Abstract: Candida albicans biofilm is a main cause of infections associated with medical devices such as catheters,contact lens and artificial joint prosthesis. The current treatment comprises antifungal chemotherapy thatpresents low success rates. Photodynamic inactivation (PDI) involves the combination of a photosensitiz-ing compound (PS) and light to generate oxidative stress that has demonstrated effective antimicrobialactivity against a broad-spectrum of pathogens, including C. albicans. This fungus senses glucose induc-ing an upregulation of membrane transporters that can facilitate PS uptake into the cell. The aim ofthis study was to evaluate the effects of glucose on methylene blue (MB) uptake and its influence onPDI efficiency when combined to a red LED with central wavelength at = 660 nm. C. albicans biofilmswere grown on hydrogel disks. Prior to PDI assays, MB uptake tests were performed with and withoutglucose-sensitization. In this system, the optimum PS administration was determined as 500 M of MBin contact with the biofilm during 30 min before irradiation. Irradiation was performed during 3, 6, 9, 12,15 and 18 min with irradiance of 127.3 mW/cm2. Our results showed that glucose was able to increaseMB uptake in C. albicans cells. In addition, PDI without glucose showed a higher viability reduction until6 min; after 9 min, glucose group demonstrated a significant decrease in cell viability when compared toglucose-free group. Taken together, our data suggest that glucose is capable to enhance MB uptake andmodulate photodynamic inactivation of C. albicans biofilm.

Palavras-Chave: glucose; methylene blue; infectious diseases; inactivation; photosensitivity; yeasts

SUZUKI, LUIS C.; KATO, ILKA T.; PRATES, RENATO A.; SABINO, CAETANO P.; YOSHIMURA, TANIA M.; SILVA, TAMIRES O.; RIBEIRO, MARTHA S. Glucose modulates antimicrobial photodynamic inactivation ofCandida albicans in biofilms. Photodiagnosis and Photodynamic Therapy, v. 17, p. 173-179, 2017. DOI: 10.1016/j.pdpdt.2016.12.003. Disponível em: http://repositorio.ipen.br/handle/123456789/27375. Acesso em: $DATA.

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Abstract: Background: Antimicrobial Photodynamic therapy (A-PDT) has been used to treat infections. Currently, microbial inactivation data is reported presenting survival fraction averages and standard errors as discrete points instead of a continuous curve of inactivation kinetics. Standardization of this approach would allow clinical protocols to be introduced globally, instead of the piecemeal situation which currently applies. Methods: To this end, we used a power-law function to fit inactivation kinetics and directly report values of lethal doses (LD) and a tolerance factor (T) that informs if inactivation rate varies along the irradiation procedure. A deduced formula was also tested to predict LD for any given survival fraction value. We analyzed the photoantimicrobial effect caused by red light activation of methylene blue (MB-APDT) and by blue light (BL) activation of endogenous microbial pigments against 5 clinically relevant pathogens. Results: Following MB- APDT, Escherichia coli and Staphylococcus aureus cells become increasingly more tolerant to inactivation along the irradiation process (T < 1). Klebsiella pneumoniae presents opposite behavior, i.e., more inactivation is observed towards the end of the process (T > 1). P. aeruginosa and Candida albicans present constant inactivation rate (T˜1). In contrast, all bacterial species presented similar behavior during inactivation caused by BL, i.e., continuously becoming more sensitive to blue light exposure (T > 1). Conclusion: The power-law function successfully fit all experimental data. Our proposed method precisely predicted LD and T values. We expect that these analytical models may contribute to more standardized methods for comparisons of photodynamic inactivation efficiencies.

Palavras-Chave: antimicrobial agents; therapy; chemotherapy; inactivation; lethal doses; light sources; data analysis; candida; methylene blue

SABINO, CAETANO P.; WAINWRIGHT, MARK; ANJOS, CAROLINA dos; SELLERA, FABIO P.; BAPTISTA, MAURICIO S.; LINCOPAN, NILTON; RIBEIRO, MARTHA S. Inactivation kinetics and lethal dose analysis of antimicrobial blue light and photodynamic therapy. Photodiagnosis and Photodynamic Therapy, v. 28, p. 186-191, 2019. DOI: 10.1016/j.pdpdt.2019.08.022. Disponível em: http://repositorio.ipen.br/handle/123456789/30437. Acesso em: $DATA.

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Abstract: Background Bovine digital dermatitis (BDD) is one of the most important diseases that effect dairy cows. Methylene blue-mediated antimicrobial photodynamic therapy (MB-APDT) emerges as a promising technique to treat superficial infections in bovines. Methods Twenty BDD lesions located at the skin horn transition of the claw of pelvic limbs of 16 cows were treated by MB-APDT, using a red LED cluster (λ = 660 nm, irradiance =60 mW/cm2, exposure time = 40 s) combined with topical application of MB at 0.01 %; or by topical application of OXY (500 mg in 20 % solution). Each lesion was treated twice with an interval of 14 days. Lesions were weekly evaluated until day 28 by clinical analysis and by histological examination on days 0 and 28. Results Both treatments led to a similar reduction of lesions area. At day 28, three lesions treated by OXY did not present completely recovery, whereas no lesions were observed in MB-APDT group. OXY resulted in a slight increase in type I and III collagen levels, while MB-APDT led to a significant increase in the total area of both collagen types. An abundant number of spirochetes were histologically observed in all lesions before treatments. On the 28th day, five lesions treated by OXY still presented a slight number of spirochetes, whereas in MB-APDT group no spirochetes were evidenced. Conclusion Our findings suggest that MB-APDT is more effective than OXY and could be used in Veterinary practice to fight BDD.

Palavras-Chave: methylene blue; antimicrobial agents; cattle; inactivation; photosensitivity; therapy; dermatitis; infectious diseases

SELLERA, FABIO P.; BARBOSA, BRUNA S.; GARGANO, RONALDO G.; RISPOLI, VIVIAN F.P.; SABINO, CAETANO P.; OLLHOFF, RUDIGER D.; BAPTISTA, MAURICIO S.; RIBEIRO, MARTHA S.; SA, LILIAN R.M. de; POGLIANI, FABIO C. Methylene blue-mediated antimicrobial photodynamic therapy can be a novel non-antibiotic platform for bovine digital dermatitis. Photodiagnosis and Photodynamic Therapy, v. 34, p. 1-8, 2021. DOI: 10.1016/j.pdpdt.2021.102274. Disponível em: http://repositorio.ipen.br/handle/123456789/32053. Acesso em: $DATA.

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Abstract: Background: Artificial insemination is widely employed in poultry, but high degrees of bacterial contamination are often observed in semen because of its passage through the cloaca. Consequently, most semen extenders for birds have antibiotics that could aggravate bacterial resistance. Methods: We evaluated the potential of antimicrobial photodynamic therapy (PDT) as an alternative to the use of antibiotics, and assessed whether changes in concentration and incubation time with methylene blue (MB), radiant exposure, and irradiance of light affect spermatozoa activity and bacteria in chicken semen. Results: Incubation with MB (< 25 µM) did not alter sperm motility, regardless of the pre-irradiation time (PIT, 1 or 5 min). Following 1 min of PIT with MB at 10 µM, samples were irradiated for 30, 60, 120, and 180 s at irradiances of 44, 29, and 17 mW/ cm² (660 nm LedBox). MB and light alone did not interfere with the analyzed parameters. However, when both factors were associated, increases in light dose led to greater reductions in sperm parameters, regardless of the irradiance used. Besides, PDT conditions that were less harmful to spermatozoa were not able to significantly reduce bacterial colonies in chicken semen. Conclusions: A failure in MB selectivity could explain unsuccessful bacterial reduction following PDT. Further research involving other photosensitizers or conjugating molecules to MB to target microbial cells is needed for PDT application in poultry breeders.

Palavras-Chave: fowl; antibiotics; reproduction; microorganisms; chickens; photodynamic therapy

NOVAES, G.A.; BLANK, M.H.; YOSHIMURA, T.M.; RIBEIRO, M.S.; PEREIRA, R.J.G. Methylene blue-mediated antimicrobial photodynamic therapy on chicken semen. Photodiagnosis and Photodynamic Therapy, v. 41, p. 1-12, 2023. DOI: 10.1016/j.pdpdt.2023.103290. Disponível em: http://repositorio.ipen.br/handle/123456789/33999. Acesso em: $DATA.

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Abstract: Background: Antimicrobial photodynamic therapy (aPDT) has been investigated as an adjunctive to periodontal treatment but the dosimetry parameters adopted have discrepancies and represent a challenge to measure efficacy. There is a need to understand the clinical parameters required to obtain antimicrobial effects by using aPDT in periodontal pockets. The aim of this study was to investigate parameters relating to the antimicrobial effects of photodynamic therapy in periodontal pockets. Material and methods: This randomized controlled clinical trial included 30 patients with chronic periodontitis. Three incisors from each patient were selected and randomized for the experimental procedures. Microbiological evaluations were performed to quantify microorganisms before and after treatments and spectroscopy was used to identify methylene blue in the pocket. A laser source with emission of radiation at wavelength of ʎ=660 nm and output radiant power of 100mW was used for 1, 3 and 5 min. One hundred μM methylene blue was used in aqueous solution and on surfactant vehicle. Results: The results demonstrated the absence of any antimicrobial effect with aqueous methylene blue-mediated PDT. On the other hand, methylene blue in the surfactant vehicle produced microbial reduction in the group irradiated for 5 min (p < 0.05). Spectroscopy showed that surfactant vehicle decreased the dimer peak signal at 610 nm. Conclusion: Within the parameters used in this study, PDT mediated by methylene blue in a surfactant vehicle reached significant microbial reduction levels with 5 min of irradiation. The clinical use of PDT may be limited by factors that reduce the antimicrobial effect. Forms of irradiation and stability of the photosensitizers play an important role in clinical aPDT.

Palavras-Chave: methylene blue; antimicrobial agents; dosimetry; clinical trials; therapy; dentistry; bacteria; irradiation; absorption

ALVARENGA, LETICIA H.; GOMES, ANA C.; CARRIBEIRO, PAMELA; GODOY-MIRANDA, BIANCA; NOSCHESE, GIOVANE; RIBEIRO, MARTHA S.; KATO, ILKA T.; BUSSADORI, SANDRA K.; PAVANI, CHRISTIANE; GERALDO, YHAGO G.E.; SILVA, DANIELA de F.T. da; HORLIANA, ANNA C.R.T.; WAINWRIGHT, MARK; PRATES, RENATO A. Parameters for antimicrobial photodynamic therapy on periodontal pocket: randomized clinical trial. Photodiagnosis and Photodynamic Therapy, v. 27, p. 132-136, 2019. DOI: 10.1016/j.pdpdt.2019.05.035. Disponível em: http://repositorio.ipen.br/handle/123456789/30018. Acesso em: $DATA.

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Abstract: Localized surface plasmon resonance (LSPR) of gold nanoparticles has been reported to increase the antimicrobial effect of the photodynamic therapy. Although silver nanoparticles (AgNPs) are an efficient growth inhibitor of microorganisms, no studies exploring LSPR of AgNPs to enhance the photodynamic inactivation (PDI) have been related. In this work, we described the LSPR phenomenon of AgNP sand investigated its interaction with riboflavin, a natural photosensitizer. We evaluated the use of AgNPs coated with pectin (p-AgNP) in riboflavin (Rb)-mediated PDI of Escherichia colt (Gram- bacteria) and Streptococcus mutans (Gram + bacteria) using a blue light-emitting diode (lambda = 455 +/- 20 nm) of optical power 200 mW. Irradiance was 90 mW/cm(2) and radiant exposure varied according to the time exposure. Uptake of Rb and p-AgNP by the cells was evaluated by measuring the supernatant absorption spectra of the samples. We observed that LSPR of p-AgNPs was able to enhance the riboflavin photodynamic action on S. mutans but not on E. colt, probably due to the lower uptake of Rb by E. colt. Taken together, our results provide insights to explore the use of the LPRS promoted by silver nanostructures to optimize antimicrobial PDI protocols.

Palavras-Chave: surfaces; plasmons; resonance; photosensitivity; inactivation; nanoparticles; silver; antimicrobial agents; light emitting diodes; bacterial spores; streptococcus; escherichia coli

RIBEIRO, MARTHA S.; MELO, LUCIANA S.A. de; FAROOQ, SAJID; BAPTISTA, ALESSANDRA; KATO, ILKA T.; NUNEZ, SILVIA C.; ARAUJO, RENATO E. de. Photodynamic inactivation assisted by localized surface plasmon resonance of silver nanoparticles: in vitro evaluation on Escherichia coli and Streptococcus mutans. Photodiagnosis and Photodynamic Therapy, v. 22, p. 191-196, 2018. DOI: 10.1016/j.pdpdt.2018.04.007. Disponível em: http://repositorio.ipen.br/handle/123456789/28994. Acesso em: $DATA.

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Abstract: Background: Methylene blue (MB)-mediated photodynamic inactivation (PDI) has shown good results in killing Candida spp. Although MB solutions are commonly used, new formulations have been designed to improve PDI. However, chemical substances in the formulation may interfere with the PDI outcome. In this sense, different methodologies should be used to evaluate PDI in vitro. Herein, we report different methodologies to evaluate the effects of PDI with an oral formulation (OF) containing 0.005% MB on Candida albicans biofilm. Methods: Biofilms were treated using the MB-OF, with 5 min pre-irradiation time and exposure to a 640 nm LED device (4.7 J/cm2). PDI was evaluated by the XTT reduction test, counting the colony forming units (CFU), a filamentation assay, crystal violet (CV) staining, and scanning electronic microscopy (SEM). Results: PDI was able to reduce around 1.5 log10 CFU/mL, even though no significant differences were noted in metabolic activity in comparison to the control immediately after PDI. A significant decrease in yeast to hyphae transition was observed after PDI, while the biofilm exhibited flattened cells and a reduced number of yeasts in SEM. The CV assay showed increased biomass. Conclusion: MB-OF-mediated PDI was effective in C. albicans biofilms, as it significantly reduced the CFU/mL and the virulence of surviving cells. The CV data were inconclusive, since the OF components interacted with the CV, making the data useless. Taken together, our data suggest that the association of different methods allows complementary responses to assess how PDI mediated by a formulation impacts biofilms.

Palavras-Chave: antimicrobial agents; methylene blue; chemotherapy; photodynamic therapy

COLLINA, GABRIELA A. da; CABRAL, FERNANDA V.; MONTEIRO, CAROLINA M.; MACHADO, GABRIELA B.; GONCALVES, JOSE M.L.A.; FREIRE, FERNANDA; PRATES, RENATO A.; RIBEIRO, MARTHA S.; PAVANI, CHRISTIANE. The importance of combining methods to assess Candida albicans biofilms following photodynamic inactivation. Photodiagnosis and Photodynamic Therapy, v. 38, p. 1-6, 2022. DOI: 10.1016/j.pdpdt.2022.102769. Disponível em: http://repositorio.ipen.br/handle/123456789/33201. Acesso em: $DATA.

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Abstract: Background: Cutaneous leishmaniasis (CL) is an important tropical neglected disease with broad geographical dispersion. The lack of effective drugs has raised an urgent need to improve CL treatment, and antimicrobial photodynamic therapy (APDT) has been investigated as a new strategy to face it with positive outcomes. Natural compounds have emerged as promising photosensitizers (PSs), but their use in vivo remains unexplored. Purpose: In this work, we investigated the potential of three natural anthraquinones (AQs) on CL induced by Leishmania amazonensis in BALB/c mice. Study Design/Methods: : Animals were infected and randomly divided into four groups: CG (control, non-treated group), G5ClSor-gL (treated with 5-chlorosoranjidiol and green LED, 520±10 nm), GSor-bL and GBisor-bL (treated with soranjidiol and bisoranjidiol, respectively, exposed to violet-blue LED, 410±10 nm). All AQs were assayed at 10 μM and LEDs delivered a radiant exposure of 45 J/cm2 with an irradiance of 50 mW/cm2 . We assessed the parasite burden in real time for three consecutive days. Lesion evolution and pain score were assessed over 3 weeks after a single APDT session. Results: G5ClSor-gL was able to sustain low levels of parasite burden over time. Besides, GSor-bL showed a smaller lesion area than the control group, inhibiting the disease progression. Conclusion: Taken together, our data demonstrate that monoAQs are promising compounds for pursuing the best protocol for treating CL and helping to face this serious health problem. Studies involving host-pathogen interaction as well as monoAQ-mediated PDT immune response are also encouraged.

Palavras-Chave: skin diseases; parasites; protozoa; light emitting diodes; luminescence; pain

DIMMER, JESICA A.; CABRAL, FERNANDA V.; MONTOYA, SUSANA C.N.; RIBEIRO, MARTHA S. Towards effective natural anthraquinones to mediate antimicrobial photodynamic therapy of cutaneous leishmaniasis. Photodiagnosis and Photodynamic Therapy, v. 42, p. 1-6, 2023. DOI: 10.1016/j.pdpdt.2023.103525. Disponível em: http://repositorio.ipen.br/handle/123456789/34086. Acesso em: $DATA.

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