Ferulic acid-loaded chitosan nanoparticles enhance radiotherapy efficacy via STAT3 suppression and caspase-8/p53 activation in Ehrlich ascites carcinoma

Radiotherapy resistance remains a major challenge in cancer treatment, necessitating novel strategies to enhance therapeutic efficacy while minimizing side effects. This study aimed to develop and evaluate ferulic acid-loaded chitosan nanoparticles (FA-ChNPs) as a multimodal radiosensitizer targeting STAT3, caspase-8, and p53 pathways in Ehrlich ascites carcinoma (EAC). FA-ChNPs were synthesized using ionic gelation and thoroughly characterized for their physicochemical properties, including size, and zeta potential through TEM and DLS. The encapsulation efficiency was verified using UV-Vis spectroscopy. In vitro drug release kinetics were evaluated using the dialysis bag method, demonstrating a sustained release profile from the nanoparticles. Biological evaluation included acute toxicity testing in healthy mice to determine the LD50 value according to OECD guidelines. For antitumor assessment, EAC-bearing mice were divided into six treatment groups (n equals 6 per group), receiving either FA-ChNPs alone (115.75mg/kg via oral gavage), gamma-irradiation alone (6Gy per week), or combination therapy. Comprehensive biological parameters were measured, including tumor progression (mass and volume), metabolic profile (total cholesterol, triglycerides, HDL-C), liver and kidney function markers (ALT, AST, ALP, creatinine, urea), oxidative stress indicators (GSH, SOD, CAT, MDA), inflammatory cytokines (TNF-alpha, IL-6, VEGF), and gene expression levels (STAT3, caspase 8, P53 via qRT-PCR). Histopathological examination of liver tissue complemented these analyses. In silico studies encompassed molecular docking with key targets (STAT3, caspase 8, TP53) and ADMET property predictions. The FA-ChNPs demonstrated favorable physicochemical properties, including a spherical morphology with an average particle size of 41.63 nm and good colloidal stability indicated by a PDI of 0.2, a zeta potential of -2.45 mV. Also, FA-ChNPs exhibited sustained in vitro drug release, contrasting with the rapid diffusion of free ferulic acid, which supports their potential for prolonged therapeutic action and a high safety margin with an LD₅₀ value of 2315mg/kg. The combination therapy demonstrated superior antitumor effects, achieving 44.3% reduction in tumor mass and 67.0% reduction in tumor volume compared to EAC controls, with corresponding tumor dimensions reduced to 1.02cm (length) × 0.74cm (width), confirming potent radiosensitization and synergistic efficacy. Biological assessments revealed significant improvements across multiple parameters: metabolic profile showed 89.2% increase in HDL-C and 29.0% decrease in triglycerides; liver and kidney function markers improved with 29.0% reduction in ALT and 39.7% decrease in creatinine; oxidative stress modulation included 87.9% increase in GSH and 32.4% reduction in MDA; inflammatory markers decreased by 40% for TNF-alpha and 51.1% for IL-6. Molecular analyses showed 70% suppression of STAT3, 108.2% activation of caspase-8, and stabilization of p53. Histopathological evaluation confirmed preserved liver architecture with minimal steatosis and inflammation. Molecular docking studies revealed strong binding affinities:-6.02 kcal/mol for STAT3, -7.31kcal/mol for CASP8, and -5.15kcal/mol for TP53. FA-ChNPs represent a groundbreaking approach to enhance radiotherapy efficacy through simultaneous pathway modulation and organoprotection. These findings support further clinical translation for treatment-resistant cancers.