Role of Liposome Type and Route of Administration in the Antitumor Activity of Liposome-Entrapped I -Fj-D-Arabinofuranosylcytosine Against Mouse L1210 Leukemia1

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[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, â€˜â€˜depotâ€•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 37Â°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 37Â°for an additional 30 mm and then equilibrated at 25Â°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 37Â°.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 37Â°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 20Â°.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 4Â°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 â€˜â€˜0,'â€˜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 37Â°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 20Â°.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). The captured amount of ama-C(the total amount of homogeneous, but they have a high surface:volume ratio drug present in the vesicle preparation as a percentage of and, hence, are severely limited for encapsulation in water the total initial amount) was determined from madioisotopic soluble drugs. Table 1 also shows that the diameters of measurements of captured [3H]ara-C which was shown to MLV liposomes become smaller following extrusion correlate with values obtained by spectroscopy at 254 nm through i .0-@tmmembranes. The majority of lipid material after lipid extraction in chloroform:methanol:water. In the experiments reported here, the capture varied from 4 to i8% for MLV and 1 to 2% for SUV depending on the Table 1 conditions. Factors which increased capture were in Capture volume and diameter range for liposomes prepared proceduresIn according to 3 different creased concentrations of ama-Cin the solution added to all cases, the lipid composition (PS:PC:CHOL, 1 :4:5), lipid the dried lipid and an increased concentration of phospho concentration (60 @xmoI/mI),andaqueousphase(300mMara-C in lipid. At 4Â°,about 0.5%/hr of ama-Cdiffused out of SUV@ 1:10 PBS) were the same. Capture and volume calculation are vesicles. In one series of experiments, ama-Cin SUV vesi basedon [3HJara-C.Capture des was stored at 4Â°for several weeks and redialyzed, and vol- Diameter cytotoxicity was determined. The loss of encapsulated ama ume (id/mg rangeâ€• C during this period was 10 to 20%, and its cytotoxicity per Preparation(nm)SUV % capture total lipid) mg was undiminished. 1.8 0.5 20-50 LUV were prepared as described previously (3i). Soni MLV (U)'@ 16.5 4.1 500-3200 cated PS vesicles were prepared in NaCI:histidine:N MLV(E) 19.3 4.8 350-1800 tmis(hydroxymethyl)methyl-2-am inoethanesulfo n ic acid a Diameters determined from measurements of electron micro buffer. The vesicles were then incubated in the presence of graphs of SUV (freeze fracture) and MLV (negative staining) as described before (29, 31). 10 mM CaCI2 for 1 hr at 37Â°and centrifuged at iO,000 x g b U, unextruded; E, extruded under pressure through 1 .0-pm for 10 mm. The pellet was mixed with 0.01 M ama-Cand nucleopore membranes.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Y. M. Rustum et a!. was recovered with minimal loss in the filtrate. Further Table 3 more, extrusion of MLV liposomes did not modify their Antitumor activity of ara-C entrapped in liposomes against relatively high encapsulation efficiency of ama-C. leukemiaL1210cells Antitumor Activity of ara-C Entrapped in Liposomes. Day0Cells (1.0 x i0@)were transplanted iv. into DBA/2J mice on Table 2 shows that in contrast to the in vitro results (25, 26), and treatment (iv.) withlater.Dosage(mg/kg ama-Cwas initiated 24 hr ama-Centrapped in negatively charged SUV showed slightly greater antitumor activities than did positively charged x Av. survival 30-day sur vivorsMLVLiposomes 1) time SUV. MLV, whether positively or negatively charged, showed much greater activity than did SUV; in both in 0/10ara-C 20 9.6Â±1.3a 0/10ara-CinSUVfree 10 9.6 Â±1.3 stances, 6 of iO mice were 30-day survivors. These effects 1/10ama-C 10 11.3Â±1.5 were obtained with a single i.p. dose of entrapped ama-C. 0/5ara-CinMLVin SUV@ 10 13.2 Â±0.8 (Free ama-Cis normally ineffective even at 1600 mg/kg given 2/10ama-C 10 16.7Â±3.4 as a single dose.) The data also show that liposomes alone 0/5ara-Cin MLV@ 10 16.5 Â±2.3 or mixed with free ama-Chad no significant effect on the 0/4a in LUV 10 9.6 Â±0.5 survival time of DBA/2J mice bearing Li2iO cells. ama-C MeanÂ±S.D. encapsulated within LUV at comparable doses was not as effective. There was no evidence of tumor in the pemitoneal somes against leukemia Li2iO when both tumor transplant cavity of mice dying after treatment with LUV, suggesting (106 cells) and treatments were carried out via the iv. route that the host toxicity after treatment with this type of is shown in Table 5. The data show that while free ama-Cdid liposome was different from that of the MLV's and SUV's. not significantly increase the survival times, ama-C en The antitumor activity of ama-C entrapped in different trapped in MLV prolonged survival. The optimal dose for types of liposomes against mice beaming L12i0 cells when MLV ama-C in a single iv. injection against Li2iO cells both cell inoculations and drug treatment were carried out given 24 hr previously is approximately 50 mg/kg. At 100 i.v. is shown in Table 3. Significant increases in life span mg/kg, the decrease in survival time may have been due to were observed when ama-C was entrapped in liposomes host toxicity. where, at iO mg/kg, 2 of 10 mice that received this treat Chart 1 outlines the p.o. antitumor activity of free ama-C ment survived for more than 30 days. SUV (10 mg/kg) only and ama-Centrapped in liposomes in an experiment against marginally increased life span, and no increase in life span L12i0 cells (106) inoculated i.v. to DBA/2J mice. The results was observed with LUV. indicate that although p.o. administration of free ama-C (10 Table 4 summarizes the results of the antitumom activity mg/kg on Day 1) was not effective, both MLV and SUV of ama-Centrapped in MLV liposomes against mice bearing containing ama-C produced a 31 and 26% increase in life different tumor loads. The data indicate that when MLV is span, respectively, over controls (MLV alone), that was used for entrapment of the drug, a dose response can be statistically significant. easily obtained. Furthermore, it was obvious that MLV were In Vivo Effect of Plain Liposomes and Liposomes Con effective against mice given injections of either i0@or i06 taming ara-C. Table 6 shows the in vivo effect of SUV and cells, with 5 of 10 and 2 of 10 mice, respectively, becoming MLV against normal HA/ICR Swiss mice. The results mdi long-term survivors at 50 mg/kg. cate that plain liposomes (containing dextrose instead of The antitumom activity of ama-C entrapped in MLV lipo ama-C)have no adverse effect to the host, even at very high levels (>5500 mg lipid per kg on Day i ). Only the unfiltered MLV preparation showed acute toxicity. The data also show Table 2 that ama-C entrapped in lipid vesicles is more toxic than is Antitumor activity of ara-C entrapped in liposomes of different the free drug. For example, the doses of ama-C lethal to 0, types and charges 50, and 100% of the mice entrapped in MLV were about DBA/2J mice were transplanted i.p. with 1.0 x and 200, 396, and 792 mg/kg on Day 1, respectively, whereas treatmentincludedDosage(ara-C)(mg/kg30-dayLiposomes was initiated 24 hr later. Only dead mice wecells,re in the calculation of average survival times.10@ free ama-C greater than 1500 mg/kg on Day 1 was required to achieve acute effect.

DISCUSSION survivalsurvivorsMLV@ on Day 1) Av. .6â€•0/10MLV 1000â€• 9.8 Â±1 Although ama-C entrapped in different types of liposomes 9.7Â±0.80/10SUV@ 1000 does not show an increase in effect against Li210 cells in 0.70/10SUV 1000 9.5 Â± vitro as compared to the free drug (25, 26), its in vivo .60/10LUV 1000 9.8 Â±1 activity after a single injection is strongly potentiated 0.90/10ara-C 1000 8.5 Â± 2.20/10ara-CinSUVfree 10 11.5 Â± against Li2iO tumor cells grown in DBA/2J mice after 16.8Â±1.82/10ara-CinSUV@ 10 liposome entrapment. 16.5Â±3.60/10ara-CinMLV 10 The present data indicate that: (a) MLV were much more 22.6Â±4.16/10ara-CinMLV@ 9 effective in trapping ama-Cthan were SUV; (b) MLV contain 21.2Â±3.86/10ara-CinLUV 9 14.6Â±1.61/10MLV 9 ing ama-C were more effective than SUV or LUV against +ara-Cfree 10 10.8Â±1.40/10 L12i0 tumors at comparable doses; (c) the antitumor activ ity of the LUV (composed of pure PS) was limited by host (S Total lipid. F) Mean Â± S.D. toxicity; (d) positively and negatively charged MLV were

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Table 4 Antitumor activity of ara-C entrapped in MLV liposomes against mouse leukemia L1210 cells latertreatmentTumor cells (1.0 x 106or 1.0 x i0@cells/mouse) were inoculated p., and 24 hr calculationofvia the i.p. route was initiated!' Only dead mice were included in the average survival time.SurvivalDosage

30-day(mg/kg treated/ on Inocu- Av. survival control suM vorsControlGroup Day 1) lum time (%) 0/10Control(PBS) 10@ 9.5 Â±1,4b 0/10ama-C(MLV1 20â€• 10@ 8.5 Â±0.9 89 0/10MLV@free 100 10@ 11.2 Â±2.8 118 0/10ara-C+ ara-C free 20 + 100 1O@ 12.8 Â±1.2 135 4/10ama-Cin MLV 100 10@ 16.8 Â±2.4k 177 5/10ara-Cin MLV 50 i0@ 24.0 Â±5.0' 253 4/iOara-Cin MLV 24 10@ 22.5 Â±6.3 237 5/10ara-CinMLVin MLV 12.5 1O@ 19.8 Â±3.5 208 0/10ara-C 6.3 10@ 17.5Â±6.3 184 0/10Control(MLV)free + MLV- 100 10' 8.6 Â±0.9 91 0/10ama-C 20 10@ 7.5Â±0.9 79 0/10ara-Cfree 100 10@ 8.4 Â±1.2 88 2/10ara-CinMLVin MLV 50 106 22.5 Â±7.3 237 25 106 18.0Â±4.2 189 0/10 a Twenty@moloflipidper20gmouse. b Mean Â± S.D.

(. Mice died with no apparent tumor in their peritoneal cavity.

Table 5 AVERAGE SURVIVAL(DAYS) + S.D Antitumor activity of free and ara-C entrapped in MLV mouse leukemia L1210(106cells) injected0Av. i.v. on Day @I .@ 0) 0 Treatment with the drug was initiated i.v. 24 hr later.on I 1 I I 1 CONTROL CMLV)__H vival treated survival /con (%)ControlGroup Dose (mg/kg) timeSum trol ARA-CjARA-C (FREE) (PBS) 0.2 mI/mouse 8.0 Â±0.7â€• Control(MLV) 20â€• 7.8Â±0.8 98 ara-Cin MLV 100 5.8 Â±0.5 74 ara-Cin MLV 50 15.4 Â±1.1 197 (MLV)ARA-C H ama-CinMLV 25 13.8 Â±1.9 176 ama-CinMLV 12.5 10.6 Â±1.5 135 ara-C in MLV 6.25 8.6 Â±0.8 110 (SUV) ama-Cfree 100 8.6 Â±0.6 110 H ama-Cfree 50 8.7 Â±1.2100 111 Chart 1. Effect of p.o. administered ama-Centrapped in liposomes on the a MeanÂ±S.D. survivaltimesof micebearingiv. Li2iO cells Â±S.D.Tumortransplants(10' b 20 @mol lipid per 20-9 mouse. cells) were carried out on Day 0, and p.o. treatment with free ama-Cor ama-C entrapped in liposomes at 10 mg/kg on Day 1 was initiated 24 hr later. Significance of differences from control were: ara-C free, p > 0.1 ; are-C equally effective in vivo ; (e) entrapped ama-Cwas active MLv),

APRIL1979 1393

Table 6 into drug-resistant tumors (30) where resistance could be Lethality of plainara-Cagainst liposomes and Iiposomes containing due to either transport defects or deletion of activating micePreparations normal HA/ICR Swiss enzyme(s). ama-Cwere of plain liposomes or liposomes containing variedfromadministered i.v. at the indicated doses in a volume 0.2 ml to 0.6 mI/20-gmouse.Dose ACKNOWLEDGMENTS

toof lethal to 50% Dose lethal We thank A. Burr, C. Wrzosek, J. Goranson, D. Millholland, and A. Lazo the micemicekg) (mg/ 100% of the for technical assistance. (mg/kg)Type

ama-CSUV- Plain ama-C Plain REFERENCES

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25. Mayhew, E., Papahadjopoulos, D., Rustum, Y. M., and Dave, C. Inhibi for introducing biologically active materials into cells. Methods Cell tion of tumor cell growth in vitro and in vivo by 1-f3-D-arabinofuranosyl Biol. 14: 23-32, 1976. cytosine entrapped within phospholipid vesicles. Cancer Res.,36: 4406- 33. Rahman, Y-E., Cemny,E. A., Tollaksen, S. L., Wright. B. J., Nance, S. L., 4411,1976. and Thomson, J. F. Liposome-encapsulated actinomycin D: potential in 26. Mayhew,E., Papahadjopoulos,D.,Rustum,Y. M., and Dave,C. Useof cancer chemotherapy. Proc. Soc. Exp. Biol. Med., 146: 1173-1176, liposomes for the enhancement of cytotoxic effects of are-C and its 1974. triphosphate. Ann. N. Y. Acad. Sci., 308: 371-386, 1978. 34. Rustum, Y. M. Metabolism and intracellular retention of i-fJ-D-arabino 27. Neerunjun,E.D., andGregoriadis,G.Tumourregressionwithliposome sylcytosine as predictors of response of animal tumors. Cancer Res., 38: entrapped asparaginase: some immunological advantages. Biochem. 543â€”549,1978. Soc.Trans.,4: 133â€”134,1976. 35. Rutman, A. J., Avadhani, G. N., and Ritter, C. Activation in vitro of 28. Papahadjopoulos,D.,and Kimelberg,H. K. Phospholipidvesicles(lipo nitrogen mustard by liposome transport. Biochem. Pharmacol. , 26: 85- somes) as models for biological membranes: their properties and inter 92, 1977. actions with cholesterol and proteins. In: S. G. Davidson (ed), Progress 36. Rutman, A. J., and Rifler, C. Transport potentiation of lipid soluble in Surface Science, Vol. 2, Part 4, pp. 141-232. Oxford, England: alkylating agents. Ann. N. Y. Aced. Sci., 308: 434â€”435,1978. Pergamon Press, Ltd., 1973. 37. Rutman, R. J., Ritter, C. A., Aradhani, N. G., and Hansel, J. Liposomal 29. Papahadjopoulos,D.,and Miller, N. Phospholipidmodelmembranes.I. potentiation of the antitumor activity of alkylating drugs. Cancer Treat. Structural characteristics of hydrated liquid crystals. Biochim. Biophys. Rep.,60: 617-618.1976. Acta,135:624-638,1967. 38. Sessa, G. , and Weissman, G. Incorporation of lysozyme into liposomes. 30. Papahadjopoulos, D., Poste, G., Vail, W. J., and Biedler, J. L. Use of A model for structure-linkedlatency.J. Biol. Chem.,245: 3295-3301, lipid vesicles as carriers to introduce actinomycin D into resistant tumor 1970. cells. Cancer Res., 36: 2988-2994, 1976. 39. Skipper,H. E., Schabel,F. M., Mellet,L. B., Montgomery,J.A., Wilkoff, 31. Papahadjopoulos, D., Vail, W. J., Jacobson, K., and Poste, G. Cochleate L. J., Lloyd, H. H., and Brockman,A. W. Implicationsof biochemical, lipid cylinders: formation by fusion of unilamellar lipid vesicles. Biochim. cytokinetic, pharmacologic and toxicologic relationships in the design Biophys. Acta, 394: 438-491 , 1975. of optimal therapeutic schedules. Cancer Chemother. Rep. , 54: 631- 32. Poste,G., Papahadjopoulos,D.,andVail,W.J. Lipidvesiclesascarriers 647, 1970.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Role of Liposome Type and Route of Administration in the Antitumor Activity of Liposome-entrapped 1- β -d-arabinofuranosylcytosine against Mouse L1210 Leukemia

Youcef M. Rustum, Chandrakant Dave, Eric Mayhew, et al.

Cancer Res 1979;39:1390-1395.

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