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Experimental Therapeutics, Preclinical Pharmacology |
1 Shaare Zedek Medical Center and
2 Hadassah Medical Center, Jerusalem, Israel and
3 ALZA Corporation, Mountain View, California
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: A small fraction of a folate-polyethylene-glycol (PEG)-distearoyl-phosphatidylethanolamine conjugate was incorporated in FTLs. Both FTLs and NTLs were PEGylated with a PEG-distearoyl-phosphatidylethanolamine conjugate to prolong circulation time. Liposomes were labeled with [3H]cholesterol hexadecyl ether with or without doxorubicin loading. Liposome levels in plasma, tissues, or ascites were assessed by the number of [3H] counts. For doxorubicin-loaded formulations, we also determined the tissue doxorubicin levels by fluorimetry. To estimate the amount of liposomes directly associated with tumor cells in vivo, we determined the [3H]radiolabel counts in washed pellets of ascitic tumor cells using the ascitic J6456 lymphoma
Results: FTLs retained the folate ligand in vivo, as demonstrated by their ability to bind ex vivo to FR-expressing cells after prolonged circulation and extravasation into malignant ascitic fluid. In comparison with NTLs, FTLs were cleared faster from circulation as a result of greater liver uptake. Despite the lower plasma levels, tumor levels of FTL-injected mice were not significantly different from those of NTL-injected mice. When NTLs and FTLs were loaded with doxorubicin, liver uptake decreased because of liver blockade, and uptake by spleen and tumor increased. When tumor-to-tissue liposome uptake ratios were analyzed, the targeting profile of FTLs was characterized by higher tumor:skin, and tumor:kidney ratios but lower tumor:liver ratio than NTLs. After a concomitant dose of free folic acid, FTLs (but not NTLs) plasma clearance and liver uptake were inhibited, indicating that accelerated clearance 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 liposome levels were significantly greater for FTL-injected mice than for NTL-injected mice, despite slightly higher levels of the latter in whole ascites.
Conclusions: Whereas folate targeting does not enhance overall liposome deposition in tumors, the targeting profile of tumor versus other tissues is substantially different and intratumor liposome distribution in ascitic tumors is affected favorably with a selective shift toward liposome association with FR-expressing cells.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10-10 M; see Refs. 8
, 9
). It is located in caveolae and participates in the cellular accumulation of folates through the process of potocytosis. In this process, receptor-bound ligand is sequestered in caveolae, internalized into postcaveolar plasma vesicles, released from the receptor via an intravesicular reduction in pH, and subsequently transported into cytoplasm for polyglutamation (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 drug delivery is supported by findings indicating up-regulation (higher expression) in many human cancers including those of the ovary, brain, kidney, lung, breast, and myeloid cells (12) . In addition, aggressive or undifferentiated tumors with advanced stage or grade appear to have an increased FR density (13 , suggesting that FR-mediated delivery may be a broad approach in cancer treatment.
In previous studies, we have investigated the in vitro binding of folate-targeted liposomes (FTLs) to tumor cells expressing FR, and the process of in vitro delivery of an anticancer drug, doxorubicin, to tumor cells via FTLs (14 , 15) . We used a conjugate of three components incorporated in the liposome bilayer to target liposomes to the FR (14) : FA, polyethylene-glycol (PEG), and distearoyl-phosphatidyl-ethanolamine (DSPE). The folate group is located at the outer end of PEG, away from the bilayer. Because methoxy-PEG-DSPE [PEG, Mr 2000; designated as mPEG(2000)] is commonly used as a liposome component to prolong liposome circulation time, we chose a longer PEG length [PEG, Mr 3350; designated as PEG(3350)] for the folate-PEG-DSPE conjugate to reduce steric interference with receptor binding. These FTLs bind avidly and are internalized by FR-expressing tumor cells, although mPEG surface coating of liposomes still interferes significantly with this process (14) . We also found that FTLs can deliver efficiently doxorubicin to tumor cells and bypass the drug efflux mechanism characteristic of multidrug resistance (15) . A critical step for evaluating the potential of FTLs as drug-delivery systems in cancer therapy is to study the fate of these systems after i.v. injection and determine whether or not they confer any advantage in tumor localization and/or intratumoral distribution of the carrier. In this study, we investigated the biodistribution of radiolabeled and doxorubicin-loaded FTLs in tumor-bearing mice and compared it with that of nontargeted liposomes (NTLs) of similar composition and size.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.5 µci/µmol phospholipid. The use of [3H]CHE is convenient for these studies because it is a stable, nonexchangeable, and nondegradable marker of liposomes (16)
, thus providing an estimate of the cumulative liposome dose in tissue. Liposomes were suspended in dextrose 5% buffered with 15 mM HEPES (pH 7.0). All formulations were analyzed for phosphorus content (Bartlett method), folate content (absorbance, A285 nm), radioactivity [counts per minute (cpm) in a ß scintillation counter] and vesicle size (dynamic laser scattering). Final phospholipid concentration was around 20 µmol/ml; folate content and [3H]CHE cpm/mol phosphorus were close to the relative input preparation ratios; mean vesicle size was in the range of 70 to 90 nm with SD <30% of the mean. In some experiments, radiolabeled FTLs and NTLs were loaded with doxorubicin using an ammonium sulfate gradient as described previously (17) . Unencapsulated doxorubicin was removed by a small Dowex resin column. The final doxorubicin/phospholipid ratio was in the range of 100–150 µg/µmol. Other parameters were similar to those for drug-free liposomes.
Animal Models and Tumors.
We used 8-wk-old SPF female BALB/c mice and Swiss CD1 athymic/nude mice in these studies. Mice were purchased from Harlan Breeding Laboratories (Jerusalem, Israel) and maintained in a SPF facility at Hadassah Medical Center with food and water ad libitum. All animal experiments were done under a protocol approved by the Hebrew University-Hadassah Institutional Review Board for use of animals in research. In some of the experiments, mice were fed a special low-folate diet (Harlan Tekled, Madison, WI) from 1 week before tumor inoculation and until mice were sacrificed for tissue distribution studies. The tumor models used here are the mouse M109 (18)
, and human KB (19)
carcinomas, and the mouse J6456 lymphoma (20)
. High FR-expressing cells were selected from these tumor cell lines as described previously for M109 and KB tumors (14)
. The characteristics of the high-FR J6456 subline will be presented in a separate report.
M109 tumor cells (106 cells/0.2 ml) or KB tumor cells (2 x 106 cells/0.2 ml) in serum-free medium suspensions were inoculated in the s.c. space of the mouse right and left flank of BALB/c or CD1 nude mice, respectively. Two to 3 weeks after 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 five mice were used for each time point of each experimental group. The interanimal variations in tumor uptake were greater than those for other normal tissues. To correct for this higher variance, each mouse was inoculated with two tumor inocula, one in each flank, so that tumor values were the mean of a greater number of samples than for other tissues, generally six or more for each time point and treatment group examined. Liposomes were injected i.v. at dose levels of 2 to 5 µmol phospholipid/mouse. When doxorubicin-loaded liposomes were used, the dose of doxorubicin was 200 µg/mouse, (equivalent to 10 mg/kg for a 20-g mouse). [3H]FA (Amersham) was also injected i.v. into tumor-bearing mice for biodistribution experiments. When a codose of FA was used in the experiment, cold FA was diluted in NaBic solution (0.84% sodium bicarbonate in 90% physiological saline) and injected i.p. at a dose of 4–5 mg/ml/mouse immediately before i.v. injection of liposomes or [3H]FA.
In the J6456 lymphoma model, BALB/c mice were inoculated i.p. with 106 J6456 cells in 0.2 ml serum-free medium. After 2 to 3 weeks, abdominal swelling developed, indicating peritoneal tumor spread and ascites, at which point liposomes were injected i.v. for the designed experiments.
Biodistribution Studies.
At scheduled time points after liposome injection, mice were anesthetized by ether or halothane inhalation, bled by eye enucleation (>1 ml of blood/mouse) and immediately sacrificed by cervical dislocation. Tumors and other indicated organs (liver, spleen, kidneys, skin) were removed, rinsed in physiological saline, weighed, and frozen at -20°C until further processing. Blood was collected in heparinized tubes and centrifuged immediately to separate plasma from blood cells. Plasma cpm were measured in a ß counter after dilution of plasma in scintillation fluid (50 µl/5 ml of Quick Safe A; Zinsser Analytic, Maidenhead, United Kingdom). Frozen tissue samples were incinerated in a Packard Sample Oxidizer, model 307 (Downer Grove, IL). The resulting radioactive water was diluted in scintillation fluid and measured in a ß counter. The number of cpm of [3H]CHE of each plasma or tissue sample was used to calculate the percentage of injected dose per milliliter of plasma or gram of tissue based on the number of cpm injected per mouse.
When mice received injections of doxorubicin-loaded, radiolabeled liposomes, the removed tissues were split in two pieces. One piece was processed for examining the radioactive counting as described above, and another piece was processed for doxorubicin content by high-performance liquid chromatography and fluorescence detection as described previously (17) .
In mice inoculated i.p. with J6456 lymphoma, ascites was collected after mice were bled and sacrificed by injecting 3 ml of PBS into the abdominal cavity of each mouse. Rinsing with a large volume of PBS is essential for obtaining a representative sample collection of fluid and cells present in the abdominal cavity. After the animal body was swung from the tail for a few seconds to mix well the buffer with the abdominal contents, the fluid collection was aspirated and immediately centrifuged to separate cells from ascitic fluid. The cells were washed twice more with PBS, counted, pelleted, and then solubilized in scintillation fluid for ß radioactive counting. Consistent with previous observations, >95% of the cells appeared to be tumor cells by light microscopy. Samples of ascitic fluid and of whole ascites, represented by the unfractionated aspirate, were also taken for radioactive counting. The amount of radioactivity expressed as phospholipid-equivalents, on the basis of the [3H]CHE/phosphate ratio in the liposome preparation, was calculated for whole ascites and for 106 ascitic cells for each mouse.
Statistical Analysis.
Nonpaired t test was used for comparison of mean liposome levels in various tissues, except for the experiments with J6456 lymphoma, in which we used the nonparametric Mann-Whitney test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Plasma levels were very low, indicating that most of the injected FA was cleared from plasma within 3 h. The following observations were also made:
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100-fold.
(b) Folate-depleted diet resulted in a sharp increase (2.5 fold) in kidney uptake of FA. This is likely to have been the result of an up-regulation of FR in the kidney tubular cells to rescue the greatest possible amount of FA from urine under conditions of folate deprivation. The change in liver uptake was minimal.
(c) Diet had an insignificant effect on tumor uptake of FA, suggesting that the degree of up-regulation or down-regulation of M109 tumor FR expression was insufficient to cause a significant change in folate uptake in vivo.
(d) A codose of cold FA competed effectively with the labeled material for tissue uptake, including the high- and low-FR tumors.
Retention of Folate Ligand by Circulating Liposomes.
Before examining further the biodistribution of FTLs, it was important to determine whether the folate ligand was retained by liposomes in vivo, particularly after they exited the blood stream and entered the interstitial fluid compartment, allowing for targeting to take place. Given the fact that PEG-3350 and folate constituted a large hydrophilic moiety, anchored into the bilayer by the DSPE lipophilic moiety, there was a possibility of dissociation 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 (<20%) from the original uptake of fresh liposomes. The ascitic fluid itself interfered slightly with binding of FTLs to target cells.
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, 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 polymer (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).
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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 in this experiment (P = 0.0436, t test), although in additional experiments focusing on the 48–72 h time point (see next section) no statistical significance was detected. Between 48 to 96 h, there was actually a drop in liposome concentration in 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 Liposomes in Tumor-Bearing Mice.
To further study the effect of folate targeting on tissue distribution and tumor uptake, additional experiments were done with PEG-coated liposomes in mice bearing s.c. implants of either the syngeneic M109-FR carcinoma or the human KB-FR carcinoma. On the basis of this and other (22)
studies indicating delayed peak tumor concentrations after administration of PEGylated liposomes, mice were 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 NTLs in both tumor models. In agreement with data presented in Fig. 4
, FTLs resulted in statistically significant lower plasma levels and greater liver uptake than NTLs (Fig. 4)
. Results from four more experiments testing liposome distribution into M109-FR-bearing mice confirmed that there was no significant difference in tumor uptake between the targeted and nontargeted preparations (data not shown).
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. Whereas liver uptake of FTLs was consistently increased, other tissues (spleen, kidney, and skin) tend to have lower levels of FTLs than NTLs. The discrepancy in uptake between the liver and spleen was notable and suggested that the increased uptake of FTLs by liver was the result of a specific receptor-mediated endocytosis process rather than nonspecific liposome clearance by the reticulo-endothelial system (RES).
Biodistribution of FTLs and NTLs Loaded with Doxorubicin.
Because the ultimate goal was to use FTLs as drug carriers, we examined the biodistribution of FTLs and NTLs loaded with doxorubicin and tracked the fate of both the radiolabeled liposomes (3H-CHE; Fig. 5A
) and that of the encapsulated drug (doxorubicin; Fig. 5B
). In the presence of doxorubicin, the biodistribution profile at 48 h after injection changed markedly: liver uptake decreased and spleen uptake increased as compared with drug-free liposomes. Tumor levels of FTLs rose by 20%, not a significant finding. Plasma levels of FTL-doxorubicin increased and were comparable with those of NTL-doxorubicin. These observations were consistent with a previous study demonstrating the clearance saturation effect of liposomal doxorubicin (23)
. RES blockade resulted in slower liposome clearance, thus erasing the advantage of NTLs over FTLs in circulation time.
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, inset) 48 h after injection and made the following observations: (a) Plasma drug-to-lipid ratios were close to 1, underscoring the stable drug retention during circulation of both liposome formulations.
(b) The liver and spleen drug-to-lipid ratios were notably lower, between 0.6 to 0.25, indicating that between 40 and 75% of the drug was released from liposomes, metabolized, and/or excreted.
(c) In tumor and skin, the drug-to-lipid ratios were in the range of 0.74–0.84, indicating that a much smaller fraction of drug (25% or less) had been metabolized and/or excreted from these tissues.
Tumor to Tissue Ratios of Liposome Uptake.
To compare the targeting profile of FTLs with that of NTLs, we examined the tumor-to-normal tissue liposome uptake ratios (Table 1)
. Tumor-to-plasma ratios are not presented; they are not pharmacodynamically relevant because the liposomes present in plasma are still in a distribution phase and their contents are not yet bioavailable. Important differences in the targeting profile of NTLs and FTLs were seen. When drug-free liposomes were examined, NTLs showed lower tumor:liver ratios but higher tumor:spleen, tumor:kidney, and tumor:skin ratios as compared with NTLs. In the KB tumor nude mouse model, the tumor:liver ratio of NTLs decreased as a result of a higher nonspecific liver uptake and was nearly equivalent to the tumor:liver ratio of FTLs. Also in nude mice, consistent with previous observations (24)
, tumor:skin ratios were noticeably lower than in BALB/c mice because of the higher liposome uptake of furless skin.
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Effect of Codose of Cold FA on Tissue Distribution Profile of FTLs.
The effect of giving a cold codose of FA to mice receiving concomitantly FTLs at 6 and 48 h after liposome injection is shown in Fig. 6
. The codose of FA significantly reduced FTL clearance from blood and FTL liver uptake to levels closer to those of NTLs. However, a remarkable finding was that the FA codose had a negligible and insignificant effect on FTL deposition in tumors. The FA codose had no effect on the clearance of NTLs (data not shown). This indicated that FTL accumulation in liver and tumor is governed by different factors. Whereas liver uptake of FTLs is likely to operate by Kupffer cell receptor-mediated endocytosis of liposomes circulating through liver sinusoids, tumor uptake first requires a passive step of extravasation, not dependent on the folate ligand and, therefore, not inhibited by FA. However, ligand-directed targeting of liposomes may still modify the intratumor distribution of liposomes, particularly increasing association with cells at the expense of the liposome pool accumulated in the extracellular fluid. This was investigated in the next set of experiments.
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indicated a significant advantage of the targeted liposomes with regard to the absolute amount of liposomes associated with tumor cells (Fig. 7B)
. Because the overall accumulation of liposomes in ascites was somewhat lower for FTLs than for NTLs (Fig. 7A)
, the relative advantage of the former in the level of association with cells was even more striking (6-fold; Fig. 7C
). There was substantial interanimal variability, stressing the need to use many animals in each experimental group. Clearly, these results indicate that in an ascitic model, where liposome movement is across a fluid cell suspension, targeting to tumor cells does take place in vivo and confers a potential cellular drug delivery advantage to FTLs over NTLs.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of folate on the liposome surface has a detrimental effect on liposome circulation time partially counteracted by the PEG coating. Therefore, the use of PEGylated liposomes appears to be an important element in the design of a folate-targeted liposome delivery system. A complementary approach to prolong circulation time of FTLs would be to reduce the molar ratio of folate-PEG-DSPE based on data published recently by Reddy et al. (27)
indicating that 0.03% (i.e., 10-fold less than the molar ratio used in our experiments) is sufficient for optimal liposome binding to tumor cell FR. That the folate residue accelerates plasma liposome clearance was also supported by the fact that a codose of free FA injected together with the liposomes blocked the clearance of FTLs without modifying that of nontargeted liposomes. A nonspecific effect of the negatively charged carboxyl group of the folate ligand seems unlikely, given its low surface concentration (0.3%) and the clearance blocking effect of a codose of soluble FA. Interestingly, the same codose of FA did not reduce tumor uptake of FTLs. There are two possible explanations for this: (a) the measured FTL accumulation in tumor is the result of passive extravasation based on the enhanced permeability and retention effect (28)
with no contribution of binding to cell FR, or (b) the higher (1000-fold) affinity of liposome multivalent binding to multiple FR on the tumor cell surface prevents displacement by free FA, as shown in previous in vitro experiments (14)
. Free FA would still effectively compete with FTLs for binding to plasma folate-binding protein because the interaction with the latter is likely to be monovalent in both cases. In any case, the FA codose may reduce liposome opsonization and slow down liposome clearance without directly affecting tumor uptake.
A critical issue in evaluating in vivo a targeted delivery system would be to demonstrate that it binds to target cells in excess of nontargeted carriers. As long as the target is not exposed to circulating liposomes, extravasation, a nonspecific process, is the rate-limiting factor of liposome localization in tumor (17)
. After extravasation, specific binding to tumor cell receptors may increase liposome retention in the tumor site. A variety of nonspecific factors, such as liposome circulation time (29)
, vascular permeability (30)
, interstitial fluid pressure (31)
, and others, may mask the contribution of specific factors on the overall liposome accumulation. However, our experiments with the ascitic J6456 lymphoma indicate that binding of FTLs to the tumor cell FR does take place in vivo and plays a significant role in the liposome biodistribution in ascitic tumors. Thus, the fraction of FTLs present in ascites taken up by tumor cells exceeds by 6-fold that of NTLs. It is unclear whether significant binding of FTLs to tumor cell FR also takes place in solid tumors. Obviously, the movement of liposomes in the extracellular space of a solid tumor is much more limited than in ascitic fluid. As a result, liposome access to tumor cells in solid tumors is probably limited to the cell layer in juxtaposition with microvessel endothelial cells (32)
. One observation that suggests binding of FTLs to tumor cell FR in the M109 and KB tumor models is the fact that tumor levels are almost identical to those of NTLs, despite evidence for enhanced RES clearance (i.e., lower plasma levels and higher liver levels). Liposome studies have established an inverse correlation between RES uptake on the one hand and plasma residence time and tumor uptake on the other hand (33)
. Therefore, we cannot rule out an increased affinity of FTLs for tumors that compensates for rapid plasma clearance and maintains high tumor levels.
The present study confirms previous observations with doxorubicin-containing liposomes (34) indicating that saturation of liver uptake becomes an important player shifting liposome biodistribution to spleen and more importantly to tumors. RES saturation yields higher FTL and NTL uptake by tumors. Of note, the tumor:skin ratio of FTLs is greater than that of NTLs in any given setting. This suggests that treatment with doxorubicin-containing FTLs may minimize skin toxicity, which is dose-limiting for Doxil, a clinical formulation of doxorubicin-containing NTLs (35) .
Altogether, these studies indicate that liposome targeting to the FR receptor has the potential means to alter liposome biodistribution with potential pharmacodynamic implications that may tilt favorably the therapeutic index. Recent encouraging reports on the therapeutic activity of cisplatin and doxorubicin encapsulated in FTLs (36 , 37) lend further support and promise to this approach.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Alberto Gabizon, Oncology Institute, Shaare Zedek Medical Ctr., POB 3235, Jerusalem, il-91031, Israel. Phone: 972-2-6555-036; Fax: 972-2-652-1431; E-mail: alberto{at}md.huji.ac.il
4 Gabizon, A., Shmeeda, H., Horowitz, A. T., and Zalipsky, S. Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugates. Adv. Drug Deliv. Rev. accepted for publication, 2004. 
Received 6/17/03; revised 8/28/03; accepted 8/28/03.
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