| Chondroitin Sulfate(CS)is a type of glycosaminoglycan composed by alternatively of D-glucuronic acid(GlcA)and N-Acetyl-D-galactosarmine(GalNac)residues,which was found in cartilage tissue of many kinds of animals.With excellent biocompatibility and biodegradability,the natural anionic glycosaminoglycan CS are widely used to conjugated with other drugs so as to build polymer-drug conjugates,which the conjugate shows a better bio-activity,better stability,lower cytotoxicity and longer half-life.Meanwhile,CS is the ligand of CD44 receptor,which has the potential on tumor active targeting with CD44 protein.Endostatin(ES)is an endogenous anti-angiogenesis drug,a peptide drug with 20 KDa molecular-weight,which was purified and isolated from the murine hemangioendothelioma.ES plays an anti-tumor activity by inhibitory the formation of new blood vessels around the tumor tissue,thereby cutting off the nutrition supply of the tumor and the metabolism pathways of waste.ES2(IVRRADRAAVP)is a short fragment found from ES,which shows about three times anti-angiogenesis activity and anti-tumor activity in vitro than that of ES.Anti-Fltl(AF)peptide is another anti-angiogenesis peptide that identified from peptide libraries.The interaction of VEGF-R1 ligands and VEGFcould be binded and prevented specifically by the AF peptide.We designed a new type of anti-angiogenesis peptide ES2-AF by solid phase synthesis technique,which contained both of ES2 and AF peptide with a linker GGGG.However,the performance of peptide ES2-AF was limited to the shorter half-life in plasma and frequent administration,which makes the patients’ compliance poor and the therapy cost high.We chemically modified the new peptide ES2-AF with CS,which can increase the stability in plasma and the targeting effect to the protein CD44.The major results of this graduation paper are as follows:1.Synthesis route and characterization assays of peptide ES2-AF and glycol-peptide CS-ES2-AF.The new anti-angiogenesis peptide of ES2-linker(GGGG)-AF was prepared and synthesized by solid phase synthesis technique and purified by HPLC,the purity can reach about 95.14%.Then the molecular weight was characterized by mass spectrum and the result corresponds with the theoretical value.Tetrabutyl ammonium salt of CS was prepared by an ion-exchange method in order to improve the poor organic solvent solubility of CS.After activated by BOP reagent,it was found that a coupling reaction occurs after CS-TBA mixed with peptide ES2-AF and DIPEA.The CS-ES2-AF conjugation was successfully synthesized and characterized by 1H-NMR.From the result of 1H-NMR analysist,there were 2 peptide molecule conjugate to the per single CS chain.2.In-vivo and iv-vitro bioactivity study of ES2-AF and CS-ES2-AF(1)A MTT assays was used to study the inhibition of ES2-AF and CS-ES2-AF on endothelial cell proliferation in vitro.The inhibition on endothelial cells are increased dose-dependently.When the peptide concentration reached 200 μg/mL,the inhibition rate of CS-ES2-AF was significantly higher than ES2-AF.(2)The inhibitory effect of CS-ES2-AF on endothelial cell migration was studied using the scratch test.The experimental results showed that ES2-AF,the mixture of CS and ES2-AF,CS-ES2-AF can inhibit the migration of endothelial cells.When the concentration ranged from 50 pg/mL to 200 μg/mL,the conjugation CS-ES2-AF displayed the best ability.The inhibition was carried out in a concentration dependent manner.(3)In vitro angiogenesis assay was carried out to evaluate inhibition of the peptide ES2-AF,the mixture of CS and ES2-AF,the glycol-peptide CS-ES2-AF on new vessel formation.We can see the CS-ES2-AF displayed the best activity in contrast with ES2-AF and the mixture at the same concentration.(4)The CAM assays was applied to determine the in vivo anti-angiogenesis activity.There was no difference between ES2-AF groups and CS-ES2-AF groups.It was not 50 μg/mL the concentration of ES2-AF until that CS-ES2-AF exhibited better obviously anti-angiogenesis activity than ES2-AF.3.Study on targeting of CS-ES2-AF(1)An ELISA ways was determined to evaluate the inhibitory effects on binding VEGF-R1 to VEGF.All experimental groups had the ability to inhibit the binding of VEGF to the VEGF-R1 receptor in a concentration-dependent manner.Compared with ES2-AF group,the same concentration of CS&ES2-AF and CS-ES2-AF significantly inhibited the binding of VEGF to its receptor,and the difference between CS-ES2-AF and ES2-AF was statistically significant.(2)The surface plasmon resonance(SPR)assay was applied to determine the targeting effect of ES2-AF and the CS-ES2-AF to CD44 protein.First,pH-scouting was indeed to perform in order to choose a suitable pH which could bind CD44 protein to CM5 chips.When the pH is 3.8,response unit of CD44 protein bond to CM5 chips reaches its maximum,and the bonding amount of protein is 394.8RU under this condition.The dissociation equilibrium constant(KD)of glycol-peptide and the peptide ES2-AF was respectively 9.895×10-9 and 3.011×10-4 mol/L.The glycol-peptide CS-ES2-AF enhanced its binding ability to CD44 protein in vitro.(3)In-vivo bio-imaging study the distribution of FITC-labeled two drugs ES2-AF-FITC and CS-ES2-AF-FITC in tumor-bearing nude mice.First the two drugs are labeled with FITC and administrated to tumor-bearing nude mice by tail intravenous injection,the photograph was taken by living image system.By contrast the distribution process of ES2-AF with CS-ES2-AF,the glycol-peptide showed a longer circulation and tumor-targeting.4.Pharmacokinetic study of ES2-AF and CS-ES2-AF.The method for determination the concentration of ES2-AF and CS-ES2-AF in plasma by fluorescence spectrophotometry was established well.Blab/c mice were administrated by intraperitoneal injection with 20mg peptide ES2-AF per kilogram weight.After analysis of pharmacokinetic parameters of ES2-AF and CS-ES2-AF by the software DAS2.0,the parameters of glycol-peptide group are improved significantly,which means CS-ES2-AF increase the plasma stability of ES2-AF.In this paper,CS-ES2-AF was synthesized successfully and characterized by 1H-NMR.The conjugate CS-ES2-AF shows better bio-activity,active targeting effect,as well as longer circulation in plasma.The peptide ES2-AF modified CS can significantly enhance the anti-angiogenesis activity and reduce the times of administration,which has excellent medical application prospects. |
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