Preparation and Characterization of Lipid-based Nanoparticles for siRNA Delivery for Purinergic Receptor Suppression in Breast Cancer Cells

Abstract

Introduction: The successful delivery of RNA payloads via lipid nanoparticles (LNP) depends on the manipulation of the ionizable lipid (IL) as an essential component. Breast cancer (BC) is the leading cause of cancer-associated death among women worldwide, and the P2X7R is a critical regulator of the purinergic receptor (PUR) in tumor proliferation, apoptosis, and metastasis. Suppression of P2X7R via short interfering RNA (siRNA) plays a unique character and is a potential therapeutic approach in BC treatment. Aim: Synthesis of IL for enhancing cellular uptake of formulated siRNA-LNP to improve the efficient delivery of siRNA-P2X7(siP2X7) for migration inhibition and apoptosis induction. Method: Synthesized IL (SIL) through a reaction of tris-amin and lauryl acrylate was obtained, and then SIL that underwent confirmed structural analyses (FT-IR, 1HNMR, and LC-MS ) was purified and incorporated into a lipid mixture solution for LNP formulation. Following LNP characterization, impurity, cytotoxicity, and uptake assays were evaluated with HPLC-ELSD analysis, MTT assay, and confocal microscopy, respectively. Finally, migration inhibition through scratch assay and apoptosis induction using flow cytometry in vitro.Results: siP2X7-LNP with excellent characterization (Z-average 126.8 nm, Zeta-potential -12.33, PDI 0.16, and encapsulation efficiency 85.35 %) achieved. Then, siP2X7-LNP cellular uptake, the optimal dose (45 pm), and incubation time (12 h) were determined. The migration inhibition extended at 48 h incubation with a 60.62% closure area on apoptosis rate on 4T-1 cells has changed (95.8%) at 24 h to late apoptosis (35%) at 48 h. Finally, P2X7R was significantly knockdowned after 48 h via western blotting assessment.Conclusion: The innovation plays a critical role in the efficient delivery of siP2X7-LNP against murine triple-negative breast cancer cells (TNBC) used for migration inhibition and apoptosis induction, resulting in efficient siRNA-LNP delivery model in BC cancer.