Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.

No abstract

In recent years, much research has been done in the field of non-ablative skin rejuvenation. This comes as a response to the continuous demand for a simple method of treating rhytides, UV exposure, and acne scars. Numerous researches involve visible light-pulsed systems (20-30 J/cm(2)). The mechanism of action is believed to be a selective heat-induced denaturalization of dermal collagen that leads to subsequent reactive synthesis (Bitter Jr., Dermatol. Surg., 26:836-843, 2000; Fitzpatrick et al., Arch. Dermatol., 132:395-402, 1996; Kauvar and Geronemus, Dermatol. Clin., 15:459-467, 1997; Negishi et al., Lasers Surg. Med., 30:298-305, 2002; Goldberg and Cutler, Lasers Surg. Med., 26:196-200, 2000; Hernandez-Perez and Ibeitt, Dermatol. Surg., 28:651-655, 2002). In this study, we suggest a different mechanism for photorejuvenation based on light-induced reactive oxygen species (ROS) formation. We irradiated collagen in vitro with a broadband of visible light (400-800 nm, 24-72 J/cm(2)) and used the spin trapping coupled with electron paramagnetic resonance spectroscopy to detect ROS. Irradiated collagen resulted in hydroxyl radicals formation. We propose, as a new concept, that visible light at the energy doses used for skin rejuvenation (20-30 J/cm(2)) produces high amounts of ROS, which destroy old collagen fibers, encouraging the formation of new ones. On the other hand, at inner depths of the skin, where the light intensity is much weaker, low amounts of ROS are formed, which are well known to stimulate fibroblast proliferation.

In the current study we compare the effect of different light sources in the visible and near infra-red (IR) range on cell stimulation. It is obvious that in order to interact with the living cell, light has to be absorbed by intracellular chromophores. In a search for chromophores responsible for photobiostimulation, endogenous porphyrins, mitochondrial and membranal cytochromes were found to be suitable candidates, as they possess absorption bands in the visible and near I.R. ranges. The above-mentioned chromophores are photosensitizers that generate reactive oxygen species (ROS) following irradiation. In our opinion the first step in photobiostimulation might be ROS formation. To confirm ROS formation by various light sources, we used the electron paramagnetic resonance (EPR) associated with spin trapping techniques. All wavelengths used (360, 630, 660, 830nm), including a broad band in the visible range (400-800nm), stimulated hydroxyl radical formation in sperm cells. Measuring the amount of OH radicals as a function of the irradiating wavelength shows that shorter wavelengths might be more effective on the cell than longer ones.

The dissociation of water needed to generate OH radicals is possibly due to intermolecular vibrational (V-V) energy transfer in water, competing with vibrational relaxation, thus leading to water dissociation. High amounts of oxygen radicals (e.g., hydroxyl groups) have a sterilization effect, whereas low concentrations of ROS stimulate fibroblasts, causing collagen and extracellular matrix formation. ROS formation may explain the wound healing effect of the Er-YAG laser in dentistry.