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

Area of Science:

• Biotechnology
• Nanomedicine
• Gene Therapy

Background:

• Diabetic nephropathy (DN) pathogenesis is significantly influenced by transforming growth factor-β1 (TGF-β1)-induced fibrosis.
• Effective non-viral delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes for gene modulation remains a critical challenge in developing DN therapies.

Purpose of the Study:

• To develop and characterize cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for efficient intracellular delivery of TGFB1-targeting CRISPR-Cas9 RNP.
• To evaluate the gene silencing efficacy of the developed NLCs for potential early-stage modulation of DN.

Main Methods:

• Hybrid NLCs were formulated using squalene, glyceryl trimyristate, and DOTAP, optimized for sub-100 nm particle size.
• CRISPR-Cas9 RNP complexes targeting human TGFB1 were assembled at 1:1 and 1:2 molar ratios and complexed with NLCs.
• Physicochemical properties were assessed using dynamic light scattering (DLS), and gene silencing was quantified via RT-qPCR and ELISA in HEK293T cells.

Main Results:

• Optimized NLCs exhibited hydrodynamic diameters between 65-99 nm with a polydispersity index (PDI) below 0.5.
• The 1:2 Cas9:sgRNA ratio demonstrated superior TGFB1 mRNA reduction (67%) compared to the 1:1 ratio (39%).
• Significant suppression of TGF-β1 protein levels was observed within 24 hours post-treatment.

Conclusions:

• DOTAP-based hybrid NLCs provide an effective non-viral platform for delivering CRISPR-Cas9 RNP, achieving substantial TGFB1 expression suppression.
• This approach shows promise for upstream modulation of profibrotic signaling in diabetic nephropathy.
• Further investigation in kidney-specific cells and in vivo models is warranted to validate therapeutic potential.