Study On Preparation Of SiRNA Loaded Nanomaterials And Their Anti-glioma Effect

Glioma are the most common primary intracranial tumors.They are highly malignant and invasive,leading to the failure of radical treatment of gliomas by traditional methods such as surgery,radiotherapy and chemotherapy.Gene therapy refers to the use of genetic engineering technology to introduce foreign genes into target cells,through the expression of foreign gene products to correct or remedy gene defects in order to achieve the purpose of treating diseases.Small interfering RNA（siRNA）is a common gene therapy drug,which degrades target mRNA by inducing RNA interference（RNAi）,thus silencing the expression of target gene.It is a promising drug for gene therapy of glioma.However,siRNA is easily degraded and difficult to enter cells,needing delivery system to work.Lipid nanoparticles（LNP）and extracellular vesicles（EV）are the most promising drug delivery vectors for siRNA.LNP is mature and has been approved by FDA for the treatment of various diseases.However,due to its inability to penetrate the blood-brain barrier and lack of targeting ability of cancer cells,it is limited to the treatment of glioma.Extracellular vesicles are expected to be an ideal carrier for glioma treatment due to their significant advantages such as the ability to penetrate the blood-brain barrier,long circulation time in vivo,low immunogenicity,homing ability and the ability to help the drugs delivered by them avoid lysosomal phagocytic pathway.In this paper,two genes STAT3 and Survivin,which are closely related to the occurrence and development of glioma were identified through literature research.The mature LNP delivery system was used to package their corresponding siRNA respectively.By comparing their anti-glioma effect,STAT3 with better anti-cancer effect was selected for follow-up study.Subsequently,EV_U251of glioma U251 was extracted and STAT3 siRNA（siSTAT3）with relatively good effect was loaded after the contents were removed.Nano drug(EVM_U251@siSTAT3)was prepared to study its targeting ability and killing activity.The main contents are as follows.（1）LNP empty carriers were successfully prepared and loaded into siSTAT3 and siSurvivin.The obtained nano particle size and surface potential were in line with expectations with good homogeneity.The drug loading rates of LNP@siSTAT3 and LNP@siSurvivin were7.80%±0.31%and 7.71%±0.38%by agarose gel electrophoresis,respectively.Both LNP@siSTAT3 and LNP@siSurvivin can significantly silence the target gene of U251 cells and selectively kill the glioma U251cells.（2）EV derived from glioma U251 cells were successfully extracted by differential gradient centrifugation.Electron microscope results showed that the extracted EV had the characteristic structure of bowl-shaped vesicles,and its particle size was between 50-200 nm.The homing ability of targeted U251 cells was verified by incubating PKH67labeled U251 source EV with U251 cells and 293T cells.The results showed that PKH67 signal in U251 cells was more than 293T cells,which successfully verified the homing ability of U251 source EV.It lays a foundation for the subsequent preparation of glioma targeted delivery system.（3）The tumogenic content of EV_U251was removed by hypotonic method,which was then collected and processed through membrane to prepare EVM_U251,and finally loaded into therapeutic siSTAT3 to prepare EVM_U251@siSTAT3 by electrolysis.The drug loading rate was3.24%±1.20%.By tracing siRNA with CY5 markers,it was proved that EVM_U251@siSTAT3 still retained the homing ability of EV_U251-targeted U251 cells.Subsequently,Lysotracker Green was used to label the lysosome of U251 cells.Results showed that EVM_U251@siSTAT3 avoided the lysosomal phagocytosis pathway and successfully delivered siRNA to the cytoplasm.Finally,we carried out cell experiments to prove its potential in treating gliomas.The results showed that EVM_U251@siSTAT3could significantly knock down the expression of STAT3 gene and produce specific killing effect on U251 cells,providing a new strategy for the treatment of gliomas.