Effetto della struttura chimica del fotosensibilizzatore e del veicolante sulla fotoinattivazione di microorganismi patogeni mediante terapia fotodinamica

Photodynamic therapy (PDT) represents a well established therapeutic modality, which was originally developed and recently approved for the treatment of a variety of solid tumours. The technique involves the combination of a tumour-localized and intrinsically non-toxic photosensitiser with harmless visible light wavelengths, which are specifically absorbed by the photosensitising agent; as a result, some hyper-reactive oxygen species are generated, which induce the specific irreversible damage of malignant cells and tissues. A novel application of PDT has been made possible by the preparation of photosensitisers whose molecule is characterized by the presence of functional groups (e. g. positively charged quaternarized nitrogens), which promote a very fast interaction with bacterial cells, hence a highly preferential inactivation of such pathogenic agents in comparison with the main constituents of host tissues, such as fibroblasts and keratinocytes. With an aim to expand the type of photosensitising dyes which can be efficaciously used as antimicrobial photodynamic agents, we decided to study the photosensitised inactivation of a well-known antibiotic-resistant Gram-positive bacterium, namely meticillin-resistant Staphylococcus aureus (MRSA), by using two non-cationic liposome-incorporated dyes, such as haematoporphyrin (HP) and chlorophyll (Chl). Liposome-delivered photosensitisers have been often adopted in anti-tumour PDT and proven to yield a higher and more selective targeting of the neoplastic lesion. Thus, it appeared of interest to investigate the effect of liposomes as carriers of the photosensitising agent on its affinity for bacterial cells and the efficiency of photoinduced bacteria killing. Toward this purpose, we selected liposomal vesicles with a different degree of fluidity at the physiological temperature, as well as with different electric charge or size. HP delivered via DOTAP liposomes induced a marked enhancement in phototoxicity of bacteria, while Chl delivered via the same liposomes had no detectable effect on the survival of MRSA cells, independently of the released amount or the nature of the vesicles. The cationic photosensitizer TDPyP used in the second part of this research was designed with the aim to increase the selectivity for bacterial cell membranes. This porphyrin was found to be efficiently incorporated into DOTAP vesicles in spite of its positive charge. Thus, when the cationic DOTAP phospholipid is used as the porphyrin carrier, an appreciable potentiation of the photocydal effect takes place. TDPyP was also included in inclusion complexes made by cationic cyclodextrin (β-CD). The inactivation of MRSA was very successful and was found to be efficient against the Gram-negative strain. The DOTAP-delivered porphyrin is recovered from the protoplasts in significantly larger amount as compared with the free TDPyP. That the plasma membrane represents a primary target of the TDPyP-photosensitised process is also in agreement with the finding that the activity of two typical marker enzymes of the MRSA plasma membrane, namely succinate and lactate dehydrogenase, is rapidly impaired during the early stages of the process photosensitised by DOTAP-delivered TDPyP. It is reasonable to hypothesize that the DOTAP vesicles or cyclodextrin behave similarly with other polycationic systems, such as poly-lysine, which primarily act as a disorganizing agent for the native three-dimensional architecture of the bacterial wall, thereby enhancing its permeability to externally added chemical agents; these can thus more easily reach the bacterial plasma membrane, where several targets of the porphyrin-photosensitised processes are present. Acanthamoeba species are responsible for opportunistic and non-opportunistic infections, which can be fatal or highly invalidating in humans and other animals, such as granulomatous amoebic encephalitis and cutaneous infections in immunocompromised individuals, and amoebic keratitis in immunocompetent individuals, respectively. The life cycle of Acanthamoeba consists of two stages, an actively feeding, dividing trophozoite and a dormant cyst. They encyst in response to adverse environmental conditions, such as food deprivation, desiccation, and changes in temperature and pH, forming a highly resistant stage endowed with a double wall containing cellulose as a major component. Antimicrobial therapy for these infections is generally empirical and patient recovery is often problematic, whereas some forms of combination therapy have proven to be more successful than single-drug therapies, because many drugs have amoebostatic but not amoebicidal activity. The development of alternative approaches in the medical and environmental control of such pathogenic protozoa is needed. Therefore we undertook a systematic investigation in order to assess the potential of PDT for the inactivation of protozoa in either the vegetative or cystic stage. In particular, our photosensitisation studies with A. palestinensis trophozoite cultures indicated the effectiveness of a tetracationic phthalocyanine (RLP068). We demonstrate that the RLP068 phthalocyanine, bearing four positively charged quaternary ammonium groups, exhibits a significant affinity for A. palestinensis even when the microorganism is in the cystic stage. After irradiation there is an appreciable inactivation in both trophozoites and cysts; observations at the optical and electronic microscope demonstrated cytoplasmatic damages of vegetative stages and wall damages of cysts. Thus, PDT appears to represent an efficient modality for the therapy of microbial infections and is characterized by a broad spectrum of action.