Initial Exploration Of IRGD Modified Cell-bound Membrane Vesicles Encapsulating Doxorubicin For Melanoma Treatment

With the gradual aging of our population,the pollution brought by industrial production and the changes of living habits brought about by the development of science and technology,the incidence rate and mortality rate of Chinese people have increased significantly.The prevention and treatment of cancer have attracted more and more attention.Doxorubicin(Dox)has been widely used as a specific drug for the treatment of tumors.However,Doxorubicin has strong toxic and side effects on various organs of human body.Therefore,researchers have put forward many solutions,and relying on drug carrier is one of the more feasible strategies.In this study,we found that cell bound membrane vesicles(CBMVs)exist on many kinds of cells.Our research group has studied the vesicles on the cell surface in many aspects,such as the identification of vesicle components,movement law and its influence on cells,and has made a preliminary exploration on the separation,purification and drug loading of this kind of vesicles.On this basis,we speculate that the modified cell surface vesicles may also be used as an effective antitumor drug targeting carrier system,so as to improve the therapeutic effect of antitumor drugs,reduce drug toxic and side effects and slow down tumor drug resistance.In order to verify this conjecture,the vesicles on the surface of human umbilical vein endothelial cells(HUVECs)were successfully isolated and purified by Triton X-100 treatment and sucrose gradient high-speed centrifugation.Then Doxorubicin was loaded into the vesicles on the cell surface by ultrasonic degradation method,and iRGD-PEG-DPSE was loaded on the vesicle surface.iRGD-Doxorubicin-cell surface vesicles(iRGD-Doxorubicin-vesicles for short)were obtained,and their entrapment efficiency and drug loading were 44.78% and 447.78679 μg/m L,respectively)。 IR and 1H NMR spectra were used to identify its components,which confirmed the success of iRGD-PEG-DPSE modification.Three fluorescence imaging of iRGD-Doxorubicin-vesicles(spontaneous fluorescence of doxorubicin,FITC fluorescence on iRGD-PEG-DPSE and Dio fluorescence labeled on vesicles)was carried out by laser confocal microscope.The co localization of fluorescence further verified the success of doxorubicin loading and iRGD-PEG-DPSE modification.The size,zeta potential and composition of iRGD-Doxorubicinvesicles nanoparticles were detected by dynamic light scattering technique and transmission electron microscope.It was found that the particle sizes of empty vesicles,Doxorubicin vesicles and iRGD-Doxorubicin-vesicles were 342 nm,396 nm and 459 nm respectively,and the diffraction pattern of iRGD-Doxorubicin-vesicles showed crystalline phase.In vitro drug release experiments showed that iRGD-Doxorubicin-vesicles had the effect of drug sustained release.In vitro cell experiments showed that doxorubicin vesicles could be recognized and phagocytized by tumor cells(mouse melanoma cell B16F10 and human hepatoma cell Hep G2),and could kill a variety of tumor cells(mouse colon cancer cell CT26.wt,mouse hepatoma cell hepa1-6,human hepatoma cell HEL,human hepatoma cell SMMC-7721,mouse melanoma cell B16F10 and human hepatoma cell Hep G2).At the same time,it can inhibit the migration of a variety of tumor cells(mouse hepatoma cell hepa1-6,mouse melanoma cell B16F10,human hepatoma cell Hep G2 and human hepatoma cell SMMC-7721).Melanoma mouse model animal experiments show that iRGD-Doxorubicin-vesicles can not only inhibit the growth of tumors(tumor volume)in mice,but also have very low toxic and side effects.The above in vitro and in vivo experimental results show that the isolated and purified cell surface vesicles can successfully load targeted peptides and load Doxorubicin and other antitumor drugs.The targeted cell surface vesicles(iRGD-Doxorubicin-vesicles)have the ability of drug sustained release.While improving the efficacy of antitumor drugs(Doxorubicin),they can also reduce tumor drug resistance and toxic and side effects,which confirms the original conjecture of this subject.In addition,we also speculate that targeting cell surface vesicles can be used as a widely used drug carrier system in the future to carry more functions,so as to diagnose and treat corresponding diseases(tumors or other diseases),but further experiments are needed to explore these aspects.