Study of potential photosensitizers for self-disinfecting surfaces using the method of antimicrobial photodynamic action

This thesis addresses the principle of antimicrobial photodynamic inactivation (PDI) as a complementary method to antibiotics or disinfection in order to deactivate pathogenic microorganisms. Herein, the prevention of settlement and growth of microorganisms on surfaces using the PDI method is a main issue with focus on the development and improvement of such surfaces. Therefore, different photosensitizers were investigated first in solution in regard to their ability to generate singlet oxygen and their phototoxic effect. Some of these photosensitizers were polymerized into different surface structures, their physical properties were clarified with spectroscopic methods and the phototoxic efficacy on the surfaces was determined. Chapter 3: In literature a rather high dark toxicity component of the photosensitizer class of phenothiazines was described and the systematics of the toxic processes were controversially discussed. Due to their wide use in PDI, the phenothiazine Methylene blue (MB) and common derivatives of this photosensitizer class were investigated in solution of H2O with direct spectroscopic methods in regard to their singlet oxygen generation and thus to clarify the singlet oxygen quantum yield. For all photosensitizers substantially smaller quantum yields as compared to values in literature were obtained via direct monitoring of singlet oxygen. This is considered as a probable effect of dimerisation or the use of indirect methods to monitor singlet oxygen without the explicit exclusion of the participation of other reactive species. Further, the attachment to Gram(+) and Gram(-) bacteria was determined quantitatively. In a second study, newly synthesized derivatives of MB were investigated in order to systemize the influence of side chains on the spectroscopic characteristics, especially the ability to generate singlet oxygen, the photostability and the phototoxic effectivity. In contrast to the commercially available dimerisation could be suppressed in some derivatives and achieving a higher photostability whilst comparable phototoxicity. Chapter 4: The phototoxic properties of the porphyrin XF73 were reported to be rather high due to good cell attachment or penetration abilities with allowing a therapeutic window. Therefore, the photophysical characteristics of this photosensitizer were investigated in H2O and in cell physiological solution with regard to aggregation and singlet oxygen generation. The results were compared to findings with TMPyP. Aggregation was enhanced in XF73, most probably due to the flexible side chains, and a stacking was further driven by the presence of 〖Cl〗^-, which is found to a high extent in cell physiological solution like PBS. The singlet oxygen generation was dramatically influenced by this stacking effect. Chapter 5: The porphyrin TMPyP was incubated in C. albicans planktonic and biofilm growing samples. Thereafter, the singlet oxygen luminescence was directly detected at 1270 nm being the first report of the involvement of singlet oxygen in these cell types with this highly sensitive, non-invasive monitoring technique. Biofilm samples were considered as complex surfaces, exhibiting different decay kinetics for singlet oxygen than their planktonic counterparts. Different experimental laser setups were used for the measurements, thus allowing irradiation of the Soret- or Q-band of the photosensitizer with different irradiation power. With an enhanced sensitivity when irradiating at 420 nm, very low photosensitizer concentrations were necessary to monitor the singlet oxygen luminescence with a good signal-to-noise ratio. A dependency of the rise and decay times of the singlet oxygen luminescence on the photosensitizer concentration used for incubation was detected. Based on these findings a model was proposed, suggesting different surroundings for TMPyP within or adherent to the cell. Chapter 6: Cellulose acetate surfaces and polyurethane-coated PMMA surfaces doped with different photosensitizers were produced and compared with regard to singlet oxygen generation and phototoxic efficacy. The generation of singlet oxygen by irradiation of the photosensitizer was proven within direct spectrally and time-resolved single-photon counting. The assignment of the rise and decay time was challenging, because the bi-exponential fit model was not sufficient for the complex surface system, which showed a multi-exponential decay manner. The presence of singlet oxygen was also monitored via an indirect method using the reaction with potassium iodide. Nevertheless, the PU-surfaces showed an effect without photosensitizer inherent, which indicates ROS generation of the material itself. The phototoxic efficacy against S. aureus was dependent on the irradiation time and the photosensitizer concentration in the material. Also, a difference in the phototoxic efficacy of the porphyrin-based and phenalenone-based photosensitizer doped in the surface material was detected, resulting in >2 〖log〗_10 reduction (> 99%) with the first and >3 〖log〗_10 reduction (> 99.9%) with the latter sample (P=50 mW〖cm〗^(-2), irradiation time 30 min)