Development of RGD-targeted gene carrier nanosystems for breast cancer treatment: In Vitro and In Vivo evaluation

ABSTRACT Objective: The primary aim of this thesis study is to develop lipid–polymer hybrid gene delivery systems (LPH-GDS) carrying plasmid DNA (pDNA) and functionalized with RGD peptides for the treatment of triple-negative breast cancer (TNBC), and to evaluate the the efficacy of these systems in vitro and in vivo. The study particularly aims to suppress tumor progression by targeting the expression of vascular endothelial growth factor (VEGF), a key mediator of tumor angiogenesis. Methods: The designed hybrid nanosystems were designed by utilizing the binding affinity between DSPE-PEG-Maleimide and thiol-functionalized cyclic RGD peptides. Physicochemical characterization was performed using dynamic light scattering (DLS) to determine particle size, zeta potential, and polydispersity index (PDI). The stability of the nanosystems in serum and against DNase I, as well as their gene release profiles in the presence of sodium dodecyl sulfate (SDS), were also evaluated. In vitro studies were conducted using the 4T1 TNBC cell line while BALB/c mouse tumor model were used to perform in vivo experiments. The levels of VEGF in tumor tissues and serum were assessed through ELISA analysis. Results: The RGD-targeted nanosystems exhibited an average particle size of 19.40 ± 0.43 nm, zeta potential of +22.8 ± 1.6 mV, and PDI value of 0.25. In vitro results demonstrated efficient transfection capability with tumor-targeting specificity. In vivo studies showed a significant reduction in tumor volume along with suppressed VEGF expression levels in the treatment group. Conclusion: The RGD peptide-targeted LPH-GTS was evaluated as a promising gene delivery platform with advanced therapy medicinal product (ATMP) potential, owing to its low cytotoxicity and high biocompatibility in 4T1 TNBC and L929 fibroblast cell lines, as well as its effective gene transfer and tumor-specific targeting capacity in breast cancer cells. Keywords: Triple negative breast cancer, targeting, gene therapy, lipid polymer hybrid nanosystem.