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<p>Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with KRAS mutations driving aggressive tumor growth and resistance to conventional therapies. Targeting KRAS directly has been challenging, necessitating alternative therapeutic approaches. This study investigates the synergistic inhibition of KRAS-driven pancreatic cancer using siRNA-loaded chitosan nanoparticles in combination with Erlotinib, an EGFR inhibitor. The optimized siRNA nanoparticle formulation exhibited a particle size of ~ 158&#xa0;nm, a PDI of 0.18, and a zeta potential of − 23.99&#xa0;mV, ensuring stability and efficient cellular uptake. In vitro siRNA release studies demonstrated sustained release (~ 65% at 24&#xa0;h), supporting prolonged gene silencing. Cellular uptake studies using flow cytometry and fluorescence microscopy confirmed significant siRNA internalization (~ 75% in PANC-1), with enhanced uptake in combination-treated cells, indicating Erlotinib-mediated endocytic modulation. Gene silencing efficiency, assessed via qPCR and Western blot, revealed that siRNA-loaded nanoparticles alone reduced KRAS expression to ~ 40%, while combination therapy further suppressed expression to ~ 20%, confirming enhanced knockdown. Western blot analysis of EGFR-related signaling proteins (p-EGFR, p-AKT, and p-ERK) indicated strong pathway inhibition (~ 25–35% protein expression in the combination group), validating dual inhibition of KRAS-driven oncogenic pathways. Cytotoxicity studies (MTT assay) showed dose-dependent reduction in cell viability, with the combination therapy reducing viability to ~ 20% at 72&#xa0;h, confirming a strong synergistic effect (CI &lt; 1). Apoptosis induction assays revealed significant enhancement in cell death (~ 70% in PANC-1), suggesting activation of the intrinsic apoptotic pathway. Additionally, migration and invasion assays demonstrated potent anti-metastatic effects, with combination therapy reducing migration (~ 78%) and invasion (~ 72%), indicating suppression of epithelial-to-mesenchymal transition (EMT) and focal adhesion kinase (FAK) signaling.</p>

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Synergistic Inhibition of KRAS-Driven Pancreatic Cancer Using siRNA-Loaded Nanoparticles with Erlotinib: A Comprehensive In Vitro Investigation

  • 期刊论文

  • 发表日期：

    2025年5月19日

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  • Dilpreet Singh

摘要

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with KRAS mutations driving aggressive tumor growth and resistance to conventional therapies. Targeting KRAS directly has been challenging, necessitating alternative therapeutic approaches. This study investigates the synergistic inhibition of KRAS-driven pancreatic cancer using siRNA-loaded chitosan nanoparticles in combination with Erlotinib, an EGFR inhibitor. The optimized siRNA nanoparticle formulation exhibited a particle size of ~ 158 nm, a PDI of 0.18, and a zeta potential of − 23.99 mV, ensuring stability and efficient cellular uptake. In vitro siRNA release studies demonstrated sustained release (~ 65% at 24 h), supporting prolonged gene silencing. Cellular uptake studies using flow cytometry and fluorescence microscopy confirmed significant siRNA internalization (~ 75% in PANC-1), with enhanced uptake in combination-treated cells, indicating Erlotinib-mediated endocytic modulation. Gene silencing efficiency, assessed via qPCR and Western blot, revealed that siRNA-loaded nanoparticles alone reduced KRAS expression to ~ 40%, while combination therapy further suppressed expression to ~ 20%, confirming enhanced knockdown. Western blot analysis of EGFR-related signaling proteins (p-EGFR, p-AKT, and p-ERK) indicated strong pathway inhibition (~ 25–35% protein expression in the combination group), validating dual inhibition of KRAS-driven oncogenic pathways. Cytotoxicity studies (MTT assay) showed dose-dependent reduction in cell viability, with the combination therapy reducing viability to ~ 20% at 72 h, confirming a strong synergistic effect (CI < 1). Apoptosis induction assays revealed significant enhancement in cell death (~ 70% in PANC-1), suggesting activation of the intrinsic apoptotic pathway. Additionally, migration and invasion assays demonstrated potent anti-metastatic effects, with combination therapy reducing migration (~ 78%) and invasion (~ 72%), indicating suppression of epithelial-to-mesenchymal transition (EMT) and focal adhesion kinase (FAK) signaling.