Laser-assisted Delivery of Molecules

into Fungal Cells

 

Fungal infections are becoming increasingly clinically relevant, but current methods to treat them are limited. Many fungi are resistant to available antifungal drugs, and other molecule-delivery methods, such as electroporation, are only designed for research techniques. Delivering molecules into fungal cells can be quite tricky due to the presence of a rigid carbohydrate cell wall. Femtosecond lasers are more frequently being used as a combinatorial method for delivering small molecules. The femtosecond laser creates ultrafast pulses of photons that interact with nanoparticles in solution, which ultimately results in the formation of pores in the cell wall or membrane, a process called photoporation. The exact mechanisms of photoporation is still being studied, but recent studies have found it is likely a combination of indirect effects such as cavitation, shockwaves, and other photo-chemical, -thermal, or -mechanical phenomena.

 

Until this point, the process of photoporation has exclusively been studied in mammalian cells. We demonstrated that in Saccharomyces cerevisiae, a single-celled yeast, this combinatorial approach was able to effectively deliver small fluorescent molecules and plasmid DNA through the cell wall. The gold nanoparticles were also synthesized through photoreduction chemistry which removed the need for harsh reducing agents and creation of cytotoxic oxidized products. Nearly 60% of cells were successfully irradiated and alive when analyzed via flow cytometry, and laser conditions where both yeast and mammalian CHO cells maintained viability. This method has potential for further preclinical optimizations for oral, vaginal, and skin mycoses.

Figure 2. Poration of S. cerevisiae and Chinese Hamster Ovary (CHO) cells. (A) Flow cytometry data of fluorescently positive viable cells under either varying laser fluence or irradiation time. (B) Confocal micrographs of photoporated S. cerevisiae and CHO cells irradiated in the presence of calcein.

Figure 1. Diagram of photoporation phenomena. When the femtosecond laser pulses activate the nanoparticle, a series of effects are triggered, such as cavitation and shock waves. These effect concuss the cells, creating pores that allow DNA and other molecules to enter.