Application Of Muscle Derived Exosome As Drug Delivery Vehicle For DMD Gene Therapy Investigation And Development Of RIP Gelonin For Antitumor Applicaion

Duchenne muscular dystrophy（DMD）is caused by a fault in an X chromosome,Approximately 1 in every 3,500 newborn boys suffer from it.This fatal fault can lead to the cease of composition of dystropin protein,and result in muscle weakness that gets worse over time because muscle cells break down and are gradually lost.Currently,cell therapy and gene therapy are the most common methods.Although the priority of antisense oligonucleotide treatment has been manifested,it still remains several unsolved problems,such as poor targeting and bioavailability,low delivery efficiency and deficiency of drug safety.Exosome is a class of nanovesicle secreted by variety of cell lines.The diameter of exosomes varies from 45 to 120 nm.Like cellular membrane,exosome has phospholipid bilayer bearing membrane proteins.Based on the cell-derived property,exosomes possess many advantages,including low immunogenicity,potential targeting,blood-brain barrier and cellular penetration.Therefore,exosomes serving as drug delivery vehicles is a promising application for gene therapy by antisense oligonucleotide.This study aims to obtain an efficient drug delivery vehicle for DMD treatment.After purification and characterization,exosomes were utilized as antisense oligonucleotide drug delivery vehicle.In order to manifest exosomes,transmission eectron microscope（TEM）,confocal laser scanning microscopy（CLSM）,Nanosight,fluorescence-activated cell sorting（FACS）and in vivo imaging system（IVIS）were applied to explore the diameter distribution,feature physiological pathways,uptake in different muscular cell lines and in vivo distribution of exosomes.Exosomes were loaded by different methods,such as electroporation and other two physical means,and the efficiency was tested by RT-PCR and PCR technology.By local injection of the drug system into tibialis anterior（TA）,the in vivo therapeutic effect was detected by immunohistochemical technique.Experimental results demonstrated that the C2C12-derived exosomes obtained by filtration and ultracentrifugation were uniformly distributed.Moreover,the physiological pathways correspond well to the literature report.Diameter of exosomes was around 110 nm and size distribution was suitable for drug delivery.TEM showed that the cup-shaped vesicle had bilayer structure,illustrating the mentioned method can preserve the intact structure of exosomes.Intracellular accumulation could be found in endosomes when colocalizing exosomes with both early and late endosome.The cellular uptake studies indicated the C2C12-derived exosomes can enter the muscular cell lines.The in vivo results showed the muscular distribution preference of C2C12-derived exosomes compared to other nano-materials.The success of drug loading demonstrated it is feasible to deliver antisense oligonucleotide by exosomes.Drug loading of antisense oligonucleotide into exosomes can be achieved by physical methods,which was effective for mediating exon skipping.By contrast,electroporation was a less effective strategy.The curative effect of drugs can be detected when locally injecting the drug-loaded exosomes produced by lyophilizated and ultrasonic vibrated into mouse muscle.In brief,the myoblast cell line C2C12-derived exosomes can be employed as a promising carrier for antisense oligonucleotide.In this study,the antisense oligonucleotied-loaded exosomes delivery system was developed to treat DMD to generate semi-functional membrane skeleton protein.With the advances of life science,unprecedented breakthroughs have been made in biotechnology.Bio-drugs have played a significant role in the development of anti-tumor drugs.Compared to small molecular drugs,such as doxorubicin（Dox）and paclitaxel（PTX）,macromolecular drugs possess very high biological activity.For example,ribosome inactivating proteins（RIP）,derived from plants,can effectively kill tumor cells by interfering with ribosome and subsequently inhibiting the synthesis of proteins at a very low dose.Gelonin is a RIP with molecular weight of 29 kDa,which is derived from the seed of gelonium multiflorum.Gelonin is a promising anticancer drug candidate.In order to enhance the therapeutic effects and increase bioavailability in Gelonin-based treatment,the studies were performed as two parts:（1）combination therapy of gelonin and Dox were employed to overcome the multidrug resistence（MDR）in breast cancer therapy;and（2）by modifying Gelonin with both low molecular weight protein（LMWP）and polyethylene glycol（PEG）.Gelonin was acquired and purified by chitin resin and Superdex 75 column.Polyacrylamide gel electrophoresis and mass spectrometry（MS）were employed for characterization of Gelonin.Cellular uptake efficiency of Dox was investigated by inverted fluorescence microscope and flow cytometry,respectively.In vivo imaging technology was utilized to investigate the drug distribution of modified protein drug in the HT1080 tumor-bearing mice.Cell apotosis was investigated by the MTT assay and flow cytometry.Western blotting was employed to detect the expression of P-gp.The antitumor therapy was conducted by peritumor injection of the drug to the tumor-bearing mice,and the side toxicity was further evaluated by histological examination.Polyacrylamide gel electrophoresis（PAGE）results showed the purified Gelonin with the molecular weight of 29 kDa.MS results of protein sequence were identical with the record of Gelonin in the IP2 database.When Dox and Gelonin were combined,the uptake efficiency of Dox by MCF-7/ADR cells was significanty increased.In vitro study demonstrated the improved therapeutic effect.For example,the apoptosis rate in the combined drugs group was much higher than the single use of DOX or Gelonin.MTT results showed that combination therapy could reduce the IC₅₀ of Dox in MCF-7/ADR cells.Western bloting results demonstrated that Gelonin could effectively reduce the expression of P-gp.Combination had more significant effect on killing tumor.Preliminary biosafty studies was investigated by histological examination and monitoring the organ coefficient.No damage on the main organs of mice was observed.The reduction of tumor weight and size demonstrated the in vivo therapeutic outcomes of the combinated drugs.In addition,Gelonin was modified by LMWP and PEG₅₀₀₀,and the modified protein drug was purified by FPLC.LMWP-PEG₅₀₀₀ modification can significantly increase Gelonin uptake in HT1080 cell line.MTT results demonstrated that toxicity of modified protein was enhanced.LMWP-PEG₅₀₀₀ modified Gelonin tended to accumulate in tumor tissues while the native Gelonin accumulated mainly in the kidney and liver.In summary,Gelonin is a highly efficient antitumor agent.The synergistical combination of Dox and Gelonin can impactfully kill the MDR tumor.LMWP-PEG₅₀₀₀ modification can improve the toxicity of Gelonin towards cancer cells and its accumulation in tumor.