Preliminary Study On Antitumor Effect Of Cell-bound Membrane Vesicles As A Drug Nanocarrier For Doxorubicin

In recent years,due to the aging of population,industrialization-induced environmental pollution,and accelerating urbanization-caused changes in lifestyles,people's tumor prevalence and mortality have been increasing significantly.The prevention and treatment of tumors,as well as the development of related drugs,have received more and more attention from scientific research teams.Doxorubicin(Dox for short)is a broad-spectrum and effective anti-cancer drug,but it has strong toxicity and side effects on the heart,liver,bone marrow,etc.For this,scientists have proposed many solutions among which the use of drug carriers is one of the more promising strategies.There are cell-bound membrane vesicles(CBMVs for short)on Surfaces of various cell types.In the early stage of this research topic,we have conducted research on other aspects of CBMVs,such as the identification,characterization,and movement laws of vesicles,and we have made preliminary explorations on the isolation and purification of this vesicles.On this basis,we speculate that cell-bound membrane vesicles may also be used as a potential anti-tumor drug carrier system to solve the toxic and side effects of doxorubicin.In order to verify the hypothesis,this project used detergent treatment and sucrosegradient high-speed centrifugation to separate and purify the vesicles from the surfaces of human umbilical vein endothelial cells(HUVECs)cultured in vitro,and loaded doxorubicin by sonic degradation method to obtain Dox-CBMVs(their encapsulation rate and drug loading were 44.77%±2.71%and 2.36%±0.05%,respectively).A dual-fluorescence imaging of the Dox-CBMVs was performed by the confocal microscope(autofluorescence of doxorubicin and Dio fluorescence labeled on the vesicles).The co-localization of fluorescence confirmed the successful loading of doxorubicin.The size and zeta potential of Dox-CBMVs nanoparticles were detected by the dynamic light scattering technology and transmission electron microscopy.We found that the unloaded vesicles and Dox-CBMVs had particle sizes of 336.87v5.08 nm and 395.87±15.78 nm,respectively.The results from in vitro drug release experiments and in vivo drug accumulation experiments show that Dox-CBMVs have a sustained drug release effect.In vitro cell experiments show that Dox-CBMVs can be recognized and phagocytosed by normal cells(HUVECs)and tumor cells(mouse colon cancer CT26.WT)and have a killing effect on HUVEC cells and various tumor cells(mouse colon cancer CT26.WT,liver cancer cell HEPA1-6,lung cancer cell LLC Lewis and human liver cancer cell SMMC-7721).The experiments on animal models of colon cancer show that Dox-CBMVs can inhibit the growth of tumors in mice(tumor volume and weight decrease significantly),inhibit cell division in tumor tissues,and promote apoptosis of tumor cells(Immunohistochemical staining of tumor tissue cross-sections:H&E staining,PH3 staining,TUNEL detection),achieving similar drug effects to free doxorubicin,indicating that Dox-CBMVs have significant antitumor drug effects.On the other hand,animal experiments show that compared with free doxorubicin,The toxic side-effects of Dox-CBMVs to the heart,liver and bone marrow are weaker(blood index and tissue section H&E staining),indicating that Dox-CBMVs can significantly reduce the toxic and side effects of doxorubicin.The above-mentioned in vitro and in vivo experiment results show that the isolated and purified cell-bound membrane vesicles can be successfully loaded with the anti-tumor drug doxorubicin,and that the cell surface vesicles after drug loading(i.e.Dox-CBMVs)have a sustained drug release ability.While maintaining the efficacy of the anti-tumor drug(doxorubicin),it can significantly reduce toxic and side effects,verifying the initial speculation of this project.In addition,we also speculate that the cell-bound membrane vesicles probably are a broad-spectrum drug carrier system for other drugs to treat corresponding diseases(tumor or other diseases),but more further experiments are needed to verify this speculation in the future.