Mechanism of photochemical internalization

Abstract

Photochemical Internalization (PCI) can revolutionize the drug or biomolecules delivery inside eukaryotic cells. This concept is grounded on the use of photosensitizers (PS), which localize mainly in endocytic vesicles membrane, it is photo-damaged and enable the release of lumem endocytic vesicles membrane into citosol. PCI has been risen the protocols efficiency in the cancer therapy. Moreover, PCI has shown the capability to improve active biomolecules delivery (gene, proteins) into intracellular environment. However, even PCI has this potential, the real application in clinical practice is very limited (some groups distributed around the world are testing this strategy). This occurs probably because there are little mechanism known about cellular processes involved in PCI, resulting in only empirical protocols. Therefore, deepening studies of PCI mechanisms will be of great value. In the beginning of graduate, the candidate for this project studied interaction between porphyrins and membranes and its relation to the photoactivity using various photophysical and biophysical techniques. The candidate featured amphiphilic molecules that interact strongly with cellular membrane thereby increasing the photodynamic activity. These studies provided the basis for proposing a project focusing on understanding the mechanisms of PCI, since it know that it is required a strong interaction and accumulation of PS in the plasma membrane in order to the drug delivery be satisfactory in PCI. Two photosensitizers, in the series studied by the applicant, showed equally efficient binding and damage to membranes. Therefore, in this project we intend to increase the knowledge about mechanism envolved in PCI and for this we will use these two oppositely charged photosensitizers TPPS2a e Cis-DiMPyP, as well as a polymer photosensitizer (PPIX covalently bound to poly-L-lysine) recently developed in the laboratory. Furthermore, internalization of antitumor drug (doxorubicin) and polymer dextran labeled with fluorescent probes (FITC-dextran 4 and 250kDa) in two cell lines (HeLa and HaCaT) will be our study models. We intend to evaluate: i) the effect of charged porphyrins (positively and negatively); ii) the difference between the release kinetics of substrates which are already in lysosomes compared with those which are provided in the extracellular environment endocytosed; iii) the effect of endocytosis inhibition by Caveolae, removing cholesterol from cytoplasmic membrane; iv) the effect of the suspended autophagic flow in PCI; v) relationship between the induction mechanisms of programmed cell death (apoptosis and autophagy) and PCI. The expected result of this work a deeper understanding of the mechanisms involved in PCI and perhaps the development of molecules that are more efficient. (AU)