Mitsuhiro Terakawa

Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave

Plasmid DNA has been successfully delivered to mammalian cells by applying a nanosecond pulsed laser-induced stress wave (LISW). Cells exposed to a LISW were selectively transfected with plasmids coding for green fluorescent protein. It was also shown that transient, mild cellular heating (approximately 43 degrees C) was effective in improving the transfection efficiency.

Targeted DNA transfection into the mouse central nervous system using laser- induced stress waves

We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

Plasmonic and Mie scattering control of far-field interference for regular ripple formation on various material substrates

We present experimental and theoretical results on plasmonic control of far-field interference for regular ripple formation on semiconductor and metal. Experimental observation of interference ripple pattern on Si substrate originating from the gold nanosphere irradiated by femtosecond laser is presented. Gold nanosphere is found to be an origin for ripple formation. Arbitrary intensity ripple patterns are theoretically controllable by depositing desired plasmonic and Mie scattering far-field pattern generators. The plasmonic far-field generation is demonstrated not only by metallic nanostructures but also by the controlled surface structures such as ridge and trench structures on various material substrates.

In vitro gene transfer to mammalian cells by the use of laser-induced stress waves: Effects of stress wave parameters, ambient temperature, and cell type

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.

Growth of high spatial frequency periodic ripple structures on SiC crystal surfaces irradiated with successive femtosecond laser pulses

We present experimentally and theoretically the evolution of high spatial frequency periodic ripples (HSFL) fabricated on SiC crystal surfaces by irradiation with femtosecond laser pulses in a vacuum chamber. At early stages the seed defects are mainly induced by laser pulse irradiation, leading to the reduction in the ablation threshold fluence. By observing the evolution of these surface structures under illumination with successive laser pulses, the nanocraters are made by nanoablation at defects in the SiC surface. The Mie scattering by the nanoablated craters grows the periodic ripples. The number of HSFL is enhanced with increasing pulse number. At the edge of the laser spot the Mie scattering process is still dominant, causing the fabrication of HSFL. On the periphery of the spot SiC substrate remains a semiconductor state because the electron density in the SiC induced by laser irradiation is kept low. The HSFL observed is very deep in the SiC surface by irradiating with many laser pulses. These experimental results are well explained by 3D FDTD (three-dimensional finite-difference time-domain) simulation.

Femtosecond laser near-field nanoablation patterning using Mie resonance high dielectric constant particle with small size parameter

We demonstrate near-field nanohole patterning using a Mie resonance, small size parameter particle for nanofabrication technology regardless of substrate’s refractive index. Maximal enhancement factor and nearly smallest spot diameter among the same size dielectric particles are simultaneously obtainable on both low-refractive-index SiO2 and high-refractive-index Si substrates with a 200 nm particle of magnetic quadrupole resonance scattering mode (n∼2.7) at 400 nm excitation wavelength. Circular nanoholes with approximately 100 nm in diameter were fabricated on both substrates using a 200 nm amorphous TiO2 particle (n=2.66+0.024i) even with lower laser fluences than a half ablation threshold of the bare substrates.

Accelerated adhesion of grafted skin by laser-induced stress wave–based gene transfer of hepatocyte growth factor

Gene therapy using wound healing-associated growth factor gene has received much attention as a new strategy for improving the outcome of tissue transplantation. We delivered plasmid DNA coding for human hepatocyte growth factor (hHGF) to rat free skin grafts by the use of laser-induced stress waves (LISWs); autografting was performed with the grafts. Systematic analysis was conducted to evaluate the adhesion properties of the grafted tissue; angiogenesis, cell proliferation, and reepithelialization were assessed by immunohistochemistry, and reperfusion was measured by laser Doppler imaging as a function of time after grafting. Both the level of angiogenesis on day 3 after grafting and the increased ratio of blood flow on day 4 to that on day 3 were significantly higher than those in five control groups: grafting with hHGF gene injection alone, grafting with control plasmid vector injection alone, grafting with LISW application alone, grafting with LISW application after control plasmid vector injection, and normal grafting. Reepithelialization was almost completed on day 7 even at the center of the graft with LISW application after hHGF gene injection, while it was not for the grafts of the five control groups. These findings demonstrate the validity of our LISW-based HGF gene transfection to accelerate the adhesion of grafted skins.

Enhanced angiogenesis in grafted skins by laser-induced stress wave-assisted gene transfer of hepatocyte growth factor.

Treatment to increase secretion of growth factors related to angiogenesis by gene transfection is a promising therapeutic solution for improving the outcome of tissue transplantation. We attempted to deliver a therapeutic vector construct carrying the human hepatocyte growth factor (hHGF) gene to skin grafts of rats using laser-induced stress waves (LISWs), with the objective of enhancing their adhesion. First we delivered the hHGF gene to rat native skin in vivo to determine the optimum gene transfer conditions. We then transferred the hHGF gene to excised rat skins, with which autografting was performed. We found that the density and uniformity of neovascularities were significantly enhanced in the grafted skins that were transfected using LISWs. These results suggest the efficacy of this method to improve the outcome of skin grafting. To our knowledge, this is the first experimental demonstration of a therapeutic efficacy based on LISW-mediated gene transfection. Since the present method can be applied not only to various types of tissues but also to bioengineered tissues, this technique has the potential to contribute to progress in transplantation medicine and future regenerative medicine.

Femtosecond laser induced periodic surface structure on poly-L-lactic acid

Laser-induced periodic surface structure (LIPSS) is one of the most remarkable nanostructures formed only by a simple procedure of laser irradiation that enables to control cell behaviors. To the best of our knowledge, however, LIPSS formation on a scaffold-usable biodegradable polymer had not been succeede d probably due to relatively-low glass transition temperature and melting temperature of such polymers. In this study, we demonstrate LIPSS formation on a poly-L-lactic acid (PLLA), a versatile biodegradable polymer which has been widely used in clinical practice. Experimental results revealed that the repetition rate of femtosecond laser is one of the key parameters for LIPSS formation on PLLA, suggesting that thermal properties and photochemical reactions should be considered. The present study expands the potential of femtosecond laser processing for fabrication of highly-biocompatible scaffold in tissue engineering.

Dielectric microsphere mediated transfection using a femtosecond laser

We demonstrate the permeabilization of cell membranes by an enhanced optical field generated under polystyrene microspheres of 1000 nm diameter excited by a femtosecond laser pulse. Fluorescent molecules and short interfering RNA (siRNA) have been successfully delivered to many cells in the irradiated area by a single 80 fs laser pulse at 800 nm wavelength in the presence of antibody-conjugated polystyrene spheres. The ratios of the cells showing permeabilization were 38% and 21% for Fluorescein isothiocyanate-dextran and siRNA, respectively, at the laser fluence of 1.06 J/cm(2). The present method has advantages both in high throughput of many cell treatments and precise processing of minute areas on cell membranes.