| ES2-AF(IVRRADRAAVPGGGGGGNQWFI)is a polypeptide with a molecular weight of 2917.51 kDa that inhibits the formation of new blood vessels.It could inhibit the proliferation and migration of endothelial cells and could specifically prevent the interaction of VEGFR1 with a series of ligands such as VEGFA,VEGFB,PIGF.Studies have shown that ES2-AF had better inhibitory effect on endothelial cell proliferation in MTT assay.In addition,ES2-AF showed significant inhibition of endothelial cell tube formation assay in vitro and neovascularization in CAM assay in vivo,respectively.However,the poor stability,poor targeting and short half-life of ES2-AF have limited wide research and application.Hyaluronic acid(HA)is a negatively charged,non-sulfated linear glycosaminoglycan.And it is composed of repeating β-N-acetylglucosamine and D-glucuronic acid units.Due to biocompatibility,biodegradability,non-immunogenicity and non-toxicity,HA has been widely used as a carrier for drug delivery.In addition,HA can bind to cell membrane surface receptors such as CD44,RHAMM,IVd4 and LEC.What’s more,CD44 receptor is highly expressed on the surface of many tumor tissue.Therefore,to obtain better bio-activity,stability and longer half-life,we designed a new peptide called ES2-AF(IVRRADRAAVPGGGGGGNQWFI).At the same time,we used HA as a modified to chemically modify ES2-AF and it was expected that conjugate would have preferable solubility,stronger targeting,longer half-life and better anti-angiogenesis effects in vivo.The main results of this paper are as follows:1.Synthesis and characterization of ES2-AF and HA-ES2-AF conjugate(1)The novel anti-angiogenic peptide ES2-linker(GGGG)-AF was synthesized by Fmoc solid phase synthesis and purified by HPLC.And the purity was more than 95%,and the structure was confirmed by 1HNRM.To improve the poor organic solvent soubility of HA,the tetrabutyl ammonium hydroxide salt(HA-TBA)of hyaluronic acid was first prepared by ion exchange method.After activated the carboxyl groups by BOP reagent,HA-TBA was mixed with ES2-AF and DIPEA to synthesize HA-ES2-AF conjugate.The HA-ES2-AF was confirmed by ’HNRM and it was determined that 75 peptides were linked to each HA chain.(2)Particle size,zeta potential and transmission electron microscopyexperiments showed that the particle sizes of ES2-AF and HA-ES2-AF were 144.13±4.36 nm and 245.4±16.59 nm,respectively.And zeta potential of ES2-AF and HA-ES2-AF was-0.49±0.013 mV and-17.7±0.2 mV,respectively.At the same time,HA-ES2-AF could form a nano-like spherical structure in the aqueous phase.2.Study on the effect of HA-ES2-AF conjugate on apoptosis and cell cycle(1)The effects of ES2-AF,HA-ES2-AF and HA&ES2-AF on the proliferation of endothelial cells were examined by CCK-8 kit.The results showed that the inhibition rates of ES2-AF,HA-ES2-AF and HA&ES2-AF at 5μg/mL,25μg/mL,75μg/mL,150μg/mL,250μg/mL,400μg/mL were(6.35%±2.70),(10.90%±2.76%),(14.62%±1.65%),(22.06%±5.57%),(28.27%±4.65%)and(39.16%±4.86%);(13.78%±1.17%)(16.12%±2.03%),(26.17%±1.63%),(31.95%±2.63%),(40.21%±2.87%)and(45.48%±2.09%);(5.56%±2.95%,(5.96)%±2.10%),(16.33%±4.28%),(20.68%±2.99%),(32.33%±8.28%),and(31,05%±0.46%).ES2-AF,HA-ES2-AF and HA&ES2-AF significantly inhibited endothelial cell proliferation in a dose-dependent manner.And compared with ES2-AF and HA&ES2-AF,HA-ES2-AF showed the best inhibition of endothelial cell proliferation.(2)ES2-AF-FTIC,HA-ES2-AF-FTIC,HA&HA-ES2-AF-FTIC were incubated with endothelial cells,respectively.Then the positive cell rate was measured using a flow cytometer.The results showed that the affinity rates of ES2-AF at concentrations of 0.5μg/mL,1μg/mL,2μg/mL,5μg/mL,10μg/mL,and 25μg/mL were(4.55%±0.61%),(22,79%±5.20%),(82.99%±3.99%),(99.61%±0.62%),(99.63%±0.06%)and(99.96%±0.01%),respectively.The positive cell rate increased in a dose-dependent manner.(3)The apoptosis of endothelial cells treated with different concentrations of HA-ES2-AF,ES2-AF and HA&ES2-AF was examined by Annexin-V method.The results showed that the apoptotic rates of ES2-AF,HA-ES2-AF and HA&ES2-AF at concentrations of 5μg/mL,25 μg/mL,50 μg/mL,and 100μg/mL were(5.44%±1.64%),(7.53±0.74%),(28.86%±1.49%),(36.64%±0.74%),(8.28%±.44%),(15.62%±0.94%),(26.85%±2.42%)(32.40%±1.61%),(4.47%±1.00%),(6.45%±1.15%),(9.36%±1.57%)and(14.8%±3.46%),respectively.And within a certain concentration range,the apoptosis rates of the cells increased when the concentrations of the drugs increased.Moreover,HA-ES2-AF conjugate were still able to significantly induce endothelial cell apoptosis after HA modified ES2-AF peptide.HA-ES2-AF retained the original biological activity.(4)The effect of HA-ES2-AF,ES2-AF on endothelial cell cycle was examined using a PI kit.The results showed that the percentage of G1,S and G2 phases of endothelial cells in the control group were(54.03%±2.77%),(35.38%±1.98%)and(10.59%±1.11%),respectively.The percentage of G1 phase,S phase and G2 phase of endothelial cells in ES2-AF group were(64.30%±7.01%),(26.22%±3.02%)and(9.48%±4.12%),respectively.And the percentage of G1 phase,S phase and G2 phase in HA-ES2-AF were(66.30%±10.04%),(25.92%±8.87%),and(7.64%±1.34%),respectively.Both ES2-AF and HA-ES2-AF could increase cell accumulation in G1 phase and decrease the proportion of cells in S phase and G2 phase.3.Stability of HA-ES2-AF conjugate(1)Study on the thermal stability of HA-ES2-AF conjugateAfter incubation for the same time at room temperature(25℃)and body temperature(37℃),the retention activity of the HA-ES2-AF and ES2-AF were examined using CCK-8.The results showed that the ES2-AF retention activity was less than 50%after incubation at 25℃ for 90 min,while the retention activity of HA-ES2-AF was still higher than 60%.After incubated at 37℃ for 90 min,the retention activities of ES2-AF and HA-ES2-AF were(40.49%±6.30%)and(53.62%±2.30%),respectively.The thermal stability of HA-ES2-AF was significantly improved compared to ES2-AF.(2)Study on the long-term stability of HA-ES2-AF conjugateThe activity ES2-AF and HA-ES2-AF was significantly reduced with multiple freeze and thaw cycles at-80℃ for one month.The results showed that the retention activity of ES2-AF and HA-ES2-AF were(46%±3.90%)and(98.55%±8.63%)for 1 day.For 31 days,the retention activity of ES2-AF was only(30.68%±4.33%),while the retention activity of the HA-ES2-AF was still(48.58%±2.13%).Compared with ES2-AF,the modified HA-ES2-AF showed higher retention activity.The results indicated that HA modification improved the long-term storage stability of ES2-AF(3)Study on the pH stability of HA-ES2-AF conjugateThe ES2-AF and HA-ES2-AF conjugate were dissolved separately in phosphate buffer with different pH values(3.5-9).The inhibitory effects on the EAHY926 cells were examined by the CCK-8 kit to evaluate the retained biological activity.The results showed that he percentages of retained activity of ES2-AF and HA-ES2-AF at pH 3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,and 9 were(6.54%± 2.10%),(16.00%±4.36%),(21.47%±1.43%),(23.71%±2.34%),(25.81%±3.91%),(27.44%±1.24%),(28.46%±1.72%),(31.22%)±2.39%),(35.31%±0.81%),(20.37%±2.97%),(14.77%±2.11%);(17.98%±2.60%),(25.69%±2.53%),(28.77%±0.48)%),(28.22%±5.61%),(32.25%±1.95%),(34.78%±2.56%),(36.36%±1.10%),(37.23%±5.72%),(36.39%±4.63%)(29.37%±4.7%),(20.07%± 4.00%),respectively.ES2-AF showed the highest activity at pH = 5.0,while the HA-ES2-AF conjugate showed higher biological activity in the range of pH 4.0-7.5.4.Study on targeting of HA-ES2-AF conjugate in vivoIn vivo imaging techniques were used to study the targeting ability of ES2-AF,HA-ES2-AF,and HA&HA-ES2-AF to tumor tissues in vivo.The results showed that ES2-AF-FITC was mainly distributed in the vicinity of cervical lymph nodesafter the tail vein injection for 2 h.However,HA-ES2-AF-FITC was mainly distributed in the abdomen and cervical lymph nodes,and weak fluorescence was observed in the tumor site.After 4 hours of administration,HA-ES2-AF-FITC had the strongest fluorescence intensity in tumor tissues compared with the other two groups.ES2-AF-FITC had a small amount of fluorescent signal at the tumor and bladder.After intravenous administration for 8 h,ES2-AF-FITC was concentrated in the kidney region,indicating that most of the ES2-AF-FITC has been metabolized.At this time,the fluorescence signal intensity and area of the HA-ES2-AF-FITC at the tumor site were significantly enhanced.After intravenous administration for 12 h,the fluorescence signal intensity and area of HA-ES2-AF-FITC and HA&HA-ES2-AF-FITC were significantly reduced in the tumor site,and a small amount of fluorescence was found in the bladder region,indicating that a small amount of metabolism began in both drugs.After intravenous administration 24 h,5 HA-ES2-AF-FITC was still present in the tumor and cervical lymph nodes,and there was also a large area of fluorescence in the bladder,indicating that the metabolic drug remained.HA-ES2-AF-FITC had a lower metabolic rate and a longer half-life in plasma and is less susceptible to hydrolysis than ES2-AF-FITC in nude mice.5.The anti-tumor effcet of HA-ES2-AF conjugate in vivoCD44 is a transmembrane glycoprotein that is widely expressed on the surface of various cells.It can participate in cell adhesion,bind to extracellular matrix or ligand and transmit information.Hyaluronic acid(HA)is the main ligand of CD44.It has been used as a targeting factor to target the surface of tumor cells that highly express CD44 receptor to improve the targeting of tumor drugs by active targeting.In this study,we used B16F10 and HepG2 tumor-bearing nude mice models to study the anti-tumor effects of HA-ES2-AF and ES2-AF in vivo.(1)The anti-tumor effect of HA-ES2-AF was assessed in vivo using B16F10 tumor-bearing nude mice.When the tumor volume reached 100-200 mm3,the tumor-bearing mice were randomly divided into 3 groups(n=6 for every group).After 14 days of continuous administration,mice were sacrificed and the tumor tissues were excised,weighed and recorded.Compared with the saline control group,the tumors of the ES2-AF-treated group and HA-ES2-AF-treated group were severely grew slowly.What s more,the mice that received HA-ES2-AF showed a significant superior growth inhibition of tumor volume compared to ES2-AF group.After the mice were sacrificed and the tumors tissue were weighed,the inhibition rate of HA-ES2-AF was 51.15%compared with that of the saline group,while the inhibition rate of the ES2-AF group was only 30.32%.The expressions of VEGF,MVD in tumor tissues of tumor-bearing mice were evaluated by immunohistochemical staining.The results showed that compared with the control group and ES2-AF group,the expression of VEGF and MVD significantly decreased in HA-ES2-AF group.(2)The anti-tumor effect of HA-ES2-AF was studied in vivo using tumor-bearing nude mice.When the tumor volume reached 100-200 mm3,the tumor-bearing mice were randomly divided into 3 groups(n=6 for every group).After 14 days of continuous administration,mice were sacrificed and the tumor tissues were excised.weighed and recorded.Compared with the saline control group,the tumors of the ES2-AF-treated group and HA-ES2-AF-treated group were severely grew slowly.What’s more,the mice that received HA-ES2-AF showed a significant superior growth inhibition of tumor volume compared to ES2-AF group.After the mice were sacrificed and the tumors tissue were weighed,the inhibition rate of HA-ES2-AF was 53.88%compared with that of the saline group,while the inhibition rate of the ES2-AF group was only 18.18%.The expressions of VEGF,MVD,Bcl-2,caspase-3,caspase-9 and cyto c in tumor tissues of tumor-bearing mice were evaluated by immunohistochemical staining.The results showed that compared with the control group and ES2-AF group,the expression of VEGF,MVD and Bcl-2 significantly decreased in HA-ES2-AF group and the expression of cyto c,caspase-3 and caspase-9 significantly increased in HA-ES2-AF group. |
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