Development of STAT-3 targeting siRNA nano-carriers for cancer therapy.

摘要

In many tumors, persistently-active signal transducer and activator of transcription 3 (STAT3) imparts several oncogenic features such as survival, proliferation, angiogenesis, and immune escape. Therefore, STAT3 targeting in cancer and cancer-exposed dendritic cells (DCs) is important for cancer therapy. Our objective is developing delivery modalities of STAT3-targeting small interfering RNA (siRNA) using lipid-modified polyethylenimine (PEI) polyplexes and poly(D,L lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), and evaluating the therapeutic outcomes in vitro and in vivo. Significant increase in siRNA condensation, protection, and cellular uptake by B16.F10 melanoma was seen by stearic-acid-modified PEI (PEI-StA) compared to unmodified PEI. Moreover, PEI-StA increased the STAT3 silencing potency of siRNA compared to PEI. STAT3 knockdown was accompanied with significant induction of interleukin-6 (IL-6) secretion and reduction of vascular endothelial growth factor (VEGF) production and cytotoxicity evidenced by increased Caspase 3 activity in vitro and in vivo, and significant inhibition in tumor growth. Analysis of tumor microenvironment showed CD3+ cells infiltration corresponding to STAT3 knockdown. The levels of CD4+ helper cells, CD8+ cytotoxic cells, and NKT cells significantly increased. DC infiltration and activation significantly increased in tumor mass following STAT3 knockdown as evidenced by high expression of CD86 and CD40. Moreover, IFN-gamma, IL-12, and TNF-alpha significantly increased following STAT3 knockdown by PEI-StA compared to PEI, suggesting Th1-type immunity. Allogenic capacity of DCs isolated from siRNA-treated mice was evidenced by the high T cell proliferation and IL-2 production in mixed lymphocytes reaction (MLR). Then, we explored STAT3 knockdown in DCs exposed to tumor derived factors (TDFs). We investigated encapsulation of siRNA complexes (PEI or PEI-StA) into PLGA NPs (PLGA-P and PLGA-PS). PLGA-P and PLGA-PS had an average diameter of ∼ 370 nm and zeta potential of ∼ -16 mV. Uptake and endosomal localization was confirmed. After TDFs exposure, DCs showed high STAT3 and low CD86 expression. STAT3 silencing by PLGA-P and PLGA-PS restored DC functionality as evidenced by upregulation of CD86, IL-12, and TNF-alpha and MLR activity. PLGA significantly reduced PEI-associated toxicity. Therefore, STAT3 targeting in B16 cells by siRNA polyplexes of PEI and PEI-StA, or in DCs by PLGA-P and PLGA-PS provide potential strategies for cancer therapy.