Despite impressive strides in light-enabled stimulation, approaches to silence targeted unmodified neurons subsets are yet to be devised. We will present a novel method to silence unmodified neurons via photothermal-mediated targeted drug delivery. Our approach uses laser-stimulation of antibody-coated gold nanoparticles to heat-activate TRPV1 (a noxious heat-sensing channel), turning this channel into a cell-specific drug-entry port. We will show that photothermal silencing blocks neuronal calcium currents and peptide release, resulting in targeted silencing of nociceptors. This approach can help to elucidate the contribution of nociceptor subpopulations to autoimmune pathologies and with further efforts can be translated into novel treatments in neuroscience.
The ability to generate 3D angiogenesis models is central for tissue engineering and drug screening applications. However, existing bio-fabrication technologies have yet to attain precise guidance of capillary networks in 3D. Here, we present our latest results in fabricating capillary networks using a novel laser-assisted bioprinting technique named Laser Induced Side Transfer (LIST). We found that LIST-printed human umbilical vein endothelial cells (HUVECs) present negligible loss of viability and maintain their abilities to migrate, proliferate and form intercellular junctions. Furthermore, we showed that LIST enabled the formation of capillary-like networks in 3D with high spatial precision (50 μm) over a large volume (1 cm3). Those networks were validated as angiogenesis assays for pro- and anti-angiogenic compounds. LIST could be widely adapted for applications requiring multiscale bioprinting capabilities, like the development of 3D drug screening models and artificial tissues.
We introduced and validated a method to miniaturize graded-index (GRIN) fiber-based optical coherence tomography (OCT) probes down to 70 μm in diameter. The probes consist in an assembly of single-mode (SM), coreless (CL), and graded-index (GRIN) fibers. We opted for a probe design enabling controlled size reduction by hydrogen fluoride etching. The fabrication approach prevents nonuniform etching for both the GRIN and SM fiber components, while it requires no probe polishing postetching. We found that the miniaturized probes present insignificant loss of sensitivity (∼1 dB) compared to their thicker (125 μm) counterparts. We also showed that their focusing capabilities remain tunable and highly predictable. The fabrication process is simple and can be carried out by using inexpensive telecom equipment. Both the fabrication process and the developed probes can benefit the prototyping of minimally invasive endoscopic tools.
Intracellular delivery of molecular cargo is the basis for a plethora of therapeutic applications, including gene therapy and cancer treatment. An efficient method to perform intracellular delivery is the photoactivation of nanomaterials that bear releasable molecular cargo. However, potential in vivo applications of this method are limited by our ability to deliver nanomaterials and light in tissue.
In this paper, we will present method to perform intracellular delivery of molecular cargo on live cells and tissue by using a reusable, needle-like optofluidic probe capable of penetrating soft tissue. The probe consists of a dual-core glass fiber, enabling simultaneous light and liquid delivery. First, we used the optofluidic probe to confine an intracellular delivery mixture, composed of 100 nm gold nanoparticles (AuNP) and membrane-impermeable calcein, in the vicinity of cancer cells and mouse retinal explants. Secondly, we delivered nanosecond (ns) laser pulses (wavelength: 532 nm; duration: 5 ns; 30-90 mJ/cm2) using the same probe and without introducing a AuNP cells incubation step.
We found that AuNP photo-activation caused localized and reversible disruption of the cell membrane, enabling calcein delivery into the cytoplasm. We measured 67% intracellular delivery efficacy and showed that the optofluidic probe can be used to treat cells with single-cell precision.
The method presented here can facilitate in vivo treatments in soft tissue of small animals (e.g. brain, retina), such us nanomaterial-assisted neuro-stimulation, transfection and tumor elimination.
Vapor nanobubbles generated around plasmonic nanoparticles (NPs) by ultrafast laser irradiation are efficient for inducing localized damage to living cells. Killing targeted cancer cells or gene delivery can therefore be envisioned using this new technology [1,2]. The extent of the damage and its non-lethal character are linked to the size of the nanobubble. Precise understanding of the mechanisms leading to bubble formation around plasmonic nanostructures is necessary to optimize the technique. In this presentation, we present a complete model that successfully describes all interactions occurring during the irradiation of plasmonics nanostructures by an ultrafast laser of various pulse widths and fluences.
Nanoavitation is caused by the interplay between heat conduction at the NP-medium interface and non-linear plasmon-enhanced photoionization of a nanoplasma in the near-field [3-5], the former being dominant for in-resonance and the latter for off-resonance irradiation. Modeling of the whole laser-nanoparticle interaction, together with the help of the shadowgraphic imaging and scattering techniques [3-5], give valuable insight on the mechanisms of cavitation at the nanoscale, leading to possible optimization of the nanostructure for bubble-based nanomedicine applications.
1- E. Boulais, R. Lachaine, A. Hatef, and M. Meunier, Journal of Photochemistry and Photobiology C: Photochemistry Reviews 17, 26-49 (2013).
2- E. Bergeron, S. Patskovsky, D. Rioux, and M. Meunier, Nanoscale 7,17836-17847 (2015).
3- E. Boulais, R. Lachaine, and M. Meunier, Nano Letters 12, 4763-4769 (2012).
4- R. Lachaine, E. Boulais, and M. Meunier, ACS Photonics 1, 331-336 (2014).
5- C. Boutopoulos, A. Hatef, M. Fortin-Deschênes, and M. Meunier Nanoscale 7,11758-11765 (2015).
Gold and silver plasmonic nanoparticles (NPs) are widely used as a contrast agent for photoacoustic (PA) imaging, taking advantage of the strong optical absorption cross-section of these particles due to their localized surface-plasmon resonance. Inspired by recent developments in ultra-high frequency wide-bandwidth transducers, we propose utilizing off-resonance ultrashort laser sources with a pulse width in the femtosecond (fs) and picosecond (ps) range to increase the efficiency of PA imaging. Also, from the fact that the laser pulse duration is shorter than the heat diffusion time of the materials, we expect practically no collateral damage of the laser irradiated biological tissues. Our preliminary studies show that irradiating the NPs with an ultrashort-pulsed laser has the potential to achieve substantially higher efficiency at generating the PA signal. Enhanced by the presence of NPs, the laser field causes a highly localized plasma nucleation around the vicinity of the NPs. Plasma relaxes through electron-ion interaction and releases a pressure wave in the surrounding medium. However, in this process, it is crucial to precisely control the heat energy absorption in the NPs to avoid their fragmentation. In this talk we present a model to simulate an optimized plasma–mediated PA signal dynamics generated from off-resonance ultrashort laser excitation (λ =800 nm, τ = 70 fs – 2 ps) of a variety of plasmonic NPs with sizes ranging from 50 nm to 100 nm.
Gold nanoparticles (AuNPs) have found numerous applications in nanomedicine in view of their robustness, ease of functionalization and low toxicity. Upon irradiation of AuNPs by a pulsed ultrafast laser, various highly localized phenomena can be obtained including a temperature rise, pressure wave, charge injection and production of nanobubbles close to the cellular membrane [1]. These phenomena can be used to manipulate, optoperforate, transfect and stimulate targeted cells [2-5]. Irradiating at 800 nm in the optically biological transparent window, we demonstrated local optoporation and transfection of cells as well as local stimulation of neurons. Two recent examples will be given: (i) Laser-induced selective optoporation of cells: The technique can be used on various types of cells and a proof of principle will be given on human cancer cells in a co-culture using functionalized AuNPs [6]. (ii) Laser-induced stimulation of neurons and monitoring of the localized Ca2+ signaling: This all optical method uses a standard confocal microscope to trigger a transient increase in free Ca2+ in neurons covered by functionalized AuNPs as well as to measure these local variations optically with the Ca2+ sensor GCaMP6s [7]. The proposed techniques provide a new complement to light-dependent methods in neuroscience. REFERENCES (by our group): (1) Boulais, J. Photochem. Photobiol. C Photochem. Rev. 17, 26 (2013); (2) Baumgart, Biomaterials 33, 2345 (2012); (3) Boulais, NanoLett. 12, 4763 (2012); (4) Boutopoulos, J. Biophotonics (2015); (5) Boutopoulos, Nanoscale 7, 11758 (2015); (6) Bergeron, Biomaterials, submitted (2015); (7) Lavoie-Cardinal, Nature Commun. submitted (2015).
Femtosecond (fs) laser generation of submicron bubbles around plasmonic nanoparticles (NPs) plays a key role in
advanced laser nanosurgery applications such as cell membrane perforation and cell transfection. In this context, we have
developed a pump-probe shadowgraphic ultrafast imaging technique capable of tracking transient bubbles generated by
fs irradiation (λ =800 nm, τ = 45 fs) of: (a) plasmonic NPs in suspensions and (b) plasmonic NPs attached to cells. The
laser fluence was systematically varied from 100 mJ/cm2 to 500 mJ/cm2 to study the effect on the bubble dynamics
generated around 100 nm gold NPs (Au NPs). The plasmonic bubble (PB) generation threshold as well as the NPs
deformation threshold were defined. Dark field (DF) imaging and scanning electron microscopy (SEM) revealed NP
clustering when 100 nm bare Au NPs were incubated with a cancer cell culture. NP clustering was correlated with PB
generation using a combined pump-probe and DF imaging approach. The clustering effect resulted in a significant 4-
times decrease to the PB generation threshold compared to single NPs. The clustering effect was further investigated by
evaluating bare and polyethylene glycol (PEG) functionalized NPs in terms of PB generation efficiency.
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