The effect of pyridylporphyrins on mitochondria

Fotodinamička terapija (PDT) je novi terapeutski pristup za liječenje tumorskih bolesti te bakterijskih, gljivičnih i virusnih infekcija. PDT uključuje preferencijalni unos fotosenzibilizatora (PS) u ciljane stanice ili tkiva, nakon čega slijedi ozračivanje tog područja vidljivom svjetlošću. Nakon apsorpcije svjetlosti, PS prelazi u pobuĎeno stanje te generira reaktivne kisikove vrste (engl. reactive oxygen species, ROS). ROS reagiraju sa staničnim komponentama te uzrokuju staničnu smrt. Porfirini su prirodni intenzivno obojeni spojevi koji se zbog mogućnosti apsorpcije vidljive svjetlosti koriste kao fotosenzibilizatori. Protutumorski učinci fotodinamičke terapije uključuju tri glavna mehanizma uništavanja tumora, a to su oštećenje tumorskih krvnih ţila i sprječavanje daljnje angiogeneze, inicijacija lokalnog upalnog i imunosnog odgovora te direktna stanična smrt posredovana ROS-om. Od samih početaka eksperimentiranja s PDT-om, uočeno je da oštećuje mitohondrije, a primjećeno je i da kationski fotosenzibilizatori imaju povećan afinitet za kolokalizaciju s mitohondrijima. Iz tog razloga, u ovom radu cilj je bio ispitati učinak kationskih amfifilnih piridilporfirina na mitohondrije. Sintetizirani su 5-(4-acetamidofenil)-10,15,20-tris(1-oksidopiridin-3- il)porfirin, 5-(4-acetamidofenil)-10,15,20-tris(N-metilpiridin-3-il)porfirin triklorid, 5-(4-oktanamidofenil)-10,15,20-tris(1-oksopiridin-3-il)porfirin te 5-(4-oktanamidofenil)-10,15,20-tris(N-metilpiridin-3-il)porfirin triklorid. Struktura spojeva je potvrĎena NMR spektroskopijom, ali NMR spektri su pokazali prisutnost nečistoća koje se nisu uspjele ukloniti, stoga ovi spojevi nisu korišteni za daljnja biološka istraţivanja. Ispitan je učinak sličnog spoja, 5-(4-oktanamidofenil)-10,15,20-tris(N-metilpiridin-3- il)porfirin triklorida. Dokazano je da testirani spoj uspješno ulazi u stanice. Pokazana je i njegova toksičnost na mitohondrije analizom markera vanjske mitohondrijske membrane, ali ne i direktno nakupljanje porfirina u mitohondrijima. Nadalje, ispitano je da li navedena oštećenja dovode do stanične smrti te je dokazano da stanice koje su tretirane porfirinom, a VII potom osvijetljene, umiru apoptozom. Dakle, u ovom radu je dokazano da testirani spoj ulazi u stanice i uzrokuje oštećenje mitohondrija bez da direktno kolokalizira s njima te u konačnici dovodi do apoptoze stanica.Photodynamic therapy (PDT) is a new therapeutic approach for the treatment of tumor diseases, but also for bacterial, fungal and viral infections. PDT involves preferential uptake of a photosensitizer (PS), in the cells / tissues, followed by irradiation of the selected region by visible light. After the absorption of light, the PS reaches an excited state and generates reactive oxygen species (ROS). ROS react with cellular components and irreversibly modify them causing cell death. Porphyrins are natural, intensely colored compounds and are used as photosensitizers due to their ability to absorb visible light. The antitumor effects of photodynamic therapy include three major mechanisms of tumor destruction: damage to the tumor blood vessels and prevention of further angiogenesis, local inflammation and immune response, and direct cell death mediated by reactive oxygen species. From the beginning of experimentation with PDT, the damage to the mitochondria has been observed. Furthermore, it is known that cationic photosensitizers have an increased affinity for targeting the mitochondria. In this work, the aim was to investigate the effect of cationic amphiphilic tripyridylporphyrins on mitochondria. 5- (4-acetamidophenyl) -10,15,20-tris (1-oxidopyridin-3-yl) porphyrin, 5- (4-acetamidophenyl) -10,15,20-tris (N-methylpyridin-3-yl) porphyrin trichloride, 5- (4-octanamidophenyl) -10,15,20-tris (1- oxopyridin-3-yl) porphyrin and 5- (4-octanamidophenyl) -10,15,20-tris (N-methylpyridin-3) -il) porphyrin trichloride were synthesized. Their structure was confirmed by NMR spectroscopy, however, NMR spectra showed also impurities that could not be removed, thus these compounds were not used for further biological research. The effect of a similar compound, 5-(4-octanamidophenyl)-10,15,20-tris(N-methylpyridin-3- yl)porphyrin trichloride, was tested. The tested compound has shown successful internalization into cells. Its toxicity to the mitochondria has also been demonstrated by the analysis of markers of the outer mitochondrial membrane, but the direct accumulation of porphyrins in the IX mitochondria has not been shown. Ultimately, it was investigated if the damage that porphyrins are causing leads to cell death and it was proven that cells treated with porphyrin, after illumination, die by apoptosis. Thus, in this work, it is proven that the tested compound is able to enter cells, causes damage to the mitochondria, but does not colocalize with the mitochondria and ultimately leads to apoptosis