肿瘤靶向D构型多肽纳米纤维作为10-羟基喜树碱载体的研究
发布时间：2018-09-04 13:58
【摘要】：自组装纳米药物载体在药物控制释放及靶向药物传输方面,已经发挥了巨大地推动作用。研究证实,除化学组成、尺寸大小及表面电位等因素外,纳米载体的形状对其在体内的代谢也具有重要影响。对于某些载体材料,纳米纤维比纳米微球表现出更少的网状内皮系统摄取和更强的肿瘤组织选择性。多肽纳米材料因生物相容性好,易连接功能基团,且可以根据需要“自下而上”地设计序列而被广泛应用。自组装多肽纳米纤维在药物传输领域具有很大的应用前景。 由天然氨基酸(L氨基酸)组成的纳米纤维在体内容易被各种蛋白酶催化降解,存在半衰期短、稳定性差等缺点,可能导致负载药物的过早释放,在一定程度上限制了其作为药物载体在体内的应用。将D构型的非天然氨基酸引入到多肽序列中,能够降低体内各种消化蛋白酶的识别和降解,比L构型的氨基酸表现出更高的稳定性。精氨酸-甘氨酸-天冬氨酸(arginine-glycine-aspartic, RGD)三肽模型是整合素αvβ3的结合基序,而整合素αvβ3过度表达于恶性肿瘤内皮细胞,对癌细胞的粘附、信号转导、迁移和生存至关重要,被广泛应用于肿瘤的诊断和靶向治疗。因此,我们设计并合成了具有肿瘤靶向性的由L构型氨基酸构成的多肽分子Nap-GFFYGRGD(用L-peptide表示)和由D构型氨基酸构成的多肽分子Nap-GDFDFDYGRGD(用D-peptide表示),将多肽分子自组装形成的纳米纤维作为疏水性抗肿瘤药物10-羟基喜树碱(10-hydroxycamptothecin, HCPT)的载体用于体内外肿瘤治疗的研究。实验结果表明：L-peptide和D-peptide经加热煮沸自然冷却后可自组装形成直径为10-20nm的纳米纤维(分别用L-fiber和D-fiber表示)；与L-fiber相比,D-fiber具有更强地抵抗蛋白酶K降解的能力,说明D氨基酸的引入提高了多肽纳米纤维的稳定性,为其体内应用奠定了基础；通过氯胺-T法对多肽进行放射性同位素125Ⅰ标记,体内分布实验结果表明L-peptide和D-peptide具有不同的体内代谢规律,L-fiber主要分布在胃中,而D-fiber主要分布于肝脏,二者在肿瘤部位均具有明显的积累；L-peptide和D-peptide可通过疏水作用与HCPT共组装形成可注射的载药纳米纤维(分别用L-fiber-HCPT和D-fiber-HCPT表示),实验表明以L-fiber和D-fiber为载体可以提高HCPT在生理环境中的溶解度,分别约51倍和46倍,并且在细胞和体内水平上提高了HCPT的抗肿瘤效果。因此,D-fiber-HCPT与L-fiber-HCPT相比具有更好的体外稳定性和体内肿瘤治疗效果。 通过本论文我们完成了一种能够对疏水性抗肿瘤药物HCPT增溶、提高其体内外抗肿瘤效果并降低毒副作用的D构型多肽纳米纤维载体的研究,为肿瘤靶向的治疗及多肽药物载体的设计提供新的思路和方法。
[Abstract]:Self-assembled nano-drug carriers have played an important role in drug delivery and drug delivery. In addition to chemical composition, size and surface potential, the shape of nano-carrier also has an important effect on its metabolism in vivo. For some carrier materials, nanofibers exhibit less reticuloendothelial uptake and stronger tumor tissue selectivity than nano-microspheres. Polypeptide nanomaterials are widely used because of their good biocompatibility, easy linking of functional groups and "bottom-up" design of sequences according to their needs. Self-assembled polypeptide nanofibers have great application prospect in the field of drug transport. Nanofibers composed of natural amino acids (L-amino acids) are easily degraded by various proteases in vivo, which may lead to the premature release of loaded drugs due to their short half-life and poor stability. To some extent, it limits its application as drug carrier in vivo. The introduction of non-natural D-shaped amino acids into polypeptide sequences can reduce the recognition and degradation of various digestive proteases in the body and exhibit higher stability than L-configuration amino acids. Arginine glycine aspartic acid (arginine-glycine-aspartic, RGD) tripeptide model is the binding motif of integrin 伪 v 尾 3, and integrin 伪 v 尾 3 overexpression in malignant tumor endothelial cells, adhesion, signal transduction, migration and survival of cancer cells is very important. It is widely used in tumor diagnosis and targeted therapy. therefore We have designed and synthesized polypeptide molecule Nap-GFFYGRGD (expressed by L-peptide) and polypeptide molecule Nap-GDFDFDYGRGD (expressed by D-peptide), which is composed of L-configuration amino acid and D-configuration amino acid, which has tumor targeting property. Fiber was used as a carrier of 10-hydroxycamptothecin (HCPT) as a hydrophobic antitumor drug in vitro and in vivo. The experimental results show that: L-peptide and D-peptide can self-assemble into nanofibers with diameter of 10-20nm (expressed as L-fiber and D-fiber, respectively) after heated and boiled naturally cooled, and D-fiber has stronger ability to resist protease K degradation than L-fiber. It shows that the introduction of D-amino acid improves the stability of polypeptide nanofibers and lays the foundation for its application in vivo. The results of in vivo distribution experiment showed that L-peptide and D-peptide had different metabolic laws in the stomach, while D-fiber mainly distributed in the liver, and both had obvious accumulation in the tumor site. L-peptide and D-peptide can co-assemble with HCPT to form injectable drug-loaded nanofibers (expressed by L-fiber-HCPT and D-fiber-HCPT respectively) through hydrophobic interaction. The experimental results show that L-fiber and D-fiber can improve the solubility of HCPT in physiological environment, about 51 times and 46 times, respectively. It also improves the anti-tumor effect of HCPT at cell and in vivo level. Therefore, D-fiber-HCPT has better in vitro stability and in vivo tumor therapy than L-fiber-HCPT. In this paper, we have completed a novel D-configuration polypeptide nanofiber carrier which can solubilize hydrophobic antitumor drug HCPT, improve its antitumor effect in vivo and in vitro, and reduce toxic side effects. To provide new ideas and methods for tumor targeting therapy and the design of polypeptide drug carrier.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
本文编号：2222310
[Abstract]:Self-assembled nano-drug carriers have played an important role in drug delivery and drug delivery. In addition to chemical composition, size and surface potential, the shape of nano-carrier also has an important effect on its metabolism in vivo. For some carrier materials, nanofibers exhibit less reticuloendothelial uptake and stronger tumor tissue selectivity than nano-microspheres. Polypeptide nanomaterials are widely used because of their good biocompatibility, easy linking of functional groups and "bottom-up" design of sequences according to their needs. Self-assembled polypeptide nanofibers have great application prospect in the field of drug transport. Nanofibers composed of natural amino acids (L-amino acids) are easily degraded by various proteases in vivo, which may lead to the premature release of loaded drugs due to their short half-life and poor stability. To some extent, it limits its application as drug carrier in vivo. The introduction of non-natural D-shaped amino acids into polypeptide sequences can reduce the recognition and degradation of various digestive proteases in the body and exhibit higher stability than L-configuration amino acids. Arginine glycine aspartic acid (arginine-glycine-aspartic, RGD) tripeptide model is the binding motif of integrin 伪 v 尾 3, and integrin 伪 v 尾 3 overexpression in malignant tumor endothelial cells, adhesion, signal transduction, migration and survival of cancer cells is very important. It is widely used in tumor diagnosis and targeted therapy. therefore We have designed and synthesized polypeptide molecule Nap-GFFYGRGD (expressed by L-peptide) and polypeptide molecule Nap-GDFDFDYGRGD (expressed by D-peptide), which is composed of L-configuration amino acid and D-configuration amino acid, which has tumor targeting property. Fiber was used as a carrier of 10-hydroxycamptothecin (HCPT) as a hydrophobic antitumor drug in vitro and in vivo. The experimental results show that: L-peptide and D-peptide can self-assemble into nanofibers with diameter of 10-20nm (expressed as L-fiber and D-fiber, respectively) after heated and boiled naturally cooled, and D-fiber has stronger ability to resist protease K degradation than L-fiber. It shows that the introduction of D-amino acid improves the stability of polypeptide nanofibers and lays the foundation for its application in vivo. The results of in vivo distribution experiment showed that L-peptide and D-peptide had different metabolic laws in the stomach, while D-fiber mainly distributed in the liver, and both had obvious accumulation in the tumor site. L-peptide and D-peptide can co-assemble with HCPT to form injectable drug-loaded nanofibers (expressed by L-fiber-HCPT and D-fiber-HCPT respectively) through hydrophobic interaction. The experimental results show that L-fiber and D-fiber can improve the solubility of HCPT in physiological environment, about 51 times and 46 times, respectively. It also improves the anti-tumor effect of HCPT at cell and in vivo level. Therefore, D-fiber-HCPT has better in vitro stability and in vivo tumor therapy than L-fiber-HCPT. In this paper, we have completed a novel D-configuration polypeptide nanofiber carrier which can solubilize hydrophobic antitumor drug HCPT, improve its antitumor effect in vivo and in vitro, and reduce toxic side effects. To provide new ideas and methods for tumor targeting therapy and the design of polypeptide drug carrier.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
【参考文献】
相关期刊论文 前1条
1 杨柯君;;全球癌症状况最新数据更新[J];上海医药;2014年02期
，本文编号：2222310
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