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9 Taylor, G. (1983)ff. MoL Graphics 1, 5-8 10 Diamond, R. (1980) In Biomolecular

Structure, Conformation, Function and Evolution (Srinivasan, R., ed.), Vol. 1, pp. 567-588, Pergamon Press

11 Jones, T. A. (1978)J. Appl. Crystallogr. 11, 268-272

12 Getzoff, E. D., Tainer, J. A., Weiner, P. K., Kollman, P. A., Richardson, J. S. and Richardson, D. C. (1985) Nature 306, 287-290

13 Perutz, M. F. and Raidt, H. (1975) Nature 255, 256-259

14 Rogers, N. K., Moore, G. B. and Stern- berg, M. J. E. (1985)J. MoL Biol. 182, 613-616

15 Weiner, P. K., Langridge, R., Blaney, J. M., Schaelfer, R. and Kollman, P. A. (1983) Proc. Natl Acad. Sci. USA 79, 3754-3758

16 Blundell, T. L., Sibanda, B. L. and Pearl, L. H. (1983) Nature 304, 273-275

17 Sibanda, B. L., BlundeU, T. L., Hobart, P. M., Fogliano, M., Bindra, J. S., Dominy, B. W. and Chirgwin, J. M. (1984) FEES Lett. 174, 103-111

18 Travers, P., Blundell, T. L., Sternberg, M. J. E. and Bodmer, W. F. (1984) Nature 310, 235-238

19 Sibanda, B. L. and Thornton, J. M. (1985) Nature 316, 170-174

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22 Morffew, A. J., Todd, S. J. P. and Snel- grove, M. J. (1983) Comput. Chem. 7, 9-16

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G., Profeta, S. and Weiner, P. (1984)J. Am. Chem. Soc. 106, 765-784

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26 Knossow, M., Daniels, R. S., Douglas, A. R., Skekel, J. J. and Wiley, D. C. (1984) Nature 311, 678-680

27 Northrop, S. H., Pear, M. R., McCammon, J. A., Karplus, M. and Takano, T. (1980) Nature 287, 659-660

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29 Honegger, A. and BlundeU, T. L. (1983) In Insulin-like Growth Factors (Spencer, E. M., ed.), pp. 93-113, Walter de Gruyter

Liposomes for drugs and vaccines Gregory Gregoriadis

Application of drugs in therapeutic and preventive medicine is marred by indiscriminate drug action and inability of drugs to reach areas in need of treatment. On the other hand, development of new, more selec- tive drugs is very expensive, lengthy and often uncertain. Recently, much attention has been given to an alternative approach, namely the use of drug delivery systems which are expected to optimize the action of drugs already in existence. One of the more promising systems is lipo- somes, microscopic spheres made of natural materials (lipids) and able to accommodate large amounts of drug. Fifteen years of liposome research have produced a great deal of knowledge of how the carrier in- teracts with the biological milieu. In turn, such knowledge has helped us to optimize liposomal drug action in situations as diverse as cancer and microbial therapy, vaccines, oral therapy and medical diagnostics. Some of these applications, especially those involving the phagocytic cells (e.g. antimicrobial therapy and vaccines) seem realistic enough to

warrant extensive support from industry.

Loss of hair, diarrhoea and suppression of the immune response in cancer chemotherapy, persistence of intracel- lular microbial infections in antimicro- bial therapy, the accumulation of toxic metals in tissues, the inconvenience and unphysiological nature o f parenteral treatment of diabetics with insulin, and the ineffectiveness of many vaccines are just some of the problems encountered in the conventional use of drugs in the treatment or prevention of disease. Explanations of these effects include indiscriminate drug action against~both target and normal tissues, inability of drugs to penetrate areas in the body requiring treatment and pre- mature drug excretion or inactivation.

Gregory Gregoriadis is at the Academic Department of Medicine, Medical Research Council Group, Royal Free Hospital School of Medicine, Pond Street, London, NW3 2QG, UK.

Ideally a drug should be selective, acting on its target (usually a popula- tion of cells) without significant ramifi- cations for the normal remainder of the host. Unfortunately, although some antibiotics are highly selective (they interfere with metabolic events unique to bacteria), most other drugs are not. Although continuous efforts are being made to improve the selectivity of drugs themselves, factors such as the mount ing costs of drug development, the complex logistics and administra- tive expense in applying drugs (especially in the developing world) and the realization that optimal drug design must in many cases await further advances in the molecular and cellular biology of disease, have stimulated research on alternative methods. These are expected to improve the action of drugs already in existence and thus to reduce costs. One major approach is to

use carrier systems 1 which can first transport drugs to where they are needed, and then be disposed of through biodegradation. What is re- markable about this approach is that one does not exchange the problems associated with the design of specific drugs with those related to endowing specificity to the carrier.

Many carrier systems presently under investigation I are ligands extracted from natural sources with an inherent ability to interact selectively with receptors on the surface of cells. For instance, antibodies will bind with exquisite specificity to relevant cell- surface antigens. A wide range of other macromolecules such as certain glyco- proteins, lipoproteins and polypeptide hormones are also highly specific for a variety of receptors.

Neither are carrier systems limited to natural materials. A considerable and ever increasing proportion of research in drug delivery is now concerned with man-made polymers, colloids and other particles ~-3. The advantage of using such systems is that one can design their structural characteristics to deter- mine their fate within the biological milieu and thus optimize their inter- action with the target. What is more, these synthetic systems can be linked to antibodies and other natural ligands which serve as target-seeking devices. A great deal of interest has been shown in liposomes 1'3, man-made carriers with multifaceted potential in drug delivery.

Liposomes: preparation and drug entrapment

About 20 years ago it was discovered that mixing dry phospholipids with water gives rise to bilayers ofphospho- lipid molecules which arrange them-

© 1985, Elsevier Science Publishers B.V., Amsterdam 0166-9430t851502,00

236

Fig. I. (a) Electron micrograph of a liposome. Liposomes can vary from about 2 5 nm to several#m diameter. Depending on the lipid composition, the liposomal surface can be neutral or positively or negatively charged. A variety of methods available for the preparation of liposomes and the entrapment of drugs are described in detail in Refs 3 and 5.

selves spontaneously to form close multilayered spherules known as lipo- somes ~'3 (Fig. 1). As they form, lipo- somes entrap water and any water- soluble solutes (e.g. drugs) that happen to be present. Lipid-soluble com- pounds, on the other hand, will be incorporated into the liposomal mem- brane. Because of their bimolecular leaflet membrane-like structure, lipo- somes were initially used (and still are) as models for the study of cell membrane biophysics. It was soon realized 4, however, that sequestration of solutes by liposomes could also be important in drug delivery. The original crude process of making liposomes was modified in numerous ways 3'5 to give newer versions of the system with high drug to lipid ratios.

Recently, high yield entrapment of drugs into liposomes was achieved by a technique 6 known as DRV (dehydra- tion-rehydration vesicles) which is simple, mild (organic solvents and sonication are avoided) and particularly suitable for large-scale use in industry (Fig. 2). It yields a freeze-dried preparation containing the liposomal lipid and drug molecules in intimate contact. Upon the controlled 6 addition of water, up to 70% of the water-soluble drug present can be entrapped into the formed liposomes 6. The suspension can then be used directly if the presence

of free (non-entrapped) drug is acceptable, or the free drug can be removed subsequently by a routine procedure such as dialysis. Prepara- tions containing only entrapped drug can be freeze-dried again 3 and the liposomal structural integrity is still apparently preserved: intact liposomes with most of their contents still entrapped are obtained upon rehydration.

Range of substances entrapped Virtually any substance, regardless of

solubility, electrical charge, size and other structural characteristics can be incorporated into liposomes provided that they do not interfere with their formation. Detergent-like substances, for instance, will prevent liposomes from forming when present above a critical concentration. Further, because incorporation into either the aqueous or the lipid phase of liposomes is passive, there is usually no need to develop different techniques for individual drugs. This is clearly illustrated 6 in the case of DRV where salts, sugars, a variety of low- molecular-weight drugs, proteins and nucleic acids, are all incorporated into liposomes by the same procedure. During the last 15 years many hundreds of drugs, including a wide range of anticancer and antimicrobial

Trends in Biotechnology, Vol. 3, No. 9, 1985

agents, proteins, enzymes, vaccines and genetic materials have been success- fully entrapped in liposomes and tested under various experimental condi- tions 1'6-9. Some of the successful uses include cure oftumour-bearing animals with liposomes carrying agents activating tumouricidal macrophagesl°, killing of microbes in bacterial and fungal infections 1~, potentiation of immune responses to otherwise ineffective or weak vaccines '~ and facilitation of adsorption of orally given insulin ~3'14 and blood clotting factors 15. Many of these agents (e.g. antibiotics, interferon, peptide hormones, factor VIII and vaccines) can now be made through recombinant DNA techniques and their availability and use (and the need for their delivery) are expected to increase considerably in the foreseeable future.

Stability of liposomes Liposomes are prepared 3 from

phospholipids such as egg yolk or soya bean lecithins. Other, semi-synthetic, phospholipids with fatty-acid chains of defined length and saturation and, increasingly now, cholesterol are also used for specific purposes. The choice of the type of phospholipid and/or the amount of cholesterol play important roles in determining liposomal stability upon storage and, as will be shown later, their fate in injected animals. Liposomal stability on storage is defined here as the extent to which a given preparation retains both its

Fig. I. (b) Representation of a unilamellar liposome showing a bilayer of phospholipid molecules with their hydrophilic heads in con- tact with water inside. Drug molecules (black dots), depending on their solubility, can be accommodated in the entrapped water space or within the lipid phase formed by the hydrophobic tails of the phospholipids.

Trends in Biotechnology, VoL 2, No. 9, 1985

original vesicle size distribution and its drug load. Instability can occur, for example, when vesicle size increases spontaneously upon standing as a result of fusion of colliding vesicles; the larger vesicles will exhibit drastically different pharmacokinetics in vivo because their size determines their clearance rates and tissue distribution; for instance, large liposomes are removed from the circulation more rapidly than smaller ones and the latter, when of a diameter of ~100 nm or less can reach the hepatic parenchymal cells ~'s'9. A second important factor in instability is that drugs of low molecular weight are likely to leak from stored liposomes.

Both aspects of liposomal stability have been investigated, especially by industrial workers 3 and both have, to a large extent, been resolved. Fusion upon standing, for instance, is reduced in small vesicles bearing a surface electric charge. Other techniques, for instance the use of additives, have been routinely used in industry for the main- tenance of colloid suspensions and, although no details have been dis- closed, they no doubt have been applied in the case ofliposomes.

Drug loss due to leakage can also be reduced considerably by the appro- priate manipulation of lipid composi- tion. Excess cholesterol in the lipo- somal membrane, for instance, packs the lipid molecules so that leakage of entrapped small molecules is mini- mized. Alternatively, fatty acid (or other lipid) derivatives of drugs can be formulated and can be incorporated into the liposomal structure in the same way as phospholipids and cholesterol. The advantage of this approach is that preparations can be lyophilized: lipo- somes reformed on rehydration retain the fatty acid derivative of the drug quantitatively. Its disadvantages include a lower drug to lipid ratio than could have been achieved with the water-soluble drug and the additional expense of synthesizing the derivative.

Owing to the small number of steps in the manufacture of drug-containing liposomes there are no serious problems in maintaining sterility ~6 throughout the procedure. For instance, in the method described in Fig. 2 essentially only one container is needed from the beginning to end. Starting with Sterile lipid and drug solutions, sterile liposomal prepara- tions can be achieved by using aseptic techniques, by irradiation or, when

Drug in water ~ . ~

Liposomes

237

/ Dilute

@ Fig. 2. Preparation of liposomnes with high-yield drug entrapment in a freeze-dried form. A solution of drug is mixed with a suspension of "empty' (water-containing) liposornes. Subsequently, the mixture is freeze-dried. The inset shows intimate contact of flattened liposomnal membrane struc- tures and of drug molecules in a dry environment. Liposomnes formed upon controlled rehydration, contain much of the original drug in the entrapped form. Separation of drug-containing liposomnes from unentrapped drug can be carried out easily if needed. For more details, see Ref. 6.

vesicles size is small enough, by filtra- tion.

Fate and behaviour of liposomes in vivo

Administration of drug-containing liposomes to animals and man has been carried out through every conceiv- able route, including intravenous, intramuscular, intradermal, intra- tracheal and enteral. A vast array of experiments ~'3's'9 has revealed many aspects of liposomai behaviour in vivo which in turn have helped us to optimize liposomal drug action for particular needs. As the amount of data amassed is too large even for a cursory discussion, only facts central to the successful application of the system in drug delivery are mentioned here.

Conventional drug-containing phos- pholipid liposomes become unstable and leaky upon contact with bloodl,S; their contents diffuse out rapidly into the circulation and the advantage of giving drugs through a delivery system is thus lost. The main culprits of such vesicle destabilization are plasma high- density lipoproteins (HDL) which remove phospholipid molecules from the bilayers 17'~8. Destabilized lipo- somes, with only a fraction of the drug originally entrapped, are removed by the tissues of the reticuloendothelial system (RES) - the liver, spleen and bone marrow. However, it is now pos- sible to render phospholipid molecules resistant to H D L attack by incorporat- ing into the liposome structure excess cholesterol and/or by making liposomes

out of 'high melting' phospholipids or phospholipids which form inter- molecular bonds with other lipids present ~9. Experiments with polar drugs have shown surprisingly high retention by these stabilized liposomes in the presence of blood even on incuba- tion for as long as two days (Table 1). When tested in vivo, such preparations show identical clearance rates for three independent vesicle markers 2°, a sure indication of liposomes retaining their structural integrity (Fig. 3). There is n ° doubt, therefore, that liposomes can be made to retain entrapped drugs quanti- tatively while in the circulation, and enter cells together with their original load. Destabilization is a disadvantage if quantitative delivery to the reticulo- endothelial tissues is required, but would allow release of drugs into the circulation.

It is well established 1'8'9 that clearance from the circulation of liposomes of large size and negative surface charge is rapid. Within minutes of intravenous injection, such liposomes are largely found in the fixed macrophages of the liver and spleen and (when made of appropriate lipids to ensure maximum drug retention in blood) constitute the vesicle of choice for the rapid delivery of drugs to these cells. Given certain liposomal lipid compositions coupled with the appropriate vesicle size (1-3 gm) and charge there can also be significant retention of liposomes by the lungs ~°. By making liposomes smaller (25 nm diameter being the smallest size possible) 3'5 one can, for

238

Table I. Retention of solutes by liposomes in the presence of blood plasma °

Solute retention (%) Liposomes 111In_bleomydn Carboxyfluorescein Sucrose Small unilamellar 99.5 (51.2) 98.0 b (49.0) 98.2 Large unilamellar 98.5 (34.5) 98.5 96.0 Multilamellar 93.7 (40.1) 96.7 b --

~Liposomes containing solutes as indicated were incubated in the presence of five volumes of blood plasma at 37°C for 24 h. Unless otherwise stated, liposomes were composed of the high-melting phospholipid distearoyl phosphatidylcholine and cholesterol in equimolar proportions. Numbers in parentheses denote values obtained from liposomes composed of equimolar egg lecithin and cholesterol. 'High-melting' phospholipids are defined here as phospholipids which have a liquid- crystalline transition temperature of above 37°C. bIncubation was carried out for 48 h.

reasons that are still not clear, prolong their circulation time to some extent. Recent work ~9 has shown that exceptionally long half-lives of over 20 hours can be achieved in the mouse if small liposomes having no net charge, a high cholesterol content and made of high-melting phosopholipids are used. Such liposomes are exceedingly stable (in terms of solute retention in the presence of blood; see Table 1). Interestingly, the more stable lipo- somes are, the longer is their half-life, and it may be that plasma components (e.g. opsonins) which adsorb onto liposomes and probably make them recognizable by the RES, cannot do so efficiently when their membranes are packed (by cholesterol, for instance) or 'solid' due to high-melting phosopholipids.

Liposomes with long half-lives not only accumulate in the liver and spleen at a much slower rate 2° (an event which gives targeted liposomes the oppor- tunity to interact with other tissues, see later), but they also exhibit a different distribution within the RES, favouring the macrophages of the bone marrow; up to 30% of a dose of long-lived liposomes can end up in this tissue 2°. In addition, liposomes smaller than approximately 100 nm can reach the parenchymal cells of the liver through the fenestrationsk Further, in animal models bearing tumours, localization (up to 18% of the injected dose/g tumour tissue) of small liposomes in the tumour has been claimed~k However, evidence 1°'2° so far with normal animals suggests that even the smallest liposomes cannot cross capillaries to enter the extravascular space.

With regard to events at the sub- cellular level, liposomes are taken up by cells through endocytosis into the lysosomal apparatus where the carrier breaks down and liberates its drug contents (Fig. 4). The contents either act within the lysosomes or in other cell

compartments, depending on the extent to which they remain active in the lysosomal milieu or their ability to diffuse through the lysosomal membranes. Work now in progress suggests that it may be possible to interfere with the lysosomotropic process to enable liposomes to present drugs to other cell areas without the hitherto obligatory step of lysosome localization 2~.

Two other routes of liposome administration, also actively explored, are the subcutaneous and oral. In the former, injected liposomes of large size disintegrate locally, probably upon attack by infiltrating macrophages 1'8'9. In contrast, smaller vesicles enter the lymphatic system and localize avidly into the lymph nodes 1'8'9. This behaviour has stimulated research on using liposomes either as reservoirs for slowly-releasing drugs (e.g. vaccines I'12) or as a means of delivering agents into the lymph nodes for imaging or treat- ment oftnmour metastases ~'8'9.

Given orally (enterally), on the other hand, liposomes are generally broken down in the gut by bile salts and (prob- ably) phospholipases ~'s'9. However, several laboratories have shown that, in spite of their vulnerability in the gut milieu, liposomes facilitate the transfer of a small proportion (< 1% of the dose) of their contents (e.g. insulin 13'14, factor VIII (Ref. 15) and some otherwise unabsorbable drugs ~) into the blood circulation. Such findings warrant fur- ther efforts in improving drug entry into the blood via liposomes, especially for situations where very little of the drug is required to enter the circulation (e.g. in administering factor VIII or some peptide hormones). To this end, liposomes of certain lipid compositions (e.g. high-melting phospholipids together with cholesterol) exhibit remarkable stability towards deter- gents. Experience in my laboratory (unpublished) shows that multilamellar

Trends in Biotechnology, Vol. 3, No. 9, 1985

liposomes composed of equimolar distearoyl phosphatidylcholine (a high- melting lecithin) and cholesterol will not liberate their entrapped solute in the presence of the membrane- solubilizing reagent, Triton X100, unless the mixture is boiled. These liposomes are likely to be considerably resistant to bile salts and may improve the proportion of drugs (e.g. peptides) surviving the gut milieu and entering the blood circulation. For drugs such as insulin, however, where variability in adsorption is unacceptable, ways must be found to ensure quantitative delivery (over 90%) into the blood by liposomes.

Appl ica t ions in medicine Interaction ofliposomes with macro-

phages in vivo within the RES, the blood stream, or granulomata in a variety of tissues, forms the basis for most of the medical applications of the system with some hope of early realization 1's'9. In this respect, lipo- somes can operate in at least two ways: (1) by introducing agents into the cells to act on undesirable residents, be it a microbe or a chemical; (2) by using agents which activate the killing or otherwise properties of the cells. In both cases remarkable results have been achieved already.

Microbial diseases Animals with intracellular parasitic

diseases such as leishmaniasis can be. cured with single small doses of

Time after injection (hours)

10(I-

75-

50-

"8 25"

10-

Fig. 3. Clearance of liposomes from the circula- tion. Mice (five animals) were injected intra- venously with small unilamellar liposomes com- posed of distearoyl phosphatidylcholine, dipal- mitoyl phosphatidylethanolamine and choles- terol(molar ratio O. 8 : 0.2 : I). Liposomes con- tained 0.2 M carboxyfiuorescein and tracer " ~ ln-labelled bleamycin in the aqueous phase and tracer [ J 4 C] distearoyl phosphatidylcholine in the lipid phase. Values are % + SD of admin- istered carbaxyfluorescein or radioactivity per total blood volume of each animal at intervals after injection.

Trends in Biotechnology, VoL 3, No. 9, 198~

relevant drugs entrapped in lipo- somes'. Larger, repeated doses of the drugs needed when given in the free form, are toxic and prevent successful treatment of the many hundred millions of humans affkcted by parasitic diseases world-wide. Whilst there are other (mostly logistic) problems as well with the treatment of such diseases in the developing world, single-dose treat- ment would certainly help enormously with both increasing curing rate and reducing costs. Other microbial diseases in which microorganisms reside intracellularly and which are also good candidates for treatment with liposomal drugs include brucellosis, tuberculosis, listeria infections and leprosy (bacterial), malaria (protozoal), and, possibly, hepatitis, yellow fever and cytomegalovirus infection (viral disease of the liver). Depending on the parasitic infections, the infected cells can be macrophages of the RES (e.g. brucellosis, tuberculosis) or hepatic parenchymal cells (e.g. malaria, hepatitis B).

Immune system Further possibilities are the

correction of metabolic and bacterio- cidal deficiencies in chronic granu- lomatous disease and the use of inter- feron and macrophage activation factors (MAFs). Interferon, one of the genetically engineered products on which industry has placed great hopes, has already been used H successfully in a liposome form to treat murine

Table 2. Antigens incorporated into lipasomes

Antigen Refs Diphtheria toxoid 26 Influenza virus haemaglutinin and

neuraminidase 7 Vesicular stomatitis virus glycolipid

extract 29 Semliki forest virus glycoproteins 30 Plasmodiumfalsiparum antigens 31 Hepatitis B surface antigen 32 Hepatitis B surface antigen

polypeptides 33 Synthetic peptide corresponding to

residues 135-155 of hepatitis B surface antigen 34

Cholera toxin 35, 36 Rubella haemaglutinin 37 Gross cell surface antigen 38 Adenovirus hexon and fiber capsid

proteins 39 Epstein-Barr virus antigen

MAgp 340 40 Human spermatozoal peptide

antigens 41 Rat transplantation antigens 42 Streptococcus mutans cell-wall antigen 43

hepatitis. Tests in experimental animals have also shown convincingly that MAFs are far more effective in pro- longing the life of, or curing tumour- bearing rodents when given via lipo- somes. For instance, in experiments with liposomal muramyl dipeptide, 74% of the treated mice were free of visible metastases. With free muramyl dipeptide only 20% of the mice were free of visible metastases ~°. The tech- nique has exciting potential for human use.

Metal detoxification Metals such as iron, lead, mercury,

cadmium and plutonium, accumulate in the RES tissues as a result of either metabolic disturbances in the body or environmental pollution. Chelation therapy, the administration of agents which interact with metals to form soluble complexes which are easily excreted, is hampered by the rapid, premature clearance of chelating agents from the body or their failure to penetrate cell membranes effectively. There are many examples 23 in the literature where chelation therapy for a variety of metal storage conditions has been improved using liposome- entrapped chelating agents. As with other drugs, liposomes retain much of the chelating agents en route to the tissue and facilitate their entry into the cells.

Adjuvants An area in which liposomes appear

particularly attractive for drug delivery is potentiation of immune response by acting as immunological adjuvants I'12. Adjuvants are agents which act in a non-specific fashion to augment the immune response to a given antigen 24. It is believed that adjuvants stimulate the proliferation and differentiation of immunocompetent cells (cells involved in the chain of events producing anti- bodies). A safe and effective adjuvant for use in human and animal immunization programs would reduce the amount of antigen (e.g. hepatitis B surface antigen or its polypeptide derivatives, diphtheria and tetanus toxolds) required for immunization with corresponding economies that are especially relevant to developing countries 24. However, adjuvants which are available today, including complete and incomplete Freund's adjuvant, bacterial endotoxins, polyanions and mineral adsorbents, can induce local

239

L¥S @~'" t

Cell m e m b r a n e

Fig. 4. Uptake of lipasomes by ceils. Endo- cytosis has been long recognized 4 as the major mechanism of liposome uptake by cells in vivo. In this simplified scheme, a lipasome containing drug molecules (dots) is endocytosed by the cell membrane. The endosome containing the lipo- some subsequently fuses with a lysosome (the digestive apparatus of cells, L YS). Within the resultant body (often known as phagolyso- some), lysosomal enzymes degrade the lipo- some and free drugs. These can either act within the lysosome or diffuse into other cell compartments or even out of the cell 7-9.

and systemic toxicity, form granulomas which persist in human subjects and pose health risks or are aesthetically unacceptable, lack efficiency or have only short-term effect. Even the vaccinia strategy 25 which employs the original smallpox vaccine virus vaccinia in a genetically engineered form containing antigens from other viruses, carries considerable risk. For instance, smallpox vaccination leads to a few encephalitis cases which, in view of the fact that smallpox has been eradicated, is unacceptable.

During early work in this labora- tory 26 it became apparent that liposomes could satisfy many of the requirements of an effective and safe adjuvant. Since liposomes are bio- degradable, interact with fLxed macro- phages avidly, exhibit great structural versatility and can be targeted to speci- fic cells in vivo (see later) they can be potentially manipulated to induce optimal antibody responses to associ- ated antigens 1'~2'27'2s. Indeed, a multi° tude of data with a number of medically relevant antigens used in immunization studies (Table 2) indicate that antibody response can often be far greater when the antigens are injected via liposomes. In addition, studies 32 with hepatitis B surface antigen have shown that immune response is cell-mediated as well, an event that plays a major role in

240

protection against most viral infec- tions 24. Successful immunization studies with liposomal antigens include the use of diphtheria toxoid, cholera toxin, hepatitis B surface antigen and polypeptides derived from it, rubella haemagglutinin and Streptococcus mutans cell-wall antigen (a potential vaccine for dental caries) (Table 2). The role of liposomes as immunological adjuvants will be strengthened further if experiments on enteral vaccines in this and other laboratories 36,43 combining liposome-entrapped anti- gens with stimulants of immune response in the gut prove successful.

Two aspects of liposomes have pre- viously inhibited enthusiasm in pharmaceutical indtistries, even for relatively 'straightforward' projects such as that of the immunological adjuvant: stability under storage and toxicity. As discussed already, liposomes can now be stored for extended periods in a stable suspension form, or be lyophilized (for example see Fig. 2). In addition, liposomes of appropriate lipid composition are not toxic~'8'9; they are toxic, for example, when sphingomyelin or phosphatidyl- serine are present (for reviews see Refs 1, 3, 7, 9 and 10). In contrast to most adjuvants, liposomes do not promote local or systemic reactions upon injec- tion ofpreimmunized animals with the entrapped antigen ~2'3~. There is also evidence that antibodies are not found against the lipid components of lipo- somes, at least in the case of lecithin 35.

Targeting ofllposomes In vitro studies

The quantitative interception oflipo- somes by the fixed macrophages of the RES has prompted workers interested in delivering drugs to other cells to develop means of endowing liposomes with target selectivity x'3'44. Cumulative evidence a'3'44 suggests that the following criteria are of particular importance for successful targeting. Firstly, the rate of uptake of liposomes by the RES must be diminished considerably so that lipo- somes can circulate in the blood long enough to ensure quantitative vesicle binding to the target. In this respect, small (~ 64 nm diameter), neutral uni- lamellar liposomes composed of high- melting phospholipids and cholesterol appear suitable ~9. Larger liposomes (accommodating much more drug than small ones) could also be targeted if interaction with the relevant cells is

very rapid and occurs quantitatively before significant interference by the RES tissues. This seems unlikely, however~ in view of the avidity with which liver and spleen remove large liposomes from the circulation. Attempts to delay clearance of large vesicles, successfully tested in animals, include saturation of the RES with empty liposomes followed by the administration of vesicles containing the drug 1. Unfortunately, such an approach, requiring the injection of large amounts of lipid may be too toxic for routine use in the clinic. Alterna- tively, it may be possible to coat the liposomal surface with agents which render liposomes less recognizable by the RES. One such agent, Poloxamer (a non-ionic surfactant of the polyoxy- ethylene polyoxypropylene series), has been used with some success in reducing the rate of clearance of nano- particles 45.

Secondly, liposomes must bear on their surface appropriate molecules (ligands) which can bind onto receptors on the surface of target cells. A variety ~'3'44 of targetting ligands have been used for this purpose, including antibodies (raised against cell surface antigens) and glycoproteins or glyco- lipids with terminal sugars which recognize their respective cell-surface receptors. Grafting of ligands onto the liposomal surface can be carried out by various techniques 3'44. These are classi- fied into those which possess anchoring hydrophobic regions enabling spon- teneous insertion of the ligand into the lipid bilayer during the preparation of liposomes, and those which require a coupling reaction between ligand and liposomes.

IgG is an example of a hydrophobic ligand: up to 20-30°7o of the immuno- globulin can be incorporated into small liposomes formed by sonication in the presence of the immunoglobulin 46 with the antigen-recognizing variable region available on the surface of the lipo- some 46. In the coupling methods, a heterobifunctional reagent is used to join appropriate groups of the ligand and of one of the liposomal lipid com- ponents 47. Regardless of how targeted liposomes are prepared, ligands are generally capable of mediating their up- take by the cell target in vitro. Uptake can vary from poor to quantitative depending on such parameters as the type of ligand, the curvature of lipo- somes and the line of cells used (e.g.

Trends in Biotechnology, VoL 3, No. 9, 1985

sufficient density of surface receptors) TM.

Thirdly, where there is a need of pharmacological action, it is usually desirable that ligand-mediated binding of liposomes to cell receptors is fol- lowed by the interiorization of the drug- containing carrier and subsequent liberation of the drug in active form. A series of experiments have now demon- strated 44'~8'49 pharmacological action on cell targets following binding, presumably upon entry of liposomes into the cells. However, other workers have shown ~ that liposome interioriza- tion by cells cannot be taken for granted once binding has occurred. Factors influencing liposome entry include the metabolic state of the cells, some of the liposomal structural characteristics (e.g. size, surface charge) and the nature of binding 44.

In vivo studies Although the ability of targeted lipo-

somes to interact with relevant cells specifically has been demonstrated mostly in vitro 1~'44, there is convincing evidence to show that targeting can also occur in vivo. A classic example is that of liposomes bearing molecules which bind to the galactose receptor in the liver. Upon injection, these liposomes are taken up selectively by all accessible tissue cells expressing the galactose receptor 5°-52 (as shown by blocking the receptor with a free ligand such that specific uptake, but not non-specific uptake, of liposomes decreases). More recently, targeting in vivo within the vascular system, was achieved using small liposomes coated with mono- clonat antibodies53: AKR mice were injected intravenously first with AKR- A cells and soon after with liposomes coated with monoclonal IgGl, specific for the Thyl . 1 antigen expressed on the surface of these cells. Liposomes and cells still in the circulation interacted to some extent. The advent ofhybridoma technology has now added further impetus in liposome targeting which could, under certain conditions, have advantages over the use of antibodies coupled to drugs directly. For instance, liposomes can accommodate large quantities of a wide range of drugs, keep them in isolation from the bio- logical milieu and bring them into con- tact with cells, potentially by a single immunoglobulin molecule per vesicle 19m. In addition, liposomes with antibodies against more than one cell-

Trends in Biotechnology, Vol. 3, No. 9, 1985

surface antigenic determinant may exhibk a firmer and more specific asso- ciation with the ceils expressing the determinants 44. However, in contrast to antibodies, liposomes, like other particulate carriers, cannot undergo transcapillary passage and are not, therefore, ideal for targeting to extra- vascular sites, at least by the intra- venous route.

Comparison with other particle- like carriers

It is difficult to compare the targeting efficiency of liposomes with that of non-liposomal part ideqike carriers, mainly because the latter have not been investigated significantly in this respect. For various reasons, including unique structural and behavioural versatility, liposomes have attracted both most of the attention of workers in drug delivery and most o f the publicity. Nonetheless, interest in systems such as nanoparticles, albumin micro- spheres and other particles made of polymeric and synthetic materials is increasing and specific applications, mainly in localized areas in the body, have been identified 54. Akhough it is probable that the variety of applications that l iposomes promise cannot be matched by any of the other systems, some o f the latter may prove easier to prepare and store. T h e mukitude of possibilities in applying drug delivery in medicine, biology, pharmacology and indeed in food and cosmetics industries means that no single carrier is likely to be a panacea for all needs. It also encourages a more realistic approach to drug delivery, namely distribution o f research efforts and resources with a wider range o f carriers.

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