CRISPR ‐Cas9 lipid nanoparticles for targeting cyclin‐dependent kinases in the tumor microenvironment

Due to the significant roles of cyclin-dependent kinases 4 and 6 (CDK4/6) in cancer progression, this study aimed to introduce clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) plasmid DNA (pDNA) encapsulated in lipid nanoparticles (LNPs) as a novel CDK4/6 inhibitor using a gene knock-out strategy for treating cancer. pDNA-LNP was prepared and characterized using a microfluidic system. The results indicated the hydrodynamic diameter of the pDNA-LNP was 90.0 ± 0.1 nm with the PDI of 0.1 and a negative zeta-potential. The cytotoxicity results demonstrated statistically significant differences at doses of 0.250, 500, and 1 μg of pDNA with the capabilities of pDNA-LNP in the induction of apoptosis, as depicted by the Annexin-V-FITC-PI method. Real-time quantitative PCR (qPCR) also indicated a significant reduction in the expression levels of both CDK4 and 6 in the cells that were treated with pDNA-LNP. The in vivo anti-tumor activities of pDNA-LNP have demonstrated that the formulation has the potential to decrease tumor size and improve survival parameters, including median survival time (MST), which was increased from 31 days for the PBS group to 51 days for the pDNA-LNP group at 0.5 μg. On the other hand, the dose of 1 μg had shown signs of toxicity, indicating the need to optimize dosing in future studies. In summary, these findings indicate that CRISPR-Cas9 encapsulated in the LNP can suppress tumor growth and offer a promising strategy for future cancer treatment approaches.