Study On Anchor Peptide-based Capture And Targeted Drug Delivery With Exosomes In Duchenne Muscular Dystrophy

Exosomes are nanovesicles with sizes of 30 to 150 nm(in diameter)that are secreted by many mammalian cells and presented in blood,urine and other body fluids.Exosomes can carry a large number of functional molecules from parent cells in the process of formation and act as intracellular messengers between cells.Based on this characteristic,exosomes are widely used in the diagnosis and treatment of various disease.Moreover,the unique features of exosomes like bilayer lipid structure,low immunogenicity,long time circulation and feasibility of modification enable exosomes to be used as delivery vehicles for large variety cargos in disease treatment.As a natural biological nano-carrier,exosomes have emerged as appealing candidate drug delivery platform in many research studies,however,obstacles such as low isolation yield,considerable complexity and potential safety concerns,and inefficient drug payload substantially hamper their therapeutic applicability in the clinical.To overcomes these limitations,in this study,we are trying to develop a new method of surface modifying exosomes,we performed invitro selection with purified CD63 second extracellular loop and screened out the anchor peptide CP05 with two rounds application enrichment.After sequencing analysis,3 candidate peptides were screened.Comparing the binding efficacy of the 3 candidate peptides with exosomes by using confocal fluorescence microscopy and flow cytometry,CP05 enable specifically binding to exosome via CD63 with over 80% binding efficacy irrespective of the origin of exosomes.Furtherly,the specific binding of CP05 to exosomes were confirmed by immunocoprecipitation.Moreover,the characteristics of exosomes including integrity of membrane,the size and invivo distribution which were modified by CP05 were detected by using electron microscopy,nanosight and invo image system.Surface modifying of exosomes by targeting moieties like M12,RVG,SP94 via CP05 directs exosomes into correspondingly tissues such as muscle,brain,tumor respectively.Exosomes painted with PMO via CP05 demonstrated a n 18-fold increase of dystrophin restoration in quadriceps in mdx after systemic treating compared to naked PMO or the CP05-PMO conjugation.CP05 also allows for simultaneous functionalization of exosomes with different moieties,including targeted PMO delivery to muscle in mdx mice by anchoring a muscle-targeting peptide and PMO on the same exosome with CP05.CP05 can anchor moieties not only to culture-derived exosomes from different cells but also to circulating exosomes from human serum.Furthermore,immobilized CP05 can specifically capture exosomes from patients' serum for diagnostic use.Overall,our study demonstrates that an exosomal anchor peptide enables direct,effective functionalization and capture of exosomes,thus providing a tool for exosome engineering,probing gene function in vivo,and targeted therapeutic drug delivery.