Therapeutic opportunities for targeted liposomal drug delivery

advanced

drug delivery reviews ELSEVIER

Advanced

Therapeutic

Drug

Delivery

opportunities

Reviews

21 (1996)

for targeted

117-133

liposomal drug delivery

Theresa M. Allen*, Elaine H. Moase Department of Pharmacology,

9-31 Medical Sciences Building. University of Alberta, Edmonton, Received

21 June

Alberta 7’6G ZH7, Canada

1996

Abstract One way to increase the therapeutic index of drugs such as anticancer drugs, which have low therapeutic indices would be by specifically targeting the drugs to the diseased cells. This can be accomplished by associating the drugs with liposomes and coupling a targeting antibody or ligand to the liposome surface. A variety of coupling methods can be used to attach antibodies or ligands to the liposomes, and, to date, the best targeting results have been obtained when the targeting moiety is attached at the terminus of a hydrophillic polymer such as polyethylene glycol (PEG). Targeted liposomes have been demonstrated to have specific binding and increased cytotoxicity to cells in vitro compared to non-targeted liposomes. The best therapeutic results in vivo have been obtained to date when the liposomes are targeted to cells easily accessible within the vasculature, or to micrometastatic cells. Treatment of more advanced solid turnouts with targeted liposomes presents a challenge to overcome the ‘binding site barrier’ at the tumour surface. Evidence is accumulating that targeting to internalizing receptors is more successful than targeting to non-internalizing receptors. Keywords: Long-circulating Ligand-mediated targeting

liposomes;

Targeted

drug delivery; Anticancer

drugs; Antibody-mediated

targeting;

1. Introduction.. ........................................................................................................................................................................ 118 2. Some developments necessary for the targeting of liposomes.. ............................................................................................. 118 2.1. Liposome size ................................................................................................................................................................ 119 2.2. Drug loading, leakage and storage.. ............................................................................................................................... 119 2.3. Long circulation half-life.. .............................................................................................................................................. 119 2.4. Efficient coupling techniques for Ab or ligands.. ........................................................................................................... 119 3. Methods for the attachment of Ab to sterically stabilized liposomes.. .................................................................................. 120 3.1. Non-covalent, biotin-avidin method .............................................................................................................................. 120 3.2. Covalent attachment of mAb at the liposome surface ................................................................................................... 121 3.3. Covalent attachment of Ab at the PEG terminus.. ........................................................................................................ 121 3.4. Other approaches .......................................................................................................................................................... 121 4. In vitro studies on ligand- or Ab-targeted liposomes ............................................................................................................ 122 4. I. Specific binding.. ............................................................................................................................................................ 122 4.2. Cytotoxicity ................................................................................................................................................................... 123 4.3. Internalization of targeted liposomes.. ........................................................................................................................... 12s S. In vivo studies on ligand- and Ab-targeted liposomes ........................................................................................................... 126 5.1. Targeting to solid turnours.. ........................................................................................................................................... 127

“Corresponding

author.

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0169-409X/96/$32.00 @ PIf SOl69-409X(96)00402-4

+ 403 4925 710: fax: 1996 Elsevier

Science

+ 403 493 8078. B.V. All rights

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5.2. Targeting within the vasculature or peritoneal CHVI~V.................................................................................. ................. 6. What are the opportunities for the USCof targctcd liposomes in viva? ................................................................................. Acknowledgments .......................................................................................... ......................................................................... References .................................................................................................................................................................................

1. Introduction Ideally, a drug destined for use in the clinic would have a high therapeutic index, which is the ratio of the drug’s efficacy (therapeutic effect) over the drug’s toxicity (side effects). In other words. pharmacologists try to develop drugs which have a good therapeutic effect with very few or no side effects. The higher the therapeutic index. the larger the margin of safety for the drug. A corollary of this for the use of chemotherapeutic drugs is the principle of selective toxicity which states that a drug should be more toxic to diseased cells than to normal cells. Many drugs, particularly chemotherapeutic drugs, have narrow therapeutic windows (low therapeutic indices) and their clinical use is compromised by dose-limiting toxic side effects. Examples of this include cardiac toxicity from the anticancer drug doxorubicin and kidney toxicity from the antifungal drug amphotericin B. If drugs having low therapeutic indices could be delivered in higher concentrations to their target cells (thereby increasing efficacy) and away from normal cells (thereby reducing toxicity), this would provide us with a means of increasing the therapeutic indices of the drugs and achieving more effective therapy. and possibly greater economic benefit. Liposomal drug delivery is one of the means being explored to achieve increased therapeutic indices for existing drugs and for some new classes of drugs whose move into the clinic has been hampered by. for example, rapid breakdown or lack of solubility. Some of the ways in which liposomes might improve the therapeutic indicts of drugs include the following: 1. Liposomes can be used as a non-toxic, biodegradable system to solubilize drugs which have low aqueous solubility. e.g. taxol [l-4] or lipophilic derivatives of cisplatin 15.61. 2. By entrapping rapidly degraded drugs in lipo-

3.

4.

5.

6.

127 12x 129 I 29

somes. the drugs can be protected from breakdown and their mean residence time in the body can be increased, e.g. cytosine arabinoside [7-lo]. By altering the biodistribution of their associated drugs. liposomes can reduce the accumulation of the drugs in sensitive tissues and decrease drug toxicity, e.g. reduction in cardiac toxicity with liposomal doxorubicin [ll1.51. Liposomes can act as sustained release systems for associated drugs, increasing the area under the time concentration curve (AUC) which is one measure of drug bioavailability, e.g. vincristine, cytosine arabinoside, doxorubicin, daunorubicin and many others [10,13.16-211. The delivery of drug to selected tissues can be improved by entrapping the drug in liposomes, taking advantage of physiologic factors which improve the localization of liposomes to the target tissues relative to free drug, e.g. increased capillary permeability which allows passive targeting of liposomes to solid tumours or areas of infection 122-281 Site-specific delivery of drugs can be improved by entrapping the drugs in liposomes which are then targeted to specific cells through the presence of antibodies or ligands on the liposome surface which bind to specific epitopes on the target cell surface.

A detailed discussion of the last point the subject of this review.

2. Some developments targeting of liposomes

necessary

will be

for the

Antibody (Ab)-mediated or ligand-mediated targeting of liposomes has been tried on a number of occasions in the past and, although targeting in vitro has been generally successful

T.M. Allen. E.H. Moase I Advanced

[29-351, targeting in vivo has been either disappointing or unsuccessful [29,31,36-381. A number of developments in liposome technology were instrumental in helping to make in vivo targeting of liposomes a reality.

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119

can be manipulated to result in a broad range of leakage rates of drugs from the liposomes which allows the liposomes to be tailored to the specific applications being pursued [19]. These technologies have been important preludes to the development of targeted liposomes.

2.1. Liposome size The ability of liposomes to extravasate and penetrate into diseased tissues is directly correlated with their size. Large liposomes are rapidly removed from circulation into tissues of macrophage origin (mononuclear phagocyte system, MPS) and do not achieve significant levels in other tissues of the body (as reviewed in [20]). Thus. they would be difficult, if not impossible, to target in vivo to cells other than the most accessible of the MPS cells, e.g. Kupffer cells, which are a natural target for liposomes. The development of techniques for making liposome populations of homogeneous small size ( I 100 nm) [39,40] has resulted in an increased ability of liposomes to penetrate into either normal tissues having sinusoidal or fenestrated epithelium or into diseased tissues having altered capillary permeability as a result of the disease process [28,41-431. This has in turn improved the chances for achieving liposome targeting in vivo to non-MPS tissues. 2.2. Drug loading, leukage and storage Even if we had been able to achieve liposome targeting in vivo, there would have been little point if the drug which we wanted to deliver either did not remain associated with the liposomes until they reached their target, or if the efficiency of drug loading was so low as to make the preparation prohibitively expensive. Some significant advances have been made in the technology of drug loading into liposomes allowing highly efficient loading and excellent drug stability. For example, many drugs are weak acids or weak bases, and these compounds can be remote-loaded into liposomes with an efficiency of close to 100% by establishing a pH [44] or chemical gradient [45] across the liposome membrane. The liposome composition, the internal pH [46] or presence of counter-ions [47]

2.3. Long circulation half-life If liposomes are to localize in significant quantities in diseased tissues such as solid tumours by means of ligand- or Ab-mediated targeting, small homogeneous size, while necessary, is not sufficient. When antibodies are attached at the surface of ‘classical’ liposomes, this dramatically increases their rate of removal from the circulation into the MPS, making it unlikely that they will be able to find and bind to their target [29.36]. A means of formulating Ab-liposomes which were long-circulating was necessary. The development of sterically stabilized liposomes (Sliposomes), in which hydrophillic polymers such as polyethylene glycol (PEG) were attached at the liposome surface [22,48-531. has provided a solution for this problem. Liposomes containing lipid derivatives of hydrophillic polymers. such as PEG-distearoylphosphatidylethanolamine (PEGDSPE), have reduced uptake into the MPS, resulting in long circulation half-lives relative to liposomes lacking PEG (as reviewed in [20]), and demonstrate dose-independent pharmacokinetics [20,22,54]).

2.4. Efficient coupling techniques ,for Ah or ligan ds

Once long-circulating liposomes had been developed it was necessary to examine methods for the efficient attachment of ligands or Ab to the liposome surface. The presence of PEG at the liposome surface could interfere with this process in either of two ways and evidence for both exists in the literature. The PEG could provide a steric hinderance to the access of Ab or ligand to the liposome surface and decrease the coupling efficiency [32,55], or, once the Ab was attached at the liposome surface, the PEG could hinder the

I20

T.M.

Alien,

E.H.

Mouse

I Advunced

Drug

21 (19%)

I17-1.Z.i

biotin-avidin method

The biotin-avidin is a non-covalent coupling method for attaching ligands or Ab at the liposome surface (Fig. 1A). Biotinylated phospholipid is included in the liposome during formation and avidin, with its four high affinity binding sites, is used to crosslink biotinylated Ab to biotinylated lipid [63,68]. This method has the advantage of simplicity and convenience, and the presence of PEG prevents liposome aggregation which has been one of the problems of this method when used with ‘classical’ liposomes [38,63,68,69]. H owever, the biotin-avidin method resulted in low Ab densities at the S-liposome surface and a low coupling efficiency [55]. Furthermore. there is some question about whether the avidin protein will be immunogenic in humans. In a variation on this method, a streptavidin lipid derivative is included in the liposomes followed by addition of biotinylated Ab

of Ab to

sterically stabilized liposomes

Prior to the development of S-liposomes, a number of methods for attaching Ab to liposomes had been described [59-671. We have examined some of these methods for their suitability for attachment of Ab to S-liposomes. In addition, we and others have developed new methods for the attachment of Ab or ligands to the terminus of the PEG in S-liposomes. A discussion of some of the highlights of the various methods follows. Fig. I (reproduced with permission from ref. [55]) is a schematic diagram of some of the coupling methods.

A.

Reviews

3.1. Non-covalent,

recognition of the Ab by its target [38,56-581. A discussion of the various methods for attaching ligands or Ab at the surface of S-liposomes, with their advantages and disadvantages, is provided below.

3. Methods for the attachment

Drliverv

1681.

Biotin-DOPE

Ah-Biotin

+

Aviclin

Biotinylated antibody

AI,-.911

+

j-

_+

Biotin-Liposomes Biotinylnfed

lQ+TzJ+

Ab-Biotin-Avidin-Biotin-Liposomes

liposomcs

N-Liposomes

k

t

k

0

RCdWXd PDP-antibody

C.

MIWI’E

lipowmcs

Imm~moliposomes

I’DP-DOPE

Ab-N 0

31

MPB-antibody

x

S--S~CHz)z-C-NH-DOPE-Liposomes

__f S-Liposomes

PDP-I’E

Fig. I. Schematic diagram of several coupling methods Hansen et al., B&him. Biophys. Acta. 1239. 133-144.

liposomcs for

attaching

1995).

lmmtmoliposomcs

ligands

or Ah to liposomes.

(Reprinted

with permission

from

T.M. Allen. E.H. Moase I Advanced

3.2. Covalent attachment of mAb at the liposome surface

A covalent thioether bond can be efficiently formed between thiol and maleimide groups at the liposome surface in this method, described several years ago by Martin and Papahadjopoulos [61]. There are two variations of this method (Fig. 1B and C). In one variation, a maleimide-derivatized phospholipid (N-(4’(-4”maleimidophenyl)butyroyl) dioleoylphosphatidylethanolamine, MPB-DOPE or n-(maleimidomethyl)cyclohexane - 1 - carboxylate - DOPE, MCC-DOPE) is included in the liposomes during formation and reacted with a thiolated Ab, formed from reacting Ab with N-succinimidyl-3(2-pyridyldithio)proprionate (SPDP) and subsequent reduction (Fig. 1B). In the other variation, the reaction is reversed with a thio-derivatized phospholipid PDP-DOPE (N-(3’-(pyridyldithio)propionoyl)-DOPE) included in the liposomes during formation and subsequently reacted with a maleimide-derivatized Ab, formed from incubation of Ab with SMPB (H-succinimidyl-4-( p-maleimidophenyl)butyrate) (Fig. 1C). In the absence of PEG in the liposomes, both of these variation result in high coupling efficiencies and high Ab densities at the liposomes surface [55]. However, in the presence of PEG, the efficiencies of the two variations are very different. When the small thiol group is at the liposome surface (Fig. lC), the PEG groups appear to sterically hinder access of the maleimide-Ab to the thiol group and coupling efficiencies are low [55]. High coupling efficiencies are retained when the MPB group is at the liposome surface (Fig. 1B) possibly because this bulky hydrophobic group causes spreading of the PEG chains allowing access of the thiolated Ab to the liposome surface [55]. A further distinction between these two variations is found in the ease of application of remote-loading methods for drugs such as doxorubicin into the liposomes. With the PDP-DOPE method (Fig. lC), doxorubicin can be remote-loaded efficiently, while for the MPBDOPE method (Fig. lB), remote-loading is variable and inefficient, likely due again to the presence of the MPB group at the liposome surface [55]. When the more hydrophillic MCC-

Drug Deliver-v Reviews 21 (1996)

DOPE is substituted loading improves.

117-133

for MPB-DOPE

121

remote-

3.3. Covalent attachment qf Ab at the PEG terminus

It has recently been demonstrated that covalent attachment of Ab at the PEG terminus is close to ideal, giving not only high coupling efficiencies and high Ab densities, but also good drug remote-loading characteristics and target recognition [55,70-741. As above, there are two variations on this method, one in which PDPPEG-DSPE is included in the liposomes and MPB-Ab is coupled to the liposomes [72] (Fig. 1E) and another variation in which the chemistry is reversed, with maleimide-derivatized PEG in the liposomes and thiolated Ab [74]. A third method for coupling Ab to the PEG terminus involves including hydrazide-PEGDSPE in the liposomes and coupling via a hydrazone bond to Ab having an oxidized carbohydrate group (via periodate) in the Fc region of the Ab [55,75]. This method is one of the simplest methods to use, and results in good drug remote-loading of the liposomes. It results in intermediate coupling efficiency and intermediate Ab densities at the liposome surface with good target recognition [55]. However, the sensitivity of some Ab to periodate treatment makes it unsuitable for all Ab [55]. Coupling at the PEG terminus has also been used by Storm et al. for attachment of proteins to the liposome surface, using DSPE-PEG77COOH incorporated into the lipid bilayer, which is subsequently activated with l-ethyl-3-3-(dimethlyaminopropyl)carbodiimide hydrochloride (EDCI) and then coupled with Glu-plasminogen [76]. In these experiments, both targeting of the liposomes to fibrin-coated surfaces and long circulation times in vivo were demonstrated. 3.4. Other approaches In yet another approach to incorporating Ab into S-liposomes, Huang and colleagues have covalently attached Ab to N-glutaryl-phosphatidylethanolamine (Ab-NGPE) in the presence of octylglucoside detergent using N-hydroxy-

122

T.M. Allen. E.H. Moose I Advunced Drug Delivrrv Kevirws 21 (1996) 117- I3.i

sulfosuccinimide as a carboxyl activation reagent. The Ab-NGPE was then incorporated into Sliposomes by detergent dialysis or reverse-phase evaporation [65.77]. These liposomes appear to have moderate Ab densities and good target recognition in the presence of GM1 or PEG of molecular weight of 2000 Da or less, but not higher molecular weight PEG [56,58,78]. However, since some unknown fraction of the AbNGPE is oriented towards the liposome interior with this method, the Ab density at the liposome surface cannot be accurately calculated. Suzuki et al. [79] have used a ‘post-coating’ method to generated PEG immunoliposomes in which Ab was first attached to liposomes lacking PEG and PEG-succinylcyteine was subsequently conjugated to the liposomes using a maleimido linker.

4. In vitro studies on ligand- or Ab-targeted liposomes Before evaluation of the therapeutic efficacy of targeted liposomes in vivo in a model system of cancer, one should first establish in vitro, using the same cell culture lines and ligands or Abs as the in vivo model. various parameters for the immunoliposomes such as specific binding. cytotoxicity of immunoliposome-entrapped anticancer drugs, and internalization (or not) of the immunoliposomes. 4.1. Spec@c binding Binding of targeted liposomes to cells is dependent upon the affinity and avidity of the targeting agent coupled to the surface of the liposomes, the surface density of the targeting agent and the method of coupling. As seen above, the chemical modification of the targeting agent required for its attachment to the liposome surface can, in some cases, result in a reduction or elimination of target binding, and in coupling methods where the ligand is attached to the liposome surface bulky hydrophillic groups can mask antigen recognition [38,55-58,801. Little or no work has been done to answer the question of whether one isotype of Ab will give higher target

binding than another (e.g. IgG, vs. IgG, vs. IgM) or whether an Ab fragment (e.g. Fab) will be better than a whole Ab. High Ab binding affinity for its target may not be desirable in all circumstances as Ab penetrability into targets such as solid tumours may be inversely related to its binding affinity because of the ‘binding site barrier’ which simply states that Abs will bind to the first target cells with which they come into contact. This will prevent the Ab (or immunoliposomes) from penetrating into the tumour interior [81]. Ab fragments often lose binding avidity with their loss of multivalent binding, and their attachment to liposomes can in theory restore multivalent binding [82], but again more work need to be done exploring these issues. As discussed above, a number of coupling methods have been developed for attaching Ab or ligands to the liposome surface or the PEG terminus of long-circulating liposomes. The ability of these liposomes to specifically bind to their target cells has been examined by a number of investigators. In our laboratory, we have examined the in vitro binding of immunoliposomes (or ligand-liposomes) to a number of different cancer cells lines including the mouse squamous cell carcinoma cell line, KLN 205, with the mAb 174 H.64 (Biomira. Inc.) attached to long-circulating immunoliposomes [32.55], the human ovarian carcinoma cell line, CaOV.3, with the mAb B43.13Rl (Biomira, Inc.) [38], the human B lymphoma, Namalwa, cell line with mAb antiCD19 [83]. and the human colon cancer. HCT1.5, with mAb 1708.82 [55]. In each of these cell lines, we could demonstrate specific binding of immunoliposomes, using either radiolabelled liposomes or flow cytometry (FACS), which could be competed out in the presence of excess free specific Ab. The increase in binding of the immunoliposomes over control liposomes (no Ab or non-specific, isotype-matched Ab) could be as high as lo-fold, but usually was in the range of 2-4-fold higher [32,55,83]. Binding of targeted long-circulating liposomes to cells had been demonstrated in a number of other models. Mori et al. [84] demonstrated a 2-fold increase in binding to normal mouse lung cells of GMI-immunoliposomes conjugated to an antibody (34A) against the lung endothelial

T.M. Allen, E.H. Moase I Advanced

thrombomodulin. The anticoagulant protein HER-2 overexpressing breast cancer cell lines, SK-BR3 or BT-474, were shown by flow cytometry to bind anti-p185HER’ PEG-immunoliposomes with comparable binding constants for the immunoliposomes as to those of free Fab’ [82]. In a ligand-targeted application, Lee and Low demonstrated that specific binding and uptake of folate-PEG-liposomes (folate conjugated to the PEG terminus) by a folate-receptor expressing human nasopharyngeal epidermal carcinoma (KB) or human cervical carcinoma cells (HeLa) [35]. In other recent experiments, specific binding of immunoliposomes (no PEG or GMl) targeted with anti-ICAMcould be demonstrated for human bronchial epithelial cells (BEA2B) and human umbilical vein endothelial cells (HUVEC) [85], and to human ovarian cancer cells (OVCAR-3) with anti-ovarian carcinoma Fab’ immunoliposomes [86]. Several laboratories have examined the effect of Ab density on the ability of liposomes to specific bind to their target cells. As might be expected, specific binding of liposomes increased with increasing Ab density, but the relationship was not a strong one for KLN-205 mouse squamous lung carcinoma cells in vitro, with large increases in Ab density resulting in only modest increases in immunoliposome binding to target cells above a minimum Ab density [55]. Similarly, for PEG-immunoliposomes conjugated at the PEG terminus to Ab 34A, the in vivo binding of the liposomes to lung was strongly dependent on Ab density below 30 Ab molecules/liposome, but no further increase in binding was observed at higher Ab densities [87]. 4.2. Cytotoxicity After specific binding of immunoliposomes to target cells can be established in vitro, the next question is whether or not this specific binding will lead to increases in cytotoxicity compared to non-targeted liposomes and to free drug. However, it should be stressed that the relevance of these results to in vivo therapeutic efficacy should be interpreted carefully. In a cell culture system, there is no opportunity for free drug to redistribute away from the cells, unlike the rapid

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in vivo redistribution (i.e. large volume of distribution) of some free drugs, e.g. doxorubicin and daunorubicin. Therefore, the cytotoxic effect for free drugs will tend to be overestimated in the cell culture experiments relative to the liposomal drugs which must be released from the liposome in order to have their effect. In vivo, on the other hand, the distribution of the liposomes tends to be confined to the central compartment and to tissues having increased permeability, e.g. solid tumours undergoing angiogenesis, and they have a small volume of distribution relative to the free drug, enhancing their in vivo efficacy relative to their in vitro efficacy. One way in which the cell culture experiments with targeted liposomes can be made to more closely mimic the in vivo situation is to wash away free drug and non-adhered liposomes after allowing a few minutes for the immunoliposomes to bind to the target cells. The washing step very roughly mimics the drug redistribution step in vivo and arguably allows a truer approximation of the comparative cytotoxicity of targeted liposomes versus free drug [32]. Two mechanisms by which cytotoxicity of targeted liposomes could occur against cells in culture may be postulated. Localized high concentrations of extracelluar drug may be produced by binding of drug-loaded targeted liposomes to the target cell surface and subsequent leakage of the drug contents with the drug entering the cell as free drug (Fig. 2). Alternatively. high intracellular drug levels might result from internalization of the bound liposomes, if they were coupled to an Ab against an internalizing epitope (Fig. 2). Cytotoxicity produced by the latter mechanism would depend on the entrapped or associated liposomal drug being able to survive the lysosomal and endosomal environment and be released from there into the cell cytoplasm from whence it could access the nucleus. Increased cytotoxicity of targeted liposomal drug, generally doxorubicin, over untargeted drug has been demonstrated in several systems. Compared to free drug, the non-targeted liposomal drug was always less active (but see comments above regarding interpretation of in vitro results) and the targeted drug could be more active, equally active or less active than the

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T.M. Allen, E.H. Mouse I Advanced

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A, Non-internplizing epitope, MDR . . cancer cell

B, Internalizing epitope, MDR cancer cell

endo

‘2

38 Fig. 2. Schematic diagram of two mechanisms cells expressing P-glycoprotein.

Non-mternalimg epitope

for cytotoxicity

free drug depending on the model system. The IC,,, of doxorubicin-loaded immunoliposomes, after l-h incubation, was nearly an order of magnitude lower (5 PM) than for non-targeted doxorubicin-loaded liposomes (43 PM) or free drug (52 PM) in the KLN 205 squamous cell carcinoma model [32]. Likewise, when doxorubiwere tin-loaded anti-p185 HER2 immunoliposomes

of Ab-targeted

liposomes

Dl

P-glycoprotein

against

multidrug

resistant

(MDR)

target

incubated with p185H”R2-overexpressing tumour cells in vitro [82], for a l-h incubation, cytotoxicity of the immunoliposomes was similar to that for free doxorubicin, but cytotoxicity for immunoliposomes coupled to a non-specific Fab’ was lo-30-fold less. For doxorubicin-loaded folate-targeted liposomes (2-h incubation), the IC,,, were 0.31 and 0.41 PM for KB and HeLa

T.M. Allen, E.H. Moase

I Advanced Drug Delivery Reviews 21 (1996) 117-133

cells, respectively, which is similar to that obtained with the free drug, but for non-targeted liposomes the values were again several fold higher (26.7 and 25.3 ,uM, respectively) [35]. In both the folate-targeted and anti-p185HER2-target liposome systems, internalization of the immunoliposomes has been demonstrated, which may explain the excellent cytotoxicity observed these models (for a discussion of the importance of internalization, see below). An interesting system for delivery of drug very specifically to the target cell is described in the work of Vingerhoeds et al. [88]. In this study, immunoliposomes bound to non-internalizing receptors were targeted to the NIH:OVCAR-3 cell line. These immunoliposomes were also surface-conjugated with the enzyme pglucuronidase. With subsequent addition of the prodrug epirubicin-glucuronide, only the prodrug in the vicinity of the targeted cell surface with bound liposomes was converted to epirubicin, the active form of the drug. An adequate surface concentration of the enzyme could be obtained such that cytotoxicity approached that of the cells incubated with free enzyme and epirubicinglucuronide [88]. Another recent in vitro model for liposome targeting is described by Khaw et. al. [89]. Cardiocytes, made experimentally hypoxic by purging with nitrogen for 24 h, were treated with immunoliposomes targeted with Fab’ fragments against intracellular cytoskeletal myosin, which would be exposed as a result of the cell membrane damage caused by hypoxia. Binding of these immunoliposomes was shown to increase cell viability over untreated cells, after a recovery period, presumably by blocking the sites where cell membrane integrity had been compromised, so that the cells could recover. This ‘targeted cell membrane sealing’ could possibly be used in conjunction with thrombolytic therapy for the treatment of myocardial infarction [89]. Targeted liposomes can deliver therapeutic molecules other than drugs. Yanagie et al. [90] described a method for boron neutron-capture therapy by delivery of a “B-compound in the internal space of anti-CEA immunoliposomes to AsPC-1 cells bearing the CEA antigen. Cells incubated with these immunoliposomes were

125

irradiated with thermal neutrons, and cell proliferation ability was suppressed, as evaluated by 3H TdR incorporation. However, this model is limited to multilamellar vesicles, rather than unilamellar liposomes, because of stability problems, so its application in vivo is limited at this moment [90]. Another interesting model has been proposed for potential treatment of oxidant injury in the premature infant lung, as a result of oxygen supplementation. Delivery of the enzyme superoxide dismutase to fetal rat lung distal epithelial (FRLE) cells was achieved by incorporation of surfactant protein-A into the membrane of pH-sensitive liposomes [91]. Cells incubated with the targeted enzyme-liposome formulations showed a 5.1-fold increase in the level of superoxide dismutase, implying that the liposomes were internalized into an acidic compartment inside the cell. In this study, delivery of the enzyme to the interior of the cell, rather than cytotoxicity, was the desired effect. 4.3. Internalization of targeted liposomes Binding of immunoliposomes to cells does not ensure delivery of the liposome contents to the interior of the cell. Diffusion of the liposome contents released at the surface of the cell from bound liposomes may result in delivery of some of the drug (contents) into cells by the same mechanism as uptake of the free drug (Fig. 2). Although the argument has been made that one can achieve locally high concentrations of drug at the cell surface by this mechanism which will result in increased drug concentrations in the cell interior relative to free drug, it has still to be convincingly demonstrated that this is so. Indeed, one could also argue that in turbulent environments in vivo, e.g. the blood stream or peritoneal cavity, the rate of diffusion and redistribution of the released drug away from the cell surface will far exceed the rate at which the drug released from bound liposomes will enter the cell. One way to ensure delivery of a high proportion of immunoliposomal contents to the cell interior is to target to an internalizing epitope which will trigger uptake of both the immunoliposomes and their contents into lysosomes and

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endosomes, where the drug contents can be released inside the cell subsequent to liposomal degradation (Fig. 2). This mechanism can be highly effective when the drug is one which escapes degradation by acid pH and lysosomal enzymes [35,82]. There are a number of ways of observing internalization. The internal space of the immunoliposome may be filled with a pH sensitive dye such as 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS) so that when the liposomes are incorporated into a lower pH internal compartment, such as an endosome, the internal pH can be determined by differences in fluorescence emission at various excitation wavelengths [92]. This method was developed as a simple assay for internalization of liposomes by macrophages. but has been used in several laboratories to confirm internalization in targeted liposome systems. HPTS-loaded apolipoprotein A-targeted liposomes were seen to become more acid over time as they were incubated with CV-1 and 5774 cells [93]. In our laboratory, asialofetuin-targeted liposomes were observed internalizing into the HBVtransfected hepatocyte cell line 2.2.15 (Ho and Allen, unpublished results). Similarly, we were able to observe internalization of anti-CD19 PEG-immunoliposomes by human B-lymphoma (Namalwa) cells [94]. The internal pH of the liposomes dropped from pH 7.4 to approximately 6.0-6.5 in these systems after incubation at 37°C. As an alternative to fluorometry to evaluate internalization, confocal microscopy can be used to view the fate of immunoliposomes loaded with a florescent dye or llorescent drug such as doxorubicin or daunorubicin after their incubation with the target cells. Folate-targeted PEG-immunoliposomes loaded with doxorubicin and then incubated with the KB cell line, a human nasopharyngeal epidermal carcinoma cell line. showed doxorubicin fluorescence present in the endocytic vesicles, nucleus and the cytoplasm when viewed under the confocal microscope [35]. In our laboratory, Namalwa cells were incubated with doxorubicin-loaded anti-CD19-PEG-immunoliposomes at 37°C. then surface labelled with free antibody and subsequently with a FITC-antibody conjugate at 4°C to visualize both the green cell surface and the red doxorubi-

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tin using the appropriate laser excitation wavelengths. Doxorubicin was found throughout the cytoplasm when doxorubicin-loaded targeted immunoliposomes were incubated with the cells, but non-targeted doxorubicin-loaded liposomes were limited to the cell surface (Lopes de Menezes and Allen, unpublished results). Confocal microscopy has also been used to visualize the internalization of rhodamine-PE labelled antip185H”R2 immunoliposomes into p18.5HER2-overexpressing tumour cells. In these experiments, unlike the previous two experiments where the targeting moiety was coupled to the PEG terminus, the Fab’ was coupled to the liposome surface and as the concentration of PEG in the liposomes increased, it was shown to interfere with liposome internalization [82]. In recent experiments. this group has coupled the Fab’ fragment to the PEG terminus and has shown no interference of PEG with internalization using this technique [74]. From the above examples, and from our personal experience, evidence is beginning to accumulate that ligands or antibodies against internalizing receptors may be the best choice for optimum targeted drug delivery. A recent report from our laboratory has demonstrated that immunoliposomal doxorubicin, targeted to the internalizing CD4 receptor on multi-drug resistant (MDR) T cells, was capable of causing a reversal of MDR in the CEM-MDR cell line [95]. Presumably, the mechanism here involves bypassing the plasma membrane and delivering the drug into a compartment where it is not readily accessible to the P-glycoprotein pump (Fig. 2).

5. In vivo studies on ligand- and Ab-targeted liposomes

A number of studies have been published recently describing in vivo applications of targeted liposomal therapy for the treatment of various experimentally-induced models of disease. Such models have had mixed success in the laboratory. For the purpose of this review, we will divide this work into liposomal targeting to solid tumours, or within the vasculature including haematological and ascitic tumours.

T.M. Allen, E.H. Moase I Advanced

5.1. Targeting to solid tumours In vivo delivery of targeted liposomes is dependent upon access of injected liposomes to all (or most) of the cells being targeted. In the case of advanced (multicellular) solid tumours, access of the targeted liposomes to the tumour cells may be limited by the degree of vascularization of the tumour and the extent of leakiness of the blood vessels nourishing the tumour. Targeted liposomes may have to pass several layers of vascular endothelium and basement membrane in order to reach the tumour cells, and in large tumours liposomes may have to pass many layers of tumour cells to reach cells deep within the Experimentally-induced peritoneal tumour. tumours, which may be more easily reached by IP injection of targeted liposomes, may be more accessible to IP injection of liposomal therapies, but treatment will again be limited in effectiveness by the size of the tumours. Ahmad and Allen [94,96] have shown effective therapeutics with doxorubicin-loaded liposomes targeted with the mAb 174H.64 against the mouse squamous cell carcinoma cell line KLN 205 in vivo. In this model, KLN 205 cells were seeded into the mouse lung by IV injection and, 3 days later, when the tumour cells had localized in the lung, the mice were treated IV with a single dose of doxorubicin-loaded mAb 174H.64targeted liposomes. This model is considered a pseudometastatic model, in that the tumour cells are injected rather than seeded from an advanced in vivo tumour and since the cells are treated at a very early stage, just as the cells are seeding into the lung, is considered to be a model for treatment of early stage metastases. Mice receiving untargeted liposomal-doxorubicin or free drug had only slight increases in mean survival times compared to controls, but the mAb-targeted liposomal doxorubicin had significantly greater mean survival times, with 40-80% long term survivors [96]. However, when treatment was delayed until 1-3 weeks after tumour injection, treatment with immunoliposomal doxorubicin using a variety of treatment schedules was not superior to non-targeted liposomes or to free drug [94]. We postulate that the immunoliposomes, because they bind to cells at

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the tumour surface, fail to gain access to tumour cells deeper in the tumour in more advanced tumours, at the multicellular stage, resulting in treatment failure. We have also described the treatment of Caov.3 xenograft tumours in nude mice with IV doxorubicin-loaded mAb B43.13-targeted liposomes [94]. These tumours are at a more advanced stage at the time of treatment and, in this model, untargeted doxorubicin-loaded liposomes were more effective in reducing the rate of xenograft growth than were the doxorubicinloaded antibody-targeted liposomes. This may again reflect a lack of access of the immunoliposomal doxorubicin to the majority of tumours cells in the tumour interior relative to the nontargeted liposomes which should have less problem passively penetrating into solids tumours undergoing angiogenesis. Many researchers have reported on the ability of non-targeted longcirculating liposomes to passively localize in solid tumours having increased capillary permeability [22,24,27]. Another possibility which may contribute to the observations is a modest decrease in circulation time of targeted versus untargeted liposomes. A recent report describes the treatment of ~185~~~’ -overexpressing breast tumour xenografts in nude mice with doxorubicin-loaded antiHER2-liposomes. Increased antitumour activity of targeted compared to non-targeted liposomes was reported but few details are available from this abstract [74]. 5.2. Targeting within the vasculature or peritoneal cavity Targeting to or within the vascular endothelium or peritoneal cavity offers uniquely accessible binding sites for IV-injected targeted liposomes. Targeting of immunoliposomes to mouse pulmonary endothelial cells was demonstrated using the mAb 34A (recently shown to target to the lung endothelial anticoagulant protein thrombomodulin) coupled to liposomes [5865,78,87]. This has been proposed as an effective means for drug delivery to many different types of lung malignancies, as it targets the lung in general rather than lung tumour cells.

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Recently, mAb 34A GM 1-immunoliposomes have been used to deliver the lipophilic prodrug dpFUdR (3’5’-0-dipalmitoyl-5-fluoro-2’-deoxyuridine) to the lung endothelium, so that the prodrug is cleaved into the active form of the drug in close proximity to the pseudometastatic (IV-injected) EMT-6 mouse mammary tumour cells, which seed into the lungs [84]. A significant therapeutic effect (%T/C value 165%) was obtained by injecting dpFUdR incorporated into mAb 34A-targeted liposomes when the mice were treated at days 1 and 3 after tumour cell injection. No therapeutic effect was seen from dpFUdR as a free drug emulsion, or incorporated into antibody-free liposomes [84]. These authors also reported that repeated injections of immunoliposomes resulted in dramatic decreased in target binding, possibly due to an immune reaction against the liposomes. The possible immunogenicity of immunoliposomes is an important cause for concern which has also been raised by Phillips et al. [97,98]. Working with the same model, Maruyama et al. [87] have conjugated the 34A Ab to the terminus of PEG and demonstrated that these liposomes were bound to lung 1.3-fold higher than when coupled to liposomes lacking PEG, and 2.6-fold higher than for PEG liposomes where the Ab was attached at the liposome surface allowing PEG to interfere with Ab recognition of its target. Torchilin et al. [99] have described the successful in vivo targeting of PEG-immunoliposomes targeted with anti-myosin Fab’ fragments to experimentally infarcted rabbit myocardium. Evidence from recent in vitro work using these immunoliposomes [X9] points toward membrane repair as a possible application for these targeted liposomes, though therapeutic studies have not yet been published. A human ovarian carcinoma model (NIH:OVCAR-3) has been developed as gross ascitic tumour with only minor solid tumour growth [ 1001. Targeting of doxorubicin-loaded liposomes was done using Fab’ fragments of the mAb OV-TL3. directed against the 0A3 antigen. which is expressed in over 90% of all human ovarian carcinomas. However, IP treatment of tumour-bearing mice with drug-loaded immunoliposomes gave no better therapeutic results than

that obtained with non-targeted doxorubicinloaded liposomes, possibly due to lack of internalization of the drug/liposome package and/or rapid leakage of the drug from the liposomes in the peritoneal environment. Following drug release, the doxorubicin is presumably rapidly redistributed resulting in a significant drop in drug concentrations in the peritoneal cavity. We have also observed rapid leakage of doxorubicin from liposomes when MPB-PE is used as the coupling lipid [55], although this problem can be resolved when the Ab is coupled at the PEG terminus. Use of an internalizing Ab in this model may result in increased efficacy for the immunoliposomes. In our laboratory, we have researched a therapeutic model for the treatment of haematological malignancies, i.e. CD19 + human B-cell lymphoma (Namalwa) cells grown in SCID mice treated with doxorubicin-loaded anti-CDl9PEG-immunoliposomes. When these cells are injected IP the cells populate the mesenteric and abdominal lymph nodes to form solid tumours, as well as grow as an ascites tumour. IV injected cells disappear from the blood stream within a few hours, and populate the bone marrow and abdominal lymph nodes. CD19 is an internalizing epitope and effective therapeutics have been achieved in both 1P [94] and IV [83] treatment models, with up to a 77% increased life span in IV-injected mice treated with doxorubicin-loaded immunoliposomes, underscoring the importance both of having an accessible target and an internalizing epitope.

6. What are the opportunities targe’ted liposomes in vivo?

for the use of

Critical to the rational use of liposomal drugs and their in vivo targeting is an understanding of the underlying cell biology of the disease and how that will affect the ability of the carrier to localize to the site of disease. The ‘binding site barrier’ appears to present a formidable obstacle to our ability to target anticaner therapy to more advanced solid tumours, but these tumours appear to be amenable to treatment with nontargeted liposomal formulations which achieve

T.M. Allen, E.H. Moase I Advanced

increased localization in the solid tumours via a mechanism of passive targeting through leaky capillaries. On the other hand, ligand- or antibody-mediated targeting within the vasculature or within the peritoneal cavity and possibly the lymph, where the targets are readily accessible, appears to be leading to success. Based on our results and the results of others to date, it would appear that the best targeting results will be obtained when liposomes are coupled to a ligand or an antibody against an internalizing receptor. These are still early days for liposome targeting, but basic research from a number of laboratories is beginning to generate some good insights into where the therapeutic opportunities mights be for targeted liposomal drug delivery.

Acknowledgments

This work is supported by the Medical Research Council of Canada (MA-9127, UI-12411) and by Sequus Pharmaceuticals Inc., Menlo Park, CA.

References [l] Sharma, A.. Mayhew, E. and Straubinger, R.M. (1993) Antitumor effect of taxol-containing liposomes in a taxol-resistant murine tumour model. Cancer Res. 53, 5877-5881. [2] Straubinger, R.M., Sharma, A., Murray. M. and Mayhew, E. (1993) Novel taxol formulations: taxolcontaining liposomes. Monographs Natl. Cancer Inst. 15, 69-78. [3] Sharma, A., Straubinger, R.M., Ojima, I. and Bernacki. R.J. (1995) Antitumor efficacy of taxane liposomes on a human ovarian tumour xenograft in nude athymic mice. J. Pharm. Sci. 84, 1400-1404. [4] Scialli, A.R.. Desesso, J.M., Rahman, A., Husain, S.R. and Goeringer, G.C. (1995) Embryotoxicity of free and liposome encapsulated taxol in the chick. Pharmacology 51, 145-151. [5] Perez-Soler, R., Francis, K., al-Baker, S., Pilkiewicz, F. and Khokhar, A.R. (1994) Preparation and characterization of liposomes containing a lipophilic cisplatin derivative for clinical use. J. Microencapsulation 11, 41-54. [6] Mori, A., Wu, S.P., Han, I., Khokhar, A.R., Perez-Soler, R. and Huang, L. (1996) In vivo antitumor activity of cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(I1) formulated in long-circulating liposomes. Cancer Chemother. Pharmacol. 37, 435-444.

Drug Delivery Reviews 21 (1996) 117-133

[71 Mayhew,

129

E., Papahadjopoulos, D., Rustum, Y.M. and Dave, C. (1976) Inhibition of tumour cell growth in vitro and in vivo by 1-R-D-arabinofuranosylcytosine entrapped within phospholipid vesicles. Cancer Res. 36, 44064411. lipoPI Kim, S. and Howell, S.B. (1987) Multivesicular somes containing cytarabine entrapped in the presence of hydrochloric acid for intracavitary chemotherapy. Cancer Treat. Rep. 71, 705-711. (91 Hong, F. and Mayhew, E. (1989) Therapy of central nervous system leukemia in mice by liposome-entrapped I-beta-D-arabinofuranosylcytosine. Cancer Res. 49, 5097-5102. C. and Chin, Y.C. [lOI Allen, T.M., Mehra, T., Hansen, (1992) Stealth liposomes: an improved sustained release system for l-beta-D-arabinofuranosylcytosine. Cancer Res. 52, 2431-2439. A.A., Shiota, R. and Papahadjopoulos, D. [111 Gabizon, (1989) Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times. J. Natl. Cancer Inst. 81, 1484-1488. WI Gabizon. A., Catane, R., Uziely, B., Kaufman, B., Safra, T., Cohen, R., Martin, F., Huang, A. and Barenholz, Y. (1994) Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes. Cancer Res. 54. 987-992. [I31 Uziely, B., Jeffers, S., Isacson, R., Kutsch, K., Wei-Tsao, D.. Yehoshua, Z., Libson, E., Muggia. F.M. and Gabizon, A. (1995) Liposomal doxorubicin: antitumor activity and unique toxicities during two complementary phase I studies. J. Clin. Oncol. 13, 1777-1785. H. [I41 Goebel, F.-D., Goldstein, D.. Goes, M., Jablonowski, and Stewart, J.S. (1996) Efficacy and safety of StealthR liposomal doxorubicin in AIDS-related Kaposi’s sarcoma. Br. J. Cancer 73, 989-994. Northfelt, D.W., Martin, F.J., Working, P., Volberding. P.A., Russell, J., Newman, M., Amantea, M.A. and Kaplan, L.D. (1996) Doxorubicin encapsulated in liposomes containing surface-bound polyethylene glycol: pharmacokinetics, tumour localization, and safety inpatients with AIDS-related Kaposi’s sarcoma. J. Clin. Pharmacol. 36, 55-63. P61 Cowens, J.W., Creaven, P.J., Greco, W.R., Brenner, D.E., Tung, Y., Ostro, M., Pilkiewicz, F., Ginsberg, R. and Petrelli, N. (1993) Initial clinical (phase I) trial of TLC D-99 (doxorubicin encapsulated in liposomes). Cancer Res. 53, 27962802. [I71 Mayer, L.D., Gelmon, K., Cullis, P.R., Boman, N., Webb, M.S., Embree, L., Tolcher, T. and Bally, M.B. Preclinical and clinical studies with liposomal vincristine., In: Progress in Drug Delivery, Biomedical Research Foundation, Tokyo, 1995. pp. 151-161. [18] Gill, P.S., Espina, B.M., Muggia, F., Cabriales, S., Tulpule, A., Esplin, J.A.. Liebman, H.A.. Forssen, E., Ross, M.E. and Levine, A.M. (1995) Phase I/II clinical and pharmacokinetic evaluation of liposomal doxorubicin. J. Clin. Oncol. 13, 996-1003. [19] Allen. T.M., Newman, MS., Woodle. M.C., Mayhew. E.

130

(201

[21]

[22]

[23]

1241

[2S]

(261

[27]

128)

[29]

[30]

1311

T.M. Allen. E.H. Mouse I Advanced and Uster, P.S. (lY95) Pharmacokinetics and antitumour activity of vincristine encapsulated in sterically stabilized liposomes. Int. J. Cancer 62, 199-204. Allen. T.M.. Hansen, C.B. and Lopes De Menezes, D.E. (199.5) Pharmacokinetics of long circulating liposomes. Adv. Drug Del. Rev. 16, 267-284. Embree. L., Gelmon. K.A., Tolcher. T., Heggie. J.R., Hudon, N.J., Dedhar, C., Bally, M.B. and Mayer. L.D. (1996) Clinical pharmacokinetics of vincristine sulphatc liposome injection (VSLI). Proc. Am. Assoc. Cancer Res. 37, 179. Papahadjopoulos. D., Allen, T.M., Gabizon. A.. Mayhew, E.. Matthay. K., Huang. S-K.. Lee. K.D.. Woodle, M.C., Lasic, D.D.. Redemann. C. and Martin. F.J. (1991) Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA 88, II 460-11 464. Gabizon, A.A. (1992) Selective tumour localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes. Cancer Res. 52, 891~ 896. Huang, S-K.. Lee. K.D., Hong, K.. Friend. D.S. and Papahadjopoulos, D. (1992) Microscopic localization ol sterically stabilized liposomes in colon carcinoma-bearing mice. Cancer Res. 52, 5135-5143. Bakker-Woudenberg, I.A.J.M., Lokerse. A.F., ten Kate. M.T. and Storm, G. (1992) Enhanced localization of liposomes with prolonged blood circulation time in infected lung tissue. Biochim. Biophys. Acta 1138, 31% 326. Bakker-Woudenberg, I.A.J.M., Lokerse. A.F.. ten Kate, M.T., Woodle, M.C. and Storm. G. (1993) Liposomcs with prolonged blood circulation and selective localization in Klebsiella pneumonia-infected lung tissue. J. Infect. Dis. 168. 164-171. Wu. N.Z., Da. D., Rudoll, T.L., Needham. D., Whorton. A.R. and Dewhirst, M.W. (1993) Increased microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumour tissue. Cancer Res. 53. 3765-3770. Gabizon, A. and Papahadjopoulos, D. (19X8) Liposomc formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. Natl. Acad. Sci. USA 85, 6949-6953. Aragnol. D. and Lesserman, L. (1986) Immune clearance of liposomes inhibited by an anti-Fc receptor antibody in vivo. Proc. Natl. Acad. Sci. LJSA 83. 269% 2703. Rahman, A., Panneerselvam, M.. Guirguis. R.. Castronovo, V., Sobel. M.E.. Abraham, K.. Daddona, P.E. and Liotta, L.A. (1989) Anti-laminin receptor antibody targeting of liposomes with encapsulated doxorubicin to human breast cancer cells in vitro. J. Nat]. Cancer Inst. 81. 1794-1800. Bank&t, R.B., Yokota, S., Ghosh, SK., Mayhew. E. and Jou, Y.H. (1989) Immunospecifc targeting of cytosinc arabinonucleoside-containing liposomes to the idiotype on the surface of a murine B-cell tumour in vitro and in vivo. Cancer Res. 49. 30-308.

Drug Delivery Reb’iews 21 (lYY6) 117-1.73 [32] Ahmad, I. and Allen, T.M. (1992) Antibody-mediated specific binding and cytotoxicity of liposome-entrapped doxorubicin to lung cancer cells in vitro. Cancer Res. 52, 4817-4820. 1331 Heath. T.D., Lopez, N.G., Piper. J.R., Montgomery. J.A., Stern, W.H. and Papahadjopoulos. D. (1986) Liposome-mediated delivery of pteridine antifolates to cells in vitro: potency of methotrexate. and its alpha and gamma substituents. Biochim. Biophys. Acta 862,72-80. (341 Moradpour, D., Compagnon, B.. Wilson, B.E., Nicolau. C. and Wands, J.R. (1995) Specific targeting of human hepatocellular carcinoma cells by immunoliposomes in vitro. Hepatology 22, 1527-1537. 1351 Lee. R.J. and Low, P.S. (1995) Folate-mediated tumour cell targeting of liposome-entrapped doxorubicin in vitro. Biochim. Biophys. Acta 1233, 134-144. [36] Debs, R.J.. Heath. T.D. and Papahadjopoulos. D. (1987) Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intravenous administration. Biochim. Biophys. Acta 901. 183-190. 1371 Matzku. S.. Krempel, H., Weckenmann, H-P.. Schirrmacher. V. Sinn, H. and Stricker. H. (1990) Tumour targeting with antibody-coupled liposomes: failure to achieve accumulation in xenografts and spontaneous liver metastases. Cancer Immunol. Immunother. 31. 285-291. 138)Allen. T.M.. Agrawal, A.K., Ahmad, I., Hansen, C.B. and Zalipsky. S. (1994) Antibody-mediated targeting of long-circulating (Stealth@) liposomes. J. Liposome Res. 4, l-25. 1391 Olson. F.. Hunt, C.A.. Szoka. F.C., Vail, W.J. and Papahadjopoulos. D. (197Y) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim. Biophys. Acta 557. 9-23. 1401 Mayer. L.D., Tai. L.C., Ko, D.S., Masin, D., Ginsberg, R.S.. Cullis. P.R. and Bally, M.B. (1989) Influence of vesicle size. lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice. Cancer Res. 49. 5922-5930. 1411 Wu. N.Z., Da, D.. Rudoll, T.I., Needham. D., Whorton. A.R. and Dewhirst. M.W. (1993) Increased microvascuIar permeability contributes to preferential accumulation of Stealth liposomes in tumour tissue. Cancer Res. 53. 3765-3770. 1421 Yuan. F. Leunig, M. Huang, S.K., Berk, D.A., Papahadjopoulos. D. and Jam, R.K. (1994) Microvascular permeability and interstitial penetration of sterically stabilized (Stealth) liposomes in a human tumour xenograft. Cancer Res. 54. 3352-3356. 1431 Huang, SK.. Martin, F.J.. Friend, D.S. and Papahadjopoulos, D. Mechanism of Stealth liposome accumulation in some pathological tissues, In: Lasic. D. and Martin, F. (Eds.), Stealth Liposomes. CRC Press. Boca Raton. Fla, 1995, pp. 119-125. 144) Mayer. L.D.. Bally. M.B. and Cullis, P.R. (1986) Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim. Biophys. Acta 857, 123-126. 1451 Bolotin, E.M., Cohen, R.. Bar. L.K., Emanuel, S.N., l*asic. D.D. and Barenholz, Y. (1994) Ammonium sul-

T.M. Allen, E.H. Moase I Advanced phate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandosomes. J. Liposome Res. 4, 455-479. (461 Boman, N.L., Mayer, L.D. and Cullis, P.R. (1993) Optimization of the retention properties of vincristine in liposomal systems. Biochim. Biophys. Acta 1152, 253258. [47] Gabizon, A., Shiota, R. and Papahadjopoulos, D. (1989) Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times. J. Nat]. Cancer Inst. 81, 1484-1488. [48] Blume, G. and Cevc, G. (1990) Liposomes for the sustained drug release in vivo. Biochim. Biophys. Acta 1029, 91-97. [49] Klibanov, A.L., Maruyama, K., Torchilin, VP. and Huang, L. (1990) Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 268, 235-237. [50] Senior, J., Delgado, C., Fisher, D., Tilcock, C. and Gregoriadis, G. (1991) Influence of surface hydrophobicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles. Biochim. Biophys. Acta 1062. 77-82. [51] Allen, T.M., Hansen, C., Martin, F., Redemann, C. and Yau-Young, A. (1991) Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Acta 1066, 29-36. [52] Allen, T.M. (1994) The use of glycolipids and hydrophillic polymers in avoiding rapid uptake of liposomes by the mononuclear phagocyte system. Adv. Drug Del. Rev. 13. 285-309. [53] Parr, M.J., Ansell, SM., Choi, L.S. and Cullis, P.R. (1994) Factors influencing the retention and chemical stability of poly(ethylene glycol)-lipid conjugates incorporated into large unilamellar vesicles, Biochim. Biophys. Acta 1195, 21-30. [54] Allen. T.M. and Hansen, C. (1991) Pharmacokinetics of Stealth versus conventional liposomes: effect of dose. Biochim. Biophys. Acta 1068, 133-141. [55] Hansen. C.B., Kao, G.Y., Moase, E.H., Zalipsky, S. and Allen, T.M. (1995) Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. Biochim. Biophys. Acta 1239, 1333144. [56] Mori, A., Klibanov, A.L., Torchilin, VP. and Huang, L. (1991) Influence of the steric barrier of amphipathic poly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett. 284, 263-266. [57] Maruyama, K., Yuda, T., Okamoto, A., Ishikura, C., Kojima. S. and Iwatsura, M. (1991) Effect of molecular weight in amphipathic polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes. Chem. Pharm. Bull. 39, 1620-1622. [58] Klibanov, A.L., Maruyama, K., Beckerleg, A.M., Torchilin, V.P. and Huang, L. (1991) Activity of amphipathic poly(ethyleneglycol) 5000 to prolong the circulation time

Drug Delivery Reviews 21 (1996) 117-133

131

of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target. Biochim. Biophys. Acta 1062, 142-148. [59] Lesserman, L.D., Barbet, J., Kourilsky, F. and Weinstein, J.N. (1980) Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A. Nature 288, 602-604. [60] Martin, F.J., Hubbell, W.L. and Papahadjopoulos, D. (1981) Immunospecific targeting of liposomes to cells: a novel and efficient method for covalent attachment of Fab’ fragments via disulfide bonds. Biochemistry 20, 4229-4238. [61] Martin, F.J. and Papahadjopoulos, D. (1982) Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting. J. Biol. Chem. 257, 286-288. [62] Chua. M-M., Fan, S-T. and Karush. F. (1984) Attachment of immunoglobulin to liposomal membrane via protein carbohydrate. Biochim. Biophys. Acta 800, 291300. [63] Loughrey, H, Bally, M.B. and Cullis, P.R. (1987) A non-covalent method of attaching antibodies to liposomes. Biochim. Biophys. Acta 901, 157-160. [64] Bogdanov, A.A., Klibanov. A.L. and Torchilin. V.P. (1988) Protein immobilization on the surface of hposomes via carbodiimide activation in the presence of N-hydorosulfosuccinimide. FEBS Lett. 231, 381-384. [65] Holmberg, E., Maruyama, K., Litzinger. D.C., Wright. S., Davis, M., Kabalka, G.W., Kennel, S.J. and Huang, L. (1989) Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions. Biochem. Biophys. Res. Commun. 165, 12721278. [66] Weiner, A.L. Chemistry and biology of immunotargeted liposomes, In: Tyle, P. and Ram, B.P. (Eds.), Targeted therapeutic systems. Marcel Dekker, Inc., New York, 1990, pp. 305-336. [67] Torchilin, V.P. and Klibanov, A.L. (1993) Coupling of ligands with liposomes membrane. Drug Target. Del. 2, 227-238. (681 Loughrey. H.C., Choi, L.S.. Cullis. I?R. and Bally, M.B. (1990) Optimized procedures for the coupling of proteins to liposomes. J. Immunol. Meth. 132, 25-35. [69] Harasym, T.O., Tardi, P., Longman. S.A.. Ansell, S.M., Bally, M.B., Cullis, P.R. and Choi, L.S. (1995) Poly(ethylene glycol)-modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes. Bioconjugate Chem. 6. 187-194. (701 Zalipsky, S., Brandeis, E.. Newman, M. and Woodle, M.C. (1994) Long circulating, cationic liposomes containing amino-PEG-phosphatidylethanolamine. FEBS Lett. 353, 71-74. [71] Zalipsky, S., Chang, J.L., Albericio, F. and Barany, G. (1994) Preparation and applications of polyethylene glyol-polystyrene graft resin supports of solid phase peptide synthesis. Reactive Polymers 22, 243-258. [72] Allen, T.M., Brandeis, E.. Hansen, C.B., Kao, G.Y. and Zalipsky, S. (1995) A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in

132

T.M. Allen, E.H. Moase I Advanced

efficient targeting to cancer cells. Biochim. Biophys. Acta 1237, 99-108. poly(ethylene glycol) I731 Zalipsky, S. (1995) Functionalized for preparation of biologically relevant conjugates. Bioconjugate Chem. 6, 150-165. [741 Kirpotin, D., Park, J.W., Hong, K., Keller, G., Benz, J. and Papahadjopoulos, D. (1996) Binding and endocytosis of sterically stabilized anti-HER2 immunoliposomes by human breast cancer cells. Proc. Am. Assoc. Cancer Res. 37. 467. function(751 Zalipsky, S. (1993) Synthesis of end-group alized polyethylene glycol-lipid conjugate for preparation of polymer-grafted liposomes. Bioconjugate Chem. 4, 296-299. G., Cevc, G., Crommelin, M.D.. Bakker(761 Blume, Woudenberg. 1.A.. Kluft, C. and Storm, G. (1993) Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochim. Biophys. Acta 1149, 180-184. [771 Klibanov, A.L. and Huang, L. (1992) Long-circulating liposomes: development and perspectives. J. Liposome Res. 2, 321-334. [781 Maruyama, K.. Kennel, S.J. and Huang, L. (lY90) Lipid composition is important for highly efficient target binding and retention of immunoliposomes. Proc. Natl. Acad. Sci. USA 87, 574445748. [791 Suzuki, S., Watanabe, S., Masuko, T. and Hashimoto. Y. (1995) Preparation of long-circulating immunoliposomes containing adriamycin by a novel method to coat immunoliposomes with poly(ethylene glycol). Biochim. Biophys. Acta 124. 9-16. PO1 Ohta, S., Igarashi, S., Honda, A., Sato, S. and Hanai, N. (1993) Cytotoxicity of adriamycin-containing immunoliposomes targeted with anti-ganglioside monoclonal antibodies. Anticancer Res. 13, 331-336. I811 Osdol, W.V.. Fujimori, K. and Weinstein, J.N. (1991) An analysis of monoclonal antibody distribution in microscope tumour nodules: consequences of a ‘binding site barrier’. Cancer Res. 51. 4776-4784. WI Park, J.W., Hong, K.. Carter, P.. Asgari, H.. Guo, L.Y.. Keller, G.A.. Wirth. C.. Shalaby, R., Kotts, C., Wood, WI., Papahadjopoulos, D. and Benz. C.C. (1995) Development of anti-p185 “FRZ immunoliposomes for cancer therapy. Proc. Natl. Acad. Sci. USA 92, 1327-1331. P31 Lopes de Menezes, D.E.. Pilarski. L.M. and Allen. T.M. (1995) Selective cytotoxicity of immunoliposomal to B lymphocytes. Proc. Am. Assoc. Cancer Res. 35, 307. WI Mori, A., Kennel. S.I., Waalkes. M.V.B., Scherphof. G.L. and Huang, L. (1995) Characterization of organ-spccitic immunoliposomes for delivery of 3’.5’-0-dipalmitoyl-5 Huoro-2’-deoxyuridine in a mouse lung metastasis model. Cancer Chemother. Pharmacol. 35. 447-456. I851 Bloemen, P.G.M., Henricks, P.A.J.. van Bloois, L.. van den Tweel, M.C.. Bloem. A.C.. Nijkamp, F.P.. Crommelin, D.J.A. and Storm. G. (1995) Adhesion molecules: a new target for immunoliposome-mediated drug delivery. FEBS Lett. 357. 140-144.

Drug Delivery Reviews 21 (1996) 117-133

WI Nassander,

U.K., Steerenberg, P.A., De Jong, W.H., Van Overveld, W.O.M., Te Boekhorst, C.M.E., Poels, L.C.. Jap, P.H.K. and Storm, G. (1995) Design of immunoliposomes directed against human ovarian carcinoma. Biochim.

Biophys.

1871 Maruyama,

Acta

K., Takizawa,

1235, 126-139. T., Yuda,

T., Kennel,

S.J..

Huang, L. and Iwatsuru. M. (1995) Targetability of novel immunoliposomes modified with amphipathic poly(ethylene glycol)s conjugated at their distal terminals to monoclonal antibodies. Biochim. Biophys. Acta 1234, 74-80.

WI Vingerhoeds, R.H.P..

M.H., Crommelin,

Haisma, H.J., Belliot, S.O., Smit. D.J.A. and Storm, G. (1996) Im-

munoliposomes as enzyme carriers (immuno somes) for antibody directed enzyme prodrug (ADEPT): optimization of prodrug activating Pharmaceut. Res. 13, 604-610.

enzymotherapy capacity.

P91 Khaw,

B-A., Torchilin, VP.. Vural, I. and Narula, J. (1995) Plug and seal: prevention of hypoxic cardiocyte death by sealing membrane lesions with antimyosin-

liposomes.

Nature Med. 1, 1195-1198. H., Tomita, T., Kobayashi, H., Fujii, Y., Takahashi, T.. Hasumi, K.. Nariuchi, H. and Sekiguchi, M. (1991) Application of boronated anti-CEA immunoliposome to tumour cell growth inhibition in in vitro boron neutron capture therapy model. Brit. J. Cancer 63. 522-526. [911 Briscoe, P.. Caniggia, I., Graves. A., Benson, B., Huang, L.. Tanswell, A.K. and Freeman, B.A. (1995) Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes. Am. Physiol. Sot. 268, L374l-380. D. (1990) [921 Daleke, D.L., Hong, K. and Papahadjopoulos, Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay. Biochim. Biophys. Acta 1024, 352-366. D. (1993) [931 Lundberg, B., Hong, K. and Papahadjopoulos, Conjugation of apolipoprotein B with liposomes and targeting to cells in culture. Biochim. Biophys. Acta 1149, 305-312. [941 Allen, T.M., Ahmad, I., Lopes de Menezes, D.E. and Moase. E.H. (1995) Immunoliposome-mediated targeting of anti-cancer drugs in vivo. Biochem. Sot. Trans. 23. 1073-1079. [951 Marjan, J., Charrois, G., Lopes de Menezes. D.E. and Allen, T.M. (1996) Antibody-mediated targeting of liposomal doxorubicin to lymphoblastic cells can reverse multidrug resistance. Proc. Am. Assoc. Cancer Res. 37, 309. M.. Samuel, J. and Allen, T.M. [961 Ahmad, I., Longenecker, (1993) Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice. Cancer Res. 53, 1484-1488. of [971 Phillips, N.C. and Emili, A. (1991) Immunogenicity immunoliposomes. Immunol. Lett. 30, 291-296. [981 Phillips. N.C., Gagne, L., Tsoukas, C. and Dahman, J. (1994) Immunoliposome targeting to murine CD4-

I901 Yanagie,

T.M. Allen, E.H. Moase I Advanced Leucocytes is dependent on immune status. J. Immunol. 152, 3168-3174. [99] Torchilin,VP., Klibanov, A.L., Huang, L., O’Donnell, S., Nossiff, N.D. and Khaw, B.A. (1992) Targeted accumulation of polyethylene glycol-coated immunoliposomes in infarcted rabbit myocardium. FASEB J. 6, 2716-2719.

Drug Delivery Reviews 21 (1996) 117-133 [loo]

133

Vingerhoeds, M.H., Steerenberg, P.A., Hendirks, J.J.G.W., Dekker, L.C., van Hoesel, Q.G.C.M., Crommelin, D.J.A. and Storm, G. (1996) Immunoliposomemediated targeting of doxorubicin to human ovarian carcinoma in vitro and in vivo. Brit. J. Cancer (in press).