Effects Of A Novel Vascular Targeting Penetrating Peptide On Glioma

Background and objectivesHuman glioma is considered to be one of the most common primary tumors in the central nervous system,with a high lethality and common recurrence.Few drugs can overcome the blood-brain barrier(BBB)to treat gliomas.In addition,histological studies have found that gliomas can drive a large number of new blood vessels through high expression of vascular endothelial growth factor and related receptors,which provides an important nutrient supply for tumor development.Therefore,inhibiting tumor angiogenesis has become one of the new treatment options for gliomas,which has great development potential.As a co-receptor of VEGFA-165,neuropilin 1(NRP1)plays an important role in enhancing the VEGFR2 signaling pathway and has become a potential target for antiangiogenic therapy.The aim of this study was to develop a dual functional peptide fused with cell-penetrating peptides(CPPs)and NRP1 targeting peptides,to penetrate the BBB and inhibit glioma angiogenesis.And we further evaluate the anti-angiogenic activity of this fusion peptide in vivo and in vitro and explore specific molecular mechanisms.Methods1.Screening NRP1 targeting peptides with strong anti-angiogenic activity in vitro.In this study,the effects of six candidate NRP1 targeting peptides on the proliferation of HUVECs were investigated using MTT assay,and whether the six candidate NRP1 targeting peptides were able to inhibit the tube formation of HUVECs in the presence of the stimulating factor VEGFA-165 using tube formation assay.2.Tat was covalently linked to the NRP1 targeting peptide through two different linkers,and then fusion peptides with stronger anti-angiogenic activity in vitro were screened.MTT assay was used to compare the effects of two fusion peptides on HUVECs proliferation,and tube formation assay were used to compare the ability of the two fusion peptides to inhibit tube formation in HUVECs in the presence of the stimulating factor VEGFA-165.At the same time,the effects of two fusion peptides on HUVECs migration were compared.3.Detection of the affinity of the fusion peptide to the NRP1 protein.The NRP1 protein was coupled to a CM5 chip,and the surface dissociation resonance(SPR)method was used to obtain the affinity dissociation constants of different peptides against the NRP1 protein.4.To evaluate the anti-angiogenic activity of the fusion peptide in vitro.MTT assay was used to detect the anti-HUVECs proliferation activity of the fusion peptide,tube formation assay was used to detect the anti-HUVECs tube formation activity of the fusion peptide in the presence of the stimulating factor VEGFA-165,and wound healing assay and Transwell chamber assay were used to detect the anti-HUVECs of the fusion peptide migration activity.Hoechst nucleic acid staining method and Annexin V-FITC/PI double staining method were used to detect the apoptosis-promoting activity of HUVECs by the fusion peptide.5.To explore the molecular mechanism of anti-angiogenic activity of fusion peptide in vitro.Western blotting was used to detect the regulation of the phosphorylation level of VEGFR2 signaling pathway-related proteins in HUVECs treated by the fusion peptide.6.Detection of the uptake of fusion peptide by bEnd.3 cells.Fluorescence microscope and flow cytometry were used to detect the uptake of FITC-labeled fusion peptide in bEnd.3 cells.7.To detect the brain distribution of the fusion peptide in vivo.Glioma U87-luc-mCherry cells were inoculated into the brain of nude mice to construct a model of orthotopic glioma in nude mice.Frozen sections of brain tissue were prepared and fluorescence scanning was performed to observe whether the FITC-labeled fusion peptide could penetrate the blood-brain barrier and accumulate in the brain tumor area by means of tail vein injection.8.To detect the inhibitory effect of fusion peptide on glioma growth in vivo.U87-luc-mCherry cells were inoculated into the brain of nude mice to establish an orthotopic glioma model.Observe the effect of fusion peptide on glioma growth using an in vivo imaging system;The changes in body weight of nude mice were recorded daily to determine whether there was significant toxicity in the treatment of different peptides;After the peptide treatment was completed,hematoxylin and erythrocytes were used to stain the brain tissue sections to further observe the size of brain tumors;CD31 antibody was used to stain the brain tissue sections to observe the tumor angiogenesis in nude mice treated with different peptides.Results1.NRP1 targeting peptide RP7 had good anti-angiogenic activity in vitro.The results of MTT assay showed that none of the six candidate NRP1 targeting peptides had significant anti-proliferative activity on HUVECs.Tube formation assay showed that the NRP1 targeting peptide of the sequence RPARPAR(RP7)showed a good ability to resist tube formation in HUVECs.Therefore,RP7 was selected as the NRP1 targeting peptide in this paper for subsequent experiments.2.Tat and linker(cysteine)fusion peptides had stronger anti-angiogenic activity in vitro.The results of MTT assay and tube formation assay showed that fusion peptides with linker cysteine or glycine have semilar anti-HUVECs proliferation activity and anti-HUVECs tube formation ability.The results of wound healing assay showed that the fusion peptide with linker as cysteine also had certain anti-HUVECs migration activity.3.The fusion peptide Tat-C-RP7 had the strongest affinity for NRP1 protein.The results of the affinity test showed that compared with RP7 and Tat,the fusion peptide Tat-C-RP7 has the strongest affinity for NRP1 protein,KD=69.7 nM.4.The fusion peptide Tat-C-RP7 showed strong anti-angiogenic activity in vitro.The results of MTT assay showed that the fusion peptide Tat-C-RP7 could significantly inhibit the proliferation of HUVECs;The results of tube formation indicated that in the presence of the stimulating factor VEGFA-165,the fusion peptide Tat-C-RP7 can significantly reduce the number of HUVECs microtubules;the results of wound healing assay and Transwell assay showed that fusion peptide Tat-C-RP7 could inhibit the migration of HUVECs;Hoechst nuclear staining and Annexin V-FITC/PI double staining showed that the fusion peptide Tat-C-RP7 could also significantly promote the apoptosis of HUVECs.5.The fusion peptide Tat-C-RP7 achieved anti-angiogenesis in vitro by down-regulating the phosphorylation levels of VEGFR2,PLCy,ERK1/2 and AKT proteins in VEGFR2-related signaling pathways.In the presence or absence of the stimulating factor VEGFA-165,the fusion peptide Tat-C-RP7 could inhibit the phosphorylation levels of VEGFR2,PLCy,ERK1/2,and AKT proteins in HUVECs,but had no effect on FAK,P38 MAPK and SRC.6.bEnd.3 cells had the strongest uptake of the fusion peptide Tat-C-RP7The results of fluorescence microscopy and flow cytometry showed that bEnd.3 cells took up FITC-labeled NRP1 targeting peptides RP7,Tat and fusion peptide Tat-C-RP7.The fusion peptide Tat-C-RP7 had the strongest uptake.7.The fusion peptide Tat-C-RP7 had the ability to penetrate the blood-brain barrier to reach the tumor site in vivo.The fluorescence scan results of frozen sections of brain tissue showed that the FITC-labeled fusion peptide Tat-C-RP7 could penetrate the blood-brain barrier and accumulate at the tumor site.8.The fusion peptide Tat-C-RP7 had the ability to inhibit glioma growth in vivoThe results of in vivo imaging and HE staining of brain tissues confirmed that the fusion peptide Tat-C-RP7 had a significant growth inhibitory effect on gliomas and had no significant effect on the body weight of nude mice.The results of CD31 immunofluorescence staining in brain tissues showed that the fusion peptide Tat-C-RP7 significantly inhibited angiogenesis in tumor areas.ConclusionWe screened the most active fusion peptide Tat-C-RP7 by in vitro activity test,which had strong anti-angiogenic activity in vivo and in vitro and the ability to penetrate the blood-brain barrier.In compared to the NRP1 targeting peptide RP7 and the cell-penetrating peptide Tat,the fusion peptide had a stronger affinity to the NRP1 protein.To further clarify its molecular mechanism,the anti-angiogenic activity of the fusion peptide Tat-C-RP7 might be achieved by down-regulating the phosphorylation levels of VEGFR2 and downstream PLC?,ERK1/2 and AKT proteins in VEGFR2-related signaling pathways in HUVECs.SignificanceConsidering on the novel anti-angiogenesis therapeutic target NRP1 and active molecules that were difficult to penetrate the blood-brain barrier,this study successfully designed and screened a dual functional fusion peptide that inhibited tumor angiogenesis and penetrated the blood-brain barrier.This provides new ideas for the application of cell-penetrating peptides and NRP1 targeting peptides in the future,and provide a research basis for antiangiogenic therapy of gliomas.