Computational design of Phe-Tyr dipeptide and preparation, characterization, cytotoxicity studies of Phe-Tyr dipeptide loaded PLGA nanoparticles for the treatment of hypertension

Phe-Tyr dipeptide which was investigated in Wakame food with greatest ACE-inhibitory activity is used as a pharmaceutical drug for the treatment of hypertension, cardiovascular diseases, and diabetic nephropathy. To improve the bioavailability of Phe-Tyr, a delivery system based on poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with Phe-Tyr (Phe-Tyr-PLGA NPs) for treating hypertension and cardiovascular diseases was prepared in this study. In the experiments, poly(lactic-co-glycolic acid) (PLGA) and Phe-Tyr dipeptide-loaded PLGA nanoparticles were prepared using the double emulsion (w/o/w) method. The characterizations of the nanoparticles were performed with a UV–vis spectrometer, the Zeta-sizer system, and FTIR spectrometer. The optimum size of the Phe-Tyr dipeptide-loaded PLGA nanoparticle was obtained with a 213.8 nm average particle size, and a 0.061 polydispersity index, −19.5 mV zeta potential, 34% of loaded and 90.09% of encapsulation efficiency. From TEM analysis, it was clearly seen that the dipeptide loaded nanoparticles had the spherical and non-aggregated morphology and Phe-Tyr dipeptide loaded-PLGA nanoparticles were obtained successfully. Cell toxicity of nanoparticles at different concentrations was assayed with XTT methods on L929 fibroblast cells. This study determined that the nanoparticles have low toxicity at lower concentration and toxicity augmented with increasing concentration of dipeptide. To analyze the effect of solvents on structure of Phe-Tyr, Molecular dynamics simulation was performed with GROMACS program and molecular orbital calculations were carried out to obtain structural and electronic properties of dipeptide. Moreover, molecular docking calculations were also employed to model and predict protein–drug interactions.

本研究中所探究的、在裙带菜（Wakame）中被证实具有最强血管紧张素转换酶（Angiotensin-Converting Enzyme, ACE）抑制活性的Phe-Tyr二肽，现已被用作治疗高血压、心血管疾病及糖尿病肾病的药物。为提升Phe-Tyr的生物利用度，本研究制备了一种基于聚乳酸-羟基乙酸共聚物（poly(lactic-co-glycolic acid), PLGA）的递送系统：该系统负载Phe-Tyr（记为Phe-Tyr-PLGA纳米粒（NPs）），用于治疗高血压与心血管疾病。实验中，本研究采用双乳化（水包油包水，w/o/w）法制备了空白PLGA纳米粒与负载Phe-Tyr二肽的PLGA纳米粒，并通过紫外-可见分光光度计、Zeta粒径分析仪（Zeta-sizer）与傅里叶变换红外光谱（FTIR）光谱仪对纳米粒进行了表征。最终得到的负载Phe-Tyr二肽的PLGA纳米粒最优参数为：平均粒径213.8 nm，多分散性指数（polydispersity index, PDI）0.061，Zeta电位-19.5 mV，载药量34%，包封率90.09%。透射电子显微镜（TEM）分析结果显示，负载二肽的纳米粒呈球形且无团聚现象，证实本研究成功制备了Phe-Tyr负载型PLGA纳米粒。本研究采用XTT法在L929成纤维细胞上检测了不同浓度纳米粒的细胞毒性，结果表明低浓度下该纳米粒毒性较低，且毒性随二肽给药浓度升高而增强。为探究溶剂对Phe-Tyr结构的影响，本研究采用GROMACS软件开展了分子动力学模拟，并通过分子轨道计算获取了该二肽的结构与电子性质；此外，本研究还通过分子对接计算对蛋白-药物相互作用进行建模与预测。