Department of Ophthalmology, the Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao, Shandong Province 266003, China.
ACS Appl Mater Interfaces. 2024 Nov 6;16(44):59777-59788. doi: 10.1021/acsami.4c11366. Epub 2024 Oct 28.
This study aims to explore the efficacy and safety of macrophage membrane-coated nanoparticles for the delivery of natamycin (NAT) in the therapy of fungal keratitis (FK). Macrophage membranes were isolated and identified by immunofluorescence staining (IFS). NAT was encapsulated into poly(lactic--glycolic acid) (PLGA). Fungal stimulated macrophage membranes (M1) or unstimulated membranes (M) were separately mixed and sonicated with PLGA nanoparticles. The biocompatible nanoparticles (PLGA-NAT, PLGA-NAT@M, and PLGA-NAT@M1) were characterized with zeta-sizer analysis, transmission electron microscopy (TEM), and Western blot. Drug encapsulation and loading efficiency and the release of NAT in the nanoparticles were detected by ultraviolet spectrophotometry. The cytotoxicity, ocular surface toxicity and irritability, and systemic safety of nanoparticles with different concentrations were assessed. In vitro, we examined the antifungal properties of the nanoparticles. The eye surface retention time, drug release, and curative effects on FK were evaluated in vitro and in vivo. IFS results showed the separation of the macrophage membrane and nucleus. The prepared nanoparticles had a typical "core-shell" structure and uniform nanometer size, and the membrane proteins were retained on the membrane allowing to exert functional effects of macrophage. The loading efficiencies of PLGA-NAT@M and PLGA-NAT@M1 were 7.6 and 6.7%, respectively. The encapsulation efficiencies of PLGA-NAT@M and PLGA-NAT@M1 were 51.2 and 41.5%, respectively. PLGA-NAT@M and PLGA-NAT@M1 could gradually release NAT and reduce the clearance of the ocular surface. Macrophage membranes enhanced the antifungal activity of PLGA-NAT. Furthermore, the membrane coated with macrophage increased the biocompatibility and decreased the corneal toxicity of nanoparticles. In vivo, PLGA-NAT@M1 significantly alleviated the severity of FK. In vitro, PLGA@M and PLGA@M1 reduced the protein levels of inflammatory cytokines after fungal stimulation. The prepared PLGA-NAT@M1 has good physical properties and biosafety. It could evade ocular surface clearance, release NAT gradually, and achieve high antifungal and anti-inflammatory efficiencies to FK. Macrophage membrane-coated nanoparticles clinically have high application potential to the treatment of FK.
本研究旨在探讨巨噬细胞膜包裹纳米粒递送达那霉素（NAT）用于真菌性角膜炎（FK）治疗的疗效和安全性。通过免疫荧光染色（IFS）分离并鉴定巨噬细胞膜。将 NAT 包封于聚乳酸-乙醇酸共聚物（PLGA）中。分别将真菌刺激的巨噬细胞膜（M1）或未刺激的细胞膜（M）与 PLGA 纳米粒混合并进行超声处理。通过纳米粒度仪分析、透射电子显微镜（TEM）和 Western blot 对生物相容性纳米粒（PLGA-NAT、PLGA-NAT@M 和 PLGA-NAT@M1）进行表征。通过紫外分光光度法检测纳米粒中药物包封和载药效率以及 NAT 的释放。评估不同浓度纳米粒的细胞毒性、眼表毒性和刺激性以及全身安全性。体外，我们检测了纳米粒的抗真菌特性。在体外和体内评估纳米粒的眼表面滞留时间、药物释放和 FK 的治疗效果。IFS 结果显示了巨噬细胞膜和核的分离。制备的纳米粒具有典型的“核壳”结构和均匀的纳米尺寸，并且保留了膜蛋白，从而发挥巨噬细胞的功能作用。PLGA-NAT@M 和 PLGA-NAT@M1 的载药效率分别为 7.6%和 6.7%。PLGA-NAT@M 和 PLGA-NAT@M1 的包封效率分别为 51.2%和 41.5%。PLGA-NAT@M 和 PLGA-NAT@M1 可以逐渐释放 NAT 并减少眼表清除。巨噬细胞膜增强了 PLGA-NAT 的抗真菌活性。此外，用巨噬细胞包被的膜增加了纳米粒的生物相容性并降低了其角膜毒性。体内，PLGA-NAT@M1 显著减轻 FK 的严重程度。体外，PLGA@M 和 PLGA@M1 降低了真菌刺激后炎症细胞因子的蛋白水平。所制备的 PLGA-NAT@M1 具有良好的物理性质和生物安全性。它可以逃避眼表清除，逐渐释放 NAT，并对 FK 达到高的抗真菌和抗炎效率。巨噬细胞膜包裹的纳米粒在临床上对 FK 的治疗具有很高的应用潜力。
