Novel meso-substituted porphyrin derivatives and its potential use in photodynamic therapy of cancer.

BACKGROUND: Photodynamic therapy (PDT) is an anticancer treatment that utilizes the interaction of light and a photosensitiser (PS), promoting tumour cell death mediated by generation of reactive oxygen species. In this study, we evaluated the in vitro photoactivity of four meso-substituted porphyrins and a porphyrin coupled to a fullerene. METHODS: The cell line employed was the LM3 mammary adenocarcinoma, and the PS with the best photokilling activity was administered to mice bearing the LM3 subcutaneously implanted adenocarcinoma. The TEMCP4+ porphyrin and its analogue TEMCC4+ chlorine contain four identical carbazoyl substituents at the meso positions of the tetrapyrrolic macrocycle and have A4 symmetry. The TAPP derivative also has A4 symmetry, and it is substituted at the meso positions by aminopropoxy groups. The DAPP molecule has ABAB symmetry with aminopropoxy and the trifluoromethyl substituents in trans positions. The TCP-C604+ dyad is formed by a porphyrin unit covalently attached to the fullerene C60. RESULTS: The PSs are taken up by the cells with the following efficiency: TAPP> TEMCP4+ = TEMCC4+ > DAPP >TCP-C604+, and the amount of intracellular PS correlates fairly with the photodamage degree, but also the quantum yields of singlet oxygen influence the PDT outcome. TAPP, DAPP, TEMCC4+ and TEMCP4+ exhibit high photoactivity against LM3 mammary carcinoma cells, being TAPP the most active. After topical application of TAPP on the skin of mice bearing LM3 tumours, the molecule is localized mainly in the stratum corneum, and at a lower extent in hair follicles and sebaceous glands. Systemic administration of TAPP produces a tumour: normal skin ratio of 31.4, and high accumulation in intestine and lung. CONCLUSION: The results suggest a potential use of topical TAPP for the treatment of actinic keratosis and skin adnexal neoplasms. In addition, selectivity for tumour tissue after systemic administration highlights the selectivity of and potentiality of TAPP as a new PS.

Photodynamic inactivation of bacteria using novel electrogenerated porphyrin-fullerene C60 polymeric films.

A porphyrin-fullerene C60 dyad (TCP-C60) substituted by carbazoyl groups was used to obtain electrogenerated polymeric films on optically transparent indium tin oxide (ITO) electrodes. This approach produced stable and reproducible polymers, holding fullerene units. The properties of this film were compared with those formed by layers of TCP/TCP-C60 and TCP/ZnTCP. Absorption spectra of the films presented the Soret and Q bands of the corresponding porphyrins. The TCP-C60 film produced a high photodecomposition of 2,2-(anthracene-9,10-diyl)bis(methylmalonate), which was used to detect singlet molecular oxygen O2((1)Δg) production in water. In addition, the TCP-C60 film induced the reduction of nitro blue tetrazolium to diformazan in the presence of NADH, indicating the formation of superoxide anion radical. Moreover, photooxidation of L-tryptophan mediated by TCP-C60 films was found in water. In biological media, photoinactivation of Staphylococcus aureus was evaluated depositing a drop with 2.5 × 10(3) cells on the films. After 30 min irradiation, no colony formation was detected using TCP-C60 or TCP/TCP-C60 films. Furthermore, photocytotoxic activity was observed in cell suspensions of S. aureus and Escherichia coli. The irradiated TCP-C60 film produced a 4 log decrease of S. aureus survival after 30 min. Also, a 4 log reduction of E. coli viability was obtained using the TCP-C60 film after 60 min irradiation. Therefore, the TCP-C60 film is an interesting and versatile photodynamic active surface to eradicate bacteria.

Mechanistic insight into the photodynamic effect mediated by porphyrin-fullerene C60 dyads in solution and in Staphylococcus aureus cells.

The photodynamic action mechanism sensitized by a non-charged porphyrin-fullerene C60 dyad (TCP-C60) and its tetracationic analogue (TCP-C60 4+) was investigated in solution and in Staphylococcus aureus cells. The ability of both dyads to form a photoinduced charge-separated state was evidenced by the reduction of methyl viologen in N,N-dimethylformamide (DMF). Moreover, the formation of superoxide anion radicals induced by these dyads was detected by the reduction of nitro blue tetrazolium. Also, photosensitized decomposition of l-tryptophan (Trp) was investigated in the presence of reactive oxygen species (ROS) scavengers. The addition of ß-carotene and sodium azide had a slight effect on reaction rate. However, photooxidation of Trp mediated by TCP-C60 was negligible in the presence of d-mannitol, while no protection was found using TCP-C60 4+. In a polar medium, these dyads mainly act by a contribution of type I pathway with low generation of singlet molecular oxygen, O2(1Δg). In S. aureus cell suspensions, an aerobic atmosphere was required for the photokilling of this bacterium. The photocytotoxicity induced by TCP-C60 was increased in D2O with respect to water, while a small effect was found using TCP-C60 4+. Furthermore, photoinactivation of microbial cells was negligible in the presence of sodium azide. The addition of d-mannitol did not affect the photoinactivation induced by TCP-C60. In contrast, S. aureus cells were protected by d-mannitol when TCP-C60 4+ was used as a photosensitizer. Also, generation of O2(1Δg) in the S. aureus cells was higher for TCP-C60 than TCP-C60 4+. Therefore, TCP-C60 appears to act in microbial cells mainly through the mediation of O2(1Δg). Although, a contribution of the type I mechanism was found for cell death induced by TCP-C60 4+. Therefore, these dyads with high capacity to produce photoinduced charge-separated state represent interesting photosensitizers to inactivate microorganisms by type I or type II mechanisms. In particular, TCP-C60 may be located in a non-polar microenvironment in the cells favoring a type II pathway, while a contribution of the type I mechanism was produced using the cationic TCP-C60 4+.

Synthesis, spectroscopic properties and photodynamic activity of two cationic BODIPY derivatives with application in the photoinactivation of microorganisms.

Two cationic BODIPYs 3 and 4 were synthesized by acid-catalyzed condensation of the corresponding pyrrole and benzaldehyde, followed by complexation with boron and methylation. Compound 3 contains methyl at the 1,3,5 and 7 positions of the s-indacene ring and a N,N,N-trimethylamino group attached to the phenylene unit, while 4 is not substituted by methyl groups and the cationic group is bound by an aliphatic spacer. UV-visible absorption spectra of these BODIPYs show an intense band at ∼500 nm in solvents of different polarities and n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water reverse micelles. Compound 3 exhibits a higher fluorescence quantum yield (ΦF = 0.29) than 4 (ΦF = 0.030) in N,N-dimethylformamide (DMF) due to sterically hindered rotation of the phenylene ring. BODIPYs 3 and 4 induce photosensitized oxidation of 1,3-diphenylisobenzofuran (DPBF) with yields of singlet molecular oxygen of 0.07 and 0.03, respectively. However, the photodynamic activity increases in a microheterogenic medium formed by AOT micelles. Also, both BODIPYs sensitize the photodecomposition of l-tryptophan (Trp). In presence of diazabicyclo[2.2.2]octane (DABCO) or D-mannitol, a reduction in the photooxidation of Trp was found, indicating a contribution of type I photoprocess. Moreover, the addition of KI produces fluorescence quenching of BODIPYs and reduces the photooxidation of DPBF. In contrast, this inorganic salt increases the photoinduced decomposition of Trp, possibly due to the formation of reactive iodine species. The effect of KI was also observed in the potentiation of the photoinactivation of microorganisms. Therefore, the presence of KI could increase the decomposition of biomolecules induced by these BODIPYs in a biological media, leading to a higher cell photoinactivation.

Synthesis, spectroscopic properties and photodynamic activity of porphyrin-fullerene C60 dyads with application in the photodynamic inactivation of Staphylococcus aureus.

A covalently linked porphyrin-fullerene C60 dyad 5 was synthesized by 1,3-dipolar cycloaddition using 5-(4-formylphenyl)-10,15,20-tris[3-(N-ethylcarbazoyl)]porphyrin, N-methylglycine and fullerene C60. Methylation of 5 was used to obtain a cationic dyad 6. Spectroscopic properties were compared in toluene, N,N-dimethylformamide (DMF) and toluene/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water reverse micelles. Absorption spectra of the dyads were essentially a superposition of the spectra of the porphyrin and fullerene reference compounds, indicating a very weak interaction between the chromophores in the ground state. The fluorescence emission of the porphyrin moiety in the dyads was strongly quenched by the attached fullerene C60 unit. The singlet molecular oxygen, O2((1)Δg), productions (ΦΔ) were strongly dependent on the solvent polarity. Similar ΦΔ values were obtained for 5,10,15,20-tetrakis[3-(N-ethylcarbazoyl)]porphyrin (TCP) in both solvents. Also, dyad 5 showed a high O2((1)Δg) generation in toluene. However, O2((1)Δg) production mediated by 5 considerably diminished in the more polar solvent DMF. Also, a high photodynamic activity involving O2((1)Δg) was found for both dyads in a simple biomimetic system formed by AOT reverse micelles. The photoinactivation ability of these dyads was investigated in Staphylococcus aureus cell suspensions. Photosensitized inactivation of S. aureus by dyad 6 exhibits a 4.5 log decrease of cell survival (99.997% cell inactivation), when the cultures are treated with 5 µM photosensitizer and irradiated with visible light (350-800 nm) for 30 min. Under these conditions, a lower photocytotoxic effect was found for 5 (3.2 log decrease). Furthermore, photoinactivation induced by 6 was higher than those obtained with the separate moieties of the dyad. Therefore, molecular structures formed by porphyrin-fullerene C60 dyads represent interesting photosensitizers to inactivate S. aureus.