Study On The Functionalization Of Exosomes As Pharmaceutical Carriers

With the in-depth development of molecular biology and genomics,a growing number of human diseases have been found to be associated with genetic abnormalities,so looking for disease susceptibility genes in order to treat disease has become a hotspots in research.Particularly in the last decade,gene therapy has been enriched and accelerated by the discovery of microRNAs(miRNA),small interfering RNA(siRNA)and long noncoding RNA(lncRNA).Since gene medicine is very unstable in vivo and easy to be degraded before geting to target organizations,finding a safe,highly effective gene carrier has become a key task for gene therapy.Viral and nonviral gene carriers are two primary gene carrier.The advantage of viral gene carriers is its high-efficient gene transfection,but it has the downside of potential carcinogenic and cytotoxicity,while nonviral gene carrier also has high-immunogenicity.Therefore,it is very insistent and necessary to construct a gene carrier with high-efficient gene transfection,tumor targeting,low immunogenicity and good biocompatibility.Exosomes are small membrane vesicles released by cells on fusion of multivesicular bodies(MVBs)with plasma membrane,which contain many biologically active compounds including miRNA and cytokines.Rencent data have demonstrated that these biologically active compounds can enter recipient cells from the donated cells by exosomes and then influence the biological behavior of recipient cells.The most striking example involved the large number of miRNA in exosomes can regulate the activation of a variety of important target genes in receptor cells.Exosome is a natural vector for transmiting nucleic acid and cytokines from one cell to another.Inspired by this,researchers have carried out many studies about exosomes as druy carrier continuously.In this paper,centering on this subject,the study has been carried out aiming at exosomes co-deliver chemotherapy drug and gene medicine and also studying how to import foreign genes into exosomes highly effective.The main work and results obtained from the researches of this paper are summarized as following:Research on exosomes co-delivering chemotherapy drug and gene medicine was experimentally carried out for the first time.We designed a recombinant fusion protein which could target Her2 protein and display on exosomes and then constructed its expressing plasmid using lentiviral vector(p-Tar-Her2-LAMP2).We obtained exosomes containing the fusion protein by over-expressing Tar-Her2-LAMP2 in HEK293T.Then chemotherapy drug and gene medicine were loaded into exosomes employed by electroporation.Finally,we evaluated the anti-tumor effect and tumor targeting of Tar-Her2-EXO/miR-21 inhibitor/5-FU in vitro and in vivo.The results showed that Tar-Her2-EXO/miR-21 inhibitor/5-FU had targeting function to tumor tissue and could focus on killing cancer cell in vitro and in vivo.Cell viability experiments in vitro and in vivo showed that Tar-Her2-EXO/miR-21 inhibitor/5-FU exhibit good inhibition function on the proliferation of HCT116 cells than Tar-Her2-EXO/miR-21 inhibitor or Tar-Her2-EXO/5-FU.A new method has been developed to import foreign genes into exosomes by biosynthesis.We constructed miR-21,siR-artificiality and miR-21 decoy lentiviral vectors separately firstly,then miR-21,siR-artificiality or miR-21 overexpressed stable cell strain based on HEK293T cells was established separately.After that the abundance of these three small RNA in the exosomes derived from the corresponding stable cell strain was analyzed.The experiment results showed that the small functional RNA overexpressed in stable cell strain can enter exosomes.We also inspected the biological function of these small functional RNA by dual-luciferase reporter gene assay.The results suggested that these small functional RNA can regulate the function of target genes in recipient cell.The exosome contained siR-PLKl(EXO-siR-PLK1)has been designed by the above biosynthesis methods and the antitumor activities have been evaluated in vitro.We firstly constructed a lentiviral vectors which can express both siR-PLK1 and PLK1 silence mutation,and then established siR-PLKl overexpressed stable cell strain based on HEK293T cells and enriched exosomes from HEK293T-siR-PLK1 culture fluid.Then we study the effect of EXO-siR-PLKl on BT474 cells in vitro.The results of RT-PCR and Western blot showed that the PLK1 could be inhibited successfully by EXO-siR-PLK1,and the flow cytometer shows that the cell cycle of BT474 cell was arrested in G2/M phrase,the apoptosis was obviously,and the proliferation of BT474 cell was inhibited.In conclusion,this study enriched the method of exosomes as a vector to carry genomic medicine and chemotherapeutics.