Physics Department, Faculty of Science, Istanbul University, Vezneciler, 34134, Istanbul, Turkey.
Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University, 34220, Istanbul, Turkey.
Daru. 2020 Jun;28(1):139-157. doi: 10.1007/s40199-019-00325-9. Epub 2020 Jan 16.
Arginine-vasopressin (AVP) is a neuropeptide and provides learning and memory modulation. The AVP (4-5) dipeptide corresponds to the N-terminal fragment of the major vasopressin metabolite AVP (4-9), has a neuroprotective effect and used in the treatment of Alzheimer's and Parkinson's disease.
The main objective of the present study is to evaluate the molecular mechanism of AVP (4-5) dipeptide and to develop and synthesize chitosan nanoparticle formulation using modified version of ionic gelation method, to increase drug effectiveness. For peptide loaded chitosan nanoparticles, the synthesized experiment medium was simulated for the first time by molecular dynamics method and used to determine the stability of the peptide, and the binding mechanism to protein (HSP70) was also investigated by molecular docking calculations. A potential pharmacologically features of the peptide was also characterized by ADME (Absorption, Distribution, Metabolism and Excretion) analysis. The characterization, in vitro release study, encapsulation efficiency and loading capacity of the peptide loaded chitosan nanoparticles (CS NPs) were performed by Dynamic Light Scattering (DLS), UV-vis absorption (UV), Scanning Electron Microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy techniques. Additionally, in vitro cytotoxicity of the peptide on human neuroblastoma cells (SH-SY5Y) was examined with XTT assay and the statistical analysis was evaluated.
The results showed that; hydrodynamic size, zeta potential and polydispersity index (PdI) of the peptide-loaded CS NPs were 167.6 nm, +13.2 mV, and 0.211, respectively. In vitro release study of the peptide-loaded CS NPs showed that 17.23% of the AVP (4-5)-NH2 peptide was released in the first day, while 61.13% of AVP (4-5)-NH2 peptide was released in the end of the 10th day. The encapsulation efficiency and loading capacity were 99% and 10%, respectively. According to the obtained results from XTT assay, toxicity on SHSY-5Y cells in the concentration from 0.01 μg/μL to 30 μg/μL were evaluated and no toxicity was observed. Also, neuroprotective effect was showed against HO treatment.
The experimental medium of peptide-loaded chitosan nanoparticles was created for the first time with in silico system and the stability of the peptide in this medium was carried out by molecular dynamics studies. The binding sites of the peptide with the HSP70 protein were determined by molecular docking analysis. The size and morphology of the prepared NPs capable of crossing the blood-brain barrier (BBB) were monitored using DLS and SEM analyses, and the encapsulation efficiency and loading capacity were successfully performed with UV Analysis. In vitro release studies and in vitro cytotoxicity analysis on SHSY-5Y cell lines of the peptide were conducted for the first time. Grapical abstract.
精氨酸加压素（AVP）是一种神经肽，可提供学习和记忆调节。AVP（4-5）二肽对应于主要加压素代谢物 AVP（4-9）的 N 端片段，具有神经保护作用，并用于治疗阿尔茨海默病和帕金森病。
本研究的主要目的是评估 AVP（4-5）二肽的分子机制，并使用改良的离子凝胶化方法开发和合成壳聚糖纳米颗粒制剂，以提高药物的有效性。对于载肽壳聚糖纳米颗粒，首次通过分子动力学方法模拟合成实验介质，用于确定肽的稳定性，并通过分子对接计算研究其与 HSP70 蛋白的结合机制。还通过 ADME（吸收、分布、代谢和排泄）分析对肽的潜在药理学特征进行了表征。通过动态光散射（DLS）、紫外可见吸收（UV）、扫描电子显微镜（SEM）、傅里叶变换红外（FT-IR）光谱技术对载肽壳聚糖纳米颗粒（CS NPs）的特性、体外释放研究、包封效率和载药量进行了表征。此外，通过 XTT 测定法研究了肽对人神经母细胞瘤细胞（SH-SY5Y）的体外细胞毒性，并进行了统计学分析。
结果表明; 载肽 CS NPs 的水动力粒径、Zeta 电位和多分散指数（PdI）分别为 167.6nm、+13.2mV 和 0.211。载肽 CS NPs 的体外释放研究表明，AVP（4-5）-NH2 肽在第一天释放了 17.23%，而在第 10 天结束时释放了 61.13%的 AVP（4-5）-NH2 肽。包封效率和载药量分别为 99%和 10%。根据 XTT 测定法获得的结果，评估了浓度为 0.01μg/μL 至 30μg/μL 的 SHSY-5Y 细胞的毒性，未观察到毒性。此外，还显示出对 HO 处理的神经保护作用。
首次使用计算系统创建了载肽壳聚糖纳米颗粒的实验介质，并通过分子动力学研究对该介质中肽的稳定性进行了研究。通过分子对接分析确定了肽与 HSP70 蛋白的结合位点。使用 DLS 和 SEM 分析监测了能够穿透血脑屏障（BBB）的制备 NPs 的大小和形态，并成功使用 UV 分析进行了包封效率和载药量的测定。首次对 SHSY-5Y 细胞系进行了载肽的体外释放研究和体外细胞毒性分析。
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