Phloretin-Conjugated Silver Nanoparticles as Novel Nano-Radiosensitizer: Synergistic Anti-Tumor Efficacy and Mechanistic Insights in Ehrlich Carcinoma Model.

INTRODUCTION: Radiotherapy remains a fundamental modality in cancer treatment; however, its efficacy is frequently compromised by inherent tumor radioresistance and the collateral damage to normal tissues at curative doses. Phloretin, a dihydrochalcone flavonoid, exhibits extensive antitumor effects in both in vitro and in vivo settings; however, its clinical application is limited due to low water solubility and rapid metabolic processing. Silver nanoparticles (AgNPs) have been recognized as agents that enhance radiation-induced oxidative damage. Accordingly, this study synthesized phloretin-conjugated AgNPs (Ph-AgNPs) and evaluated their potential as a safe, tumor-selective radiosensitizer in a murine model of solid Ehrlich carcinoma. METHODS: Ph-AgNPs were synthesized through an environmentally friendly reduction process and subsequently characterized utilizing UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering, and transmission electron microscopy. The acute oral toxicity (LD₅₀) and hepatorenal safety profiles were assessed in healthy Swiss albino mice. Mice bearing solid Ehrlich ascites carcinoma (EAC) were randomly divided into six groups (n = 6 per group) for antitumor evaluation: Normal control, Normal+Ph-AgNPs, EAC (control), EAC + γ-irradiation (γ-IR; 6 Gy), EAC + Ph-AgNPs (98.5 mg), and EAC + Ph-AgNPs + γ-IR. The parameters assessed were tumor growth, blood indices, serum liver transaminases (ALT, AST, ALP), renal function (urea and creatinine levels), biomarkers of oxidative stress in the tumor tissue (GSH, SOD and MDA), signaling pathways associated with apoptosis (p-AMPK, Bax and Bcl-2) and cell cycle regulators (CDK2/CDK6/p53), evaluated by biochemical assays, quantitative real-time PCR (RT-qPCR) or western blot analysis. Molecular docking studies were also conducted to visualize the binding interactions of phloretin with CDK2, CDK6, and p53. RESULTS: Ph-AgNPs were monodisperse spheres with an average diameter of 85.89 nm and a zeta potential of -12.35 mV, maintaining stability in physiological media. At the therapeutic dosage, Ph-AgNPs exhibited no hepatorenal toxicity in normal mice and significantly mitigated the transient elevations in hepatic and renal markers induced solely by irradiation. The combined application of Ph-AgNPs and γ-irradiation yielded the most pronounced tumor suppression, resulting in a 59.7% reduction in mean tumor mass and a 69.6% decrease in tumor volume compared to EAC controls. This outcome was associated with GSH and SOD depletion, increased MDA levels, elevated p-AMPK levels, and an increased Bax/Bcl-2 ratio, along with coordinated downregulation of CDK2 and CDK6 and upregulation of p53, as confirmed at the protein level. Docking analyses indicated strong, energetically favorable interactions of phloretin within the active sites of CDK2, CDK6, and p53. DISCUSSION: Ph-AgNPs appear to enhance tumor sensitivity to radiation through multiple mechanisms, including concurrent oxidative stress, mitochondrial apoptosis, and p53-mediated cell-cycle arrest. CONCLUSION: This study finds that Ph-AgNPs are nano-radiosensitizers that can be combined with γ-ray irradiation, are well tolerated, and significantly enhance the therapeutic efficacy of γ-ray-mediated therapy for solid EAC tumors. This rationale underpins our multi-mechanistic approach, which combines the enhanced phloretin delivery and radiosensitizing properties of AgNPs to radiosensitive and radioresistant cells.