LIST: A Newly Developed Laser-assisted Cell Bioprinting Technology.

Cell bioprinting technologies aim to fabricate tissue-like constructs by delivering biomaterials layer-by-layer. Bioprinted constructs can reduce the use of animals in drug development and hold promise for addressing the shortage of organs for transplants. We recently introduced a laser-assisted drop-on-demand bioprinting technology termed Laser Induced Side Transfer (LIST). This technology can print delicate cell types, including primary neurons. This bioprinting protocol includes the following key steps: cell harvesting, bio-ink preparation, laser setup priming, printing, and post-printing analysis. This protocol includes a detailed description of the laser setup, which is a rather unusual setup for a biology lab. This should allow easy reproduction by readers with basic knowledge of optics. Although we have focused on neuron bioprinting, interested readers will be able to adapt the protocol to bioprint virtually any cell type.

Development and ex-vivo validation of 36G polyimide cannulas integrating a guiding miniaturized OCT probe for robotic assisted subretinal injections.

We introduced and validated a method to encase guiding optical coherence tomography (OCT) probes into clinically relevant 36G polyimide subretinal injection (SI) cannulas. Modified SI cannulas presented consistent flow capacity and tolerated the typical mechanical stress encountered in clinical use without significant loss of sensitivity. We also developed an approach that uses a micromanipulator, modified SI cannulas, and an intuitive graphical user interface to enable precise SI. We tested the system using ex-vivo porcine eyes and we found a high SI success ratio 95.0% (95% CI: 83.1-99.4). We also found that 75% of the injected volume ends up at the subretinal space. Finally, we showed that this approach can be applied to transform commercial 40G SI cannulas to guided cannulas. The modified cannulas and guiding approach can enable precise and reproducible SI of novel gene and cell therapies targeting retinal diseases.

Nanophotonics Enable Targeted Photothermal Silencing of Nociceptor Neurons.

The sensory nervous and immune systems work in concert to preserve homeostasis. While this endogenous interplay protects from danger, it may drive chronic pathologies. Currently, genetic engineering of neurons remains the primary approach to interfere selectively with this potentially deleterious interplay. However, such manipulations are not feasible in a clinical setting. Here, this work reports a nanotechnology-enabled concept to silence subsets of unmodified nociceptor neurons that exploits their ability to respond to heat via the transient receptor potential vanilloid type 1 (TRPV1) channel. This strategy uses laser stimulation of antibody-coated gold nanoparticles to heat-activate TRPV1, turning this channel into a cell-specific drug-entry port. This delivery method allows transport of a charged cationic derivative of an N-type calcium channel blocker (CNCB-2) into targeted sensory fibers. CNCB-2 delivery blocks neuronal calcium currents and neuropeptides release, resulting in targeted silencing of nociceptors. Finally, this work demonstrates the ability of the approach to probe neuro-immune crosstalk by targeting cytokine-responsive nociceptors and by successfully preventing nociceptor-induced CD8+ T-cells polarization. Overall, this work constitutes the first demonstration of targeted silencing of nociceptor neuron subsets without requiring genetic modification, establishing a strategy for interfering with deleterious neuro-immune interplays.

Neuro-Immunity and Gut Dysbiosis Drive Parkinson's Disease-Induced Pain.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting 1-2% of the population aged 65 and over. Additionally, non-motor symptoms such as pain and gastrointestinal dysregulation are also common in PD. These impairments might stem from a dysregulation within the gut-brain axis that alters immunity and the inflammatory state and subsequently drives neurodegeneration. There is increasing evidence linking gut dysbiosis to the severity of PD's motor symptoms as well as to somatosensory hypersensitivities. Altogether, these interdependent features highlight the urgency of reviewing the links between the onset of PD's non-motor symptoms and gut immunity and whether such interplays drive the progression of PD. This review will shed light on maladaptive neuro-immune crosstalk in the context of gut dysbiosis and will posit that such deleterious interplays lead to PD-induced pain hypersensitivity.

A Smart Vitrector Equipped by a Fiber-Based OCT Sensor Mitigates Intentional Attempts at Creating Iatrogenic Retinal Breaks During Vitrectomy in Pigs.

Purpose: The occurrence of iatrogenic retinal breaks (RB) in pars plana vitrectomy (PPV) is a complication that compromises the overall efficacy of the surgery. A subset of iatrogenic RB occurs when the retina (rather than the vitreous gel) is cut accidentally by the vitrector. We developed a smart vitrector that can detect in real-time potential iatrogenic RB and activate promptly a PPV machine response to prevent them. Methods: We fabricated the smart vitrectors by attaching a miniaturized fiber-based OCT sensor on commercial vitrectors (25G). The system's response time to an iatrogenic RB onset was measured and compared to the literature reported physiologically limited response time of the average surgeon. Two surgeons validated its ability to prevent simulated iatrogenic RB by performing PPV in pigs. Note that the system is meant to control the PPV machine and requires no visual or audio signal interpretation by the surgeons. Results: We found that the response time of the system (28.9 ± 6.5 ms) is 11 times shorter compared to the literature reported physiologically limited reaction time of the average surgeon (P < 0.0001). Ex vivo validation (porcine eyes) showed that the system prevents 78.95% (15/19) (95% confidence interval [CI] 54.43-93.95) of intentional attempts at creating RB, whereas in vivo validation showed that the system, prevents 55.68% (30/54) (95% CI 41.40-69.08), and prevents or mitigates 70.37% (38/54) (95% CI 56.39-82.02) of such attempts. A subset of failures was classified as "early stop" (i.e., false positive), having a prevalence of 5.26% (1 /19) in ex vivo tests and 24.07% (13/54) in in vivo tests. Conclusions: Our results indicate the smart vitrector can prevent iatrogenic RB by providing seamless intraoperative feedback to the PPV machine. Importantly, the use of the smart vitrector requires no modifications of the established PPV procedure. It can mitigate a significant proportion of iatrogenic RB and thus improve the overall efficacy of the surgery. Translational Relevance: Potential clinical adoption of the smart vitrector can reduce the incidence of iatrogenic RB in PPV and thus increase the therapeutic outcome of the surgery.

Bioprinting of Adult Dorsal Root Ganglion (DRG) Neurons Using Laser-Induced Side Transfer (LIST).

Cell bioprinting technologies aim to fabricate tissuelike constructs by delivering biomaterials layer-by-layer. Bioprinted constructs can reduce the use of animals in drug development and hold promise for addressing the shortage of organs for transplants. Here, we sought to validate the feasibility of bioprinting primary adult sensory neurons using a newly developed laser-assisted cell bioprinting technology, known as Laser-Induced Side Transfer (LIST). We used dorsal root ganglion neurons (DRG; cell bodies of somatosensory neurons) to prepare our bioink. DRG-laden- droplets were printed on fibrin-coated coverslips and their viability, calcium kinetics, neuropeptides release, and neurite outgrowth were measured. The transcriptome of the neurons was sequenced. We found that LIST-printed neurons maintain high viability (Printed: 86%, Control: 87% on average) and their capacity to release neuropeptides (Printed CGRP: 130 pg/mL, Control CGRP: 146 pg/mL). In addition, LIST-printed neurons do not show differences in the expressed genes compared to control neurons. However, in printed neurons, we found compromised neurite outgrowth and lower sensitivity to the ligand of the TRPV1 channel, capsaicin. In conclusion, LIST-printed neurons maintain high viability and marginal functionality losses. Overall, this work paves the way for bioprinting functional 2D neuron assays.

Drop-on-demand cell bioprinting via Laser Induced Side Transfer (LIST).

We introduced and validated a drop-on-demand method to print cells. The method uses low energy nanosecond laser (wavelength: 532 nm) pulses to generate a transient microbubble at the distal end of a glass microcapillary supplied with bio-ink. Microbubble expansion results in the ejection of a cell-containing micro-jet perpendicular to the irradiation axis, a method we coined Laser Induced Side Transfer (LIST). We show that the size of the deposited bio-ink droplets can be adjusted between 165 and 325 µm by varying the laser energy. We studied the corresponding jet ejection dynamics and determined optimal conditions for satellite droplet-free bioprinting. We demonstrated droplet bio-printing up to a 30 Hz repetition rate, corresponding to the maximum repetition rate of the used laser. Jet ejection dynamics indicate that LIST can potentially reach 2.5 kHz. Finally, we show 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. Sample preparation is uncomplicated in LIST, while with further development bio-ink multiplexing can be attained. LIST could be widely adapted for applications requiring multiscale bioprinting capabilities, such as the development of 3D drug screening models and artificial tissues.

Etching-enabled extreme miniaturization of graded-index fiber-based optical coherence tomography probes.

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.

A needle-like optofluidic probe enables targeted intracellular delivery by confining light-nanoparticle interaction on single cell.

Intracellular delivery of molecular cargo is the basis for a plethora of therapeutic applications, including gene therapy and cancer treatment. A very efficient method to perform intracellular delivery is the photo-activation of nanomaterials that have been previously directed to the cell vicinity and bear releasable molecular cargo. However, potential in vivo applications of this method are limited by our ability to deliver nanomaterials and light in tissue. Here, we demonstrate intracelullar delivery using a needle-like optofluidic probe capable of penetrating soft tissue. Firstly, 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. Secondly, we delivered nanosecond (ns) laser pulses (wavelength: 532 nm; duration: 5 ns) using the same probe and without introducing a AuNP cells incubation step. The 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. Finally, we demonstrated targeted delivery in tissue (mouse retinal explant) ex vivo. We expect that this method can enable nanomaterial-assisted intracellular delivery applications in soft tissue (e.g. brain, retina) of small animals.

Multiscale modeling of plasmonic enhanced energy transfer and cavitation around laser-excited nanoparticles.

Nanoscale bubbles generated around laser-excited metallic nanoparticles are promising candidates for targeted drug and gene delivery in living cells. The development of new nanomaterials for efficient nanobubble-based therapy is however limited by the lack of reliable computational approaches for the prediction of their size and dynamics, due to the wide range of time and space scales involved. In this work, we present a multiscale modeling framework that segregates the various channels of plasmon de-excitation and energy transfer to describe the generation and dynamics of plasmonic nanobubbles. Detailed comparison with time-resolved shadowgraph imaging and spectroscopy data demonstrates that the bubble size, dynamics, and formation threshold can be quantitatively predicted for various types of nanostructures and irradiation parameters, with an error smaller than the experimental uncertainty. Our model in addition provides crucial physical insights into non-linear interactions in the near-field that should guide the experimental design of nanoplasmonic materials for nanobubble-based applications in nanomedicine.

Photon-induced generation and spatial control of extreme pressure at the nanoscale with a gold bowtie nano-antenna platform.

Precise spatial and temporal control of pressure stimulation at the nanometer scale is essential for the fabrication and manipulation of nano-objects, and for exploring single-molecule behaviour of matter under extreme conditions. However, state-of-the-art nano-mechanical transducers require sophisticated driving hardware and are currently limited to moderate pressure regimes. Here we report a gold plasmonic bowtie (AuBT) nano-antennas array that can generate extreme pressure stimulus of ∼100 GPa in the ps (10-12 s) time scale with sub-wavelength resolution upon irradiation with ultra-short laser pulses. Our method leverages the non-linear interaction of photons with water molecules to excite a nano-plasma in the plasmon-enhanced near-field and induce extreme thermodynamic states. The proposed method utilizes laser pulses, which in contrast to micro- and nano-mechanical actuators offers simplicity and versatility. We present time-resolved shadowgraphic imaging, electron microscopy and simulation data that suggest that our platform can efficiently create cavitation nano-bubbles and generate intense pressure in specific patterns, which can be controlled by the selective excitation of plasmon modes of distinct polarizations. This novel platform should enable probing non-invasively the mechanical response of cells and single-molecules at time and pressure regimes that are currently difficult to reach with other methods.

Rational Design of Plasmonic Nanoparticles for Enhanced Cavitation and Cell Perforation.

Metallic nanoparticles are routinely used as nanoscale antenna capable of absorbing and converting photon energy with subwavelength resolution. Many applications, notably in nanomedicine and nanobiotechnology, benefit from the enhanced optical properties of these materials, which can be exploited to image, damage, or destroy targeted cells and subcellular structures with unprecedented precision. Modern inorganic chemistry enables the synthesis of a large library of nanoparticles with an increasing variety of shapes, composition, and optical characteristic. However, identifying and tailoring nanoparticles morphology to specific applications remains challenging and limits the development of efficient nanoplasmonic technologies. In this work, we report a strategy for the rational design of gold plasmonic nanoshells (AuNS) for the efficient ultrafast laser-based nanoscale bubble generation and cell membrane perforation, which constitute one of the most crucial challenges toward the development of effective gene therapy treatments. We design an in silico rational design framework that we use to tune AuNS morphology to simultaneously optimize for the reduction of the cavitation threshold while preserving the particle structural integrity. Our optimization procedure yields optimal AuNS that are slightly detuned compared to their plasmonic resonance conditions with an optical breakdown threshold 30% lower than randomly selected AuNS and 13% lower compared to similarly optimized gold nanoparticles (AuNP). This design strategy is validated using time-resolved bubble spectroscopy, shadowgraphy imaging and electron microscopy that confirm the particle structural integrity and a reduction of 51% of the cavitation threshold relative to optimal AuNP. Rationally designed AuNS are finally used to perforate cancer cells with an efficiency of 61%, using 33% less energy compared to AuNP, which demonstrate that our rational design framework is readily transferable to a cell environment. The methodology developed here thus provides a general strategy for the systematic design of nanoparticles for nanomedical applications and should be broadly applicable to bioimaging and cell nanosurgery.

Cell perforation mediated by plasmonic bubbles generated by a single near infrared femtosecond laser pulse.

We report on transient membrane perforation of living cancer cells using plasmonic gold nanoparticles (AuNPs) enhanced single near infrared (NIR) femtosecond (fs) laser pulse. Under optimized laser energy fluence, single pulse treatment (τ = 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. AuNP clustering increased drastically the bubble generation efficiency, thus enabling an effective laser treatment using low energy dose in the NIR optical therapeutical window.

Cell-specific optoporation with near-infrared ultrafast laser and functionalized gold nanoparticles.

Selective targeting of diseased cells can increase therapeutic efficacy and limit off-target adverse effects. We developed a new tool to selectively perforate living cells with functionalized gold nanoparticles (AuNPs) and near-infrared (NIR) femtosecond (fs) laser. The receptor CD44 strongly expressed by cancer stem cells was used as a model for selective targeting. Citrate-capped AuNPs (100 nm in diameter) functionalized with 0.01 orthopyridyl-disulfide-poly(ethylene glycol) (5 kDa)-N-hydroxysuccinimide (OPSS-PEG-NHS) conjugated to monoclonal antibodies per nm(2) and 5 µM HS-PEG (5 kDa) were colloidally stable in cell culture medium containing serum proteins. These AuNPs attached mostly as single particles 115 times more to targeted CD44(+) MDA-MB-231 and CD44(+) ARPE-19 cells than to non-targeted CD44(-) 661W cells. Optimally functionalized AuNPs enhanced the fs laser (800 nm, 80-100 mJ cm(-2) at 250 Hz or 60-80 mJ cm(-2) at 500 Hz) to selectively perforate targeted cells without affecting surrounding non-targeted cells in co-culture. This novel highly versatile treatment paradigm can be adapted to target and perforate other cell populations by adapting to desired biomarkers. Since living biological tissues absorb energy very weakly in the NIR range, the developed non-invasive tool may provide a safe, cost-effective clinically relevant approach to ablate pathologically deregulated cells and limit complications associated with surgical interventions.

Dynamic imaging of a single gold nanoparticle in liquid irradiated by off-resonance femtosecond laser.

Plasmonic nanoparticles can lead to extreme confinement of the light in the near field. This unique ability of plasmonic nanoparticles can be used to generate nanobubbles in liquid. In this work, we demonstrate 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. We have employed combined ultrafast shadowgraphic imaging, in situ dark field imaging and dynamic tracking of AuNP Brownian motion to ensure the study of individual AuNPs/nanolenses under multiple fs laser pulses. We demonstrate 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 for future development of in vivo AuNP-assisted laser nanosurgery and theranostic applications, where AuNP fragmentation should be avoided to prevent side effects, such as cytotoxicity and immune system's response. The experimental results have been correlated with theoretical modeling to provide an insight to the AuNP-safe cavitation mechanism as well as to investigate the deformation mechanism of the AuNPs at high laser fluences.

A photosynthetic biosensor with enhanced electron transfer generation realized by laser printing technology.

One of the limits of current electrochemical biosensors is a lack of methods providing stable and highly efficient junctions between biomaterial and solid-state devices. This paper shows how laser-induced forward transfer (LIFT) can enable efficient electron transfer from photosynthetic biomaterial immobilized on screen-printed electrodes (SPE). The ideal pattern, in terms of photocurrent signal of thylakoid droplets giving a stable response signal with a current intensity of approximately 335 ± 13 nA for a thylakoid mass of 28 ± 4 ng, was selected. It is shown that the efficiency of energy production of a photosynthetic system can be strongly enhanced by the LIFT process, as demonstrated by use of the technique to construct an efficient and sensitive photosynthesis-based biosensor for detecting herbicides at nanomolar concentrations.

Detection of DNA mutations using a capacitive micro-membrane array.

The detection of DNA hybridization using capacitive readout and a biosensor array of ultrathin Si membranes is presented. The biosensor exploits the ability of the ultrathin membranes to deflect upon surface stress variations caused by biological interactions. Probe DNA molecules are immobilized on the membrane surface and the surface stress variations during hybridization with their complementary strands force the membrane to deflect and effectively change the capacitance between the flexible membrane and the fixed substrate. The sensor array comprises 256 such sensing sites thus allowing the concurrent sensing of multiple DNA mutations. The biosensor and its performance for the detection of complementary DNA strands are demonstrated using beta-thalassemia oligonucleotides. The experimental results show that the presented sensors are able to detect DNA hybridization and to discriminate single nucleotide mismatches.