Study Of Gene SiRNA Loaded Mesoporous Polymer Nanospheres For Tumor Therapy

The study in Anti-tumor has always been a hot spot in the field of biomedicine,and drugs and siRNA have also attracted more attention as an anticancer therapeutic agent currently.One of the advantages of siRNA is that it can avoid the toxic effects of drug during the process of treatment and is superior to drug in the treatment of drug-resistant tumors.This thesis aims to construct a loading system using Bcl-2 siRNA as the therapeutic agent and mesoporous polymer nanospheres as carriers through layer-by-layer self-assembly,which can treat drug-resistant breast cancer efficiently.Up to date,mesoporous polymer nanospheres have not been reported as a carrier of siRNA.Breast cancer cells are highly drug-resistant and have high expression of Bcl-2 genes.Therefore,we hoped to construct a new therapeutic system in order to obtain good treatment effect in tumors.In this thesis,mesoporous polymer nanospheres(MPNs)were prepared by soft template synthesis as carrier for siRNA.In the experiment,self-assembled polymerization and hydrothermal treatment at 200°C were used to prepare mesoporous nanospheres with spherical and uniform structure.Then mesoporous nanospheres were modified as cationic nanocarriers.The loading rate under different small molecule-modified nanospheres and the release conditions under different pH and with or without macromolecule layer on the nanocarriers surface were investigated respectively.In the cellular study,the biocompatibility of the carrier and the loading system was examined using the MTT method and fluorescence microscopy.Flow cytometry and fluorescence microscopy were used to investigate and compare the effect of different loading systems on endocytosis and apoptosis of cancer cells.The expression of mRNA and protein in cancer cells was investigated by RT-PCR and Western blot.In the animal experiment,the nude mouse tumor model was constructed and the target molecule folic acid(FA)was introduced to investigate the therapy effect by inhibition of tumor and histopathology examination.The experimental results showed that the synthesized mesoporous polymer nanospheres have spherical structure,regular and uniform size less than 200nm and good dispersibility.After modification,the morphology of the nanospheres remained intact and there was no apparent destruction.Nanospheres can be gradually degraded under the action of various enzymes,which has laid the foundation for their application in vivo.Different cationic molecules had different influence on the efficiency of loading siRNA for mesoporous polymer nanospheres.The loading rate of modified nanospheres using small molecules PEI(Mw=600)was as high as 37.2%.In the case of pH=7 or a macromolecular encapsulation layer on the nanospheres,the gene release rate was slower than that under other conditions.Cellular experiment results showed that the carrier and the loading system had good biocompatibility and no cytotoxicity.Coating macromolecule PEI(Mw=13000)could increase the endocytosis efficiency,and the fluorescence intensity of PEI coating group was higher than other groups.The apoptotic rates of OMPNs-PEI~1@siRNA and OMPNs-PEI~1@siRNA@PEI~2 co-cultured with MCF-7 cells for 48 hours were 13.99%and 28.7%respectively.The apoptotic rate also increased with the increase of co-culture time and siRNA dose,showing the time-and dose-dependence.The results of RT-PCR and WB experiments showed that OMPNs-PEI~1@siRNA@PEI~2 group could effectively silence gene expression,the inhibition rates of mRNA and protein expression were 74%and 50%,respectively,which were much higher than those of the control group and showed a good gene silencing effect.The results from the animal experiments showed that the treatment effect of OMPNs-PEI~1@siRNA@PEI~2@FA group on MCF-7 tumor was significantly higher than that of saline group and pure siRNA group.In this group,the tumor growth rate was lower and the tumor volume was smaller.The therapy extended the life cycle of nude mice and the tumor inhibition rate reached 67%.Compared with the OMPNs-PEI~1@siRNA group,the advantages of the PEI coating and the target-factor FA could also be viewed,and the treatment effect of OMPNs-PEI~1@siRNA@PEI~2@FA group was much better.The histopathological results showed that the OMPNs-PEI~1@siRNA@PEI~2@FA group had significantly less visceral tissue damage than the other three groups,demonstrating the superiority of our constructed load system.