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

使用激光激活纳米颗粒将抗真菌 dsRNA 递送至酵母和丝状真菌中

• 批准号: 10360291
• 负责人: Luz Adriana Avila Flores
• 金额: $ 44.51万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360291
• 关键词: Address; Adhesions; Adverse drug effect; Affect; Animals; Antifungal Agents; Apoptosis; Area; Arthrodermataceae; Biotechnology; CRISPR/Cas technology; Candida albicans; Candida auris; Cell Culture Techniques; Cell Death; Cell Wall; Cell membrane; Cells; Cellular Structures; Chemotherapy-Oncologic Procedure; Chitin; Clinical; Collaborations; Creativeness; DNA delivery; Data; Development; Discipline; Dose; Double-Stranded RNA; Drug Interactions; Dyes; Effectiveness; Electroporation; Ergosterol; Essential Genes; Evaluation; Exclusion; Exposure to; Fostering; Fungal Genes; Genes; Glass; Glucans; Glycoproteins; Goals; Growth; HIV; Human; Immunocompromised Host; In Vitro; Infection; Infectious Skin Diseases; Interdisciplinary Study; Laboratories; Lasers; Lead; Liposomes; Literature; Mammalian Cell; Mannans; Medical; Medicine; Membrane; Methodology; Methods; Microbial Biofilms; Molds; Mucous Membrane; Multi-Drug Resistance; Mycoses; Nature; Necrosis; Nucleic Acids; Opportunistic Infections; Oral; Organ Transplantation; Outcome; Oxidative Stress; Pathogenicity; Pathway interactions; Peptides; Perforation; Pharmaceutical Preparations; Physiologic pulse; Polymers; Process; Publishing; RNA; RNA Interference; Reporting; Research; Research Project Grants; Resistance; Resistance development; Risk; Saccharomyces; Saccharomyces cerevisiae; Saccharomycetales; Science; Shock; Skin; Students; System; Techniques; Technology; Testing; Time; Tissues; Toxic effect; Transcript; Translations; Trichophyton; Vagina; Vesicle; Virus Diseases; Work; Yeast Model System; Yeasts; aqueous; base; clinically relevant; effective therapy; fungus; gene function; gene gun; graduate student; innovation; iron oxide; irradiation; keratinocyte; knock-down; molecular carrier; monolayer; nanoGold; nanoparticle; novel; novel strategies; older patient; pathogenic fungus; side effect; tool; undergraduate student; wound

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.

这个多学科的研究项目将开发一种新的方法来提供抗真菌药物， 相关真菌的研究。我们的长期目标是推进新的方法 治疗由多药耐药酵母菌引起的真菌感染，特别是皮肤和粘膜的真菌感染 如耳念珠菌或嗜酸性皮肤真菌如红色毛癣菌。本提案的目的 是开发双链RNA（dsRNA）作为抗真菌剂;其中dsRNA将靶向并抑制 导致细胞死亡的重要真菌基因的翻译。然而，dsRNA不能容易地穿透真菌， 细胞壁，其由几丁质、葡聚糖、甘露聚糖和糖蛋白组成。核心假设是， 根据我们自己的初步数据，我们可以在真菌细胞中产生短暂的断裂， 使用由用红外线照射金纳米颗粒（AuNPs）产生的空化和冲击波来形成壁 飞秒（fs）激光脉冲。其基本原理是，抑制这些必需基因将导致 降低真菌的生长和/或生存力。与此同时，没有已知的机制， 细胞可以对dsRNA产生抗性。拟议研究的目标将通过以下三个方面实现： 独立的具体目标：1。通过激光活化纳米颗粒的修饰递送致死dsRNA （LANP）在C. albicans和T.红色; 2.纳米颗粒尺寸和组成对纳米颗粒的影响的评估 通过局域网进行通信;以及3.评估LANP在人角质形成细胞中的毒性，以及在 抑制真菌生物膜。这项建议是创新的，因为它使用了一种完全不同的方法， 通过使用（a）dsRNA作为抗真菌剂和（B）激光活化纳米颗粒来促进抑制真菌细胞 dsRNA的细胞内递送。这项研究是有意义的，因为它可以导致新的发展， 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基因编辑系统递送至真菌细胞，并可广泛用于 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. 使用RNAi，这在芽殖酵母中几乎没有探索过。最后，这一问题的多面性 研究将培养学生的创造力，并鼓励学生在不同学科之间进行合作。