhD in the field of Biomaterial Science from the same university. Gert Storm was appointed as Professor (Drug Targeting Chair) at the University of Utrecht in 2000 and, in 1999, as Adjunct Professor at the Department of Pharmaceutics, Royal School of Pharmacy, Copenhagen, Denmark. His research interests are in the fields of biopharmaceutics and drug targeting and he acts as a consultant to a number of pharmaceutical companies. Professor Storm is a member of the editorial (advisory) board of the J. Drug Targeting, J. Liposome Research, S.T.P. Pharma Sciences and Eur. J. Pharm. Sciences and is Special Features 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. 19­31. 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. 137­180. 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 P K Gupta and G A Brazeau), Interpharm Press, Denver, Colorado, US, pp. 337­354.



Drug Delivery


Targeting with Liposomes

`Conventional' Liposomes

drugs. The remainder of this article will focus on targeting to tissues upon parenteral administration ­ intravenously, intratumourally or in cavities such as the intraperitoneal cavity. To fully appreciate the potential, but also the limitations of the drug-targeting concept with liposomes injected directly into the bloodstream, it is essential to understand those pathophysiological and anatomical conditions that control the fate of colloidal particles in the body. In addition, it is important to appreciate the opportunities that surface modifications offer in directing liposomes to their target. Figure 1 depicts schematically the four basic types of liposome `surface make-up'. Non-surface-modified liposomes either disintegrate in the bloodstream (`fluid state' liposomes) or they circulate and are picked up predominantly by macrophages (Kupffer cells) in the liver and also in the spleen. The rate and extent of their uptake depends on bilayer rigidity, liposome size and dose (small liposomes with rigid bilayers tend to circulate for a number of hours). This predominant uptake by macrophages has been used to deliver antimicrobial agents effectively to these macrophages when they are infected with intracellular pathogens. Dramatic improvements in therapeutic potential have been reported.5,6 Another therapeutic goal that has been identified is the possibility of delivering immuno-modulating agents (for example muramyl dipeptide or tripeptide) to enhance the antitumoural and antiviral activity of macrophages.

`Stealth' Liposomes

Liposomes can be used for site-specific delivery of

Figure 1: Schematic Representation of Four Major Liposome Types

Conventional Stealth



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

Figure 2: Principle of Drug Targeting with Immunoliposomes


Drug containing immunoliposome

Target cell

Table 1: Reasons to Use Liposomes as Drug Carriers Solubilisation Protection Duration of action Directing potential Internalisation Liposomes may solubilise lipophilic drugs that would otherwise be difficult to administer intravenously. 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. Liposomes can prolong drug action by slowly releasing the drug in the body. Targeting options change the distribution of the drug through the body. Liposomes are endocytosed or phagocytosed by cells, opening up opportunities to use `liposome-dependent drugs'. Lipidbased structures (not necessarily liposomes) are also able to bring plasmid material into the cell through the same mechanism (non-viral transfection systems). Liposomes can be used as adjuvants in vaccine formulations.


Adapted from Storm and Crommelin (1998).2


5. D J A Crommelin and H Schreier (1994), "Liposomes", Colloidal Drug Delivery Systems (Ed. J Kreuter), Marcel Dekker Inc., pp. 73­190. 6. R M Schiffelers, G Storm and I A J M Bakken-Woudenberg, "Liposome-encapsulated amioglycosides in preclinical and clinical studies", J. Antimicrob. Chemother., 48 (2001), pp. 333­344.



Non-target cell

No immunospecific binding

Two important observations were made and reported in the 1980s regarding the fate of intravenously administered liposomes. First, it was found that the endothelial lining of `healthy' blood vessels forms an efficient barrier to liposomal escape from the blood circulation upon intravenous administration. Only in sinusoidal tissue is escape


Liposomes ­ Successful Carrier Systems for Targeted Deliver y of Drugs

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

1. Passive targeting Extracellular contents release 2. Active targeting 2. Transfer of lipophilic compounds


6. TAT­peptidemediated translocation

5. Intracellular fusion

3. Liposome internalisation 4. Membrane fusion

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

possible for small liposomes. However, in a number of solid tumours and at sites of inflammation, the endothelium is more permeable and allows extravasation of small liposomes. This results in accumulation of liposomes at the tumour site and at inflammatory sites (for example bacterial infections and arthritic joints), but, in general, for conventional liposomes, removal from the circulation is too fast to benefit from this escape mechanism. Thus, long circulation times of liposomes are required to take full advantage of this `leaky endothelium' effect. This brings us to the second important finding. Coating liposomes with PEG reduces the rate of uptake by macrophages (`stealth' effect) and leads to a prolonged presence of liposomes in the circulation and consequently ample time for these liposomes to escape from the circulation through a leaky endothelium. This stealth principle has been used to develop the successful doxorubicin-loaded liposome product that is currently marketed as Doxil® or Caelyx® for treatment of solid tumours. Recently, impressive therapeutic improvements were described by using corticosteroid-loaded liposomes in experimental arthritic models. By far the most attention regarding the application of long-circulating liposomes has been on their potential to escape from the blood

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.

circulation. However, these long-circulating liposomes may also act as a reservoir for prolonged release of a therapeutic agent. Woodle, et al. reported on a remarkably long pharmacological action of vasopressin when formulated in longcirculating liposomes.7

Liposomes with `Homing' Devices

An important consideration is how to make

7. M C Woodle, G Storm, M S Newman, J L Jekot, L R Collins, F C Martin and F C Szoka, "Prolonged systemic delivery of peptide drugs by long-circulating liposomes: illustation with vasopressin in the Brattleboro rat", Pharmaceutical Research, 9 (1992), pp. 260­265.



Drug Delivery


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

designed to reach fibrin clots specifically (plasminogen has an affinity for fibrin) in order to deliver fibrinolytics. More recently, reports have appeared on arginineglycine-aspartic acid (RGD) peptide-driven targeting of liposomes to endothelial cells in order to block angiogenesis. 9 Saccharide-directed targeting has also been described, for example the use of saccharide antennae (including galactose) to direct liposomes to hepatocytes. Targeted liposomes should have ready access to the target site and should not be taken up by macrophages before encountering their target tissue or cells. Therefore, nowadays, stealth technology is often combined with attachment of a homing device to the terminal end of the PEG chain that is exposed to the aqueous medium. Specific attachment of the targeted liposomes to their target has been successful; however, hard and convincing experimental data on therapeutic advantages is scarce.

Prospects of Liposome Drug Targeting Strategies

Bystander effect




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

Prodrug Active drug


There has been investigation into how liposome targeting can lead to tissue-specific therapeutic effects. Upon interaction with the target cell, a number of approaches have been proposed and some of these have proven to offer therapeutic advantages in animal models. The more successful approaches so far are discussed as follows.

Strategy One (see Figures 3 and 4)

Tumour cell

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.

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

The immunoliposomes are interacting with a cell surface receptor that is endocytosed, leading to immunoliposome internalisation upon immunoliposome­cell interaction. For a successful action of the liposome-associated drug, escape from the endosome is often required as many drugs are inactivated when the endosome matures from the endosomal state into a lysosome. For endosomal escape, fusogenic peptides (often derived from viruses such as the influenza virus) have been proposed, or, alternatively, pH-dependent liposomes are used that destabilise the endosomal membrane when the pH level drops (see Figure 3). The liposome structure that is now required comprises several components with their specific functions: the liposome as carrier, the antibody as homing device, PEG as stealth coat, the


8. E Mastrobattista, G A Koning and G Storm, "Immunoliposomes for targeted delivery of antitumor drugs", Advanced Drug Delivery Reviews, 40 (1999), pp. 103­127. 9. E Mastrobattista, D J A Crommelin, J Wilschut and G Storm, "Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells", J. Liposome. Res., in press (2002).


Liposomes ­ Successful Carrier Systems for Targeted Deliver y of Drugs

fusogen as endosomal escape tool and (last but not least) the drug. This drug preferably belongs to the category `liposome-dependent drugs', because maximal advantage can then be obtained from the drug targeting approach. An example of a liposomedependent drug is the A chain of diphtheria toxin. Without a carrier to deliver this compound into the cytosol, it is inactive. Only in the cytosol does it exert its extremely high toxicity by blocking ribosome activity efficiently (see Figure 4).

Strategy Two (see Figure 5)

enzyme is delivered. Site-specific delivery of the enzyme 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 to the target site on one targeted immunoliposome (see Figure 6).


An effective strategy developed by Scherphof, et al.8 is based on the selective binding of immunoliposomes that contain a lipophilic prodrug. This prodrug is transferred selectively from the cellbound immunoliposomes into the target cell. Subsequently, the prodrug is converted in the lysosome into the active drug. From the lysosome, it leaks into the cytoplasm and maybe even to the outside of the cell, causing a so-called `bystander effect' (see Figure 5).

Strategy Three (see Figure 6)

Considering the complexity of these three targeting strategies and the strong desire in the pharmaceutical world `to keep things simple', wellestablished technologies such as liposome stealth technologies and other existing drug-carrying lipid complexes are preferred in the short run. Only in cases where these `simple' solutions fail and new strategies for life-threatening diseases are on the drawing board will the industry further develop those strategies that have been successful in an academic setting. Every component of such complex structures as depicted in Figure 4 should be chosen carefully to fit into the delivery strategy, and challenges will be encountered regarding reproducibility, stability and upscaling methodologies. s

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

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