Vapour nanobubbles – the golden ticket to the biofilm center

The hindered penetration of antibiotics through biofilms is one of the reasons for the alarming increase in bacterial tolerance to antibiotics and innovative approaches are required to prevent a true post-antibiotic era. One approach is interfering with the dense biofilm architecture in order to improve the penetration of antibiotics towards the deeper cell layers in biofilms. In this paper, we aimed to locally disrupt the biofilm integrity and improve the diffusion of antibiotics by nanoscale laser-induced bubbles (so-called vapour nanobubbles) created deep within the biofilm.

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Nov 08, 2018
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The story began 6 years ago, when we started using laser-induced vapour nanobubbles (VNB) to temporarily permeabilize the cell membrane of mammalian cells for the intracellular delivery of all kinds of exogenous molecules 1,2. In this technique, gold nanoparticles (AuNP) were deposited on the cell membrane of living cells, and after pulsed laser irradiation, nanosized pores were created in the membrane by the physical force exerted by VNB emerging from the photothermally heated AuNP.  Macromolecules, such as nucleic acids or proteins, in the surrounding cell medium could be introduced into living cells without noticeable toxicity. It did not take long to realize that the photoporation technique could deliver exogenous compounds in selected subpopulations of cells, even down to the single cell level 3,4. The spatio-controlled feature of this technique can be appreciated in Figure 1, where HeLa cells were photoporated with FITC-dextran (10 kDa) in such a way that fluorophores were only delivered into those cells corresponding to the drawing of Albert Einstein. 

Figure 1, adapted from Xiong, R. et al. Fast spatial-selective delivery into live cells. J. Control. Release 266, 198–204 (2017) 3.

At the same time we were also working to improve drug delivery to biofilms, mostly by nanomedicines, as a way to enhance drug penetration deep into dense biofilms. After finding out that nanoparticles below 0.1 µm are small enough to penetrate into dense biofilms 5, soon the idea arose that it could be possible to administer AuNP to biofilms and interfere with the biofilm structural integrity by the physical force exerted by laser-induced VNB. Due to the fine precise control of laser light, and the localized action of VNB, the technique would be highly spatio-controlled and, therefore, would have the potential to be applied to specific infected areas in the body. First proof-of-concept experiments were carried out on the gram-negative bacterium Burkholderia multivorans. Dark-field microscopy images showed that the localized shockwaves induced by VNB could subtly but significantly expand the space between sessile cells, which resulted in 80 times improvement of the efficacy of the antibiotic tobramycin.

Encouraged by these positive results, we selected two other pathogens that were more related to biofilm-related wound infections, as this would be a potential target application: Pseudomonas aeruginosa and Staphylococcus aureus. In both types of biofilms we observed similar effects, namely a VNB-induced biofilm disruption and increased tobramycin potency (about 25 times). Upon application of multiple laser pulses, VNB could be repeatedly formed, and biofilms became even more dispersed with further enhancement of tobramycin’s efficiency up to three orders of magnitude. Using fluorescent model molecules we could confirm that laser-induced VNB indeed cause a substantial increase in the penetration of molecules deep into the biofilm. As more cells become exposed to a high concentration of the applied antibiotic, this explains why it’s killing efficiency could be substantially enhanced. Importantly, while the laser-induced VNB increase the space between cells, we could show that bacteria are not released from the biofilm, which otherwise would give cause for concern to induce sepsis in clinical applications. 

In this era of rising antimicrobial resistance our findings show that, besides from searching for new and improved antimicrobial drugs, there is much to be gained even with existing drugs by improving their penetration into biofilms. By harnessing the power of laser light and nanotechnology, laser-induced VNB are an elegant new way to reach that goal. This work is a clear example of a fruitful cross-fertilization between two distinct fields in life sciences research, being the “nanotechnology field” on the one hand (team of Prof. Dr. Kevin Braeckmans, Biophotonic Imaging Group, Laboratory for General Biochemistry and Physical Pharmacy) and the “microbiology field” on the other hand (team of Prof. Dr. Tom Coenye, Laboratory of Pharmaceutical Microbiology). We are convinced that in order to tackle the highly complex health threats the world is facing today - such as cancer and bacterial resistance – multidisciplinary approaches will be key to arrive at innovative solutions!

Eline Teirlinck 1,2,

Prof. Dr. Kevin Braeckmans 1,2,3

Prof. Dr. Tom Coenye 4

1 Laboratory of General Biochemistry and Physical Pharmacy, University of Ghent, Ghent, Belgium, Centre for Nano- and Biophotonics, Ghent, Belgium, 3Université de Lille, IEMN UMR 8520 and Lab. Phys. Lasers Atomes & Mol. UMR 8523, Villeneuve d’Ascq, France, 4 Laboratory of Pharmaceutical Microbiology, University of Ghent, Ghent, Belgium,

You can find the link for the manuscript here: Laser-induced vapour nanobubbles improve drug diffusion and efficiency in bacterial biofilms

References:

1.           Xiong, R. et al. Comparison of Gold Nanoparticle Mediated Photoporation: Vapor Nanobubbles Outperform Direct Heating for Delivering Macromolecules in Live Cells. ACS Nano 8, 6288–6296 (2014).

2.           Xiong, R. et al. Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging. Nano Lett. 16, 5975–5986 (2016).

3.           Xiong, R. et al. Fast spatial-selective delivery into live cells. J. Control. Release 266, 198–204 (2017).

4.           Xiong, R. et al. Selective Labeling of Individual Neurons in Dense Cultured Networks With Nanoparticle-Enhanced Photoporation. Front. Cell. Neurosci. 12, 80 (2018).

5.           Forier, K. et al. Probing the size limit for nanomedicine penetration into Burkholderia multivorans and Pseudomonas aeruginosa biofilms. J. Control. Release 195, 21–28 (2014).


Go to the profile of Kevin Braeckmans

Kevin Braeckmans

Full professor, Ghent University

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