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.
