• Abstract and Figures
• Public Full-text

[CANCER RESEARCH 39, 1390-1395, April 1979] 0008-5472/79/0039-0000$02.OO Role of Liposome Type and Route of Administration in the Antitumor Activity of Liposome-entrapped I -fJ-D-arabinofuranosylcytosine against Mouse L1210 Leukemia1 Youcef M. Rustum,2 Chandrakant Dave, Eric Mayhew, and Demetrios Papahadjopoulos Departments of Experimental Therapeutics (V. M. R., C. D.J Medicine A (V. A.) and Experimental Pathology fE. M. , D. P.), Roswell Park Memorial Institute, Buffalo, New York 14263 ABSTRACT delivery system (12). The idea was that drugs entrapped in the aqueous space(s) inside liposomes could be delivered Studies were undertaken to evaluate the antitumor activ to the site of action with greater selectivity and/or less ity of i-f3-D-amabinofuranoSylcytoSine (ama-C)entrapped in degradationthanforthe freedrug (iO).Theirabilityto liposomes of different size and net surface charge. Lipo potentiate the pharmacological efficacy of various drugs in somes were composed of phosphatidylcholine and choles vitro against mammalian cultured cells (32) enhanced their terol mixed with either phosphatidylserine (negative prospects as a drug delivery system. Studies using lipo charge) or stearylamine (positive charge). Multilamellam somes as carriers for anticancer drugs in vivo have so far vesicles were much more effective in trapping ama-Cthan shown mixed results (7, 1i , 17, i8, 21-27, 33, 35-37). were small unilamellam vesicles. Irrespective of liposome However, there has not as yet been a sustained and system size, ama-Centrapped in positively charged liposomes was atic effort to optimize conditions and examine in detail slightly more effective in inhibiting the growth of mouse which liposomal properties give maximal antitumom effects leukemia Li210 cells in culture compared to free ama-C or ina givensystem. ama-Centrapped in negatively charged liposomes. The me Mayhew et al. (25, 26) and Kobayashi et al. (22, 23) have suIts in vivo indicate that: (a) multilamellar vesicles contain shown that anti-tumor activity of ama-C,3an effective agent ing ama-C were more effective than were small or large against some experimental tumor models (5, 34, 39) and unilamellar vesicles composed of pure phosphatidylsemine against human leukemia (3, 4, 6, 8, 9, 13, 14), can be against Li2iO tumors at comparable doses; (b) the antitu enhanced by encapsulation of the drug in liposomes. This momactivity of the type of large unilamellar vesicles used study was carried out to investigate further the efficacy of was limited by host toxicity; (c) positively and negatively ama-C entrapped in different types of liposomes against charged multilamellam vesicles were equally effective in leukemia Li2iO and to determine if ama-C entrapped in vivo ; (d) entrapped ama-Cwas effective against Li 210 tu liposomes is effective when administered i.v. and p.o. as moms at both 10@and 106 initial tumor load; (e) entrapped well as i.p. ama-Cwas active against Li210 tumors when tumor trans plant and treatment were both carried out by the iv. route; (f) mice treated p.o. with entrapped ama-Cshowed increased MATERIALS AND METHODS survival times; and (g) acute toxicity tests in mice indicated Preparation of Phospholipid Vesicles. Two series of that for liposomes without the drug the dose lethal to 50% vesicles were made, those containing ama-C and control of the mice was greater than 5 g/kg (total lipid per body vesicles containing dextrose and buffer only. All chemicals weight). The same tests indicated that encapsulated ama-C were prepared or obtained as described in detail previously was more toxic than was the free drug. The mechanisms of (26,29). the antitumor action of liposome-entrapped ama-Care not Negatively charged vesicles, chloroform methanol solu clear, and the evidence could be explained by some type of tions of PS and PC, were mixed with CHOL (molar ratio, ‘‘depot―system without any assumption of a specific deliv PS:PC:CHOL, 1:4:5) and evaporated to dryness under nitro emy system to tumor cells. gen and reduced pressure. Ten to 300 Mmol of lipid were used in different experiments. The dried lipid was sus INTRODUCTION pended (usually 60 @.tmol/ml) in an aqueous solution nor mally containing one-tenth strength PBS- and 0.3 M ama-C Phospholipid vesicles (liposomes) were originally devel or similar concentration of dextrose and vortexed at 37°for oped as a model membrane system (i , 2). However, it soon io mmor longeruntilall lipidwassuspended.Dextrose became clear that their capacity to entrap ions (6) and small containing liposomes (plain) were used as controls. MLV (28) and large (19, 38) molecules along with their low were further shaken in a reciprocal shaker (i stroke/sec) at permeability presented considerable advantages as a drug 37°for an additional 30 mm and then equilibrated at 25°for 30 mm. The suspensions were then diluted to 2.5 to 5.0 , This investigation was supported in part by Grants CA-18420, CA-i 8921, 3 The abbreviations used are: ara-C, 1-f3-D-arabinofuranosylcytosine; PS, Contract CM 77118, and a Core Program Grant CA-i3038 from the National phosphatidylserine; PC, phosphatidylcholine; CHOL, cholesterol; MLV, mul Cancer Institute. tilamellar vesicles; SUV, small unilamellar vesicles; LUV, large unilamellar 2 To whom requests for reprints should be addressed, at Roswell Park vesicles; MLv- , free multilamellar lipid vesicles; PBS- , calcium- and magne Memorial Institute, 666 Elm Street, Buffalo, N.Y. 14263. sium-free 0.01 M phosphate-buffered saline; PBS, phosphate-buffered sa Received July 13, 1978: accepted January 12, 1979. line. i 390 CANCER RESEARCH VOL. 39 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Antitumor Activity of Liposome-entrapped ara-C volumes with PBS . For preparation of filtered MLV lipo shaken with glass beads at 37°.EDTA and NaOH were somes, the diluted suspensions were passed through i .0- added to give final concentration of iS mM and pH 7.0, @munipome (nucleopore) filters (25-mm diameter; Bio-Rad respectively. The suspension was allowed to equilibrate for Laboratories, Richmond, Calif.). Both filtered and unfiltered 30 mm at 37°andthen was diluted with iOO mM NaCI buffer preparations were then centrifuged at 100,000 x g for 30 and centrifuged at 48,000 x g for 20 mm. The pellet was mm at 20°.The pellets, usually containing more than 85% washed 3 times in 0.9% NaCI solution buffer and was finally of the lipid material, were resuspended in 0.Oi M PBS and resuspended in 0.01 M PBS dialyzed overnight at 4°against iOO volumes of PBS. The Determinations of entrapped ama-C were made as de concentration of the lipid in the final suspension was scribed for SUV and MLV. Unless otherwise indicated, all usually the same as the original (60 to 80 @tmol/ml)except antitumor activity studies were carried out using 0.22-Mm for the in vivo toxicity experiments where the pellet was filtered liposome preparations. resuspended in a minimal volume of PBS resulting in lipid AntitumorActivity.FemaleDBA/2Jmiceweighing18 to concentration i 50 to 200 .tmol/ml for filtered and 220 to 22 g were used throughout these studies. Randomized mice 280 @tmol/mlfor unfiltered preparations. were inoculated i.p. or i.v. (tail vein) with either i0@or i06 Suv were preparedas before(26)by sonicatingan cells on Day ‘‘0,'‘and treatment was initiated 24 hr later aliquot of the MLV suspension immediately after the initial (Day 1)by i.p.,iv.,orp.o.route.Administrationp.o.was vortexing in a closed glass tube under nitrogen placed in a carried out by stomach intubation in unanesthetized ani sonicating bath (Laboratory Supply Co. ; Model T-80-80-IRS) mals with the use of a 22-gauge needle with a small, at 37°for 1 hr. All liposome preparations were used within stainless steel ball tip. Control mice were treated with either 24 hr from time of preparation unless otherwise stated. The 0.i ml 0.9% NaCI solution or an equivalent volume of sonicated suspension was dialyzed against 100 volumes of liposomes per g of body weight. In calculating the average PBS ,changed every30 mm, 5 or6 timestoeliminatenon survival time, only the mice dead on or before Day 30 were vesicle-associated ama-C.In some experiments, the dialysis includedinthecalculation. step was substituted for by passage of vesicle suspension In Vivo Effects of Liposomes. Swiss mice weighing 18 to through Sephadex G-50 columns to remove extravesicular 20 g were fasted for 24 hr prior to the initiation of treatment. drug. Any large multilamellar vesicles remaining were elim Mice were then given iv. injections of liposomes. The mated by centmifugation of the suspension at 100,000 x g weight of individual mice was followed daily during the first for 1 hr at 20°.After preparation, vesicles were either used week and then weekly thereafter. immediately or stored in PBS . If stored prior to use, the vesicles were redialyzed to remove any drug that had leaked RESULTS from vesicles into the PBS. Positively charged vesicles were prepared similar to neg Size and Efficiencyof ara-C Encapsulationby Different ative vesicles except that steamylamine was used in place of Types of Liposomes. Table 1 shows the size distribution of PS (molar ratio, SA:PC:CHOL 1:3:3) and changes in some liposomes unextruded and extruded through 1.0-jtm nu methodological details as described for negative vesicles cleopome membranes. The size of the vesicle was monitored above. All liposome preparations were used within 24 hr by negative stain and freeze fracture electron microscopy. from time of preparation unless otherwise stated. The sonicated unilamellar vesicles are very small structures, Tracers used were [3HJara-C (Amersham/Seamle; 24 Ci/ ranging approximately from 20 to 50 nm and are relatively mmol).

Load more

  7

Copy Link