Gold Nanoparticles Combined With Recombinant Human Endostatin Induced Tumor Vascular Normalization

Angiogenesis is a process by which vessels are formed through preexisting ones,and this plays a key role in the progression of solid tumors.This process is initiated by the wealth of pro-angiogenic factors produced by tumor cells in response to a hypoxic microenvironment.Excessive production of pro-angiogenic factors leads to an imbalance between pro-and anti-angiogenic signals,which results in structurally and functionally abnormal vessels characterized by dilatation,tortuosity,high permeability,risk of recurrence or metastasis,and reduction of radiotherapy or chemotherapy effect.Moderate anti-angiogenic therapies are considered to regulate tumor vascular morphology and function and improve the efficiency of antitumor therapy.However,the duration of vascular normalization is transient.Thus,monitor the process from start to finish is crucial and the identification of markers that indicate normalization may be useful to determine the optimal schedules on which to administer anti-tumor therapies.Objective: First,to investigate the targeting of gold nanoparticles(AuNPs)combined with anti-angiogenesis agents,and detect the time window of tumor vascular normalization and anti-tumor effect of 5-FU.Second,observe the effect of AuNPs on tumor vascular normalization and the inhibition of metastasis,and to explore its related mechanisms.Finally,observe the changes of Thrombospondin-1 during the time window of tumor vascularization.Methods: In the first section,the characterization of AuNPs with recombinant human endostatin(rhES)was identified by ultraviolet-visible spectrophotometry(UV-vis spectrum),dynamic laser scattering(DLS)and field emission transmission electron microscopy(FETEM).The dose of rhES in tumors,pericyte coverage(?-SMA / CD31),blood perfusion(FITC-Lectin / CD31),vascular permeability(FITC-Dextran / CD31)and the expression of hypoxia inducible factor-1?(HIF-1?)in tumor tissue were measured by immunofluorescence technique.5-FU was given in the normalization window and the concentration of 5-FU in tumor tissue was detected by high performance liquid chromatography(HPLC).The tumor size and median survival time(MST)of tumor bearing model was observed.In the second section,tumor vascular permeability(FITC-Dextran)and perfusion(FITC-Lectin)were measured by immunofluorescence,the epithelial-mesenchymal transition(EMT),vascular endothelial growth factor(VEGF)and its receptor(VEGFR2)and alleviation of tumor hypoxia(pimetanazole staining)were detected by immunohistochemistry.The effect of AuNPs on migration of tumor cells and endothelial cells was detected by wound healing assay,and the the cell viability were detected by CCK-8 method.The expression of MMP2,c-Myc,Vimentin,E-Cadherin,ZO-1 and SPARC in tumor vascular normalization and metastasis were observed by Western blotting.In the third section,the normalization of tumor blood vessels was identified as as described above,the change of thrombospondin-1in peripheral blood during time window was measured by enzyme-linked immunosorbent assay(ELISA).Results: The accumulation of rhES in tumor significantly increased after combined with AuNPs,and maintained a long time compared with the control.Immunofluorescence showed normalization of tumor blood vessels from day 4 to day 8 after AuNPs-rhES administration,the coverage of pericyte(?-SMA)and FITC-Lectin perfusion were increased,the deposition of FITC-Dextran around the blood vessels and the expression of HIF-1? in tumor tissue were decreased.Compared with monotherapy,combination with AuNPs-rhES significantly increased 5-FU localization to the tumor sections.5-FU monotherapy and 5-FU combined with AuNPs-rhES reduced growth significantly,however,tumors receiving combination therapy exhibited a more significant reduction in growth compared to tumours receiving 5-FU monotherapy.Our results from the lectin analysis revealed that tumor blood perfusion significantly increased after Au NPs treatment compared with controls,and the ratio of lectin+ to CD31 expression indicated vessels with regular perfusion.Leakage of injected dextran into tumor vasculature remarkably decreased after AuNPs treatment.The immunohistochemical staining showed that the AuNPs treatment significantly reduced tumor hypoxia,and down-regulated the expression of Vimentin and up-regulated the expression of E-Cadherin in the tissue.AuNPs reduce the extent of lung metastasis in vivo,and suppress the migration of HUVECs and B16F10 cells in vitro.On the contrary,data from the CCK-8 test indicated that the AuNPs had no effect on the cell viability.In addition,AuNPs inhibits the expression of MMP2 in endothelial cells,but promotes the expression of ZO-1,inhibits the expression of MMP2 and c-Myc in tumor cells,reverses the process of EMT.RT-qPCR showed that AuNPs reduced the expression of Snail-1,Snail-2,Twist-1,ZEB-1 and ZEB-2.Finally,we found that the expression of plasma thrombospondin-1 decreased significantly during normalization of tumor vascular normalization(day 2-8),and then increased after the closure of normalization window.Conclusion:1.AuNPs can be used as a nano-drug delivery system to target transport rhES into tumor tissue.2.The combination of AuNPs with rhES promoted the normalization of tumor blood vessels,and increased the effect of chemotherapy.3.AuNPs inhibit EMT in tumor cells by down-regulating MMP-2,c-Myc,SPARC and EMT related genes.4.AuNPs inhibit endothelial cell migration and angiogenesis by down-regulating MMP-2 and enhance vascular wall integrity by up regulating the expression of ZO-1.5.The change of circulating thrombospondin-1 is consistent with the hypoxia in the time window of tumor vascular normalization,and it is expected to be an indicator to predict the normalization of tumor blood vessels.