多靶点抗血管新生肽ES2-AF的透明质酸化修饰及修饰物的生物活性、靶向性及药代动力学研究

发布时间：2018-06-05 16:16

本文选题：ES2-AF + 化学修饰　；参考：《山东大学》2017年硕士论文

【摘要】：内皮抑素(Endostatin,ES)是一个分子量为20kDa的多肽,最早是从小鼠血管内皮瘤中分离出来的,成为首个被发现的内源性抑制血管新生的细胞外基质片段,同时也是被研究最多的内源性血管新生抑制剂。ES中有一段可高效抑制内皮细胞增殖、迁移的序列ES2(60-70,IVRRADRAAVP),其体内抑制新生血管生成的活性约为完整ES序列的3倍。Anti-fltl肽(GNQWFI,AF)是从位置扫描合成肽组合文库中通过一个包含纯化的重组VEGF及嵌合受体的扫描系统遴选出来的一种VEGFR1选择性的六肽。AF肽可特异地与VEGFR1结合,从而抑制VEGFR1与一系列配体,如VEGFA、VEGFB、PIGF等的相互作用。与其他VEGFR1的单克隆抗体或RNA拮抗剂不同,AF肽是一种能够通过较低生产成本简易合成的非免疫原性的六肽,这些优点决定了其适合在临床应用,但是存在水溶性较差的缺点。透明质酸(Hyaluronic acid,HA)是一种从牛眼玻璃体中发现的、带负电荷的、非硫酸化的线性糖胺聚糖,重复二糖单元为β-N-乙酰氨基葡萄糖和D-葡萄糖醛酸,其生物可降解和高载药性的特点使其广泛应用于医药领域。HA可通过与细胞及细胞间质的受体作用调节肿瘤细胞侵袭、肿瘤细胞的分化、成纤维细胞中病毒的转化、胚胎组织形成等过程。有四种HA特定受体存在于细胞膜表面——CD44、RHAMM(Receptor for HA-Mediated Motility)、IVd4 和 LEC,这之中在肿瘤部位表达较多的是CD44。AF和ES2在活性方面既有相似又有不同,如果将这两个肽的序列融合在同一个肽中,有望获得更好的生物活性、更好的稳定性和更长的半衰期。为此,我们设计了序列为IVRRADRAAVPGGGGGGNQWFI的肽链,将其命名为ES2-AF,并利用可以靶向肿瘤部位高表达的CD44受体的HA对其进行化学修饰,期待修饰后的多肽溶解性更好、靶向性更强、半衰期更长,从而更好的在体内更好的发挥抗新生血管生成作用。本课题研究内容和取得的主要成果如下:1..ES2-AF的合成与表征采用Fmoc固相合成方法合成了一种ES2-linker(GGGG)-AF肽,由高效液相色谱进行纯化,纯度均可达95%以上,并进行了结构的确证。2.HA-ES2-AF的合成与表征由于HA溶于水但不溶于有机溶剂,为了下一步的偶联反应,首先用四丁基氢氧化铵对其进行离子交换,形成可溶于DMSO的HA-TBA结合物。HA-TBA分子经卡特缩合剂(BOP)活化羧基后,与ES2-AF、DIPEA(N,N-二异丙基乙胺)反应得到HA-ES2-AF结合物。经1H NMR核磁结果分析表明ES2-AF成功被HA修饰,连接率93.75%,平均每HA链上连结60个ES2-AF肽。3.ES2-AF与HA-ES2-AF的生物活性研究3.1 ES2-AF及HA-ES2-AF的体外抗新生血管生成活性研究(1)采用MTT法评价了抗内皮细胞增殖作用,ES2与ES2-AF在体外均具有抑制内皮细胞增殖的作用,并呈现随浓度增加的趋势。当ES2-AF浓度分别为5μg/mL、50 μg/mL、1000μg/mL、2000μg/mL、500μg/mL 时,其对内皮细胞增殖的抑制率分别为(11.37%±6.09%)、(13.63%±4.92%)、(17.61%±5.59%)、(16.74%±4.57%)、(27.49%士5.56%)。经 HA 修饰后的结合物 HA-ES2-AF 也在体外表现出了一定的抑制内皮细胞增殖的作用,当其肽浓度分别为表5 μg/mL、50μg/mL、100μg/mL、200μg/mL、5000μg/mL时,其对内皮细胞增殖的抑制率分别为(6.67%±4.92%)、(12.01%±8.13%)、(16.96%±5.23%)、(24.37%±8.97%)、(36.40%±7.65%)。(2)采用Transwell小室法测定了抑制内皮细胞转移的作用。以PBS作为空白对照,当药物肽浓度均为200 μg/mL时,AF、ES2与ES2-AF三组发生细胞转移的个数分别为(229±11)、(254±4)、(255±13)。AF、ES2 与 ES2-AF 三组药物对于内皮细胞转移都有一定的抑制作用,但是差异不大,说明三组实验药物对于肿瘤转移的抑制作用并没有很大差异。当肽浓度均为200μg/mL时,ES2-AF、HAES2-AF混合物、HA-ES2-AF结合物三组发生细胞转移的个数分别为(255±13)、(202±24)、(170±7),HA-ES2-AF 表现出了最强的抑制内皮细胞转移的作用,具体机制仍有待进一步研究。(3)利用管腔形成实验研究了 ES2-AF、HA-ES2-AF等对内皮细胞EAhy926形成血管的影响。用PBS作为空白对照,AF与ES2在各浓度时抑制体外内皮细胞管腔形成的作用相差不大,但ES2-AF对于内皮细胞管腔的形成的抑制作用明显高于AF和ES2。在肽浓度均为25 μg/mL、100 μg/mL、200 μg/mL时,AF组的节点数分别为(99± 19)、(75± 17)、(63± 12),ES2组的节点数分别为(67± 14)、(72±3)、(61±5),HA-ES2-AF 组的节点数分别为(32±3)、(17±6)、(19±5)。由此可见,HA-ES2-AF对内皮细胞管腔形成的抑制作用在所有给药组中是最强的,说明利用HA对多肽的化学修饰提高了其抑制内皮细胞官腔形成的能力。(4)采用ELISA方法根据抗原抗体结合原理测定了 AF、ES2、ES2-AF及HA-ES2-AF对于VEGF及其受体VEGFR1(Flt-1)结合的抑制能力。ES2与ES2-AF均表现出比AF更强的抑制能力,其中ES2-AF的抑制作用略强于ES2。HA-ES2-AF在 5 μg/mL～200 μg/mL 范围内的相对值分别为(83.24%±0%)、(55.01%±0.07%)、(51.04%士0.09%)、(41.25%±0.01%)、(37.76%±0.03%),说明 HA-ES2-AF对VEGF及其受体VEGFR1(Flt-1)结合的抑制能力呈浓度依赖性提高。较高浓度时HA-ES2-AF抑制VEGF及其受体VEGFR1(Flt-1)结合的能力明显高于ES2-AF肽。3.2 ES2-AF及HA-ES2-AF的体内抗新生血管生成活性研究在鸡胚绒毛尿囊膜(CAM)实验中,与生理盐水组进行相比,AF在实验剂量下对CAM血管生成没有明显抑制作用;ES2-AF组在5 μg/mL、25 μg/mL、50 μg/mL时新生血管面积百分比分别为(32.03%±0.10%)、(22.84%±0.19%)、(19.26%±0.03%),表现出比ES2更高的体内抗血管生成的生物活性并呈现剂量相关。HA与ES2-AF混合物中HA的加入并没有对ES2-AF在动物体内的抑制CAM血管生成产生较大影响。而HA-ES2-AF组在5 μg/mL、25 μg吨/mL、50 μg/mL时新生血管面积百分比分别为(9.67%±0.03%)、(10.12%±0.01%)、(13.10%±0.04%),与ES2-AF及混合物组相比抑制CAM血管生成的活性明显提高。4.HA-ES2-AF的靶向能力研究4.1体外与CD44受体结合能力研究通过表面等离子共振(SPR)技术研究了 HA-ES2-AF在体外对肿瘤细胞表面受体CD44的结合能力。以HA为阳性对照,研究了 HA-ES2-AF、ES2-AF与CD44蛋白之间的相互作用关系。筛选合适的pH将CD44蛋白锚定于CM5芯片表面,使样品流过芯片,通过Biacore T200的分析软件得出以下三种样品与CD44蛋白结合的 KD值:HA 为 4.198×10-11 mol/L,HA-ES2-AF 为 4.779×10-10mol/L,ES2-AF为3.011×10-4mol/L,即样品与CD44结合能力的排列顺序为:HAHA-ES2-AF》ES2-AF,经过HA的修饰HA-ES2-AF结合物对细胞表面受体CD44的结合能力较修饰前的肽有十分显著的提升,具有潜在的体内肿瘤部位靶向能力。4.2体内组织分布研究通过活体成像技术研究了 HA-ES2-AF在体内组织分布情况。将ES2-AF与HA-ES2-AF用FITC标记,分别给药于荷黑色素瘤裸鼠,采用小动物活体成像仪进行拍照记录。通过比较发现HA-ES2-AF-FTIC在裸鼠体内分布代谢时间较长,即HA-ES2-AF-FITC的体内血浆半衰期较长,证明了化学修饰有助于增强多肽的稳定性,这与后面药代动力学实验的结果一致;分布于肿瘤部位的药物增多,表明经HA修饰的ES2-AF-FITC在荷瘤裸鼠体内对于肿瘤部位有一定的靶向性,证明了糖基化修饰有助于提高多肽的靶向分布能力,这与SPR实验的结果相符合。5.HA-ES2-AF的药代动力学研究本实验选用Wistar大鼠作为体内药物代谢动力学的模型,通过比较药代动力学参数发现,与ES2-AF相比,HA-ES2-AF在大鼠体内的循环时间明显延长,半衰期由2.79 h延长至18.07 h,平均滞留时间MRT由4.68 h增加至13.18 h,药时曲线下面积AUC0-∞也由2887.80 mg/L·h增大至10694.70 mg/L·h,血浆清除率CL也有所下降。较长时间的维持高血药水平有助于减少给药剂量或降低给药频率。本研究取得的成果和结论主要有:(1)成功固相合成了纯度在95%以上的ES2-AF肽,其体内外抗新生血管生成活性高于ES2或AF。(2)成功完成HA对ES2-AF的化学修饰及结构表征,平均每HA链上连结60 个 ES2-AF肽。(3)HA-ES2-AF表现出比ES2-AF和HAES2-AF混合物更强的抑制内皮细胞增殖与迁移的能力、更强的抑制VEGF与受体结合的能力,以及更强的抑制CAM新生血管生成的能力。(4)利用SPR和活体成像技术发现,HA-ES2-AF比ES2-AF具有更强的CD44结合能力,并表现出一定的体内肿瘤部位靶向性和更长的体内分布代谢时间。(5)HA-ES2-AF的体内半衰期是ES2-AF的6.48倍,有助于减少给药剂量或降低给药频率。
[Abstract]:Endostatin (ES) is a polypeptide of molecular weight of 20kDa. It was first isolated from the mouse endothelioma and became the first endogenous extracellular matrix fragment to inhibit angiogenesis, and it was also the most studied endogenous angiogenesis inhibitor,.ES, which could effectively inhibit the proliferation of endothelial cells. The migration sequence ES2 (60-70, IVRRADRAAVP), the 3 times.Anti-fltl peptide (GNQWFI, AF) that inhibits the angiogenesis activity of the neovascularization in the complete ES sequence (GNQWFI, AF) is a VEGFR1 selective six peptide.AF peptide selected from the position scanning synthetic peptide library through a purified recombinant VEGF and chimeric receptor. Unlike VEGFR1, which inhibits the interaction of VEGFR1 with a series of ligands, such as VEGFA, VEGFB, PIGF, etc., unlike other VEGFR1 monoclonal antibodies or RNA antagonists, AF peptide is a non immunogenic six peptide that can be easily synthesized through lower production costs. These advantages determine that it is suitable for clinical application, but there is water. Hyaluronic acid (HA) is a kind of negative charged, non sulfated linear glycosaminoglycan found in the vitreous body of the bull's eye, and repeated two sugar units are beta -N- acetylglucosamine and D- glucuronic acid. Their biodegradability and high drug resistance are widely used in pharmaceutical field.HA. The receptor action of cell and cell interstitium regulates the invasion of tumor cells, differentiation of tumor cells, transformation of viruses in fibroblasts, and embryonic tissue formation. There are four kinds of HA specific receptors that exist on the surface of the cell membrane - CD44, RHAMM (Receptor for HA-Mediated Motility), IVd4 and LEC, which are more expressed in the tumor site. CD44.AF and ES2 are both similar and different in activity. If the sequences of these two peptides are fused into the same peptide, better bioactivity, better stability and longer half-life are expected. To this end, we designed a peptide sequence of IVRRADRAAVPGGGGGGNQWFI, named ES2-AF, and can be used to target tumor. HA, a highly expressed CD44 receptor, modifies it chemically. It is expected that the modified peptides have better solubility, stronger targeting and longer half-life, and thus better play the role of anti angiogenesis in the body. The contents of this study and the main achievements of this study are as follows: the synthesis and characterization of 1..ES2-AF by solid phase synthesis of Fmoc Methods a kind of ES2-linker (GGGG) -AF peptide was synthesized and purified by high performance liquid chromatography with the purity of up to 95%. The structure confirmed the synthesis and characterization of.2.HA-ES2-AF because HA was dissolved in water but insoluble in organic solvent. For the next step of coupling reaction, four butyl hydrogen hydroxide was first used to exchange it. After activating the carboxyl group.HA-TBA molecule soluble in DMSO HA-TBA binding agent (BOP), the HA-ES2-AF binding was obtained with ES2-AF, DIPEA (N, N- two isopropyl ethylamine). The 1H NMR NMR results showed that ES2-AF successfully was modified and the connection rate was 93.75%. The average biological activity of 60 peptides connected to each chain was found. Study on the antiangiogenesis activity of 3.1 ES2-AF and HA-ES2-AF in vitro (1) the proliferation of endothelial cells was evaluated by MTT. Both ES2 and ES2-AF had the effect of inhibiting the proliferation of endothelial cells in vitro, and showed a tendency to increase with concentration. When ES2-AF concentration was 5 mu g/ mL, 50 u g/mL, 1000 mu g/mL, 2000 mu g/mL, 500 um g/mL, The inhibition rate of endothelial cell proliferation was (11.37% + 6.09%), (13.63% + 4.92%), (17.61% + 5.59%), (16.74% + 4.57%) and (27.49% 5.56%). The binding substance HA-ES2-AF modified by HA also showed a certain inhibitory effect on endothelial cell proliferation in vitro, when its peptide concentration was 5 g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL, respectively. The inhibition rate of the proliferation of endothelial cells was (6.67% + 4.92%), (12.01% + 8.13%), (16.96% + 5.23%), (24.37% + 8.97%) and (36.40% + 7.65%). (2) the inhibitory effect of inhibition of endothelial cell metastasis was measured by Transwell chamber method. PBS was used as empty white control. When the concentration of drug peptide was 200 g/mL, AF, ES2 and ES2-AF three groups were fine. The number of cell metastases was (229 + 11), (254 + 4), (255 + 13).AF, ES2 and ES2-AF three had a certain inhibitory effect on endothelial cell metastasis, but the difference was not significant. It showed that there was no significant difference in the inhibitory effect of the three groups of experimental drugs on tumor metastasis. When the peptide concentration was 200 g/mL, ES2-AF, HAES2-AF mixture, HA-ES2- The number of cell metastases in the three groups of AF conjugates was (255 + 13), (202 + 24) and (170 + 7), and HA-ES2-AF showed the strongest inhibitory effect on endothelial cell metastasis. The specific mechanism still remained to be further studied. (3) the effect of ES2-AF, HA-ES2-AF and so on on the vascular formation of endothelial cell EAhy926 was studied. PBS was used as an empty space. In white control, the inhibitory effect of AF and ES2 on the formation of endothelium cells in vitro was not very different, but the inhibitory effect of ES2-AF on the formation of endothelium cells was significantly higher than that of AF and ES2. at the concentration of 25 mu g/mL, 100 g/mL and 200 u g/mL, the number of nodes in AF group was (75 + 17), (63 + 12), and the nodes of ES2 group were divided. Not (67 + 14), (72 + 3), (61 + 5), the number of nodes in group HA-ES2-AF was (32 + 3), (17 + 6), (17 + 6), (19 + 5). Thus, the inhibitory effect of HA-ES2-AF on the formation of endothelium cells was the strongest in all drug groups, indicating that the ability of using HA to modify the peptide to inhibit the formation of endothelial cells was improved. (4) the use of ELISA method The inhibition ability of AF, ES2, ES2-AF and HA-ES2-AF to VEGF and its receptor VEGFR1 (Flt-1) binding ability.ES2 and ES2-AF were determined by the antigen antibody binding principle. The inhibitory effect of ES2-AF was stronger than that of ES2.HA-ES2-AF in the range of 5 micron to 200 micron (83.24% + 0%), (55.01% + 0.07%), (51.04% 0.09%), (41.25% + 0.01%), (37.76% + 0.03%), indicating that HA-ES2-AF has a concentration dependence on the inhibition of VEGF and its receptor VEGFR1 (Flt-1) binding. The ability of HA-ES2-AF to inhibit VEGF and its receptor VEGFR1 (Flt-1) binding at higher concentrations is significantly higher than that of ES2-AF peptide.3.2 ES2-AF and HA-ES2-AF in vivo. The activity study in the chick embryo chorioallantoic membrane (CAM) experiment, compared with the saline group, AF did not significantly inhibit the angiogenesis of CAM at the experimental dose, and the percentage of the new blood vessel area in the ES2-AF group was (32.03% + 0.10%), (22.84% + 0.19%) and (19.26% + 0.03%) at 5 UU g/mL, 50 u g/mL, and (19.26% + 0.03%), showing higher than ES2. The biological activity of anti angiogenesis in the body and the addition of HA in the mixture of dose related.HA and ES2-AF did not have a significant effect on the inhibition of ES2-AF in the inhibition of CAM angiogenesis in the animals. The percentage of the HA-ES2-AF group was (9.67% + 0.03%), (10.12% + 0.01%), respectively (10.12% + 0.01%), (13.10% + 0) (13.10% + 0) at 5 mu g/mL, 9.67% tons /mL and 50 micron g/mL. 4%), compared with ES2-AF and mixture group, inhibition of the activity of CAM angiogenesis significantly improved the targeting ability of.4.HA-ES2-AF. 4.1 in vitro and CD44 receptor binding ability study through surface plasmon resonance (SPR) technique to study the binding ability of HA-ES2-AF to the surface receptor CD44 of tumor cells in vitro. With HA as positive control, the study of HA-ES2 The interaction between -AF, ES2-AF and CD44 protein. Select the appropriate pH to anchor the CD44 protein on the surface of the CM5 chip and flow the sample through the chip. Through the analysis software of Biacore T200, the KD values of the following three samples are combined with the CD44 protein: HA is 4.198 x 10-11 mol/L. That is, the sequence of the binding ability of the sample and CD44 is HAHA-ES2-AF>ES2-AF, the binding ability of the HA modified HA-ES2-AF binding to the cell surface receptor CD44 is significantly improved, and the potential target of the tumor location in the body of the body,.4.2 in vivo, is studied by the living imaging technique for the study of HA-ES2-A The tissue distribution of F in the body. ES2-AF and HA-ES2-AF were marked with FITC, and the drug was given to nude mice with melanoma, respectively. The metabolism time of HA-ES2-AF-FTIC in nude mice was longer, that is, the plasma half-life of HA-ES2-AF-FITC in the body was longer, which proved that chemical modification was helpful. To enhance the stability of the polypeptide, this is in accordance with the results of the pharmacokinetic experiment in the rear. The increase of the drug distribution at the tumor site indicates that the HA modified ES2-AF-FITC has a certain target to the tumor site in the nude mice bearing the tumor. It is proved that glycosylation modification helps to improve the target distribution of the polypeptide, which is the result of the SPR experiment. The pharmacokinetics of.5.HA-ES2-AF in this experiment selected Wistar rats as a model of pharmacokinetics in vivo. By comparing pharmacokinetic parameters, it was found that compared with ES2-AF, the cycle time of HA-ES2-AF in rats was obviously prolonged, the half life period was extended from 2.79 h to 18.07 h, and the average retention time MRT increased from 4.68 h to 13. .18 h, the area AUC0- infinity under the drug time curve was also increased from 2887.80 mg/L. H to 10694.70 mg/L. H, and the plasma clearance rate CL decreased. The long time maintenance of high blood drug level was helpful to reduce the dosage or reduce the frequency of drug delivery. The results and conclusions of this study were mainly: (1) a successful solid-phase synthesis of more than 95% of the ES2-AF peptide, The activity of anti angiogenesis in vivo and in vivo was higher than that of ES2 or AF. (2), which successfully completed the chemical modification and structural characterization of HA to ES2-AF. On average, 60 ES2-AF peptides were linked to each HA chain. (3) HA-ES2-AF showed a stronger inhibition of endothelial cell proliferation and migration than ES2-AF and HAES2-AF, and stronger inhibition of the binding ability of VEGF to the receptor. And stronger inhibition of CAM angiogenesis. (4) using SPR and living imaging techniques, HA-ES2-AF has a stronger CD44 binding capacity than ES2-AF, and shows a certain target targeting and longer body metabolism time in the body. (5) the half life of HA-ES2-AF is 6.48 times that of ES2-AF and helps to reduce the dosage of the drug. Dose or decrease the frequency of drug delivery.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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