Fluorinated Polylysine-Enabled Transmucosal Delivery Of CRISPR-Cas9 System To Target DAD1 Gene For The Intravesical Instillation Therapy Of Bladder Cancer

Objective: Bladder cancer is one of the common tumors in the urinary system.Intravesical instillation therapy is an important treatment method for bladder cancer.This study intends to screen target gene can be used for intravesical instillation therapy of bladder cancer,construct fluoropolylysine/CRISPR-Cas9 nano drug system targeting the selected gene,and explore its feasibility and safety in intravesical instillation therapy of bladder cancer.Methods: This study focused on the abnormal glycosylation modification of bladder cancer,and screened out the potential new target DAD1 gene for the treatment of bladder cancer through bioinformatics analysis.Combined with CRISPR-Cas9 gene editing technology,the CRISPR-Cas9 plasmid(Cas9-sgDAD1)that can target DAD1 gene was constructed.Fluorine chain was modified on polylysine(PLL)by chemical reaction between amino group and carboxyl group.Fluoropolysine(PLLF)can selfassemble into nano micelles and combine with Cas9-sgDAD1 to form nanoparticles.The knockdown effect of PLLF/Cas9-sgDAD1 nanoparticles on DAD1 was evaluated by RT-qPCR and WB experiments.The anti-tumor effect of PLLF/Cas9-sgDAD1 nanoparticles was evaluated by CCK-8 test,PI staining and flow cytometry.The mucosal permeability of PLLF/Cas9-sgDAD1 nanoparticles was evaluated by 3D cell ball,Ussing-Chamber experiment and intravesical instillation.The mechanism of cell apoptosis induced by targeting DAD1 was explored by RNA-sequence,and was verified by WB experiment.Mouse model of bladder carcinoma in situ was successfully constructed.The anti-tumor effect of PLLF/Cas9-sgDAD1 nanoparticles and the knockdown effect on DAD1 gene through intravesical instillation were evaluated by HE staining,immunohistochemical staining,TUNEL staining and WB experiment.Results: The results of bioinformatics analysis and immunohistochemical staining of bladder tissue chip HBla U108Su01 showed that the expression of DAD1 gene in bladder cancer tissue was significantly higher than that in normal bladder tissue,and the higher the expression level,the worse the prognosis of bladder cancer patients.Knockdown of DAD1 gene with small interfering RNA(si RNA)significantly inhibited the proliferation and migration of bladder cancer cells,but had no effect on normal bladder epithelial cells.Fluorinated polylysine(PLLF)can self-assemble into nano micelles,and its particle size is 218.7 ± 13.8 nm.PLLF and Cas9-sgDAD1 plasmid can form stable nanoparticles through positive and negative electric attraction.The size of nanoparticles is 185 ± 3.4 nm.Under the conditions of different N/P ratios(0.5,0.75,1,2,4,8),the particle size of nanoparticles is stable at about 200 nm.PLLF/Cas9-sgDAD1 nanoparticles can penetrate into the interior of 3D cell spheres to play an antitumor role,and can penetrate the bladder mucosa to complete the effective delivery of Cas9-sgDAD1 plasmid.PLLF/Cas9-sgDAD1 nanoparticles can effectively reduce the expression of DAD1 gene and play an anti-tumor role in vivo and in vitro.The phosphorylation levels of ERK,JNK and P38,key signal transduction proteins of mitogen activated protein kinase(MAPK)signaling pathway,were inhibited after DAD1 knockdown,which participated in the regulation of apoptosis of bladder cancer cells.Intravesical instillation of PLLF/Cas9-sgDAD1 nanoparticles can effectively inhibit the growth of tumor in the mouse model of bladder cancer in situ,without causing local inflammatory reaction and toxic reaction of heart,liver,spleen,lung and kidney.Conclusion: DAD1 gene can be used as a new target for the treatment of bladder cancer.PLLF can efficiently deliver the Cas9-sgDAD1 plasmid in vivo and in vitro,which can provide new options for the intravesical instillation therapy of bladder cancer.