Abstract
Purpose: :
For a successful treatment of infectious keratitis Photodynamic Inactivation (PDI) of bacteria presents a novel alternative to conventional antibiotic therapy. To evaluate the possibility of PDI for bacterial pathogens in the cornea the diffusion of the photosensitizer (PS) chlorin e6 (Ce6) was assessed in individual layers of corneal tissue in porcine eyes.
Methods: :
Chlorin e6, dissolved in sodium chloride, was evenly distributed on to porcine corneas using a gel forming agent (sodium polyacrylate). Series of measurements with different contact times and concentrations were carried out. After removal of the photosensitizer, tissue sections (8 µm) were prepared and investigated by fluorescence microscopy with an excitation wavelength of 405 nm. With this set-up, fluorescence intensity at 670 nm was spatially resolved whereof the penetration depth could be determined. To simulate the penetration in diseased or injured cornea tissue the epithelium was removed before application of chlorin e6. In addition, chemical and thermal modifications on the surface of the cornea were induced to analyze the impact on diffusion processes of the photosensitizer.
Results: :
After an exposure period of 15 min and concentrations in the region of 400 μM the photosensitizer was sufficiently absorbed for PDI of bacteria. Chemical injuries, such as acid and base, implied an increase of the penetration depth into the tissue. From the measured values of fluorescence intensity it was possible to determine the maximum concentration reached in the stroma. It turned out that this concentration almost reached the applied concentration. As a result of measurements on the distribution of the PS in cornea the diffusion coefficients of chlorin e6 were calculated.
Conclusions: :
Fluorescence measurements were used to determine accurately the penetration depth and the concentration of the PS chlorin e6 into the cornea. In the real treatment situation, the epithelium has to be removed. Under those conditions concentrations < 1 mM of chlorin e6 were sufficient to reach a tissue penetration of the active ingredient to the depth of ~ 500 µm.
Keywords: cornea: stroma and keratocytes • depth • photodynamic therapy