Advanced Drug Delivery Reviews 56 (2004) 1177 1192 www.elsevier.

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Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acidPEG conjugates
Alberto Gabizon *, Hilary Shmeeda, Aviva T. Horowitz, Samuel Zalipsky
Oncology Department, Shaare Zedek Medical Center, Hebrew University School of Medicine, Jerusalem, Israel ALZA Corporation, Mountain View, CA, USA Received 6 October 2003; accepted 5 January 2004

Abstract Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combine long-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a number of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs. D 2004 Elsevier B.V. All rights reserved.
Keywords: Folate; Liposome; Targeting; Chemotherapy; Murine tumor model; PEGylation

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Folate-targeted liposomes (FTL) versus nontargeted liposomes (NTL) 1.2. FTL versus nonliposome-based folate-targeted systems . . . . . . . FR expression and tumor models . . . . . . . . . . . . . . . . . . . . Formulation issues . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Achieving prolonged circulation time . . . . . . . . . . . . . . . 3.2. Optimization of the PEG-folate conjugate concentration . . . . . . 3.3. PEG steric interference with binding . . . . . . . . . . . . . . . 3.4. Insertion versus conventional incorporation of folate PEG lipid into . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178 1178 1179 1180 1181 1181 1181 1182 1183

2. 3.

* Corresponding author. Oncology Department, Shaare Zedek Medical Center, POB 3235, Jerusalem 91031, Israel. Tel.: +972-2-655-5036; fax: +972-2-652-1431. E-mail address: alberto@md.huji.ac.il (A. Gabizon). 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.01.011

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A. Gabizon et al. / Advanced Drug Delivery Reviews 56 (2004) 11771192 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183 1183 1184 1185 1186 1188 1188 1189 1190 1190 1190 1190

In vitro studies with FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . 4.1. Kinetics of liposome binding to tumor cells . . . . . . . . . . . . . . . 4.2. Delivery of doxorubicin encapsulated in FTL to tumor cells . . . . . . . . 4.3. In vitro cytotoxicity of doxorubicin encapsulated in FTL . . . . . . . . . 5. Pharmacokinetics and tissue distribution studies with FTL-encapsulated drugs . . . 6. Therapeutic effects of FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . 6.1. Folate-targeted PEGylated (STEALTHR) liposomal doxorubicin (FTL-Dox) 6.2. Folate-targeted PEGylated (STEALTHR) liposomal cisplatin (FTL-cisplatin) 7. Toxicity of cytotoxic drugs encapsulated in FTL . . . . . . . . . . . . . . . . . 8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The rationale for cancer targeting with folate ligands attached to the liposome surface is based on two layers. First, there is a common layer to all folatetargeted systems which relates to the choice of the tumor cell folate receptor (FR) as the target. FR upregulation or over-expression is commonly associated with a broad variety of tumor types including solid and hematological malignancies, and it appears to be more frequently observed in advanced stages of cancer [1]. How specific and frequent is FR overexpression in cancer cells to justify its choice as target is discussed in other articles of this issue of Adv. Drug Deliv. Rev. and will not be addressed here. The second layer contains elements unique to liposomal and perhaps other nanoparticulate drug carrier systems and will be addressed here. The strength of the folate-targeted liposome approach stems from conceptual advantages over two alternative approaches: nontargeted liposomal systems and nonliposome-based folate-targeted systems. 1.1. Folate-targeted liposomes (FTL) versus nontargeted liposomes (NTL) A schematic illustration of the folate liposome targeting concept is presented in Fig. 1. Long-circulating liposomes, such as polyethylene glycol (PEG) coated liposomes (also known as STEALTHR liposomes) [2], tend to accumulate in tumors as a result of increased microvascular permeability and defective lymphatic drainage, a process also referred to as the enhanced permeability and retention (EPR) effect [3]. This is a passive and nonspecific process of liposome

extravasation that is statistically improved by the prolonged residence time of liposomes in circulation and repeated passages through the tumor microvascular bed [4]. However, except for rare instances, tumor cells are not directly exposed to the blood stream. Therefore, for an intra-vascular targeting device to access the tumor cell FR, it must first cross the vascular endothelium and diffuse into the interstitial fluid. Experimental data with antibody-targeted liposomes and FTL indicate that liposome deposition in tumors is similar for both targeted and nontargeted systems [5 7], supporting the hypothesis that extravasation is indeed the rate-limiting step of liposome accumulation in tumors. However, once liposomes have penetrated the tumor interstitial fluid, binding of targeted liposomes to FR may occur thus shifting the intra-tumor distribution from the extracellular compartment to the tumor cell compartment, as shown recently for a mouse ascitic tumor [7]. Binding to tumor cells may be followed by internalization of liposome contents via folate-receptor mediated endocytosis (Fig. 1). Retrograde movement of liposomes into the blood stream, if any, will be reduced for liposomes with binding affinity to a tumor cell receptor. Obviously, when the parameter of drug delivery is considered, there will always be a combination of in situ release from an extracellular liposome depot and intra-cellular release from internalized liposomes. Therefore, the theoretical advantages of FTL over NTL are related to a shift of liposome distribution to the tumor cell compartment, delivery of liposomal contents to an intra-cellular compartment in liposomeassociated form, and, possibly, prolonged liposome retention in the tumor. On the negative side, the main disadvantage of a targeted system to a cancer cell

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Fig. 1. Schematic drawing illustrating the concept of folate targeting of liposomes to tumor cells. The blue dots represent the liposomal folate ligands. The red dots represent the drug molecules encapsulated in the liposome water phase. The various steps involved in the targeting process are numerically designated from 1 to 6. Steps 1 3 are common to nontargeted and targeted liposomes. Steps 4 6 are specific to FTL. (1) Liposomes with long-circulating properties increase the number of passages through the tumor microvasculature. (2) Increased vascular permeability in tumor tissue enables properly downsized liposomes to extravasate and reach the tumor interstitial fluid. (3) Drug is gradually released from liposomes remaining in the interstitial fluid and enters tumor cells as free drug to exert a cytotoxic effect. (4) Other liposomes bind to the FR expressed on the tumor cell membrane via the folate ligand. Because of the limited diffusion capacity of liposomes, binding is likely to be limited to those tumor cells in closest vicinity to blood vessels. (5) Liposomes are internalized by tumor cells via FRME. (6) Internalized liposomes release their drug content in the cytosol enabling the drug to exert its cytotoxic effect.

receptor such as FR is the difficulty of a large nanosize assembly, such as FTL, to penetrate a solid tumor mass, specially considering the high interstitial fluid pressure that is often present in tumor masses of clinically detectable size [8]. 1.2. FTL versus nonliposome-based folate-targeted systems Liposomal systems offer an elegant drug delivery amplification system. Each liposome vesicle carries a drug cargo usually in the order of 103 104 molecules. For instance, in the case of a STEALTHR liposome formulation known as Doxil, there are between 10,000 and 15,000 doxorubicin molecules per vesicle [9], and these may be targeted with the help of as few ligands as 10 per vesicle, i.e. a 100 1000-fold delivery amplification factor when the drug:ligand ratio is considered. Another theoretical advantage of liposomal systems is that their size far exceeds the critical

glomerular filtration threshold. Therefore, unlike low molecular weight folate-targeted complexes, FTL do not have access to the luminal side of kidney tubular cells where FR is expressed, thereby sparing kidneys of massive FR-mediated liposomal drug delivery and subsequent toxicity [10]. One of the disadvantages of ` -vis small drug folate conjugates is that FTL vis-a liposomes are bulky structures that are difficult to internalize by nonphagocytic cells. The best characterized pathway of liposome internalization, mediated by clathrin-coated pits, often leads to sequestration of liposome contents within the lysosome compartment. An alternative pathway of endocytosis, known as potocytosis, may operate for receptors associated with cell caveolae or lipid rafts, such as FR [10], and facilitate access to the cytosol via acidic endosomes bypassing lysosomes. It is well established that FTL enter cells by FR-mediated endocytosis (FRME) [11]. In addition, experimental data with folate-targeted, pH-sensitive liposomes are consistent with liposome

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transit through an acidic vesicle compartment [12]. A connection between post-caveolar or post-raft endosomes and lysosomes is possible, since markers of the clathrin-coated pit pathway and folate conjugates have been shown to co-localize in the same cell organelles [13]. Thus, an important fraction of internalized liposomes may end up in lysosomes. The cell trafficking of liposomes following FRME needs to be better understood, specially since intra-cellular trafficking of small molecular weight folate conjugates may be different from that of nanoparticles with multimeric binding such as FTL.

2. FR expression and tumor models A prerequisite for investigation of any targeted system is the availability of tumor models with stable overexpression of the target receptor. Routine cell culture conditions expose tumor cells to high folate concentrations so that even if a fresh tumor explant overexpresses FR, in vitro culture may gradually cause downregulation of FR. The standard approach we have used to generate a FR-overexpressing cell line is to cultivate the cells in a folate-free culture medium. FR upregulation is a common response of cells grown in a folate-depleted environment. Many tumor cell lines respond to folate-depleted culture conditions with upregulation of FR. This is generally a reversible process, i.e. when folate supplies are restored FR is downregulated [14]. Therefore, FRoverexpressing cell lines should be maintained in folate-free medium. The addition of 10% nondialyzed serum to folate-free medium results in a sub-physiologic concentration of folic acid (3 nM) under which these cell lines maintain high FR expression [14].

We have studied several animal tumor models overexpressing the FR, including mouse M109 carcinoma and its multidrug-resistant cells (MDR) subline, M109R [14], the human KB carcinoma [15], and the mouse J6456 lymphoma [16]. High FR (HiFR)expressing cells have been selected from these tumor cell lines as previously described for M109 and KB tumors [14]. A high FR-expressing J6456 subline was similarly obtained by a single in vivo passage of tumor cells followed by repeated in vitro passage in a folate-free culture medium. Baseline information on the uptake of free folic acid and on the effect of folate-depleted diet on receptor expression in vivo is obviously of great importance in the testing of FTL. Since we found that folate binding by the M109 tumor was not affected by the diet within the short time frame required for a tissue distribution study, experiments with this tumor model and with the KB human carcinoma (another well-established model of inducible and stable high FR expression [11,13,16]) proceeded with animals on normal diet. In contrast, J6456 lymphoma quickly downregulated FR in animals with a normal, folateenriched diet (Table 1). Therefore, experiments with the J6456-HiFR should be carried out preferably in animals maintained on a folate-depleted diet. The results of folic acid uptake and targeted versus nontargeted liposomal uptake in the J6456-HiFR tumor, presented in Table 1, point to a 30-fold drop in radiolabeled folate in cells from mice fed a normal, folate-enriched, diet, and to a 3 12-fold increase in liposome uptake when FTL are compared to NTL. The importance of using a folate-depleted diet in in vivo experiments with folate-targeted systems has been questioned. Clearly if tumor FR expression is quickly downregulated under a folate-rich diet, then

Table 1 Folic Acid (F.A.) and liposome uptake of J6456 and J6456-HiFR tumor cell linesa Cells/source J6456 (parental line) J6456-HiFR (in vitro F.A.-depleted medium) J6456-HiFR (mice on normal diet) J6456-HiFR (mice on F.A.-depleted diet)
3 H-F.A. fmole/106 cells 3

H-CHE-NTL pmole/106 cells

H-CHE-FTL pmole/106 cells

2F1 14,675 F 1403 186 F 127 5660 F 931

215 F 12 286 F 29 Not done 430 F 8

199 F 23 3719 F 340 Not done 1470 F 133

a J6456 cells (parental line) obtained from in vitro passage using standard (folate-rich) culture medium. J6456-HiFR cells were obtained from either in vitro passage in F.A.-depleted medium or in vivo passage in mice on normal diet or F.A.-depleted diet. Cells incubated at 37 jC for 30 min in the presence of radiolabeled F.A. (0.1 pmol/ml), and for 24 h in the presence of 3H-CHE (cholesterol hexadecyl ether) labeled NTL or FTL (300 nmol phospholipid/ml). Results are expressed as fmol F.A. per million cells, or pmol phospholipid per million cells.

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the use of a special folate-depleted diet is necessary. In our experience, the folate-depleted diet in specific pathogen-free (SPF) mice exposed to chemotherapy causes serious weight loss and is problematic for longterm therapeutic experiments lasting several weeks or months after treatment has been completed. The approach we have generally adopted is to put mice on a folate-depleted diet shortly before tumor inoculation and put them back on a normal diet 1 week after the last treatment is administered. FTL have been cleared from circulation and their interactions, if any, with the tumor FR should be over.

circulation. In addition, optimal drug retention is critical to ensure delivery of an intact drug payload upon reaching the target cell. For drugs encapsulated in the water phase of liposomes, stable retention can be achieved by using high Tm (>37 jC) phospholipids and cholesterol. Therefore, the formulations we have used are STEALTHR type and consist of fully hydrogenated soybean PC, cholesterol, and PEG DSPE conjugate. Vesicle size is tailored to V 100 nm by sequential extrusion to ensure that there is no physical hindrance to extravasation and internalization [9]. 3.2. Optimization of the PEG-folate conjugate concentration In a liposome coated with PEG polymers, a reasonable strategy is to present the folate ligand at the outer end of the PEG chain. Folate has been coupled to amino-PEG DSPE [19] mainly through the gamma-carboxyl to PEG DSPE as seen in Fig. 2 and as described before [11,14,20]. The affinity of the result-

3. Formulation issues 3.1. Achieving prolonged circulation time It has been well established that prolonged circulation is a prerequisite for tumor accumulation of liposomes [17,18]. PEGylated liposomes are the best basis for a formulation that confers a long half-life in

Fig. 2. Structures of various lipopolymers discussed in this review: mPEG DSPE, Folate PEG DSPE, and the disulfide-linked cleavable lipopolymer, mPEG DTP DSPE. Note that approximately 80% of the folate moieties are linked as shown, through the gamma carboxyl, while the remaining 20% are alpha-carboxyl linked analogs, as determined by HPLC assay of carboxypeptidase G-treated conjugate [Ref. 14]. Degree of polymerization, n = 45 for derivatives of mPEG of molecular weight 2000 Da; n = 75 for derivatives of PEG of 3350 Da.

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ing conjugate to FR is about 10-fold lower than unconjugated FA [14]. However, since liposome binding is multivalent, i.e. mediated through several ligands, its overall affinity for a target cell is the product of the individual affinities of the ligands participating in binding. This is why the affinity of FTL for FR-expressing cells is much higher, and nearly a 1000-fold greater concentration of soluble folic acid is needed to compete effectively with FTL for binding to FR [14,21]. Because of the flexibility of the PEG chain, a small number of folate residues on the liposome surface may be sufficient to enable liposome binding to the cell FR. In STEALTHR liposomes, the molar ratio of PEG DSPE is approximately 5%, probably far more than the amount of ligand we would need on the liposome surface to secure an optimal chance of binding to FR. Based on earlier work from the laboratory of Low and colleagues [11,20] and our subsequent studies [14,21], it appears that a molar fraction of 0.2 0.5% folate PEG DSPE is sufficient for effective interaction with the cell membrane FR. The rest of liposomal PEG would be in the form of the standard methoxy(m)PEG DSPE conjugate. However, a recent study [22] testing a wide range of PEGfolate concentrations indicates that optimal binding is obtained with low levels of 0.03%, about 10-fold less than those commonly used in previous studies. The authors hypothesize that, at high surface density, a folate folate interaction prevents folate binding to the receptor [22]. 3.3. PEG steric interference with binding In our early studies, we observed that mPEG (2000) DSPE significantly interferes with the binding and uptake of liposomes targeted with 0.5% folate PEG(2000) DSPE [14]. Therefore, we increased the PEG length in the folate conjugate to MW 3350, a change that resulted in a major improvement of the targeting effect. Even then, interference with binding to FR is not entirely overcome (as shown below in Fig. 3A). One option, not yet tested, is to extend further the PEG length of the folate conjugate. Excluding PEG DSPE from the liposomes adversely affects their in vivo circulation time (as shown below in Fig. 8) and is, therefore, not an option. One alternative strategy to avoid the interference of PEG

Fig. 3. (A) Effect of substituting mPEG DSPE (PEG) with mPEG DTP DSPE (thiolytically cleavable lipopolymer, PEG-SS) on liposome binding to M109R-HiFR tumor cells. In vitro incubation of 3H-CHE labeled liposome preparations at phospholipid concentration of 300 nmol/ml in the absence or presence of 1 mM cysteine for 24 h. Test conditions were as previously reported for liposome cell binding assays [14]. (B) Effect of substituting mPEG DSPE (PEG) with mPEG DTP DSPE (thiolytically cleavable lipopolymer, PEG-SS) on plasma clearance of FTL. 3H-CHE labeled preps (2 Amol phospholipid per mouse) were injected i.v. into BALB/c mice (n = 4 8 per group), and animals were sacrificed after 24 h. Average of two experiments. All differences are significant by oneway Anova with Bonferroni post-test at P < 0.001 level, except for FTL PEG-SS vs. FTL w/o PEG, which is not significant.

coating on binding and uptake of FTL while maintaining its shielding effect on circulating liposomes is to design a cleavable PEG lipid [23,24]. This has been done using a conjugate with a thiolytically cleavable disulfide linked mPEG dithiodipropionate (DTP) DSPE. As illustrated in Fig. 2, this lipopolymer contains DTP as a linking moiety between the PEG and lipid components [23]. Ideally, cleavable PEG should be sufficiently stable in plasma. Gradual cleavage and release of PEG

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will take place in the extracellular fluid or upon contact with cell membrane components. This will enable avid binding and internalization of FTL by FR-expressing cells. Fig. 3A shows the results of an in vitro experiment testing binding of FTL coated with cleavable mPEG DTP DSPE to FR-expressing cells in the absence or presence of cysteine. There is a clear enhancement of liposome binding to cells in the presence of cysteine when mPEG DTP DSPE is included in the FTL formulation. In contrast, control FTL prepared with standard, noncleavable mPEG DSPE show poor binding and no effect of cysteine. However, in vivo, FTL prepared with mPEG DTP DSPE were cleared much faster than control FTL (Fig. 3B) indicating that this particular lipopolymer is too labile and is not entirely stable in circulation. New cleavable lipopolymers with increased stability were recently prepared [25] and await testing. Although this approach is a promising one, it still requires optimization. 3.4. Insertion versus conventional incorporation of folate PEG lipid into liposomes Another important aspect of formulation is whether the folate ligand can be inserted into preformed liposomes as opposed to conventional incorporation during liposome preparation. The latter procedure requires the ligand-PEG lipid to be co-mixed with other liposome components during the initial step of liposome preparation. Recently it was demonstrated that incubation of pure ligand-PEG lipids with liposomes results in their clean insertion into the outer leaflet of the liposomal bilayer [26]. This has several advantages from the pharmaceutical point of view (reviewed by Zalipsky et al. Ref. [27]): (i) FTL could be prepared without modifying the production line of a commercial liposomal formulation; (ii) folate targeting can be applied to a variety of liposomal drug formulations; (iii) folate ligand will be inserted only in the outer leaflet of the bilayer relevant for targeting and thus will not be buried in the liposomal interior, and will not be able to interact with the encapsulated drug; (iv) provided that pure ligand-PEG lipid is used, the insertion into preformed liposomes is the only method of preparation of ligand-bearing PEGliposomes that completely avoids incorporation of any extraneous residues [27]. Considering that fo-

late PEG DSPE [14] is a well-characterized pure conjugate, all four advantages of the insertion approach can potentially apply. Ligand insertion has been reported for antibody targeted liposomes with satisfactory yields [28]. We have tested the folate PEG DSPE insertion method with two PEGylated liposomal formulations: DOXILR (STEALTHR liposomal doxorubicin) and SPI-77 (STEALTHR liposomal cisplatin), and obtained a high rate of ligand association (range 62 94%) with liposomes, resulting in a final concentration of 0.35 0.55% of folate PEG DSPE in mol% of total phospholipid [29]. A recent report has also demonstrated an efficient yield for post-insertion of folate PEG lipophilic conjugates into preformed liposomes loaded with doxorubicin [30].

4. In vitro studies with FTL-encapsulated drugs In vitro observations may give a false assessment of a targeting strategy using liposomes or other carriers due to the complexity of the in vivo setting and the enormous drug pharmacokinetic changes caused by the use of particulate drug carriers [9]. Nonetheless, in vitro studies are still important in assessing the ability of targeted liposomes to interact with FR-expressing cells and to deliver bioavailable drug into the relevant cellular compartments. 4.1. Kinetics of liposome binding to tumor cells Fig. 4 depicts the effects of liposome concentration and incubation time on the in vitro binding of FTL labeled with 3H-cholesterol hexadecyl ether (3H-CHE) to M109-HiFR cells. Although an increase in liposome uptake with time of incubation is clearly seen for all three lipid concentrations tested (30, 100, and 300 nmol/ml), the steepest slope, a 3-fold increase within 20 h, was obtained with the lowest concentration. In addition, at any given incubation time, the highest relative uptake of liposomes was observed with the lowest concentration. These results indicate that saturation of liposome uptake begins at 100 nmol/ml and possibly at lower concentrations. Furthermore, the kinetics are consistent with recycling of receptors, enabling a gradual, albeit slower rise of liposome uptake with time.

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gaining access to the nucleus may originate from destabilized membrane-bound liposomes. For an example of the nuclear localization of doxorubicin (Dox) in KB tumor cells after exposure to FTL-Dox, see Fig. 5. The doxorubicin transfer to the nucleus is clearly drug-specific since, when similar FTL were loaded with rhodamine, the fluorescent label remained in the cytoplasm. Perhaps, the most attractive feature of doxorubicin-loaded FTL was the ability to deliver doxorubicin to MDR cells as effectively as to the parental, doxorubicin-sensitive cells. As seen in Fig. 6, resistant cells accumulated much less drug than sensitive cells when exposed to free Dox. In contrast, a similar level of drug uptake was observed in both types of cells when exposed to FTL-Dox. The in vitro uptake of NTL-Dox was negligible. This and other studies with FTL [20,31,32] support the hypothesis that FR-mediated drug delivery is an effective approach to deliver anthracyclines to tumor cells. Furthermore, the bypass of the P-glycoprotein (Pgp) efflux pump suggests a potential role of FTL in overcoming drug resistance [21].

Fig. 4. Kinetics of tumor cell liposome uptake in vitro: M109-HiFR cells were incubated in the presence of 3H-CHE labeled FTL. Test conditions were as previously reported for liposome cell binding assays [14]. (A) Effect of phospholipid concentration. (B) Effect of incubation time.

4.2. Delivery of doxorubicin encapsulated in FTL to tumor cells Our studies on in vitro delivery of doxorubicin by FTL are described in detail in Goren et al. [21]. FTL loaded with doxorubicin are taken up by FR-bearing cells and the drug is swiftly transferred from the intracellular compartment to the nucleus, indicating that a significant fraction of the drug is released from liposomes in the cytosol and redistributes to the nucleus due to its known affinity for DNA. Although there is strong evidence from confocal microscopy that a substantial fraction of the drug enters cells in liposomal form, it cannot be ruled out that part of the drug

Fig. 5. Confocal fluorescence microscope picture of KB-HiFR tumor cells after 2-h in vitro exposure to 10 AM FTL-Dox. Doxorubicin fluorescence (orange) is readily recognized in the nucleus sparing the nucleolus. A rim of membrane-bound fluorescent liposomes is also seen. Test conditions were as previously reported for confocal microscope observations of liposomal doxorubicin cell uptake [21].

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Fig. 6. Enhanced Dox delivery via FTL to M109R-HiFR (MDR+) tumor cells. Tumor cells were exposed to free or liposomal drug as FTL or NTL for 1 h, at a Dox concentration of 10 AM. Drug not associated with cells was washed out by centrifugation. Cellassociated drug was extracted and measured as previously described [21].

4.3. In vitro cytotoxicity of doxorubicin encapsulated in FTL As shown in a number of studies [20,31,32], drug delivered by FTL is always more cytotoxic than drug

delivered by NTL. When FTL-Dox is compared to free Dox, the results are variable with small differences in both directions. This is likely due to variations in drug bioavailability that may result from liposome formulation, drug loading method, saturation of liposome uptake, and cell-dependent liposome uptake pathways. In our studies [21], we found a slight advantage for free drug with lower IC50 than that for FTL-Dox. This finding was difficult to reconcile with the higher drug levels measured in cells exposed to FTL-Dox as compared to cells exposed to free Dox, suggesting that part of the drug delivered by FTL is not internalized and/or remains sequestered on the cell surface or within intra-cellular compartments. To test the full cytotoxic potential of FTL-Dox in a longer assay exposing tumor cells to the in vivo milieu, we performed a Winn assay. In this assay, the cells are exposed to the drug in various forms in vitro. After a short exposure (1 2 h), the cells are washed to remove any noncell-associated material, counted, and inoculated in the animal. The number of animals developing tumors and the timing of tumor development provide an indication of the cytotoxic activity exerted by the treatment upon direct in vitro

Fig. 7. Winn assay to examine in vivo the cytotoxic effect of FTL-Dox after in vitro exposure of M109R-HiFR cells. M109R-HiFR cells incubated for 2 h in the presence of free Dox, FTL-Dox (w/o mPEG), or NTL-Dox (DOXILR), and then washed to remove any nonassociated drug/liposome. Mice were inoculated with 106 cells in the footpad. Curves show the probability of preventing tumor development. FTL-Dox was significantly more effective than free Dox or NTL-Dox. Untreated vs. Free Dox P = 0.0075; Free Dox vs. NTL-Dox P = 0.0313; Free Dox vs. FTL-Dox, P = 0.0250; Doxil vs. FTL-Dox P < 0.0001 (log rank test).

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exposure to tumor cells. As seen in one representative experiment (Fig. 7), FTL-Dox was significantly more effective than free drug, and certainly more than the nontargeted formulation, in agreement with the in vitro drug delivery data shown in Fig. 6. Thus, we conclude that FTL is capable of delivering Dox to tumor cells at high levels with potential biologic activity superior to free drug and NTL.

5. Pharmacokinetics and tissue distribution studies with FTL-encapsulated drugs As mentioned above, PEG coating interferes with the uptake of FTL. However, PEG coating is critical for the long circulation time of liposomes, and this is in turn a prerequisite for liposome accumulation in tumors, as can be seen when the plasma clearance of PEGylated FTL is compared to that of nonPEGylated FTL after i.v. injection in mice Fig. 8. Therefore, in most of our in vivo experiments we use FTL formulated with mPEG DSPE at approximately 4.7% molar ratio and folate PEG DSPE at approximately 0.3% molar ratio. The PEG tether used for the folate conjugate is longer (3.35 K) than that of mPEG DSPE (2 K) in an effort to reduce the interference of the latter with FR-mediated cell uptake [14]. The pharmacokinetics of FTL in rats is shown in Fig. 9. In comparison

to NTL of similar composition, FTL showed a faster clearance despite the fact that both formulations were PEGylated. The accelerated clearance of FTL may be the result of direct liposome uptake by the liver FR. Alternatively, binding of the plasma folate binding protein, a soluble form of FR, to circulating liposomes could result in opsonization and liposome tagging for removal by the reticulo-endothelial system by nonspecific mechanisms. There was a slight, additional acceleration of clearance of FTL when rats were put on folate-deficient diet, owing to higher uptake in liver and particularly in spleen (Fig. 9 inset), suggesting upregulation of FR. Clearly, the most relevant biodistribution data are those addressing the comparative fate of FTL and NTL in tumor-bearing mice [7]. Fig. 10 depicts the results of such a biodistribution experiment comparing FTL with NTL in tumor (M109-HiFR)bearing mice. The main conclusions of our recently published biodistribution studies [7] are: i) FTL retain the folate ligand in vivo, even after prolonged circulation and extravasation into malignant ascitic fluid. ii) Liver uptake of FTL is greater and faster in comparison to NTL, resulting in lower plasma levels of the former (Fig. 10). iii) Tumor levels of FTL-injected mice in mouse M109 and human KB models are not significantly different from those of NTL-injected mice (Fig. 10), although kinetically there is a trend for greater FTL deposition in the tumor at early time points (6 h) and greater NTL deposition at late time points (48 72 h). iv) Liver uptake of FTL is significantly reduced by concomitant injection of a large dose of free folic acid. However, tumor levels of FTL remain unaffected by such a co-dose of folic acid suggesting that liposome accumulation in tumors is dictated by liposome extravasation rate rather than by binding to FR. v) In an ascitic tumor model that enables discrimination between the tumor cell compartment and the extracellular fluid, tumor cell-associated liposome levels were significantly greater for FTL-injected mice than for NTL-injected mice, indicating that folate targeting shifts liposome distribution from the tumor extracellular space towards association with FR-expressing tumor cells.

Fig. 8. Plasma and tumor levels of 3H-CHE labeled liposomes 24 h after i.v. injection of 2 Amol phospholipid per mouse in M109-FR bearing mice. Higher plasma and tumor levels were observed after injection of PEGylated FTL (FTL with PEG) as compared to nonPEGylated FTL (FTL w/o PEG). Differences in plasma and tumor levels between FTL with PEG and FTL w/o PEG were statistically significant, P = 0.0021 and P = 0.0114 (t-test), respectively.

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Fig. 9. Pharmacokinetics of FTL in rats fed normal or folate-deficient diet. Data were obtained from the mean of two experiments, with four rats (Simonsen Albino, Gilroy, CA) per group in each experiment. Liposomes radiolabeled with a 67Ga-deferoxamine complex encapsulated in the liposome water phase were prepared as previously described and injected into the rat tail vein [39] at a dose of 6 8 Amol per rat. Figure inset shows tissue levels 24 h after injection.

Fig. 10. Tissue distribution of NTL and FTL (2 Amol per mouse) 48 h after i.v. injection in M109-HiFR tumor-bearing BALB/c female mice. There were five mice per group, and two subcutaneous tumor implants per mouse. Liposomes were radiolabeled with 3H-CHE. Plasma, spleen and kidney levels of NTL were significantly higher than those of FTL at P levels of 0.0001, 0.0144, and 0.0017, respectively, while liver levels of FTL were significantly higher than those of NTL at P < 0.0001. Tumor and skin levels were not significantly different.

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The in vivo tumor uptake of FTL loaded with boron-containing compounds has also been investigated in the M109 and KB FR-expressing tumor models by Lee and colleagues. Their results show similar [6] or slightly greater [33] levels for FTL as compared to NTL peaking at 24 h after injection.

6. Therapeutic effects of FTL-encapsulated drugs 6.1. Folate-targeted PEGylated (STEALTHR) liposomal doxorubicin (FTL-Dox) We have examined the activity of FTL-Dox in three tumor models. Initially, we tested the M109-HiFR tumor. NTL-Dox (DOXILR) and FTL-Dox were both highly and equally effective against this tumor achieving a high percentage of cures and a major improvement in activity over free Dox (unpublished data). The second model tested was the M109R-HiFR tumor. This tumor has an MDR phenotype conferring resistance to doxorubicin. Given our in vitro data indicating that folate-mediated drug delivery can bypass the MDR

efflux pump, and enhance drug uptake, we reasoned that FTL-Dox will be more active than free drug and NTL-Dox in vivo. However, here again, FTL-Dox and NTL-Dox were equally active and both were more active than free drug (Fig. 11). These experiments were done in mice under a normal, folate-enriched diet. In a third model, J6456-HiFR cells which quickly downregulate the FR in animals under normal diet, were tested with FTL-Dox in this under a folate-depleted diet (Fig. 12). The use of folate-depleted diet was complicated by the increased sensitivity of these mice to toxic effects of chemotherapy. In fact, NTL-Dox (DOXILR) was highly toxic in animals under a folate-depleted diet (100% deaths). FTL-Dox was toxic to a small fraction (30%) of the animals while retaining a strong antitumor activity. The reason for this difference is still unclear although it is conceivable that the small amount of folate present in conjugate of the FTL-Dox formulation can rescue the animals from lethal toxicity. At any rate, the toxicity issue precludes a net assessment of a therapeutic advantage of FTL-Dox. A recently published study on the therapeutic efficacy of FTL-Dox of similar composition to

Fig. 11. Therapeutic test of FTL-Dox in mice inoculated with M109R-HiFR (MDR+ tumor). 106 cells were implanted into the footpad of BALB/c male mice. Formulations were injected i.v. on days 9, 17, 35, and 42 at a dose of 8 mg/kg Dox (total 32 mg/kg). There were 10 mice per group (free Dox, NTL-Dox, FTL-Dox). Curves show the probability of tumor growth control and survival. Free Dox vs. NTL-Dox or FTLDox, P < 0.0001; NTL-Dox vs. FTL-Dox, not significant (log rank test).

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Fig. 12. Therapeutic test of FTL in mice inoculated with J6456-HiFR and fed a folate-deficient diet. BALB/c female mice were inoculated i.p. with 106 J6456-FR lymphoma cells. Seven days later, mice were treated i.v. with 10 mg/kg, NTL-Dox (DOXIL), or FTL-Dox. Survival was recorded and analyzed by log rank test. Mice were fed a folate-deficient diet from 1 week before tumor inoculation till 1 week after treatment. All mice treated with NTL-Dox died of toxicity. Only 3/10 mice treated with FTL-Dox died of toxicity. The difference between NTL-Dox and FTL-Dox is significant by log rank test at P = 0.0032.

STEALTHR points to significantly greater tumor inhibition as compared to NTL-Dox in the KB carcinoma model growing in nude mice fed a folate-free diet [34]. However, there are two methodological issues requiring cautious interpretation: the drugs used in that study were administered i.p. instead of i.v., a factor that may distort the pharmacokinetics of liposomal vehicles, and the doxorubicin dose (10 mg/kg 6 injections) is far above the mouse LD50 (reviewed in [35]). Another published study on the therapeutic efficacy of FTL-Dox deals with upregulation of FR-h expression1 in acute myelogenous leukemia with alltrans retinoic acid to render these cells more sensitive to the targeted agent [36]. In these mouse ascites leukemia models, FTL-Dox was more efficacious than NTL-Dox using, as above, the i.p. treatment route.

6.2. Folate-targeted PEGylated (STEALTHR) liposomal cisplatin (FTL-cisplatin) A formulation of FTL-cisplatin was prepared in our laboratory by post-insertion of folate PEG DSPE into a PEGylated liposomal cisplatin formulation (SPI-77, provided by ALZA) and tested in early clinical studies [37]. SPI-77 is definitely less toxic than free cisplatin, but, at the same time, it appears to be significantly less active in several tumor models [38] thus rendering its therapeutic potential of limited value. Preliminary results have been presented [29] indicating that the folate-targeted preparation is more effective than the nontargeted one, yet not more effective than free cisplatin in the M109-HiFR tumor model. Further studies with FTLcisplatin are ongoing. There are marked differences between the FTLcisplatin and FTL-Dox formulations: in the former, the drug-to-lipid ratio is f 5-fold lower, and the drug release rate is much slower [38]. From the data gathered on these two formulations in folate-targeted form, it is evident that properties of the basic formu-

1 FR-h, a receptor of lower affinity as compared to FR-a, is often expressed in CD34+ bone marrow cells in inactive form and in some leukemias in active form. In most tumors and epithelial tissues, dealt with in this review, FR-a is expressed.

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lation have important implications on pharmacologic performance and that conclusions cannot be extrapolated from one formulation to another.

7. Toxicity of cytotoxic drugs encapsulated in FTL Except for strictly phase-specific drugs, liposome encapsulation generally reduces toxicity of cytotoxic drugs, such as doxorubicin and cisplatin [35]. Limited information is available on the toxicity of these drugs when delivered by FTL. The presence of FR in normal tissues such as liver and kidney may raise the concern of toxicity to these tissues when liposomal drugs are targeted with folate. As seen in the previous section, our experience from therapeutic trials with Doxil suggests that FTL-Dox is tolerated at least as well as NTL-Dox. Similar results have been obtained when FTL-cisplatin is compared to SPI-77, both preparations being several-fold less toxic than cisplatin [29]. Other published therapeutic studies with FTL and doxorubicin have not reported on any unexpected toxicities. The results of tissue distribution studies suggest that enhanced toxicity to kidney is unlikely since FTL show actually a slightly decreased uptake by kidney as compared to NTL [7]. This is not a surprising finding given the fact that the FR of kidney tubular cells is located in the luminal (urinary) side which can only be accessed by molecules undergoing glomerular filtration [10], meaning that this compartment is clearly inaccessible to intact liposomes. Thus, although the data are still preliminary, the concerns that folate targeting may worsen liposomal drug toxicity appear to be unfounded.

addressed are related to the optimal density of folate ligands on the liposome surface and to the interference of PEG coating with FRME. They may have an impact on in vivo liposome clearance and liposome interaction with tumor cells. Studies evaluating the toxicity of various dose levels of drug-loaded FTL and comparing it with that of their nontargeted counterparts are also required. Finally, therapeutic studies that (i) cover a broad range of tumor models and dose levels and (ii) address the issue of modulation of FR expression by dietary folate content and/or treatment need yet to mature. In agreement with the principle that liposome extravasation is the rate-limiting step of tumor localization, the current body of data does not support the claim that folate targeting increases to a sizable extent the overall liposome concentration in subcutaneouslygrowing tumor implants. However, folate targeting may still lead to significant pharmacodynamic changes with improvement of the therapeutic index by shifting drug distribution from the extracellular to the intracellular tumor compartment while systemic toxicity is left unchanged or even reduced.

Acknowledgements This work was supported in part by the Israel Science Foundation, by the Israel Society against Cancer, and by ALZA Corporation (Mountain View, CA). We wish to thank the technical help all along these studies of Dina Tzemach, and Lidia Mak (Shaare Zedek Medical Center). We also wish to acknowledge Charles Engbers and Mary Newman (ALZA Corp., and formerly SEQUUS Pharmaceuticals) for performing the rat pharmacokinetic experiments.

8. Concluding remarks Although the use of liposomes as passive devices for drug delivery in cancer has recently taken a firm hold in our standard clinical armamentarium, the concept of ligand-mediated active liposome targeting still needs further experimental proof of validity in animal models and surely in clinical trials. A number of important questions remain open and need further testing before the added value of folate targeting to liposome delivery can be thoroughly evaluated at a preclinical level. Formulation issues that need to be References
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