Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 16 3 2026 09 30 pH-Sensitive Folate-Targeted Liposomal Vitamin D Reprograms EZH2/STAT3 Axis and Induces Apoptosis, ROS, and Autophagy in MCF-7 Breast Cancer Cells 550 561 10.34172/apb.46791 EN Soheil Abbaspour-Ravasjani https://orcid.org/0000-0001-7541-1638 Rahim Shahvalizadeh https://orcid.org/0000-0002-9940-5539 Malahat Safavi https://orcid.org/0009-0001-6883-0188 Mina Afrashteh Nour https://orcid.org/0000-0001-6781-1092 Mahdi Talebi https://orcid.org/0000-0002-3633-2280 Maryam Mohammadi Hamed Hamishehkar https://orcid.org/0000-0001-9905-0662 Journal Article 10.34172/apb.46791 2025 11 20 2026 06 22 Introduction: Vitamin D3 (Vit D3) possesses pleiotropic anticancer activity; however, its clinical application is limited by poor bioavailability and nonselective cellular uptake. This study aimed to develop folate (FA)-targeted, pH-sensitive liposomes for enhanced delivery of Vit D3 to breast cancer cells and to evaluate their effects on the VDR/EZH2/STAT3 signaling axis. Methods: Vit D3-loaded pH-sensitive liposomes were formulated and characterized for particle size, zeta potential, encapsulation efficiency, stability, and pH-responsive release. Cellular uptake, cytotoxicity, clonogenicity, apoptosis, reactive oxygen species (ROS) generation, autophagy, cell-cycle distribution, RT-qPCR, and Western blotting were evaluated in MCF-7 cells. Results: The optimized formulations exhibited nanoscale particle size, high encapsulation efficiency, physicochemical stability, and enhanced acidic pH-triggered release. FA-targeted liposomes significantly increased cellular uptake and cytotoxicity compared with free Vit D3 and non-targeted liposomes, with IC50 values of 15, 11, and 7.2 IU·mL-1, respectively. Vit D3-PEG/lipo-FA induced a 3.2-fold upregulation of VDR expression while suppressing EZH2 and STAT3 expression by 60% and 50%, respectively. In addition, treatment increased Caspase-3, LC3B, and NOX4 expression, promoted apoptosis and ROS generation, altered cell-cycle progression, and markedly reduced clonogenic survival. Western blot analysis confirmed these molecular alterations at the protein level. Conclusion: FA-targeted pH-sensitive liposomal delivery significantly enhances the anticancer efficacy of Vit D3 in MCF-7 cells by modulating the VDR/EZH2/STAT3 axis and activating apoptosis-, autophagy-, and ROS-associated pathways, highlighting its potential as a targeted nanotherapeutic strategy for breast cancer treatment.