Functionalized Caprolactone-Polyethylene Glycol Based Thermo-Responsive Hydrogels of Silibinin for the Treatment of Malignant Melanoma

Abstract

Purpose: Silibinin, is a natural compound, which has shown anticancer activity in various malignancies. In this study, we evaluated the anticancer effects of silibinin in B16-F10 melanoma cells and developed a novel thermoresponsive hydrogel for local delivery of this compound. Method: A thermoresponsive hydrogel loaded with silibinin was prepared using triblock copolymers of poly[(alpha-benzyl carboxylate-epsilon-caprolactone)-co-(alpha-carboxyl-epsilon-caprolactone)](ran)-b-PEG-b -[(alpha-benzyl carboxylate-epsilon-caprolactone)-co-(alpha-carboxyl-epsilon-caprolactone)](ran) (PCBCL-b-PEG-b-PCBCL), namely PolyGel (TM), and compared with a Pluronic F-127 formulation of silibinin. Sol-gel transition temperature of hydrogels was measured by inverse flow method and modulated differential scanning calorimetry (MDSC). Silibinin loading efficiency was measured by HPLC. The MTT and clonogenic assays were used to assess the cytotoxicity and anti-proliferative effects of silibinin on B16-F10 melanoma cells. Flow cytotmetry was used to quantify the induced level of apoptosis and measure the intracellular level of activated STAT3 (pSTAT3) following silibinin treatment in B16.F10 cells. The effects of silibinin on the activation of oncogenic proteins were also evaluated by western blot. Results: Silibinin inhibited cell proliferation (IC50 = 67 mu M), provoked cell cycle arrest, induced apoptosis, suppressed key oncogenic pathways (i.e STAT3 and MEK/ERK), and enhanced the cytotoxic effects of doxorubicin in B16-F10 cells. Both PolyGel (TM) and Pluronic F-127 hydrogels were effective in loading silibinin. A lower drug release pattern within 24h, fitting first- order release kinetics, was observed for the release of silibinin from both gels compared to free drug. PolyGel (TM) demonstrated enhanced percutaneous absorption of silibinin through increasing mouse skin intracellular lipid fluidity as documented by DSC of skin following PolyGel (TM) use. Silibinin loaded in PolyGel inhibited the growth of B16-F10 cells (IC50 = 30 mu M) and effectively suppressed pSTAT3 activity in B16-F10 cells at 10 mu M. Conclusion: Our results imply a great potential for PolyGel (TM) formulations of silibinin for local treatment of malignant melanoma.