| Chondroitin sulfate(CS)is an endogenous mucopolysaccharide with good biocompatibility.The basic structure is composed of disaccharide units(glucuronic acid and N-acetylgalactosamine)alternately connected.Chondroitin sulfate is widely used as a drug carrier due to its unique physical and chemical properties,biodegradability,and non-toxicity.In addition,chondroitin sulfate can bind to the CD44 receptor that is highly expressed on the surface of tumor cells,which has the potential for receptor-mediated targeted therapy.The peptide ES2-AF is a new type of anti-angiogenesis peptide,which is obtained by connecting the peptide ES2(IVRRADRAAVP)and peptide AF.The function of ES2 is to inhibit the proliferation and migration of endothelial cells.The function of AF is to block the neovascular pathway.There are some shortcomings of peptide ES2-AF such as poor stability and short half-life.Therefore,the improvement of peptide drugs have become a hot research area.Cisplatin(CDDP)is a chemotherapy drug for the treatment of cancer.CDDP can bind to the DNA of tumor cells,inhibit the replication of DNA and induce the apoptosis of cells.Cisplatin has been widely used due to its wide adaptability,strong self-action and synergistic effect with a variety of chemotherapy drugs.However,cisplatin has some disadvantages limited its further applications such as strong toxicity,poor water solubility,and poor targeting.Tumor cells form a unique microenvironment due to their strong metabolism,which is different from normal tissues and is conducive to their own growth and development.The environment has the characteristics of low pH,low oxygen,and high reducing substances.Compared to the normal tissues,tumor tissues contain a large amount of reduced glutathione(GSH).This study connected CS with peptide ES2-AF by reducing sensitive bond,hoping to achieve better targeting effect and controlled release of drugs while improving the poor stability and short half-life of peptide ES2-AF.In addition,in order to achieve the peptide ES2-AF with chemotherapy drugs cisplatin(CDDP)synergistic treatment,CDDP is loaded with the CS-Cys-ES2-AF.When the drug arrives at the tumor site,the reduction-sensitive bond is fractured and the nanoparticles are disintegrated due to the unique tumor microenvironment.Subsequently,the cisplatin and peptide ES2-AF are released.Therefore,the drug could inhibit endothelial cell proliferation and kill tumor cells,achieving the effect of tumor inhibition.The major results of this graduation paper are as follows:(1)Synthesis and characterization of peptide ES2-AF and glycol-peptide CS-Cys-ES2-AFThe antiangiogenic peptide ES2-AF was synthesized by solid phase synthesis and purified in high performance liquid phase.The purity was above 95%.CS-CysES2-AF,a reduction-sensitive polymer,was synthesized by a two-step amidation reaction using cystamine as the link arm.The CS-Cys-ES2-AF conjugation was successfully synthesized and characterized by 1H NMR.The 1H NMR showed that 10 peptides were linked to each CS chain.After the chemical modification using CS,the particle size of CS-Cys-ES2-AF was 218.13±4.72 nm.The average zeta potentials of CS-Cys-ES2-AF was-15.20±3.50 mV.Meanwhile,CS-Cys-ES2-AF presented a spherical structure in water.(2)Bioactivity evaluation of ES2-AF and CS-Cys-ES2-AF in vitroThe effects of ES2-AF and CS-Cys-ES2-AF on endothelial cell proliferation were detected by CCK8 kit.The inhibition rates of ES2-AF and CS-Cys-ES2-AF at 5?g/mL,25 ?g/mL,50 ?g/mL,100 ?g/mL,200?g/mL,500 ?g/mL was(9.58%±1.48%),(12.19%±2.01%),(13.05%±74%),(22.63%±1.62%),(24.46%±3.31%)and(29.12%±4.66%);and(11.76%±3.35%),(15.59%±3.51%),(18.20%±3.77%),(24.07%±1.87%),(27.21%±1.81%)and(38.21%±2.82%),respectively.When the ES2-AF concentration reached 500 ?g/mL,the inhibitory effect of CS-Cys-ES2-AF on endothelial cell proliferation was significantly better than the ES2-AF.According to the above results,ES2-AF and CS-Cys-ES2-AF displayed an inhibitory effect on cell proliferation in a dose-dependent manner.The migration effect of ES2-AF and CS-Cys-ES2-AF on endothelial cells was studied by scratch method.The results showed that the migration of endothelial cells was inhibited by concentration dependence in all groups.When the concentration of ES2-AF was 100 ?g/mL,200 ?g/mL and 500 ?g/mL,the migration rates of endothelial cells were(53.90%±2.82%),(42.02%±3.05%)and(34.37%±1.09%).After CS modification,the migration rates of endothelial cells were(35.73%±4.00%),(2728%±3.92%)and(25.70%±0.88%).Therefore,the inhibitory effect of CS-Cys-ES2-AF on endothelial cell migration was significantly better than the ES2-AF.The tube formation assay was used to evaluate the effect of ES2-AF and CS-Cys-ES2-AF on endothelial cell tubulogenesis.The number of branches of ES2-AF and CS-Cys-ES2-AF at 100 ?g/mL,200 ?g/mL,500 ?g/mL was(152.00±26.09),(143.00±4.84)and(119.11±9.51);(110.11±8.50),(103.89±8.01)and(90.00±7.00),respectively.When the ES2-AF concentration reached 200 ?g/mL and 500 ?g/mL,the inhibitory effect of CS-Cys-ES2-AF on endothelial cell tube formation was significantly better than the ES2-AF.(3)Preparation and characterization of cisplatin(CDDP)-loaded CS-Cys-ES2-AFUsing CS-Cys-ES2-AF as the carrier material,cisplatin was loaded by coordination with the carboxyl group on the CS chain,and the conjugate(CDDP&CS-Cys-ES2-AF)was prepared successfully.Subsequently,the drug loading(DL)and encapsulation rate(EE)were determined.DL was 4.41%±0.36%,and EE was 33.05%±0.95%.The particle size,Zeta potential and particle size morphology of CDDP&CS-Cys-ES2-AF under the optimal prescription were determined.The particle size of CDDP&CS-Cys-ES2-AF was 231.07±15.51 nm.The Zeta potential of CDDP&CS-Cys-ES2-AF was-14.13±1.34 mV.(4)Bioactivity evaluation of CDDP&CS-Cys-ES2-AF in vitroCCK8 kit was used to determine the effect of CDDP&CS-Cys-ES2-AF on endothelial cells proliferation and melanoma cells proliferation.The inhibition rates of CDDP&CS-Cys-ES2-AF on endothelial cells and melanoma cells proliferation at 5?g/mL,25 ?g/mL,50?g/mL,100?g/mL,200?g/mL,500 ?g/mL were(12.52%±2.75%),(16.06%±1.58%),(20.65%±3.20%),(30.20%±3.31%),(32.22%±0.96%)and(45.19%±2.97%);(10.65%±2.31%),(16.52%±1.10%),(19.84%±1.44%),(36.45%±3.67%),(42.07%±3.38%)and(74.73%±2.42%),respectively.Compared with the inhibitory effects of different concentrations of CS-Cys-ES2-AF on endothelial cell proliferation,it was found that when the concentration(as ES2-AF concentration)is 200?g/mL and 500 ?g/mL,the inhibition effect of CDDP&CS-Cys-ES2-AF on endothelial cell proliferation is significantly stronger than CS-Cys-ES2-AF.Therefore,CDDP&CS-Cys-ES2-AF significantly inhibited the proliferation of endothelial cells and melanoma cells,presenting a concentration-dependent inhibition.The inhibitory effect of CDDP&CS-Cys-ES2-AF on endothelial cells and melanoma cells migration was investigated using the scratch test.When the CDDP&CS-Cys-ES2-AF concentration reached 100 ?g/mL,200?g/mL and 500?g/mL,the endothelial cells and melanoma cells migration rate were(35.20%±0.53%),(33.23%±1.26%)and(22.73%±4.16%);(34.02%±1.44%),(19.18%±2.27%)and(15.47%±0.50%),respectively.Compared with the inhibitory effect of different concentrations of CS-Cys-ES2-AF on endothelial cell migration,no significant difference was found.According to the above results,CDDP&CS-Cys-ES2-AF showed concentration-dependent inhibition of endothelial cells and melanoma cells migration.The tube formation assay was used to evaluate the effect of CDDP&CS-Cys-ES2-AF on endothelial cell tubulogenesis.When the CDDP&CS-Cys-ES2-AF concentration reached 100 ?g/mL,200 ?g/mL and 500?g/mL,the number of branches was(113.00±1.73),(95.67±7.64)and(89.00±3.61).CDDP&CS-Cys-ES2-AF displayed an inhibitory effect on tube formation in a dose-dependent manner.Meanwhile,compared with the inhibitory effect of different concentrations of CS-Cys-ES2-AF on the formation of endothelial cells,no significant difference was found.(5)Targeting research of CS-Cys-ES2-AF and CDDP&CS-Cys-ES2-AFThe targeting effect of CS-Cys-ES2-AF and CDDP&CS-Cys-ES2-AF was studied by using surface plasmon resonance(SPR)method.The equilibrium dissociation constants(KD)of CS-Cys-ES2-AF and CDDP&CS-Cys-ES2-AF were 2.09 × 10-8 and 1.18 × 10-6 respectively,while ES2-AF had no obvious binding to CD44.The binding ability to CD44 protein was enhanced after the ES2-AF was modified by CS.In this study,the CS-Cys-ES2-AF and CDDP&CS-Cys-ES2-AF were successfully prepared.Many assays in vitro were carried out,we found that the two drugs had significant bioactivity and strong targeting in vitro.Meanwhile,this study also realized the synergistic treatment of anti-angiogenesis drugs and chemotherapy drugs,laying a foundation for the development of anti-tumor drug. |
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