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[CANCER RESEARCH 43, 2927-2934, June 1983] 0008-5472/83/0043-0000$02.00 Effects of Liposome Dose and the Presence of Lymphosarcoma Cells on Blood Clearance and Tissue Distribution of Large Unilamellar Liposomes in Mice1 Marma Ellens, Henriette W. M. Morseli, Bert H. J. Dontje, Dharamdajal Kalicharan, Caesar E. Hulstaert, and Gerrit L. Scherphof2 Laboratory of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 KZ [H. E., H. W. M. M., B. H. J. D., G. L S.] and Centre for Medical Electron Microscopy, University of Groningen, Oostersingel 69", 97J3 EZ [D. K., C. E. H.], Groningen, The Netherlands ABSTRACT Depression of reticuloendothelial activity has been suggested to serve as a means to reduce liver and spleen uptake of Large unilamellar liposomes (50 to 500 ^mol of lipid per kg) liposomes and to prolong the circulation time of the vesicles (1, were injected i.v. or i.p. into normal and lymphosarcoma-bearing 12, 13). The rate of disappearance of liposomes from the blood mice. The percentage of the dose remaining in the blood and decreases with increasing dose (33), indicating that the clearance that accumulated in liver, spleen, and various other organs was mechanism is a saturable process. Concomitantly, one would measured 4 hr after injection. The results indicate that liposomes expect the proportion of liposomes taken up by the liver to cause a dose-dependent saturation of the hepatic and splenic decrease with increasing dose, but conflicting results have been clearance capacities. One day after injection of 106 lymphosar- reported on this subject (12, 33). Tumor cells may influence coma cells, the capacity of the tumor-bearing mice to eliminate reticuloendothelial activity (3), but thus far little attention has liposomes from the blood (in a 4-hr period) was inhibited 30 to been paid to the possible effects of the presence of tumor cells 50%. This could be ascribed to a decreased activity of the on the elimination of liposomes from the blood. reticuloendothelial system caused by the tumor cells, as was We confirmed that large liposome doses result in prolonged indicated by the simultaneous inhibition of carbon clearance. Six circulation time in normal as well as in tumor-bearing mice, after days after injection of the lymphosarcoma cells, the elimination both i.v. and i.p. administration. During this study, we observed, of liposomes from the blood in tumor-bearing mice was restored however, a profound influence of the presence of lymphosar to the value in normal mice. The possible involvement of tumor coma cells in the animals on blood clearance and organ distri cells in the uptake of liposomes by the liver was investigated bution of the liposomes. In addition, we obtained direct evidence morphologically after i.v. injection of peroxidase-containing lipo of the lack of in vivo liposome uptake by lymphosarcoma cells somes into lymphosarcoma-bearing mice. Liposome-entrapped situated in the liver, despite their direct accessibility from the peroxidase activity was never observed in the tumor cells. The bloodstream. results presented here indicate that the lymphosarcoma cells do not directly participate in the hepatic accumulation of liposomes, MATERIALS AND METHODS although their mere presence may have significant indirect effects on the elimination of liposomes from the blood and on their tissue Liposome Preparation. Sphingomyelm (from bovine brain) and cho distribution. lesterol were obtained from Sigma Chemical Co., St. Louis, Mo. Phos- phatidylserine, purified from bovine brain as described by Papahadjopou- los and Miller (24), was a gift from Dr. J. C. Wilschut of our laboratory. INTRODUCTION 125l-Labeled PVP3 (specific activity, 38 /iCi/mg; M, 30,000 to 40,000) was purchased from the Radiochemical Centre, Amersham, England. Unipore The potential clinical usefulness of liposomes as a system for polycarbonate filters were from Bio-Rad Laboratories, Richmond, Calif. the controlled delivery of therapeutic agents has been discussed Sepharose CL-2B and Ficoll (M, 70,000) were obtained from Pharmacia in a number of recent reviews (15, 17, 23). After i.v. or i.p. Fine Chemicals, Piscataway, N. J. Horseradish peroxidase Grade II 3,3'- injection, a substantial proportion of the liposomes is found to diaminobenzidine tetrahydrochloride Grade II were from Sigma. The accumulate in the liver and spleen. Liposomes can be taken up diammonium salt of 2,2'-azinodi(3-ethylbenzthiazolinsulfonic acid) was by tissue macrophages (mainly located in liver and spleen) as from Boehringer/Mannheim, Mannheim, Germany. has been demonstrated for Kupffer cells in the liver (27, 37), but REV were prepared essentially as described by Szoka and Papa- relatively little is known about any factors which may affect such hadjopoulos (35). Sphingomyelin, cholesterol, and phosphatidylserine uptake. In cancer chemotherapy, only a few successful applica (molar ratio, 4:5:1) were dispersed in diethyl ether to give a concentration of 20 /¿molof total lipid per ml (the diethyl ether was distilled in the tions of liposomes as a drug carrier system have been reported presence of sodium bisulfite immediately before use). The aqueous phase (11, 14, 19, 21, 25), and the presently available evidence sup (150 mM NaCI:5 mw Tris-HCI, pH 7.4) contained 126l-labeled PVP (2.6 ports the hypothesis that the therapeutic effects from a liposome- mg/ml with varying specific activity). Per ml of the organic phase, 0.3 ml encapsulated cytotoxic drug are due to a delayed elimination of the aqueous phase was added, and the mixture was sonicated under from the blood and a sustained release of the drug from the nitrogen in a bath-type sonicator (Branson). After formation of a homo liposomes, rather than to a selective uptake of the encapsulated geneous suspension of inverted micelles, the organic phase was re drug by the tumor cells (16, 29). moved on a rotary evaporator, and the vesicles thus formed were extruded through a polycarbonate membrane with 0.4-Mm pores (36). 1This research was supported by the Netherlands Cancer Foundation Koningin Wilhelmina Fonds, Project GUKC llx. 1To whom requests for reprints should be addressed. 3 The abbreviations used are: PVP. polyvinyl pyrrolidone; REV, reverse-phase evaporation vesicles; PBS, phosphate-buffered saline. JUNE 1983 2927 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1983 American Association for Cancer Research. H. E//er>sefal. Nonencapsulated 125l-labeled PVP was separated from encapsulated the carbon concentration was measured by determining the light absor- material by gel filtration on Sepharose CL-2B. Void volume fractions bance at 620 nm. The phagocytotic index was calculated according to containing the liposomes were pooled and, if necessary, concentrated in the method of Donald and Tennent (8). an Amicon concentration cell (XM-300 filter). Vesicle lipid concentrations Microscopic Studies. Horseradish peroxidase-containing REV were were determined by lipid phosphorus assay according to the method of injected i.v. into 3-month-old (C57BL x Gr) F, mice. The mice received Bartlett (4), as modified by Böttcher ef al. (5). Liposomes prepared this 2.8 MfTiolof liposomal lipid containing 0.13 mg of peroxidase. Two types way had an encapsulated volume of 4 to 5 liters per mol of phospholipid. of controls were included: (a) mice receiving neither liposomes nor For encapsulation of peroxidase, a solution of 30 mg of horseradish peroxidase; and (o) mice receiving liposomes mixed with free peroxidase. peroxidase per ml of 150 ITIMNaCI:5 rriM Tris-HCI pH 7.4, was used as After 2 hr, the mice were anesthetized with ether, and the liver was the aqueous phase. Peroxidase-containing liposomes were separated perfused in situ (flow rate, 2 ml/min) at room temperature via the portal from nonentrapped enzyme by gel chromatography on Sepharose CL- vein, first with cacodylate buffer (0.1 M with 2% PVP and 0.4% NaNO2, 2B. Peroxidase activity associated with the liposomes was measured by pH 7.4) for 1 min to remove blood, subsequently with 2% glutaraldehyde a modification of the method of Steinman and Cohn (34), as described in 0.1 M cacodylate buffer for 7 to 8 min to fix the tissue, and finally by Roerdink era/. (28). The diammonium salt of 2,2'-azinodi(3-ethylbenz- again with the cacodylate buffer to remove the fixative. Vibratome thiazolin sulfonic acid) was used as a chromogen. Latency of the en sections (30 to 40 Mm; Oxford vibratome sectioning system) were trapped enzyme was defined as: thoroughly washed with PBS (137 mw NaCI, 2.6 FTIMKCI, 6.4 HIM Na2HPO4, and 1.4 mKH2PO4, pH 7.4) and incubated, for the ultrastruc Total enzyme activity - Free enzyme activity tural demonstration of peroxidase reaction with 0.075% 3,3'-diamino- x 100% Total enzyme activity benzidine tetrahydrochloride and 0.001% H2O2 in PBS, for 30 min in the dark at 37° (10). The sections were washed in PBS and cacodylate Total and free enzyme activities refer to the activities in the presence buffer (0.1 M with 6.8% sucrose, pH 7.4) and postfixed in 1% OsO4 in and absence of 1% Triton X-100, respectively. Latency for the vesicles cacodylate buffer, dehydrated in an ethanol series, and embedded in used in this study was 94%. Epon 812. Semithin (1 ßm)and ultrathin sections were cut (LKB ultra- Animals. Three-month-old female C57BL x Gr F, mice were obtained tome). Semithin sections were stained with toluidine blue. From these locally. A B-lymphosarcoma, which originated spontaneously in a C57BL sections, areas were selected for ultrathin sectioning. Ultrathin sections mouse (7), was transplanted weekly by i.p. injection of 106 tumor cells. were stained with uranyl acetate and lead citrate and examined in a Seven days after inoculation, the spleen was significantly enlarged (from Philips EM 300 electron microscope.

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