新型姜黄素纳米胶束滴眼液的制备及体内外性质研究

目的　制备新型姜黄素纳米胶束滴眼液，检测其理化性质，并观察其生物学性质。方法　通过溶剂蒸发-水化法制备姜黄素纳米胶束滴眼液，分别测定其粒径、包封率和稳定性；通过眼局部刺激实验方法考察纳米胶束的细胞毒性和细胞摄取作用；采用眼局部刺激实验方法测定姜黄素纳米胶束滴眼液的体内眼局部刺激性；以兔眼表给予花生四烯酸溶液建立眼局部炎症模型，评价纳米胶束的抗炎活性。结果　使用聚己内酰胺-聚乙酸乙烯酯-聚乙二醇聚合物成功构建姜黄素纳米胶束滴眼液，粒径为（５０．１±１．０）ｎｍ，包封率为（９９．３７±０．７６）％；储存稳定性分析结果显示，１２周后储存在４℃质量比为１８:１的姜黄素纳米胶束滴眼液药物含量仍有（９８．１３±０．９７）％，而质量比１５:１的药物含量为（８９．３２±１．５９）％。２５℃条件下的实验结果与４℃结果相似，因此最佳处方质量比为１８:１。制备的姜黄素纳米胶束呈橙红色透明状，优化处方后的纳米胶束粒径、多分散系数和Ｚｅｔａ电位分别为（５０．１±１．０）ｎｍ、０．０７９±０．０１１和－（１．８３±０．６７）ｍＶ，包封率为（９９．３７±０．７６）％。姜黄素溶液在ｐＨ６．０至ｐＨ７．４的不同条件下，降解速度分别是对应姜黄素纳米胶束降解速度的２４．９倍、２８．４倍、４４．８倍、７３．７倍、３１５．０倍和６６２．１倍。体外细胞实验结果显示浓度为５００．００μｇ·ｍＬ－１的姜黄素纳米胶束孵育细胞４８ｈ后，细胞存活率为８６．８９％；４．５ｍｇ·ｍＬ－１姜黄素纳米胶束滴眼液孵育细胞１ｈ后未发现细胞毒性。兔眼局部刺激性实验结果显示，姜黄素纳米胶束滴眼液、玻璃酸钠滴眼液（１ｍｇ·ｍＬ－１）和空白ＰＢＳ３组实验在不同时间点的临床评分均在０～２的范围内，属于无刺激性等级。体内角膜渗透性结果显示，姜黄素纳米胶束组的荧光强度均显著强于姜黄素溶液组。抗炎活性结果显示姜黄素纳米胶束表现出显著的剂量依赖性抗炎活性。结论　局部聚合物构建的姜黄素纳米胶束滴眼液显著改善了姜黄素的体内外及生物学性质，有望开发成为一种新型有效的眼用姜黄素制剂。

Studies on a new nanomicelle curcumin ophthalmic solution :preparation and characterizations in vitro/in vivo

Objective To prepare a novel nanomicelle curcumin ophthalmic solution and investigate its physicochemical properties as well as biology properties in cells/arumals level. Methods Nanomicelle curcumrn ophthalmic solution was prepared by a solvent evaporation-hydration method , and its mean diameter , encapsulation efficiency and stability were detected, respectively. The cytotoxicity and cellular uptake of the nanomicelle were tested by cellular experiments in vitro. The ocular irritation tests in vivo were investigated by using eye comprehensive score. The eye local inflammatory models were set up by instilling arachidonic acid solution in rabbit eyes to evaluate the anti-inflammatory activity of nanomicelle. Results Nanomicelle curcumin ophthalmic solution were successfully prepared by using polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer ( PVCL-PVA-PEG) with the mean diameter and encapsulation efficiency of ( 50. I + 1. 0) nm and ( 99. 37 +0. 76) % . The remaining drugs of nanomicelle curcumin with 18 : I and 15 : I mass ratio were (98. 13 + 0. 97 )% and ( 89. 32+ 1. 59 ) % after 12 weeks. The experimental results under 25 9C were sinular to those under 4 9C ,so the best mass ratio was 18 : 1. The curcumin nanomicelle was orange red and transparent , the nanomicelle particle size ,polydispersity index and Zeta potential were (50. I + 1. 0) nm ,0. 079 + 0. 011 and - ( 1. 83 + 0. 67 ) mV . and the encapsulation efficiency was ( 99. 37 + 0. 76 ) % . Under different conditions of pH 6. 0 to pH 7. 4 , the degradation rate of curcumin solution was 24. 9 , 28. 4 , 44. 8 , 73. 7 , 315. 0 . 662. I times respectively , which correspond to the degradation rate of curcumin nanoparticles. The cellular experiments in vitro showed that the cell viability were 86. 8g% as the concentration of 500 Vg . mL -l after cell incubation of 48 hours.and there was no cytotoxicity after cell incubation of I hour with 4. 5 mg . mL -l nanomicelle curcunun. To determine the ocular tolerance in rabbits ’ eyes , the values of the clinical scores were 0 - 2 at different time points in all three groups. 4. 5 mg . mL -l curcunun exhibited powerful anti-inflammatory efficiency as the anti-inflammatory activity results displayed. Conclusion Nanomicelle curcunun that fabricated with PVCL-PVA-PEG are greatly improved the curcumin ’s biology properties in vivo/in vitro , and can be a promising topical delivery system for the ocular adnunistration of curcunun.