基于薄荷醇“引药”/麦胚凝集素/穿膜肽TAT共修饰的姜黄素核-壳同载脂质纳米混悬的协同角膜促透及葡萄膜炎抑制作用机制

It is still one formidable obstacle to improving the intraocular drug delivery efficiency. Based on the theory of "Drug-Guide" in traditional Chinese medicine (TCM), the menthol that can enhance the permeability of drugs, and the cell-penetrating peptide TAT which is capable of penetrating across the plasma membrane are used as transcorneal enhancers. And wheat germ agglutinins (WGA) are utilized as the specific mucoadhesive with transmembrane mucins in cornea epithelial cells. In this study, a novel ocular drug carrier, the menthol based/WGA/TAT peptides conjugated lipid nanosuspensions loaded with curcumin simultaneously in core-shell, is designed to offer the following benefits. Firstly, the association of enhancers with nanocarriers will promote drug transcorneal delivery dramatically, and thus to increase the drug concentration in the intraocular tissues. Secondly, the specific cytoadhesion of WGA and the electrical reaction between cationic TAT peptides and negative proteins in corneal epithelium will prolong the drug residence time. Lastly, after the curcumin nanosuspensions being incorporated into the inner phase of liposomes, with the curcumin and menthol loaded in the lipid bilayer, the loading efficiency of liposemes will be improved, and the drug release rate will be depressed. In order to understand the retention properties of nanocarriers on the ocular surface, the transcorneal penetration characters and its mechanisms, as well as anti-inflammatory effects, the nanocarriers will be fully investigated. In conclusion, the nanocarriers constructed in this study would be an efficient intraocular drug delivery system, and these mechanisms would be elucidated detailedly, which will provide necessary evidence to develop innovative TCM preparations for ophthalmology and also promote the application process of TCM.

高效跨眼部给药屏障的药物递送，一直是制剂学领域亟待解决的难题。本课题借鉴中医药“引药”理论，以具有药引促透作用的薄荷醇与高效穿膜能力的TAT肽为角膜促渗剂，麦胚凝集素WGA为角膜上皮层中跨膜黏蛋白的特异性黏附剂，开拓性地设计一种薄荷醇“引药”/WGA/TAT共修饰姜黄素核-壳同载脂质纳米混悬剂。其一利用两种促渗剂与纳米载体的协同作用大幅提高药物的角膜透过效率；其二协同利用WGA的黏附作用与TAT的静电作用增加眼表滞留性，进一步促透；其三将姜黄素纳米混悬负载于脂质体（脂质双分子壳层同时载姜黄素、薄荷醇）的内水相核中，既提高脂质体载药量，又延缓释药。在复合载体构建基础上，对其黏附性能、角膜促透作用及机制、葡萄膜炎抗炎机制进行研究。本课题的开展有望实现药物高效跨角膜递送，并阐明薄荷醇角膜促透及姜黄素的葡萄膜炎抗炎机制，为新型眼用中药制剂的开发应用提供实验基础和理论依据，进一步推动中医药应用进程。

高效跨眼部给药屏障的药物递送，一直是制剂学领域亟待解决的难题。本项目以具有促透作用的薄荷醇与高效穿膜能力的TAT肽为角膜促渗剂，麦胚凝集素WGA为角膜上皮细胞的特异性黏附剂，设计并制备一种高角膜透过效率、长眼表滞留性的多功能纳米载体—薄荷醇“引药”/WGA/TAT共修饰姜黄素核-壳同载脂质纳米混悬剂（WGA/TAT-FB/Cur-LNC）。课题主要对纳米载体黏附性能、角膜促透作用及机制、眼部抗炎作用进行了研究。制备的WGA/TAT-FB/Cur-LNC纳米载体平均粒径为167.7 ± 3.35 nm，呈单峰分布，zeta电位为22.17 ± 1.08 mV。TEM照片显示其呈近球形，粒子之间无聚集现象。Cur的包封率为68.94 ± 0.74%，FB的包封率为71.91 ± 4.48%。细胞毒性及HET-CAM刺激性实验结果显示，纳米载体对HCE-T细胞几乎无毒性作用，生物相容性较好。通过HCE-T细胞摄取、3D细胞球模型和房水药代动力学评价，证实了WGA、TAT功能化纳米载体（WGA/TAT-LNC）的角膜穿透促进性能及粘附能力。细胞摄取机制实验表明，WGA/TAT-LNC诱导细胞内钙离子浓度和膜电位增加。细胞内化途径为小窝蛋白和网格蛋白介导的内吞作用，同时依赖于WGA/TAT-LNC与细胞膜之间的静电相互作用和特异性粘附作用，摄取过程存在一定程度的能量依赖性。用WGA/TAT-LNC处理的HCE-T细胞的F-actin图像显示细胞间的紧密连接打开。在体眼表滞留性结果显示，给药后连续冲洗 1 h，WGA/TAT-FB/Cur-LNC中FB的滞留率分别是FB/Cur-LNC和FB/Cur-Sol的1.80和3.87倍，表明WGA/TAT-FB/Cur-LNC增加药物的眼表滞留时间；氟比洛芬在房水中的药代动力学表明，WGA/TAT-LNC提高了药物跨角膜转运的生物利用度。此外，在兔眼部症炎模型中，WGA/TAT-FB/Cur-LNC显着抑制泪液和房水中的PGE2，IL-6和TNF-α产生，增强了药物的抗炎作用。本项研究结果表明，WGA/TAT- LNC是一种高效跨角膜递送的药物载体，可延长眼表滞留时间，促进药物经角膜递送。同时，本研究为新型眼用中药制剂的开发应用提供理论支撑，并进一步推动中医药现代化应用进程。