靶向感染微环境的生物响应HBc-两性霉素B抗真菌作用机制研究

The clinical application of amphotericin B was limited by its severe adverse reactions, such as renal toxicity and hemolysis. Base on Hepatitis B Virus core protein, we constructed and expressed a pH sensitive and fungal immunogenicity protein drug carrier (Advanced HBc) for encapsulating Amphotericin B (Advanced HBc-amphotericin B). We found that Advanced HBc-Amphotericin B could release Amphotericin B in response to acidic environment and be delivered by neutrophils after being swallowed up by them rapidly. The immune recognition of fungi by host macrophages could be enhanced by Advanced HBc-amphotericin B. Our study revealed that Advanced HBc-amphotericin B exhibited stronger fungicidal activity and little hemolytic activity in vitro. Advanced HBc-amphotericin B displayed enhanced anti-fungal effects and lower renal toxicity in vivo. On the basis of the preliminary work, we will further study the mechanism of Advanced HBc-amphotericin B targeting to infectious organ depend on neutrophils and the mechanism of enhancing the immune response against fungi. Our study will finally clarify the cellular and molecular mechanisms of enhanced anti-fungal and reduced toxic effect of Advanced HBc-amphotericin B. Therefore, our study will provide experimental evidence and theoretical basis for further promoting its clinical application.

肾毒性、溶血等严重不良反应限制抗真菌药物两性霉素B在临床上的应用。本项目前期基于乙肝病毒核心蛋白（HBc）序列表达构建了pH敏感及真菌免疫原性药物载体(Advanced HBc)。该载体包封两性霉素B形成的纳米颗粒（Advanced HBc-两性霉素B）在体外能响应酸性环境释放药物、迅速被中性粒细胞吞噬并随之迁移，并可增强宿主巨噬细胞对真菌的免疫识别；体外研究表明Advanced HBc-两性霉素B杀菌活性显著增强、溶血效应基本消失；动物体内研究表明其药效作用显著增强、肾毒性显著降低。本项目拟在前期工作基础上进一步研究Advanced HBc-两性霉素B依赖中性粒细胞靶向感染部位、响应酸性感染微环境释放药物的机制，以及该纳米颗粒增强宿主抗真菌感染免疫应答的机制；明确Advanced HBc-两性霉素B药效增强、毒性降低的细胞及分子机制，为进一步促进其转化为临床应用提供实验依据和理论基础。

近年来临床上免疫功能低下人群显著增多，侵袭性真菌感染的发生率明显上升，常见致病真菌特别是念珠菌感染的发生率已经升至医院内感染的第四位。侵袭性真菌感染病死率高达40%，已经成为严重威胁人类健康的疾病。两性霉素B（Amphotericin B，AmB）作为多烯类抗生素主要用于治疗侵袭性真菌感染，有很强的抗菌活性，目前仍被喻为治疗真菌感染的“黄金标准”。由于两性霉素B能够与哺乳动物肾小管上皮细胞以及红细胞膜上的胆固醇发生非选择性地结合，引起肾毒性、溶血等较为严重的不良反应，并且两性霉素B溶解性低，导致其在体液内分散性差，也影响了其抗真菌感染药效学作用。虽然两性霉素B脂质体制剂能够在一定程度上改善该药物引起的不良反应，但是仍然不能完全解决上述临床问题。.本项目前期基于两性霉素B具有肾毒性和溶血效应等严重不良反应而限制其临床应用的问题，构建并重组表达了真菌免疫原性的蛋白质药物载体Advanced HBc，并进一步利用其包封两性霉素B形成纳米（Advanced HBc-两性霉素B, AmB Np）。本课题进一步研究了AmB Np的性质，进而通过体内和体外实验阐明了AmB Np的减毒增效作用。本课题系统探讨了AmB Np对免疫细胞的影响，发现AmB Np能够通过中性粒细胞迁移使其在感染微环境中，并在中性粒形成NET过程中将AmB释放。本课题探索了AmB Np减毒、增效的机制，为进一步促进其临床转化应用提供实验依据和理论基础。