Janice R. Perussi

Inativação fotodinâmica de microrganismos

Photodynamic Therapy uses photosensitive dyes and visible light that, combined in the presence of oxygen, produce cytotoxic species that cause tumor death. Microorganisms such as bacteria, fungi, yeasts and viruses (including HIV) can also be inactivated by visible light after treatment with an appropriate photosensitizer as an alternative low cost treatment for localized infections, viral lesions such as acnes, and fungical skin lesions for example. Besides, Photodynamic Inactivation can be used for sterilization of blood and its subproducts for clinical use, in the treatment of drinking water as well as in antimicrobial detoxification of foods.

Photodynamic inactivation of biofilm: taking a lightly colored approach to stubborn infection

Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents.

Aggregation of meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin in its free base, Fe(III) and Mn(III) forms due to the interaction with DNA in aqueous solutions: Optical absorption, fluorescence and light scattering studies

Interactions of the water soluble meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin (TMPyP) in its free base, Mn(III) and Fe(III) forms with DNA in aqueous solutions have been studied by optical absorption, fluorescence and resonance light-scattering (RLS) spectroscopies. Optical absorption and fluorescence measurements have demonstrated the presence of three different species of TMPyP free base and its Mn(III) form in DNA solutions. These species correspond to free porphyrin monomers, monomers bound to DNA and porphyrin aggregates formed on the surface of DNA molecules. This assignment correlates very well with the RLS data. Aggregation reduces the fluorescence of the TMPyP free base. Fe(III)TMPyP also forms aggregates, however, more than three species exist in the presence of DNA due to the equilibria between its free and bound monomers and μ-oxo dimers. The degree of aggregation of Mn(III) and Fe(III) forms of TMPyP is higher than that of its free base. One of the possible explanations of this fact lies in the competition between intercalation and aggregation of TMPyP free base in the process of its binding to DNA; the intercalation of porphyrin should reduce its degree of aggregation. For the Mn(III) and Fe(III) TMPyP forms this competition does not exist as they do not intercalate.

Hypericin encapsulated in solid lipid nanoparticles: Phototoxicity and photodynamic efficiency

The hydrophobicity of some photosensitizers can induce aggregation in biological systems, which consequently reduces photodynamic activity. The conjugation of photosensitizers with nanocarrier systems can potentially be used to overcome this problem. The objective of this study was to prepare and characterise hypericin-loaded solid lipid nanoparticles (Hy-SLN) for use in photodynamic therapy (PDT). SLN were prepared using the ultrasonication technique, and their physicochemical properties were characterised. The mean particle size was found to be 153 nm, with a low polydispersity index of 0.28. One of the major advantages of the SLN formulation is its high entrapment efficiency (EE%). Hy-SLN showed greater than 80% EE and a drug loading capacity of 5.22% (w/w). To determine the photodynamic efficiency of Hy before and after encapsulation in SLN, the rate constants for the photodecomposition of two (1)O2 trapping reagents, DPBF and AU, were determined. These rate constants exhibited an increase of 60% and 50% for each method, respectively, which is most likely due to an increase in the lifetime of the triplet state caused by the increase in solubility. Hy-SLN presented a 30% increase in cell uptake and a correlated improvement of 26% in cytotoxicity. Thus, all these advantages suggest that Hy-loaded SLN has potential for use in PDT.

Fluorescence Studies of Extracellular Hemoglobin of Glossoscolex paulistus in Met Form Obtained from Sephadex Gel Filtration

Abstract Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus in the met form presents an unique band at pH 7.0 and two bands at pH 9.0 as a result of alkaline dissociation. SDS-PAGE of the intact protein obtained at pH 7.0 is very similar to that for the oxyhemoglobin. Chromatography at pH 9.0 indicates complete dissociation of the oligomeric protein into two low molecular weight fractions corresponding to the trimers and monomers, showing that the oxidized extracellular hemoglobin is less stable than the oxyhemoglobin with respect to alkaline dissociation. Fluorescence quantum yields of different fractions obtained in the chromatography, as well as extinction coefficients at 280 nm and 415 nm, were estimated and compared to human methemoglobin. The fluorescence data are consistent with the high content of aromatic residues in G. paulistus hemoglobin. The increase in the fluorescence quantum yield upon both alkalinization and dissociation was correlated with the reduction of intramolecular quenching but the exposure of tryptophan residues to the solvent did not changed significantly as occurs for the oxy form. The intact native protein has a quantum yield of 0.36% at pH 7.0, increasing to 1.89% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 1.1% at pH 7.0 increasing to 1.43% at pH 9.0. The maximum emission wavelength of the intact protein (330 nm) is consistent with tryptophan residues being relatively buried; they become more fluorescent upon dissociation into smaller subunits but not more exposed since the wavelength of maximum emission is essentially unchanged at pH 9.0. In the monomer, the tryptophan residues also remain buried inside the protein molecule at pH 9.0 (328 nm). The dependencies of fluorescence quantum yields on the pH show in a clear way the hemichrome transitions observed by optical absorption spectroscopy indicating that the formation of two types of hemichromes accompany the distinct increase in fluorescence quantum yield. One type of hemichrome is irreversibly formed around pH 7.5–8.0 and a second reversible hemichrome is formed above pH 9.5–10.0. They are associated with the bis-imidazole low spin hemichrome and with a high spin hemichrome where the weakening of the iron bond to proximal histidine takes place. Addition of cyanide to the metHb solution produces the cyanomet form of the protein which is considerably more stable towards alkaline dissociation and presents a smaller change in quantum yield as a function of pH. Circular dichroism suggests that the formation of hemichromes is not accompanied by significant protein denaturation.

Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus.

Sephadex G-200 chromatography of the extracellular hemoglobin from the giant earthworm G. paulistus in the met form presents a single peak at pH 7.0 and two peaks at pH 9.0 as a result of alkaline dissociation. SDS-PAGE shows that the polypeptide chains are very similar to those observed for the oxy form and the two peaks at pH 9.0 correspond to the trimer contaminated by linkers and monomers which seems to be quite pure. The aquomet acid form is stable as an oligomer of molecular mass 3.1 x 10(6) Da only in a narrow pH range around neutrality. Increasing the pH above 7.5 leads to an irreversible transition from aquomet to hemichrome I which is the low-spin bis-imidazole complex. At pHs above 9.5-10.0 a second reversible transition takes place from hemichrome I to hemichrome II, a high-spin complex which is associated with the weakening and possible disruption of the proximal Fe--N histidine bond. Thus, increase in pH above 8.0 induces changes in the heme pocket that involve both the distal and proximal sides of the heme. EPR measurements show a very sharp decrease of the aquomet high-spin signal in the range of pH 7.0-8.0 and a very small low-spin signal even at liquid helium temperatures. The transition to hemichrome I is also accompanied by the loss of heme optical activity monitored by CD, which is consistent with the weakening of heme--globin interaction. Hemichrome I in the presence of cyanide gives the typical cyanometHb derivative which has a transition to a hemichrome at much higher pHs. This observation suggests that the dissociation of the oligomer in alkaline medium as well as the stability of the heme on the proximal side, depend both upon the ligand present at the sixth coordination position on the distal side. Hence, we believe that hemi(hemo)chrome formation in G. paulistus Hb and other invertebrate hemoglobins is a common phenomenon, not associated with protein denaturation, which may provide a fine tuning mechanism to control subunit interactions through changes in the distal side of the heme pocket.

Ionization and binding equilibria of papaverine in ionic micelles studied by 1H NMR and optical absorption spectroscopy

The binding of the vasodilator drug papaverine (PAV) to micelles of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS), cationic cetyltrimethylammonium chloride (CTAC) and anionic sodium dodecylsulfate (SDS) in aqueous solution was studied by 1H NMR and electronic absorption spectroscopy. In the presence of HPS or CTAC, the apparent pK(a) of PAV decreased by about 2 units, while it increased by about 2 units upon binding to SDS. However, the chemical shift patterns of both protonated (PAVH+) and deprotonated (PAV0) forms of PAV are not sensitive to the type of surfactant. The association constants were estimated as 5 +/- 2 M(-1) for PAVH+-CTAC, 8 +/- 3 M(-1) for PAVH+-HPS, (7 +/- 2) x 10(5) M(-1) for PAVH+-SDS, and 1.5 x 10(3) to 3.0 x 10(3) M(-1) for the complexes of PAV0 with all three types of micelles. Using these data, an electrostatic potential difference on the micelle-water interface was calculated as 150 +/- 10 mV for CTAC, 140 +/- 10 mV for HPS and - 140 +/- 10 mV for SDS. The results suggest that PAV aromatic rings are located in the hydrophobic part of the micelle. The electrostatic attraction or repulsion of the protonated quinoline nitrogen and surfactant headgroups changes the affinity of PAV to micelles and, thus, shifts the ionization equilibrium of PAV. The electrostatic potential of HPS micellar surface is determined by the cationic dimethylammonium headgroup fragment, whereas the anionic sulfate fragment attenuates the effective charge of HPS headgroup.

Binding of the Mn(III) complex of meso-tetrakis(4-N-methyl-pyridimumyl) porphyrin to DNA. Effect of ionic strength

Interactions of the water-soluble Mn(III) complex of meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin (Mn(III)TMPyP) with DNA in aqueous solutions at low (0.01 M) and high (0.2 M) ionic strengths have been studied by optical absorption, resonance light scattering (RLS) and 1H NMR spectroscopies. Optical absorption and RLS measurements have demonstrated that in DNA solutions at low ionic strength the Mn(III)TMPyP form aggregates, which are decomposed at DNA excess. At high ionic strength the aggregation was not observed. We explain this effect by assuming that upon increase in ionic strength, Mn(III) TMPyP dislocates from the DNA sites, which produces better conditions for the porphyrin aggregation, to sites where the aggregation is hindered. The 1H NMR data demonstrated that the aggregation observed at low ionic strength reduces the paramagnetism of Mn(III)TMPyP. This phenomenon was not observed at the high ionic strength in the absence of aggregation.

Fluorescence studies of extracellular hemoglobin of Glossoscolex paulistus obtained by gel filtration

Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus presents a unique band at pH 7.0 and several bands at pH 9.0 as a result of alkaline dissociation. SDS-polyacrylamide gel electrophoresis (PAGE) of the intact protein obtained at pH 7.0 shows the existence of five different bands with molecular weights of 12 ± 1, 26 ± 3, 28 ± 2, 34 ± 1 and 53 ± 1 kDa. In the presence of β-mercaptoethanol, six distinct bands are obtained with molecular weights 13 ± 1, 14.8 ± 0.3, 15.8 ± 0.2, 16.6 ± 0.2, 35.1 ± 0.3 and 40.5 ± 0.5 kDa. Reduction of disulfide bonds of a trimer of molecular weight 53 kDa leads to the appearance of monomeric subunits of 14.8, 15.8 and 16.6 kDa. The molecular weights obtained from chromatography in Sephadex G-200 at pH 9.0 are different from those from SDS-PAGE. An intense band at 110 ± 12 kDa due to the tertiary complex of two disulfide-linked trimers and monomer chain (trimer + monomer), (abcd)2, is observed in addition to a small amount of high molecular weight fraction in the exclusion volume, as well as poorly resolved trimer (42 ± 2 kDa) and monomer (15 ± 2 kDa) bands. Fluorescence quantum yields of different fractions obtained in the chromatography as well as extinction coefficients at 280 and 415 nm were estimated and compared with human hemoglobin. The fluorescence data presented are consistent with the high content of aromatic residues in the G. paulistus hemoglobin. The increase in the fluorescence quantum yields upon both alkalinization and dissociation were correlated with the exposure of tryptophan residues to the solvent. The intact native protein has a quantum yield of 0.25% at pH 7.0, assigning 14% to tryptophan solution at pH 7.0 and increasing to 0.8% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 0.8% at pH 7.0, increasing to 2.1% at pH 9.0. The maximum emission wavelength of the intact protein (327 nm) is consistent with tryptophan residues buried, quenched and becoming more exposed and more fluorescent upon dissociation into smaller subunits (332-335 nm). In the monomer the tryptophan residues remain buried inside the protein molecule at pH 9.0.

Correlation of cytotoxicity and depth of necrosis of the photoproducts of photogem

Photodynamic therapy (PDT) is an approved modality for cancer treatment, which involves the administration of a photosensitive drug (PS) that is selectively accumulated in neoplastic tissues and their vasculature and subsequently can be activated with light at the appropriate wavelength to generate reactive molecular species that are toxic to tissues. In PDT, a great part of the used PS suffers degradation by light (photobleaching) that involves a decrease in the absorption and intensity of fluorescence of the photosensitizer as well as photoproduct formation evidenced by the appearance of a new absorption band. In this study, we investigated the correlation of cytotoxicity and depth of necrosis of Photogem and its photoproducts obtained previously by irradiation at 514 and 630 nm. The cytotoxicity for degraded Photogem decreases with the previous irradiation time of Photogem solution suggesting that the photoproducts of Photogem are less cytotoxics than the original formulation. A transition between the necrosed epithelium and healthy epithelium of normal liver of rats after irradiation at 630 nm was observed with irradiated and nonirradiated PS. It is observed that the depth of necrosis only at irradiation dose of 150 J/cm2 in both concentrations is greater for Photogem followed by Photogem degradated previously at 514 and then at 630 nm. The results obtained suggest that the threshold of necrosis values is lower for Photogem followed by its photoproducts formed, suggesting that the photoproducts present a low photodynamic activity. If the photosensitizer degradation happens at the same time as tumor destruction, the drug degradation can be complete before reaching the threshold of necrosis; then it is very important to control the drug concentration and light intensity of irradiation during PDT.