KEYWORDS: In vitro testing, Laser therapeutics, Laser irradiation, Plasma, Laser energy, Laser tissue interaction, In vivo imaging, Pulsed laser operation, Defense and security, Medical research
Laser-mediated gene transfection is very attractive as a new method for targeted gene therapy because of its high spatial controllability of laser energy. Previously, we demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISW). In this study, we investigated the experimental conditions to increase transfection efficiency in vitro. By applying single-pulse LISW, transfection efficiency was increased with increasing laser fluence. Increase in the number of laser pulses increased transfection efficiency for laser fluences up to 1.3 J/cm2, but at higher fluences (>1.7 J/cm2), efficiency showed saturation tendency. Temperature dependence of transfection efficiency was also investigated.
We previously demonstrated in vitro that the simultaneous application of cellular heating and a laser-induced stress wave (LISW) enhanced the uptake of porfimer sodium (Photofrin) by cells. In this study, we attempted to apply this technique to gene transfer to cultured cells. LISW and/or a transient mild (~43°C) heating been applied to deliver plasmid coding for green fluorescent protein (EGFP) to NIH-3T3 cells. It was found that simultaneous application of an LISW and the heating significantly increased the transfection efficiency by a factor of 2.5 when compared with that for the cells treated with an LISW alone.
In order to enhance the efficacy of photodynamic therapy for skin diseases, we previously demonstrated that the simultaneous application of heating and a laser-induced stress wave (LISW) accelerated skin permeabilization of porfimer sodium (Photofirn) in in-vivo experiments. In this study, to investigate the uptake of Photofrin by cells within skin, we carried out in-vitro experiments using fibroblast-like cells. Immediately after replacing culture medium with 43 degrees C phosphate buffered saline solution of Photofrin in a well, the cells were exposed to a LISW that was generated by irradiation of a nanosecond laser pulse onto a target attached on the back of the well. Uptake of Photofrin within the cells was observed with a confocal laser scanning microscope, while the cells were stained with DAPI (4',6-diamidino-2-phenylindole) to evaluate the survival rate. The largest uptake was observed for the simultaneous application (heating + LISW) with high survival rates (more than 95%). These results suggested that Photofrin was transported into fibroblasts within skin. In this method, irradiation of low-fluence laser was effective to enhance uptake, and therefore optical fiber can be used for laser delivery.
The skin permeability of most of drugs is low, because the stratum corneum works as a solid barrier. It is required, therefore, to enhance the skin permeability for transdermal delivery of drug. To investigate the effects of heating skin and exposing skin to a laser-induced stress wave (LISW) on the drug permeability, we tried to deliver porfimer sodium into rat’s skin. The experiment was performed under the four different skin conditions; exposing to a LISW (case 1); heating (case 2); exposing to a LISW and heating (case 3); control (case 4). It was observed in all cases except the case 4 (control), the drug permeated into the dermis through the epidermis. The deepest penetration was obtained in the case 3 (a LISW plus heating). It was suggested that heating increased the fluidity of the lipid bilayers in the stratum corneum, and therefore the drug permeability might be enhanced.
A COP (cyclic olefin polymer)-coated silver hollow waveguide was used to transmit 1064-nm nanosecond laser pulses that irradiated a metal target to produce an ultrasound wave (stress wave) through plasma formation on the target. Porcine gastric and myocardium tissues, on which a solution of rhodamine-B isothiocyanate-dextran (10 kDa) was dropped, were exposed to the stress wave. Much deeper drug penetration was observed for the tissues exposed the stress wave than for the tissues without stress wave exposure.
We investigated in vitro the mechanism of myocardium tissue ablation with nanosecond pulsed laser at the visible and near-infrared wavelengths. In experiments, porcine myocardium tissue was used as sample. It was found that the ablation rate at 1064 nm was larger than that at 532 nm in spite of lower absorption coefficient at 1064 nm than that at 532 nm for the tissue. During ablation the laser-induced optical emission intensity was measured and it was correlated with the ablation depth. Ablated tissue samples were fixed and stained, and histological analysis was performed with an optical microscope and a polarization microscope. For the 1064-nm laser-ablated tissues thermal damage was very limited, although damage that was presumably caused by mechanical effect was observed. The optical emission intensity during the 1064-nm laser ablation was higher than that during the 532-nm laser ablation at the same laser intensity. And for the 1064-nm laser ablation the ablation threshold was nearly equivalent to the optical emission. Based on these experimental results, we concluded that with the 1064-nm laser light, the tissue removal was achieved through a photodisruption process. Application of 1064-nm, nanosecond pulsed laser photodisruption to transmyocardial laser revascularization (TMLR) was discussed.
With a Q-switched Nd:YAG laser (the fundamental band and its harmonics), ablation characteristics have been investigated in vitro for porcine myocardium tissue. At a constant laser intensity of approximately 2.0 GW/cm2, the laser pulses irradiated the tissue and the depth of the ablation hole formed was measured. After the laser irradiation, the tissue was histologically analyzed with an optical microscope and a polarization optical microscope to evaluate the laser-induced damage in the tissue. It was found that the ablation rate for a 1064-nm laser irradiation was comparably high to that for a 355-nm laser irradiation, and the ablation efficiency was lower for a 266-nm laser irradiation than for a 355-nm laser irradiation. During the ablation, strong plasma formation was observed for 266 nm and 1064 nm, while the laser-induced optical emission was dominated by fluorescence for 355 nm. The histology showed that the thermal damage thickness decreased with decreasing the wavelength for 266, 355, and 532 nm, but for 1064 nm thermal damage was very limited although a certain extent of mechanical damage was observed. Based on these experimental results, the ablation mechanism for each laser wavelength and the optimum laser wavelength for the TMLR (transmyocardial laser revascularization) are discussed.
To investigate the optimum laser condition for transmyocardial laser revascularization, ablation characteristics of porcine myocardium tissue have been measured in vitro in the spectral region from UV to near IR (230 - 1064 nm). In experiments, a nanosecond, tunable optical parametric oscillator and a Q-switched Nd:YAG laser (the fundamental band and its 2nd, 3rd, and 4th harmonics) were used for ablation. Measurement of ablation hole depth and diameter, and histological analysis with an optical microscopy were performed. It was found that the ablation efficiency increased and the thermal damage threshold of the tissue decreased with decreasing the wavelength. However, at relatively high fluences (approximately 5 J/cm2), unexpected deep ablation was obtained with a 1064-nm laser light, where not thermal but acoustic damage was observed near the walls of the ablation holes. Because intense laser- induced plasma was observed in this case, the shock wave would contribute to removal and/or damage of the tissue.
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