Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma.

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Porphyrin nanoemulsion for antimicrobial photodynamic therapy: effective delivery to inactivate biofilm-related infections.

The management of biofilm-related infections is a challenge in healthcare, and antimicrobial photodynamic therapy (aPDT) is a powerful tool that has demonstrated a broad-spectrum activity. Nanotechnology has been used to increase the aPDT effectiveness by improving the photosensitizer's delivery properties. NewPS is a simple, versatile, and safe surfactant-free nanoemulsion with a porphyrin salt shell encapsulating a food-grade oil core with promising photodynamic action. This study evaluated the use of NewPS for aPDT against microorganisms in planktonic, biofilm, and in vivo models of infected wounds. First, the potential of NewPS-mediated aPDT to inactivate Streptococcus pneumoniae and Staphylococcus aureus suspensions was evaluated. Then, a series of protocols were assessed against S. aureus biofilms by means of cell viability and confocal microscopy. Finally, the best biofilm protocol was used for the treatment of S. aureus in a murine-infected wound model. A high NewPS-bacteria cell interaction was achieved since 0.5 nM and 30 J/cm2 was able to kill S. pneumoniae suspension. In the S. aureus biofilm, enhanced efficacy of NewPS-aPDT was achieved when 100 µM of NewPS was applied with longer periods of incubation at the light dose of 60 J/cm2. The best single and double-session protocol reduced 5.56 logs and 6.03 logs, respectively, homogeneous NewPS distribution, resulting in a high number of dead cells after aPDT. The in vivo model showed that one aPDT session enabled a reduction of 6 logs and faster tissue healing than the other groups. In conclusion, NewPS-aPDT may be considered a safe and effective anti-biofilm antimicrobial photosensitizer.

Lung surfactant negatively affects the photodynamic inactivation of bacteria-in vitro and molecular dynamic simulation analyses.

In the context of the rapid increase of antibiotic-resistant infections, in particular of pneumonia, antimicrobial photodynamic therapy (aPDT), the microbiological application of photodynamic therapy (PDT), comes in as a promising treatment alternative since the induced damage and resultant death are not dependent on a specific biomolecule or cellular pathway. The applicability of aPDT using the photosensitizer indocyanine green with infrared light has been successfully demonstrated for different bacterial agents in vitro, and the combination of pulmonary delivery using nebulization and external light activation has been shown to be feasible. However, there has been little progress in obtaining sufficient in vivo efficacy results. This study reports the lung surfactant as a significant suppressor of aPDT in the lungs. In vitro, the clinical surfactant Survanta® reduced the aPDT effect of indocyanine green, Photodithazine®, bacteriochlorin-trizma, and protoporphyrin IX against Streptococcus pneumoniae. The absorbance and fluorescence spectra, as well as the photobleaching profile, suggested that the decrease in efficacy is not a result of singlet oxygen quenching, while a molecular dynamics simulation showed an affinity for the polar head groups of the surfactant phospholipids that likely impacts uptake of the photosensitizers by the bacteria. Methylene blue is the exception, likely because its high water solubility confers a higher mobility when interacting with the surfactant layer. We propose that the interaction between lung surfactant and photosensitizer must be taken into account when developing pulmonary aPDT protocols.

Photobleaching Kinetics and Effect of Solvent in the Photophysical Properties of Indocyanine Green for Photodynamic Therapy.

Indocyanine green is an attractive molecule for photodynamic therapy due to its near infrared absorption, resulting in a higher tissue penetration. However, its quantum yields of the triplet and singlet state have been reported to be low and then, reactive oxygen species are unlikely to be formed. Aiming to understand the ICG role in photodynamic response, its photobleaching behavior in solution has been studied under distinct conditions of CW laser irradiation at 780 and 808 nm, oxygen saturations and solvents. Sensitizer bleaching and photoproduct formation were measured by absorption spectroscopy and analyzed using the PDT bleaching macroscopic model to extract physical parameters. ICG photobleaching occurs even at lower oxygen concentrations, indicating that the molecule presents more than one way of degradation. Photoproducts were produced even in solution of less than 4 % oxygen saturation for both solvents and excitation wavelengths. Also, the amplitude of absorption related to J-dimers was increased during irradiation, but only in 50 % PBS solution. The formation of photoproducts was enhanced in the presence of J-type dimers under low oxygen concentration, and the quantum yields of triplet and singlet states were one order of magnitude and two times higher, respectively, when compared to ICG in distilled H2 O.

Nanostructure-Driven Indocyanine Green Dimerization Generates Ultra-Stable Phototheranostics Nanoparticles.

Indocyanine green (ICG) is the only near-infrared (NIR) dye approved for clinical use. Despite its versatility in photonic applications and potential for photothermal therapy, its photobleaching hinders its application. Here we discovered a nanostructure of dimeric ICG (Nano-dICG) generated by using ICG to stabilize nanoemulsions, after which ICG enabled complete dimerization on the nanoemulsion shell, followed by J-aggregation of ICG-dimer, resulting in a narrow, red-shifted (780 nm→894 nm) and intense (≈2-fold) absorbance. Compared to ICG, Nano-dICG demonstrated superior photothermal conversion (2-fold higher), significantly reduced photodegradation (-9.6 % vs. -46.3 %), and undiminished photothermal effect (7 vs. 2 cycles) under repeated irradiations, in addition to excellent colloidal and structural stabilities. Following intravenous injection, Nano-dICG enabled real-time tracking of its delivery to mouse tumors within 24 h by photoacoustic imaging at NIR wavelength (890 nm) distinct from the endogenous signal to guide effective photothermal therapy. The unprecedented finding of nanostructure-driven ICG dimerization leads to an ultra-stable phototheranostic platform.

Photodynamic Inactivation of Microorganisms Using Semisynthetic Chlorophyll a Derivatives as Photosensitizers.

In this study, we describe the semisynthesis of cost-effective photosensitizers (PSs) derived from chlorophyll a containing different substituents and using previously described methods from the literature. We compared their structures when used in photodynamic inactivation (PDI) against Staphylococcus aureus, Escherichia coli, and Candida albicans under different conditions. The PSs containing carboxylic acids and butyl groups were highly effective against S. aureus and C. albicans following our PDI protocol. Overall, our results indicate that these nature-inspired PSs are a promising alternative to selectively inactivate microorganisms using PDI.

Strategies to Improve the Antimicrobial Efficacy of Photodynamic, Sonodynamic, and Sonophotodynamic Therapies.

BACKGROUND AND OBJECTIVES: This work evaluated antimicrobial photodynamic therapy (PDT), sonodynamic therapy (SDT), and the association of both therapies (sonophotodynamic therapy [SPDT]), mediated by curcumin (Cur) against Staphylococcus aureus biofilm. Next, additional strategies for these treatments were assessed. MATERIALS AND METHODS: S. aureus biofilms received PDT, SDT, and SPDT, mediated by Cur (80 µM), LED light (450 nm), and 1 MHz ultrasound. The same treatments were also performed adding a strategy: Cur with sodium dodecyl sulfate (SDS), Cur with potassium iodide (KI) or a pre-treatment with ultrasound. Cell viability was determined and biofilm architecture was evaluated under confocal microscopy. RESULTS: SPDT was more effective to inactivate the bacteria than PDT and SDT. SDS achieved the greatest viability reductions, followed by KI and ultrasound pre-treatment. Confocal images revealed biofilm disruption and a reduced number of cells in all treatments. However, SPDT exhibited a pronounced effect and it was greater using SDS. CONCLUSION: SPDT was more effective and additional strategies potentiated its effectiveness. Lasers Surg. Med. © 2021 Wiley Periodicals LLC.

Epigallocatechin Gallate Enhances MAL-PDT Cytotoxic Effect on PDT-Resistant Skin Cancer Squamous Cells.

Photodynamic therapy (PDT) has been used to treat certain types of non-melanoma skin cancer with promising results. However, some skin lesions have not fully responded to this treatment, suggesting a potential PDT-resistant phenotype. Therefore, novel therapeutic alternatives must be identified that improve PDT in resistant skin cancer. In this study, we analyzed the cell viability, intracellular protoporphyrin IX (PpIX) content and subcellular localization, proliferation profile, cell death, reactive oxygen species (ROS) detection and relative gene expression in PDT-resistant HSC-1 cells. PDT-resistant HSC-1 cells show a low quantity of protoporphyrin IX and low levels of ROS, and thus a low rate of death cell. Furthermore, the resistant phenotype showed a downregulation of HSPB1, SLC15A2, FECH, SOD2 and an upregulation of HMBS and BIRC5 genes. On the other hand, epigallocatechin gallate catechin enhanced the MAL-PDT effect, increasing levels of protoporphyrin IX and ROS, and killing 100% of resistant cells. The resistant MAL-PDT model of skin cancer squamous cells (HSC-1) is a reliable and useful tool to understand PDT cytotoxicity and cellular response. These resistant cells were successfully sensitized with epigallocatechin gallate catechin. The in vitro epigallocatechin gallate catechin effect as an enhancer of MAL-PDT in resistant cells is promising in the treatment of difficult skin cancer lesions.

Overall Results for a National Program of Photodynamic Therapy for Basal Cell Carcinoma: A Multicenter Clinical Study to Bring New Techniques to Social Health Care.

Along the past years, a national program to implement photodynamic therapy (PDT) for nonmelanoma skin cancer (NMSC) was performed over the Brazilian territory. Using a strategy involving companies, national bank, and medical partners, equipment, medication, and protocols were tested in a multicenter study. With results collected over 6 years, we could reach a great deal of advances concerning the use of PDT for skin cancer. We present the overall reached results of the program and discuss several aspects about it, including public politics of treatment. A discussion about advantages of this technique within conditions of health care is placed, comparing PDT with surgery, including an analysis about the implementation of PDT in countries in development as Brazil, considering not only technical but social aspects, as the distribution of medical doctor in the Brazilian territory. The program resulted in a huge dissemination of PDT in Brazil and many countries in Latin America, in a partnership among public politics, universities, companies, and hospitals and clinics and in the insertion of national technologies as option to treat NMSC. Consequence of the program is mainly the continuation of the use of PDT in Brazil and many countries in Latin America.

Vascular Effects of Photodynamic Therapy with Curcumin in a Chorioallantoic Membrane Model.

Photodynamic Therapy (PDT) is a treatment that requires light, a photosensitizing agent, and molecular oxygen. The photosensitizer is activated by light and it interacts with the oxygen that is present in the cellular microenvironment. The molecular oxygen is transformed into singlet oxygen, which is highly reactive and responsible for the cell death. Therefore, PS is an important element for the therapy happens, including its concentration. Curcumin is a natural photosensitizer and it has demonstrated its anti-inflammatory and anti-oxidant effects that inhibit several signal transduction pathways. PDT vascular effects of curcumin at concentrations varying from 0.1 to 10 mM/cm² and topical administration were investigated in a chick Chorioallantoic Membrane (CAM) model. The irradiation was performed at 450 nm, irradiance of 50 mW/cm² during 10 min, delivering a total fluence of 30 J/cm². The vascular effect was followed after the application of curcumin, with images being obtained each 30 min in the first 3 h, 12 h, and 24 h. Those images were qualitatively and quantitatively analyzed with a MatLAB®. Curcumin was expected to exhibit a vascular effect due to its angio-inhibitory effect. Using curcumin as photosensitizer, PDT induced a higher and faster vascular effect when compared to the use of this compound alone.

Effects of phototherapy plus physical training on metabolic profile and quality of life in postmenopausal women.

A cluster of metabolic abnormalities are markedly higher among postmenopausal women. The present study evaluated the effects of infrared light emitting diode (LED) during treadmill training on multiple metabolic markers, body fat, dietary habits and quality of life in postmenopausal women. Forty-five postmenopausal women aged 50-60 years were randomly assigned to one of three groups, and of these, 30 women successfully completed the full study. The three groups were as follows: (i) the LED group, which performed treadmill training associated with phototherapy (n = 10); (ii) the exercise group, which carried out treadmill training only (n = 10); and (iii) the sedentary group, which neither performed physical training nor underwent phototherapy (n = 10). Training was performed over a period of six months, twice a week for 45 min per session at 85-90% of maximal heart rate (HRmax), which was obtained during a progressive exercise testing. The average HR and velocity during treadmill training were 144 ± 9 bpm and 5.8 ± 1.3 km/h for both trained groups. The irradiation parameters were 100 mW, 39 mW/cm2 and 108 J/cm2 for 45 min. Anthropometric data, skinfolds thickness, biochemical exams (lipid profile, glucose and insulin levels), dietary habits and quality of life were evaluated. The sum of skinfolds significantly improved in the exercise and sedentary groups (p < 0.05). Additionally, there was an improvement in lipid profile, particularly, total cholesterol and low-density lipoprotein, which reduced significantly for all groups (p ˂ 0.05). However, intake of saturated fats was significantly reduced in the sedentary group only (p < 0.05). The quality of life improved in the LED group only, with a significant reduction in the total WHQ score (p < 0.05). Physical training with or without phototherapy may improve the metabolic profile. In addition, phototherapy together with treadmill training prevented an increase in subcutaneous fat and facilitated an improved quality of life in postmenopausal women.

Interstitial PDT using diffuser fiber-investigation in phantom and in vivo models.

Photodynamic therapy (PDT) has been used for local treatment of several types of tumors. Light penetration of biological tissue is one limiting factor in PDT, decreasing the success rates of the treatment of invasive and solid tumors. In those cases, a possible solution is to use interstitial PDT, in which both diffuser optical fibers are inserted into the tumor. The uniformity of the diffuser emission plays a crucial role in planning the delivery of the appropriate light fluence and in ensuring treatment success. In this study, we characterized a diffuser optical fiber concerning its homogeneity. We showed that the diffuser emission can be inhomogeneous and that the necrosis generated by interstitial PDT using such a diffuser for illumination is asymmetrical in volume as a result. This observation has relevant consequences in achieving success in PDT and phototherapies in general, as the delivered light fluence depends on adequate previous knowledge of the irradiation profile.

A Multicenter Clinical Study of Expected and Unexpected Side Reactions During and After Skin Cancer Treatment by Photodynamic Therapy.

Photodynamic therapy (PDT) has been widely used for oncologic indications, especially nonmelanoma skin cancer such as superficial and nodular basal cell carcinoma (BCC). We present a multicenter clinical study conducted between 2012 and 2014 analyzing the adverse reactions during and after PDT with a standardized protocol in 866 lesions. A total of 728 patients with positive clinical and histopathological diagnosis for BCC with up to 2 cm diameter were treated. The procedure consisted of curettage and topical application of cream containing 20% methyl 5-aminolevulinate. The illumination (630 nm and 150 J/cm2) was performed 3 hours after the cream application. The expected and unexpected effects observed were pain, healing, and inflammatory reactions. The pain intensity was correlated with the anatomical localization of the lesion. The patients reported a higher intensity of pain in lesions located on the head and neck rather than on the trunk and limbs. The number of sessions also influenced the pain response. A total of 83% of patients showed perfect healing and the other 17% presented abnormal healing. PDT plays an important role in BCC because of its low cost, ease of use, and low rate of side effects.

In vitro evaluation of photodynamic therapy using curcumin on Leishmania major and Leishmania braziliensis.

Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 µg/ml up to 7.8 µg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm(2) at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.

Photobiological characteristics of chlorophyll a derivatives as microbial PDT agents.

Chlorin-e6 (chl-e6) and a hydrogenated derivative (chl-e6H) were semi-synthesized, and their photophysical properties and photodynamic activity against Escherichia coli, Staphylococcus aureus and Candida albicans evaluated. Methyl pheophorbide-a (Mepheo-a) was obtained from S. maxima using methanolic extraction with acid catalysis (CH3OH­H2SO4). Chlorin-e6 was prepared from Mepheo-a by basic hydrolysis with H2O­acetone and NaOH. Hydrogenated Chlorin-e6 was synthesized by a similar procedure starting from the hydrogenated methyl pheophorbide-a (Mepheo-aH). Photophysical studies were performed in order to determine the singlet oxygen quantum yield of chl-e6H which is higher than that of chl-e6. The microorganism inactivation of chl-e6 and chl-e6H was investigated at two concentrations and three fluence levels. Both chl-e6 and chl-e6H showed microorganism inactivation against Gram-positive bacteria and a fungus.

Pixel-level classification of pigmented skin cancer lesions using multispectral autofluorescence lifetime dermoscopy imaging.

There is no clinical tool available to primary care physicians or dermatologists that could provide objective identification of suspicious skin cancer lesions. Multispectral autofluorescence lifetime imaging (maFLIM) dermoscopy enables label-free biochemical and metabolic imaging of skin lesions. This study investigated the use of pixel-level maFLIM dermoscopy features for objective discrimination of malignant from visually similar benign pigmented skin lesions. Clinical maFLIM dermoscopy images were acquired from 60 pigmented skin lesions before undergoing a biopsy examination. Random forest and deep neural networks classification models were explored, as they do not require explicit feature selection. Feature pools with either spectral intensity or bi-exponential maFLIM features, and a combined feature pool, were independently evaluated with each classification model. A rigorous cross-validation strategy tailored for small-size datasets was adopted to estimate classification performance. Time-resolved bi-exponential autofluorescence features were found to be critical for accurate detection of malignant pigmented skin lesions. The deep neural network model produced the best lesion-level classification, with sensitivity and specificity of 76.84%±12.49% and 78.29%±5.50%, respectively, while the random forest classifier produced sensitivity and specificity of 74.73%±14.66% and 76.83%±9.58%, respectively. Results from this study indicate that machine-learning driven maFLIM dermoscopy has the potential to assist doctors with identifying patients in real need of biopsy examination, thus facilitating early detection while reducing the rate of unnecessary biopsies.

Complete photodynamic inactivation of Pseudomonas aeruginosa biofilm with use of potassium iodide and its comparison with enzymatic pretreatment.

Pseudomonas aeruginosa, a gram-negative bacterium, accounts for 7% of all hospital-acquired infections. Despite advances in medicine and antibiotic therapy, P. aeruginosa infection still results in high mortality rates of up to 62% in certain patient groups. This bacteria is also known to form biofilms, that are 10 to 1000 times more resistant to antibiotics compared to their free-floating counterparts. Photodynamic Inactivation (PDI) has been proved to be an effective antimicrobial technique for microbial control. This method involves the incubation of the pathogen with a photosensitizer (PS), then, a light at appropriated wavelength is applied, leading to the production of reactive oxygen species that are toxic to the microbial cells. Studies have focused on strategies to enhance the PDI efficacy, such as a pre-treatment with enzymes to degrade the biofilm matrix and/or an addition of inorganic salts to the PS. The aim of the present study is to evaluate the effectiveness of PDI against P. aeruginosa biofilm in association with the application of the enzymes prior to PDI (enzymatic pre-treatment) or the addition of potassium iodide (KI) to the photosensitizer solution, to increase the inactivation effectiveness of the treatment. First, a range of enzymes and PSs were tested, and the best protocols for combined treatments were selected. The results showed that the use of enzymes as a pre-treatment was effective to reduce the total biomass, however, when associated with PDI, mild bacterial reductions were obtained. Then, the use of KI in association with the PS was evaluated and the results showed that, PDI mediated by methylene blue (MB) in the presence of KI was able to completely eradicate the biofilm. However, when the PDI was performed with curcumin and KI, no additive reduction was observed. In conclusion, out of all strategies evaluated in the present study, the most promising strategy to improve PDI against P. aeruginosa biofilm was the use of KI in association with MB, resulting in eradication with 108 log bacterial inactivation.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro.

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes.

Necrosis response to photodynamic therapy using light pulses in the femtosecond regime.

One of the clinical limitations of the photodynamic therapy (PDT) is the reduced light penetration into biological tissues. Pulsed lasers may present advantages concerning photodynamic response when compared to continuous wave (CW) lasers operating under the same average power conditions. The aim of this study was to investigate PDT-induced response when using femtosecond laser (FSL) and a first-generation photosensitizer (Photogem) to evaluate the induced depth of necrosis. The in vitro photodegradation of the sensitizer was monitored during illumination either with CW or an FSL as an indirect measurement of the PDT response. Healthy liver of Wistar rats was used to evaluate the tissue response. The photosensitizer was endovenously injected and 30 min after, an energy dose of 150 J cm(-2) was delivered to the liver surface. We observed that the photodegradation rate evaluated via fluorescence spectroscopy was higher for the FSL illumination. The FSL-PDT produced a necrosis nearly twice as deep when compared to the CW-PDT. An increase of the tissue temperature during the application was measured and was not higher than 2.5 °C for the CW laser and not higher than 4.5 °C for the pulsed laser. FSL should be considered as an alternative in PDT applications for improving the results in the treatment of bulky tumors where higher light penetration is required.