Development of KRAS-Targeting siRNA and Chemotherapy Co-Delivery Chitosan NPs for Enhanced Treatment of Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers due to its aggressive progression, late-stage diagnosis, and limited treatment options. These clinical challenges are compounded by intrinsic resistance to chemotherapy and the high prevalence of activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS). To address these barriers, we developed a chitosan-based nanoparticles platform for the co-delivery of paclitaxel (PTX) and small interfering RNA (siRNA) targeting a prevalent KRAS variant, G12D (CP-PTX-KRAS G12D siRNA) to pancreatic cancer cells. The CP-PTX conjugate was synthesized via carbodiimide-mediated coupling between pegylated chitosan and carboxyl-modified PTX, then complexed with siRNA through electrostatic interactions to form dual-functional nanoparticles.The optimized formulation exhibited a compact size (~26.5 nm) and a moderately negative zeta potential (−9.8 mV), indicating stable siRNA loading and favorable physicochemical properties for intracellular delivery. The platform was initially validated using PolyIC, a double-stranded RNA and firefly luciferase siRNA to optimize formulation parameters and assess delivery efficiency. Cytotoxicity assays in KRAS G12D-mutant PANC-1 cells revealed dose-dependent viability reductions with single-agent PTX and KRAS siRNA, exhibiting IC50 values of 0.5470 µM and 0.2784 µM, respectively. Co-treatment at fixed PTX:siRNA molar ratios (1:0.2, 1:0.5, 1:1, 1:2) significantly enhanced cytotoxicity compared to single agents. CP-PTX-siRNA nanoparticles achieved consistently lower total IC50 values than corresponding free drug combinations across all tested ratios. At the 1:0.2 ratio, the nanoparticles formulation showed a total IC50 of 0.0921 µM (vs. 0.1328 µM for free drug), with a CI value of 0.317, indicating strong synergy. Similar trends were observed for other ratios, with all CI values <1, supporting synergistic interactions between PTX and siRNA when co-delivered via nanoparticles. These findings demonstrate the potential of CP-PTX-siRNA nanoparticles as an effective co-delivery platform for chemo-gene therapy in KRAS-driven pancreatic cancer. This system offers a modular and biocompatible approach to overcoming drug resistance and genetic drivers of malignancy, supporting its further development for translational cancer therapy.