Synthesis, characterization, and cellular investigation of three smart polymeric nanoparticles as efficient plasmid CRISPR (pCRISPR) delivery vehicles

Purpose. Gene therapy is a strategy to provide therapeutic advantages by modifying genes, and CRISPR/Cas9 is the simplest and the most efficient gene editing technology. Appropriate smart nanocarriers are a promising way to deliver these gene editing tools into cells efficiently. Methods. In this study, three novel smart nanocarriers were prepared for CRISPR/Cas9 delivery into PC12 cells. We designed a smart polyplex using synthetic redox-responsive polyethyleneimine (rPEI) made by crosslinked PEI ∼ 2 KD and pCRISPR using electrostatic interactions. Then, to avoid unwanted interactions with blood ingredients three natural polysaccharides with negative charge, hyaluronic acid (HA), chondroitin sulfate (CS), and alginate(ALG) have been used separately as outer shells. Additionally, nanocarriers were characterized in terms of zeta potential, size distribution, and loading efficiency. Finally, the cytotoxicity of nanocarriers and GFP gene expression were evaluated. Results. The average size of the nanocarriers with outer coats of HA, ALG, and CS was around 47, 66.5, and 309 nm, respectively. Furthermore, these three nanocarriers indicated a high loading efficiency, high capacity for cellular uptake (>90%), and no significant toxicity. The pCRISPR expression amount was estimated up to 21%. Conclusion. These redox-responsive polymeric nanocarriers suggest novel and efficient carriers for CRISPR/Cas9 delivery.