Delivery of anti-fungal dsRNA into yeast and filamentous fungi using Laser-Activated Nanoparticles

这一多学科研究项目将开发一种新的方法，将抗真菌药物输送到医学上 相关真菌使用超短激光脉冲和纳米颗粒。我们的长期目标是推进新的方法 治疗由耐多药酵母菌引起的真菌感染，特别是皮肤和粘膜感染 如金黄色念珠菌或顽固性皮肤癣菌，如红色毛癣菌。这项提议的目的是 是开发双链RNA(DsRNA)作为抗真菌药物；其中dsRNA将靶向并抑制 真菌必需基因的翻译导致细胞死亡。然而，dsrna不能轻易地穿透真菌。 细胞壁，由甲壳素、葡聚糖、甘露聚糖和糖蛋白组成。核心假设，即 根据我们自己的初步数据得出的结论是，我们可以在真菌细胞中产生瞬时休息 利用红外线照射金纳米颗粒(AuNPs)产生的空化和冲击波的墙 飞秒(飞秒)激光脉冲。其基本原理是抑制这些基本基因会导致 真菌的生长和/或生存能力下降。同时，目前还没有已知的真菌通过什么机制 细胞可能对dsRNA产生抵抗力。拟议研究的目标将通过三个阶段实现 独立的特定目标：1.通过激光激活的纳米颗粒通过光敏作用传递致命性dsRNA (LANPs)在白色念珠菌和红色毛滴虫中；2.评估纳米颗粒的大小和组成对 3.评估LANP对人角质形成细胞的毒性，以及在 抑制真菌生物膜。这项提议是创新的，因为它使用了一种完全不同的方法来选择性地 通过使用(A)dsRNA作为抗真菌剂和(B)激光激活的纳米颗粒来阻碍真菌细胞 DsRNA在细胞内的传递。这项研究具有重要的意义，因为它可以引导新的 治疗口腔、阴道和皮肤真菌病的策略。这种方法也可以成为一种新的实验室工具 将DNA、dsRNA或CRISPR/Cas9基因编辑系统输送到真菌细胞，可广泛用于 不同的生物技术领域来研究和调节基因功能。我们还将验证C. 白念珠菌使用RNAi，这在萌芽酵母中几乎没有被探索过。最后，这件事的多面性 研究将培养创造力，鼓励学生在不同学科之间进行合作。

This multidisciplinary research project will develop a new method to deliver antifungal agents to medically relevant fungi using ultrashort laser pulses and nanoparticles. Our long-term goal is to advance new approaches to treat fungal infections, particularly of the skin and mucous membranes, caused by multi-drug resistant yeasts such as Candida auris or recalcitrant dermatophytes such as Trichophyton rubrum. The objective of this proposal is to develop double-stranded RNA (dsRNA) as an antifungal agent; where the dsRNA will target and inhibit the translation of essential fungal genes leading to cell death. However, dsRNA cannot easily penetrate the fungal cell wall, which is composed of chitin, glucans, mannans, and glycoproteins. The central hypothesis, which was formulated on the basis of our own preliminary data, is that we can generate transient breaks in the fungal cell wall using cavitation and shock waves that result from irradiating gold nanoparticles (AuNPs) with infrared femtosecond (fs) laser pulses. The rationale is that suppression of these essential genes would lead to decreased growth and/or viability of the fungi. At the same time, there is no known mechanism by which fungal cells could develop resistance against dsRNA. The objective of the proposed research will be achieved by three independent specific aims: 1. Delivery of lethal dsRNA through photoporation via Laser-Activated Nanoparticles (LANPs) in C. albicans and T. rubrum; 2. Evaluation of the effect of nanoparticle size and composition on photoporation via LANPs; and 3. Evaluation of LANP toxicity in human keratinocytes, and effectiveness in inhibiting fungal biofilms. This proposal is innovative because it uses an entirely different approach to selectively impede fungal cells by using (a) dsRNA as an antifungal agent and (b) laser activated nanoparticles to facilitate the intracellular delivery of dsRNA. This research is significant because it can lead to the development of new strategies to treat oral, vaginal and skin mycoses. This method can also become a new laboratory tool for the delivery of DNA, dsRNA, or CRISPR/Cas9 gene editing systems to fungal cells, and can be widely used in different biotechnology areas to study and modulate gene function. We will also validate essential genes in C. albicans using RNAi, which has been barely explored in budding yeast. Finally, the multifaceted nature of this research will foster creativity and encourage student collaboration within different disciplines.