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Final Report Summary - LIGHT2NANOGENE (Cellular bioengineering by plasmonic enhanced laser nanosurgery)

The Light2NanoGene project aims to the development and optimization of a novel technique for nanosurgery of living cells. The technique, named plasmonic enhanced laser nanosurgery, combines the advantages of two rapidly expanding research and technological fields, namely plasmonics and ultrafast lasers, to build a versatile tool capable of performing high throughput cell nanosurgery. The main innovative goal of the project is the optical fiber integration of the plasmonic nanosurgery tool towards in-vivo (i.e., living subject) applications. In-vitro cell transfection (i.e., introduction of exogenous material (siRNA, fluorescence molecules) through the membrane of breast cancer stem cells (CSCs)) is the specific nanosurgery application of the Light2NanoGene project.
The work plan is organized in five work packages, aiming to the development and optimization of the plasmonic nanoparticles assisted laser nanosurgery technology. Briefly, the research methodology is organized towards the accomplishment of three main objectives: (a) Selection of the most efficient plasmonic material for laser nanosurgery by employing theoretical modelling and experimental investigations of their interactions with laser pulses. (b) Intracellular delivery of exogenous material (fluorescence molecules, siRNA) into living cancer cells using optimal nanomaterials. (c) Integration of the developed technology towards a fiber-based, portable and low-cost laser system for cell nanosurgery.
So far the following results have been achieved (published or submitted for publication results as on September 2nd, 2016):
The interaction of ultrafast lasers with nanoparticles can lead to nanoscale localization of energy deposition in liquid and eventually to nanobubble generation. Nanobubbles are transient, nanoscale carriers of dynamic energy that find applications in cancer cell killing and cell transfection. The detection and characterization of nanobubbles is challenging due to their extreme spatiotemporal properties. In the framework of the Light2NanoGene project, an advanced imaging method for studying ultrafast laser-nanoparticle interactions and nanobubbles in a single particle level was developed for the first time [CV: J4. C. Boutopoulos et al., Nanoscale, 7 11758-11765 (2015)]. The technique enables the (a) detection and quantification of nanobubble (radius ≥ 500 nm, lifetime ≥ 30 nm) and (b) in-situ monitoring of the nanoparticle integrity upon laser irradiation. Compared to existing detection systems, the method allows for acquiring data sets from single nanoparticles for the first time, which is crucial to validate nanobubble theoretical modelling frameworks. In fact, we used the method to (a) screen a large library of nanoparticles towards the optimization of the experimental settings for cancer cell perforation [CV: J1. R. Lachaine, C. Boutopoulos et al., Nano Letters, 16 3187-3194 (2016)], and (b) to develop and validate a multiscale theoretical model, aiming to predict and optimize nanobubble dynamics for biomedical applications [paper under review in Nature Photonics]. This research work has provided new insights in the physical mechanisms of ultrafast laser-nanoparticle interactions, which are of great importance for scaling up the biomedical applications of nanobubbles.
Furthermore, we demonstrated with single-particle monitoring that 100 nm gold nanoparticles (AuNPs) irradiated by off-resonance femtosecond (fs) laser in the tissue therapeutic optical window (λ = 800 nm), can act as a durable nanolenses in liquid and provoke nanocavitation while remaining intact [Boutopoulos, C.; Hatef, A.; Fortin-Deschênes, M.; Meunier, M. Nanoscale 2015, 7 (27), 11758–11765]. We showed that 100 nm AuNPs can generate multiple, highly confined (radius down to 550 nm) and transient (life time < 50 ns) nanobubbles. The latter is of significant importance towards in vivo application of the AuNP-assisted laser nanosurgery, where AuNP fragmentation should be avoided to prevent side effects, such as cytotoxicity and immune system’s response.
In [CV: J3. C. Boutopoulos et al, Journal of Biophotonics, 9 26-31 (2016)], we demonstrated reversible perforation of the membrane of human breast cancer cells using nanobubbles generated by AuNPs enhanced single near infrared (NIR) femtosecond (fs) laser pulse. We used significantly lower energy dose comparing to previous studies involving multiple pulse irradiation. Under optimized laser energy fluence, single pulse treatment (pulse width = 45 fs, λ = 800 nm) resulted in 77% cell perforation efficiency and 90% cell viability. Using dark field and ultrafast imaging, we demonstrated that the generation of submicron bubbles around the AuNPs is the necessary condition for the cell membrane perforation. The latter has been a major finding towards the understanding of the cell membrane perforation mechanism and the implementation of an effective laser therapeutic tool for safe in vivo gene therapy treatments.
Work has been performed towards the development of a fiber-based, portable and low-cost laser system for cell nanosurgery. The reader is referred to the Light2NanoGene web site for latest news and publications regarding to this topic. (http://cboutop.com/light2nanogene/)

Related information

Reported by

THE UNIVERSITY COURT OF THE UNIVERSITY OF ST ANDREWS
United Kingdom

Subjects

Life Sciences
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