负载Ferrostatin-1的脱铁蛋白脂质体在真菌性角膜炎中的作用研究

Ferrostatin-1-Loaded Apotransferrin Liposomes for the Treatment of Fungal Keratitis

摘要: Ferrostatin-1 (Fer-1)是一种常见的铁死亡抑制剂，具有抗炎、抗氧化潜能，但其存在水溶性差、体内半衰期短等问题限制了临床应用。本研究旨在构建一种负载Fer-1的脱铁蛋白修饰的纳米脂质体(Fer@Tf-LP)，评估其抗真菌、抗炎活性及生物安全性，为真菌性角膜炎的治疗提供新策略。方法：采用薄膜水化法制备包裹Fer-1的脂质体(Fer@LP)，通过后插入法偶联脱铁蛋白至脂质体表面(Fer@TF-LP)，并利用粒度电位分析仪、透射电子显微镜对Fer@LP和Fer@TF-LP的理化性质进行评估。采用CCK-8细胞增殖实验评价Fer@LP和Fer@TF-LP的药物毒性。通过最低抑菌浓度实验(MIC)等考察Fer@LP和Fer@TF-LP的抗真菌性能。烟曲霉菌感染人角膜上皮细胞(HCECs)后，分别给予Fer-1、Fer@LP、Fer@TF-LP处理，采用逆转录聚合酶链式反应(RT-PCR)和酶联免疫吸附实验(ELISA)检测促炎细胞因子IL-6及IL-1β mRNA和蛋白的表达水平。结果：成功制备了负载Fer-1的脱铁蛋白脂质体，平均粒径为180.6 ± 1.88 nm，TEM结果显示Fer@LP和Fer@TF-LP均具备典型的球形纳米结构。CCK-8结果显示，在实验浓度范围内，Fer@Tf-LP对HCECs活力无明显影响，表明其具有良好的生物安全性。MIC实验结果显示，Fer@Tf-LP在45 μg/mL浓度下对烟曲霉菌的抑制率即超过50%，而游离Fer-1达到同等抑制效果所需浓度为90 μg/mL，Fer@TF-LP对烟曲霉菌的抑菌活性优于Fer-1。此外，与游离Fer-1相比，同等浓度的Fer@Tf-LP处理能更显著地下调感染细胞中IL-6及IL-1β的mRNA和蛋白表达水平。结论：负载Fer-1的脱铁蛋白脂质体(Fer@Tf-LP)兼具高效抗真菌及抗炎特性，且安全性良好。该纳米递药系统为改善真菌性角膜炎的治疗效果提供了新的思路。

Abstract: Ferrostatin-1 (Fer-1) is a well-established inhibitor of ferroptosis with documented anti-inflammatory and antioxidant properties. However, its clinical translation is hindered by poor aqueous solubility and a short in vivo half-life. The present study aimed to develop a Fer-1-loaded nanoliposome modified with apotransferrin (Fer@Tf-LP) and to evaluate its antifungal and anti-inflammatory efficacy, as well as its biosafety, thereby offering a potential therapeutic strategy for fungal keratitis. Fer-1-encapsulated liposomes (Fer@LP) were prepared using the thin-film hydration method. Apotransferrin was subsequently conjugated onto the liposomal surface via a post-insertion technique to generate Fer@Tf-LP. The physicochemical characteristics of both formulations were assessed using dynamic light scattering and transmission electron microscopy. Cytotoxicity was evaluated in human corneal epithelial cells (HCECs) using the CCK-8 assay. Antifungal activity against Aspergillus fumigatus was determined through minimum inhibitory concentration (MIC) testing and assessment of conidial germination and hyphal growth. In an A. fumigatus infection model of HCECs, the expression levels of the pro-inflammatory cytokines IL-6 and IL-1β were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) following treatment with free Fer-1, Fer@LP, or Fer@Tf-LP. The results demonstrated that Fer@Tf-LP was successfully fabricated, displaying a spherical morphology with an average particle size of 180.6 ± 1.88 nm. CCK-8 assays confirmed that Fer@Tf-LP exhibited no significant cytotoxicity against HCECs within the tested concentration range, indicating favorable biocompatibility. Notably, Fer@Tf-LP exhibited superior antifungal activity, achieving over 50% inhibition of A. fumigatus growth at 45 μg/mL, whereas free Fer-1 required a concentration of 90 μg/mL to achieve a comparable effect. Furthermore, Fer@Tf-LP significantly suppressed conidial germination and hyphal proliferation. In infected HCECs, treatment with Fer@Tf-LP at equivalent concentrations resulted in a more pronounced downregulation of IL-6 and IL-1β mRNA and protein levels compared to free Fer-1. In conclusion, the Fer-1-loaded apotransferrin liposome (Fer@Tf-LP) demonstrates potent antifungal and anti-inflammatory activities with a robust safety profile. This nanoscale drug delivery system presents a promising and innovative approach for improving the therapeutic outcomes of fungal keratitis.