Delivery of Doxorubicin to Solid Tumors using Thermosensitive Liposomes

Abstract

Liposomes are lipid vesicles with an aqueous interior size between 50 nm and 200 nm in diameter. Liposomal drugs currently in clinical use are characterized primarily by their decreased side effects rather than improved therapeutic potency. Significant improvements in the efficacy of liposomal drug therapy may be obtained using thermosensitive liposomes (TSL) in combination with local hyperthermia (HT). TSL release their content at a specific formulation-dependent gel to liquid-crystalline phase transition temperature. At this temperature the membrane's permeability increases by several orders of magnitude. The purpose of present work was preparation of TSL loaded with doxorubicin (Dox) and investigation of their effect on B-16 mouse melanoma and Ehrlich (line ELD) carcinoma in combination with HT. TSL were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, cholesterol and α-tocopherol acetate in molar ratios of 9:1:0.02:0.2:0.2 (composition 1); 9:1:0.1:0.5 (composition 2); 9:1:0.2:0.75 (composition 3); 9:1:0.3:1 (composition 4); 9:1:0.4:1.2 (composition 5). The extrusion was performed through 200 nm polycarbonate membranes at 50 °C. Dox was loaded into TSL by ammonium ion gradient. Drug-to-lipid ratio was 0.13:1 (w/w). Dox-TSL were lyophilized for better stabilization with 4 % sucrose added as a cryoprotectant. Vesicle size was measured using Nicomp-380 Submicron Particle Sizer. Dox-TSL were separated from untrapped Dox by gel filtration. In biological experiments tumors were transplanted in the shank muscle of mice. Dox-TSL or free Dox were injected in retroorbital sinus of animals at doses of 9 and 4.5 mg Dox per kg body weight. HT treatment of shank with transplanted tumor was performed at 43 °C for 30 min using water bath. Efficacy of Dox encapsulation in TSL of compositions 4 and 5 was 60 % (diameter of vesicles was 175 ± 10 nm). TSL of compositions 2 and 3 encapsulated 88 % and 86 % of Dox, respectively (diameter of vesicles was 160 ± 10 nm). TSL of composition 1 trapped 88-94 % of Dox (diameter of vesicles was 175 ± 15 nm). The composition 1 has been chosen for preparation of lyophilized drug, which has been assessed in biological experiments. The doubling time of B-16 melanoma was 9 days after heating on a background of Dox injection at dose of 9 mg/kg, while heating of tumors after injection of Dox-TSL at doses of 4.5 and 9 mg/kg increased tumor doubling time up to 12 and 16 days, respectively. The doubling time of Ehrlich carcinoma increased from 3 days in the control group up to 14 days for the group of mice administered 9 mg/kg of Dox-TSL followed by HT in 15-20 min. Encapsulation of Dox in TSL has resulted in decrease of its toxicity as judged by animal survival. Thus, Dox-TSL in combination with HT has shown more efficiency than free Dox in suppression of tumor growth.