Photodynamic inactivation (PDI) is a rapidly developing antimicrobial technology which combines a non-toxic photoactivatable dye or photosensitizer (PS) in combination with harmless visible light of the correct wavelength to excite the dye to its reactive triplet state that will then generate reactive oxygen species (ROS) that are highly toxic to cells. Buckminsterfullerenes are closed-cage molecules entirely composed of sp2 hybridized carbon atoms and although their main absorption is in the UV, they also absorb visible light and have a long-lived triplet state. When C60 fullerene is derivatized with cationic functional groups it forms molecules that are more water-soluble and can mediate PDT efficiently upon illumination, and moreover cationic fullerenes can selectively bind to microbial cells. In this report we describe the synthesis and characterization of several new cationic fullerenes. Their relative effectiveness as broad-spectrum antimicrobial photosensitizers against Gram-positive, Gram-negative bacteria, and a fungal yeast was determined by quantitative structure function relationships.

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.

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.

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.