Soheil Abbaspour-Ravasjani
1,2 
, Rahim Shahvalizadeh
2 , Malahat Safavi
3 , Mina A frashteh Nour
4,2 , Mahdi Talebi
5 , Maryam Mohammadi
6, Hamed Hamishehkar
2,7* 1 Student Research Committee, Department of Medical Nanotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
2 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3 Department of Food Industry Science and Engineering, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
4 Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
5 Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
6 Department of Food Science and Engineering, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
7 Research Center of New Material and Green Chemistry, Khazar University, 41 Mehseti Street, AZ1096, Baku, Azerbaijan
Abstract
Background: 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.