Antibody-directed liposomes as drug-delivery vehicles

Advanced Drug Delivery Reviews, 3 (1989) 343-389 343 Elsevier

ADR 00026

Antibody-directed liposomes as drug-delivery vehicles

Stephen Wright and Leaf Huang Department of Biochemistry, University of Tennessee, Knoxville, TN, U.S.A.

(Received February 5, 1988) (Accepted February 15, 1988)

Key words: Liposome; Drug targeting; Antibody-liposome conjugation; Liposome-cell interaction; Li- posome targeting

Contents

Summary ................................................................................................................. 344

I. Introduction ................................................................................................... 345

II. Preparation of antibody-targeted liposomes .......................................................... 347 1. Covalent conjugation to pre-formed liposomes ................................................ 349 2. Incorporation of lipophilic antibodies ............................................................ 352

III. Immunoliposome binding and drug delivery in vitro ............................................... 353 1. Binding to immobilized antigens ................................................................... 353

Abbreviations: RES, reticuloendothelial system; SUV, small unilamellar vesicle; PE, phosphatidylethanolamine; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; SPDP, N-hydroxysuccinimidyl- 3-(2-pyridyldithio)propionate; DTP, dithiopyridine; MPB, N-[4-(p-maleimidophenyl)butyryl]; LUV, large unilamellar vesicle; REV, reverse-phase evaporation vesicle; SATA, succinimidyl-S-acetylthioacetate; SAMSA, S-acetylmercaptosuccinic anhydride; AETA, aminoethylthioacetyl; NHSP, N-hydroxysuccinimide ester of palmitic acid; FITC, fluorescein isothiocyanate; MLV, multilamellar vesicle; MTX, methotrexate; DNP-cap, N-dinitrophenylamino caproyl; CF, carboxyfluorescein; DOPC, dioleoyl- phosphatidylcholine; TNP, trinitrophenyl moiety; PHC, palmitoyl homocysteine; CHEMS, cholesteryl hemisuccinate; DOPE, dioleoylphosphatidylethanolamine; RET, resonance energy transfer; OA, oleic acid; ANTS, 1-aminonaphthalane-3,6,8-trisulphonic acid; DPX, N,N'-p-xylylenebis (pyridinium bromide); NBD, 7-nitro-2,1,3-benzoxadiazol-4-yl; ADP, adenosine diphosphate; HSV, herpes simplex virus; TK, thymidine kinase; PEP, phosphoenolpyruvate; CK, carboxykinase; CAT, chloramphenicol acetyltransferase; AFP, anti-human c~-fetoprotein; MHC, major histocompatibility complex; DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; TPE, phosphatidylethanolamine transphosphatidylated from egg phosphatidylcholine

Correspondence: L. Huang, Department of Biochemistry, Biology Business Office, University of Ten- nessee, M407 Waiters Life Sciences Building, 125 Austin Peay Boulevard, Knoxville, TN 37996-0840, U.S.A.

0169-409X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

344 S. WRIGHT AND L. HUANG

2. Drug delivery to cells in culture ................................................................... 358

IV. Controlled release immunoliposomes .................................................................. 363 1. Acid-sensitive liposomes ............................................................................. 363 2. Temperature-sensitive liposomes .................................................................. 369 3. Target-sensitive liposomes ........................................................................... 370

V. Animal studies with immunoliposomes ................................................................ 372

VI. Conclusions .................................................................................................... 377

VII. Future perspectives .......................................................................................... 378

Acknowledgements .................................................................................................... 379

References ............................................................................................................... 379

Summary

The field of l iposome targeting has developed rapidly in the past 10 years as a multidisciplinary approach to the controlled delivery of bioactive molecules. Li- posomes as delivery vehicles offer several benefits over the administration of free compounds as therapeutic devices. Advantages such as the ability to encapsulate a wide variety of polar, nonpolar, and amphipathic agents make the l iposome an attractive delivery device for many applications. Encapsulation protects the drug from metabolic degradation and protects host tissue from non-specific effects until their arrival at the site of consumption. The large relative carrying capacity of the l iposomes permits the transfer of thousands of drug molecules to a given cell. Re- gardless of the type of l iposome or its route of administration, the pr imary con- sumer of either empty or drug-laden liposomes are the highly phagocytic members of the reticuloendothelial system. Thus, except for these 'natural ' target cells, liposomes remain relatively non-specific transporters. Conjugation of targeting ligands such as antibodies to the drug or drug carrier confers specificity of that complex for a certain cell or organ expressing the targeted antigenic determinant. However , direct conjugation of drug with antibody has met with limited success, due in part to the required chemical coupling between the drug and antibody. Al- ternatively, by first entrapping a native drug into a liposome by a non-perturbing method, followed by conjugation of antibodies to the surface of the liposome, some of the advantages of either independent approach may be realized without many of the disadvantages. The pros and cons of this general approach have been de- bated during the past decade in many comprehensive reviews and monographs that have been published on the subject of l iposome targeting. The pr imary emphasis of the current effort is to focus on one particular strategy for liposome targeting, i.e., the use of antibodies as vector molecules for targeted liposome drug delivery systems. In the sections that follow we try to describe the progress realized and the pitfalls and the limitations encountered with this approach to drug targeting.

ANTIBODY-DIRECTED LIPOSOMES AS DRUG-DELIVERY VEHICLES 345

I. Introduct ion

Perhaps the foremost problem of contemporary chemotherapy is the lack of specificity of a drug for its target organ and the profound side effects resulting from administration of the large dosages needed to reach the target site at acceptable levels. The large variety of potent chemotherapeutic agents that have been developed in the last two decades have shown great potential but are too frequently just as toxic to the host cells as they are to the target cells. The desirability of a system which can mediate the site-specific delivery of drugs is not new. In fact, it was the recognized father of chemotherapy, Paul Ehrlich, who over 80 years ago envi- sioned the ability of drugs to be selectively taken up at infected sites. He later foresaw the use of "a carrier by which to bring therapeutically active groups to the organ in question" [1]. The number and diversity of 'magic bullets' and drug carriers that have since been devised are too numerous to catalog here and several reviews and monographs have appeared on the topic [2-5]. All of the rapidly emerging technologies in the field of drug targeting are primarily designed for one purpose, to concentrate the drug or other bioactive molecule at the target site with minimal effects on surrounding normal tissue. Optimally, the role of the targeting vehicle is to act as a guidance system which allows the drug or other cytodestruc- tive element to home in on its target from an otherwise random distribution. Since at this time we have no intelligent homing devices with an a priori knowledge of which direction the target lies, we must settle on a system of 'close encounters'. Under this scenario, the targeted drug or drug carrier wanders about the body with its 'key' projecting outward until it encounters the 'lock' for which it was designed. Once this specific interaction has occurred, presumably at the cell surface, then the drug may elicit its response either topically or after internal processing by the target cell. Presumably, no response will commence upon bumping into a non-targeted cell, which, in our analogy has either no 'lock' or at least the wrong 'lock' for our roaming 'key'.

Among the many schemes devised for controlled drug delivery, two quite different approaches have proved particularly promising. One approach involves direct targeting of the drug by conjugation with a ligand having a distinct affinity for a particular cell or tissue. Such ligands might include hormones, carbohydrates, lectins, peptides, and receptor agonists or antagonists and immunoglobulins. An- tibody targeting is a type of ligand-mediated targeting which is dependent on a ligand-receptor interaction to mediate the binding of drug to target cells. Immu- noglobulins are ideal targeting molecules because of their specificity and high affinity for distinct targets and the relative ease with which antibodies can be prepared against a variety of different antigen molecules. Further, with the advent of the technology to produce monoclonal antibodies, clonal populations of antibody can now be generated against very specific epitopes of the target antigen. This technology now also includes the ability to produce human monoclonal antibodies [6]. Conjugation of drugs to antibodies for therapeutic applications has been only partially successful (reviewed in Ref. 7). Many times the drug's activity is altered as the result of the new bonding and there may not be mechanisms operable for


cleavage of this bond which is often required for drug activity. Alternatively, concentration of a drug within microreservoirs can be considered another approach to targeting in that the cells which take up and process the microreservoirs are affected to a greater extent than the surrounding tissue. In addition, encapsulation isolates the drug from the internal milieu until the carrier either starts to leak or releases its contents following cellular uptake. Microreservoirs include microcap- sules, red blood cells and their ghosts, and lipid vesicles. Lipid vesicles or liposomes have been widely used as delivery devices for ions, proteins, genetic material, and drugs ranging from anticancer, antibacterial, antifungal and antiviral to immunoregulators (see Refs. 8-17 for reviews).

The potential of liposomes as microscopic carriers was first realized following the discovery by Bangham and colleagues [18] that these synthetic vesicular structures could entrap water-soluble compounds. However, these devices are not limited to encapsulation of hydrophilic agents; water-insoluble compounds such as amphipathic drugs can also be successfully incorporated into liposomes. These hydrophobic agents tend to partition into the nonpolar fatty-acyl region of the membrane bilayer where they reside in equilibrium with the phospholipids. Liposomes have been used to entrap a wide variety of compounds ranging from simple metal ions to enzymes and DNA plasmids. Benefits of using liposomes in drug delivery schemes include the ability to encapsulate a large number of drug molecules into a biodegradable reservoir which protects the drug from metabolism until it reaches its destination. In addition, the pharmacokinetics and biodistribution of the liposomes-associated drug are different from those of the free drug. This may be particularly beneficial for coadministration of multiple drugs in combination regi- mens.

Although there have recently been many procedures devised for the efficient encapsulation of a variety of materials into liposomes, the concurrent development of the necessary technology needed to direct the carrier to specific targets has lagged far behind. However, if substances entrapped in liposomes are to be delivered successfully to selected sites, then efficient targeting techniques must be developed. Several different approaches have been used to target liposomes to specific organs or cells. The most direct approach was to deposit the drug-bearing liposomes directly to the site where therapy is desired. Since this method is sometimes impossible and rarely practical, other liposome-targeting strategies had to be devised. Administration of liposomes intravenously or subcutaneously invariably results in their accumulation in liver and spleen or the lymphatics, respectively. This strategy of natural targeting can be utilized for treatment of pathologies localized to the reticuloendothelial system (RES) but can represent a significant barrier for delivery to other organ systems.

A combination of two different approaches that have been mentioned above, vector or ligand conjugation and liposome encapsulation, would have many advantages over either method alone; the ligand-conferring specificity and the liposome-contributing volume capacity. For example, addition of a vector molecule such as an antibody to the liposome surface would facilitate the specific binding of the liposome to its target cell where it can potentially release thousands of drug


molecules as the result of a single binding event. The efficacy of using antibodies as biospecific targeting agents is well established and it was perhaps inevitable that they be utilized to give some direction to the inert lipid carriers. These antibody- targeted liposomes, or immunoliposomes as we shall hereafter refer to them, have been the subject of previous reviews [9,19-22]. In the sections which follow we will describe first the procedures devised to bring antibody and liposome together and then the in vitro and in vivo studies which have evaluated its utility as a drug delivery vehicle.

II. Preparation of antibody-targeted liposomes

There have been many methods developed over the past decade for the conjugation proteins to l iposomes and the reader is advised to consult any of several recent thorough review [9,23,24]. Many of these coupling approaches are idealized in Fig. 1. The most basic approach to antibody immobilization on liposomes is simple adsorption. Adsorpt ion can be viewed as either an intentional coupling method or the undesirable side reaction of a more specific coupling procedure. As the first procedure devised for targeting liposomes with antibodies, Weissmann and

Acylated antibody ~ Thiolated antibody

y Fab' (~ ~--"~ Protein A Fc Phospholipid ~ Biotin

Avidin

Fig. 1. Possible methods of conjugation of antibodies to the liposome surface (left to right). Binding of thiolated antibody to derivatized PE; binding of antibody to phospholipid through Schiff base reduction or glutaraldehyde conjugation; incorporation of fatty acid-derivatized antibody; binding of Fab' to derivatized PE; indirect antibody conjugation through binding to protein A which has been thiolated and bound to derivatized PE, indirect binding of biotinylated antibody to biotin-PE mediated by

avidin.

348

TABLE I

EXAMPLES OF ANTIBODY-CONJUGATED LIPOSOMES

S. WRIGHT AND L. HUANG

Antibody Protein modification Liposome composi- Conjugation method Refs. tion

IgG,F(ab')2 none PC/Chol / Ganglio- Reduced Schiff base 40,41 side 45:45:10 between oxidized gan- (REV) glioside and protein.

Polyclonal none PC/Chol/PE Glutaraldehyde cross- 36 antibody 6:2:2 (SUV) linking between

amines. Mohoclonal SPDP thiolation PC/Chol/DTP-PE Disulfide 46 antibody 11.3:6.6:0.2 (SUV) Rabbit Fab' reduction PC/Chol/DTP-PE Disulfide 45

9:10:1 (REV) reduction PC/Chol/MPB-PE Thioether 49

10:9.5:0.5 (REV) SPDP thiolation PC/Chol/MPB-PE Thioether 47

64:35:1 (SUV) SAMSA thiolation PC/Chol/DCP/IA- Disulfide 34

(AETA)2-PE 2:1.5:0.2:0,2 (SUV)

",/-globulin SATA thiolation PC/Chol/DCP/ Thioether 54 MPB-PE 19:16:4:1 (REV) PC (SUV)

Rabbit Fab'

(Protein A)

Rabbit IgG

Beuce-Jones IA-DL-PE alkyla- monomer tion

F(ab')2 citraconylation and conjugation to PE with CDI

Monoclonal IgG NHSP acylation

PC/Chol 2:1 (SUV)

PE/PHC 8:2 or PC/Chol 2:1 (SUV or modified REV)

Spontaneous incorpo- 62 ration of protein into preformed liposomes. Spontaneous incorpo- 37,38 ration of protein into preformed liposomes. Detergent or etherme- 66-68,71 diated incorporation with dialysis.

Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; DCP, dicetyl phosphate; Chol, cholesterol; DTP, dithiopyridine; MPB, (maleimidophenyl) butyryl; IA, iodoacetyl; AETA, amino- thioacetyl; DL, dansyllysyl; CDI, carbodiimide; SAMSA, S-acetylmercaptosuccinic anhydride; SATA, Succinimidyl-S-acetyl thioacetate; NHSP, N-hydroxysuccinimide ester or palmitic acid.

co-workers [25,26] observed that aggregated immunoglobul ins would coat and partially insert into l iposomes with the Fc region exposed to the surrounding me- dium. Significant adsorption of antibodies to small unilamellar vesicles (SUV's) can be achieved ei ther during [27-29] or after [26,30-33] their sonication. Adsorp t ion was greater for small [30] negatively charged [28] l iposomes suggesting that inser- tional or ionic mechanisms were involved. Sonication of the ant ibody in the presence or absence of lipid had no significant effect on their binding capacity [28]. Adsorp t ion has recently been re-emphasized by Senior et al. [30] following their detect ion of large non-specific binding of native ant ibody to l iposomes which had been pre-derivat ized for covalent conjugat ion with thiolated antibodies. F rom 34 to 89% of added IgG could be bound on the vesicle surface and there was no leak-


age of vesicle contents reported. Thiolated IgG exhibits very little non-specific binding to SUV's ]34]. This method is attractive in its simplicity and the adsorp- tively coated liposomes exhibit considerable stability even in the presence of serum [27,28,30]. However, the mode of attachment is non-specific and the coating efficiency is highly variable. Even though Huang and Kennel [28] found no large in- terspecies differences in the adherent capacity of normal IgG, there may be differences in IgG subtype for a given liposome [30].

There are two major approaches which have been developed for the specific and controlled coating of liposomes with antibody. The first approach is to derivatize lipid, typically the amino group of phosphatidylethanolamine (PE), which, following incorporation into the liposome, will covalently attach antibody which is subsequently applied. The second major approach is to convert an otherwise hydrophilic protein antibody molecule into an amphipathic molecule which can then non- covalently intercalate into the liposomal bilayer. We will discuss briefly only some of the more popular techniques along with their most recent modifications. Ex- amples of these procedures are listed in Table I.

H.1. Covalent conjugation to pre-formed liposomes

One of the first successful attempts at covalent attachment of antibody to liposomes was achieved by glutaraldehyde conjugation. The traditional use of glutaraldehyde has been for cell fixation in which proteins are effectively cross-linked via their amino groups. Under the proper conditions, glutaraldehyde can also be used for controlled conjugation of amino groups between antibody and PE. Following their earlier success with liposome immobilized a-chymotrypsin [35], Torchilin and co-workers [36] used glutaraldehyde to conjugate polyclonal Ig to PE-containing SUV's. These immunoliposomes retained antigen binding capacity both in vitro and in vivo and although up to 60% coupling efficiency was achieved, the net antibody incorporation was low. Potential undesirable side reactions would be homocou- piing between proteins or between liposomes which would limit its general use- fulness.

Another fundamental conjugation method involves coupling of protein carbox- ylic groups with the primary amine of PE on the liposome. Coupling between antibody and liposome has been achieved with a water soluble carbodiimide (EDCI) [37]. A major drawback is potential protein-protein cross-linking but protection of the protein amines by prior citraconylation minimizes this side reaction ]37,38]. Further, this method is inefficient and produces unstable liposomes which retain little of entrapped contents [37,38]. More recently, Kung and Redemann [39] have used carboxyacyl derivatives of PE for conjugation to protein amines using EDCI. PE was derivatized with C4 to C18 dicarboxylic acids which provide spacer arms of various chain lengths. Maximum antibody incorporation was found for 1,12-do- decanedicarboxylic acid (C16). Incorporation was dependent upon the spacer length and the shortest derivative, succinic (C6), produced no detectable protein binding.

Protein attachment to liposomes can also be achieved via simple Schiff base formation under the proper conditions [40--42]. Oxidation by periodate of the car-

350 S. W R I G H T A N D L. H U A N G

bohydrate moieties on glycolipid-containing liposomes to the corresponding al- dehydes facilitates Schiff base formation with protein amines. The conjugate is then stabilized by subsequent reduction with sodium borohydride or sodium cyanobo- rohydride. Coupling efficiencies of up to 200 Ixg IgG per p.mol lipid has been achieved [40,41]. These liposomes showed greater binding capacity to target cells than immunoliposomes produced by earlier conjugation techniques. Potential drawbacks of this technique are that 9-20 mol% of glycolipid must be included in the liposome and that periodate addition to the pre-formed liposomes may oxidize the fatty-acyl chains of the lipid and/or the contents within the liposome. In addition, up to a 60% reduction of immunoreactivity of antibodies following periodate oxidation has been observed [42]. Aldehyde groups can alternatively be generated from the carbohydrate groups on the constant region of the heavy chain of immunoglobulins by mild oxidation with periodate or glucose oxidase [43]. Ad- dition of oxidized antibody to liposomes containing a hydrazide group resulted in up to 535 ~g of IgMs per i~mol phospholipid [43].

One of the most popular conjugation methods utilizes the heterobifunctional agent N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate (SPDP) [44]. In this scheme, SPDP is reacted with liposomal-PE to produce the dithiopyridine derivative, DTP-PE, which can then react by disulfide interchange with any protein having a free thiol [44-47]. Immunoglobulin Fab' fragments are thus potential coupling partners with yields up to 600 Ixg Fab' per ~mol lipid [45]. Typically 1-5 mol% of DTP-PE is incorporated into the liposome with complete retention of entrapped molecules [44-47]. If intact antibody is used (following SPDP modification) then greater than 60% of antibody-conjugated liposomes could be precipi- tated by Staphylococcus aureus indicating exposed antibody Fc regions on the liposome surface. Conjugation of antibody Fab' via the free SH is thus disadvan- tageous for in vivo applications because if the Fc portion remains intact and exposed, the immunoliposomes can be trapped by Fc receptor-bearing cells present in the host. Advantages of this method are a lack of homocoupling between proteins or liposomes, provision of a spacer arm, and the stability of liposomal-PE- DTP and antibody-DTP. The primary disadvantage is that the disulfide product is susceptible to reductive cleavage by endogenous thiols and would therefore be generally unsuitable as an in vivo targeting vehicle.

Goundalkar et al. [48] demonstrated that derivatized fatty acid could also be used as an anchor for antibody conjugation to pre-formed liposomes. Stearylamine was reacted with SPDP and then incorporated into liposomes. Thiolated antibody could then be conjugated to the liposomes with a 24-32% binding efficiency. Approx. 20% of an encapsulated drug leaked out during the latter binding reaction. In 1982, Martin and Papahadjopoulos [49] introduced a new sulfhydryl-reactive lipid derivative N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE). When reacted with a thiol-bearing antibody or Fab' fragment, a stable thioether bond is formed between the two by sulfhydryl addition across the maleimide double bond. Coupling efficiency is 20-30%, producing up to 600 I~g Fab' per ~xmol lipid corresponding to about 3000 Fab' per 0.2 Ixm vesicle. The advantage of this protocol over that with SPDP is that the conjugation is essentially irreversible and


the covalently linked product is stable in serum [49]. For most of the earlier targeting strategies, SUV's were most often employed. However, large unilamellar vesicles (LUV's) such as those produced by the reverse-phase evaporation technique [50] have larger internal volume and better trapping efficiencies. Reverse- phase evaporation vesicles (REV's) conjugated to antibody with this derivative are stable and do not aggregate or leak [49]. In addition, they exhibit good binding and retention of contents even in 50% serum [49]. However, depending upon the lipid composition, high concentrations of the derivative can perturb membranes. Coupling of Fab' to liposomes with 5 mol% MPB-PE caused increases in size, po- lydispersity and resulted in up to 95% loss of entrapped carboxyfluorescein [51]. Others have reported that up to 10 mol% MPB-PE could be used without any ad- verse effects on bilayer structure [49]. Again, these discrepancies are probably largely due to differences in lipid composition and methods used for preparation of liposomes.

MPB-PE or DTP-PE have been used with success for conjugation of Fab' which has a free thiol available for conjugation following cleavage of F(ab')2 with dithiothreitol (DTT). If the protein has no free sulfhydryl groups available, then it can be thiolated with a variety of agents (Table II). In fact, SPDP was originally designed to introduce sulfhydryl groups into proteins for reversible protein-protein conjugation [52] and was therefore the early choice to thiolate antibodies [44,46,47]. It is necessary that soluble F(ab')2 or Ig-DTP be first reduced, the products of which are unstable and must be used immediately. In 1983, Dun- can et al. [53] introduced a new thiolating agent, succinimidyl-S-acetylthioacetate (SATA). This agent has the advantage that deprotection of the sulfhydryl is ac- complished with hydroxylamine at neutral pH and does not necessitate removal of strong maleimide-reactive reducing agents prior to addition to derivatized liposomes. SATA has been used to conjugate up to 300 Ixg Ig per p~mol lipid into MPB- PE sensitized liposomes [54]. S-acetylmercaptosuccinic anhydride (SAMSA) is very similar to SATA but alters the electrostatic properties of the protein. Traut 's reagent (2-iminothiolane) [55] has been used to thiolate many proteins including monoclonal antibody [56] and has the advantage that no deprotection is required.

There have been several novel methods developed for the indirect coupling of

TABLE II

METHODS OF INTRODUCTION OF THIOLS INTO PROTEINS

Protein Thiolation reagent Activating agent Refs.

Polyclonal (rabbit) N-succinimidyl-3-(2-pyridyldi- dithiothreitol (DTT) 52 antibodies thio)propionate (SPDP) Monoclonal IgG SPDP DTT 44,46 Protein A SPDP DTT 44,47 ~ - g lobu l i n Succinimidyl-S-acetyl thioacetate hydroxylamine 53,54

(SATA) IgG S-acetylmercaptosuccinic anhy- hydroxylamine 34

dride (SAMSA) Monoclonal IgG 2-Iminothiolane (Traut's reagent) none 55,56


antibody with liposomes. These systems are designed primarily for targeting of liposomes to cells with derivatized antibody already bound to their surface. Two principal schemes have been utilized based upon the affinity between protein A of S. aureus and antibody Fc, and the affinity of biotin for avidin. Leserman and co- workers [44,57,58] conjugated protein A to liposomes after both had been derivatized with SPDP. Protein A-bearing liposomes are added to cells which had been pre-treated with the targeting antibody resulting in specific binding of the liposomes of the cells with the newly added Fc. A similar mechanism is employed with the biotin-avidin system [59-61]. Typically, liposomes and antibody are both derivatized with biotin. Target cells bound with biotinylated antibody are then treated with avidin followed by the biotinylated liposomes. Avidin has the capacity to bind up to four biotin molecules with very high affinity, thus serving to cross-link the two biotinylated components. Streptavidin has recently been used to avoid the non- specific binding properties of native avidin [59].

H.2. Incorporation of lipophilic antibodies

The second major approach to incorporate antibody into liposomes is to add a hydrophobic moiety to the antibody which facilitates its association with the amphipathic bilayer. Sinha and Karush [62] were the first to attempt this approach utilizing PE and the hydrophobic anchor. An alkylating phospholipid derivative, N-(N-iodoacetyl-N-dansyllysyl)-PE, was used to derivatize Bence-Jones mono- mers which then spontaneously attached to either preformed liposomes or red cell ghosts. However, this iodoacetyl derivative of PE is not an effective crosslinking agent for coupling thiolated protein into pre-formed liposomes [34]. Addition of one or two aminoethylthioacetyl (AETA) groups as spacers between iodoacetyl and PE groups makes it a reasonably efficient surface target for conjugation with thiolated antibody [34]. Periodate oxidation of the carbohydrate unit of phosphatidylinositol to a free aldehyde permits Schiff base formation between protein and phospholipid [63]. Derivatized protein can then be incorporated into liposomes by detergent dialysis. Yet another approach utilizes a polysaccharide derivative of cholesterol as the anchoring molecule [64]. The polysaccharide used in these studies, pullulan, enhanced the stability of the immunoliposomes without affecting its target specificity.

The most commonly used nonpolar anchor is a fatty acid such as palmitic acid. Two different protocols have been developed for conjugating fatty acid onto antibody. The N-hydroxysuccinimide ester of palmitic acid (NHSP) is one popular choice for modifying antibody to make it more lipophilic. Huang, A. et al. [65] were the first to use this agent for incorporation of monoclonal antibody into liposomes. The antibody is first reacted with NHSP with the extent of derivatization controlled by the input ratio of NHSP to Ig and/or the reaction time [66]. Optimal coupling stoichiometry for incorporation into detergent dialysis liposomes was 4-5 palmitoyl chains per IgG [66-68]. Under these conditions, about 90% of the acyl chains were located on the heavy chain of the antibody. Antigen binding capacity of the antibody was reduced 3-4-fold but the liposomes exhibited good target cell


specificity [65-68]. Slow infusion of palmitoyl IgG in detergent into a suspension of DPPC liposomes (DOC:PC = 60:1, mol/mol) at their main phase-transition temperature resulted in up to 52% incorporation [70]. Acylated antibody can also be incorporated into modified REV liposomes [71]. Antibodies modified by this technique have recently been shown to stabilize otherwise unstable lipid assem- blies into forming liposomes [72]. The major benefit of this targeting scheme is that all procedures may be carried out in the absence of harsh coupling reagents and that potential homocoupling is not a concern.

Utilizing palmitic chloroanhydride to acylate their protein, Torchilin et al. [73] were able to mediate protein incorporation either during or subsequent to liposome formation. The advantage of this latter approach is that it can be completed in the absence of detergent although incorporation is slow and of low efficiency [73].

III. Immunoliposome binding and drug delivery in vitro

111.1. Binding to immobilized antigens

The various techniques that were developed to conjugate antibodies to the liposome surface were devised for the ultimate goal of enhancing drug delivery. Ef- fective drug delivery with liposomes will involve three phases: transport through blood and interstitium to the target site, binding to target antigens on the cell surface, and internalization and intracellular release of encapsulated drug. We will first discuss the in vitro situation in which the model is tested under near ideal conditions thus involving only the latter two aspects, binding and internalization.

The initial step in any effort at liposome targeting is to evaluate the specificity of binding between carrier and target. The addition of antibody to the liposome surface greatly enhances its avidity for antigen bearing cells, but sometimes at the expense of also increasing the nonspecific binding to nontarget cells. Therefore it is usually necessary to compare the interaction between immunoliposomes and its target with either the immunoliposome and a nonantigenic cell or between the target cell and a nonspecific immunoliposome.

The first reported successful attempt at antibody-mediated targeting was achieved by Gregoriadis and Neerunjun [29]. Sonicated liposomes containing 111I-labeled bleomycin were coated with adsorbed 125I-labeled IgG raised against whole cells. When anti-HeLa cell antibodies were used, the uptake by HeLa cells of both la- bels was 25-fold greater than that for fibroblasts. Specific binding of antibody-coated liposomes to targeted fibroblasts and AKR-A cells was also demonstrated [29].

Targeting to human erythrocytes was examined with periodate conjugated immunoliposomes. Up to 8000 liposomes (0.2 ~m SUV) coated with 143 anti-human erythrocyte rabbit F(ab')2 molecules could bind to each human erythrocyte [40]. This represents binding of about 80% of the added immunospecific liposomes compared to less than 1% binding of added nonspecific immunoliposomes. Im- munospecific targeting to sheep erythrocytes has also been reported for liposomes coupled with palmitic acid esterified antibodies [69]. Liposome binding to the sheep

354

TABLE III

DELIVERY WITH pH-SENSITIVE IMMUNOLIPOSOMES

S. WRIGHT AND L. HUANG

Liposome Antibody Entrapped agent Target c e l l Comments Refs.

PE/PHC 8:2 palmitoyl anti- calcein L-929 very efficient de- 83 (modified REV) H2K k livery, blocked

at low temp or with lysosomotropic agents

PE/OA 8:2 palmitoyl anti- ara-C MTX L-929 only drugs in 87 (modified REV) H2K k targeted pH-sen-

sitive immunoliposome were beneficial, prely- sosomal delivery

PE/OA 8:2 palmitoyl anti- DT-A L-929 only targeted 88 (DRV) H2K k pH-sensitive im-

munoliposomes were toxic, blocked by free antibody or immunoliposome

PE/Chol/OA palmitoyl anti- plasmid DNA L-929 ( tk) targeting en- 94 4:4:2 (detergent H2K k (adsorbed) hanced transfor- dialysis) mation PE/Chol/OA palmitoyl anti- plasmid DNA RDM-4 efficient in vivo 92 4:4:2 (detergent H2K k (adsorbed) delivery, con- dialysis) trolled expres-

sion

red blood cells was fast (within 1 min) and produced some changes in morphology in the cells.

The effectiveness of monoclonal antibody for liposome targeting to cultured cells was demonstra ted by Huang et al. [65]. Using an N-hydroxysuccinimide ester of palmitic acid, monoclonal antibody to the mouse histocompatibility antigen H-2K k was acylated and incorporated into phosphatidylcholine (PC) liposomes (0.09 Ixm) by detergent dialysis. These liposomes showed specific binding to H-2K k expressing L-929 cells but not to A-31 cells which expressed the H-2K d antigen. For either native liposomes or liposomes derivatized with nonspecific antibody there was no significant binding to either cell type reported. The liposome preparat ion was het- erogeneous with respect to antibody density and only those liposomes which had greater than 67 antibody molecules per liposome became cell associated. Subse- quent studies indicated that l iposome-associated antibody containing 4-5 palmitoyl chains per IgG had an apparent dissociation constant 6-7-fold higher than native antibody [66]. Optimal incorporation of these antibodies into liposomes was achieved using an initial lipid/protein ratio of 10/1 (w/w). These liposomes which had only about 48 IgG molecules per liposome demonstrated saturable binding to L-929 cells. At saturation there was about 5 times as much antibody associated to


liposome-treated cells as there was to cells incubated with free underivatized antibody. These acylated antibodies could also be efficiently (over 80%) incorporated into REV vesicles (0.15-0.45 p~m) which had a high drug capture efficiency [71]. Immunoliposome (200 IgG per liposome) binding to RDM-4 non-adherent lymphoma cells (H-2K k) was saturable with respect to antibody or lipid binding and could be blocked by free anti-H-2K k IgG. When incubated at 4°C, about 90% of the immunoliposomes taken up by RDM-4 cells could be removed by proteases but only 30% could be released from cells incubated at 37°C [74]. This suggests that at 4°C most of the liposomes are surface bound and not internalized as they are at the higher temperature. Cytochalasin B or a combination of 2-deoxyglucose and NaN 3 could inhibit the uptake at 37°C. Specific intracellular delivery to target but not control cells was demonstrated with encapsulated carboxyfluorescein (an impermeable aqueous fluorophore) which was diluted and dequenched when released into the cytosol subsequent to liposome internalization and processing at 37°C (but not at 4°C) [74].

Indirect targeting with protein A conjugated liposomes also proved to be an effective means to achieve binding to target cells [44]. Human leukocytes were shown to bind with protein A liposomes (SPDP conjugated) only in the presence of anti- human 13-2-microglobulin antibodies. Direct conjugation of this IgG to liposomes (via SPDP) also produced selective binding to human but not mouse cells [44]. In- corporation of up to 4.4 mol dithiopyridine per mol IgG had no appreciable effect on binding via Fab or Fc regions [46]. Only one molecule of coupled antibody per liposome was apparently sufficient to mediate liposome binding to either target cells or to S. aureus .

In contrast to immunoliposomes in which both Fab and Fc portions are exposed, liposomes conjugated with Fab' will bind only to antigen and not to Fc receptor- bearing cells. Martin and co-workers [45] conjugated antihuman erythrocyte Fab' fragments to SPDP-derivatized reverse phase vesicles (6000 Fab' per 0.2 p~m vesicle). Binding of up to 5000 vesicles per erythrocyte was specific for human cells and was moderately stable in the presence of 50% serum. Serum stability could be improved with an irreversible conjugation method utilizing MPB-PE as the lipid anchor [49]. Vesicles retained their aqueous contents during conjugation and binding [45,49]. Immunoliposomes could also be targeted to erythrocytes with anti-glycophorin A antibodies [75]. Binding was increased 80-fold and was inhibited only by soluble anti-glycophorin and not its Fab' fragment. Targeting of liposomes with anti-rat erythrocyte F(ab')2 was found to enhance their binding to erythrocytes in rat blood [76]. The permeability properties of the liposomes were not affected by binding to target cells.

For any antibody-targeting strategy to succeed there must be an epitope on the surface of the intended cell which is somehow unique so that molecules can be directed to or processed by that cell specifically. The field of immunoliposome targeting as it has developed has been directed primarily at, although not exclusively for, antitumor therapy. However, there has been little effort to target these carriers specifically to tumor cells. Most of the in vitro delivery studies with model systems, although utilizing tumor cell lines, have been based upon targeting di-


rected toward epitopes such as Fc receptors or histocompatibility antigens. This is not for a lack of targetable sites on tumor cells. There are presently dozens of molecules which have been identified as tumor-specific or more frequently as tumor- associated antigens, and most of these are surface molecules to which antibodies have been generated. These targets may include modified or overexpressed proteins or modified glycolipids. Transformation-associated glycolipids may be the product of either incomplete synthesis resulting in accumulated precursors or due to induction of synthesis of foreign glycolipids. Urdal and Hakamori [61] indirectly targeted antibody or antibody-associated liposomes to the tumor-associated glycolipid ganglio-N-triosylceramide found on L5178c127 or Kirsten virus-transformed 3T3 cells. Coupling between antibody and liposome was dependent upon the high-affinity binding of avidin to biotin. Cells were first incubated with biotinylated anti-glycolipid antibody which was then followed by addition of drug-containing liposomes to which had been conjugated either avidin or biotin (plus free avidin as the cross-linker in the latter case). Avidin was covalently coupled to glycolipid-containing liposomes by periodate oxidation, whereas biotin was incorporated by using biotinyl-PE. In the presence of free avidin as cross-linker, the antibody-treated L5178c127 cells were not at all sensitive to actinomycin D encapsulated in biotin-liposomes, although they were sensitive to the targeted drug, biotinyl neocarzinostatin. Neocarzinostatin was believed to be cytotoxic without being internalized [61] based upon previous reports that its effects can be mediated at the surface of the cell [77]. Transformed 3T3 cells were only moderately sensitive to the actinomycin-D liposomes although it was demonstrated that they did bind to the target cells. This suggests that either the bound liposomes were not internalized or if they were, then the contents were not effectively delivered into the cytosol.

Torchilin and colleagues [78,79] have examined the potential of antibody-conjugated liposomes to bind to extracellular matrices. Antibodies against fibronectin or laminin were modified by the palmitoylchloride procedure [73] and incorporated into PC liposomes with trace amounts of [3H]cholesterol or [14C]cholesterol oleate (lipid markers) by detergent dialysis. Using protein monolayers as the target, anti-laminin or antifibronectin immunoliposomes bound with nearly the same specificity and affinity to the glycoproteins as the free antibody. This targeting scheme offers the possibility of targeting to areas of vascular damage where proteins of the extracellular matrix are exposed. Deendothelialization and exposure of subendothelial components may be the consequence of trauma, surgery or various cardiovascular pathologies. These events invariably lead to platelet aggregation and thrombus formation at the site of damage which could represent a threat to patients at risk for coronary thrombosis. In fact, perfusion studies with partially denuded arterial segments demonstrated in situ that immunoliposome targeting was feasible for these applications [79]. Liposomes which were conjugated with human anti-collagen type-I antibodies (biotin-avidin coupling) could be visualized in denuded areas of the vessels [61]. Although drug delivery to luminal lesions was not attempted in these studies, the potential for liposome delivery of anti-platelet or related drugs is apparent.


Since most immunoliposomes have several antibody molecules attached to their surface, it might be expected that, depending upon the antigen density, a single liposome could be bound to the target cell at several points. Even derivatized antibodies have high lateral diffusional mobilities within the liposome bilayer [80]. It is therefore plausible that subsequent to the initial binding event, additional antibodies may diffuse into the area and form multipoint attachments or multivalent binding. This might be expected to appear as an increase in the binding affinity of the intact liposome. In fact, that is what is observed experimentally [43,45,81]. Fluorescence quenching of fluorescein by anti-fluorescein antibodies was utilized by Heath et al. [43] to examine possible multivalent interactions between liposome-associated antibody (periodate conjugated and only 40% active) and target cells. Binding to fluorescein isothiocyanate (FITC)-labeled erythrocytes (108 FITC/RBC) was rapid (within 5 min at 37°C) and was insensitive to antibody density above 230 active binding sites per liposome. They found that there was up to a 1000-fold greater affinity for immunoliposomes (K a = 109 M -1) than free antibody (Ka = 106 M -~) for the target cells over a wide range of cell densities although at higher cell densities apparent hemagglutination reduced the available binding sites [43].

Quantitative analysis of immunoliposome binding to antigens immobilized on polystyrene microtitre plates has been reported by Klibanov et al. [81]. Liposomes conjugated with palmitoyl-anti-IgG (with 40% of protein being trypsin sensitive) bound to an antigen monolayer with an apparent dissociation constant of (1.5-5).10 -9 M liposomes (for a 100 nm liposome particle of 105 lipid molecules). For native antibody the K d was 2.5 • 10 .8 M antibody, 10-fold lower than for the immunoliposomes (which had about 30 exposed antibodies per liposome). The mere 10-fold enhancement of binding affinity conferred by liposome incorporation is much lower than the 1000-fold difference reported by Heath et al. [42] but is due to the different ways the authors express their values. Heath et al. relate protein association constants (intrinsic vs. functional) whereas Klibanov et al. [81] were relating the dissociation constants of binding particles (antibodies vs. liposomes). In either situation it can be concluded that a drug encapsulated in an antibody- targeted liposome would have a greater probability of interacting with the target cell than it would have if simply attached directly to the antibody.

In studies where the antigen density on target cells was manipulated there was not a simple relationship between liposome binding and antigen density. Lipo- somes incorporating variable amounts of palmitic acid derivatized antibody (anti- H-2K k IgG) [65] were incubated with RDM-4 lymphoma cells in which the number of surface H-2K k molecules was controlled by treatment of cells with protein- ase k [82]. For liposomes (90 nm diameter) with 0 to 55 total antibodies incorporated, binding of the lipid component (labeled with 3H-cholestanyl ether) of antibody-associated liposomes to cells of any antigen density was proportional to surface density of antibody on the liposome. However, binding of immunoliposomes with high antibody density did not vary considerably between cells with 8.7 • 103 and 6.4 • 1 0 4 sites per cell, although as the antibody density was lowered the difference became larger. Therefore, immunoliposomes with low antibody density


may be better at discriminating between high and low antigen density cell populations.

Another potential mechanism for enhancing cell specificity through multipoint binding is to target to multiple antigens on a given cell. This would be of advantage for cells which have a unique combination of antigens rather than a specific single antigen for targeting. Liposomes conjugated with antiglycophorin A antibodies have been shown to interact with the human leukemia cells line K562 either through their Fc receptors or by binding to glycophorin [75]. Binding of immunoliposomes was only partially blocked by excess anti-glycophorin Fab' or excess human IgG and was completely prevented only when both types of competing antibodies were present. In addition, both ligands were needed to block delivery of cytotoxic drugs to these cells [75]. This scheme may be especially useful for delivery to tumor cells which shed surface antigen which could block binding of liposomes targeted only to that antigen. The idea of multitargeted binding has also been studied from a different approach by Trubetskoy et al. [83]. An indirect targeting protocol was used where binding of biotinylated liposomes to protein monolayers bound with different biotinylated antibodies was mediated by avidin. Addition of antifibrinogen, antifibronectin, or anti-LDL antibodies to an immobilized mixture of target proteins resulted in binding of fewer liposomes than if all three antibodies were present simultaneously. The effects were subadditive. An alternative to the use of multiple antibodies against individual targets might be found in the use of hybrid antibodies. The recent generation of these biospecific antibodies having independent Fab"s permits simultaneous binding to two different antigens [84].

111.2. Drug delivery to cells in culture

Some of the earliest studies utilizing immunoliposomes were aimed at delivery to enzyme-deficient cells as a model for treatment of lysosomal storage diseases. For example, multilamellar vesicles (MLV's) containing horseradish peroxidase and coated with non-specific heat-aggregated IgM were given to dogfish phagocytes which are normally low in peroxidase activity [25]. These cells bound the liposomes via their FC receptors, endocytosed the complex, and were subsequently able to express peroxidase activity. This was the first demonstration of effective delivery of liposome contents to a target cell. This method was also found to be effective for the in vitro introduction of hexosaminidase A into leukocytes of Tay- Sachs patients [26].

Leserman and co-workers utilized a similar targeting scheme to examine the ability of liposomes to deliver cytotoxic drugs to three fundamentally different types of cells [85]. Antibody Fc receptors are rapidly internalized by the highly phagocytic tumor cell line P388D1 but not by the nonphagocytic P388 line. Liposomes containing the antifolate drug methotrexate (MTX) and the antigenic lipid N-di- nitrophenylaminocaproyl-PE (DNP-cap-PE) were mixed with target cells that had been pre-incubated with anti-DNP antibodies which became cell associated via their Fc region. Unlike cells that were either non-phagocytic (P388) or receptor nega-


tive (EL4), the P388D1 cells were much more susceptible to the MTX encapsulated in the immunoliposomes than to the free drug as determined by [3H]deoxyuridine incorporation [85]. Although it was the initial premise that drug delivery to the cell was imminent if one could just achieve tight binding of the liposome to the target cell, a comparison between the P388 and the P388D1 data suggested otherwise. It was apparent that an active means of internalization of surface-bound liposomes was required.

The requirement for factors other than binding were also demonstrated by Weinstein and co-workers [86]. In this study the binding of liposomes to human lymphocytes was mediated by bivalent antibody which was directed against surface components common to the two opposing structures (trinitrophenyl moiety). The use of the self-quenching dye carboxyfluorescein (CF) entrapped inside the liposomes permitted quantitative determination of liposome binding and, more importantly, permitted one to distinguish between delivery of entrapped marker to the cell cytosol from simple binding. Although there were from 5000 to 14000 di- oleoylphosphatidylcholine (DOPC) SUV's bound to each cell, there was no release of contents into the lymphocytes. Since the dye itself is relatively imper- meant, it was concluded that the liposomes were not interacting with the target cell beyond the initial binding step. For efficient cytoplasmic delivery of contents, the liposomes must either fuse at the cell surface or be internalized where it can eventually release its contents directly into the cell interior during cellular processing. The target antigen used in this early study was a trinitrophenyl moiety (TNP) which had been randomly incorporated into the lymphocyte membrane. Failure to achieve delivery was probably due to exclusion of the TNP-antibody-liposome complex from endocytic vacuoles. Thus, even though the liposome may be tightly bound to the cell, if there is no additional processing to accommodate drug transfer, then the system will be of little use as a therapeutic modality. However, this limited interaction can be utilized for cytological studies where fiuorophore-containing liposomes can be used as a sensitive means to immunolabel cells [87,88].

The introduction of efficient methods of antibody coupling to liposomes generated great expectations for the therapeutic potential of immunoliposomes. Pre- vious studies up to this point had utilized the Fc portion of the antibody most frequently to target the liposomes to cells bearing Fc receptors. In 1981 Leserman et al. [89] targeted liposomes to murine histocompatibility antigens by covalent attachment of antibodies to SUV's using SPDP. Binding and delivery was followed by encapsulating CF and MTX in the liposomes, respectively. It was found that upon targeting to mitogen-induced spleen blast cells there was not a direct cor- relation between the number of liposomes bound and the effect of encapsulated MTX. Liposomes targeted to other histocompatibility antigens were more effective at drug delivery than those targeted to H-2K k, even though more H-2Kk-tar - geted liposomes were bound to the cells. The lysosomotropic amine, NH4C1, inhibited the effects of encapsulated but not free drug, suggesting that endocytosis was the mechanism of uptake. Therefore, if this type of liposome is bound to a surface component which is not actively internalized then it will not be an effective delivery vehicle.


Using the same targeted liposomes, this group utilized this system not so much for its therapeutic potential, but more as a means to measure endocytosis apart from surface binding. In studies designed to examine the fate of MHC-encoded determinants, they observed differential endocytosis by T and B lymphocytes of an identical specific surface antigen [90]. Liposomes containing MTX and targeted to cells expressing H-2K or surface molecules of molecular weight 94 000 and 180 000 were much more cytotoxic for T cells than B cells. However, even though T-cells extensively internalized the antibody-targeted liposomes, there was no enhancement of MTX cytotoxicity by encapsulation in targeted liposomes as compared to the free drug. Each cell type was equally sensitive to free drug. These results suggest that for effective chemotherapy with antibody-coated liposomes, targeting to an antigen which has a much higher internalization rate than other surface molecules may be sufficient to generate selective toxicity. Thus, targeting with antibodies to tumor-specific antigens might not be necessary if the endocytic rate was simply higher for a common antigen on the tumor cell.

Rather than generate a large panel of specifically targeted immunoliposomes, an indirect targeting strategy was devised in which liposomes coated with protein A from S. aureus could be used against cells already targeted with free antibody [57,89,91,92]. The primary advantage of this approach is that a single population of liposomes can be used against a variety of target antigens. For DPPC liposomes containing 34% cholesterol and 1% DTP-PE, up to 15 protein A molecules could be conjugated to SUV's following SPDP modification [91]. As before, it was found that B-cells were insensitive to liposome-entrapped MTX even though they had a greater surface density of the target antigen, H-2K k, than T-cells which were quite sensitive to these cytotoxic liposomes. However, in addition to displaying cell specificity the effects were also antigen specific. If the liposomes were instead targeted to I-EK molecules on the B-cells, then MTX induced inhibition of 3H-dUrd was observed [91]. The two cell types were equally sensitive to free MTX but only the T cells were more sensitive to encapsulated over free drug, regardless of target antigen. Using gold-labeled protein A bound to surface-attached antibody, Machy et al. [93] have recently shown that T-lymphocytes spontaneously internalize 20-40% of their MHC-encoded class I molecules in a process involving coated pits and coated vesicles. No internalization was observed in B cells. These observations point out that internalization of a given surface antigen may be drastically different between cell types and if endocytosis is to be relied upon as the principal means of uptake, then more consideration should be given as to the turnover rate of target antigen than to the surface density of the antigen.

The rate of endocytosis of liposomes bound to MHC-encoded proteins can be controlled by drugs that interfere with energy metabolism, microfilament organi- zation or phospholipase activity [58]. Intracellular release of CF from liposomes bound to cells via protein-A-antibody was inhibited by lysosomotropic NH4C1, the metabolic poison phenylarsenoxide, and the Ca2+-dependent enzyme phospholipase A:. There was also a partial requirement for extracellular Ca 2+ for endocytosis of liposomes. More importantly from a drug delivery standpoint, they were able to demonstrate that the calmodulin antagonist trifluoperazine enhanced the


apparent rate of endocytosis. Increasing internalization augments the effects of cytotoxic liposomes.

There are strict limitations on the size of particles that can be taken up by endocytosis and liposome size has been shown to be a critical factor in determining the extent of liposomal drug delivery. LUV and SUV conjugated to anti-Thy 1.1 antibody were tested for their ability to deliver MTX-y-asp to either AKR or R1.1 T-lymphoma cells [94]. Although either type of liposome bound to the target cells, the LUV's (0.45 txm) failed to enhance the drug effects for either cell type whereas SUV's (0.053 jxm) were 22-40 times more effective than the free drug. The role of liposome size in drug delivery was also examined for cultured tumor cells using the indirect targeting strategy with protein-A conjugated liposomes. For liposomes of 80, 200 and 400 nm entrapping MTX or CF there was an inverse relationship between liposome size and the ability to deliver a cytotoxic drug to cells targeted with anti-H-2K k IgG [92]. This did not correlate with cell binding as there was more CF associated with the larger liposomes than there was for the smaller liposomes bound to cells. The enhanced cytotoxicity of the small liposomes was thought to result from size restrictions of the endocytic vesicles such that only the smaller liposomes could be engulfed. The average size of coated pits are estimated at about 150 nm in diameter so that liposomes smaller than this should be readily accepted. In fact, there was no difference in drug delivery for liposomes of 60 or 80 nm in diameter [92]. In addition, RDM-4 cells or lymphoblasts did not process immunoliposomes of 200 nm or larger although L-929 cells were sensitive to the larger (200 and 400 nm) liposomes, although the smaller carriers were still more effective [92]. This may suggest that the endocytic size restrictions are cell-type dependent. Moreover, fibroblasts such as L929 cells, are known to internalize their Class-I molecules via uncoated cell surface vesicles and tubular invaginations rather than coated pits [95].

The antigen selectivity of antibody-coated liposomes can be implemented for immunoselection protocols. Machy et al. [57] have recently used protein-A conjugated cytotoxic liposomes to select for cells lacking a specific surface marker which had been tagged with antibody. In the presence of anti-H2K k IgG RDM-4 cell mutants which no longer expressed the H-2K k determinant were not sensitive to protein-A-coated liposomes containing MTX whereas the wild-type cells were eliminated within 48 h. Some of the mutant cells could be induced to augment their expression of H-2K k in the presence of interferon-y [57]. Therefore, the use of interferon-y/ could improve the efficacy of liposomes targeted to interferon-sensitive cell-surface molecules, although interferon-insensitive mutants which lack the target molecule would not be affected and could continue development.

A novel targeting strategy employs conjugation of antigen to the liposome surface for targeting to sensitized lymphocytes [96]. A palmitylated nanopeptide of myelin basic protein was incorporated into liposomes containing MTX and incubated in vitro with T-lymphocytes obtained from guinea pigs sensitized to the antigen. Targeted cells were significantly more susceptible to the drug than nontargeted cells.

One of the goals of targeted drug delivery is to develop a system which mini-


mizes the toxicity of a drug to nontargeted host tissue. When using liposomes, host toxicity could arise from leakage of drug from the liposome during transit to its target. One way of overcoming this problem other than enhancing the serum stability of the liposome is to encapsulate an agent which would be nontoxic to cells even if it did leak out. Heath and co-workers introduced the use of so-called liposome-dependent cytotoxic agents in 1983 [97]. These drugs are thus classified because they are very poorly permeable and must be delivered into the cytosol by indirect routes, presumably via endocytosis. Methotrexate-~-asparate (MTX-~-asp) is an example of a liposome-dependent drug. MTX is a folate analog which is rapidly taken up by cells by an energy-dependent facilitated transport mechanism [98]. MTX-~/-asp is a derivative of MTX which is equipotent in its inhibition of dihy- drofolate reductase but has an influx Km of at least 100-fold greater than that of the parent compound [99]. As a result of its low permeability, MTX-~/-asp in its free form is 200-fold less toxic to L1210 cells than MTX. Liposomes containing MTX-~-asp were targeted to L-929 cells with anti-H2K k antibodies producing a 6-12-fold enhancement of binding. The cytotoxicity of MTX-~-asp was increased 10-fold (IC5o -- 0.066 ~M) over the free drug (IC50 = 0.66 ~M) when encapsulated in the appropriately targeted liposome. Further, the cytotoxic effects were inhibited by antibody-coated empty vesicles or NH4C1, suggesting internalization via an endocytic route. Even more pronounced effects of targeting with this drug are observed if the liposomes are conjugated with protein A rather than with antibody directly [100]. In addition, for antibody-pretreated AKR/J SL2 cells the IC5o for MTX-~-asp encapsulated in protein A liposomes was 9-fold lower than for liposomes conjugated with goat anti-mouse immunoglobulin. For antibody-directed protein A liposomes, cytotoxicity was proportional to the antibody concentration and incubation time. lgG 1 was about 15-fold less effective than the other three murine isotypes at mediating protein A liposome delivery. Although it could take up to 4-8 h for liposome binding to plateau, up to one-third of the cell-associated liposomes could be internalized after 2 h at 37°C [100]. Internalization could be followed by removing any surface-bound liposomes by acidification to pH 3 and quantitating the remaining liposome markers. Cytotoxicity was proportional to the amount internalized and only subsaturating amounts of liposomes were needed.

Another liposome-dependent cytotoxic agent recently introduced by Heath et al. [101] is 5-fluoroorotate. This drug is a derivative of 5-fluoruracil and has no known transport pathway. Drug encapsulated in large, negatively charged liposomes was demonstrated to be 14-35-fold more cytotoxic to L929 or CV1-P cells than free drug [101].

A novel type of cytocidal immunoliposome, the phototoxic liposome, has recently been developed which has the advantage of being selectively activated subsequent to binding [102]. Phototoxic LUV's were prepared by reverse-phase eva- porization of a PC/cholesterol mix containing 2.5 mol% of 3-palmitoyl-2-[1- pyrenedecanoyl]-L-alpha-PC and a small amount of MPB-PE for antibody conjugation. Human T lymphocytes treated with the phototoxic liposomes targeted to a surface protein lost their ability to proliferate in response to phytohemagglu- tinin. The primary advantage of this system is that the liposomes need not be en-


docytosed although they believe that endocytosis and subsequent damage to lysosomes may contribute to their mode of action. Cytotoxicity can be selective because the phototoxic drug is only active at the site of irradiation.

IV. Controlled release liposomes

Controlled release liposomes refer to those liposomes which are designed to release entrapped aqueous agents in a controlled manner through environmental manipulations. Controlled release hposomes are a type of special function liposome which can be used for localized drug delivery even in the absence of ligand- mediated targeting. The concept of controlled drug release from liposomes implies that delivery can be directly manipulated by the design of the mechanism mediating release. Examples of this include liposomes which release upon encountering an area which has been locally heated or those which release after entering a mildly acidic environment. Although either type of selective release occurs independent of direct targeting, these liposomes which can actually be triggered to release their aqueous contents under certain environmental conditions represent a type of physical targeting of liposome. One such type of physical targeting is found with pH- sensitive liposomes. These liposomes are constructed such that when certain membrane components become protonated under low pH conditions the membrane structure is altered resulting in increased permeability and/or enhanced fusogenic activity. A similar system which responds to heat rather than pH are the temperature-sensitive liposomes and these will be discussed in a later section.

IV.1. Acid-sensitive liposomes

All of the initial studies and much of the recent work with antibody-targeted liposomes have been based upon liposomes composed of PC or PC/cholesterol. These compositions are frequently utilized primarily because of their high degree of stability, particularly in the presence of serum components. However, this liposome composition may not be the best choice for efficient release of entrapped drugs upon arriving at their target. It is now well established that endocytosis is the primary mechanism of liposome internalization but it is not known at which point subsequent to the initial uptake that the liposomes are able to deliver their contents to the cytosol. As the endocytic vesicle containing the membrane-bound liposome pinches off from the plasma membrane it now assumes the role of a targeted carrier itself which is destined for the lysosomes. However, soon after departing from the cell surface, the endosome or transport vesicle starts to undergo many changes which will continue until it eventually fuses with the lysosome where its remaining contents can be completely hydrolyzed. One of the early events of this transition is an acidification of the endosome interior, reaching a pH of approx. 5.0--6.5 [103,104]. The lysosomal pH is even lower and many drugs such as cytosine arabinoside may be acid labile [105] and if delivered all the way to the lysosomes are frequently inactivated under its degradative conditions [74]. There- fore, release of the drug out of the liposome and endosome from a pre-lysosomal


site is necessary for the efficient delivery of this type of drug. Liposomes which are pH sensitive have been developed to meet this need for selective release.

Liposomes made pH-sensitive were first described by Yatvin et al. in 1980 [106]. The pH-dependent trigger which was used was the N-acylamino acid palmitoyl homocysteine (PHC). When PHC was incorporated into PC liposomes with encapsulated calcein, an isothermal pH-induced leakage of the dye could be demonstrated. The underlying mechanism was originally thought to be thiolactone ring formation by homocysteine which is known to occur at low pH. However, subsequent studies suggested that electrostatic headgroup interactions or lateral phase separations of the components were more likely involved [107,108]. Headgroup composition, acyl chain length, and the amount of acylamino acid incorporated were all important for the pH sensitivity [108]. Internalization of these PC-based liposomes into acidic compartments should facilitate leakage and release of contents into the vacuole. However, for most applications the drug must then escape to the cytosol to reach its target. Some drugs are more likely to penetrate through the endosomal membrane than others. For example, weakly anionic drugs such as MTX could leak more rapidly when located in acidic environment because the drug would have a higher permeability when neutralized by protons. Drugs which are less permeable could remain sequestered inside the endosome/lysosome unless there is an alternative mechanism for release.

Fusion of the liposome with the endosomal membrane would mediate direct release of vesicle contents into the cytosol. Although many studies using PC-based liposomes have demonstrated the efficacy of drug delivery with liposomes and immunoliposomes, superior delivery might be achieved by using liposome compositions which are more prone than PC to undergo fusion with endosomal membranes at reduced pH. Generation of fusion-competent liposomes which were pH- sensitive was achieved by incorporating a titratable component into liposomes composed of PE rather than PC. Unsaturated PE does not form stable bilayer vesicles at neutral pH [109-111] but rather assumes the hexagonal (HII) phase. How- ever, PE can be induced to form bilayers by the addition of molecules such as phospholipids [112,113}, fatty acids [114-116], acylated amino acids [107,117,118], or cholesterol derivatives [118-126]. There are many other stabilizing molecules available (proteins and other phospholipids) but the above stabilizing molecules are of particular importance for pH-sensitive liposomes because all are weakly anionic at neutral pH but upon entry into an acidic environment they become neutralized and lose their bilayer stabilizing ability. When these PE liposomes become physically unstable they start to leak and/or become fusion competent due to the phase transition towards the H~I phase. In studies utilizing cholesteryl hemisuccinate (CHEMS) for membrane stabilization, Szoka and co-workers [119,121] demonstrated that PE/CHEMS liposomes aggregate and then leak in a lipid concentration-dependent manner in response to acidification to pH 5 and below.

The ability of protein-free liposomes composed of DOPE and at least 20 mol% PHC to undergo rapid pH-dependent fusion was first demonstrated by Connor et al. [107]. Using electron microscopy, gel filtration and resonance energy transfer (RET), these authors observed apparent fusion between SUV's within a pH range


similar to that found in the endosome (pH <6.5). In addition, these liposomes (PE/PHC = 8:2) were very permeable to calcein under the same conditions. How- ever, addition of 40 mol% cholesterol significantly reduced the acid-induced leakage with minimal effects on fusion [107]. Others have shown that addition of cholesterol to PE-containing liposomes can actually increase its fusion capacity [122]. The results with PHC-stabilized liposomes are in contrast to pH-sensitive liposomes with oleic acid (OA) as the stabilizer which were found to be quite leaky under similar conditions. Liposomes composed of DOPE/chol/OA (4:4:2) leaked more than 85% of entrapped calcgln within 5 min at pH 5.5 [114]. Liposomes composed of PE prepared by transphosphatidylation of egg PC (TPE) and OA (7:3 mole ratio) have been shown to aggregate, become destabilized, and fuse below pH 6.5 [115]. These acid-sensitive liposomes (DOPE/OA, 7:3) have been shown to mediate cytoplasmic delivery of calcein. As evidenced by diffuse intracellular fluorescence [123] up to 30 mol% cholesterol could be incorporated into the liposome without inhibiting cytoplasmic delivery. PC/OA liposomes were stable at pH 4-8 and were unable to achieve cytoplasmic delivery of the dye. Acid respon- sive liposomes which are fusogenic can also be prepared with bioactive lipids such as arachidonic acid and 1-oleoyl-2-acetylglycerol [124]. Liposomes composed of mixed chain egg PE/OA (7:3) became destabilized when the pH was lowered to less than 6.5 or upon addition of calcium [115]. Characteristics of destabilization included rapid proton-induced aggregation of liposomes coincident with release of internal aqueous contents and membrane fusion.

Fusion between two liposomes involves bilayer-bilayer contact followed by con- solidation of the two opposed membranes into a continuous bilayer which involves mixing of lipid components and intermixing of the aqueous contents from the two liposomes. The initial contact between liposomes leads to aggregation into larger structures which can be followed by light scattering or turbidity measurements. The two aspects of actual fusion can be monitored with the use of aqueous or lipid markers which are fluorescently labeled. Intermixing of separately entrapped aqueous markers 1-aminonaphthalane-3,6,8-trisulfonic acid (ANTS) and N,N'-p- xylylenebis (pyridinium bromide) (DPX) results in quenching of the fluorescent signal from ANTS by DPX upon vesicle fusion [125]. A leakage assay can also be developed with these markers by following the dequenching of ANTS by DPX when they leak out from liposomes from which they have been coencapsulated [115,119]. For the RET lipid mixing assay a donor/acceptor pair of labeled lipids 7-nitro-2,1,3- benzoxadiazol-4-yl-PE (NBD-PE, acceptor) and No(lissamine rhodamine B sul- fonyl)-PE (Rh-PE, donor) are each incorporated into one population of liposomes such that there is significant NBD-PE quenching by Rh-PE. When fused with an unlabeled population the probes are diluted and dequenched resulting in fluorescence signal enhancement [126]. Development of these novel biophysical techniques have been very important in elucidating the probable mechanism of action of this type of liposome.

An alternate type of pH-sensitive liposome has recently been described in which the destabilization characteristics apparently do not involve membrane fusion. Li- posomes composed of N-succinyldioleoyl-PE and DOPE (3:7) start to leak at pH


5 and below but do not fuse [112]. Release was believed to result from bilayer destabilization induced by pH-dependent packing defects of the lipids and electrostatic interactions between the stabilizing molecules. The mechanism of delivery by pH-sensitive liposomes is dependent upon the phase behavior of the liposome which in turn is based on the liposome composition. Addition of targeting molecules to the bilayer may alter its phase dynamics and thus its ability to encapsulate, retain and specifically release its contents.

However, a recent report describing the preparation of biotin-bearing pH-sensitive liposomes suggests that these liposomes can also be effectively targeted while retaining both their contents and phase behavior [127]. Incorporation of less than 1 mol% biotinaminocaproyl-PE into PE/cholesterol/OA (7:3:3) liposomes did not significantly change the pH-dependent leakage profile. These liposomes could be agglutinated with acetylated avidin and bound with very high affinity to avidin- coated microtitre plates.

Liposomes sensitive to pH can also be conjugated with acylated antibody without altering their permeability response to acidification [116], although calcium- induced fusion activity was reduced by antibody incorporation [128]. However, targeting of calcein-containing pH-sensitive liposomes (PE/PHC, 8:2) with acylated anti-H-2K k IgG resulted in delivery of the dye into the cytoplasm of L-929 ceils [117]. Dye was dispersed throughout the cell when given with pH-sensitive immunoliposomes but showed only punctate patterns for pH-insensitive immunoliposomes (PC based). A cytoplasmic calcein concentration of 50 ixM was determined using a microscope-associated photometer. This concentration repre- sented dye transfer from approx. 1000 liposomes per cell, indicating a very efficient delivery from approx. 50% of the internalized liposomes.

The therapeutic potential of antibody-conjugated acid-sensitive liposomes has been demonstrated in vitro by the efficient target-specific delivery of antitumor drugs and cytotoxins. Connor and Huang [118] selectively delivered MTX or ara- C to L-929 cells with anti-H-2K k targeted liposomes (56 IgG per 130 nm liposome) composed of DOPE/OA (8:2). Moreover, targeted pH-sensitive liposome delivery was more efficient at cell killing than either that of the free drugs or targeted PC liposomes. Pretreatment with chloroquine or NH4C1, each a weak base which neu- tralizes acidic organelles, inhibited cytoplasmic delivery of calcein [117] or ara-C [118] by the immunoliposomes, indicating liposome uptake into base-sensitive organelles, presumably endocytic vesicles.

A tryptic fragment of diphtheria toxin, fragment A (DTA), is a potent inhibitor of protein synthesis. DTA is normally impermeable to cells because it lacks the fragment-mediating cell binding and entry and therefore is not cytotoxic as a free protein. However, upon gaining entry into the cell interior elongation factor 2 is selectively inactivated by a reaction involving the adenosine diphosphate (ADP)- ribosyl transferase activity of the catalytic A fragment leading to cell death [129,130]. It has been determined that the effects are stoichiometric, i.e., only one toxin molecule is sufficient to kill the cell [131]. The characteristics of low permeability and high lethality make DTA a natural candidate for delivery with liposomes. In early studies using proteoliposomes containing Sendai glycoproteins,


Okada and co-workers [131,132] demonstrated enhanced cytotoxicity of DTA, presumably as the result of direct liposome-cell fusion. Using the fusogenic pH- sensitive immunoliposomes Collins and Huang [116] have successfully delivered the DTA to murine cells which are normally toxin-resistant. DTA was encapsulated into DOPE/OA (8:2) vesicles by a modified dehydration/rehydration procedure [133]. Acid-sensitive liposomes conjugated with anti-H-2K k but not non-targeted or targeted pH-insensitive liposomes were able to deliver DTA specifically to L- 929 cells as determined by inhibition of 3H-leucine incorporation. The effects could be blocked by pretreating the cells with NH4C1 or chloroquine.

The potential of gene therapy that has been realized in the past few years [134] has led to intensive efforts in the development of techniques for the efficient delivery of DNA into cells. The currently available methods such as microinjection, electroporation, chromosome-mediated insertion, microcell-mediated gene transfer, DNA-mediated gene transfer, infection with recombinant RNA or DNA vi- ruses, spheroblast fusion, and cell fusion are considered inefficient and relatively non-specific. Moreover, none of these methods are acceptable for in vivo transfection. Liposomes have also been utilized to introduce nucleic acids into several different cell types, although with minimal benefits compared to conventional methods. Effective liposome-mediated transfer has been reported into bacteria [135,136], fungal [137], and animal cells [138-147]. Transfer is frequently initiated by the addition of a membrane fusogen such as DMSO or PEG. The established ability to introduce drugs or proteins into cells with pH-sensitive immunoliposomes suggests that this system may also be applicable for the specific delivery of other macromolecules such as plasmids via similar mechanisms. Using such a system, Wang and Huang [145-147] have demonstrated efficient delivery and expression of exogenous DNA to targeted cells both in vitro and in vivo. A plasmid (pPCTK-6A) containing the herpes simplex virus thymidine kinase gene (HSV-TK) was encapsulated in acid-sensitive liposomes (DOPE/cholesterol/OA, 4:4:2 mole ratio) targeted with acylated IgG against the routine H-2K k histocompatibility complex [145]. Liposomes prepared by detergent dialysis with a mean diameter of 290 nm entrapped approx. 20% of the added DNA, corresponding to about 3-5 8.2 kb plasmids per liposome. So that the expression of this gene could be regulated following incorporation into the host genome, the endogenous promoter was removed and replaced with a promoter containing a cAMP regulatory region [148]. Therefore, by adjusting the cAMP levels the introduced promoter from a rat phosphoenolpyruvate carboxykinase (PEP-CK) gene would turn on or off the transcription of HSV-TK. Since all mammalian cells normally have the salvage enzyme thymidine kinase, tk- mutant mouse L cells were used as the target cells so that gene expression could be followed by monitoring regulated TK activity. An- tibody-targeted liposomes which were pH-sensitive were about 8-fold more effective at restoring thymidine kinase activity to the Ltk- cells than pH-insensitive immunoliposomes [145]. The presence of antibody increased the effectiveness of the pH-sensitive liposomes 6-fold compared to those not targeted. Expression of enzyme activity was under the control of cAMP indicating that TK activity was the result of transformation and not reversion of the mutant. When long term trans-

368 S. W RI GHT AND L. H U A N G

formation by pH-sensitive immunoliposomes was compared with that by a traditional method such as calcium phosphate transfection, the liposomes were found to produce a transformation efficiency of 9% vs. 1% for the salt precipitation method. Liposome administration was also much less toxic to the cells than incubation with high concentrations of calcium phosphate. In addition, DNA blot hybridization studies of long-term transformants indicated that the method of delivery may dictate at which site the plasmid DNA integrates into the host genome [145]. Efficient transfection by pH-sensitive immunoliposomes was also observed if DNA was simply adsorbed to the surface of preformed liposomes rather than encapsulated within during liposome formation [147]. Induced kinase activity was elevated 30-fold for entrapped and 20-fold for adsorbed compared to free DNA. Efficient liposome-mediated transfection with adsorbed DNA has recently been described by others [149]. Unless cells susceptible to passive targeting are the targets, then antibody conjugation should further increase the delivery efficiency.

Huang and co-workers have also found that targeted pH-sensitive liposomes could be used to deliver DNA to lymphoma cells transplanted to the peritoneal cavity of nude mice [146]. A 4.6 kb plasmid (pBB0.6-CAT) containing the Esch- erichia coli chloramphenicol acetyltransferase (CAT) gene under the control of the PEP-CK promoter used previously was encapsulated in pH-sensitive immunoliposomes. RDM-4 lymphoma cells (H-2K k positive) which had been injected i.p. into immunodeficient mice were able to take up about 20% of an i.p. injected dose of immunoliposomes. When activated with 8-bromo-cAMP and 3-isobutyl-l-meth- ylxanthine, CAT-specific activity in these cells transformed with the liposomes was 12-fold higher than that in the liver and 5-fold higher than that detected in the spleen. No activity was found in peritoneal macrophages although these cells accumulated a significant amount of liposomal lipid. PC-based immunoliposomes (pH- insensitive) produced only one-fourth the activity in target cells that pH-sensitive vesicles could. These results have important implications for gene therapy and cancer therapy for they demonstrate that genetic material can be effectively delivered in vivo to targeted cells with little incorporation into the genome of nontargeted host cells. The ability to deliver DNA to cells in vivo is the greatest advantage of using liposomes over other methods.

Mechanistically, the premise that pH-sensitive liposomes fuse with the endosome membrane and release their contents directly into the cytosol is most strongly supported by the studies demonstrating intracellular delivery of macromolecules which are too large to permeate across intact membrane [116,145]. Alternatively, simultaneous endosome and liposome membrane rupture is conceivable which leads to dispersal of entrapped contents into the cytosol. This mechanism should also proceed with empty pH-sensitive liposomes, yet no cytotoxicity is observed with these liposomes as one might expect following rupture of the acidic endosomes. However, the ability to deliver DNA adsorbed to the surface of pH-sensitive immunoliposomes [147] suggests that this mechanism is possible.

The rate and extent of endocytosis and cellular processing including sorting and recycling of internalized targeted liposomes has not been examined. Gruenberg and Howell [150] have recently reported that an internalized transmembrane protein


(G protein of VSV) resides for less than 5 min in a fusion-competent endosome. The fusion-competent compartment was determined to be the early endosome where sorting of the protein for recycling or degradation occurs, which, for the G protein, was 50% in either direction. Visualization of endosomes reveal that they move bidirectionally along linear tracks of microtubules by discontinuous salta- tians until they cluster along with lysosomes in the perinuclear region of the mi- crotubule-organizing center [151].

The mechanism and dynamics of PE-phase behavior and the role of Hn formation in bilayer destabilization has been examined in detail by Szoka and co- workers [119,120,152,153] and recently reviewed by Siegel [154]. It was observed that pH-dependent liposome destabilization and leakage was mediated by liposome aggregation [119,120]. Moreover, they reported that destabilization is greater between two different kinds of liposomes than between pure pairs [120] which may have relevance for interaction between liposomal and cellular membranes.

IV.2. Temperature-sensitive immunoliposomes

Other schemes have been developed which also rely on the dynamics of lipid phase behavior and are of particular use for cells with inherently low endocytic activity and thus inappropriate candidates for pH-sensitive liposomes. Utilization of temperature-sensitive liposomes was one of the first attempts at physical targeting of liposomes. Taking advantage of the well-known observation that the permeability of liposomes is highest at the temperature at which they undergo a phase change from gel to liquid-crystalline [155], Yatvin and co-workers [156] examined the ability of liposomes to release entrapped dye contents as a function of temperature. DPPC was chosen for these studies because it has a phase-transition temperature (41°C) which can be reached with only mild hyperthermia to the animal. Multilamellar vesicles of DPPC leaked 85% of drug when heated near their phase-transition temperature in the presence of serum. The transition temperature is also sensitive to the number of lamellae in the liposome, the presence of serum, and the addition of any other lipids or hydrophobic components [157]. For example, the behavior of this system in the presence of amphipathic drugs will be drug-dependent because of the potential effects of membrane-interacting drugs on the phase behavior of the liposomes [158,159]. The drug delivery potential of this system was initially ascertained by the enhanced ability to kill E. coli with neo- mycin encapsulated in DPPC/DSPC (3:1) vesicles when heated to 41°C [156]. Ma- gin and Weinstein and co-workers [157,160,161] have demonstrated the ability of heat-sensitive liposomes to deliver MTX to tumor cells following local hyperthermia. This therapy is somewhat selective in that only the liposomes in the locally heated area release their drugs. The effectiveness of heat-sensitive liposomes can be enhanced by targeting with antibody which will bring the liposome into direct contact with the cell surface prior to release of its contents. Heat-sensitive immunoliposomes were prepared by Sullivan and Huang [70] by injection of palmitic acid-derivatized IgG into a suspension of DPPC near its phase-transition temperature. Although incorporation of antibody changed the temperature at which dye


Fig. 2. Proposed mechanism of release from heat-sensitive immunoliposomes. Phase boundaries formed by the co-existence of gel and fluid-phase lipids at the phase-transition temperature facilitates leakage

of soluble drugs from liposomes bound to the cell surface.

release became maximal in buffer, in the presence of 5% serum the release temperature was the same (40°C) for bare or conjugated liposomes. Moreover, immunoliposomes which were bound to target cells retained their ability to release entrapped contents upon heating [70,162]. Using this protocol they demonstrated that 3H-uridine encapsulated in heat-sensitive immunoliposomes could be taken up more efficiently by the target cells than free nucleoside [162]. Since uptake could be mostly eliminated with nucleoside transport inhibitors the mechanism of delivery was presumed to involve surface release from bound liposomes producing a locally high concentration of substrate which was then taken up by the normal transport routes (see Fig. 2).

IV.3. Target-sensitive immunoliposomes

Target-sensitive immunoliposomes as described by Ho et al. [72] represent a composite of pH-sensitive and heat-sensitive liposomes. These liposomes are constructed much like their pH-sensitive counterparts in that they are based on PE, yet they have no stabilizers other than the targeting molecule, such as palmitoyl IgG. However, like the heat-sensitive liposomes, the site of delivery is at the cell surface. Thus, therapeutically, these liposomes were also designed for drug delivery to cells with either generally low endocytic activity or poorly internalized sur-


face targets. Although they have similar potential targets the suggested mechanism of release for this system is quite novel and different from those which are temperature sensitive. One of the initial observations important for the development of this system was by Taraschi et al. [163] who reported that PE liposomes stabilized by incorporated glycoproteins could be destabilized and induced back into their native non-bilayer structures (Hit) following their binding to lectins. Other amphipathic molecules such as the lipid hapten DNP-cap-PE has also been found to stabilize D O P E when incorporated at a level of greater than 12 mol% [164]. If these antigenic liposomes were loaded with quenched calcein and added to immobilized anti-DNP antibodies they leaked in an antibody concentration-dependent manner [164]. The mechanism of release was presumed to be multivalent binding between liposome and antibody leading to contact capping of the stabilizer at the binding site. This redistribution of the stabilizing hapten is believed to result in reorganization of the membrane lipids toward the H n phase which produced highly permeable areas where the stabilizer had vacated (see Fig. 3). Similar find- ings were obtained with glycophorin-stabilized PE liposomes which could be dis- rupted when treated with trypsin [165]. It is of interest that glycophorin can stabilize D O P E whereas if added to DOPC it increases bilayer permeability [166].

Antibody-dependent target-sensitive liposomes were introduced when it was found that under certain well-defined conditions fatty acid-derivatized IgG could itself stabilize D O P E [72]. Liposomes composed of PE and acylated IgG against the gD envelope protein of HSV loaded with calcein exhibited target-induced lysis

o o

Fig. 3. Proposed mechanism of release from target-sensitive immunoliposomes. Subsequent to binding to surface antigens the stabilizing acylated antibodies undergo contact capping which leads to lipid instability in the vacated regions. The PE-based liposome then is susceptible to contact-dependent destabilization which pushes the bilayer toward the hexagonal equilibrium phase which is characterized by rapid leakage from intermediate states. Drugs released from this pericellular site at locally high con-

centrations are then taken up by cellular transport systems.


when incubated with HSV [167] or HSV-infected L cells [72]. In addition, these liposomes were able to deliver specifically ara-C or acyclovir to virus-infected cells resulting in enhanced antiviral activity compared to the free drug [168]. As before, the mechanism was thought to involve multivalent binding followed by pericellular lysis. Uptake of surface-released drug was apparently through the nucleoside carrier and could be effectively blocked by specific nucleoside-transport inhibitors. Kinetic and ultrastructural studies implied that collision among bound liposomes was essential for liposome destabilization [169]. This system also has potential for the development of liposome-based homogenous immunoassays [170-172].

V. Animal studies with immunoliposomes

One of the first obstacles encountered with liposome administration in vivo is stability of the particle in blood. Successful drug delivery with immunoliposomes requires that the drug remains with its carrier until it reaches the target site, at which point it should dissociate from its carrier so that it may then unite with the effector molecule which is the ultimate target. There are a variety of interactions between liposome and blood components which can disrupt the integrity of the liposome resulting in leakage and premature loss of the drug to be delivered. Thor- ough reviews on this topic have been presented by Scherphof et al. [173], Juliano et al. [174], and Bonte and Juliano [175].

The physical properties and ultimate fate of blood-borne liposomes can be in- fluenced by interactions with serum components such as albumin, immunoglobulins, clotting factors, fibronectin, and C-reactive protein [176,177]. However, the most prominent means of liposome destabilization in blood is interaction with serum lipoproteins. Scherphof and co-workers [178] have shown that the phospholipid constituents of liposomes can actually be extracted by high-density lipoproteins re- suiting in leakage of entrapped contents. The extent of leakage depends upon the HDL/phospholipid ratio [173,177]. Although the mechanism of interaction is still unresolved, it appears that phospholipid transfer to HDL or an HDL-like particle is multi-phasic with at least one saturable component. In addition, a small amount of phospholipid transfer out of liposomes can also be mediated by low and/or very low density lipoproteins [177].

It is evident that the ability of serum proteins to interact with the liposome is heavily dependent upon the bilayer physical state. What effects the addition of antibody has on the physical properties of the liposome depends upon the method of conjugation. Due to packing considerations, coupling to liposomal lipids might not be expected to be as perturbing as the incorporation of multiacylated antibodies. Liposomes with protein coupled to glycolipids by the periodate procedure were just as stable (nonleaky) in buffer as liposomes treated with periodate alone [41]. In contrast, incorporation of peptides modified with palmitic acid into liposomes results in the alteration of several bilayer properties [179]. Sullivan and Huang [70] observed that incorporation of palmitoyl-IgG into DPPC altered the rate and extent of heat-induced carboxyfluorescein release. When heated in buffer, the temperature for maximal rate of dye release was lowered and the total amount of dye


released was decreased from 80 to 45%. In the presence of 5% serum the release temperature was now the same for both free and antibody-coated liposome although the extent of dye release was still lower for the latter group. Liposomes with Fab' fragments attached via DTP-PE [45] or MPB-PE [49] are relatively nonleaky in the presence of serum. The mechanism of serum-induced bilayer disrup- tion for antibody-conjugated liposomes has not been examined in any detail. It is expected that the lipid components of targeted liposomes are susceptible to the same attack by serum proteins that the antibody-free liposomes are. However, the presence of the foreign antibodies on the surface of the liposome increases its an- tigenicity and thus becomes more susceptible to complement-mediated lysis [180]. Furthermore, antibody attached to liposomes could fix complement itself and bring about a complement-mediated lysis [180].

The interactions between liposomes and serum components are governed to a large extent by the size and composition of the liposome. Inclusion of 30-50 mol% cholesterol in the liposome enhances its stability in serum and is now routinely included in most liposome preparations to be used in the presence of serum [181]. Cholesterol increases liposomal stability by increasing the lipid packing density which eliminates any phase boundaries (and phase transitions) and thus decreases potential interactions with serum lipoproteins. Glycolipids can reduce plasma-induced leakage in proportion to their sialic acid content [182]. The effects of adding both cholesterol and gangliosides are synergistic with in vitro half-lives in serum of up to 24 h obtainable for SUV's at 37°C (PC/chol/Gx= 10:5:1) [180]. Even greater serum stability has been reported for sphingomyelin/cholesterol mixtures [176]. Incorporation of PS [182], sulfatides [182], or sphingomyelin [183], altering phospholipid structure [184], or utilizing polymerizable lipids [176,185] can also enhance serum stability. Allen and Chonn [186] have recently demonstrated that LUV composed of ganglioside GM 1 and sphingomyelin had significantly reduced uptake by RES components in vivo. In addition, LUV's retain their contents longer than SUV's in the presence of plasma [182] although others have reported that SUV's exhibit much longer half lives in blood than MLV's [187,188]. The small radius of curvature of SUV's influences bilayer packing and thus susceptibility to disintegration by serum proteins.

Fatty acid-stabilized pH-sensitive immunoliposomes (PE/OA) are unstable in serum where they will spontaneously aggregate and release entrapped calcein [189,190]. Fatty acids may not be an appropriate choice as a PE-stabilizer for in vivo studies, since it can be removed from the liposome by serum components [113]. For example, oleic acid is known readily to partition out of liposomes in the presence of albumin [191]. Albumin is known to induce leakage of inulin from PC liposomes [192] and it can also rapidly destabilize PE/OA liposomes (Collins, D. and Huang, L., unpublished observations). However, the pH-sensitive liposomes are apparently stable in vivo if cholesterol is also incorporated [146]. Rather than adding a third component such as cholesterol to enhance serum stability, substi- tution with a different pH trigger such as a double-chain amphiphile [112,193] can also be used for the preparation of serum stable acid-sensitive liposomes [193].

Although serum stability is an important parameter for liposome carriers, this

374 s. WRIGHT AND L. HUANG

does not necessarily mean that more stable liposomes are more effective carriers in general. For some applications where sustained blood levels of drug is the goal, then slow controlled release from the liposome is desired. In fact, liposomes can act as small continuous infusion devices because encapsulated drugs generally have a longer plasma half-life than the free drugs [189]. A slow release mechanism would be particularly beneficial for the delivery of cell cycle-dependent drugs and where a reduced frequency of administration is desired. The ability to anchor the liposome by antibody targeting can effectively prolong the drug effects at the target site. Slow release of acyclovir from liposomes targeted with antibody to herpes virus-infected corneal cells in culture has been shown to be an effective means of inhibiting virus replication [194,195] although its effectiveness as a topical treatment in vivo has yet to be demonstrated.

Liposome encapsulation has previously been found to be a means by which to reduce some of the major side effects of potent chemotherapeutic agents. The dose- limiting cardiotoxicity of adriamycin can be attenuated by entrapment in liposomes of cardiolipin [196,197] or other anionic phospholipids [198-200] resulting in an overall enhancement of its therapeutic index. Encapsulation of the very toxic anti-fungal agent amphotericin B increases its therapeutic index [201] apparently by mediating selective drug transfer to fungal cells [202]. In spite of the many promising in vitro studies demonstrating the efficacy of liposomes as drug delivery devices, the widely anticipated benefits of liposome use in vivo have not, in general, been realized. There are of course exceptions such as for the successful treatment of Leishmaniasis with liposome encapsulated antimonials [203,204] and the delivery of amphotericin B for systemic fungal infections [201,205-207]. Although the selectivity of treatment with these liposome-encapsulated drugs is high, attempts at further enhancing their therapeutic index by surface targeting has not been reported.

There have been very few reports on the in vivo use of immunoliposomes for delivery of chemotherapeutic drugs. This apparently stems from the problem of compartmentalization. In general, the distribution of liposomes is restricted to the compartment in which they are administered. For example, to extravasate from the circulation, liposomes injected intravenously must be transported through or around capillary endothelial cells where they will then usually encounter the basal lamina as a second barrier [4,207]. Only particles much smaller than limit vesicles are capable of escaping out of the circulation through sinus discontinuities between the endothelial cells [208,209]. However, in the liver and spleen SUV's are able to penetrate the relatively large sinusoids (no basement membrane) and ac- cumulate in both parenchymal and non-parenchymal cells. These openings into the space of Wisse [210] are about 100 nm so that there is a size restriction here as elsewhere because MLV's are taken up less readily then SUV's [194]. Extravasa- tion elsewhere should be possible, since leukocytes are known to cross unruptured capillaries through the ameboid movement involved in diapedesis. The apparent inability of liposomes to escape the circulation is a major impediment for their use against extravascular targets such as solid tumors. Effort must now be focused on targetable sites confined to the cardiovascular system for which targeted tiposomes


may be of benefit as therapeutic or diagnostic agents. Alternate routes of administration have not been examined so closely as the in-

travenous approach. Liposomes injected s.c. are rapidly drained to the regional lymph node where, depending upon size, they may enter into the circulation [211]. Liposomes injected into the subarachnoid space have been shown to prolong the drug retention time significantly with no display of undesirable toxic effects (Huang, L., unpublished results). Intraperitoneal tumors have been a favorite target for in vivo studies of liposome targeting [146,212]. Administration of pH-sensitive immunoliposomes i.p. to mice carrying RDM-4 lymphoma cells as an ascites tumor results in accumulation of approx. 30% of the injected dose in ascites cells [146]. Targeting increased the uptake by non-adherent ascites cells (primarily RDM-4 cells) 2-fold without altering accumulation by adherent cells. The next largest site of uptake was the stomach with approx. 15%. Injection of either non-targeted pH- sensitive liposomes into tumor-bearing animals or targeted pH-sensitive liposomes into tumor-free animals resulted in significant uptake by the spleen which was even greater than that found in the ascites cells. Antibody targeting increased accumulation in the heart 3-5-fold. Liver, lung and kidney each accumulated less than 5% of the injected dose of any liposome type.

One of the first attempts at using antibody-targeted liposomes for anti-tumor therapy in an animal model was reported by Hashimoto et al. [212]. Liposomes were prepared by immobilizing an IgMs fragment against a mouse mammary tumor-associated antigen on SUV's containing actinomycin D and were found to be selectively cytotoxic in vitro. Intraperitoneal injection of these chemoimmunoli- posomes into mice with an i.p. transplanted tumor (MM46) resulted in more effective therapy than either free drug or BSA-coated drug-containing liposomes [212]. However, the relevancy of the i.p.-i.p, system as a general in vivo model is questionable and reflects primarily the potential of the vehicle for local therapy. The authors also examined potential systemic effects by i.v. administration of liposomes targeted against a subcutaneous MM46 tumor [212]. A single i.v. dose of liposomes for treatment of tumors implanted 4 days earlier was found to be moderately effective in reducing tumor weights in mice compared to the effects of control liposomes. A more significant tumor reduction was obtained if the mice were pre-dosed with unmodified MLV's prior to the injection of the antibody-conjugated liposomes. These effects were attributed to specific binding to the tumor cells based upon their in vitro results. However, since extravasation of the targeted liposomes was unlikely the effects were more probably due to sustained serum levels of drug from the liposome. Although blood clearance values for liposomes were not determined, the ability of the animal to clear colloidal carbon from the circulation was inhibited by MLV predosing [208]. Circulating macrophages and res- ident macrophages in each organ are apparently the primary cell types responsible for liposome uptake at these sites [213]. The augmented effects on targeted cells that can be achieved with MLV predosing is apparently due to saturation of phagocytic cells so that their capacity to engulf the immunoliposomes is impaired. Blockage of RES components by predosing with dextran sulfate [213,214], SUV's [215], REV's [216] and MLV's [217] have been demonstrated effective in altering


the circulation time and organ distribution of liposomes. Rather than predosing with lipid for RES blockade Aragnol and Leserman [218]

have evaluated the ability of an anti-Fc receptor antibody to influence the distribution of injected liposomes in the presence of accelerated immune elimination. They demonstrated that it is possible in vivo to enhance Fc receptor-mediated uptake of ligand-bearing liposomes by the use of liposome-specific monoclonal antibodies or to reduce Fc receptor-mediated uptake with a monoclonal antibody to the Fc receptor. They also evaluated the influence of passive or elicited anti-DNP antibodies on the circulation and tissue distribution of i.v. injected DNP-bearing liposomes. These liposomes had increased uptake to the liver which could be ab- rogated by injection of an anti-Fc receptor antibody.

Gregoriadis et al. [219] have recently examined the ability of anti-Thyl IgG- bearing liposomes to interact with circulating target AKR-A cells which had been injected by the same route. They concluded that antibody-targeted liposomes are cleared more rapidly than bare liposomes from the circulation of mice which had or had not been injected with the target cells. Radioactive and fluorescent markers from antibody-bearing liposomes were cleared from target-bearing animals faster than from the control animals. Approx. 15% of the injected dose interacted with the circulating AKR-A cells. Konno et al. [220] recently examined the therapeutic potential of antibody targeted liposomes against a solid tumor in vivo. Anti-human alpha-fetoprotein (AFP) monoclonal antibody were conjugated to SUV's (via SPDP/MBS) composed of PC/chol/PA and loaded with adriamycin for targeting to AFP-positive human hepatoma cells (Li-7) maintained in nude mouse as xeno- transplanted subcutaneous tumors [222]. Reduction in tumor weight and histo- pathological evidence indicated that the drug-loaded liposomes with immobilized antibody were therapeutically more effective than unconjugated liposomes. Treat- ment was initiated by administration of three i.v. injections of liposomes 10-14 days after inoculation when tumor weight exceeded 100 rag. Tumor (Li-7) reduction with the immunoliposomes was enhanced by targeting with tumor-specific antibodies whereas antibody specificity made no difference for immunoliposomes given against an AFP-negative strain (MX1).

The ability of immunoliposome to reach target cells depends on the extent of RES uptake and accessibility of the target within that compartment. Encapsula- tion of chemotherapeutic agents in targeted liposomes can alter its biodistribution and, depending upon its dose-limiting toxicity, the maximum tolerated dose. For example, ADR dosages of up to 7.5 mg per kg can be tolerated in encapsulated form when administered i.v., since the normal toxic distribution of the drug to the heart is reduced [220]. However, the lower uptake by the heart that was reported was due to the encapsulation and could not be attributable to the presence of antibody on the liposome. In fact, the addition of the antibody to the liposome did not significantly alter the drug levels in serum, spleen, lung, kidney, and most no- tably, the tumor. Liver accumulation was found to be initially higher for the targeted liposome which lasted up until about 8 h postinjection. In addition, previous studies have indicated that protein-coated liposomes [221] and antibody-coated liposomes [222] are cleared faster from the circulation to the liver than unconju-


gated liposomes [221,223]. Injection of antibody conjugated liposomes into antigen-negative mice circulated for the same period as control liposomes whereas they rapidly disappeared from the plasma in mice expressing the targeted determinant [223]. Debs et al. [222] found that antibody-mediated enhancement of liver uptake resulted in decreased uptake in a number of organs outside the RES. The liposomes used were coated with an antibody against a murine lymphoid differentia- tion antigen (Thy 1.1) present in blood, thymus, lymph nodes and spleen of Thy 1.1 mice (AKR-J). Uptake of antibody-coated SUV's by lymph nodes of AKR-J mice was enhanced 7.4-fold by using antigen-specific antibody and uptake at this site was 3 times that in antigen-negative mice (AKR-Cu). Predosing with a large amount of unmodified liposomes prior to i.v. injection of the targeted liposomes reduced liver uptake of the coated liposomes but did not alter uptake by the lymph nodes.

Murine biodistribution of antibody-conjugated pH-sensitive liposomes has been examined by Connor and Huang [189] and was found to be essentially the same as for antibody-free liposomes. Lipos0mes labeled with 3H-cholestanyl ether (lipid marker) and t25I-tyraminyl-inulin (aqueous marker) were injected into the tail vein of antigen-positive (C3H) or antigen negative (Balb.c) mice. Acid-insensitive liposomes (PC/OA) which were antibody-free or coated with palmitoyl-anti-H-2K k IgG displayed similar clearance rates and organ distribution in either type of mouse. However, in contrast to pH-insensitive liposomes which were taken up primarily by liver and spleen, a significant amount of 3H-CE from pH-sensitive liposomes was found in the lung as well as in the liver. After about 6 h the marker was dis- tributed the same as for the pH-insensitive profile. The presence of antibody on the PE/OA liposomes resulted in a slightly faster liver accumulation which was not antigen-dependent.

VI. Conclusions

(1) There have now been developed a sufficient number and diversity of procedures for targeting of liposomes with antibodies or antibody fragments such that this aspect of the targeted delivery problem is no longer a major concern. More- over, most of these methods are relatively nonperturbing with respect to encapsulated drugs.

(2) Delivery of anticancer drugs by immunoliposomes to cells in vitro has been shown by several groups to be advantageous in many aspects over simple drug encapsulation or administration of free drug. Delivery of molecules such as toxins and plasmids is also augmented by immunoliposome encapsulation.

(3) Although binding to the target antigen is a prerequisite, it alone is not sufficient for immunoliposome delivery into the cell. The efficiency of drug delivery by antibody-targeted liposomes depends upon the method of antibody targeting, surface density and internalization of the target antigen, and the physical properties of the liposome, including size, charge, and phase behavior.

(4) Special function or controlled release immunoliposomes such as those which are pH-sensitive or temperature-sensitive are now available which can mediate de-

378 S. W RI GHT AND L. H U A N G

livery either at intracellular or pericellular sites. The mechanism of action of these phase-sensitive, polymorphic liposomes is just beginning to be understood.

(5) Although antibody targeting can apparently enhance delivery in vivo, the same problems of compartmentalization and RES uptake that exist for non-targeting liposome carriers also remain for the targeted carriers. However, the stability of immunoliposomes in serum can be controlled by using appropriate liposome constituents.

VII. Future perspectives

Perhaps the most significant barrier yet to be surmounted in the field of liposomal drug delivery is the rapid scavenging by components of the RES. This problem applies to non-targeted as well as targeted liposomes. For antibody-conjugated liposomes, RES capture suggests that those liposomes which are available initially to interact with their target and fail to do so on their first pass will probably be engulfed before encountering the target for a second time. Although some progress has been achieved in reducing RES uptake through measures such as liposome modification or RES saturation by predosing, liposome capture by RES components remains a formidable problem. The recent success in RES avoidance by utilizing a combination of membrane stabilizers and/or size modifications suggests the possibility of overcoming this barrier.

A second major obstacle faced by liposome-based drug therapy is anatomical compartmentalization. Even though liposomes can be administered through a variety of routes the fact that these subcellular sized structures cannot permeate ep- ithelial and/or endothelial boundaries severely limits their utility particularly for disseminated solid tumors. However, some macromolecular ligands are able to be taken up, delivered to the other side of the cell and released by a process of transcytosis. The intriguing idea that liposomes may be capable of crossing some of these boundaries by transcytosis must be vigorously pursued.

The development of novel controlled release liposomes has given new impetus into the area of drug delivery using targeted liposomes. Although using immunoliposomes which are sensitive to temperature or pH appears to be promising for drug delivery, they are not idealized carriers. For pH-sensitive immunoliposomes in particular much more must be done to elucidate the precise mechanism of intracellular delivery so that the system may be optimized as a delivery vehicle. The ability of antibody-targeted pH-sensitive liposomes to efficiently deliver macromolecules such as plasmids (discussed below) in addition to smaller drugs indicates that these liposomes can be widely utilized as a drug delivery vehicle. These studies open up the possibility of using DNA as a new class of drug. For example, genes coding for toxic protein products or antisense RNA would be cytotoxic when delivered to and expressed by the target cell. Genes coding for secreted products such as peptide hormones or proteins used as vaccines could be used.

Perhaps the brightest future for immunoliposome delivery lies in its unique ability to deliver DNA to specific cells in vivo. Use of DNA as a chemotherapeutic drug offers at least the following three advantages over the use of the conventional


drugs such as the small molecular-weight drugs, peptides and proteins: (a) the ac- t ion of gene is highly amplified th rough transcript ion and translation. Theoret i - cally a few copies of a gene are sufficient to p roduce significant biological modifications to a given cell; (b) the expression of a gene can be tightly regulated by choosing an appropr ia te p romote r and enhancers ; (c) if the D N A leaks out of the l iposome carrier it would be rapidly des t royed by the serum nucleases, and thus produce minimal toxicity to the normal cells. Target-specific immunoliposomes have now offered the oppor tun i ty for this novel class of chemotherapeut ic drug.

Much progress has been made iff the past decade toward an unders tanding of what role l iposomes may play as a drug delivery device. It is now clear that liposomes will not be the universal magic bullet as was earlier thought . More practical applications of l iposomes are now being pursued. Many l iposome-based drug for- mulat ions are soon scheduled for clinical trials backed by a newly spawned com- petit ive industry.

Acknowledgements

W o r k done in this labora tory is suppor ted by a contract f rom L i p o G e n , Inc. , N I H grants No. CA24553 and AI25834. L .H. was a recipient of a Research Career D e v e l o p m e n t A w a r d f rom N I H (CA00718). S.W. is a recipient of an N I H fellow- ship 1 F32 CA08442-01. We thank Carolyn Drake for excellent technical assist- ance.

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Antibody-directed liposomes as drug-delivery vehicles

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