An important reason for the decreased sensitivity of bacteria towards antibiotics is their capability to form so-called biofilms. The increased tolerance of sessile cells is multifactorial and includes reduced penetration rate and potency of antibiotics through dense biofilms. Strategies that have the ability to interfere with the integrity of biofilms and allowing a better penetration of antimicrobial agents are highly sought after. In this work, we explore the potential of laser-induced vapor nanobubbles (VNB) formed from plasmonic nanoparticles irradiated with nanosecond pulsed laser light to locally disturb biofilm integrity and improve antibiotics diffusion. Our results show that biofilms of both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria can be locally disrupted by the pressure waves from laser-induced VNB inside the biofilms. Most importantly, VNB-mediated biofilm disruption increased tobramycin efficacy up to 1-3 log orders of magnitude, depending on the treatment regimens and type of organism. In addition, we explored the use of VNB to enhance the efficacy of a broad range of antimicrobials used for treating wound infections, towards a first potential clinical application of the technique. Our results confirm that VNB-mediated biofilm disruption is an effective technique to enhance the activity of those antimicrobials that experience hindered diffusion in biofilms. Future work will extend the evaluation of this novel concept towards more complex multi-species biofilms and in vitro wound models before going to in vivo evaluations.
Macromolecular agents such as nucleic acids and proteins need to be delivered into living cells for therapeutic purposes. Among physical methods to deliver macromolecules across the cell membrane, laser-induced photoporation using plasmonic nanoparticles is a method that is receiving increasing attention in recent years. By irradiating gold nanoparticles bound to the cell membrane with laser light, nanosized membrane pores can be created. Pores are formed by localized heating or by vapour nanobubbles (VNBs) depending on the incident laser energy. Macromolecules in the surrounding cell medium can then diffuse through the transiently formed pores into the cytoplasm. While both heating and VNBs have been reported before for permeabilization of the cell membrane, it remains unclear which of both methods is more efficient in terms of cell loading with minimal cytotoxicity. In this study we report that under condition of a single 7 ns laser pulse VNBs are substantially more efficient for the cytosolic delivery of macromolecules. We conclude that VNB formation is an interesting photoporation mechanism for fast and efficient macromolecular delivery in live cells.
There is considerable interest in using Quantum Dots (QDs) as fluorescent probes such for cellular imaging due to unique advantages in comparison with conventional molecular dyes. However, cytosolic delivery of QDs into live cells remains a major challenge. Here we demonstrate highly efficient delivery of PEG-coated QDs into live cells by means of laser-induced vapour nanobubbles. Using this procedure we succeeded in high-throughput loading of ~80% of cells while maintaining a cell viability of ~85%.
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