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ELSEVIER International Journal of Pharmaceutics 154 (1997) 123 140internationaljournal ofpharmaceuticsReviewLiposomes in drug delivery: progress and limitationsAmarnath Sharma *, Uma S. SharmaDepartment qf Experimental Therapeutics, Roswell Park Cancer Institute, and Department of Pharmaceutics, SUN Y at Buffalo,Buffalo, NY, USAReceived 12 December 1996; receivedin revised form 26 March 1997; accepted 30 April t997AbstractLiposomes are microparticulate lipoidal vesicles which are under extensive investigation as drug carriers forimproving the delivery of therapeutic agents. Due to new developments in liposome technology, several liposome-based drug formulations are currently in clinical trial, and recently some of them have been approved for clinical use.Reformulation of drugs in liposomes has provided an opportunity to enhance the therapeutic indices of variousagents mainly through alteration in their biodistribution. This review discussesthe potential applications of liposomesin drug delivery with examples of formulations approved for clinical use, and th

e problems associated with furtherexploitation of this drug delivery system. 1997 Elsevier Science B.V.Keywords: Liposome; Drug delivery; Doxorubicin; Amphotericin B1. IntroductionRecent advances in biomedical science andcombinatorial chemistry have resulted in the de-sign and synthesis of hundreds of new agents withpotential activity against a wide range of thera-peutic targets in vitro. However, most of thesenew drugs fail to live up to their potential in theclinic. For instance, although there are numerous* Corresponding author. Tel.: + 1 716 8455754; fax: +1716 8458857; e-mail: [email protected]

anticancer agents that are highly cytotoxic totumor cells in vitro, the lack of selective antitu-mor effect in vivo precludes their use in clinic.One of the major limitations of antineoplasticdrugs is their low therapeutic index (TI), i.e. thedose required to produce anti-tumor effect is toxicto normal tissues. The low TI of such drugs maybe due to: (i) their inability to achieve therapeuticconcentrations at the target site (solid tumors); (ii)nonspecific cytotoxicity to critical normal tissuessuch as bone marrow, renal, GI tract and cardiactissue; and/or (iii) problems associated with for-0378-5173/97/$17.00 1997 Elsevier Science B.V. All rights reserved.

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126 A. Sharma, U.S. Sharma/ International Journal of Pharmaceutics 154 (1997) 123-140drugs. Thus, the predominant mechanism behindintracellular delivery of drugs by liposomes maymainly depend on their composition, as depictedin Fig. 2.Liposomes can be classified in terms of compo-sition and mechanism of intracellular delivery intofive types as: (i) conventional liposomes (CL); (ii)pH-sensitive liposomes; (iii) cationic liposomes;(iv) immunoliposomes; and (v) long-circulatingliposomes (LCL). The typical composition andcharacteristics for these types of liposomes arelisted in Table 1.2.2. On the basis of sizeThe liposome size can range from very small(0.025 /~m) to large (2.5 /~m) vesicles. Further-more, liposomes may have single or multiple bi-layer membranes (Fig. 1). The vesicle size is acritical parameter in determining circulation half-life of liposomes, and both size and number ofbilayers influence the extent of drug encapsulationin the liposomes.

On the basis of their size and number of bilay-ers, liposomes can also be classified into one ofthree categories: (i) multilamellar vesicles (MLV);(ii) large unilamellar vesicles (LUV); and (iii)small unilamellar vesicles (SUV). The size andcharacteristics of these types of liposomes arelisted in Table 2.ical states above and below the To. The lipids arein a rigid, well-ordered arrangement ('Solid' gel-like phase) below the To, and in a liquid-crys-talline ('Fluid') phase above the To. The fluidity ofliposome bilayers can be altered by using phos-pholipids with different T~ which in turn can vary

from -20 to 90C depending upon the lengthand nature (saturated or unsaturated) of the fattyacid chains. Presence of high T~ lipids (Tc > 37C)makes the liposome bilayer membrane less fluid atthe physiological temperature and less leaky. Incontrast, liposomes composed of low T~ lipids(T~ < 37C) are more susceptible to leakage ofdrugs encapsulated in aqueous phase at physio-logical temperatures. The fluidity of bilayers mayalso influence the interaction of liposomes withcells: liposomes composed of high Tc lipid appearto have a lower extent of uptake by RES, com-pared to those containing low Tc lipid (Gabizon

and Papahadjopoulos, 1988). Incorporation ofcholesterol into lipid bilayer can also affect thefluidity of the membranes. At high concentrations(> 30 molar %), cholesterol can totally eliminatephase transition and decrease the membrane fluid-ity at a temperature > Tc, which makes the lipo-somes more stable and less leaky after systemicadministration.3.2. Surface charge3. Parameters which influence in vivo behavior of


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liposomesLiposome formulations of various drugs can beoptimized in terms of drug content, stability, de-sirable biodistribution patterns, and cellular up-take by altering their physicochemical parameters.These parameters include fluidity of bilayer mem-brane, surface charge density, surface hydration,and size (Straubinger et al., 1993). The effects ofthese parameters on the physical and biologicalperformance of liposomes are described below.3.1. Bilayer fluidityLipids have a characteristic phase transitiontemperature (To), and they exist in different phys-The nature and density of charge on the lipo-some surface are important parameters which infl-uence the mechanism and extent of liposome-cellinteraction. Both of these parameters can be al-tered by changing the lipid composition. Lack ofsurface charge can reduce physical stability ofsmall unilamellar liposomes (SUV) by increasingtheir aggregation (Sharma and Straubinger, 1994).Moreover, neutral liposomes do not interact sig-nificantly with cells, and in such cases, the drugmay mainly enter cells after being released from

liposomes extracellularly (Sharma et al., 1993a).On the other hand, high electrostatic surfacecharge could promote liposome-cell interaction.It has been shown that negatively charged lipo-somes are predominantly taken up by cellsthrough coated-pit endocytosis (Straubinger et al.,


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A. Sharma, U.S. Sharma//International Journal of Pharmaceutics 154 (1997) 123 140 1291983) as depicted in Fig. 2. Studies have alsoshown that negative charge density influences theextent of liposome interaction with cells (Sharmaet al., 1993a). Negative surface charge may notonly increase intracellular uptake of liposomes bytarget cells but by the same mechanism acceleratetheir plasma clearance after systemic administra-tion (Gabizon et al., 1990). Negatively chargedliposomes may also release their contents in thecirculation and/or extracellularly after interactionwith blood components and tissues. Unlike nega-tively charged liposomes, cationic liposomes havebeen proposed to deliver contents to cells byfusion with cell membranes (Felgner et al., 1994).3.3. SurJiwe hydrationInclusion of a small fraction (5-10 mole%) ofcompounds bearing hydrophilic groups such asmonosialoganglioside (GM~), hydrogenated phos-phatidylinositol (HPI) and polyethylene glycolconjugated with lipid (PEG-DSPE), in the bilayermembrane reduces the interaction of liposomes

with cells and blood components and makes lipo-somes sterically stabilized (Allen and Chonn,1987; Allen et al., 1985; Gabizon and Papahad-jopoulos, 1988; Papahadjopoulos et al., 1991). Asmentioned earlier, the presence of hydrophilic sur-face coating offers steric hindrance to opsoninadsorption on bilayer which further reduces therate of liposome uptake by the cells of RES (Allenet al., 1991). Thus, the sterically stabilized lipo-somes are more stable in biological environmentand can exhibit up to 10-fold higher circulationhalf-lives than liposomes without hydrophilic sur-face coating (Klibanov et al., 1991; Lasic et al.,

1991).3.4. Liposome sizeLiposome size is one of the main parameterswhich determines the fraction cleared by the RES(Harashima et al., 1994). Small liposomes (_< 0.1/Lm) are opsonized less rapidly and to a lowerextent compared to large liposomes (>0.1 /~m)and therefore, the rate of liposome uptake byRES increases with the size of the vesicles. Reduc-tion in liposome size has also been correlated withincreased accumulation in tumor tissue. This maypartly be due to longer circulation half-life ofsmall liposomes compared to large liposomes. In

addition, small liposomes can extravasate by pas-sive convective transport through the tumor capil-laries much more easily than large liposomes.Tumor capillaries are generally more permeablethan normal capillaries and fluid can leak throughgaps in the vasculature along with plasmaproteins, other macromolecules and microparticu-lates such as liposomes. Extravasation of lipo-somes depends on their size and occurs passively;the driving force behind this is the difference


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between intravascular hydrostatic and interstitialpressures (Yuan et al., 1994). Liposome size canbe reduced by sonication, extrusion, or mi-crofluidization of MLV or LUV.3.5. Method of preparationLiposomes of different sizes and characteristicsusually require different methods of preparation.The most simple and widely used method forpreparation of MLV is the thin-film hydrationprocedure in which a thin film of lipids is hy-drated with an aqueous buffer at a temperatureabove the transition temperature of lipids. Thedrug to be encapsulated is included either in theaqueous hydration buffer (for hydrophilic drugs)or in the lipid film (tbr lipophilic drugs). Thin-filmhydration method produces a heterogeneous pop-ulation of MLV (1 5/~m diameter) which can besonicated or extruded through polycarbonatefilters to produce small (up to 0.025 /~m) andmore uniformly sized population of SUV.Although thin-film hydration is a simple tech-nique, one of the major disadvantages of thismethod is its relatively poor encapsulation effi-ciency (5 15%) of hydrophilic drugs. Moreover,

reduction of liposome size further decreases theamount of encapsulated drug. MLV with highentrapment efficiency (up to 40%) can be preparedby freeze-drying preformed SUV dispersion in anaqueous solution of the drug to be encapsulated(Ohsawa et al., 1984). The encapsulation effi-


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130 A. Sharma, U.S. Sharma / International Journal of Pharmaceuties 154 (1997) 123-140ciency of MLV can also be increased by hydratinglipid in the presence of an organic solvent (Papa-hadjopoulos and Watkins, 1967; Gruner et al.,1985).Several methods have been developed for thepreparation of large, unilamellar vesicles (LUV),including solvent (ether or ethanol) injection, de-tergent dialysis, calcium induced fusion, and re-verse-phase evaporation (REV) techniques. SUVcan be prepared from MLV or LUV by sonica-tion (using probe sonicator) or extrusion (passagethrough a small orifice under high pressure).In the methods described above, an amphiphilicionizable drug which exhibits lipophilic and hy-drophilic properties depending on the pH of thesolution, may not be encapsulated with high effi-ciency because the drug molecules can diffuse inand out of the lipid membrane. Thus, the drugwould be difficult to retain inside liposomes.However, these type of drugs can be encapsulatedinto preformed liposomes with high efficiency (up

to 90%) using the 'active loading' technique (Clercand Barenholz, 1995). In the 'active loading'method, the pH in the liposome interior is suchthat the unionized drug which enters the liposomeby passive diffusion is ionized inside the liposome,and ionized drug molecules accumulate in theliposome interior in high concentrations due totheir inability to diffuse out through the lipidbilayer. For example, doxorubicin and epirubicinmay be entrapped in preformed SUV with highefficacy using 'active loading' methods (Mayer etal., 1990; Mayhew et al., 1992).For a detailed review of methods of liposome

preparation see Szoka and Papahadjopoulos(1980), Deamer and Uster (1980); Martin (1990)and New (1990).4. Applications of liposomes in drug deliveryNew drug delivery systems such as liposomesare developed when an existing formulation is notsatisfactory and reformulation offers superiortherapeutic efficacy and safety over the existingformulation. Indeed, liposome formulations ofsome drugs have shown a significant increase intherapeutic efficacy and/or therapeutic indices inpreclinical models and in humans, compared totheir non-liposomal formulations. The therapeutic

applications of liposomes generally fall into sev-eral categories briefly described below.4.1. Formulation aidHydrophobic drugs such as cyclosporin andpaclitaxel are usually formulated in surfactantsand organic co-solvents for systemic administra-tion in humans. These solubilizers may cause tox-icity at the doses needed to deliver the drug. Incontrast, liposomes are made up of lipids whichare relatively non-toxic, non-immunogenic, bio-


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compatible and biodegradable molecules, and canencapsulate a broad range of water-insoluble(lipophilic) drugs. Currently, liposomes or phos-pholipid mixtures are being used as excipients forpreparing better-tolerated preclinical and clinicalformulations of several lipophilic, poorly water-soluble drugs such as amphotericin B. In preclini-cal studies, liposomes have been evaluated as avehicle for the delivery of paclitaxel and itsanalogs as an alternative to the cremophor/ethanol vehicle (Sharma, 1994; Sharma et al.,1993; 1995). Paclitaxel liposomes were able todeliver the drug systemically and increase thetherapeutic index of paclitaxel in human ovariantumor models (Sharma et al., 1997, 1995).4.2. Intracellular drug deliveryDrugs with intracellular targets/receptors arerequired to cross the plasma membrane for phar-macological activity. Liposomes can be used toincrease cytosolic delivery of certain drugs such asN-(phosphonacetyl)-L-aspartate (PALA) whichare normally poorly taken up into cells (Heathand Brown, 1989-1990). PALA is taken up intothe tumor cells through fluid-phase endocytosis

(pinocytosis) and it diffuses out into the cyto-plasm as the endosome pH drops (White andHines, 1984). However, pinocytosis is very limitedin its efficiency. Liposomal delivery of drugswhich normally enter the cells by pinocytosis canbe very effective (Sharma, 1994) because lipo-somes can contain greater concentrations of drugcompared to the extracellular fluid and the endo-


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A. Sharma, U.S. Sharma / International Journal of Pharmaceutics 154 (1997) 123-140 131cytosis process by which negatively charged lipo-somes are predominantly taken up by the cells, ismore efficient than pinocytosis. For example, thepotency of PALA encapsulated liposomes was upto 500-fold greater against human ovarian tumorcell lines than that of free PALA (Sharma et al.,1993a).4.3. Sustained release drug deliverySustained release systems are required for drugssuch as cytosine arabinoside (Ara-C) that arerapidly cleared in vivo and require plasma con-centrations at therapeutic levels for a prolongedperiod for optimum pharmacological effects. It isnow possible to design sustained release liposomeformulations with an extended circulation half-lifeand an optimized drug release rate in vivo. Forexample, Ara-C encapsulated in LCL is effectiveas a prolonged release system in the treatment ofmurine L1210/C2 leukemia (Allen et al., 1992).Conventional liposomes which localize by phago-cytosis in the cells of RES may also act as a

sustained release depot by slowly leaking drugsfrom RES into the general circulation.4.4. Gene therapyA number of systemic diseases are caused bylack of enzymes/factors which are due to missingor defective genes. In recent years, several at-tempts have been made to restore gene expressionby delivery of the relevant exogenous DNA orgenes to cells (reviewed by Crystal, 1995).Cationic liposomes (Table 1) have been consid-ered as potential non-viral human gene deliverysystem (Felgner et al., 1987; Felgner and Ringold,1989; Felgner et al., 1995; Gao and Huang, 1995).

They are usually composed of a cationic lipidderivative and a neutral phospholipid (DOPE).The latter is required by certain cationic lipids toform stable liposomes. Some of the widely usedcationic liposome formulations are: lipofectin(DOTMA:DOPE, 1:1); lipofectamine (DOSPA:DOPE, 3:1); transfectace (DDAB:DOPE, 1:3); cy-tofectin (DMRIE:DOPE); transfectam (DOGS)and DC-cholesterol. The negatively charged ge-netic material (e.g., plasmid) is not encapsulatedin liposomes but complexed with cationic lipidsby electrostatic interactions. Plasmid-liposomecomplexes are thought to enter the cell by fusion

with the plasma or endosome membrane (Fig. 2).Allovectin-7, a gene transfer product is cur-rently in clinical trials (phase I/II) as an im-munotherapeutic agent for the treatment ofmetastatic melanoma, renal cell and colorectalcarcinoma (Table 3). Allovectin-7 is composed ofa plasmid containing the gene for the major histo-compatibility complex antigen HLA-B7 with fl-2microglobulin formulated with the cytofectin(DMRIE:DOPE). The ongoing clinical trials


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have indicated that intralesional injection ofAllovectin-7 can be performed safely and havedemonstrated antitumor activity in some patients(Nabel et al., 1995).Plasmid-liposome complexes have many ad-vantages as gene transfer vehicles over viral-basedvectors (Crystal, 1995): (i) these complexes arerelatively nonimmunogenic because they lackproteins; (ii) liposomes or lipid complexes can beused for transfection of large-sized genetic mate-rial; and (iii) viruses, unlike plasmid-liposomecomplexes, may replicate and cause infections.However, there are several problems limiting theapplication of liposomes as a gene delivery sys-tem. Some of these problems are discussed in theSection Section 5.5.4.5. Site-avoidance deliveryDrugs used in the treatment of diseases likecancer usually have a narrow therapeutic index(TI) and can be highly toxic to normal tissues.The toxicity of these drugs may be minimized bydecreasing delivery to critical normal organs. Ithas been shown that even a small reduction indistribution of the drug to critical organs by

encapsulation in liposomes can significantly re-duce the drug toxicity (Szoka, 1991). Liposomesare taken up poorly by tissues such as heart,kidney, and GI tract, which are major sites fortoxic side-effects of a variety of antineoplasticdrugs. Thus, liposome formulation may improvethe TI by altering the biodistribution of drugaway from drug sensitive normal tissues. For


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A. Sharma, U.S. Sharma / International Journal of Pharmaceutics 154 (1997) 123 140 133Table 4Liposomeand lipid-basedproductsapproved for humanuseProduct drug Formulation Company Indication/target CountryDoxilVM(a) Doxorubicin Liposomes(LCL) SequusPharmaceuticals, KaposisarcomaInc., CA in AIDSLiposomes(CL) NeXstarPharmaceutical, SeriousfungalInc., CO infectionsLiposomes(LCL) NeXstarPharmaceutical, KaposisarcomaInc., CO in A1DSLipid complex SequusPharmaceutical, SeriousfungalInc., CA infectionsLipid complex The LiposomeCompany, SeriousfungalNJ infectionsAmbisomeTM AmphotericinBDaunoX-DaunorubicinomeTM citrateAmphocilTM AmphotericinBAbelcetTM AmphotericinBUSA and EuropeAround the worldin 24countries

USA and EuropeAsia and EuropeUSA and EuropeaMarketedas CaelyxTM in Europe.4.6.1. Passive targetingThis approach for liposome drug delivery ex-ploits the natural tendency of certain cells such asKupffer cells in the liver, and circulatingmacrophages of RES to phagocytose foreign mi-croparticulates such as liposomes. Conventionalliposome (CL) formulations of drugs and im-munostimulators have been successfully used fortargeting the cells of RES, and exhibit significant

improvement in the TI of the drugs (reviewed byAlving, 1991). In clinical trials, systemic adminis-tration of CL containing muramyl peptide deriva-tives caused enhancement in the tumoricidalproperties of monocytes in patients with recurrentosteosarcoma (reviewed by Killion and Fidler,1994). Liposomes have also been used to enhancethe antigenicity of certain molecules for new vac-cine formulations (Section Section 4.8). Further-more, CLs have also been employed for targetingof immunosuppressive drugs to lymphatic tissuessuch as the spleen. In a preclinical model, anincrease in immunosuppressive activity, i.e. a de-

lay in heart transplant rejection was observed withCL-encapsulated methylprednisolone (Binder etal., 1994).4.6.2. Active targetingActive targeting of liposome encapsulated drugsmay be accomplished by coupling specific anti-bodies to vesicles (immunoliposomes). Immuno-liposomes containing diphtherin toxin (DT) havebeen shown to provide protection against thenon-specific toxicity of DT during cancer


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chemotherapy (Vingerhoeds et al., 1996). Long-circulating immunoliposomes (hydrophilic poly-mer-coated vesicles bound to antibodies and


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134 A. Sharma, U.S. Sharma / International Journal of Pharmaceutics 154 (1997) 123-140(iii) drugs can also reach their intracellular targetby diffusion after release from immunoliposomesassociated with target tissue (Fig. 2). Therefore,unlike antibodies-drug conjugates, in some casesimmunoliposomes may not have to undergo re-ceptor mediated-endocytosis to deliver their con-tents intracellularly. The limitations of activetargeting of drugs using liposomes are discussedin the Section Section 5.4.4. 7. IntraperitoneaI administrationDirect administration of antineoplastic agentsinto the intraperitoneal (i.p.) cavity has been pro-posed to be therapeutically advantageous forcancers that develop in or metastasize to theperitoneal cavity (Dedrick et al., 1978). Intraperi-toneal chemotherapy has been somewhat unsuc-cessful for free drugs because of relatively fastclearance of the drugs from the i.p. cavity result-ing in lowered concentrations at the site of ac-tion (Markman et al., 1989). However, theclearance of liposomes from the peritoneal cavity

is significantly slower than that of free drug andtherefore, higher drug concentrations can beachieved in the proximity of the target site forextended periods of time with the use of lipo-some formulations. Furthermore, reformulationof erosive drugs in liposomes has been shown toreduce local drug toxicity such as dermal toxicityof doxorubicin (Forssen and Tokes, 1983). Anincrease in TI of paclitaxel in liposomes after i.p.administration (Sharma et al., 1996) may also bedue to a reduction in local (abdominal) toxicityof the drug.Crommelin, 1995). The tendency of liposomes to

interact with macrophages in RES is exploited inthis approach (passive targeting).The mechanism by which liposomes cause in-creases in antigens' immune response is not fullyunderstood. However, augmentation of liposomaladjuvanticity can be achieved by co-administra-tion of liposome encapsulated antigen with otheradjuvants such as lipid A, lipopolysaccharides,muramyldipeptide and interleukin (IL-2) (Grego-riadis, 1990; Gregoriadis and Panagiotidi, 1989).Furthermore, antibody-mediated targeting ofliposomal to antigen-presenting cells may alsoimprove immunostimulatory effects (Alving,

1991). The influence of physicochemical proper-ties of the liposomes such as charge density,membrane fluidity and epitope density, on theimmune response of the antigen has been exten-sively studied (reviewed by Kersten and Crom-melin, 1995). For instance, liposomeformulations of inactivated encephalomyocarditisand Semliki Forest viruses were significantlymore immunogenic when charged phospholipidswere used compared to neutral lipids (Kraai-


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jeveld et al., 1984). The phase transition tempera-ture (To) of the lipids also appears to influenceimmunogenicity. For example, immunogenicityof haptens was higher in liposomes composed oflipids with a high Tc than in those with a low T~(Yasuda et al., 1977).Recently, the first liposome-based vaccine(liposomes containing inactivated hepatitis Avirions) was approved for human use in Switzer-land and currently, several other liposome-basedvaccines are in clinical trials (Gregoriadis, 1995).4.8. Immunological adjuvants in vaccinesLiposomes can encapsulate antigens in theiraqueous space or incorporate in the bilayer de-pending on the lipophilicity of the antigen. Lipo-somes were first used as immunologicaladjuvants in order to enhance the immune re-sponse to encapsulated diphtheria toxoid (Allisonand Gregoriadis, 1974). Since then, liposomeshave been used as nontoxic adjuvants with bacte-rial, viral, protozoan, tumor and other antigens(Alving, 1991; Gregoriadis, 1995; Kersten and5. Limitations of liposome technologyAs described above, liposomes have a great

potential in the area of drug delivery. However,liposome-based drug formulations have not en-tered the market in great numbers so far. Someof the problems limiting the manufacture anddevelopment of liposomes have been stability is-sues, batch to batch reproducibility, sterilizationmethod, low drug entrapment, particle size con-trol, production of large batch sizes and short


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A. Sharma, U.S. Sharma /International Journal of Pharmaceutics 154 (1997) 123 140 135circulation half-life of vesicles. Some of these is-sues such as short half-life have been resolvedresulting in increased numbers of clinical trials(Table 3) and new approvals (Table 4). Some ofthe remaining problems are discussed in detailbelow.5.1. StabilityOne of the major problems limiting the wide-spread use of liposomes is stability--both physi-cal and chemical. Depending on their compo-sition, the final liposome formulations may haveshort shelf-lives partly due to chemical and physi-cal instability. Chemical instability may be causedby hydrolysis of ester bond and/or oxidation ofunsaturated acyl chains of lipids. Physical insta-bility may be caused by drug leakage from thevesicles and/or aggregation or fusion of vesicles toform larger particles. Both of these processes(drug leakage and change in liposome size) influ-ence the in vivo performance of the drug formula-tion, and therefore may affect the therapeutic

index of the drug. For instance, large liposomesmay be rapidly cleared by RES leading to sub-therapeutic plasma concentrations of the drug andreduced AUCs (area under the plasma concentra-tion-time curve). Physical instability may alsooccur due to partitioning out of a hydrophobicdrug from the bilayer into the solvent on standing(or long term storage). Some of the stability prob-lems may be overcome by lyophilization in whichthe final liposome product is freeze-dried with acryoprotectant (mostly a sugar like Trehalose)and is reconstituted with vehicle immediatelyprior to administration. Lyophilization increases

the shelf-life of the finished product by preservingit in a relatively more stable dry state. Someliposome products on market or in clinical trialsare provided as a lyophilized powder. For exam-ple, AmBisomeTM,the first liposome product to bemarketed in several countries is supplied as alyophilized powder to be reconstituted with sterilewater for injection. Recently, lyophilized pacli-taxel-liposome formulations have been developedwhich show good stability (Sharma et al., 1997;Straubinger et al., 1995).5.2. SterilizationIdentification of a suitable method for steril-

ization of liposome formulations is a major chal-lenge because phospholipids are thermolabile andsensitive to sterilization procedures involving theuse of heat, radiation and/or chemical sterilizingagents. The method available for sterilization ofliposome formulations after manufacture is filtra-tion through sterile 0.22 ,urn membranes. How-ever, filtration is not suitable for large vesicles(>0.2 /zm) and also is not able to removeviruses. Sterilization by other approaches such as


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7-irradiation and exposure to chemical sterilizingagents are not recommended because they cancause degradation of liposome components andmay leave toxic contaminants (Zuidam et al.,1995). Suitable methods for sterilization of lipo-some formulations are being explored by severalgroups. For instance, it has been shown thatunder certain conditions, liposomes with ther-mostable, lipophilic drugs could be sterilized byautoclaving without substantial loss of contentsand/or degradation of phospholipids (Zuidam etal., 1993).5.3. Encapsulation efficiencyLiposome formulation of a drug could only bedeveloped if the encapsulation efficiency is suchthat therapeutic doses could be delivered in areasonable amount of lipid, since lipids in highdoses may be toxic and also cause non-linear(saturable) pharmacokinetics of liposomal drugformulation. Some new approaches that providehigh encapsulation efficiencies for hydrophilicdrugs have been developed. For instance, activeloading of the amphipathic weak acidic or basicdrugs in empty liposomes can be used to increase

the encapsulation efficiency (Mayer et al., 1986;Clerc and Barenholz, 1995). However, active load-ing is not suitable for hydrophobic drugs such aspaclitaxel for which encapsulation efficiency is< 3 mole% mainly due to the low affinity of drugfor the lipid bilayers (Sharma et al., 1997; Sharmaand Straubinger, 1994).


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136 A. Sharma, U.S. Sharma / International Journal of Pharmaceutics 154 (1997) 123-1405.4. Active targetingOne of the major limitations of active targetingusing ligand-directed immunoliposomes has beentheir rapid clearance due to non-specific uptakeby the cells of RES. The development of LCLconjugated with ligands has revived interest in thisfield since LCL are not as rapidly cleared by RES.However, many problems still remain to be over-come. For instance, foreign immunoglobulin-lig-ands conjugated to immunoliposomes may induceimmunogenicity and increase clearance on subse-quent exposure. The ligand (antibodies) conju-gated with liposomes may increase the liposomesize and reduce extravasation and thus could limittargeting to intravascular targets (Gregoriadis,1995). It has been shown that size of LCI may beincreased in the blood circulation by interactionof the antibodies with serum components, whichin turn can increase their size-dependent uptakeby spleen (Emanuel et al., 1996). Moreover, im-munoliposome enter the cells by endocytosis (Fig.

2) and if liposome contents are not released fromthe endosome, they would ultimately be degradedin the lysosomes (Section 5.6). This would only betrue for drugs sensitive to lysosomal enzymes.5.5. Gene therapyA number of technical problems have to beovercome before cationic liposome-mediatedtransfection can be fully exploited. For instance,liposomes are significantly less efficient than viral-vectors in their transfection ability. Furthermore,the DNA-lipid complexes are not stable in termsof particle size (Gao and Huang, 1995; Hong etal., 1997) for long periods of time. In addition,

there is lack of in vivo targeting after systemicadministration, and the toxicity of cationic lipidslimits the administered dose of the DNA lipidcomplex. Plasmid-liposomes complexes may bemore suited to delivery of genetic material bylocal administration.5.6. Lysosomal degradationOnce the liposome has reached the target cell,the efficacy is determined not only by the amountof drug associated with the cell, but also by theamount of drug reaching the 'target molecule'inside the cells. Immunoliposomes may deliver thedrug to the cells selectively but the pharmacologi-

cal activity depends on the ability of intact drugto diffuse into cytoplasm from the endosomes insufficient amounts.6. Liposome formulations in marketLiposome and lipid-complex formulations ofdoxorubicin, daunorubicin and amphotericin B(AmB) have been approved for clinical use inseveral countries (Table 4). Anthracycline gly-cosides such as doxorubicin and daunorubicin arehighly effective antineoplastic drugs, however,


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they can cause severe cardiac toxicity in humans.For free doxorubicin, the potential for develop-ment of irreversible cardiomyopathy is the majordose-limiting factor which restricts the life-timecumulative drug dose in humans to 550 mg/m 2.Long-circulating liposome formulations of an-thracyclines have been shown to improve the TIof the drugs against a variety of solid tumors bynot only reducing cardiac toxicity but also in-creasing drug accumulation in tumors (Gabizon,1994; Forssen et al., 1996). Doxorubicin is anideal candidate for encapsulation in liposomessince it can be encapsulated with high efficiencyinto liposomes using an active loading method(Mayer et al., 1990). The first liposome productapproved for use in USA was DoxilTM, a LCLformulation of doxorubicin. DoxilTM exhibited upto 8-fold increased circulation half-life comparedto free drug (Gabizon, 1994) and a lower inci-dence of side-effects presumably by avoiding highpeak concentrations of the free drug. Encapsula-tion of doxorubicin in liposomes has increased theTI and made possible dose escalation mainly byreducing the dose-limiting cardiac toxicity.

Amphotericin B (AmB) is an antifungal agentused for the treatment of serious systemic fungalinfections. However, AmB therapy is associatedwith high rates of serious side effects which in-clude nephrotoxicity, thrombophlebitis, hypoka-lemia and anemia (de Marie et al., 1994). Theseside effects limit the dose levels which can


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A. Sharma, U.S. Sharma / International Journal of Pharmaceutics 154 (1997) 123 140 137be achieved (0.7-1.5 mg/kg of Fungizone TM)andare the major reason for failure or discontinuationof therapy. Liposome-and lipid-based formula-tion of AraB have been shown to have superior TIover the deoxycholate-based formulation (Fungi-zoneTM) mainly due to a decrease in the doselimiting nephrotoxicity. The incidence of otherside-effects is also lower; in some studies these(primarily hypokalemia) ranged from 10 to 20%(Hay, 1994; Ng and Denning, 1995). The reduc-tion in toxicity may be due to selective transfer ofAmB from lipid bilayers or complexes to thefungus (target site), thus decreasing the interac-tion of drug with human cell membranes (Julianoet al., 1987). The reduction in the extent andfrequency of side effects such as nephrotoxicityhas allowed for escalation of AmB doses.In summary, this article reviewed the possibleapplications of liposomes and discussed, in brief,some problems associated with formulation anddevelopment. An encouraging sign is the increas-

ing number of clinical trials involving liposomeand lipid-based products (Table 3). With thenewer developments in the field, several compa-nies are actively engaged in expansion and evalua-tion of liposome products for use in anticancerand antifungal therapy and for prophylaxis (vac-cines) against diseases. Further refinements in theliposome technology will spur the full-fledgedevolvement of liposomes as drug carriers.7. AbbreviationsDORIEDOPEDOSPA

DOTAPDOTMAFLGM1HPILCILCLLUVMLVOAPCPEPG

RESPEG-DSPESUVTI1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bro-midedioleoylphosphatidylethanolamine2,3-dioleoyloxy-N-(2(spermine


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carboxamido)-ethyl)-N,N-dimethyl-1-propanaminiumfluoroacetate1,2-dioleoyloxy-3-(trimethylam-monio) propaneN-{1-(2,3-dioleoyloxy)propyl}-N,N,N-trimethyl ammoniumchloridefusogenic liposomesmonosialogangliosidehydrogenated phosphatidyl-inositollong-circulating immunolipo-someslong-circulating liposomeslarge unilamellar vesiclesmultilamellar vesiclesoleic acidphosphatidylcholinephosphatidylethanolaminephosphatidylglycerolreticuloendothelial systempolyethylene glycol conjugatedwith distearoyl PE

small unilamellar vesiclestherapeutic indexAmBCLCHEMSCholDC-cholDDABDOGSDMRIEamphotericin Bconventional liposomescholesteryl hemisuccinate

cholesterol3BN (N',N'-dimethy-laminoethane)carbamoyl-cholesteroldimethyl-dioctadecyl ammoniumbromidedioctadecyldimethyl ammoniumchloride1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammo-nium bromideAcknowledgementsAuthors would like to thank Drs Eric Mayhew

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