ANTICANCER RESEARCH 21: 2577-2584 (2001) Accumulation of Vinblastine into Transfersomes and Liposomes in Response to a Transmembrane Ammonium Sulfate Gradient and their Cytotoxic / Cytostatic Activity In Vitro* H. MASWADEH\ c. DEMETZOS2, K. DIMAS3, s. HATZIANTONIOU\ A. GEORGOPOULOS\ M. RALLIS\ P. DALLASl and G. PAPAIOANNOUl School of Pharmacy, 1 Department of Pharmaceutical Technology, 2 Department of Pharmacognosy, Panepistimiopolis, Zografou ]5771, University of Athens, 3 Department of Biochemistry and Molecular Biology, Oncology Research Center, "St. Savvas" Hospital, Athens, Greece Abstract. Vinblastine was encapsulated into liposomes com- posed from lipids dimiristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC), with cholesterol and transfersomes with sodium cholate prepared by the thin film hydration method. The percentage of vinblastine encapsulation, the stability of transfersomes and liposomes and the rate of release of encapsulated vinblastine at 37'C were studied. The results showed that encapsulation of vinblastine into liposomes was higher than 98% at a drug/phospholipid molar ratio from 0.17 to 0.18, while encapsulation of vinblastine into transferso- mes varied from 50% to 80% at a drug/phospholipid molar ratio from 0.05 to 0.09. The retention of drug in liposomes and in transfersomes was found to be time/dependent. The retention of drug in transfersomes compared to the liposomes was reduced due to the presence of sodium cholate which caused destabili- zation and reduced the main phase transition temperature Trnof the PC bilayers. The cytotoxic/cytostatic activity of the two lipo- some formulations and the two transfersome formulations with or without encapsulated vinblastine were examined against nine human cell lines and the parameters GIso, TGI, LCso were estimated according to the NCI protocol. Free DPPC/sodium cholate liposomes found to exhibit strong antiproliferative activity in contrast to the other three free liposomal formulations (DPPC/Cholesterol, DMPC/cholestero4 DMPC/sodium cholate). On the other hand, vinblastine encapsulated into the liposomes found to exhibit 20-fold less activity on average, in the three parameters calculate compare to the free vinblastine. *This work is a part of H. Maswadeh Ph.D. thesis. Correspondence to: Dr. C. Demetzos, School of Pharmacy, De- partment of Pharmacognosy, Panepistimiopolis, Zografou 15771, University of Athens, Greece. e-mail: demetzos@gaJenos. pharm.uoa.gr. Key Words: Vinblastine, liposomes, transfersomes, sodium cholate, cytotoxic / cytostatic activity. 0250-7005/2001 $2.00+.40 Vinca alkaloids are one of the most important classes of the antineoplastic drugs. Vincristine and vinblastine, were iso- lated from the periwinkle plant Catharanthus roseus G. Don (Apocynaceae). The mechanism of action of vinca alkaloids is well established. They bind to spindle microtubules and arrest cells in metaphase. Vinblastine (Figure 1) is used in the treatment of leukemia and Hodgkin,s disease, breast carci- noma Wilms tumor, Ewing,s sarcoma and small cell lung cancer, either alone or in combination with other chemo- therapeutic agents (1, 2). Bile acids and salts are amphiphilic physiological substances and they have been widely used in studies on the detergent-membrane interaction (3). Previous work has shown that transfersomes (which contain sodium cholate) are sufficiently flexible to pass even through the pores which are appreciably smaller than their own size and may bring their encapsulated agents through the biological permeation barriers (4). The aim of this work was to inve- stigate some factors that affect the percentage of vinblastine encapsulation into liposomes and transfersomes by using a transmembrane ammonium sulfate gradient method as well as to study the effect of sodium cholate on the drug release in buffer at 3TC and the cytotoxic/cytostatic activity of the two liposomal formulations, compared to the cytotoxic/cytostatic activity of the two transfersomal formulations against nine human leukemic cell lines. It should be noted that vinblastine has never been encapsulated into transfersomes by a trans- membrane ammonium sulfate gradient method and also that this formulation has never been examined against human leukemic cell lines in vitro. Materials and Methods Materials. Dimiristoylphosphatidy1choline (DMPC), dipalmitoylpho- sphatidy1choline (DPPC), and cholesterol were obtained from Avanti Polar lipids Inc. (AL.USA). Vinblastine as Vinblastine sulfate was used as in its commercial form (Vinblastine sulfate, Pharmachemie B. V., Haarlem, Netherlands). Organic solvents i.e chloroform (CHCI3) and methanol. (MeOH) were spectroscopic grade. Ammonium sulfate, TES (N -tris[HydroxymethyIJmethyl- 2-aminoethanesulfonic acid), triethanol- 2577