Maurício da Silva Baptista

Photodynamic antimicrobial chemotherapy (PACT) for the treatment of malaria, leishmaniasis and trypanosomiasis

A photodynamic effect occurs when photosensitiser molecules absorb light and dissipate the absorbed energy by transferring it to biological acceptors (usually oxygen), generating an excess of reactive species that are able to force cells into death pathways. Several tropical diseases present physiopathological aspects that are accessible to the application of a photosensitiser and local illumination. In addition, disease may be transmitted through infected blood donations, and many of the aetiological agents associated with tropical diseases have been shown to be susceptible to the photodynamic approach. However, there has been no systematic investigation of the application of photoantimicrobial agents in the various presentations, whether to human disease or to the disinfection of blood products or even as photo-insecticides. We aim in this review to report the advances in the photoantimicrobial approach that are beneficial to the field of anti-parasite therapy and also have the potential to facilitate the development of low-cost/high-efficiency protocols for underserved populations.

Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways†

Here, 10 guidelines are presented for a standardized definition of type I and type II photosensitized oxidation reactions. Because of varied notions of reactions mediated by photosensitizers, a checklist of recommendations is provided for their definitions. Type I and type II photoreactions are oxygen‐dependent and involve unstable species such as the initial formation of radical cation or neutral radicals from the substrates and/or singlet oxygen (1O2 1∆g) by energy transfer to molecular oxygen. In addition, superoxide anion radical ( O2·− ) can be generated by a charge‐transfer reaction involving O2 or more likely indirectly as the result of O2‐mediated oxidation of the radical anion of type I photosensitizers. In subsequent reactions, O2·− may add and/or reduce a few highly oxidizing radicals that arise from the deprotonation of the radical cations of key biological targets. O2·− can also undergo dismutation into H2O2, the precursor of the highly reactive hydroxyl radical ( ·OH ) that may induce delayed oxidation reactions in cells. In the second part, several examples of type I and type II photosensitized oxidation reactions are provided to illustrate the complexity and the diversity of the degradation pathways of mostly relevant biomolecules upon one‐electron oxidation and singlet oxygen reactions.

Effects of ionic strength on the antimicrobial photodynamic efficiency of methylene blue

Antimicrobial photodynamic therapy (APDT) may become a useful clinical tool to treat microbial infections, and methylene blue (MB) is a well-known photosensitizer constantly employed in APDT studies, and although MB presents good efficiency in antimicrobial studies, some of the MB photochemical characteristics still have to be evaluated in terms of APDT. This work aimed to evaluate the role of MB solvents ionic strength regarding dimerization, photochemistry, and photodynamic antimicrobial efficiency. Microbiological survival fraction assays on Escherichia coli were employed to verify the solutions influence on MB antimicrobial activity. MB was evaluated in deionized water and 0.9% saline solution through optical absorption spectroscopy; the solutions were also analysed via dissolved oxygen availability and reactive oxygen species (ROS) production. Our results show that bacterial reduction was increased in deionized water. Also we demonstrated that saline solution presents less oxygen availability than water, the dimer/monomer ratio for MB in saline is smaller than in water and MB presented a higher production of ROS in water than in 0.9% saline. Together, our results indicate the importance of the ionic strength in the photodynamic effectiveness and point out that this variable must be taken into account to design antimicrobial studies and to evaluate similar studies that might present conflicting results.

Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue.

The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical symptoms, the results indicate that defects in the repair/tolerance of oxidatively generated DNA lesions are not sufficient to explain these severe clinical features of certain XP patients.

Quenching of Singlet Molecular Oxygen, O2(1Δg), by Dipyridamole and Derivatives

Dipyridamole (DIP) is known for its vasodilating and antiplatelet activity, exhibiting also a potent antioxidant effect, strongly inhibiting lipid peroxidation. This effect has been studied in mitochondria and a correlation between the DIP derivatives’ structure, the ability to bind to micelles and biological activity has been suggested. In the present work, the quenching of singlet molecular oxygen, O2(1Δg), by DIP and RA47 and RA25 derivatives was analyzed in acetonitrile (ACN) and aqueous acid solutions. Laser flash photolysis excitation of methylene blue (MB) was made at 532u2003nm and monomol light emission of O2(1Δg) was monitored at 1270u2003nm. Bimolecular quenching constants in ACN are consistent with an efficient physical quenching, presenting values a bit lower than the diffusion limit (ktu2003=u20033.4–6.8u2003×u2003108u2003m−1u2003s−1). The quenching process probably occurs via reversible charge transfer with the formation of an exciplex. Calculation of ΔGet associated with O2(1Δg) quenching corroborates with uncompleted electron transfer. In aqueous acid solutions (pHu2003=u20033.0), the kt values for DIP and derivatives are 20‐fold smaller when compared with ACN. The electrochemical properties of DIP in ACN are characterized by two consecutive one‐electron processes with half‐wave oxidation potentials of 0.30 and 0.67u2003V vs saturated calomel electrode (SCE). However, in an aqueous acid medium, a single oxidation wave is observed involving a two‐electron process (0.80u2003V vs SCE). Therefore, O2(1Δg) quenching is consistent with electrochemical data.

Antimicrobial photodynamic therapy for caseous lymphadenitis abscesses in sheep: Report of ten cases.

Department of Internal Medicine, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 7-Cidade Universitaria, 05508-270 Sao Paulo, SP, Brazil Santos Metropolitan University, Av. Prof. Antonio Manoel de Carvalho, 3935-Morro da Nova Cintra, 11080-300 Santos, SP, Brazil Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87-Cidade niversitaria, 05508-900 Sao Paulo, SP, Brazil Center for Lasers and Applications, Av. Lineu Prestes, 2242-Cidade Universitaria, 05508-000 Sao Paulo, SP, Brazil Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Avenida Prof. Lineu Prestes 748-Cidade Universitaria, 05508-900 Sao Paulo, P, Brazil

Local clinical phototreatment of herpes infection in São Paulo.

The clinical use of topical photodynamic therapy in herpes simplex lesions in São Paulo is presented and discussed. Although previous attempts utilising this type of approach in the USA were discontinued in the early 1970s due to several presentations of post-treatment Bowens disease, none of the cases from the clinic presented here have displayed any complications on follow-up. In addition, lesion recrudescence periods are generally much longer than with conventional approaches. This is thought to be due to improvements in the treatment protocol, viz. use of the non-toxic photosensitisers methylene blue and Hypericum perforatum extract in place of proflavine and neutral red in the original trials, differences in photosensitisation pathway and illumination of the treatment site with red rather than fluorescent/UV light. Post-treatment cosmesis is also excellent.

Plasmid DNA damage induced by singlet molecular oxygen released from the naphthalene endoperoxide DHPNO2 and photoactivated methylene blue

To investigate oxidative lesions and strand breaks induction by singlet molecular oxygen (1O2), supercoiled-DNA plasmid was treated with thermo-dissociated DHPNO2 and photoactivated-methylene blue. DNA lesions were detected by Fpg that cleaves DNA at certain oxidized bases, and T4-endoV, which cleaves DNA at cyclobutane pyrimidine dimers and apurinic/apyrimidinic (AP) sites. These cleavages form open relaxed-DNA structures, which are discriminated from supercoiled-DNA. DHPNO2 or photoactivated-MB treatments result in similar plasmid damage profile: low number of single-strand breaks or AP-sites and high frequency of Fpg-sensitive sites; confirming that base oxidation is the main product for both reactions and that 1O2 might be the most likely intermediate that reacts with DNA.

Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5±1.8 Ǻ, in contrast to only 2±1.5 Ǻ usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca(2+) the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na(+) was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors.

Photodynamic Therapy in the Treatment of Osteomyelitis

Osteomyelitis is a bone infection that may initiate due to a local trauma or infection or it may originate due to infections occurring elsewhere in the body (microorganisms are transported by the bloodstream in this case). The bone may be predisposed to infection because of trauma or because of a medical condition like diabetes. The microorganisms responsible for the infection are usually pyogenic bacteria. The most frequent infectious agents are in order of prevalence Staphylococcus aureus, Streptococcus (groups A and B), Haemophilus influenzae, and Enterobacter species [1].