Effects of antimicrobial photodynamic therapy with photodithazine® on methicillin-resistant Staphylococcus aureus (MRSA): Studies in biofilms and experimental model with Galleria mellonella.

Staphylococcus aureus infections are a severe health problem due to the high mortality rate. Conventional treatment of these infections is via the administration of antibiotics. However, its indiscriminate use can select resistant microorganisms. Thus, it is necessary to develop alternatives for antibiotic therapy. Antimicrobial Photodynamic Therapy (aPDT), a therapeutic method that associates a photosensitizer (PS), a light source with adequate wavelength to the PS, interacts with molecular oxygen generating reactive oxygen species responsible for cell inactivation, is a viable alternative. This work aimed to analyze, in vitro and in vivo, the action of aPDT with PS Photodithazine® (PDZ) on the methicillin-resistant S. aureus (MRSA) strain. In the in vitro method, the S. aureus biofilm was incubated with PDZ at 50 and 75 µg.mL-1 for 15 min, adopting the light dose of 25, 50, and 100 J/cm2. In addition, PS interaction, formation of reactive oxygen species (ROS), bacterial metabolism, adhesion, bacterial viability, and biofilm structure were evaluated by scanning electron microscopy. Subsequently, the strain was inoculated into models of Galleria mellonella, and the survival curve, health scale, blood cell analysis, and CFU/mL of S. aureus in the hemolymph were analyzed after aPDT. In the in vitro results, bacterial reduction was observed in the different PDZ concentrations, highlighting the parameters of 75 µg.mL-1 of PDZ and 100 J/cm2. As for in vivo results, aPDT increased survival and stimulated the immune system of G. mellonella infected by S. aureus. aPDT proved effective in both models, demonstrating its potential as an alternative therapy in treating MRSA bacterial infections.

Molecular effects of photodynamic therapy with curcumin on Leishmania major promastigotes.

Leishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.

Photodynamic therapy of cationic and anionic BSA-curcumin nanoparticles on amastigotes of Leishmania braziliensis and Leishmania major and Leishmania amazonensis.

Cutaneous leishmaniasis is a neglected disease prevalent in tropical countries, and conventional treatment can cause several serious side effects. Photodynamic therapy (PDT) can be considered a promising treatment alternative, as it is non-invasive therapy that has no side effects and uses accessible and low-cost substances, such as curcumin. This study evaluated the PDT response with cationic and anionic BSA nanoparticles encapsulated with curcumin in macrophages infected with L. braziliensis, L. major, and L. amazonensis. The nanoparticle system was characterized using a steady-state technique, scanning electron microscopy (SEM) study, and its biological activity was evaluated using macrophage cell lines infected with different Leishmania species. All spectroscopy measurements demonstrated that BSA curcumin (BSACur) has good photophysical properties, and confocal microscopy shows that macrophages and protozoa internalized the nanoparticles. The viability test demonstrated that at low concentrations, such as 0.1, 0.7, and 1.0 µmol. L-1, there was a decrease in cell viability after PDT application. Furthermore, a decrease in the number of parasites recovered was observed in the PDT groups. The results allowed us to conclude that curcumin loaded into BSA nanoparticles may have potential application in drug delivery systems for PDT protocols, demonstrating reduced cell viability at lower concentrations than free curcumin.

Adverse effects of topical photodynamic therapy in rosacea - Case report.

Rosacea is a chronic and inflammatory skin condition, with relapses being a common characteristic. Its treatments are based on cosmetics, drugs, and the application of procedures based on high-powered light. Photodynamic Cosmetic Therapy (PCT) combines light, a photosensitizer (PS), and molecular oxygen present in tissues, generating photochemical reactions capable of causing tissue and vascular destruction, stimulating tissue repair. We report a case with an adverse effect caused by applying PCT, using 2 % 5-aminolevulinic acid (ALA 2 %), and irradiated with amber LED light associated with infrared radiation for the control of rosacea. A patient with subtype II rosacea underwent PCT treatment of 3 sessions at 21-day intervals, being evaluated using photographic images and Wood's lamp. In the first session of the therapy, an exacerbated inflammatory process was observed. Such an adverse event is estimated to be as a result of the patient using ointment containing corticosteroids for a short period. With the use of medications, it was possible to recover the appearance of the skin thoroughly, and after 21 days, the treatment sessions were performed again. Despite the complication that affected the patient in this study, positive effects were found after the pharmacological therapeutic measures were adopted.

Fourier Transform Infrared Spectroscopy (FT-IR) of Pseudomonas aeruginosa post photodynamic therapy with Curcumin in vitro.

Alternative therapies against pathogens are under intense investigation because of their increasing resistance to antibiotics. Photodynamic therapy (PDT) is one such alternative that has shown promising results. However, for the widespread use of PDT, it is essential to decipher underlying mechanisms, so as to improve PDT's therapeutic applications. Because of this, we have studied biochemical changes in pathogen Pseudomonas aeruginosa, a medically important bacteria that has developed antibiotic resistance, after PDT with curcumin photosensitizer. Results show a drastic decrease in α-helix protein and increased disordered and ß-sheet secondary structure proteins in P. Aeruginosa post-PDT compared to control. Interestingly, these biochemical changes differ from PDT of pathogens Leishmania braziliensis and Leishmania major with photosensitizer methylene blue. This observation underlines the need for extensive studies on PDT of different pathogens to understand mechanisms of action and develop better PDT strategies.

Antimicrobial photodynamic therapy with curcumin on methicillin-resistant Staphylococcus aureus biofilm.

Healthcare-Associated Infections (HAI) effect approximately 1.5 million individuals worldwide. Among the causes of HAIs in Latin America, Staphylococcus aureus presents a severe danger due to its rapid spread and ease of developing antibiotic resistance. Upon acquiring methicillin resistance, it receives the classification Methicillin-Resistant Staphylococcus aureus (MRSA), responsible for 40 to 60% of HAIs. The increase in resistant microorganisms led to the search for alternative methods, such as antimicrobial Photodynamic Therapy (aPDT), forming Reactive Oxygen Species (ROS), leading bacterial cells to death. The objective of this work was to evaluate in vitro the antimicrobial action of PDT with curcumin in MRSA biofilm. The strains were induced to form biofilm and incubated with curcumin for 20 min, irradiated with LED (Light Emitting Diode) 450 nm, at 110 mW/cm2, 50 J/cm2 for 455 s, subsequently counting the Colony Forming Units, Scanning Electron Microscopy (SEM) micrographs, Confocal Microscopy images, Resazurin dye test, ROS quantification to assess the effect of PDT on biofilm. The results show that PDT with curcumin reduced the biofilm growth of the MRSA strain. In addition, confocal microscopy showed that curcumin was internalized by S. aureus in the cells at the concentration used, and when isolated, curcumin and the irradiation parameter did not show cytotoxicity. The study demonstrated that the PDT in the established parameters reduced the growth of the MRSA strain biofilm, making it a relevant alternative possibility for the inactivation of this strain.

Gelatin nanoparticles via template polymerization for drug delivery system to photoprocess application in cells.

Photodynamic therapy (PDT) is a clinical treatment based on the activation of light-absorbing photosensitizers (PS) to generate reactive oxygen species, which are toxic to the targeted disease cells. Because most PS are hydrophobic with poor water solubility, it is necessary to encapsulate and solubilize PS in aqueous conditions to improve the photodynamic action for this compound. In this work, gelatin-poly(acrylic acid) nanoparticles (PAA/gelatin nanoparticles) via template polymerization for incorporation aluminum chloride phthalocyanine (ClAlPc) as a model drug for PDT application were developed. Biocompatible core-shell polymeric nanoparticles were fabricated via template polymerization using gelatin and acrylic acid as a reaction system. The nanoparticulate system was studied by scanning electron microscopy, steady-state, and their biological activity was evaluated using in vitro cancer cell lines by classical MTT assay. The obtained nanoparticles had a spherical shape and DLS particle size were determined further and was found to be around 170 nm. The phthalocyanine-loaded-nanoparticles maintained their photophysical behaviour after encapsulation. It is found that ClAlPc can be released from the nanoparticles in a sustained manner with a small initial burst release. In vitro cytotoxicity revealed that ClAlPc-loaded nanoparticles had similar cytotoxicity to free ClAlPc with mouse melanoma cancer cell line (B16-F10). In vitro photoeffects assay indicated that the nanoparticle formulation was superior in anticancer effect to free ClAlPc on mouse melanoma cancer cell line B16-F10. The results indicate that ClAlPc encapsulated in gelatin-poly(acrylic acid) nanoparticles are a successful delivery system for improving photodynamic activity in the target tissue.

Photodynamic effect of protoporphyrin IX in gliosarcoma 9l/lacZ cell line.

Photodynamic Therapy (PDT) is an oncologic treatment, producing reactive oxygen species (ROS) to induce the death of cancer cells. This study aimed to evaluate the action of PDT on gliosarcoma cells, using protoporphyrin IX as PS by incubation with the precursor aminolevulinic acid (ALA). An LED device was used with a light dose of 10 J/cm². The success of the therapy proved to be dependent on the concentration of ALA, and an incubation time of 4 h required for an effective response. Cell death was prevalent due to necrosis when assessed 18 h post-PDT. ALA proved to be an option to PDT in cells of the 9 L/lacZ, with the protocol tested.

Zinc pthalocyanine loaded poly (lactic acid) nanoparticles by double emulsion methodology for photodynamic therapy against 9 L/LacZ gliosarcoma cells.

Development delivery systems, such as nanoparticles, represent a growing area in biomedical research. Nanoparticles (NP) were prepared using a double-emulsion method to load zinc(II) phthalocyanine (ZnPc). NP were obtained using poly (lactic acid) (PLA). ZnPc is a second generation of photosensitizer used in photodynamic therapy (PDT). ZnPc loaded PLA nanoparticles (NPLA-ZnPc) were prepared by double-emulsion method, characterized and available in cellular culture. The mean nanoparticle size presented particle size was 384.7 ± 84.2 nm with polydispersity index (PDI) of 0.150 ± 0.015, and the encapsulation efficiency was of 83%. The nanoparticle formulations presented negative zeta potential values (-27.5 ± 1.0 mV), explaining their colloidal stability. ZnPc loaded nanoparticles maintain its photophysical behavior after encapsulation. Photosensitizer release from nanoparticles was sustained over 168 h with a biphasic ZnPc release profile. An in vitro phototoxic effect in range of 80% was observed in 9 L/LacZ gliosarcoma cells at laser light doses (10 J cm-2) with 3.0 µg mL-1 of NPLA-ZnPc. All the physical-chemical, photophysical and photobiological measurements performed allow us to conclude that ZnPc loaded PLGA nanoparticles is a promising drug delivery system for PDT.

Efficiency of Antimicrobial Photodynamic Therapy with Photodithazine® on MSSA and MRSA Strains.

Staphylococccus aureus is a ubiquitous and opportunistic bacteria associated with high mortality rates. Antimicrobial photodynamic therapy (aPDT) is based on the application of a light source and a photosensitizer that can interact with molecular oxygen, forming Reactive Oxygen Species (ROS) that result in bacterial inactivation. This study aimed to analyze, in vitro, the action of aPDT with Photodithazine® (PDZ) in methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains. The strains were incubated with PDZ at 25, 50, 75, and 100 mg/L for 15 min and irradiated with fluences of 25, 50, and 100 J/cm2. The internalization of PDZ was evaluated by confocal microscopy, the bacterial growth by counting the number of colony-forming units, as well as the bacterial metabolic activity post-aPDT and the production of ROS. In both strains, the photosensitizer was internalized; the production of ROS increased when the aPDT was applied; there was a bacterial reduction compared to the control at all the evaluated fluences and concentrations; and, in most parameters, it was obtained complete inactivation with significant difference (p < 0.05). The implementation of aPDT with PDZ in clinical strains of S. aureus has resulted in its complete inactivation, including the MRSA strains.

Evaluation of the Photodynamic Therapy with Curcumin on L. braziliensis and L. major Amastigotes.

Cutaneous leishmaniasis (CL) is a neglected disease prevalent in tropical countries with the ability to cause skin lesions. Photodynamic therapy (PDT) represents a specific and topical option for the treatment of these lesions. This study evaluated the response of macrophages infected with L. braziliensis and L. major to PDT with curcumin. Curcumin concentrations were evaluated in serial dilutions from 500.0 to 7.8 µg/mL using LED (λ = 450 ± 5 nm), with a light dose of 10 J/cm2. The Trypan blue viability test, ultrastructural analysis by scanning electron microscopy (SEM), mitochondrial polarity by Rhodamine 123 (Rho 123), curcumin internalization by confocal microscopy, and counting of the recovered parasites after the PDT treatment were performed. The lowest concentrations of curcumin (15.6 and 7.8 µg/mL) presented photodynamic inactivation. Cell destruction and internalization of curcumin in both macrophages and intracellular parasites were observed in microscopy techniques. In addition, an increase in mitochondrial membrane polarity and a decrease in the number of parasites recovered was observed in the PDT groups. This study indicates that PDT with curcumin has the potential to inactivate infected macrophages and might act as a basis for future in vivo studies using the parameters herein discussed.

CELLULAR AND METABOLIC CHANGES AFTER PHOTODYNAMIC THERAPY IN LEISHMANIA PROMASTIGOTES.

Leishmaniasis is a zoonotic disease, regarded by WHO as a public health problem that has presented a significant increase in the recent years. Conventional treatment is toxic and leads to serious side effects. Photodynamic therapy has been studied as a treatment to cutaneous leishmaniasis. This study aimed to evaluate the cell viability, morphological changes, type of cell death, production of reactive oxygen species, and changes in the mitochondrial membrane and DNA fragmentation in Leishmania braziliensis and Leishmania major promastigotes. Confocal microscopy was used to quantify the fluorescence emitted by JC-1, Annexin V, and propidium iodide reagents. The trypan blue exclusion test was used to evaluate the viability of the cells, the mitochondrial activity was verified with MTT, and the morphological changes were analyzed for SEM and DNA damage using the comet assay. PDT using curcumin at 500, 125, and 31,25 µg/mL decreased the viability of the parasites and induced changes in the mitochondrial membrane potential. The production of reactive oxygen species was dose-dependent and was observed only in the groups submitted to PDT. DNA damage was also observed in the parasite cells. The morphology of the cells was affected mainly at the highest curcumin concentration, resulting in rounded cells with a shortened flagellum. When the type of cell death was analyzed, the prevalence of apoptosis was noted. The results support the use of curcumin as photosensitizer in PDT against Leishmania promastigotes in the treatment for cutaneous leishmaniasis.

Analysis of the effects of Photodynamic therapy with Photodithazine on the treatment of 9l/lacZ cells, in vitro study.

Gliosarcoma is an aggressive brain tumor. Photodynamic Therapy (PDT) is a treatment that can be used for various cancers of the CNS. The aim of this study was to analyze the effects of PDT with Photodithazine (PDZ) in the treatment of gliosarcoma, using 9 L/lacZ cells and serial concentrations of 200 µg/mL to 3.1 µg/mL of PDZ. The samples were divided into two groups: dark and light (10 J/cm²). The PDZ was internalized along all the cytoplasmic extension. Viability tests demonstrated a reduction in viable cells after PDT. The production of ROS was concentration-dependent and PDZ was found in mitochondria and lysosomes, presenting a discrete connection with α-tubulin. However, this structure is likely damaged, evidenced by changes in the morphological analysis. Thus, according to the parameters of this study, PDZ proved to be an interesting PS in PDT for the treatment of gliosarcoma, with the inherent limitations of an in vitro study.

Biochemical changes in Leishmania braziliensis after photodynamic therapy with methylene blue assessed by the Fourier transform infrared spectroscopy.

Photodynamic therapy (PDT) with photosensitizer methylene blue was applied to Leishmania braziliensis, and Fourier transform infrared (FTIR) spectroscopy was used to study biochemical changes in the parasite after PDT in comparison to untreated (C), only irradiation (I), and only photosensitizer (PS). Spectral analysis suggests increase in lipids, proteins, and protein secondary structures in PDT compared with C and decrease in nucleic acids and carbohydrates. Interestingly, these trends are different from PDT of Leishmania major species, wherein lipids decrease; there are minimal changes in secondary structures and increase in nucleic acids and carbohydrates. The study thus suggests possibility of different biomolecular players/pathways in PDT-induced death of L. braziliensis and L. major.

Analysis of the effect of photodynamic therapy with Fotoenticine on gliosarcoma cells.

Gliosarcoma is a highly aggressive malignant neoplasm and a histopathological variant of wild-type glioblastoma multiforme isocitrate dehydrogenase (HDI). The current standard treatment consists of chemotherapy, radiotherapy and surgical resection, however, despite advances in these techniques, the patient's prognosis remains unfavorable. Photodynamic therapy (PDT) is a noninvasive technique that has been highlighted as an alternative form of cancer treatment because it does not present the side effects associated with systemic treatments. The objective of this study was to evaluate the cell viability and the intracellular localization of photosensitizer (PS) chlorin e6 Fotoenticine in 9L/lacZ cells. Therefore, tests of cytotoxicity, morphology, and location of PS were performed. The viability test showed no cytotoxicity in the dark at all concentrations and 100 % cell death at the highest concentrations after PDT. The mitochondrial activity test showed a reduction in all groups after PDT. The production of reactive oxygen species (ROS) was higher in the PDT groups and dependent on the PS concentration. Morphological analysis after PDT showed apparent cytoplasmic destruction in all the tested concentrations, with the presence of rounded cells due to the loss of their extensions and absence of nuclear alterations. The PS accumulation in the mitochondria and cytoskeleton was observed by the confocal microscopy; however, there is no evidence of its internalization in the lysosomes. It was concluded that PDT with Fotoenticine is a promising alternative therapy showing decreased cell viability, increased ROS production and adequate localization to trigger cell death.

Preparation of gelatin nanoparticles by two step desolvation method for application in photodynamic therapy.

Gelatin nanoparticles have recently been receiving considerable attention because they offer a good option as release systems due to their low cost, biocompatibility, biodegradability and its application in several types of formulations. This study aim was to evaluate the potential application of gelatin nanoparticles entrapping a photosensitizer in Photodynamic Therapy. Gelatin nanoparticles were studied by steady-state techniques and the biological activity evaluated by in vitro MTT assay. The particles were spherical in shape exhibiting a 273 nm diameter with a low tendency to aggregate. The loading efficiency was 76%. Photosensitizer photophysical properties were shown to be preserved after GN encapsulation. The cells viability obtaining 85% cells death compared with control. The results demonstrate that gelatin nanoparticles can be successfully applied for photosensitizers encapsulation or other active drugs and be used as an optimal medium for a variety of bioactive materials, which can also be encapsulated by the proposed method.

Methylene blue internalization and photodynamic action against clinical and ATCC Pseudomonas aeruginosa and Staphyloccocus aureus strains.

Bacterial infections have been a major challenge to health. Increasing resistance to antimicrobial agents, according to World Health Organization, could be the major cause of death until 2050. Photodynamic therapy emerges as an alternative in microbial inactivation, due to its selectivity and to decreasing or dismissing antibiotic use. This study aimed at evaluating, in vitro, the internalization of the Methylene Blue and its photodynamic activity against a clinical and ATCC strain of Pseudomonas aeruginosa and Staphyloccocus aureus. Thus, the strains were incubated with MB in concentrations of 100, 300 e 500 µg/ml and then irradiated with a LED (±660 nm) at fluence of 10 and 25 J/cm2. The MB internalization was evaluated using a confocal microscope (Zeiss LSM 700), to capture the MB and the DAPI (for DNA staining). It was possible to observe that the MB was internalized by the bacterial cells, in all concentrations tested. The CFU/ml count demonstrated significant reduction (p ≤ 0,01) at the average 5.0 logs comparing with control group for the two species in all the tested concentrations. In conclusion, the strains tested were capable of internalizing the MB. PDT with MB was able to decrease the growth of the tested strains in vitro, being a promising alternative to the future treatment of infections caused by these species.

Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis.

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500µg/ml, in serial dilution, until 7,8µg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost.