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Advanced Drug Delivery Reviews 56 (2004) 1177 – 1192 www.elsevier.com/locate/addr Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid–PEG 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 phospholipi
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  Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid–PEG 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 chemotherapeuticagents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached tothe outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combinelong-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although anumber 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 enhancesthe cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary andappear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting canconfer 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11781.1. Folate-targeted liposomes (FTL) versus nontargeted liposomes (NTL) . . . . . . . . . . . . . . . . . . . . . . . 11781.2. FTL versus nonliposome-based folate-targeted systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11792. FR expression and tumor models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11803. Formulation issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11813.1. Achieving prolonged circulation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11813.2. Optimization of the PEG-folate conjugate concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11813.3. PEG steric interference with binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11823.4. Insertion versus conventional incorporation of folate–PEG–lipid into liposomes . . . . . . . . . . . . . . . . . . 11830169-409X/$ - see front matter   D  2004 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2004.01.011* 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).www.elsevier.com/locate/addr Advanced Drug Delivery Reviews 56 (2004) 1177–1192  4. In vitro studies with FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11834.1. Kinetics of liposome binding to tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11834.2. Delivery of doxorubicin encapsulated in FTL to tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 11844.3. In vitro cytotoxicity of doxorubicin encapsulated in FTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11855. Pharmacokinetics and tissue distribution studies with FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . . . . . 11866. Therapeutic effects of FTL-encapsulated drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11886.1. Folate-targeted PEGylated (STEALTH R ) liposomal doxorubicin (FTL-Dox) . . . . . . . . . . . . . . . . . . . 11886.2. Folate-targeted PEGylated (STEALTH R ) liposomal cisplatin (FTL-cisplatin) . . . . . . . . . . . . . . . . . . . 11897. Toxicity of cytotoxic drugs encapsulated in FTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11908. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190 1. Introduction The rationale for cancer targeting with folateligands attached to the liposome surface is based ontwo layers. First, there is a common layer to all folate-targeted systems which relates to the choice of thetumor cell folate receptor (FR) as the target. FR upregulation or over-expression is commonly associ-ated with a broad variety of tumor types includingsolid and hematological malignancies, and it appearsto be more frequently observed in advanced stages of cancer  [1]. How specific and frequent is FR over-expression 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 secondlayer contains elements unique to liposomal and perhaps other nanoparticulate drug carrier systemsand will be addressed here. The strength of thefolate-targeted liposome approach stems from concep-tual advantages over two alternative approaches: non-targeted liposomal systems and nonliposome-basedfolate-targeted systems. 1.1. Folate-targeted liposomes (FTL) versus non-targeted liposomes (NTL) A schematic illustration of the folate liposometargeting concept is presented in Fig. 1. Long-circu- lating liposomes, such as polyethylene glycol (PEG)coated liposomes (also known as STEALTH R  lip-osomes) [2], tend to accumulate in tumors as a result of increased microvascular permeability and defectivelymphatic drainage, a process also referred to as theenhanced permeability and retention (EPR) effect  [3].This is a passive and nonspecific process of liposomeextravasation that is statistically improved by the prolonged residence time of liposomes in circulationand repeated passages through the tumor microvascu-lar bed [4]. However, except for rare instances, tumor  cells are not directly exposed to the blood stream.Therefore, for an intra-vascular targeting device toaccess the tumor cell FR, it must first cross thevascular endothelium and diffuse into the interstitialfluid. Experimental data with antibody-targeted lip-osomes and FTL indicate that liposome deposition intumors is similar for both targeted and nontargetedsystems [5–7], supporting the hypothesis that extrav- asation is indeed the rate-limiting step of liposomeaccumulation in tumors. However, once liposomeshave penetrated the tumor interstitial fluid, bindingof targeted liposomes to FR may occur thus shiftingthe intra-tumor distribution from the extracellular compartment to the tumor cell compartment, as shownrecently for a mouse ascitic tumor  [7]. Binding to tumor cells may be followed by internalization of liposome contents via folate-receptor mediated endo-cytosis (Fig. 1). Retrograde movement of liposomes into the blood stream, if any, will be reduced for liposomes with binding affinity to a tumor cell recep-tor. Obviously, when the parameter of drug delivery isconsidered, there will always be a combination of insitu release from an extracellular liposome depot andintra-cellular release from internalized liposomes.Therefore, the theoretical advantages of FTL over  NTL are related to a shift of liposome distribution tothe tumor cell compartment, delivery of liposomalcontents to an intra-cellular compartment in liposome-associated form, and, possibly, prolonged liposomeretention in the tumor. On the negative side, the maindisadvantage of a targeted system to a cancer cell  A. Gabizon et al. / Advanced Drug Delivery Reviews 56 (2004) 1177–1192 1178  receptor such as FR is the difficulty of a large nano-size 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 deliveryamplification system. Each liposome vesicle carries adrug cargo usually in the order of 10 3  –10 4 molecules.For instance, in the case of a STEALTH R  liposomeformulation known as Doxil, there are between10,000 and 15,000 doxorubicin molecules per vesicle[9], and these may be targeted with the help of as fewligands as 10 per vesicle, i.e. a 100–1000-fold deliv-ery amplification factor when the drug:ligand ratio isconsidered. Another theoretical advantage of liposo-mal systems is that their size far exceeds the criticalglomerular filtration threshold. Therefore, unlike lowmolecular weight folate-targeted complexes, FTL donot have access to the luminal side of kidney tubular cells where FR is expressed, thereby sparing kidneysof massive FR-mediated liposomal drug delivery andsubsequent toxicity [10]. One of the disadvantages of  FTL vis-a`-vis small drug–folate conjugates is that liposomes are bulky structures that are difficult tointernalize by nonphagocytic cells. The best charac-terized 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 withcell caveolae or lipid rafts, such as FR  [10], and facilitate access to the cytosol via acidic endosomes bypassing lysosomes. It is well established that FTLenter cells by FR-mediated endocytosis (FRME) [11]. In addition, experimental data with folate-targeted, pH-sensitive liposomes are consistent with liposome Fig. 1. Schematic drawing illustrating the concept of folate targeting of liposomes to tumor cells. The blue dots represent the liposomal folateligands. The red dots represent the drug molecules encapsulated in the liposome water phase. The various steps involved in the targeting processare 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 graduallyreleased from liposomes remaining in the interstitial fluid and enters tumor cells as free drug to exert a cytotoxic effect. (4) Other liposomes bindto 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) Internalizedliposomes release their drug content in the cytosol enabling the drug to exert its cytotoxic effect.  A. Gabizon et al. / Advanced Drug Delivery Reviews 56 (2004) 1177–1192  1179  transit through an acidic vesicle compartment  [12]. Aconnection between post-caveolar or post-raft endo-somes and lysosomes is possible, since markers of theclathrin-coated pit pathway and folate conjugates have been shown to co-localize in the same cell organelles[13]. Thus, an important fraction of internalized lip-osomes may end up in lysosomes. The cell traffickingof liposomes following FRME needs to be better understood, specially since intra-cellular traffickingof small molecular weight folate conjugates may bedifferent from that of nanoparticles with multimeric binding such as FTL. 2. FR expression and tumor models A prerequisite for investigation of any targetedsystem is the availability of tumor models with stableoverexpression of the target receptor. Routine cellculture conditions expose tumor cells to high folateconcentrations so that even if a fresh tumor explant overexpresses FR, in vitro culture may graduallycause downregulation of FR. The standard approachwe have used to generate a FR-overexpressing cellline is to cultivate the cells in a folate-free culturemedium. FR upregulation is a common response of cells grown in a folate-depleted environment. Manytumor cell lines respond to folate-depleted cultureconditions with upregulation of FR. This is generallya reversible process, i.e. when folate supplies arerestored FR is downregulated [14]. Therefore, FR- overexpressing cell lines should be maintained infolate-free medium. The addition of 10% nondialyzedserum to folate-free medium results in a sub-physio-logic concentration of folic acid (3 nM) under whichthese cell lines maintain high FR expression [14].We have studied several animal tumor modelsoverexpressing the FR, including mouse M109 carci-noma and its multidrug-resistant cells (MDR) subline, M109R  [14], the human KB car cinoma [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 KBtumors [14]. A high FR-expressing J6456 subline wassimilarly obtained by a single in vivo passage of tumor cells followed by repeated in vitro passage ina folate-free culture medium.Baseline information on the uptake of free folicacid and on the effect of folate-depleted diet onreceptor 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 bythe diet within the short time frame required for atissue distribution study, experiments with this tumor model and with the KB human carcinoma (another well-established model of inducible and stable highFR expression [11,13,16]) proceeded with animals on normal diet. In contrast, J6456 lymphoma quicklydownregulated FR in animals with a normal, folate-enriched diet (Table 1). Therefore, experiments with the J6456-HiFR should be carried out preferably inanimals maintained on a folate-depleted diet. Theresults of folic acid uptake and targeted versus non-targeted liposomal upt ake 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 inliposome uptake when FTL are compared to NTL.The importance of using a folate-depleted diet in invivo experiments with folate-targeted systems has been questioned. Clearly if tumor FR expression isquickly downregulated under a folate-rich diet, then Table 1Folic Acid (F.A.) and liposome uptake of J6456 and J6456-HiFR tumor cell lines a  Cells/source  3 H-F.A.fmole/10 6 cells 3 H-CHE-NTL pmole/10 6 cells 3 H-CHE-FTL pmole/10 6 cellsJ6456 (parental line) 2 F 1 215 F 12 199 F 23J6456-HiFR (in vitro F.A.-depleted medium) 14,675 F 1403 286 F 29 3719 F 340J6456-HiFR (mice on normal diet) 186 F 127 Not done Not doneJ6456-HiFR (mice on F.A.-depleted diet) 5660 F 931 430 F 8 1470 F 133 a  J6456 cells (parental line) obtained from in vitro passage using standard (folate-rich) culture medium. J6456-HiFR cells were obtainedfrom 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  j Cfor 30 min in the presence of radiolabeled F.A. (0.1 pmol/ml), and for 24 h in the presence of   3 H-CHE (cholesterol hexadecyl ether) labeled NTLor FTL (300 nmol phospholipid/ml). Results are expressed as fmol F.A. per million cells, or pmol phospholipid per million cells.  A. Gabizon et al. / Advanced Drug Delivery Reviews 56 (2004) 1177–1192 1180
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