| Background and objective:The prevalence of ovarian cancer ranks 7th among female cancers.Because there are no obvious symptoms in the early and middle stages,they are generally found to be advanced.Generally,surgery and chemotherapy are the the treatment of ovarian cancer.In the case of repeated medication,drug resistance gradually develops,which is one of the main reasons for the poor prognosis in patients.Drug-resistant cancer cells always appear to highly express P-glycoprotein(P-gp).The P-glycoprotein expressed by the drug-resistance-related gene MDR1 is an ATP-dependent protein,which is available to pump the drugs out of the cytoplasm,reduces the concentration of the drug in cancer cells,thereby inducing drug resistance.In this study,Doxorubicin-resistant ovarian cancer cell SKOV3(SKOV3/Dox)was developed to study ovarian cancer drug resistance.Nano-delivery system using extracellular vesicles as carriers has also been one of the hot spots in recent years.Compared with animal-derived extracellular vesicles,the extracellular vesicles extracted from plants are more productive as a drug carrier.Compared with artificial nanocarriers,plant-derived extracellular vesicles with subcellular structure have more affinity for tumor cells,but whether plant-EVs can overcome tumor cell resistance is rarely studied.It has been reported that animal-derived extracellular vesicles can enter cells through endocytosis or membrane fusion,to further avoid the role of P-gp as a drug pump,and can be used for tumor resistance research.In this study,lemon-derived extracellular vesicles were used as a carrier,and heparin-cRGD conjugates were targeted to modify lemon-derived extracellular vesicles containing doxorubicin(HR-EVs-Dox,EHRD),which increased doxorubicin accumulations in drug-resistant ovarian cells thus improving its killing effect,so as to further study the therapeutic capacity of cRGD modified plant-derived extracellular vesicles drug-delivery system in vivo.Results1.The results of dynamic light scattering,ultraviolet spectrum scanning,transmission electron microscopy and other experiments comfirmed the extraction of lemon-derived extracellular vesicles and the successful synthesis of EHRD.2.Western blot results showed that the expression of P-gp in the drug-resistant cell SKOV3/Dox induced by doxorubicin was 35.16±1.1 times of SKOV3.(P<0.001)The IC50 of SKOV3/Dox was 4.27±0.5 times that of SKOV3.3.SKOV3-luc/Dox,a drug-resistant strain SKOV3/Dox transfected with firefly luciferase gene,was used to construct an in situ ovarian cancer animal model.Tumors in vivo have certain resistance to Dox.4.The results of cell viability assays and cell uptake experiments showed that lemon-derived extracellular vesicles as a carrier could enhance the uptake of nanodrugs by tumor cells,combined with cRGD which strongly targeted to integrinαvβ3,could show the strongest killing effect in SKOV3/Dox and SKOV3.5.Application of endocytosis inhibitors is for studying the endocytosis pathway of EHRD.The results showed that lemon EVs as a drug carrier can increase the drug entry pathway,which mainly enters the cell through the caveolin-dependent endocytosis pathway.6.The results about circulation times of Cy7-labeled lemon-derived extracellular vesicles and drugs loaded through lemon-derived extracellular vesicles in the blood showed that lemon-derived extracellular vesicles as a drug carrier could enhance the stability of drugs in the blood.7.The results of in vivo distribution indicated that lemon-derived extracellular vesicles mainly accumulated in the liver of xenograft models,followed by kidney,tumor and spleen;heparin-cRGD mainly accumulates in the liver,kidney and tumor of the body,compared with the former,heparin-cRGD had greater fluorescence intensity at the tumor site.Conclusion1.EHRD showed the strongest cell entry ability and killing effect in the drug-resistant human ovarian cell line SKOV3/Dox.2.Lemon EVs as drug carriers can enter drug-resistant cells through various endocytosis pathways,which is conducive to evading the role of P-gp.3.The nanoparticles modified by cRGD can enhance the active targeting of the drug,which specifically enhances the concentration of the drug in the target cell. |
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