Abstract 4473: Liposomal nanoparticle mediated gene modulation for the treatment of melanoma in murine models

Abstract Background: The Wnt/β-catenin pathway plays a significant role in melanoma progression, including the suppression of tumor-specific immunity. Inhibition of this pathway represents a promising therapeutic strategy. Our study investigates the potential of liposomal nanoparticles (LNPs) for delivering Wnt/β-catenin signaling pathway-inhibiting mIR-124 plasmids to melanoma cells. Materials and Methods: Cationic liposomes were synthesized using the thin-film hydration method. Liposomes were mixed and incubated with a full-length cloned miR-124 plasmids vector (pDNA) at a 10:1 lipid-to-pDNA ratio to allow nanoparticle (NPs) formation referred to as lipoplex or (miR-124-NP). The size distribution and zeta potential of the LNPs were measured using dynamic light scattering (DLS). Nanoparticle morphology was assessed using transmission electron microscopy (TEM). B16-N2 melanoma cells were seeded and treated with miR-124-NP and blank liposomes (10 µg lipid). Cytotoxicity and transfection efficiency were evaluated at 24- and 48-hours post-treatment using immunofluorescence and flow cytometry. Female C57BL/6 mice, bearing B16-N2 tumors in both flank regions, were assigned to 1) Blank Liposomes B-NP, 2) miR-124-NP (n=12 tumors per group). On day 13 post-tumor inoculation, the mice received three intratumoral injections of miR-124-NP (40 µg DNA per injection) at 3-day intervals. Tumor volume was measured daily using caliper measurements. The delivery of nanoparticles to the tumors was monitored by an in vivo imaging system (IVIS), detecting GFP signals at multiple time points. At 24 hours post-injection, mice were euthanized, and tumors were harvested for further analysis via flow cytometry and RT-PCR to assess transfection efficiency. Results: DLS and TEM analysis confirmed that the miR-124-NP exhibited proper size (180 nm), charge (16.66 mV), and structure. The miR-124-NP formulation (1:10 μg plasmid: lipid ratio) successfully transfected B16-N2 melanoma cells without causing toxicity. Immunofluorescence and flow cytometry data verified the transfection was successful, with 40% efficiency. In vivo imaging using IVIS demonstrated successful delivery of the plasmid to tumor cells. In vivo, miR-124-NP treatment significantly reduced tumor volumes compared to the blank liposome control, with the tumor growth curve showing a marked decreased total tumor burden to up to 50% through day 20 post-inoculation. RT-PCR analysis revealed that miR-124-NP treatment led to decreased expression of SLUG and SNAIL, along with a reduction in vimentin levels, indicating a modulation of epithelial-mesenchymal transition (EMT) processes. Conclusion: LNPs showed high retention in B16-N2 cells and were non-toxic in vitro. In vivo miR-124-loaded LNPs effectively transfected tumor cells, modulating beta-catenin signaling and EMT gene expression, leading to reduction in tumor burden in C57BL/6 mice. Citation Format: Poonam Yadav, Santosh K. Mandal, Bill Tang, Rahul A. Sheth. Liposomal nanoparticle mediated gene modulation for the treatment of melanoma in murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4473.