DOTAP-Based Hybrid Nanostructured Lipid Carriers for CRISPR-Cas9 RNP Delivery Targeting TGFB1 in Diabetic Nephropathy

Background: Diabetic nephropathy (DN) is largely driven by transforming growth factor-β1 (TGF-β1)-mediated fibrosis. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes offer precise gene disruption, yet effective non-viral delivery remains a challenge. This study developed cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for intracellular delivery of TGFB1-targeting RNP as an early-stage platform for DN gene modulation. Methods: A single-guide RNA (sgRNA) targeting human TGFB1 was assembled with Cas9 protein (1:1 and 1:2 molar ratios). Hybrid NLCs comprising squalene, glyceryl trimyristate, and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were formulated via optimized emulsification-sonication to achieve sub-100 nm particles. Physicochemical properties, including polydispersity index (PDI), were assessed via dynamic light scattering (DLS), while silencing efficacy in HEK293T cells was quantified using quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Results: Optimized NLCs achieved hydrodynamic diameters of 65-99 nm (PDI < 0.5) with successful RNP complexation. The 1:2 Cas9:sgRNA formulation produced the strongest gene-editing response, reducing TGFB1 mRNA by 67% (p < 0.01) compared with 39% for the 1:1 ratio. This translated to a significant reduction in TGF-β1 protein (p < 0.05) within 24 h. Conclusions: DOTAP-based hybrid NLCs enable efficient delivery of CRISPR-Cas9 RNP and achieve significant suppression of TGFB1 expression at both transcriptional and protein levels. These findings establish a promising non-viral platform for upstream modulation of profibrotic signaling in DN and support further evaluation in kidney-derived cells and in vivo renal models.