Anti-Angiogenesis Targeting Therapy Of Malignant Tumor Through Nanoparticles Based On RGD Oligopeptides

Background and ObjectivesAngiogenesis plays a key role in the growth and metastasis of primary malignant solid tumor.In recent years,anti-angiogenesis targeting therapy for tumor vascular endothelial cells have assisted radiotherapy and chemotherapy to improve the survival rate of patients with tumor.Because of the characteristics of low adverse reaction,anti-angiogenenic therapy has become a hot spot in basic and clinical research.Integrin alpha v and beta 3 is highly expressed in the surface of various tumor cells and vascular endothelial cell,which plays a significant role in tumor angiogenesis.Some researches showed that RGD oligopeptides can be competitively binding with integrin receptor on the surface of tumor,which can prevent the adhesion between tumor cells and matrix and therefore inhibit the formation of tumor angiogenesis,tumor growth and migration.In recent years,exosome,which derived from cell or organism,and self-assembly nanometer vector based biological macromolecular,demonstrated the characteristic of nanoscale including excellent stability,biocompatibility,easy to modify on the surface,high permeability and retention(EPR effect)in tumor tissues.Those characteristics can prolong the circulation time in body and reduce side effects of drugs,which present glorious application prospect.In this study,we designed two kinds of anti-angiogenesis targeting nanoparticles based on RGD oligopeptides with different strategies and conducted the anti-angiogenesis therapeutic study of nasopharyngeal carcinoma and ovarian cancer individually.Methods and Results1.Screening and verifying anti-angiogenesis therapeutic targets of nasopharyngeal carcinoma.Methods:Elisa assay was used to screen anti-angiogenesis therapeutic targets of nasopharyngeal carcinoma(Hsa-miR-18a and EBV-miR-BART10-5p),q-PCR,microtubule formation,chicken embryo chorioallantoic membrane and matrix plug assays were applied to verify the screened result.Results:Hsa-miR-18a and EBV-miR-BART10-5p were successfully screened and verified as anti-angiogenesis therapeutic targets of nasopharyngeal carcinoma.2.Construction of the anti-angiogenic nanoparticle based on RGD oligopeptides and exosome.Methods:Co-transfected method was used to construct a targeting nano-carrer(iRGD-exo-antagomiRs)carrying antagomiRs based on RGD oligopeptides and exosome.Dynamic light scattering(DLS)and transmission electron microscopy(TEM)were used to characterize the nanoparticle.Results:We successfully constructed targeting nanoparticle based on RGD oligopeptides and exosome:iRGD-exo-antagomiRs.3.iRGD-exo-antagomiRs were applied to anti-angiogenic therapy for nasopharyngeal carcinoma.Methods:Microtubule formation,chicken embryo chorioallantoic membrane,matrix plug,subcutaneous tumor of nude mice and immunohistochemistry assays were applied to detect the anti-angiogenesis therapeutic effect of iRGD-exo-antagomiRs upon nasopharyngeal carcinoma in vitro and in vivo.Results:iRGD-exos-antagomiRs presented an excellent anti-angiogenesis therapeutic effect upon nasopharyngeal carcinoma in vitro and in vivo.4.Regulatory mechanism of anti-angiogenesis therapeutic effect of iRGD-exoantagomiRs upon nasopharyngeal carcinoma.Methods:Bioinformatics websites and luciferase reporter gene experiment were applied to predict and detect target genes of Hsa-miR-18a and EBV-miR-BART10-5p.Western blot assay was used to detect the changes of downstream regulated network following interference and recovery of target gene.Results:Hsa-miR-18a and EBV-miR-BART10-5p co-targeted and regulated the Spry3 gene,which promoted the activation of angiogenic regulation network in downstream,and thereby regulated the formation of angiogenesis in nasopharyngeal carcinoma.5.cRGD-NPs2 was applied for anti-angiogenic therapy of ovarian cancer.Methods:Self-assembly targeting nanoparticle based on RGD oligopeptides and biomacromolecule(cRGD-NPs2)was constructed through chemical method in water.MTT and flow cytometry uptake assays were used to screen out preferable nanoparticle.Microtubule formation,matrix plug,subcutaneously tumor in nude mice and immunohistochemical assays were applied to detect the biological activity of cRGD-NPs2 in vitro and in vivo.3D culture,transwell,bordern and western blot assays were applied to detect influence upon VM of cRGD in ovarin cancer.Results:The self-assembled targeting nano-carrier(cRGD-NPs2)based on RGD oligopeptides and biomacromolecule was successfully constructed,and cRGD-NPs2 presented significantly anti-angiogenesis therapeutic activity of ovarian cancer in vitro and in vivo.cRGD inhibits vaseulogenic mimicry formation by down-regulating uPA expression and reducing EMT in ovarian cancer.Conclusion1.Hsa-miR-18a and EBV-miR-BART10-5p were successfully screened and verified as anti-angiogenesis therapeutic targets of nasopharyngeal carcinoma.2.Nanoparticles based on RGD oligopeptides and exosome(iRGD-exo-antagomiRs)were successfully constructed.3.iRGD-exo-antagomiRs presented the inhibited effect of angiogensis in nasopharyngeal carcinoma both vitro and in vivo.4.The regulation mechanism of iRGD-exo-antagomiRs against the formation of angiogenesis in nasopharyngeal carcinoma is to regulate the expression of Spry3 gene and its angiogenic regulation network in downstream,and thereby promoted the formation of angiogenesis in nasopharyngeal carcinoma.5.Nanoparticles based on RGD oligopeptides and biomacromolecule(cRGD-NPs2)were successfully constructed.cRGD-NPs2 is able to prohibit the formation of angiogenesis in ovarian cancer both in vitro and in vivo.cRGD inhibits vasculogenic mimicry formation by down-regulating uPA expression and reducing EMT in ovarian cancer.