Proteomic Investigation over the Antimicrobial Photodynamic Therapy Mediated by Rose Bengal Against Staphylococcus aureus

In order, understanding the antimicrobial action of photodynamic therapy and how this technique can contribute to its application in the control of pathogens. The objective of the study was to employ a proteomic approach to investigate the protein profile of Staphylococcus aureus after antimicrobial photodynamic therapy mediated by rose bengal (RB-aPDT). S. aureus was treated with RB (10 nmoL L⁻¹) and illuminated with green LED (0.17 J cm⁻²) for cell viability evaluation. Afterward, proteomic analysis was employed for protein identification and bioinformatic tools to classify the differentially expressed proteins. The reduction in S. aureus after photoinactivation was ~2.5 log CFU mL⁻¹. A total of 12 proteins (four up-regulated and eight down-regulated) correspond exclusively to alteration by RB-aPDT. Functionally, these proteins are distributed in protein binding, structural constituent of ribosome, proton transmembrane transporter activity and ATPase activity. The effects of photodamage include alterations of levels of several proteins resulting in an activated stress response, altered membrane potential and effects on energy metabolism. These 12 proteins required the presence of both light and RB suggesting a unique response to photodynamic effects. The information about this technique contributes valuable insights into bacterial mechanisms and the mode of action of photodynamic therapy.     

Hiporfin‐Mediated Photodynamic Therapy in Preclinical Treatment of Osteosarcoma

This study was carried out to investigate the anti‐tumor effect and mechanism of hiporfin‐mediated photodynamic therapy (hiporfin‐PDT) in osteosarcoma. We found that hiporfin accumulated mainly in the cytoplasm of osteosarcoma cells in a time and concentration‐dependent manner. Hiporfin‐PDT inhibited the proliferation, induced apoptosis and produced cell cycle arrest at G2M in osteosarcoma cell lines. Hiporfin‐PDT increased the expression of cleaved‐caspase‐3, cleaved PARP‐1, Bax and RIP1 while it decreased the expression of Bcl‐2; in addition, low concentration of hiporfin increased LC3 conversion. Furthermore, cell death caused by hiporfin‐PDT could be rescued by Nec‐1 but not by Z‐VAD‐FMK. Production of reactive oxygen species was increased after hiporfin‐PDT. In vivo studies showed a significant decrease in tumor volume and weight after hiporfin‐PDT in all three tumor mouse models investigated (subcutaneous and orthotopic). Histological analysis showed widespread cell apoptosis and necrosis after treatment. Immunohistochemistry also showed upregulation of cleaved‐caspase‐3 and downregulation of Bcl‐2 after hiporfin‐PDT. These results indicate that hiporfin‐PDT exhibits a killing effect in osteosarcoma both in vitro and in vivo, which is associated with apoptosis and necroptosis, while autophagy plays a protective role. All these findings shed light on a potential future clinical use for hiporfin in the treatment of osteosarcoma.     

Design of Photosensitizing Agents for Targeted Antimicrobial Photodynamic Therapy

Photodynamic inactivation of microorganisms has gained substantial attention due to its unique mode of action, in which pathogens are unable to generate resistance, and due to the fact that it can be applied in a minimally invasive manner. In photodynamic therapy (PDT), a non-toxic photosensitizer (PS) is activated by a specific wavelength of light and generates highly cytotoxic reactive oxygen species (ROS) such as superoxide (O²⁻, type-I mechanism) or singlet oxygen (¹O₂*, type-II mechanism). Although it offers many advantages over conventional treatment methods, ROS-mediated microbial killing is often faced with the issues of accessibility, poor selectivity and off-target damage. Thus, several strategies have been employed to develop target-specific antimicrobial PDT (aPDT). This includes conjugation of known PS building-blocks to either non-specific cationic moieties or target-specific antibiotics and antimicrobial peptides, or combining them with targeting nanomaterials. In this review, we summarise these general strategies and related challenges, and highlight recent developments in targeted aPDT.     

Cationic Chalcogenoviologen Derivatives for Photodynamic Antimicrobial Therapy and Skin Regeneration

A series of water-soluble cationic chalcogenoviologen-based photosensitizers for photodynamic antimicrobial therapy (PDAT) is reported. The Se-containing derivatives (SeMV²⁺) 5 b and 6 b showed good antimicrobial activities due to the presence of chalcogen atoms and a cationic scaffold. The former efficiently enhanced the generation of reactive oxygen species (ROS), and the latter facilitated the ROS delivery to bacteria, resulting in their death. Interestingly, alkyl-modified photosensitizers showed higher antimicrobial activities than commonly reported photosensitizers with quaternary ammonium (QA) groups. In particular, the SeMV²⁺ ( 6 b ) with excellent antibacterial activities efficiently promoted the healing of infected wounds in mice. Simple yet novel, nontoxic and biocompatible chalcogenoviologens provided a promising strategy to develop new efficient photosensitizers for photodynamic antimicrobial therapy and skin regeneration.     

Preclinical Evaluation of Cetuximab and Benzoporphyrin Derivative-Mediated Intraperitoneal Photodynamic Therapy in a Canine Model

Peritoneal carcinomatosis (PC) can occur as an advanced consequence of multiple primary malignancies. Surgical resection, radiation or systemic interventions alone have proven inadequate for this aggressive cancer presentation, since PC still has a poor survival profile. Photodynamic therapy (PDT), in which photosensitive drugs are exposed to light to generate cytotoxic reactive oxygen species, may be an ideal treatment for PC because of its ability to deliver treatment to a depth appropriate for peritoneal surface tumors. Additionally, epidermal growth factor receptor (EGFR) signaling plays a variety of roles in cancer progression and survival as well as PDT-mediated cytotoxicity, so EGFR inhibitors may be valuable in enhancing the therapeutic index of intraperitoneal PDT. This study examines escalating doses of benzoporphyrin derivative (BPD)-mediated intraperitoneal PDT combined with the EGFR-inhibitor cetuximab in a canine model. In the presence or absence of small bowel resection (SBR) and cetuximab, we observed a tolerable safety and toxicity profile related to the light dose received. Additionally, our findings that BPD levels are higher in the small bowel compared with other anatomical regions, and that the risk of anastomotic failure decreases at lower light doses will help to inform the design of similar PC treatments in humans.     

Metronomic Photodynamic Therapy as a New Paradigm for Photodynamic Therapy: Rationale and Preclinical Evaluation of Technical Feasibility for Treating Malignant Brain Tumors¶

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low‐dose PDT using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX(PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA‐mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate, sodium salt nick‐end labeling positive) cells as compared with an acute, high‐dose regimen (ALA‐αPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectro‐fluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor‐to‐brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light‐emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.     

Photodynamic Action of Methylene Blue on the Paramecium Membrane

An electrophysiological study of photodynamic action on the Paramecium membrane was carried out. In the presence of methylene blue (MB), light-spot stimulation of an anterior and a posterior part induced a depolarization and a hyperpolarization of the membrane, respectively. Under voltage-clamping, the anterior stimulation induced an inward current, while the posterior stimulation induced an outward current. The amplitudes of these currents were dependent on the membrane potential. When K⁺ channels were blocked with Cs⁺ and tetraethylammonium (TEA⁺), the posterior outward current was inhibited, while the anterior inward current was not inhibited. Intracellular application of the Ca²⁺ chelator, 1,2 -bis (2-aminophenoxy) ethane- N,N,N',N' -tetraacetic acid (BAPTA) also inhibited the posterior outward current, but the anterior inward current was unaffected. These results suggest that photodynamic action on the Paramecium membrane primarily opens the Ca²⁺ channels and the following influx of Ca²⁺ activates the Ca²⁺-dependent K⁺ channels localized mainly on the posterior part of the membrane.     

Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review

Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi‐/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi‐/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.     

Effects of Photodynamic Therapy Using Hematoporphyrin Monomethyl Ether on Experimental Choroidal Neovascularization

Hematoporphyrin monomethyl ether (HMME) is a novel and promising second-generation porphyrin-related photosensitizer for photodynamic therapy (PDT). To study the effects of HMME PDT on choroidal neovascularization (CNV) in rats, the PDT was performed 20 min after HMME bolus injection, which was investigated prior to the PDT by fluorescence microscopy with laser-induced CNV, and delivered at an irradiance of 400, 600 and 1000 mW cm⁻² corresponding to a fluence of 36, 54, 90 J cm⁻² in PDT plan I (15 mg kg⁻¹ HMME). In PDT plan II (30 mg kg⁻¹ HMME), the laser had a constant irradiance of 600 mW cm⁻², which was delivered for 60, 90 or 150 s, to also achieve total energy doses of 36, 54 or 90 J cm⁻². CNV closure rates assessed by fluorescein angiography and histologic damage to treated areas of choroid and retina varied as a function of the dose of HMME and of the activating light energy fluence. Endothelial cell labeled by platelet/endothelial cell adhesion molecule-1 presented treated CNV lesions that were significantly reduced in size (P < 0.01). It can be concluded that PDT using HMME can effectively occlude CNV. HMME is a potentially useful photosensitizer for the reduction in CNV size of irradiated areas.     

The Effects of Photodynamic Therapy on Human Neutrophil Migration Using Bacteriochlorin a

Abstract— Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils, indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.     

Disinfect Porphyromonas gingivalis Biofilm on Titanium Surface with Combined Application of Chlorhexidine and Antimicrobial Photodynamic Therapy

Various antimicrobial modalities have been proposed to treat peri‐implantitis but resulted in limited outcomes. The aim of this in vitro study was to evaluate the disinfection efficacy of combined application of chlorhexidine digluconate (CHX) and antimicrobial photodynamic therapy (aPDT) of titanium surfaces previously contaminated with Porphyromonas gingivalis biofilm. P. gingivalis biofilms were grown on 32 polished and 32 sandblasted large‐grit acid‐etched (SLA) titanium surfaces. Titanium disks were allocated into four groups as follows: (1) immersed in phosphate‐buffered saline (PBS), (2) immersed in 0.2% CHX, (3) application of aPDT and (4) immersed in 0.2% CHX and subsequent aPDT. Residual bacteria were determined by microbial culture analysis and by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) imaging. Combination protocol (CHX+ aPDT) was the most effective in eradicating P. gingivalis (P < 0.05) on both polished and SLA surfaces. There was no significant difference in the number of remaining P. gingivalis between polished titanium disks and the SLA ones in four groups (P > 0.05). Under the limitation of this study, combined technique of preceding application of CHX and subsequent aPDT was shown to be an efficient method in reducing P. gingivalis numbers in both polished and SLA titanium surfaces.     

Susceptibility of Ureaplasma urealyticum to Methylene Blue‐Mediated Photodynamic Antimicrobial Chemotherapy: An in vitro Study

The aim of this study was to detect the susceptibility of Ureaplasma urealyticum to methylene blue‐mediated photodynamic antimicrobial chemotherapy (PACT). Three U. urealyticum strains including the standard serotype 1 and 5, and a clinically collected strain were used in this study. Strains were first incubated in 96‐well culture plates in the presence of methylene blue with decreasing concentrations (from 1 to 0.015625 mg mL^?1) for 20 or 60 min, and then submitted to irradiation with a light‐emitting diode laser with a power density of 100 mW cm^?2 for 8, 17, 34 or 68 min. Regrowth of the strains was performed soon after irradiation. A significant inactivation effect was observed after PACT. Longer incubation time induced more extensive inactivation of U. urealyticum. No difference in response to PACT was observed between the two biovars of U. urealyticum. It was concluded that PACT had a significant inactivation effect on U. urealyticum, and it might be a promising alternative treatment for resistant U. urealyticum infections.     

Hydrogen Peroxide Enhances the Antibacterial Effect of Methylene Blue‐based Photodynamic Therapy on Biofilm‐forming Bacteria

Recently, increased attention has been focused on endoscopic disinfection after outbreaks of drug‐resistant infections associated with gastrointestinal endoscopy. The aims of this study were to investigate the bactericidal efficacy of methylene blue (MB)‐based photodynamic therapy (PDT) on Pseudomonas aeruginosa (P. aeruginosa), which is the major cause of drug‐resistant postendoscopy outbreak, and to assess the synergistic effects of hydrogen peroxide addition to MB‐based PDT on biofilms. In planktonic state of P. aeruginosa, the maximum decrease was 3 log₁₀ and 5.5 log₁₀ at 20 and 30 J cm^?2, respectively, following MB‐based PDT. However, the maximum reduction of colony forming unit (CFU) was decreased by 2.5 log₁₀ and 3 log₁₀ irradiation on biofilms. The biofilm formation was significantly inhibited upon irradiation with MB‐based PDT. When the biofilm state of P. aeruginosa was treated with MB‐based PDT with hydrogen peroxide, the CFU was significantly decreased by 6 log₁₀ after 20 J cm^?2, by 7 log₁₀ after 30 J cm^?2 irradiation, suggesting significantly higher efficacy than MB‐based PDT alone. The implementation of the combination of hydrogen peroxide with MB‐based PDT through working channels might be appropriate for preventing early colonization and biofilm formation in the endoscope and postendoscopy outbreak.     

Enhanced Fluorescence Imaging and Photodynamic Cancer Therapy Using Hollow Mesoporous Nanocontainers

Here we show that “off‐on” type of photodynamic therapy agents could be developed using hollow mesoporous silica nanoparticles (HMSNPs), which can be used not only for enhancing delivery of photosensitizers to cancer cells but also for enabling switchable optical properties of the photosensitizers. Fluorescence and singlet oxygen generation of the photosensitizer‐loaded HMSNP are turned off in its native state. In vitro cell studies showed that this HMSNP‐based “off‐on” agent may have potential utility in selective fluorescence detection and photodynamic therapy of cancers.     

Disinfection of Cariogenic Pathogens in Planktonic Lifestyle,Biofilm and Carious Dentine with Antimicrobial Photodynamic Therapy

Antimicrobial photodynamic therapy (aPDT) has been recommended for clinical application. Its antibacterial effect on bacteria remained in dentinal tubule was seldom investigated. Here, we evaluated the antibacterial effects of aPDT on Streptococcus mutans (S. mutans) and Lactobacillus acidophilus (L. acidophilus) in planktonic lifestyle, biofilm and carious dentine. Mono-species biofilms or dentinal caries formed on human dentine slices or slabs. Bacterial suspension, biofilms and dentine caries were treated with 0.1 mg mL⁻¹ toluidine Blue O followed by irradiation with a light emission diode (λ − 635 ± 10 nm; 500 mW; 31.5 J cm⁻²; 60 s) and 0.12% chlorhexidine (CHX), respectively. Residual bacteria were determined by microbial culture analysis and scanning electron microscopy (SEM). One-way analysis of variance (ANOVA) was performed to detect the significance of the variables. Both treatments significantly reduced the number of L. acidophilus in planktonic state, biofilm and carious dentine (P < 0.05). For S. mutans, CHX was only bactericidal against suspension (P < 0.05), while aPDT was effective on both suspension and biofilm (P < 0.05) while not for dentin caries (P > 0.05). Under the experimental conditions assessed, aPDT could be an alternative disinfection method for superficial layer of caries cavity. Its disinfection on bacteria in dentinal tubule of deep layer was deficient.     

Flow Injection Photoamperometric Investigation of Ascorbic Acid Using Methylene Blue Immobilized on Titanium Phosphate

Abstract

Methylene blue (MB) was incorporated into titanium phosphate (TiP) after pretreatment of TiP with the gas, n‐butyl amine. The dye is strongly retained and not easily leached from the layered host matrix. The adsorbed MB on TiP was used to prepare modified carbon paste electrodes (MCPE), which were studied voltammetrically and in amperometric flow injection (FI) mode for the electrocatalytic oxidation of ascorbic acid (AA). The electrochemical behavior of the immobilized dye was investigated with cyclic voltammetry, at a pH 7.0 phosphate buffer containing 0.5 M KCl, at different potential scan rates. The MB immobilized on the support underwent a quasi‐reversible electrochemical redox reaction. A homemade flow‐through electrochemical cell with a suitable transparent window for irradiation of the electrode surface was constructed and used for amperometric FI studies. The photoamperometric‐FI conditions were optimized for sensitivity and reproducibility at a flow rate of 1.5 mL/min, a transmission tubing length of 25 cm, a sample injection volume of 100 µL, and a constant applied potential of +100 mV vs. SCE. The calibration curve for AA was linear over the concentration range from 1.0×10^?6 to 2.5×10^?5 mol l^?1 for both amperometric and photoamperometric studies. But the slope of the photoelectrocatalytic FIA procedure was improved about 52% compared with those obtained without irradiation. The results obtained for AA determination in some pharmaceutical products are in good agreement with those obtained using the procedure involving the reaction between triiodide and AA.     

Preclinical Assessment of Hypocrellin B and Hypocrellin B Derivatives as Sensitizers for Photodynamic Therapy of Cancer: Progress Update

Abstract— Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomelic form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 [2-( N,N -dimethylami-no)-propylamine-HB]. Extinction coefficients at 630 nm (φ₆₃₀) are 6230, 6190 and 4800, respectively; and ¹O₂ quantum yields, φ, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA-R1 permanently ablates EMToVEd tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhi-murium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.     

The Effects of Thrombocytopenia on Vessel Stasis and Macromolecular Leakage after Photodynamic Therapy Using Photofrin

Abstract— Several studies have reported thrombus formation and/or the release of specific vasoactive eicosanoids, suggesting that platelet activation or damage after photodynamic therapy (PDT) may contribute to blood flow stasis. The role of circulating platelets on blood flow stasis and vascular leakage of macromolecules during and after PDT was assessed in an intravital animal model. Sprague-Daw-ley rats bearing chondrosarcoma on the right hind limb were injected intravenously (i.v.) with 25 mg/kg Photofrin 24 h before light treatment of 135 J/cm² at 630 nm. Thrombocytopenia was induced in animals by administration of 3.75 mg/kg of rabbit anti-rat platelet antibody i.v. 30 min before the initiation of the light treatment. This regimen reduced circulating platelet levels from 300000/mm³ to 20000/mm³. Reductions in the luminal diameter of the microvasculature in normal muscle and tumor were observed in control animals given Photofrin and light. Venule leakage of macromolecules was noted shortly after the start of light treatment and continued throughout the period of observation. Animals made thrombocytopenic showed none of these changes after PDT in either normal tissues or tumor. The lack of vessel response correlated with the absence of thromboxane release in blood during PDT. These data suggest that platelets and eicosanoid release are necessary for vessel constriction and blood flow stasis after PDT using Photofrin.     

Toward Precise Antitumoral Photodynamic Therapy Using a Dual Receptor-Mediated Bioorthogonal Activation Approach

Targeted delivery and specific activation of photosensitizers can greatly improve the treatment outcome of photodynamic therapy. To this end, we report herein a novel dual receptor-mediated bioorthogonal activation approach to enhance the tumor specificity of the photodynamic action. It involves the targeted delivery of a biotinylated boron dipyrromethene (BODIPY)-based photosensitizer, which is quenched in the native form by the attached 1,2,4,5-tetrazine unit, and an epidermal growth factor receptor (EGFR)-targeting cyclic peptide conjugated with a bicycle[6.1.0]non-4-yne moiety. Only for cancer cells that overexpress both the biotin receptor and EGFR, the two components can be internalized preferentially where they undergo an inverse electron-demand Diels–Alder reaction, leading to restoration of the photodynamic activity of the BODIPY core. By using a range of cell lines with different expression levels of these two receptors, we have demonstrated that this stepwise “deliver-and-click” approach can confine the photodynamic action on a specific type of cancer cells.