Study On Construction Of Nucleic Acid Nanoparticles Loading SiRNA-AKR1C3 And Targeting Inhibition Of Castration-resistant Prostate Cancer

Prostate cancer is a common and significant clinical problem among men worldwide.In developed western countries,cancer has the highest incidence and the second highest mortality.In China,with the aging of the population and changes in dietary structure,the incidence of prostate cancer is increasing year by year,which has already been a common malignant tumor that seriously endangers the health of middleaged and elderly men in China.Inevitably,prostate cancer will progress to castrationresistant prostate cancer(CRPC)after treated with hormone deprivation therapy(ADT)The main mechanism is that the androgen signaling pathway is reactivated directly or indirectly.Aldehyde-ketone reductase 3(AKR1C3)is located at the end of the androgen synthesis pathway and is the key enzyme for the last two steps of the synthesis of testosterone and dihydrotestosterone in tumor cells.Therefore,it is the key enzyme for tumor cells to obtain androgen synthesis capacity after ADT and the key factor in reactivating the androgen signaling pathway to promote prostate cancer progressing to CRPC.It is a new strategy to target AKR1C3 for the prevention and treatment of CRPC by down-regulating the expression of AKR1C3 to reduce the synthesis of androgens in prostate cancer cells.Recently,researchers have made progress in study of the inhibitor of AKR1C3 and have developed multiple variety of AKR1C3 inhibitors.However,for the shortcomings of AKR1C3 inhibitors,such as low tissue selectivity,poor specificity and highly toxicity,the prospect of these clinical application is not promising.Consequently,the research of tumor-specific AKR1C3 inhibitors has become urgent.It has been possible to solve the problem above due to the development of RNA interference technology and targeted nano-transport system.Objective: Nucleic acid nanoparticles were prepared through combining the targeted nano-transport system and RNA interference technology,can target the prostate cancer cells,and inhibit the AKR1C3 protein and down-regulate cell proliferation.The characteristics of the nucleic acid nanoparticles were characterized,and the stability,targeting and effectiveness of the nucleic acid nanoparticles were evaluated at the cellular level.The mechanism of the effect of the nucleic acid nanoparticles is preliminary explored.Methods and results:? In the experiment,targeted nucleic acid nanocarriers were synthesized that consist of a positron polymer nanomaterial carrier,containing PAMAM and PEG,and PSMA-aptamer,that target prostate cancer cells.Then combining si RNA-AKR1C3 by electrostatic bonding.Nucleic acid nanoparticles(PAMMA-PEG-PSMA-aptamer / si RNA-AKR1C3)were successfully constructed and characterized.Explored through SRB experiments and AGE(agarose gel electrophoresis)experiment,the optimal range for cells of the nucleic acid nanocarriers were PAMAM-PEG-PSMAaptamer=1:1.5:0.5 and N/P=40.The components of the nucleic acid nanoparticles were identified by nuclear magnetic resonance proton spectroscopy.The characteristic peaks of PAMAM and PEG was still remained and the MAL could react with PSMA-aptamer.The morphology of the nucleic acid nanoparticles observed by transmission electron microscopy proved that nanoparticles were spherical when observed as circle.the particle size and zeta potential of the nucleic acid nanoparticles were measured.The results showed that the zeta potential got smaller approaching 0m V and the particle size increased after PAMAM combined with PEG,but a tight state appeared after synthesis of PSMA-aptamer and si RNA loading,and the particle size gradually decreased to about 453.9nm.? Based on the successfully synthesized prostate cancer targeting nucleic acid nanoparticles(PAMMA-PEG-PSMA-aptamer / si RNA-AKR1C3),their targeting and effectiveness were tested at the cellular level.The results showed that the nucleic acid nanoparticles could target PSMA-positive prostate cancer and depend on the time and concentration.When the concentration was constant,there were more drugs swallowed into the cells within 4h than 1h.Along with the concentration increasing,the drugs swallowed into the cells increased gradually.LNCa P cells with high expression of AKR1C3 under castrated environment were constructed to simulate castration-resistant prostate cancer.SRB assay was used to detect cell proliferation.After transfection with AKR1C3,the proliferation rate of LNCa P cells was increased by 26.58 ± 13.41%.The proliferation of ells was inhibited by nucleic acid nanoparticles.The suppression rate of LNCa P cells was 22.31 ± 7.64%,and 22RV1 cells was 4.47 ± 2.3%.Ed U flow cytometry was used to detect cell proliferation further.After transfection with AKR1C3,the proliferation rate of LNCa P cells was increased by 83.52 ± 11.38%.The proliferation of cells was inhibited by the action of nucleic acid nanoparticles.The suppression rate of LNCa P cells was 36.49 ± 1.07%,and 22RV1 cells was 52.07 ± 13.84%.At the effective concentration,nucleic acid nanoparticles could significantly down-regulate AKR1C3 protein and cyclin D1 protein.In LNCa P cells,inhibition rate was 71.44% and 54.56%,and in 22RV1 cells,inhibition rate was 22.24% and 31.84%.The experiments above confirmed that nucleic acid nanoparticles could effectively suppress prostate cancer cell proliferation by down-regulating AKR1C3 protein.Nucleic acid nanoparticles were incubated with cells at a safe cell concentration,significantly down-regulated the AKR1C3 protein and cyclin D1 protein,and effectively inhibited prostate cancer cell proliferation.Conclusion:The combination of nanomedicine and RNA interference technology has been successfully used to synthesis the nucleic acid nanoparticles and is a targeting specific si RNA interference system(PAMMA-PEG-PSMA-aptamer / si RNA-AKR1C3)which can target prostate cancer and made AKR1C3 as a therapeutic target.Then verify its safety and the effectiveness to inhibit the proliferation of castration-resistant prostate cancer cells at the cellular level.This study will provide theoretical and experimental basis for the innovative application of si RNA-AKR1C3 and the treatment of CRPC.