Chang Che-Yi, Wang Ming-Chen, Miyagawa Takuya, Chen Zhi-Yu, Lin Feng-Huei, Chen Ko-Hua, Liu Guei-Sheung, Tseng Ching-Li
Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei; Department of Biomedical Engineering, Chung Yuan Christian University, Taoyuan.
Department of Biomedical Engineering, Chung Yuan Christian University, Taoyuan.
Int J Nanomedicine. 2016 Dec 30;12:279-294. doi: 10.2147/IJN.S114754. eCollection 2017.
Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αβ integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αβ. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.
角膜新生血管化（NV）会破坏视觉功能，引发包括失明在内的眼部疾病。因此，角膜NV的治疗对公众健康具有重大影响。表没食子儿茶素-3-没食子酸酯（EGCG）具有抗血管生成作用，被选作抑制剂用于治疗人血管内皮细胞以治疗角膜NV。本研究合成了一种精氨酸-甘氨酸-天冬氨酸（RGD）肽-透明质酸（HA）共轭复合物包被的明胶/EGCG自组装纳米颗粒（GEH-RGD NPs），用于靶向人脐静脉内皮细胞（HUVECs）上的αβ整合素，并使用角膜NV小鼠模型评估这种用作眼药水的纳米药物的治疗效果。通过氢核磁共振和傅里叶变换红外光谱证实了HA与RGD通过羧基和胺基进行了共轭。GEH-RGD NPs的平均直径为168.87±22.5 nm，带正电荷（19.7±2 mV），EGCG负载效率高达95%。透射电子显微镜获取的GEH-RGD NPs图像显示为球形且壳结构约为200 nm。在纳米制剂中观察到约30小时后有30%左右的缓释模式。表面等离子体共振证实GEH-RGD NPs能特异性结合整合素αβ。体外细胞活力测定表明，与EGCG或非RGD修饰的纳米颗粒相比，GEH-RGD在低EGCG浓度（20μg/mL）时能有效抑制HUVEC增殖。此外，GEH-RGD NPs能显著抑制HUVEC迁移，抑制率低至58%，持续24小时。在角膜NV小鼠模型中，用GEH-RGD NP眼药水治疗后，在碱烧伤的角膜中观察到血管数量减少且变细。总体而言，本研究表明GEH-RGD NPs已成功开发并合成，作为具有特异性靶向能力的血管内皮细胞抑制剂。此外，它们可用于眼药水以抑制角膜NV小鼠的血管生成。
