伏立康唑纳米银复合膜对烟曲霉菌性角膜炎的疗效及安全性

摘要

背景 伏立康唑具有良好的抗真菌活性,但目前临床上所用剂型的生物利用度并不理想.改良伏立康唑眼用剂型并提高药物的生物利用度对于改善真菌性角膜炎的预后,减少药物不良反应具有重要意义. 目的 研究伏立康唑纳米银/聚合物复合材料在治疗真菌性角膜炎过程中的缓释作用、药物疗效和安全性.方法 选择6～8周龄雌性SPF级C57BL/6小鼠210只,其中30只用于药物的生物相容性验证,180 只小鼠用于疗效观察,均以左眼作为实验眼.药物的生物相容性研究中将30只小鼠按照随机数字表法分为正常对照组、纳米银/羧基石墨烯/壳聚糖季铵盐复合物(CS-ETA/Ag/GO)组和包被伏立康唑的纳米银/羧基石墨烯/壳聚糖季铵盐复合物(CS-ETA/Ag/GO/Vor)组,分别将自行构建的CS-ETA/Ag/GO膜和CS-ETA/Ag/GO/Vor膜贴敷于小鼠左眼角膜,分别于贴敷后1d、7d行角膜组织的常规组织病理学检查.药物疗效观察实验中,应用随机数字表法将180只小鼠随机分为模型对照组、CS-ETA/Ag/GO组和CS-ETA/Ag/GO/Vor组.3个组小鼠均用33G注射器针头于左眼角膜基质内注射5×107 CFU/ml烟曲霉菌悬浮液2.0μl建立烟曲霉菌性角膜炎小鼠模型,CS-ETA/Ag/GO组和CS-ETA/Ag/GO/Vor组小鼠分别在模型眼角膜贴敷相应的药膜.分别于造模后第1、3、5、7天用裂隙灯显微镜对术眼角膜炎症进行评分及组织病理学检查,采用真菌平板并计算角膜载菌量;采用real-time PCR法检测角膜组织中炎性因子TNF-α mRNA和IL-1β mRNA的相对表达量.结果 CS-ETA/Ag/GO膜和CS-ETA/Ag/GO/Vor膜贴敷于正常小鼠角膜后1～7d,经组织病理学检查均未见异常.CS-ETA/Ag/GO/Vor组各时间点小鼠的角膜炎症评分明显低于模型对照组和CS-ETA/Ag/GO组,3个组间和不同时间点角膜炎症评分的总体差异均有统计学意义(F分组=237.29,P=0.00;F时间=260.33,P=0.00).角膜组织病理学检查显示,模型对照组小鼠角膜水肿,部分角膜基质溶解坏死,第7天部分小鼠角膜穿孔.CS-ETA/Ag/GO组于造模后角膜炎症表现轻于模型对照组,随造模后时间的延长,角膜炎症逐渐减轻,而各个时间点CS-ETA/Ag/GO/Vor组小鼠角膜炎症反应最为轻微,造模后第7天角膜炎症接近痊愈.造模后CS-ETA/Ag/GO/Vor组小鼠角膜载菌量最低,且随着时间延长,载菌量逐渐减少,各组不同时间点角膜载菌量的总体差异比较差异均有统计学意义(F分组=113.15,P=0.00;F时间=126.52,P=0.00).CS-ETA/Ag/GO/Vor组小鼠角膜中IL-1β mRNA和TNF-α mRNA相对表达量均明显低于模型对照组和CS-ETA/Ag/GO组,其相对表达量均于造模后第5天达峰,第7天明显下降.3个组间小鼠在造模后不同时间点角膜中相对表达量的差异均有统计学意义(IL-1β:F分组=189.90,P=0.00;F时间=108.56,P=0.00;TNF-α:F分组=82.55,P=0.00;F时间=44.36,P=0.00). 结论 CS-ETA/Ag/GO/Vor药物缓释膜通过伏立康唑和银离子的协同作用共同抑制真菌活性,减轻真菌诱导的角膜炎症反应.CS-ETA/Ag/GO/Vor药物缓释膜贴敷于角膜后生物相容性好,未发现角膜组织的毒性反应.%Background Voriconazole is the traditionally used antifungal agent,but its ophthalmic form is unsatisfactory.A novel ophthalmic drug delivery system with biomedical devices may be of promising for the prognosis of fungal keratitis.Objective This study was to investigate the sustained release,therapeutic effect and biocompatibility of effect and quaternized chitosan functionalized with carboxylated graphene and nano-silver and voriconazole (CS-ETA/Ag/GO/Vor) for fungal keratitis.Methods This study complied with the Regulations for the Administration of Affair Concerning Experimental Animals of State Science and Technology Commission.Two hundred and ten SPF female C57BL/6 mice were selected with the age 6-8 weeks for the biocompatibility experiment (30 mice) and therapeutic observation of CS-ETA/Ag/GO/Vor (180 mice).CS-ETA/Ag/GO and CS-ETA/Ag/GO/Vor were attached on the normal corneas of mice and compared with the normal mice to assess the histopathological changes.Aspergillus fumigatus-infected mouse models were established in the left eyes of 180 mice by intrastromally injection of 2.0 μl Aspergillus fumigatus suspension with the density of 5 × 107 CFU/ml,then the mice were randomized into the model control group,CS-ETA/Ag/GO group and CS-ETA/Ag/GO/Vor group,and the corresponding membrane were attached the central corneas in different groups.In 1 day,3,5,7 days after modeling,the corneas were examined under the slit lamp microscope and scored,and corneal sections were prepared for the histopathological examination.Fungal activity was confirmed by plate counts,and real-time PCR was employed to assay the relative expressions of interleukin-1β (IL-1β) mRNA and tumor necrosis factor-α (TNF-α) mRNA in the corneas.Results No morphological abnormality was seen in the corneas in the normal control group,CS-ETA/Ag/GO group and CSETA/Ag/GO/Vor group.Corneal inflammatory score was significantly lower in the CS-ETA/Ag/GO/Vor group in various time points,with a significant differences among the groups and time points (Fgroup =237.29,P=0.00;Ftime =260.33,P=0.00).The edema,necrosis or perforation of cornea were seen in the model control group,and slighter inflammatory response in the CS-ETA/Ag/GO group,and corneal edema was gradually disappear in the CS-ETA/Ag/GO/Vor group.The corneal fungal loads were highest in the model control group and lowest in the CS-ETA/Ag/GO/V or group,with significant differences among the three groups and various time points (Fgroup =113.15,P =0.00;Ftime =126.52,P=0.00).The relative expressions of IL-1β mRNA and TNF-α mRNA in the corneas peaked in the fifth day after modeling in all of the three groups,and the expression levels of IL-1β mRNA and TNF-α mRNA in the corneas were lowest in the CS-ETA/Ag/GO/Vor group,showing significant differences among the groups and time points (IL-1β:Fgroup =189.90,P =0.00;Ftime =108.56,P =0.00;TNF-α:Fgroup =82.55,P =0.00;Ftime =44.36,P =0.00).Conclusions CS-ETA/Ag/GO/Vor delivery system plays an anti-fungal activity in fungal keratitis by the synergistic effect of voriconazole and Ag+.In addition,CS-ETA/Ag/GO/Vor appears to have a good safety after topical application.