Triptolide-loaded Exosomes For The Treatment Of Ovarian Cancer

ObjectiveTo investigate the effect of triptolide-loaded exosomes(TP-Exos)on ovarian cancer and macrophages in vitro and in vivo.Methods1.SKOV3-exosomes(SK-Exos)were collected by ultracentrifugation and ultrafiltration centrifugation.TP-Exos was constructed by sonication and ultrafiltration centrifugation.2.SK-Exos and TP-Exos were characterized by transmission electron microscopy,western blotting,nanoparticle-tracking analysis and high-performance liquid chromatography.3.Tumor cell targeting assay,MTT assay and Brd U cell proliferation assay were used to investigate the suitability of SK-Exos as drug delivery carriers.4.MTT assay,cell proliferation assay and apoptosis experiment were used to study the effect of TP-Exos on ovarian cancer in vitro.The effect of TP-Exos on ovarian cancer in vivo was evaluated by constructing ovarian cancer subcutaneous transplanted tumor model,monitoring the tumor volume of mice and the TUNEL staining assay.The toxicity of TP-Exos in vivo was evaluated by liver and kidney function and histopathology of major organs(heart,liver,spleen,lung,kidney and ovary).5.The effects of TP-Exos on macrophages in vitro were detected by macrophage model construction experiment,macrophage uptake exosome assay,MTT assay and ELISA assay.Constructing ovarian cancer subcutaneous transplanted tumor model,ELISA assay and immunofluorescence co-localization assay were used to detect the effect of TP-Exos on macrophages in vivo.Results1.SK-Exos were cup-shaped and that TP-Exos had an elliptical structure.The sizes of SK-Exos and TP-Exos were 129.6 ± 1.9 nm and 159.9 ± 2.7nm,respectively.CD9 and CD81 were expressed in both exosome types.The encapsulation efficiency of TP in TP-Exos was 76.5% ± 1.8%.2.SK-Exos did not promote the proliferation of SKOV3 cells in the concentration range of 0.2-40 μg/ml.PKH26 labeled exosomes(PKH26-Exos)could uptake by SKOV3 cells,and Dir labeled exosomes(Dir-Exos)could be enriched to the tumor site of tumor bearing mice.3.The cytotoxic and apoptotic effects on SKOV3 cells of TP-Exos were weaker than those of TP,and tumor cell proliferation inhibition and tumor growth inhibition were stronger than that of TP.TP-Exos had no effect on the level of BUN and Cr,but significantly increased the level of AST and ALT.TP-Exos had no obvious pathological damage to heart,lung,kidney and ovary,but had damage to liver and spleen.4.The function of the induced macrophage model was basically the same as that of macrophages in vivo.The ability of M0 macrophages to uptake of fluorescent labeled exosomes was significantly higher than that of M1,M2 macrophages.The toxicity of TP-Exos to the three types of macrophages was significantly weaker than that of free TP.TP-Exos could inhibit the secretion of IL-10 and promote the secretion of IL-12 by M0,M2 macrophages,while it could inhibit the secretion of IL-12 and promote the secretion of IL-10 by M1 macrophages in vitro.TP-Exos could significantly inhibit the secretion of IL-10 and TGF-β and promote the secretion of IL-12 and TNF-α in vivo.In addition,there was no significant change in the number of M1 and M2 macrophages in tumor tissue after TP-Exo treatment.Conclusions1.SK-Exos were successfully extracted.It has natural tumor targeting and has no effect on cell proliferation,indicating that SK-Exos may serve as drug delivery agents for tumor cells in vivo and in vitro.2.TP-Exos were successfully prepared.It has the general characteristics of exosomes and good drug encapsulation efficiency.TP-Exos has good anti-ovarian cancer effect in vivo and in vitro,but it has toxic effect on liver and spleen,suggesting that further optimization of TP-Exos is needed to attenuate the damage to liver and spleen.3.Three types of macrophage models were successfully constructed.TP-Exos could not only be phagocytized by macrophages(M0 > M2 > M1),but also attenuate the toxicity of TP on macrophages.TP-Exos may have the ability to promote M1 macrophage formation in vitro and in vivo.