In Vivo Fate of Folate-Targeted Polyethylene-Glycol Liposomes in Tumor-Bearing Mice

Vol. 9, 6551–6559, December 15, 2003 Clinical Cancer Research 6551

Alberto Gabizon,1 Aviva T. Horowitz,2 higher tumor:skin, and tumor:kidney ratios but lower tu- Dorit Goren,2 Dina Tzemach,1 Hilary Shmeeda,1 mor:liver ratio than NTLs. After a concomitant dose of free and Samuel Zalipsky3 folic acid, FTLs (but not NTLs) plasma clearance and liver uptake were inhibited, indicating that accelerated clearance 1Shaare Zedek Medical Center and 2Hadassah Medical Center, Jerusalem, Israel and 3ALZA Corporation, Mountain View, California was mediated by the folate ligand. Surprisingly tumor uptake was not significantly affected by a codose of folic acid. In the J6456 ascitic tumor model, tumor cell-associated ABSTRACT liposome levels were significantly greater for FTL-injected Purpose: To compare the in vivo tissue distribution of mice than for NTL-injected mice, despite slightly higher folate-targeted liposomes (FTLs) injected i.v. in mice bear- levels of the latter in whole ascites. ing folate receptor (FR)-overexpressing tumors (mouse Conclusions: Whereas folate targeting does not enhance M109 and human KB carcinomas, and mouse J6456 lym- overall liposome deposition in tumors, the targeting profile phoma) to that of nontargeted liposomes (NTLs) of similar of tumor versus other tissues is substantially different and composition. intratumor liposome distribution in ascitic tumors is af- Experimental Design: A small fraction of a folate-poly- fected favorably with a selective shift toward liposome asso- ethylene-glycol (PEG)-distearoyl-phosphatidylethanolamine ciation with FR-expressing cells. conjugate was incorporated in FTLs. Both FTLs and NTLs were PEGylated with a PEG-distearoyl-phosphatidylethanolamine conjugate to prolong circulation time. Liposomes INTRODUCTION were labeled with [3H]cholesterol hexadecyl ether with or Receptor-mediated endocytosis pathways can be exploited without doxorubicin loading. Liposome levels in plasma, for specific targeting of liposomes and intracellular delivery of tissues, or ascites were assessed by the number of [3H] liposome contents (1, 2). Coupling liposomes to a ligand, that is counts. For doxorubicin-loaded formulations, we also deter- directed to an over-expressed receptor in cancer cells and that mined the tissue doxorubicin levels by fluorimetry. To esti- normally undergoes endocytosis, is a strategy that can improve mate the amount of liposomes directly associated with tumor selectivity and facilitate access of liposomes to the intracellular cells in vivo, we determined the [3H]radiolabel counts in compartment. Folic acid (FA) is one of the well-studied target- washed pellets of ascitic tumor cells using the ascitic J6456 ing ligands used for this strategy. Macromolecules and particu- lymphoma late carriers, conjugated to FA are successfully recognized by Results: FTLs retained the folate ligand in vivo,as folate receptors (FRs) and internalized into cells via folate- demonstrated by their ability to bind ex vivo to FR-express- receptor mediated endocytosis (3–5). Cell surface receptors for ing cells after prolonged circulation and extravasation into FA are overexpressed across a broad spectrum of human tumors malignant ascitic fluid. In comparison with NTLs, FTLs (6, 7). The FR is a glycosyl-phosphatidylinositol-anchored gly- ϳ Ϫ10 were cleared faster from circulation as a result of greater coprotein with high affinity for the FA vitamin (Kd 10 M; liver uptake. Despite the lower plasma levels, tumor levels of see Refs. 8, 9). It is located in caveolae and participates in the FTL-injected mice were not significantly different from cellular accumulation of folates through the process of potocy- those of NTL-injected mice. When NTLs and FTLs were tosis. In this process, receptor-bound ligand is sequestered in loaded with doxorubicin, liver uptake decreased because of caveolae, internalized into postcaveolar plasma vesicles, re- liver blockade, and uptake by spleen and tumor increased. leased from the receptor via an intravesicular reduction in pH, When tumor-to-tissue liposome uptake ratios were ana- and subsequently transported into cytoplasm for polyglutama- lyzed, the targeting profile of FTLs was characterized by tion (10, 11). The receptor is then recycled to the cell surface. The lack of immunogenicity and relatively simple chemistry of FA make folate-receptor mediated endocytosis a very useful tool in specific drug targeting. The relevance of FR as a useful target for tumor-specific Received 6/17/03; revised 8/28/03; accepted 8/28/03. drug delivery is supported by findings indicating up-regulation Grant support: This work was supported by research grants from the (higher expression) in many human cancers including those of Israel Science Foundation (Jerusalem, Israel) and ALZA Corporation (Mountain View, CA). the ovary, brain, kidney, lung, breast, and myeloid cells (12). In The costs of publication of this article were defrayed in part by the addition, aggressive or undifferentiated tumors with advanced payment of page charges. This article must therefore be hereby marked stage or grade appear to have an increased FR density (13, advertisement in accordance with 18 U.S.C. Section 1734 solely to suggesting that FR-mediated delivery may be a broad approach indicate this fact. Requests for reprints: Alberto Gabizon, Oncology Institute, Shaare in cancer treatment. Zedek Medical Ctr., POB 3235, Jerusalem, il-91031, Israel. Phone: In previous studies, we have investigated the in vitro bind- 972-2-6555-036; Fax: 972-2-652-1431; E-mail: [email protected]. ing of folate-targeted liposomes (FTLs) to tumor cells express-

ing FR, and the process of in vitro delivery of an anticancer described previously (17). Unencapsulated doxorubicin was re- drug, doxorubicin, to tumor cells via FTLs (14, 15). We used a moved by a small Dowex resin column. The final doxorubicin/ conjugate of three components incorporated in the liposome phospholipid ratio was in the range of 100–150 ␮g/␮mol. Other bilayer to target liposomes to the FR (14): FA, polyethylene- parameters were similar to those for drug-free liposomes. glycol (PEG), and distearoyl-phosphatidyl-ethanolamine Animal Models and Tumors. We used ϳ8-wk-old SPF (DSPE). The folate group is located at the outer end of PEG, female BALB/c mice and Swiss CD1 athymic/nude mice in

away from the bilayer. Because methoxy-PEG-DSPE [PEG, Mr these studies. Mice were purchased from Harlan Breeding Lab- 2000; designated as mPEG(2000)] is commonly used as a lipo- oratories (Jerusalem, Israel) and maintained in a SPF facility at some component to prolong liposome circulation time, we chose Hadassah Medical Center with food and water ad libitum. All animal experiments were done under a protocol approved by the a longer PEG length [PEG, Mr 3350; designated as PEG(3350)] for the folate-PEG-DSPE conjugate to reduce steric interference Hebrew University-Hadassah Institutional Review Board for with receptor binding. These FTLs bind avidly and are internal- use of animals in research. In some of the experiments, mice ized by FR-expressing tumor cells, although mPEG surface were fed a special low-folate diet (Harlan Tekled, Madison, WI) coating of liposomes still interferes significantly with this proc- from 1 week before tumor inoculation and until mice were ess (14). We also found that FTLs can deliver efficiently doxo- sacrificed for tissue distribution studies. The tumor models used rubicin to tumor cells and bypass the drug efflux mechanism here are the mouse M109 (18), and human KB (19) carcinomas, characteristic of multidrug resistance (15). A critical step for and the mouse J6456 lymphoma (20). High FR-expressing cells evaluating the potential of FTLs as drug-delivery systems in were selected from these tumor cell lines as described previ- cancer therapy is to study the fate of these systems after i.v. ously for M109 and KB tumors (14). The characteristics of the injection and determine whether or not they confer any advan- high-FR J6456 subline will be presented in a separate report. 6 tage in tumor localization and/or intratumoral distribution of the M109 tumor cells (10 cells/0.2 ml) or KB tumor cells (2 ϫ 6 carrier. In this study, we investigated the biodistribution of 10 cells/0.2 ml) in serum-free medium suspensions were inoc- radiolabeled and doxorubicin-loaded FTLs in tumor-bearing ulated in the s.c. space of the mouse right and left flank of mice and compared it with that of nontargeted liposomes BALB/c or CD1 nude mice, respectively. Two to 3 weeks after (NTLs) of similar composition and size. inoculation, mice injected with M109 or KB cells developed palpable solid tumors. Mice used in these biodistribution experiments had tumors weighing between 10 to 299 mg. Three to MATERIALS AND METHODS five mice were used for each time point of each experimental Liposome Preparation. Liposome formulations were group. The interanimal variations in tumor uptake were greater prepared by standard methods of thin lipid film hydration and than those for other normal tissues. To correct for this higher polycarbonate membrane extrusion down through 0.05-␮m variance, each mouse was inoculated with two tumor inocula, pores as reported previously (14). Hydrogenated soybean phos- one in each flank, so that tumor values were the mean of a phatidyl-choline was from Avanti (Birmingham, AL) or Lipoid greater number of samples than for other tissues, generally six or (Ludwigshafen, Germany), cholesterol was from Sigma (St. more for each time point and treatment group examined. Lipo- Louis, MO), and mPEG(2000)-DSPE was a gift from ALZA somes were injected i.v. at dose levels of 2 to 5 ␮mol phospho- (Mountain View, CA). Folate-derivatized PEG(3350)-DSPE lipid/mouse. When doxorubicin-loaded liposomes were used, was synthesized at ALZA as described previously (14). FTLs the dose of doxorubicin was 200 ␮g/mouse, (equivalent to 10 were composed, on a molar ratio basis, of 55% hydrogenated mg/kg for a 20-g mouse). [3H]FA (Amersham) was also injected soybean phosphatidyl-choline, 40% cholesterol, 4.7% i.v. into tumor-bearing mice for biodistribution experiments. mPEG(2000)-DSPE, and 0.3% of folate-PEG(3350)-DSPE. When a codose of FA was used in the experiment, cold FA was NTLs were identical to FTLs except for 5% of mPEG(2000)- diluted in NaBic solution (0.84% sodium bicarbonate in 90% DSPE and no folate-PEG-DSPE. These preparations were ra- physiological saline) and injected i.p. at a dose of 4–5 mg/ml/ diolabeled with [3H]cholesterol-hexadecyl ether (3H-CHE; Am- mouse immediately before i.v. injection of liposomes or ersham, Buckinghamshire, United Kingdom) at a specific ratio [3H]FA. of ϳ0.5 ␮ci/␮mol phospholipid. The use of [3H]CHE is con- In the J6456 lymphoma model, BALB/c mice were inoc- venient for these studies because it is a stable, nonexchangeable, ulated i.p. with 106 J6456 cells in 0.2 ml serum-free medium. and nondegradable marker of liposomes (16), thus providing an After 2 to 3 weeks, abdominal swelling developed, indicating estimate of the cumulative liposome dose in tissue. Liposomes peritoneal tumor spread and ascites, at which point liposomes were suspended in dextrose 5% buffered with 15 mM HEPES were injected i.v. for the designed experiments. (pH 7.0). All formulations were analyzed for phosphorus con- Biodistribution Studies. At scheduled time points after

tent (Bartlett method), folate content (absorbance, A285 nm), ra- liposome injection, mice were anesthetized by ether or halo- dioactivity [counts per minute (cpm) in a ␤ scintillation counter] thane inhalation, bled by eye enucleation (Ͼ1 ml of blood/ and vesicle size (dynamic laser scattering). Final phospholipid mouse) and immediately sacrificed by cervical dislocation. Tu- concentration was around 20 ␮mol/ml; folate content and mors and other indicated organs (liver, spleen, kidneys, skin) [3H]CHE cpm/mol phosphorus were close to the relative input were removed, rinsed in physiological saline, weighed, and preparation ratios; mean vesicle size was in the range of 70 to 90 frozen at Ϫ20°C until further processing. Blood was collected in nm with SD Ͻ30% of the mean. heparinized tubes and centrifuged immediately to separate In some experiments, radiolabeled FTLs and NTLs were plasma from blood cells. Plasma cpm were measured in a ␤ loaded with doxorubicin using an ammonium sulfate gradient as counter after dilution of plasma in scintillation fluid (50 ␮l/5 ml

Fig. 1 Biodistribution of [3H]folic acid (3H-FA) 3 h after i.v. injection into tumor-bearing [M109-folate receptor (M109-FR) or M109] BALB/c mice. A, tissue distribution in mice fed normal or folate-depleted diet. Statistical analysis (t test): M109-folate receptor versus M109 (regardless of diet), P ϭ 0.0201; liver with folate-depleted diet versus liver with normal diet, P ϭ 0.0193; kidney with folate-depleted diet versus kidney with normal diet, P Ͻ 0.0001. B, effect of cold codose of folic acid (3H-FA ϩ FA; 5 mg/mouse i.p. in NaBic solution, immediately before i.v. [3H]folic acid). All comparisons were statistically significant at P Ͻ 0.0001 (t test).

of Quick Safe A; Zinsser Analytic, Maidenhead, United King- RESULTS dom). Frozen tissue samples were incinerated in a Packard Biodistribution of Radiolabeled Free FA. As a first Sample Oxidizer, model 307 (Downer Grove, IL). The resulting step, it was important to examine the biodistribution of the radioactive water was diluted in scintillation fluid and measured ligand, FA, in soluble form. For this, we injected i.v. [3H]la- ␤ 3 in a counter. The number of cpm of [ H]CHE of each plasma beled FA (500,000 cpm/mouse) into BALB/c mice bearing s.c. or tissue sample was used to calculate the percentage of injected implants of M109-FR tumors and of the low-FR-expressing dose per milliliter of plasma or gram of tissue based on the M109 parental tumor. In a separate experiment, mice received number of cpm injected per mouse. an additional large codose of unlabeled (cold) FA (4–5 mg) by When mice received injections of doxorubicin-loaded, ra- the i.p. route. The results obtained in mice on normal diet and diolabeled liposomes, the removed tissues were split in two mice on folate-depleted diet, and with or without a codose of pieces. One piece was processed for examining the radioactive unlabeled (cold) free FA, are shown in Fig. 1 (A and B). Plasma counting as described above, and another piece was processed levels were very low, indicating that most of the injected FA for doxorubicin content by high-performance liquid chromatog- was cleared from plasma within 3 h. The following observations raphy and fluorescence detection as described previously (17). were also made: In mice inoculated i.p. with J6456 lymphoma, ascites was collected after mice were bled and sacrificed by injecting 3 ml (a) There was an increased uptake in the M109-FR tumor of PBS into the abdominal cavity of each mouse. Rinsing with (2.5-fold in mice on normal diet). This is expected, although the a large volume of PBS is essential for obtaining a representative ratio in FR expression between the M109-FR and M109 cell sample collection of fluid and cells present in the abdominal lines is much greater, ϳ100-fold. cavity. After the animal body was swung from the tail for a few (b) Folate-depleted diet resulted in a sharp increase (ϳ2.5 seconds to mix well the buffer with the abdominal contents, the fold) in kidney uptake of FA. This is likely to have been the fluid collection was aspirated and immediately centrifuged to result of an up-regulation of FR in the kidney tubular cells to separate cells from ascitic fluid. The cells were washed twice rescue the greatest possible amount of FA from urine under more with PBS, counted, pelleted, and then solubilized in scin- conditions of folate deprivation. The change in liver uptake was tillation fluid for ␤ radioactive counting. Consistent with previ- minimal. ous observations, Ͼ95% of the cells appeared to be tumor cells (c) Diet had an insignificant effect on tumor uptake of FA, by light microscopy. Samples of ascitic fluid and of whole suggesting that the degree of up-regulation or down-regulation ascites, represented by the unfractionated aspirate, were also of M109 tumor FR expression was insufficient to cause a taken for radioactive counting. The amount of radioactivity significant change in folate uptake in vivo. expressed as phospholipid-equivalents, on the basis of the (d) A codose of cold FA competed effectively with the [3H]CHE/phosphate ratio in the liposome preparation, was cal- labeled material for tissue uptake, including the high- and culated for whole ascites and for 106 ascitic cells for each low-FR tumors. mouse. Retention of Folate Ligand by Circulating Liposomes. Statistical Analysis. Nonpaired t test was used for com- Before examining further the biodistribution of FTLs, it was parison of mean liposome levels in various tissues, except for important to determine whether the folate ligand was retained by the experiments with J6456 lymphoma, in which we used the liposomes in vivo, particularly after they exited the blood stream nonparametric Mann-Whitney test. and entered the interstitial fluid compartment, allowing for

only a minor part of the difference in plasma levels (data not shown). As seen in Fig. 3B and described below, FTLs accumulated in the liver at much greater levels than NTLs within 24–48 h after injection, indicating that the liver accounted for most of the difference in plasma clearance. This was possibly related either to recognition of the folate ligand and opsonization by plasma folate binding protein or to direct recognition of liposomal folate by the liver FR. Tumor uptake (Fig. 3B) showed different kinetics from liver uptake. During the first 6 h after injection, FTLs had a slight advantage over NTLs in tumor accumulation. This was reversed at 48 h in favor of NTLs. These differences in tumor uptake reached statistical significance in favor of NTLs at 48 h Fig. 2 Folate-targeted liposomes retain folate ligand and folate-recep- ϭ tor binding ability after in vivo passage. Results of in vitro liposome in this experiment (P 0.0436, t test), although in additional binding (40 nmol/ml phospholipid) to M109-folate receptor cells: experiments focusing on the 48–72 h time point (see next [3H]cholesterol-hexadecyl ether-labeled liposomes were recovered from section) no statistical significance was detected. Between 48 to ␮ ascitic fluid 48 h after i.v. injection (5 mol phospholipid/mouse). 96 h, there was actually a drop in liposome concentration in Control liposomes were either untreated or incubated in the presence of ascitic fluid. tumor for both FTLs and NTLs, which was probably related to a label dilution effect attributable to tumor growth with minimal input from the liposome-depleted blood compartment. Effect of Folate Targeting on Tissue Distribution of targeting to take place. Given the fact that PEG-3350 and folate Liposomes in Tumor-Bearing Mice. To further study the constituted a large hydrophilic moiety, anchored into the bilayer effect of folate targeting on tissue distribution and tumor uptake, by the DSPE lipophilic moiety, there was a possibility of dis- sociation of the entire FA-PEG-DSPE construct from the liposome structure under in vivo conditions (21). We chose to address this issue using an ascitic mouse model in which i.p. tumor inoculation resulted in production of ascites with increased vascular permeability of the peritoneal membrane. After i.v. injection liposomes extravasate gradually into the peritoneal cavity. We recovered ascitic fluid rich in liposomes and used in vitro binding to high FR-expressing cells for testing. As seen in Fig. 2, FTLs were capable of binding to target cells after a 48-h in vivo passage with only a minor decrease (Ͻ20%) from the original uptake of fresh liposomes. The ascitic fluid itself inter- fered slightly with binding of FTLs to target cells. Incorporation of Folate-PEG-DSPE in Liposome Accel- erates the Plasma Clearance of PEG-Coated Liposomes. As seen in Fig. 3A, FTLs were cleared faster than PEG-coated NTLs,4 indicating that the folate residue anchored with PEG(3350) on the liposome surface retained, to a partial degree, the ability to interact with factors accelerating liposome clearance from plasma despite the presence of liposome mPEG(2000) coating. Conjugating folate to a shorter PEG poly- Յ mer (Mr 2000) to reduce folate exposure and prolong circulation time was not attempted because previous in vitro data had indicated a major interference with the ability of liposome to interact with the FR (14). FTLs prepared without mPEG coating were rapidly cleared from circulation, their plasma levels being 9-fold lower than those of PEGylated FTLs 24 h after injection (data not shown). Although blood cells, mostly WBCs, were found to take up substantially more FTLs than NTLs, blood cells accounted for

Fig. 3 A, plasma clearance of folate-targeted liposomes (FTL) and 4 Gabizon, A., Shmeeda, H., Horowitz, A. T., and Zalipsky, S. Tumor nontargeted liposomes (NTL) in BALB/c mice; dose, 2 ␮mol phospho- cell targeting of liposome-entrapped drugs with phospholipid-anchored lipid/mouse. B, liver and tumor (M109-folate receptor) uptake of folate- folic acid-PEG conjugates. Adv. Drug Deliv. Rev. accepted for publi- targeted liposomes (FTL) and nontargeted liposomes (NTL); dose, 2 cation, 2004. ␮mol phospholipid/mouse.

Fig. 4 Biodistribution of radiolabeled folate-targeted liposomes (FTL) and nontargeted liposomes (NTL) in tumor-bearing mice. A, M109-folate receptor (M109-FR)-bearing BALB/c mice sacrificed 65 h after i.v. injection of 5 ␮mol phospholipid/mouse. B, KB-folate receptor (KB-FR)-bearing CD1 nude mice sacrificed 72 h after i.v. injection of 4 ␮mol phospholipid/mouse. Statistical analysis (t test): differences were significant for plasma (A: P ϭ 0.0038; B: P ϭ 0.0299) and liver (A: P ϭ 0.0021; B: P ϭ 0.0009). All other comparisons were not significant.

additional experiments were done with PEG-coated liposomes clearance, thus erasing the advantage of NTLs over FTLs in in mice bearing s.c. implants of either the syngeneic M109-FR circulation time. carcinoma or the human KB-FR carcinoma. On the basis of this On the basis of the [3H]CHE and doxorubicin results, we and other (22) studies indicating delayed peak tumor concen- obtained normalized drug-to-lipid ratios (see Fig. 5B, inset)48h trations after administration of PEGylated liposomes, mice were after injection and made the following observations: sacrificed 2–3 days after liposome injection. As shown in Fig. 4 (A and B), the tumor localization of FTLs was similar to that of (a) Plasma drug-to-lipid ratios were close to 1, underscor- NTLs in both tumor models. In agreement with data presented ing the stable drug retention during circulation of both liposome in Fig. 4, FTLs resulted in statistically significant lower plasma formulations. levels and greater liver uptake than NTLs (Fig. 4). Results from (b) The liver and spleen drug-to-lipid ratios were notably four more experiments testing liposome distribution into M109- lower, between 0.6 to 0.25, indicating that between 40 and 75% FR-bearing mice confirmed that there was no significant differ- of the drug was released from liposomes, metabolized, and/or ence in tumor uptake between the targeted and nontargeted excreted. preparations (data not shown). (c) In tumor and skin, the drug-to-lipid ratios were in the Data on normal tissue uptake of FTLs and NTLs are also range of 0.74–0.84, indicating that a much smaller fraction of presented in Fig. 4. Whereas liver uptake of FTLs was consis- drug (ϳ25% or less) had been metabolized and/or excreted from tently increased, other tissues (spleen, kidney, and skin) tend to these tissues. have lower levels of FTLs than NTLs. The discrepancy in Tumor to Tissue Ratios of Liposome Uptake. To com- uptake between the liver and spleen was notable and suggested pare the targeting profile of FTLs with that of NTLs, we that the increased uptake of FTLs by liver was the result of a examined the tumor-to-normal tissue liposome uptake ratios specific receptor-mediated endocytosis process rather than non- (Table 1). Tumor-to-plasma ratios are not presented; they are specific liposome clearance by the reticulo-endothelial system not pharmacodynamically relevant because the liposomes pres- (RES). ent in plasma are still in a distribution phase and their contents Biodistribution of FTLs and NTLs Loaded with Doxo- are not yet bioavailable. Important differences in the targeting rubicin. Because the ultimate goal was to use FTLs as drug profile of NTLs and FTLs were seen. When drug-free liposomes carriers, we examined the biodistribution of FTLs and NTLs were examined, NTLs showed lower tumor:liver ratios but loaded with doxorubicin and tracked the fate of both the radio- higher tumor:spleen, tumor:kidney, and tumor:skin ratios as labeled liposomes (3H-CHE; Fig. 5A) and that of the encapsu- compared with NTLs. In the KB tumor nude mouse model, the lated drug (doxorubicin; Fig. 5B). In the presence of doxorubi- tumor:liver ratio of NTLs decreased as a result of a higher cin, the biodistribution profile at 48 h after injection changed nonspecific liver uptake and was nearly equivalent to the tumor: markedly: liver uptake decreased and spleen uptake increased as liver ratio of FTLs. Also in nude mice, consistent with previous compared with drug-free liposomes. Tumor levels of FTLs rose observations (24), tumor:skin ratios were noticeably lower than by ϳ20%, not a significant finding. Plasma levels of FTL- in BALB/c mice because of the higher liposome uptake of doxorubicin increased and were comparable with those of NTL- furless skin. doxorubicin. These observations were consistent with a previous Significant changes in the tissue-uptake ratios were seen study demonstrating the clearance saturation effect of liposomal when doxorubicin-containing liposomes were compared with doxorubicin (23). RES blockade resulted in slower liposome drug-free liposomes. These changes occurred with both NTLs

Fig. 5 Biodistribution of radiolabeled folate-targeted liposomes (FTL) and nontargeted liposomes (NTL) loaded with doxorubicin (Dox). M109-folate receptor (M109-FR)-bearing BALB/c mice sacrificed 48 h after i.v. injection of 200 ␮g doxorubicin/mouse, equivalent to ϳ10 mg/kg weight and 2 ␮mol phospholipid/mouse. Results pooled from two identical experiments with similar results. A, results based on [3H]cholesterol-hexadecyl ether (3H-CHE) radiolabel. B, results based on doxorubicin; inset table shows normalized drug-to-lipid ratios for the various tissues examined. Statistical analysis (t test): each experiment was analyzed separately, with the highest P value being presented. Differences between nontargeted liposomes and folate-targeted liposomes were significant for liver (A: P ϭ 0.0010; B: P ϭ 0.0167) and spleen (A: P ϭ 0.0051; B: P ϭ 0.0019). All other comparisons were not significant.

and FTLs and appeared to be the result of liver saturation (22) on FTL deposition in tumors. The FA codose had no effect on increasing the tumor:liver ratios. When liver became saturated, the clearance of NTLs (data not shown). This indicated that FTL the spleen compensatory uptake resulted in paradoxical reduc- accumulation in liver and tumor is governed by different factors. tion of the tumor:spleen ratios. Tumor:skin ratios were not Whereas liver uptake of FTLs is likely to operate by Kupffer affected by the presence of doxorubicin in agreement with the cell receptor-mediated endocytosis of liposomes circulating passive, nonsaturable liposome uptake of these tissues (23) through liver sinusoids, tumor uptake first requires a passive characterized mainly by deposition in the extracellular space step of extravasation, not dependent on the folate ligand and, (25). therefore, not inhibited by FA. However, ligand-directed target- Effect of Codose of Cold FA on Tissue Distribution ing of liposomes may still modify the intratumor distribution of Profile of FTLs. The effect of giving a cold codose of FA to liposomes, particularly increasing association with cells at the mice receiving concomitantly FTLs at 6 and 48 h after liposome expense of the liposome pool accumulated in the extracellular injection is shown in Fig. 6. The codose of FA significantly fluid. This was investigated in the next set of experiments. reduced FTL clearance from blood and FTL liver uptake to Intratumor Distribution of FTLs. To quantify the frac- levels closer to those of NTLs. However, a remarkable finding tion of liposomes bound to tumor cells as opposed to that in was that the FA codose had a negligible and insignificant effect extracellular fluid, we chose an ascitic tumor model of a high FR-expressing tumor, the J6456-FR lymphoma. This tumor cell line was selected from parental J6456 by in vitro culture in Table 1 Tumor:tissue uptake ratios for liposome biodistribution folate-depleted medium. A total of 106 J6456-FR cells were based on percent of injected dose per gram tumor/tissuea injected i.p. into BALB/c mice. Because the FR of J6456-FR Uptake ratios cells would have been rapidly down-regulated in vivo when mice were fed the normal, folate-enriched diet, these experi- Tumor/ Tumor/ Tumor/ Tumor/ Tumor Nb Liver Spleen Kidney Skin ments were done in mice fed a folate-depleted diet. After 2–3 weeks, when abdominal swelling was noticed, mice received i.v. M109-FRc NTL 5 0.93 1.25 3.20 6.65 injections of radiolabeled FTLs and NTLs. Three days later, FTL 5 0.66 1.50 3.91 9.90 mice were bled and sacrificed, and ascites were collected as NTL-Dox 2 1.86 0.61 ND 8.54 indicated in “Materials and Methods.” A comparison of the FTL-Dox 2 1.14 0.40 ND 13.34 results obtained with FTLs and NTLs (Fig. 7) indicated a KB-FR NTL 1 0.55 1.24 1.79 1.44 significant advantage of the targeted liposomes with regard to FTL 1 0.46 1.66 3.19 2.48 the absolute amount of liposomes associated with tumor cells a Liposome dose: 2–5 ␮moles phospholipid per mouse. Results (Fig. 7B). Because the overall accumulation of liposomes in based on [3H]cholesterol-hexadecyl ether radiolabel, 48–72 h after i.v. ascites was somewhat lower for FTLs than for NTLs (Fig. 7A), injection. the relative advantage of the former in the level of association b N, number of experiments used for calculation of mean tumor: with cells was even more striking (ϳ6-fold; Fig. 7C). There was tissue uptake ratios. c FR, folate receptor; NTL, nontargeted liposome; FTL, folate- substantial interanimal variability, stressing the need to use targeted liposome; Dox, doxorubicin; ND, not determined. many animals in each experimental group. Clearly, these results

Fig. 6 Effect of a codose of folic acid (F.A.) on the biodistribution of folate-targeted liposomes (FTL) in M109-folate receptor (M109-FR)-bearing BALB/c mice. A, 6 h after liposome injection; B, 48 h after liposome injection. Statistical analysis (t test) of FTL with and without codose of folic acid: A, plasma (P ϭ 0.0365), liver (P ϭ 0.0003), M109-FR in tumor (not significant); B, plasma (P ϭ 0.0110), liver (P ϭ 0.0171), and tumor M109-FR (not significant).

indicate that in an ascitic model, where liposome movement is the short time window required for a tissue distribution study, across a fluid cell suspension, targeting to tumor cells does take experiments with this tumor model and with the KB human place in vivo and confers a potential cellular drug delivery carcinoma, another well-established model of inducible and advantage to FTLs over NTLs. stable high FR expression (26), proceeded with animals on normal diet. In contrast, the J6456 lymphoma quickly down- DISCUSSION regulates FR in animals with a normal, folate-enriched diet Without adequate information on the in vivo tissue distri- (unpublished data). bution of FTLs in animal models, it is difficult to address The presence of folate on the liposome surface has a rationally experiments aimed at testing the therapeutic potential detrimental effect on liposome circulation time partially coun- of liposome targeting with folate ligands. The purpose of this teracted by the PEG coating. Therefore, the use of PEGylated study was to evaluate whether or not folate targeting has an liposomes appears to be an important element in the design of a impact on liposome biodistribution and, specifically, on tumor folate-targeted liposome delivery system. A complementary ap- uptake. Baseline information on the uptake of free FA and on proach to prolong circulation time of FTLs would be to reduce the effect of folate-depleted diet on receptor expression in vivo the molar ratio of folate-PEG-DSPE based on data published is obviously of great importance. Because we found that folate recently by Reddy et al. (27) indicating that 0.03% (i.e., 10-fold binding by the M109 tumor was not affected by the diet within less than the molar ratio used in our experiments) is sufficient

Fig. 7 In vivo enhancement of liposome binding to ascitic J6456 lymphoma cells by folate-targeted delivery 72 h after i.v. injection of folate-targeted liposomes (FTL) and nontargeted liposomes (NTL)5␮mol phospholipid/mouse. A, liposome accumulation in whole ascites. B, liposome accumulation in ascitic tumor cells. C, fraction of liposomes in ascites bound to tumor cells. Results of statistical analysis (Mann-Whitney test) is shown in the figure. Each point represents the data for an individual mouse. Horizontal bars represent median values.

for optimal liposome binding to tumor cell FR. That the folate bicin-containing FTLs may minimize skin toxicity, which is residue accelerates plasma liposome clearance was also sup- dose-limiting for Doxil, a clinical formulation of doxorubicin- ported by the fact that a codose of free FA injected together with containing NTLs (35). the liposomes blocked the clearance of FTLs without modifying Altogether, these studies indicate that liposome targeting to that of nontargeted liposomes. A nonspecific effect of the neg- the FR receptor has the potential means to alter liposome bio- atively charged carboxyl group of the folate ligand seems un- distribution with potential pharmacodynamic implications that likely, given its low surface concentration (0.3%) and the clear- may tilt favorably the therapeutic index. Recent encouraging ance blocking effect of a codose of soluble FA. Interestingly, the reports on the therapeutic activity of cisplatin and doxorubicin same codose of FA did not reduce tumor uptake of FTLs. There encapsulated in FTLs (36, 37) lend further support and promise are two possible explanations for this: (a) the measured FTL to this approach. accumulation in tumor is the result of passive extravasation based on the enhanced permeability and retention effect (28) ACKNOWLEDGMENTS with no contribution of binding to cell FR, or (b) the higher We thank Lidia Mak and Moshe Bronstein for technical help. (ϳ1000-fold) affinity of liposome multivalent binding to mul- tiple FR on the tumor cell surface prevents displacement by free REFERENCES FA, as shown in previous in vitro experiments (14). Free FA 1. Sapra, P., and Allen, T. M. 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Alberto Gabizon, Aviva T. Horowitz, Dorit Goren, et al.

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