Development of Injectable PEGylated Liposome Encapsulating Disulfiram for Colorectal Cancer Treatment

Disulfiram (DS), an anti-alcoholism medicine, shows strong anti-cancer activity in the laboratory, but the application in clinics for anti-cancer therapy has been limited by its prompt metabolism. Conventional liposomes have shown limited ability to protect DS. Therefore, the aim of this study is to develop PEGylated liposomes of DS for enhanced bio-stability and prolonged circulation. PEGylated liposomes were prepared using ethanol-based proliposome methods. Various ratios of phospholipids, namely: hydrogenated soya phosphatidylcholine (HSPC) or dipalmitoyl phosphatidylcholine (DPPC) and N-(Carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG₂₀₀₀) with cholesterol were used. DS was dissolved in the alcoholic solution in different lipid mol% ratios. The size of the resulting multilamellar liposomes was reduced by high-pressure homogenization. Liposomal formulations were characterized by size analysis, zeta potential, drug loading efficiency and stability in horse serum. Small unilamellar vesicles (SUVs; nanoliposomes) were generated with a size of approximately 80 to 120 nm with a polydispersity index (PDI) in the range of 0.1 to 0.3. Zeta potential values of all vesicles were negative, and the negative surface charge intensity tended to increase by PEGylation. PEGylated liposomes had a smaller size (80-90 nm) and a significantly lower PDI. All liposomes showed similar loading efficiencies regardless of lipid type (HSPC or DPPC) or PEGylations. PEGylated liposomes provided the highest drug biostability amongst all formulations in horse serum. PEGylated DPPC liposomes had t_1/2 =77.3 ± 9.6 min compared to 9.7 ± 2.3 min for free DS. In vitro cytotoxicity on wild type and resistant colorectal cancer cell lines was evaluated by MTT assay. All liposomal formulations of DS were cytotoxic to both the wild type and resistant colorectal cancer cell lines and were able to reverse chemoresistance at low nanomolar concentrations. In conclusion, PEGylated liposomes have a greater potential to be used as an anticancer carrier for disulfiram.