Silk Fibroin-Modified Liposome/Gene Editing System Knocks out the PLK1 Gene to Suppress the Growth of Lung Cancer Cells

Abstract

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR–Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR–Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy.

A quaternary ammonium-based nanosystem enables delivery of CRISPR/Cas9 for cancer therapy

“…, using siRNA to knockdown the expression 29–31 and the CRISPR/Cas9 technique to achieve its knockout. 32,33 In this study, a CRISPR/Cas9 delivery system was developed for the delivery of Cas9/PLK1 sgRNA RNP complexes (denoted as RNP PLK1 ) to improve genome editing efficiency (Scheme 1). The delivery system consisted of a quaternary ammonium-functionalized poly( l -lysine) (Qua-PLL) and a cholesterol-modified PEG (Chol-PEG), which were self-assembled with negatively charged RNP PLK1 to form a ternary complex (Qua-PLL/Chol-PEG/RNP PLK1 , abbreviated as QNP/RNP PLK1 ).…”