Abstract LB-8: A novel remote loading method with solubility gradient to encapsulate effevtive amount of taxanes into liposomes.
Authors: Shigehiro Tsukasa , Kasai Tomonari , Mizutani Akifumi , Murakami Hiroshi , Mikuni Katsuhiko , Mandai Tadakatsu , Hamada Hiroki , Seno Masaharu 
Published: August 2014

Targeted liposomal drug delivery is considered to enhance the therapeutic effects of anti-cancer agents because high amount of drug could be administered with least side effects. However, water-insoluble hydrophobic and non-weak base chemical compounds such as paclitaxel (PTX) are not suitable for efficient encapsulating. We conferred water-solubility of PTX by coupling glucose at 7-OH to produce 7-alpha-glucosyloxyacetylpaxcitacel (gPTX). gPTX showed enhanced water-solubility with the 40 % ethylene glycol (EG) in PBS and with the CEP buffer (Cremophor EL and Ethanol in PBS). The maximal solubility of gPTX in these solvents was 1 and 20 mg/ml, respectively. We prepared gPTX encapsulated liposome (gPTX-L) with remote loading method by solubility gradient between 40 % EG and CEP buffer. gPTX efficiently encapsulated into liposomes. The encapsulation efficiency and loading efficiency were 72.9 % and 8.5×10−2 drug mol / initial lipid mol. Then, trastuzumab was conjugated on the surface of gPTX-L to prepare immunoliposome (gPTX-IL) for targeting HER2 overexpressing cancer cells. The particle size of gPTX-IL was under 150 nm and Z potential was -3.07 mV. We evaluated IC50 values by MTT assay after 72 hours exposure. The IC50 values of PTX, gPTX, gPTX-L and gPTX-IL for SK-BR-3 cells were 5.5, 18.9, 6.6 and 5.3 nM, respectively. gPTX-IL exhibited the highest toxicity in these paclitaxel formulations. Similarly, the IC50 values for HT-29 cells were 1.3, 11.0, 7.6 and 6.8 nM, respectively. We then evaluated the time required for each formula to inhibit the growth at the half maximal level (IT50) at the minimum concentration to show maximal cytotoxicity (IC100) after 72 hours exposure. gPTX-IL showed the shortest IT50 values of 1.6 and 1.1 h in SK-BR-3 and HT-29 cells respectively. We then evaluated in vivo distribution of liposome and immunoliposome encapsulating HSA conjugated Cy5.5 in ICR-nu/nu mice bearing HT-29 cell tumors. Both liposome and immunoliposome accumulated at the tumor site, but immunoliposome exhibited less accumulation at liver than liposome. Based on these results, we then evaluated the anti-cancer efficacy of the formula in vivo using tumor bearing ICR-nu/nu mice. While gPTX-L and free trastuzumab did not inhibit tumor growth, gPTX-IL decreased tumor volume and inhibited tumor growth with low side effects at the dose of 150 mg/kg gPTX, which was the lethal dose of naked gPTX exhibiting 25% survival rate of mice one week after the injection of gPTX. Collectively, the gPTX-encapsulated immunoliposome with novel encapsulation strategy should be an excellent candidate of highly efficient drug delivery system of cancer treatment targeting HER2.

Citation Format: Tsukasa Shigehiro, Tomonari Kasai, Akifumi Mizutani, Hiroshi Murakami, Katsuhiko Mikuni, Tadakatsu Mandai, Hiroki Hamada, Masaharu Seno. A novel remote loading method with solubility gradient to encapsulate effevtive amount of taxanes into liposomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-8. doi:10.1158/1538-7445.AM2013-LB-8