CRISPR/Cas9 targeting liposomes knocked down multidrug resistance proteins in brain endothelial cells as a model to predict potential pharmacoresistance

This investigation aimed to use CRISPR-Cas9 gene-editing to knock down P-glycoprotein (P-gp) expression and then establish a feasible cell line to evaluate the potential pharmacoresistance of therapeutic agents mediated by efflux. A cationic liposome was prepared as a smart bomb by conjugating with a peptide-based targeting ligand (THRPPMWSPVWP), specifically binding to transferrin receptors at the blood-brain barrier (BBB), and then formed a nanocomplex with P-gp knockdown CRISPR/Cas9 plasmid. Higher uptakes of targeted and stable li-posomes in bEND.3 cells were observed compared to non-peptide conjugated ones (p < 0.05). The P-gp trans-porters were successfully knocked down by the cell-nontoxic CRISPR/Cas9 targeted liposomes and P-gp associated ATP activities were higher in the transfected cells (p < 0.05). Functional studies of knocked down cells were evaluated by using prototypical P-gp substrates rhodamine 123 and doxorubicin. More accumulation of rhodamine 123 and higher cytotoxic sensitivity of doxorubicin was observed in the transfected cells as compared with those in the wild-type cells.

Automated summary

This study aimed to knock down P-glycoprotein (P-gp) expression through CRISPR-Cas9 gene-editing and establish a viable cell line for evaluating the pharmacoresistance of therapeutic agents mediated by efflux. A cationic liposome was prepared as a smart bomb by conjugating with a peptide-based targeting ligand that specifically bound to transferrin receptors at the blood-brain barrier (BBB), and then formed a nanocomplex with the P-gp knockdown CRISPR/Cas9 plasmid. The cell-friendly CRISPR/Cas9 targeted liposomes successfully knocked down the P-gp transporters and increased P-gp associated ATP activities in the transfected cells. Functional studies of the knocked down cells showed enhanced accumulation of rhodamine 123 and increased cytotoxic sensitivity to doxorubicin.