Effect of Liposome Composition and Other Factors on the Targeting of Liposomes to Experimental Tumors: Biodistribution and Imaging Studies1
Total Page:16
File Type:pdf, Size:1020Kb
(CANCER RESEARCH SO.6371-6378. October I. 1990] Effect of Liposome Composition and Other Factors on the Targeting of Liposomes to Experimental Tumors: Biodistribution and Imaging Studies1 Alberto Gabizon,2 David C. Price, John Huberty, Robert S. Bresalier, and Demetrios Papahadjopoulos Cancer Research Institute ¡A.(j., I). P.] and Department of Radiology, [D. C. P., J. H.J, L'nirersity of California, San Francisco, California 9414}; (iastroinlestinal Research Laboratories, I eteram Administration Medical Center, and Department of Medicine, I 'nirersity of California, San Francisco, California 94121 [R. S. B.J; and Liposome Technology Inc., Mento Park, California 94025 ¡A.CiJ ABSTRACT temperature, cholesterol, and careful size control result in in hibition of RES uptake with concomitant enhancement of We have examined the distribution of radiolabeled liposomes in tumor- tumor uptake (5). bearing mice after i.v. injection. Two mouse tumors (B16 melanoma, In this report, we describe tissue distribution and imaging J6456 lymphoma) and a human tumor (LS174T colon carcinoma) inoc ulated i.m., S.C.,or in the hind footpad were used in these studies. When studies with transplantable mouse and human tumor models various liposome compositions with a mean vesicle diameter of ~ 100 nm using 3 different radiolabels of liposomes. The findings here were compared using a radiolabel of gallium-67-deferoxamine, optimal indicate that the concentration of liposome-encapsulated radio- tumor localization was obtained with liposomes containing a phosphati- labels in tumors is well above that of most other tissues and dylcholine of high phase-transition temperature and a small molar frac approximates the values obtained in the liver. The evidence tion of monosialoganglioside or hydrogenated phosphatid)linositol (HPI). gathered strongly supports the validity of a direct tumor target At 24 h after injection, average values of tumor uptake higher than 10% ing approach with liposomes. of the injected dose per g and liver-to-tumor ratios close to 1 were reproducibly obtained. Increasing the molar fraction of HPI from 9% to 41% of the total phospholipid resulted in enhancement of liver uptake MATERIALS AND METHODS and decrease of tumor uptake. Methodological aspects that influence Preparation of Liposomes. The sources of materials used in this study- vesicle size appear to affect significantly liposome localization in the tumor. However, varying the phospholipid dose within a 10-fold range are the same as reported previously (5). The main fatty acid components of the hydrogenated phospholipids used were 66% stéarateand 33% caused only minor changes in the percent of injected dose recovered in palmitate for phosphatidylinositol, and 85% stéarateand15% palmitate the tumor. A high uptake by tumors was also observed using other radiolabels ¡|'II|imiliuand indium-111-labeled bleomycin ("'In-Bleo)| in for phosphatidylcholine. Liposomes were prepared by thin lipid film monosialoganglioside- and HPI-containing liposomes. In the case of '"In- hydration followed by repeated extrusions through polycarbonate mem Bleo, encapsulation in liposomes resulted in ~20- to 40-fold increase in branes of defined pore size (0.05 urn) as described previously (5). For hydration, we used isotonic solutions of saline (pH range, 6.0-7.0) with tumor accumulation of the radiolabel at 24 h after injection. The marked either DF (20-25 mivi), Bleo (5-15 units/ml), or ['Hjinulin (250 MCi/3 localization of liposomes in the mouse footpad inoculated with tumor as opposed to the contralateral mock-injected footpad was also documented ml). The mean vesicle size obtained with this methodology was in the by imaging experiments with gallium-67-deferoxamine and '"In-Bleo- range of 70 to 120 nm with a SD not larger than 30% of the Gaussian labeled liposomes. These results support the contention that some gly- mean as measured by dynamic laser scattering. In some cases, where colipid-containing liposomes previously shown to have long circulating indicated, liposomes were prepared by solvent injection (6) in the following way: lipids were weighed and dissolved in a 7:3 mixture of half-lives accumulate significantly in a variety of tumors and are prom ethanohdimethyl sulfoxide: the lipid solution was warmed to 60°Cand ising tools for the delivery of anti-tumor agents. injected through a 21- to 23-gauge needle, at a rate of =10 ml/min, into the bulk water phase (consisting of a isotonic solution of NaCl and INTRODUCTION 20 mM DF), which was kept at 60°Cand constantly stirred up; the organic solvents were diluted to 15% of the total volume, thus enabling Liposomes have been used as carriers of cytotoxic drugs with liposome formation. Ethanol, dimethyl sulfoxide, and unencapsulated a strategy based on reduction of toxicity and/or passive delivery DF were removed by repeated cycles of cartride dialysis (Diaflo hollow- to liver-infiltrating tumors (1, 2). The fast and dominant uptake fiber cartridge: Amicon, Danvers, MA) against isotonic saline or glu of liposomes by the RES' (3, 4) has prevented so far the cose 5%. Liposomes were concentrated =5-fold during the last dialysis adoption of a more direct strategy based on selective homing step. Liposomes were used either without any further manipulations or of liposomes to tumors. Targeting of liposomes to tumors after additional extrusion through 0.05 ^m-pore polycarbonate mem requires, most importantly, a prolonged circulation half-life of branes and Sephadex G-75 gel filtration to remove any DF released by intact liposomes which, in turn, depends on a reduction of the the extrusion procedure. Phospholipid concentration was determined by a phosphate assay rate of clearance by the RES. In addition, there is a need to (7, 8). Liposomes were stored either under argon or in vacuum-sealed minimize the leakage of liposome contents during their pro tubes at 5°Cand tested within 1 month after preparation. longed stay in the blood stream. Recent developments have Radiolabeling of Liposomes. The method of labeling preformed li shown that the inclusion of some glycolipids in the liposome posomes containing DF with 67Ga citrate has been described in detail bilayer coupled with phospholipids of high phase-transition (9). Labeling preformed liposomes containing Bleo (Bristol-Myers, Syracuse, NY) with '"InCI (New England Nuclear, Boston. MA) was Received 3/5/90: accepted 6/15/90. done following a similar technique. Briefly, 20 n\ of a 1 mg/ml solution 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 of oxine in ethanol or oxine hemisulfate in saline was added to an accordance with 18 U.S.C. Section 1734 solely to indicate this fact. aliquot of 100 to 200 jiCi of '"InCI and incubated at 60T for 15 min. 1Work supported until November 1987 by a grant from the National Cancer Bleo-containing liposome suspensions (5-15 (<mnl phospholipid/ml) Institute (CA-35340) and subsequently by Liposome Technology. Inc.. and the were incubated for 1 h at 60°Cwith 2 /¿Ci'"In-oxine per ¿irnol American Cancer Society. 1To whom requests for reprints should be addressed, at Department of phospholipid. Removal of unencapsulated '"In-oxine was achieved by Oncology. Hadassah Medical Center. P.O. Box 12.000. Jerusalem. Israel 91120. passing the liposome suspension through an anion-exchange resin *The abbreviations used are: Bleo. bleomycin; Ch. cholesterol; DF, deferox- (AG 1X2, acetate form, 200-400 mesh size; Bio-Rad, Emeryville, CA) amine: DSPC. distearoylphosphatidylcholine: GM|. monosialoganglioside; HPI, in the same way as done for 67Ga-oxine (9). This method resulted in hydrogenated phosphatidylinositol: PC. phosphatidylcholine: PC. phosphatidyl- glycerol; RES. reticulocndothelial system; c'cID/g. percentage of injected dose per >80% encapsulation of '"In in Bleo-containing liposomes. Previous g. work had shown that '"In is translocated from the '"In-oxine complex 6371 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. LIPOSOME TARGETING TO TUMORS to Bleo with an efficiency close to 100% as assessed by separation on same phospholipid head group (dipalmitoylphosphatidylgly- thin-layer chromatography. cerol-DSPC versus PG-PC). Liposomes containing phospho- Tritium-labeled inulin (New England Nuclear) was encapsulated in lipids of widely different phase transition temperature (PG and liposomes by adding it to the hydration buffer (physiological saline). DSPC) resulted in low tissue recoveries probably as a result of The rest of the procedure was as described above for the thin lipid film/ extrusion method (5); 2.8% of the initial amount of [3H]inulin was phase separation and leakage of the label (5). Of note, skin entrapped in GMi-DSPC-Ch liposomes. uptake was several-fold higher in mice given injections of lipo Animals and Tumors. Age-matched C57BL/6 and BALB/c inbred somes with extended circulation time and high tumor uptake. female mice from Simonsen Laboratories (Gilroy, CA) and NCR-NU No such increase was observed in kidneys, intestine, and car athymic nude outbred female mice from the National Cancer Institute cass. The highest uptake values per g tissue were seen in the (Frederick, Ml)) were used in these studies. The mouse B16 melanoma spleen. However, given its small weight, the absolute uptake of was inoculated either i.m. into the hind leg (IO5 cells) or into the hind spleen was still below that of liver and skin for most formula footpad (2.5 x 10" cells) of syngeneic C57BL/6 mice. The mouse J6456 lymphoma (10) was inoculated into the hind leg (10'' cells) of syngeneic tions tested. BALB/c mice. The human LS174T colon carcinoma (11) was trans Biodistribution of (J\I,-containing Liposomes in Nude Mice planted by s.c. injection of IO7cells into the flank of nude mice. Cells Bearing the LS174T Human Colon Carcinoma were injected in serum-free RPMI-1640 medium (GIBCO, New York, NY).