Re-self-assemble Heparin-based Nanoparticles For Glioma Therapy Through Both Anti-proliferation And Anti-angiogenesis

Objective:This research aimed to fabricate the re-self-assemble heparin-based nanoparticles which can across the blood-brain barrier and exert the efficacy of anti-glioma cell proliferation,tumor endothelium-dependent vascular and vasculogenic mimicry,and estimate its biological activity with in vitro and in vivo experiments,and explore its mechanism.The work was hoped to provide novel thought for the drug research and development of glioma,and establish the basis for clinical application.Methods:The re-self-assemble heparin-based nanoparticles(H-S-R NPsl)was verified by nuclear magnetic resonance spectroscopy(1H NMR),ultraviolet spectrophotometer(UV),BCA protein quantification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MOLDI-TOF).Transmission Electron Microscope(TEM)and dynamic light scattering(DLS)were used to investigate the characterization of H-S-R NPs in aqueous solution and blood-mimic condition.The in-vitro blood-brain barrier model was used to study the blood-brain barrier penetration ability of H-S-R NPs under different conditions.The in vitro ability of targeting,anti-proliferation and anti-microtubule formation of H-S-R NPs on EphA2 and ?v?3 overexpressed glioma cells and neovascular endothelial cells were investigated by flow cytometry,confocal microscopy,CCK8 and three-dimensional culture,respectively.Construct the subcutaneous/intracranial glioma-bearing mice model to investigate the biodistribution and in vivo anti-glioma effect of H-S-R NPs.The mechanism of H-S-R NPs was studied by immunohistochemistry analysis of Ki67 and CD34/PAS.HE staining was used to evaluate the in vivo safety of H-S-R NPs.Results:The results of 1H NMR,UV,BCA protein quantification and MOLDI-TOF confirmed the successful preparation of H-S-R NPsl,the content of SWL and cRGD peptides were 12.57%and 7.61%(w/w),respectively.TEM and DLS showed that H-S-R NPs were about monodisperse spherical particles with a particle size of 164 ± 16 nm in water and a smaller particle size of 63 ± 11 nm in a blood-mimic environment.The results of in vitro blood-brain barrier model indicated that the smaller particle size favors its ability to cross blood-brain barrier.The excellent cellular uptake of single-targeted H-S 1,H-R 1 and dual-targeted H-S-R NPsl by U87,U251 and HUVEC cells that overexpress EphA2 and ?v?3 was confirmed by flow cytometry and confocal microscopy,while the low EphA2 and?v?3 expression HEB cells showed relative lower uptake.CCB-8 assay showed that H-S-R NPsl had better killing effect on EphA2 and ?v?3 overexpressing cells than H-S 1,H-R 1 and H-S-R NPs2,and all the conjugates had no significant killing effect on HEB cells.The results of three-dimensional culture showed that H-S-R NPs1 showed better effect on microtubule formation than H-S 1 and H-R 1 on U87,U251 and HUVEC.The results of in vivo distribution showed that H-S-R NPs1 was quickly accumulated in U251 subcutaneous glioma(0.5 hours),reached a peak at 12 hours,and then gradually decayed.the ex vivo imaging and histological sections of confocal microscopy confirmed that H-S-R NPsl could across the blood-brain barrier to accumulate in the glioma tissues.The results of in vivo tumor inhibition experiments show that,H-S-R NPsl in subcutaneous/glioma tumor bearing mice have a good inhibition of glioma growth and prolong the survival of tumor-bearing mice.The results showed that the expression rate of Ki67(20.39 ± 2.57%)in H-S-R NPs1 treatment group was significantly lower than that in control group(89.69 ± 3.74%)and H-S 1 treatment group(58.78 ± 4.19%),while the amounts of endothelial-dependent vessels(CD34 +)and vasculogenic mimicry(CD34-/PAS +)in the tumor of H-S-R NPsl treatment group were significantly less than those in the control group(62.33 ± 9.5)and H-S 1 treatment group(30.67 ± 2.89).HE staining showed that H-S-R NPsl was not found to have significant organ toxicity.Conclusion:The re-self-assemble heparin-based nanoparticles prepared in this study are ideal candidate for the treatment of glioma.