Enhancing the efficiency of 5-aminolevulinic acid-mediated photodynamic therapy using 5-fluorouracil on human melanoma cells

Background5-Aminolevulinic acid-mediated photodynamic therapy (ALA–PDT) is an effective and noninvasive modality for treatment of several types of non-melanoma skin cancers. This in-vitro study attempted to know whether the killing effect of ALA–PDT on the human melanoma cells (Mel-Rm cell line) could be increased by the presence of 5-fluorouracil (5-FU).MethodsTo evaluate the effect of ALA–PDT in combination with 5-FU on viability of human melanoma Mel-Rm cells, the cells incubated with 5-ALA and 5-FU for 3 h in nontoxic concentrations, and subsequently illuminated with a 630 nm light-emitting diode array. The cells viability and cytotoxicity determined by mitochondrial activity and lactate dehydrogenase assays.ResultsCombination of ALA–PDT and 5-FU (FU–ALA–PDT) showed a considerable growth inhibition according to the results of MTT assay compared to ALA–PDT. The results of LDH assay also showed a cytotoxicity effect in ALA–PDT; however, the FU–ALA–PDT showed no significantly enhancement in cytotoxicity compared to ALA–PDT using LDH assay.ConclusionThe Mel-Rm cells incubation with 5-FU before PDT enhances the efficiency of 5-Aminolevulinic acid-mediated photodynamic therapy.     

A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm

BackgroundAntimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm.Material and methodsEnterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630 nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm.Results8log₁₀ reduction in bacterial count was observed in Enterococcus faecalis while 3log₁₀ in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy.ConclusionWe conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells.     

Effects of ALA-PDT on the macrophages in wound healing and its related mechanisms in vivo and in vitro

BackgroundSeveral studies have suggested the effectiveness of photodynamic therapy (PDT) for wound healing. Macrophages are critical immune cells necessary for regulated inflammation during wound repair. However, the available information regarding the effects of PDT on macrophages during cutaneous wound healing remains insufficient. This study aimed to further investigate these aspects in vivo and in vitro.MethodsMouse full-thickness wound models were used as the study samples to investigate the therapeutic effects and mechanisms of 5-aminolevulinic acid (ALA) PDT. Wound healing rate, granulation tissue formation, local inflammation, M1/M2 macrophages differentiation, were measured at different time points treated by ALA-PDT. The polarization of macrophages induced by ALA-PDT was further evaluated in vitro using PCR and western blot analysis.ResultsALA-PDT could promote formation of granulation tissue, increase inflammatory infiltration and activate M1 macrophages in the early stage of injury. While, ALA-PDT could also facilitate absorption of granulation tissue, inhibit inflammatory infiltration and enhance M2 macrophages polarization in the later stage of wound repair. In vitro, ALA-PDT could modulate the ratio of M2 polarization to M1 polarization via NF-κB signaling pathway.ConclusionsALA-PDT topical application stimulates wound healing by regulating formation of granulation tissue, inflammatory process and M1/M2 macrophages differentiation. The study places a preliminary theoretical basis for topical ALA-PDT to be administered clinically in cutaneous wounds healing.     

Pain during topical photodynamic therapy - comparing methyl aminolevulinate (Metvix®) to aminolaevulinic acid (Ameluz®); an intra-individual clinical study

BackgroundActinic keratoses are often treated by photodynamic therapy. However, the main side effect of this treatment is pain during and shortly after illumination.ObjectivesTo evaluate, in an intra-individual study, whether the pain response differ in treatment of actinic keratoses in scalp and forhead, using branded methyl aminolevulinate (MAL) and aminolaevulinic acid (ALA).Materials and methodsPatients with mild to moderate actinic keratoses on forehead and scalp were treated with methyl aminolaevulinate (MAL)-PDT and aminolaevulinic acid (ALA)-PDT on two similar areas of forehead and scalp. The pain response were measured using visual analogue scale ranging from 0 to 10 during the illumination and 30 min after the treatment.ResultsFourteen patients completed treatment to MAL and ALA-PDT. We found no significant difference in pain intensity between MAL and ALA-PDT, neither during the treatment (p-value = 1) nor 30 min after the treatment (p-value of 0.19).ConclusionsThis intra-individual study demonstrate no significant difference between the pain response during PDT using methyl aminolevulinate and aminolaevulinic acid.     

Efficacy and safety of ALA-PDT in treatment of diabetic foot ulcer with infection

BackgroundDiabetic foot ulcers (DFUs) with infection is a major clinical issue, as the infection not only potentially devastate the wound healing, but also is the factor that most often leads to amputation. Nevertheless, traditional antibiotic treatment is often insufficient to clear the infection, which could lead to side effects. Photodynamic therapy (PDT) has broad-spectral antibacterial activity. Meanwhile, it is difficult to induce antibiotic resistance. Here, we aim to evaluate the safety and efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in the treatment of DFUs with infection.MethodsIn our study, 5 diabetic patients with infectious DFUs were diagnosed by pathological examination and the depth of wound was examined by X-Ray. All patients’ wounds were firstly irradiated with 20% ALA-PDT (635 nm, 100 J/cm², 80 mW/cm²) using the red LED to control the infection. Treatment will be combined with debridement if there is granulation necrosis or secretion on the wound surface. PDT sessions were performed weekly in all patients until healing was achieved. All patients were followed up for 0.6–1.2 years after treatment.ResultsIn 5 patients, the DFUs with infection was completely controlled by ALA-PDT. There was no recurrence of DFUs in the follow-up of 0.9 years (range, 0.6–1.2 years) after the treatment.ConclusionsALA-PDT treatment for DFUs with infection show successful outcomes and might ultimately avoid amputation.     

Rutin-gallium complex mediated antimicrobial photodynamic therapy: An in vitro studies against Streptococcus mutans biofilms

BackgroundDiscoloration of teeth following antimicrobial photodynamic therapy (aPDT) is a serious concern. Common photosensitizers are colored, and access to a photosensitizer that does not leave color on the teeth or is the same color as the enamel and dentin is highly demanded. The physicochemical characterization, anti-virulence, and antimicrobial effects of a novel rutin-gallium (III) (Rt-Ga) complex as novel photosensitizer are presented herein.Materials and MethodsPhotophysical properties and cytotoxicity of the Rt-Ga complex were evaluated in comparison with the parental rutin. Intracellular reactive oxygen species (ROS) generation following Rt-Ga complex-mediated aPDT was measured using the fluorescent 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) method. The anti-biofilm effects of Rt-Ga complex-mediated aPDT on Streptococcus mutans were assessed using a colorimetric assay. The virulence‑associated gtfB gene expression was assessed following Rt-Ga complex-mediated aPDT by quantitative real‑time PCR.ResultsThe photophysical properties of the Rt-Ga complex revealed a significant blue-shift in absorption (60 nm shift) and increased extinction coefficient (4100 M −1 cm −1; at λmax = 450 nm). Average (± SEM) DCF fluorescence intensities in an arbitrary unit (A.U.) were 7.1 ± 0.9, 4.1 ± 0.5, and 1.7 ± 0.3 for 10.0 μM of Rt-Ga complex-mediated aPDT, 7.5 μM of Rt-Ga complex-mediated aPDT, and 10.0 μM of Rt-Ga complex alone, respectively. The corresponding DCF fluorescence intensities were 710% (P = 0.001), 410% (P = 0.001), and 170% (P = 0.02) of the untreated S. mutans as the control group (1.0 ± 0.1 A.U.), respectively. The novel Rt-Ga complex-mediated aPDT exhibited no toxicity against primary human gingival fibroblast cells, a dose dependent decrease in S. mutans biofilm cell survival and virulence were observed (91.4% and 11.7-fold, respectively).ConclusionThe Rt-Ga complex-mediated anti-virulence and antimicrobial photodynamic effects were superior to the one caused by rutin alone making the Rt-Ga complex a more promising photosensitizer than the parent material.     

The effect of hypoxia on photodynamic therapy with 5-aminolevulinic acid in malignant gliomas

BackgroundGlioblastoma (GBM) is a high-grade, poor prognosis tumor that is resistant to standard treatment. The presence of a small number of glioma stem cells (GSCs) surviving in the harsh microenvironment is responsible for their refractoriness. This study aimed to investigate the effect of a hypoxic environment on the sensitivity of GSCs to photodynamic therapy with 5-aminolevulinic acid (ALA-PDT).Materials and methodsSix human GSC lines, Mesenchymal types HGG13, HGG30, HGG1123, and Proneural types HGG146, HGG157, HGG528, were divided into two groups: normoxia (O₂ 21%)-cultured cells (Normoxia-GSCs), and hypoxia (O₂ 5%)-cultured cells (Hypoxia-GSCs). To compare the effects of different oxygen partial pressures on photoporphyrin Ⅸ (PpⅨ) biosynthetic activity, PpⅨ biosynthetic enzyme and transporter expression levels were examined by qRT-PCR; the intracellular PpⅨ concentration was determined using flow cytometry. Additionally, the sensitivity of these two groups of cells to ALA-PDT was evaluated in vitro.ResultsHypoxia-GSCs showed higher mRNA levels of FECH (ferrochelatase), which is required for iron synthesis to convert PpⅨ to heme, compared with Normoxia-GSCs. Flow cytometry revealed that the accumulation of PpⅨ in Hypoxia-GSCs reduced upon incubation with ALA. However, Hypoxia-GSCs showed less reduction in sensitivity to ALA-PDT than Normoxia-GSCs.ConclusionHypoxia-GSCs had lower intracellular PpⅨ accumulation than Normoxia-GSCs due to increased gene expression of FECH, and that their sensitivity to ALA-PDT was reduced less, despite accumulating lower concentrations of PpⅨ. ALA-PDT is a potentially effective therapy for hypoxia-tolerant GSCs that exist in hypoxia at 5% oxygen concentration.     

Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: An in vitro study

BackgroundIn this study, the photoinactivation of 5-aminolevulinic acid (ALA) has been investigated on Candida albicans biofilms in vitro.MethodsAfter culture and proliferation of Candida albicans biofilms in vitro, the metabolic activity was confirmed using XTT reduction assay. Then, the suitable incubation time and concentration of ALA were determined by measuring PpIX accumulation quantities. Photosensitivity of the biofilms treated with ALA solution was studied in optical doses of 50, 100, 200 and 300 J/cm² while light irradiation was applied by a red light semiconductor. Finally, rapid immunofluorescence staining method using the LIVE/DEAD FungaLight Yeast Viability Kit and XTT assay were conducted to visualize and quantify the antifungal effect of ALA-PDT on Candida albicans biofilms.ResultsA 5 h incubation time and 15 mM ALA concentration were determined for this study. Photoinactivation of ALA-PDT on Candida albicans biofilms showed a significant increase of protoporphyrin IX (PpIX) in the biofilms. The metabolic activity of Candida albicans biofilms tread with ALA-PDT confirmed the inhibition efficacy compared with control groups. Upon radiation at 300 J/cm², cells in Candida albicans biofilms were 74.45% inhibited.ConclusionsPpIX can be absorbed in biofilm-grown Candida albicans in vitro and under appropriate parameters, photochemistry can be triggered by light in combination with ALA and inhibits Candida albicans biofilms effectively.     

Development of a novel Schiff base derivative for enhancing the anticancer potential of 5-aminolevulinic acid-based photodynamic therapy

5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), is now widely used for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various cancers. Recently, we found that treatment of cancer cells with the Schiff base derivative TX-816 along with ALA could significantly increase the efficacy of ALA-PDT. This enhancing effect of TX-816 on ALA-PDT is attributed to 3,5-dichlorosalicylaldehyde (DCSA), a molecule produced by the degradation of TX-816. Similar to TX-816, DCSA significantly enhances the effect of ALA-PDT. Furthermore, DCSA could restore the sensitivity of cancer cells that acquired resistance to ALA-PDT. These results indicate that DCSA, as well as TX-816, is a potent lead compound for the development of an ALA-PDT sensitizer. TX-816 might be a useful compound for designing prodrug-type ALA-PDT enhancers.     

The in vitro effect of antimicrobial photodynamic therapy with indocyanine green on Enterococcus faecalis: Influence of a washing vs non-washing procedure

IntroductionThe purpose of this study was to evaluate the in vitro effect of washing and non-washing of indocyanine green (ICG) as photosensitizer (PS) on bacterial count, biofilm formation, development and degradation of Enterococcus faecalis.MethodsThe anti-bacterial, anti-biofilm formation, anti-biofilm development and biofilm degradation of anti-microbial photodynamic therapy (aPDT) against E. faecalis was determined at concentrations of 3 to 2000 μg/mL of ICG, subject to 18 J/cm² dose of diode laser (808 nm) in washing and non-washing producers. Bacterial viability measurements and biofilm assays were evaluated by broth microdilution method and crystal violet assays, respectively.ResultsICG-mediated aPDT, using 25 to 2000 μg/mL and 50 to 2000 μg/mL showed significant reduction in E. faecalis growth when compared to the control in non-washing and washing producers, respectively (P < 0.05). Also, ICG-mediated aPDT showed a significantly inhibitory effect on biofilm formation of E. faecalis in concentration of 6 to 2000 μg/mL and 100 to 2000 μg/mL in non-washing and washing groups (P < 0.05). The biofilm development was inhibited by concentrations of 12 to 2000 μg/mL and 100 to 2000 μg/mL in non-washing and washing groups. The biofilm degradation increased from concentrations of 12 to 2000 μg/mL and 250 to 2000 μg/mL in non-washing and washing groups, respectively.ConclusionThis study shows that the application of ICG should be accompanied by laser irradiation without being washed out to achieve better result for bacterial count reduction and anti-biofilm effects.     

Effects of 5-aminolevulinic acid photodynamic therapy on TLRs in acne lesions and keratinocytes co-cultured with P. acnes

ObjectiveTo investigate the effect of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on the expression of Toll like receptors (TLRs) in human keratinocytes and its role in acne treatment.MethodsTLR2 and TLR4 expression in acne lesions before and after ALA-PDT were examined by immunohistochemical assay. Primary keratinocytes were obtained from acne lesions, co-cultured with P. acnes and then treated with ALA-PDT using red or blue LED. Cytokines production were examined by ELISA, TLR2 and TLR4 gene expression by real-time PCR, and TLR2 and TLR4 protein expression by Western-blot assay.ResultsThe overexpression of TLR2 and TLR4 in acne lesion were detected, which became negative or weaker after ALA-PDT. The infection of P. acnes in keratinocytes could significantly increase the levels of early inflammatory cytokines (e.g. IL-1α, TNF-α and IL-8) (P < 0.05). Such responses could be inhibited by ALA-PDT. P. acnes infection could also significantly increase TLR2 and TLR4 expressions in keratinocytes (P < 0.05), which could be down-regulated by ALA-PDT.ConclusionsALA-PDT could inhibit innate immune responses in keratinocytes treated with P. acnes via TLRs pathways.     

Efficacy and therapeutic reactions of tri-needle-pretreatment combined with painless ALA-PDT for the treatment of moderate-to-severe acne vulgaris: A randomized controlled trial

Background: Our clinical experience suggests that pretreatment of the original lesions may be crucial for enhancing the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) in moderate-to-severe acne vulgaris. We performed this randomized controlled trial (RCT) to validate this observation.Methods: Efficacy and therapeutic reactions between tri-needle-pretreatment with ALA-PDT (TP-PDT) and conventional ALA-PDT without pretreatment (NP-PDT) were compared. In TP-PDT group, tri-needle-pretreatment was performed using comedone extractors, fire needles, or plum-blossom needles, according to the lesion type. In the TP-PDT group, 5% ALA cream was applied to lesions 30 min before illumination (LED red light: 633±10 nm, 40 mW/cm², 150 J/cm²). In the NP-PDT group, 5% ALA cream was applied 1 h before illumination (60 mW/cm², 72 J/cm²). Patients underwent four sessions, at 1-week intervals. The efficacy was evaluated as the proportion of patients achieving a remarkable effective rate, based on the reduction in the number of lesions. A numeric rating scale was used to assess the severity of pain, erythema, and edema.Results: Forty-eight patients completed the trial. The proportion of remarkable effective rate was significantly greater and the pain score was significantly lower for the TP-PDT than NP-PDT group. The edema score was significantly higher for the TP-PDT than NP-PDT group. There was no difference in erythema scores between the two groups.Conclusion: The tri-needle-pretreatment can improve the efficacy of ALA-PDT, without an increase in pain, for the treatment of moderate-to-severe acne vulgaris. These qualities make the TP-PDT a promising gold standard pretreatment for ALA-PDT for acne vulgaris.     

Effects of photodynamic therapy on Streptococcus mutans and enamel remineralization of multifunctional TiO2-HAP composite nanomaterials

BackgroundAs photosensitizer and photocatalyst, titanium dioxide (TiO₂) can produce a photodynamic reaction for antibacterial treatment. This study aims to explore a Titanium dioxide/nano-hydroxyapatite (TiO₂-HAP) composite combined with the dental curing lamp (385–515 nm) in clinical which could inhibit the dental plaque biofilm formed by Streptococcus mutans (S. mutans) and promote the enamel surface remineralization simultaneously.MethodsX-ray Diffraction (XRD) and high resolution transmission electron microscope (HRTEM) were used to detect the characterization of TiO₂-HAP composite nanomaterials. Photodynamic properties of TiO₂-HAP were detected by Diffuse reflectance spectrum (DRS) and fluorescence spectroscopy. Bacterial growth was measured by reading the absorbance of bacterial cultures and confocal microscope was used to observe the biofilm removal ability of nanomaterials. The ability of TiO₂-HAP to promote enamel remineralization was measured by Scanning electron microscope (SEM).ResultsThe OD 600 of S. mutans was 0.76 in the control group and 0.13 in group of TiO₂-HAP with exposure to light-emitting diode (LED) (150 mW/cm²) for 5 min, suggesting its sustained antibacterial potency and inhibition of the metabolic activity of dental plaque microcosm biofilm. Also, the release of calcium and phosphorus ions in TiO₂-HAP can promote enamel mineralization simultaneously. After 15 days of remineralization, the Ca/P ratio of demineralized enamel surface increased from 1.28 to 1.67, which was similar to that of normal enamel.ConclusionsThe TiO₂-HAP exhibit a promising anti-bacterial activity and remineralization capacity which can prevent the occurrence of caries to the greatest extent and promote the biomimetic mineralization of dental tissues.     

Periopathogens differ in terms of the susceptibility to toluidine blue O-mediated photodynamic inactivation

BackgroundThe main goal of periodontal therapy is to eliminate the infection spreading in periodontium. Antimicrobial photodynamic therapy may be applied in order to eradicate pathogens remaining in periodontal tissues after conventional mechanical debridement, to improve the treatment results. The aim of this in vitro study was to evaluate the susceptibility of selected key periopathogens to toluidine blue O-mediated photodynamic inactivation and the influence of photosensitizer’s concentration and light dose on the effectiveness of this process.MethodsFollowing bacterial strains were used in the experiments: Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomyctemecomitans ATCC 33384, Fusobacterium nucleatum ATCC 10953. Toluidine blue O (TBO) was used in concentration ranging from 0.004 to 0.5 mg/mL. Irradiation was performed by a non-laser red light source.ResultsComplete eradication of P. gingivalis was obtained upon the application of TBO in the concentration of 0.1 mg/mL and the highest light dose. A, actinomycetemcomitans was, in turn, not susceptible to photodynamic inactivation regardless of the dosimetric parameters applied. High viability reductions were also obtained for F. nucleatum, however no complete eradication. The effectiveness of photodynamic inactivation of susceptible periopathogens was dependent on the light dose and photosensitizer’s concentration.ConclusionsPeriopathogens differ in terms of their susceptibility to photodynamic inactivation. Antimicrobial PDT may be valuable in the treatment of those cases of periodontal disease, in which P. gingivalis is a dominating pathogen. Microbiological examination prior to clinical application of aPDT may be recommended.     

The effects of ALA-PDT on microbiota in pilosebaceous units of patients with severe acne: A metagenomic study

Background5-aminolevulinic acid mediated photodynamic therapy (ALA-PDT) is increasingly used to control severe acne. However, its impact on skin microbiota remains uncertain.ObjectivesWe aimed to compare the makeup, diversity, and function of the microbiota in pilosebaceous units of patients with severe acne before and after ALA-PDT.MethodsA longitudinal cohort study was performed on 11 participants with severe facial acne. All patients were given 5%ALA-PDT every two weeks for three sessions in total. The contents of lesions were sampled for metagenomic sequencing at baseline and two weeks after the first ALA-PDT session.ResultsCutibacterium acnes was the most dominant species followed by Staphylococcus epidermidis and Pseudomonas fluorescens. Treatment with ALA-PDT led to clinical improvements in acne severity concurrent with a significant reduction in the relative abundance of C. acnes, while P. fluorescens increased significantly after ALA-PDT. No significant change was identified in other species. ALA-PDT administration was associated with an increased microbiota diversity and reductions in the relative abundance of the functional genes involved in energy metabolism and DNA replication.ConclusionsALA-PDT plays a therapeutic role by killing C. acnes, increasing P. fluorescens and the microbiome diversity, while inhibiting the function of microbiota in pilosebaceous units of severe acne.     

In-Vitro Use of Verteporfin for Photodynamic Therapy in Glioblastoma

BackgroundStummer et al. established fluorescence-guided surgery (FGS) for glioblastoma (GBM) using 5-aminolevulinic acid (5-ALA). Its metabolite, protoporphyrin IX (PPIX), is also a photosensitizer and can be used for photodynamic therapy (PDT) using a laser beam of 635 nm. The porphyrin derivate verteporfin (VP) was discovered to have properties to penetrate the brain, pharmacologically target glioma cells, and is approved for PDT of choroidal neovascularization in wet age-related macular degeneration at 689 nm.ObjectiveTo elucidate whether GBM cell lines are susceptible to PDT with second-generation photosensitizer VP.MethodsHuman glioma cell lines LN229, HSR-GBM1, and a low-passage patient-derived GBM cell line P1 were treated with variable concentrations of VP for 24 h, followed by PDT at 689 nm using a diode laser light. Cell viability was measured using the MTT assay and VP uptake was measured using a desktop cytometer.ResultsSignificantly higher cell death following PDT with VP compared to VP treatment alone or no treatment was detected in all cell models (LN229, HSR-GBM1, P1). Flowcytometric measurements revealed a concentration-dependent cellular uptake of VP after 24 h incubation up to 99% at 10 µM (HSR-GBM1).ConclusionThis study demonstrates that PDT with VP causes cell death in GBM cells at marginal concentrations. Additionally, red spectrum fluorescence was detected at therapeutic concentrations in all cell lines, validating the cellular uptake of VP in GBM cells. VP, therefore, is not only a potential drug for targeting GBM pharmacologically but can be used as an optical imaging dye in surgery and photosensitizer to make GBM susceptible to PDT.     

Fluorescence evaluations for porphyrin formation during topical PDT using ALA and methyl-ALA mixtures in pig skin models

BackgroundPhotodynamic Therapy (PDT) using Aminolevulinic acid (ALA) and derivative molecules as topical medication and as a precursor of protoporphyrin (PPIX), is limited due to low permeation through skin or efficiency in porphyrin production. This behavior affects the production and homogeneity of PPIX distribution on superficial skin and in the deeper skin layers. Many authors propose alternatives to solve this such as, modification in the ALA and derivativemolecules, modifying the chemical properties of emulsion external phase or incorporating a delivery system to the emulsion. The goal of this study is to discuss what proportion of ALA and Methyl aminolevulinate (MAL) on mixtures increase the amount and uniformity of PPIX formation at superficial skin by fluorescence evaluations.MethodsThe study was conducted in vivo using a pig skin model. PPIX production was monitored using fluorescence spectroscopy and widefield fluorescence imaging on skin surface. 20% of ALA and MAL cream were done mixing the following proportions: ALA, M2 (80% ALA–20% MAL), M3 (60% ALA–40% MAL), M4 (50% ALA–MAL), M5 (40% ALA–60% MAL), M6 (20% ALA–80% MAL) and MAL.ResultsMixtures M3, M4, and M5 showed the most PPIX production on skin by widefield fluorescence imaging and fluorescence spectroscopy in 3 h of incubation. These results suggest that 50% of ALA and MAL in the same mixture increase the PPIX production in amount, homogeneity and time production when compared to ALA and MAL. This has a positive impact on photodynamic damage optimizing the PDT treatment.     

The effect of indocyanine green loaded on a novel nano-graphene oxide for high performance of photodynamic therapy against Enterococcus faecalis

BackgroundRecently developed photodynamic therapy (PDT) has gained attention for achieving effective root canal disinfection. Using an optimized nontoxic photosensitizer (PS), such as indocyanine green (ICG), is an imperative part of this technique. Therefore, the objective of the current study was to improve ICG photodynamic properties through incorporation of ICG into nano-graphene oxide (NGO) in order to produce NGO-ICG as a new PS and also to assess the antimicrobial effects of NGO-ICG against Enterococcus faecalis after photodynamic therapy.Materials and methodsNGO-ICG was synthesized based on oxidation of graphite flakes and direct loading of ICG onto NGO. NGO-ICG formation was confirmed using the Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), and UV–vis spectrometry. The antimicrobial and anti-biofilm potential of NGO-ICG-PDT against E. faecalis was assessed via colony forming unit and crystal violet assays, respectively.ResultsFT-IR, SEM and UV–vis spectrometry confirmed successful synthesis of NGO-ICG containing 200 μg/mL of ICG. NGO-ICG-PDT at an energy density of 31.2 J/cm² showed a significant reduction (2.81 log) in the count of E. faecalis (P < 0.05). NGO-ICG-PDT significantly reduced the biofilm formation ability of E. faecalis up to 99.4% (P < 0.05). The overall antimicrobial and anti-biofilm potential of NGO-ICG-PDT was higher than PDT based on ICG (1000 μg/mL) (47% and 21%, respectively).ConclusionBecause NGO-ICG-PDT showed a significant reduction in the number and biofilm formation ability of E. faecalis at low ICG concentrations (200 μg/mL), it could be a new approach to adjuvant treatment of endodontic infections.     

Effects of photodynamic therapy in the treatment of high-grade vaginal intraepithelial lesions following hysterectomy and HPV infection

BackgroundNon-invasive treatment therapy, such as 5-Aminolevulinic acid photodynamic therapy (ALA-PDT), has gained attention for its effect on select cervical and vaginal lesions. To investigate the effect of ALA-PDT on high-grade vaginal intraepithelial lesions (HG VAIN) after hysterectomy and high-risk human papillomavirus (HR-HPV) infection, in this study, we evaluated the clinical efficacy and safety of ALA-PDT in 23 patients with HG VAIN following hysterectomy and HPV.Methods23 patients with HG VAIN after hysterectomy were selected for photodynamic therapy, and the therapeutic effect, adverse reactions, recurrence rate and HPV clearance rate were analyzed respectively. The patients were followed up at 3, 6, 9, and 12 months after ALA-PDT. HPV, thinprep cytologic test (TCT) and reid colposcopic index (RCI) score should be performed 3 months after treatment. When the RCI score is higher than or equal to 3, a colposcopy biopsy should be conducted, the additional ALA-PDT should be continued if residual lesions were detected. When the RCI score is lower than or equal to 2, HPV and TCT should be reviewed every 3 months.ResultsAfter 3 months of photodynamic therapy, 21 patients were cured, with the cure rate of 91.3% (21/23). Two patients (8.7%) had residual lesions, which had degraded compared with the previous. After treatment, the overall HR-HPV clearance rate was 56.5% at 3 months, 65.2% at 6 months, 69.5% at 9 months, and 74% at 12 months. No obvious adverse reactions were found during and after treatment. Moreover, no recurrence occurred during the whole follow-up period.ConclusionsCompared with other therapies, ALA-PDT is a novel therapy with non-invasive procedure in HG VAIN after hysterectomy. ALA-PDT can be used for the treatment of HG VAIN after hysterectomy, moreover, it can not only effectively clear HR-HPV, but also can prevent the recurrence and progression of vaginal lesions caused by persistent HR-HPV infection.     

Antifungal effect of a new photosensitizer derived from BODIPY on Candida albicans biofilms

BackgroundPhotodynamic therapy (PDT) may be an alternative treatment of Candida albicans (C. albicans) infections. The aim of this study was to investigate the antifungal effect of PDT mediated by a new photosensitizer (PS) derived from BODIPY (BDP-4L) on C. albicans biofilms.MethodsC. albicans biofilms were incubated with BDP-4L of different concentrations and then irradiated at the light doses of 1.8, 3.6, 5.4, 7.2 and 9.0 J/cm². XTT reduction assay was conducted to determine the PS concentration and PDT parameters. Confocal light scanning microscopy (CLSM) and scanning electron microscope (SEM) were used to visualize and quantify the effect of BDP-4L on C. albicans biofilms after PDT.ResultsC. albicans biofilms were inactivated in light dose-dependent and PS concentration-dependent manners using BDP-4L as PS. Without irradiation, no inactivation effect was observed when PS concentrations varied from 5 μM to 80 μM. 40 μM PS with 3.6 J/cm² irradiation resulted in a significant reduction of 83.8% in biofilm metabolic activities. CLSM assay demonstrated that cell viability was obviously inhibited by 82.6%. SEM images revealed ruptured and rough cell surface, indicating increased cell membrane permeability after PDT.ConclusionsOur results suggested that BDP-4L mediated PDT exhibited a favorable antifungal effect on C. albicans biofilms.