| Endostatin(ES)is a peptide of 20 kDa molecular weight which was isolated from the murine hemangioendothelioma.Endostatin is the first discovered endogenous anti-angiogenesis fragment of extracellular matrix and also the endogenous angiogenesis inhibitor which was most studied.There is a short fragment(IVRRADRAAVP)found from endostatin named ES2,its anti-angiogenesis activity in vivo is about 3 times that of ES.Anti-flt 1 peptide(GNQWFI)was identified from peptide libraries contains recombinant VEGFs and chimeric receptors.Anti-fltl peptide binds to VEGFR1 could specifically prevent the interaction of VEGFR1 and VEGFR1 ligands(like VEGFA,VEGFB and PIGF).Different from other anti-VEGFR1 antibodies,the non-immunogenic anti-fltl peptide can be easily synthesized.But the poor water-solubility limited its application.Hyaluronic acid(HA)is a linear glycosaminoglycan which was firstly found in bovine vitreous body,its structure is composed of repeating N-acetyl-D-glucosamine and D-glucuronic disaccharide units.By reacting with receptors of cells and intercellular substance,HA mediates many procedures such as tumor cell migration,tumor cellular differentiation,formation of embryonic tissue.There are four particular receptors for HA,such as CD44,RHAMM,IVd4 and LEC,during which CD44 is often highly expressed on the surface of tumor tissues.We designed a ES2-AF peptide(IVRRADRAAVPGGGGGGNQWFI),which contained the amino sequences of both ES2 and AF peptide.We look forward to better bio-activity,stability and longer half-life.Further,we chemically modified the new designed peptide with HA which can target to CD44.The main results of this paper are as follows:1.Synthesis and characterization of ES2-AF A peptide of ES2-linker(GGGG)-AF was synthesised by a Fmoe method and purified by HPLC,the purity can reach more than 95%.Then the structure was characterized.2.Synthesis and characterization of HA-ES2-AFTo improve the poor organic solvent solubility of HA,tetrabutyl ammonium salt of HA was synthesized by an ion-exchange method.After activated by BOP reagent,HA-TBA was mixed with ES2-AF and DIPEA to have a coupling reaction.The result from 1H NMR analysis verificated that ES2-AF peptide was successfully modificated by HA,the bio-conjugation is 93.75%,the number of conjugated peptide molecule was 60 per single HA chain.3.Study of ES2-AF and HA-ES2-AF on the bio-activity3.1 In vitro anti-angiogenesis of ES2-AF and HA-ES2-AF(1)A MTT assay was used to measure anti-endothelial cells proliferation activity.Anti-endothelial cells proliferation activity of novel neovascularization peptide ES2-AF was better than ES2 and increased in a dose-dependent manner.As the concentration of ES2-AF was 5 ?g/mL,50 ?g/mL,100 ?g/mL,200 ?g/mL,500 ?/mL,the inhibition on endothelial cells was respectively(11.37%±6.09%),(13.63%±4.92%),(17.61%±5.59%),(16.74%±4.57%),(27.49%±5.56%).After HAylation the HA-ES2-AF exhibited inhibitory efficacy on endothelial cell proliferation.As the peptide concentration was 5?g/mL,50 ?g/mL,100 ?g/mL,200 ?g/mL,500 ?g/mL,the inhibition rate of HA-ES2-AF was(6.67%±4.92%),(12.01%± 8.13%),(16.96%±5.23%),(24.37%±8.97%),(36.40%±7.65%)respectively.(2)We performed a transwell assay to measure anti-endothelial cells migration activity.PBS was performed as blank control.As the peptide concentration was 200?g/mL,the amount of migration cells of AF,ES2 and ES2-AF was respectively(229±11),(254±4),(255±13).Though AF,ES2 and ES2-AF can all inhibit migration of endothelial cell,but there was no significant difference between them.When the peptide concentration was 200 ?g/mL,the amount of migration cells of ES2-AF,HA&ES2-AF,HA-ES2-AF was respectively(255±13),(202±24),(170±7).HA-ES2-AF displayed the best anti-endothelial cells migration activity.(3)A tube formation assay was carried out to evaluate inhibition of ES2-AF,HA-ES2-AF on new vessel formation.PBS was performed as blank control.There was no significant difference during the inhibition on tube formation of AF and ES2,but ES2-AF showed much higher efficacy.As the peptide concentration was 25,100,200?g/mL,the numbers of branches of AF,ES2 and ES2-AF was respectively(3213),(17±6),(19±5).We can see that HA-ES2-AF displayed the best inhibitory in contrast with ES2-AF and mixture of HA and ES2-AF.It is illustrated that chemical modification could significantly improve the ability of inhibiting tube formation.(4)An ELISA assay was used to evaluate the inhibition effect on binding VEGFR1 to VEGF.ES2 and ES2-AF exhibited more efficient inhibitory than AF,as well as the effect of ES2-AF was a little higher than ES2.At the peptide concentration range of 5?g/mL?200 pg/mL,the relative value of HA-ES2-AF was respectively(83.24%±0),(55.01%± 0.07),(51.04%±0.09),(41.25%±0.01),(37.76%0.03).It means that HA-ES2-AF exhibited inhibitory on binding VEGFR1 to VEGF in a dose-dependent manner.At high peptide concentrations the inhibition effect of HA-ES2-AF on binding VEGFR1 to VEGF was excellently higher than that of ES2-AF peptide.3.2 In vivo anti-angiogenesis acitivity of ES2-AF and HA-ES2-AFCAM assay was used to determine anti-angiogenic activity in vivo.To contrast with saline,AF showed no significant inhibition on chicken chorioallantoic membrane in experimental doses.Percentage of new vessels was respectively(32.03%±0.10),(22.84%±0.19),(19.26%±0.03),when the concentration of ES2-AF was 5,25,50?g/mL.Obviously.ES2-AF exhibited better anti-angiogenic activity than ES2 and displayed in a dose-dependent manner.It did not make a great change after the mixture of HA and ES2-AF.After chemical modification,percentage of new vessels of HA-ES2-AF was respectively(9.67%±0.03),(10.12%±0.01),(13.10%±0.04)as the peptide concentration was 5,25.50 ?g/mL.So we can infered that modification enhanced the inhibitory activity of ES2-AF.4.Study on targeting of HA-ES2-AF4.1 Binding ability to CD44 in vitroThe surface plasmon resonance(SPR)assay was performed to evaluate the targeting properties of HA,ES2-AF and HA-ES2-AF to CD44,HA was set to be positive control.pH-scouting was performed to choose a suitable pH which can bind CD44 protein to CM5 chips.The dissociation equilibrium constant(KD)of HA,HA-ES2-AF and ES2-AF was respectively 4.198×10-11,4.779×10-10,3.011×10-4 mol/L.The conjugation of HA to ES2-AF greatly increased its binding ability to CD44 in vitro.4.2 In vivo study of tissue distribution of HA-ES2-AFWe labled ES2-AF and HA-ES2-AF with FITC and administered to tumor-bearing nude mice.The bio-images were recorded by a IVIS kinetics system.By contrast with ES2-AF,HA-ES2-AF showed a longer circulation time in nude mice.The more fluorescence intensity in tumor area indicated that the HA-ES2-AF showed tumor targeting to some extent.5.Pharmacokinetic study of HA-ES2-AFWistar rats were used in the pharmacokinetic study.After analysis of pharmacokinetic parameters of ES2-AF and HA-ES2-AF,half-life of HA-ES2-AF was speculated lengthened to 18.07 h from 2.79 h,MRT was increased to 13.18 h from 4.68 h,AUC0-? was also increased to 10694.70 mg/L·h from 2887.80 mg/L·h.Longer maintenance of high blood levels is excepted to reduce the clinical dose or frequency of administration.Main results and conclusions were as follows:(1)ES2-AF peptide was successfully prepared by a Fmoc method and the purity can reach more than 95%.Its angiogenesis effect was higher than ES2 or AF.(2)Chemical modification of HA to ES2-AF was successfully carried out and the novel conjugate was characterized.Sixty peptides were conjugated to each HA chain.(3)Compared to ES2 and ES2-AF,HA-ES2-AF exhibited higher inhibition effect on endothelial cell proliferation and migration,inhibition effect on VEGF binding to its receptor and inhibition effect on CAM new vessels.(4)After conjugation to HA,the novel conjugate HA-ES2-AF showed high binding ability to CD44 in vitro and targeting to tumor tissue in vivo.(5)In vivo half-life of HA-ES2-AF is 6.48 folds that of ES2-AF.This will be beneficial for reducing the administration dose and decreasing administration frequency. |
PDF Full Text Request