Establish And Evaluate Of NEDD4-siRNA Targeted Delivery System

China's annual report on cancer registration shows that cancer(ie,malignant tumor)is the primary cause of human death.Tumor treatment has become an important issue urgently to be solved.The traditional tumor treatment methods include radiation therapy,cytotoxic drug therapy and surgical treatment.In recent years,tumor-targeted drugs for specific targets have been developed.In August 2018,FDA approved the first siRNA-interfering drug targeting the thyroxine transporter(TTR)gene for the treatment of adult patients with hereditary ATTR amyloidosis This makes small nucleic acid therapeutics become a hot research topicAt present,in the incidence of several common tumors in the clinic,colon cancer has become the third most common malignant tumor in the world and the fourth leading cause of death.This study established a simple preparation process,reliable results,suitable for small nucleic acid targeted delivery carriers.It is divided into passive targeting carriers(siRNA liposomes)and active targeting carriers(siRNA-HA liposomes)for the treatment of colorectal cancer,and in vitro effects of these two carriers are studied accordingly.The details are as follows(1)determine the process used by single-factor process screening,and then use DOE design(24-1+4 center point)to construct blank liposomes;using DOTAP,DSPC,Chol and DSPE-PEG2000,according to particle size,PDI and Zeta potential as indicators to determine the best prescription composition of blank liposomes(2)study on siRNA-loaded liposomes.DOTAP has a cationic effect that can bind siRNA through static electricity;Chol can fill the gaps in the lipid bilayer and keep the structure stable;DSPE-PEG2000 makes liposomes have a long circulation;HA has the effect of targeting CD44 cells.Because passively targeted liposomes are simple to prepare and the process is reproducible,while HA actively targets liposomes due to load HA,the zeta potential is negatively charged and the drug loading is low,so two delivery carriers were investigated simultaneously to put forward two ideas By comparing the encapsulation rate,particle size,PDI and Zeta potential,the composition of the final passive targeting vector(siRNA liposome)and active targeting vector(siRNA-HA liposome)was determined(3)Electron microscopic observation of final siRNA liposomes and siRNA-HA liposomes,encapsulation efficiency check,serum stability and long-term stability inspection,and finally cytotoxicity and flow cytometry apoptosis tests.Test results,prepared siRNA liposome and siRNA-HA liposome,the electron microscope observation particle size were 130.75 ± 0.87nm and 192.53 ± 4.29nm,siRNA-HA liposome can clearly observe HA target,;The results of cytotoxicity and flow cytometry test showed that it has gene silencing function on LOVO cells,with an efficiency of about 60%.The resulting vector was demonstrated to be capable of delivering small nucleic acids.