Delivery of Methylene Blue and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate from cross-linked poly(vinyl alcohol) hydrogels: A potential means of photodynamic therapy of infected wounds

Poly(vinyl alcohol)–borate complexes were evaluated as a potentially novel drug delivery platform suitable for in vivo use in photodynamic antimicrobial chemotherapy (PACT) of wound infections. An optimised formulation (8.0% w/w PVA, 2.0% w/w borax) was loaded with 1.0 mg ml⁻¹ of the photosensitisers Methylene Blue (MB) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Both drugs were released to yield receiver compartment concentrations (>5.0 μg ml⁻¹) found to be phototoxic to both planktonic and biofilm-grown methicillin-resistant Staphylococcus aureus (MRSA), a common cause of wound infections in hospitals. Newborn calf serum, used to simulate the conditions prevalent in an exuding wound, did not adversely affect the properties of the hydrogels and had no significant effect on the rate of TMP-mediated photodynamic kill of MRSA, despite appreciably reducing the fluence rate of incident light. However, MB-mediated photodynamic kill of MRSA was significantly reduced in the presence of calf serum and when the clinical isolate was grown in a biofilm. Results support the contention that delivery of MB or TMP using gel-type vehicles as part of PACT could make a contribution to the photodynamic eradication of MRSA from infected wounds.     

Development of novel oral formulations prepared via hot melt extrusion for targeted delivery of photosensitizer to the colon

Colon‐residing bacteria, such as vancomycin‐resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit^®‐based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso‐tetra(N‐methyl‐4‐pyridyl)porphine tetra‐tosylate (TMP), or 5‐aminolevulinic acid hexyl‐ester (h‐ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen‐releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6‐h period. For TMP and h‐ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon‐residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.     

Advances in photodynamic antimicrobial chemotherapy

Photodynamic therapy (PDT) and photodynamic antimicrobial chemotherapy (PACT) combine light and photosensitizers to treat cancers and microbial infections, respectively. In PACT, the excitation of a photosensitizer drug with appropriate light generates reactive oxygen species (ROS) that kill pathogens in the proximity of the drug. PACT has considerably advanced with new light sources, biocompatible photosensitizers, bioconjugate methods, and efficient ROS production. The PACT technology has evolved to compete with or replace antibiotics, reducing the burden of antibiotic resistance. This review updates recent advances in PACT, with special references to light sources, photosensitizers, and emerging applications to microbial infestations. We also discuss PACT applied to COVID-19 causing SARS-CoV-2 treatment and disinfecting food materials and water. Finally, we discuss the pathogen selectivity and efficiency of PACT.     

delta-Aminolaevulinic acid mediated photodynamic antimicrobial chemotherapy on Pseudomonas aeruginosa planktonic and biofilm cultures

To demonstrate photodynamic antimicrobial chemotherapy (PACT) against planktonic and biofilm cultures of Pseudomonas aeruginosa, using photoporphyrin IX which could be endogenously synthesized by administrating delta-aminolaevulinic acid (delta-ALA), and a light emitted diode (LED) array to photoactivate the photosensitizer. P. aeruginosa suspended cells or biofilms, grown on a rotating disk reactor, were treated by different concentrations of delta-ALA in the dark for 1 h, followed by LED irradiation for various time. Regrowth experiments were conducted by placed PACT-treated disks back to a sterile reactor. Viable cells were determined by serial dilution and plate counts. Both P. aeruginosa planktonic and biofilm cells were inhibited by PACT with light doses or photosensitizer concentrations increasing. Treatments of planktonic cells with 10 mM delta-ALA and incident dose 240 J cm(-2) or 7.5 mM ALA and incident dose 360 J cm(-2) led to completely photoinactivation. No viable biofilm cells were found after treatment of 20 mM delta-ALA and incident dose 240 J cm(-2). However, regrowth was observed once PACT-treated biofilms were put back to a sterile reactor. Regrowth could be prevented only if biofilm samples were treated PACT twice. delta-ALA-mediated PACT on P. aeruginosa planktonic and biofilm cells was effective, though the detailed mechanism still required further investigation.     

光动力抗菌光敏剂的研究进展

光动力抗菌化学疗法是一种结合光敏剂分子和可见光产生的活性氧物种杀灭病原微生物的抗感染治疗方法.活性氧物种能够与致病菌中的多种生物活性分子反应,这一特性使得微生物不易对该方法产生耐药性,这也是该方法近年来备受关注的主要原因.本文重点介绍了近年来光动力抗菌化学疗法领域新型光敏剂药物的研究进展,包括卟啉类衍生物、BODIPY化合物、共轭聚合物和钌多吡啶配合物.     

In vitro photodynamic inactivation of Cryptococcus neoformans melanized cells with chloroaluminum phthalocyanine nanoemulsion

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 μm) and light doses (e.g. 10 J cm(-2)) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C. neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.     

Effect of Dosimetric and Physiological Factors on the Lethal Photosensitization of Porphyromonas gingivalis in vitro

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using tolui-dine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (³H-TBO). In the presence of TBO, a light dose-dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 µg/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8–8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 µg/mL), compared to the other two phases.     

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.     

Elaboration of chitosan-coated nanoparticles loaded with curcumin for mucoadhesive applications

Polycaprolactone (PCL) nanoparticles decorated with a mucoadhesive polysaccharide chitosan (CS) containing curcumin were developed aiming the buccal delivery of this drug. These nanoparticles were prepared by the nanoprecipitation method using different molar masses and concentrations of chitosan and concentrations of triblock surfactant poloxamer (PEO-PPO-PEO), in order to optimize the preparation conditions. Chitosan-coated nanoparticles showed positive surface charge and a mean particle radius ranging between 114 and 125 nm, confirming the decoration of the nanoparticles with the mucoadhesive polymer, through hydrogen bonds between ether and amino groups from PEO and CS, respectively. Dynamic Light Scattering (DLS) studies at different scattering angles and concentrations have shown that the nanoparticles are monodisperse (polydispersity indices were lower than 0.3). The nanoparticle systems were also examined with Nanoparticle Tracking Analysis (NTA), and the results were in good agreement with those obtained by DLS. Colloidal systems showed mean drug content about 460 μg/mL and encapsulation efficiency higher than 99%. Finally, when coated with chitosan, these nanoparticles show a great ability to interact with mucin indicating also their suitability for mucoadhesive applications.     

Study of germ tube formation by Candida albicans after photodynamic antimicrobial chemotherapy (PACT)

Due to the augmented number of immunocompromised patients, the infections associated to the pathogen of the genus Candida have increased dramatically in the recent years. In order to proliferate, Candida albicans can produce a germ tube formation extending from the cells. The germ tube formation is a transition state from budding to hyphal cells, and represents an essential stage for virulence. In this work we studied the effect of the photodynamic antimicrobial chemotherapy (PACT), a potential antimicrobial treatment on the germ tube formation by C. albicans. Germ tube formation was induced by goat serum after different treatments with Methylene blue (MB) and Laser (683nm). Our results demonstrated that photodynamic therapy using MB, as a photosensitizing drug; inhibits both the growth and the germ tube formation by C. albicans. Thus, our results suggest the possibility that Methylene blue, combined with light in a specific wavelength, can be used as a promising novel antifungal agent.     

Photodynamic Inactivation of Conidia of the Fungi Metarhizium anisopliae and Aspergillus nidulans with Methylene Blue and Toluidine Blue

Antimicrobial photodynamic treatment (PDT) is a promising method that can be used to control localized mycoses or kill fungi in the environment. A major objective of the current study was to compare the conidial photosensitization of two fungal species (Metarhizium anisopliae and Aspergillus nidulans) with methylene blue (MB) and toluidine blue (TBO) under different incubation and light conditions. Parameters examined were media, photosensitizer (PS) concentration and light source. PDT with MB and TBO resulted in an incomplete inactivation of the conidia of both fungal species. Conidial inactivation reached up to 99.7%, but none of the treatments was sufficient to achieve a 100% fungicidal effect using either MB or TBO. PDT delayed the germination of the surviving conidia. Washing the conidia to remove unbound PS before light exposure drastically reduced the photosensitization of A. nidulans. The reduction was much smaller in M. anisopliae conidia, indicating that the conidia of the two species interact differently with MB and TBO. Conidia of green and yellow M. anisopliae mutants were less affected by PDT than mutants with white and violet conidia. In contrast to what occurred in PBS, photosensitization of M. anisopliae and A. nidulans conidia was not observed when PDT was performed in potato dextrose media.     

Development and validation of a solid-phase extraction gas chromatography–mass spectrometry method for the simultaneous quantification of methadone, heroin, cocaine and metabolites in sweat

A sensitive and specific method is presented to simultaneously quantify methadone, heroin, cocaine and metabolites in sweat. Drugs were eluted from sweat patches with sodium acetate buffer, followed by SPE and quantification by GC/MS with electron impact ionization and selected ion monitoring. Daily calibration for anhydroecgonine methyl ester, ecgonine methyl ester, cocaine, benzoylecgonine (BE), codeine, morphine, 6-acetylcodeine, 6-acetylmorphine (6AM), heroin (5-1000 ng/patch) and methadone (10-1000 ng/patch) achieved determination coefficients of >0.995, and calibrators quantified to within +/-20% of the target concentrations. Extended calibration curves (1000-10,000 ng/patch) were constructed for methadone, cocaine, BE and 6AM by modifying injection techniques. Within (N = 5) and between-run (N = 20) imprecisions were calculated at six control levels across the dynamic ranges with coefficients of variation of <6.5%. Accuracies at these concentrations were +/-11.9% of target. Heroin hydrolysis during specimen processing was <11%. This novel assay offers effective monitoring of drug exposure during drug treatment, workplace and criminal justice monitoring programs.     

Chitosan nanoparticles for antimicrobial photodynamic inactivation: characterization and in vitro investigation

The growing resistance to antibiotics has rendered antimicrobial photodynamic inactivation (PDI) an attractive alternative treatment modality for infectious diseases. Chitosan (CS) was shown to further potentiate the PDI effect of photosensitizers and was therefore used in this study to investigate its ability to potentiate the activity of erythrosine (ER) against bacteria and yeast. CS nanoparticles loaded with ER were prepared by ionic gelation method and tested for their PDI efficacy on planktonic cells and biofilms of Streptococcus mutans, Pseudomonas aeruginosa and Candida albicans. The nanoparticles were characterized for their size, polydispersity index and zeta potential. No toxicity was observed when planktonic cells and biofilms were treated with the nanoparticles in the dark. However, when the cells were exposed to light irradiation after treatment with free ER or ER/CS nanoparticles, a significant phototoxicity was observed. The antimicrobial activity of ER/CS nanoparticles was significantly higher than ER in free form. The particle size and incubation time of the nanoparticles also appeared to be important factors affecting their PDI activity against S. mutans and C. albicans.     

The Development of Ru(II)-Based Photoactivated Chemotherapy Agents

Photoactivated chemotherapy (PACT) is a novel cancer treatment method that has drawn increasing attention due to its high selectivity and low side effects by spatio-temporal control of irradiation. Compared with photodynamic therapy (PDT), oxygen-independent PACT is more suitable for treating hypoxic tumors. By finely tuning ligand structures and coordination configurations, many Ru(II) complexes can undergo photoinduced ligand dissociation, and the resulting Ru(II) aqua species and/or free ligands may have anticancer activity, showing their potential as PACT agents. In this mini-review, we summarized the progress in Ru(II)-based PACT agents, as well as challenges that researchers in this field still face.     

Selective ther cleavage in the aporphine series. Conversion of (S)-bulbocapnine into (S)-corytuberine and (S)-corydine methyl ether.

Bulbocapnine methyl ether ( 2 ), on treatment with boron halides, affords the aporphine-1,2-diol ( 3 ), the novel aporphines 5 and 6 or the phenanthrene derivative 11 depending on the reaction conditions. 3 can be further transformed into corydine methyl ether ( 4 ); 6 has been converted to corytuberine ( 8 ). Similarly, dehydrobulbocapnine methyl ether 9 was converted to 10 .     

Synthesis, photophysical properties and in vitro photodynamic activity of axially substituted subphthalocyanines

A new series of subphthalocyanines substituted axially with an oligoethylene glycol chain [SPcB(OCH(2)CH(2))(n)OH, n = 3 (2) or 4 (3)] or a p-phenoxy oligoethylene glycol methyl ether chain [SPcBOC(6)H(4)(OCH(2)CH(2))(n)OCH(3), n = 2 (4) or 3 (5)] have been synthesised by substitution reactions of boron subphthalocyanine chloride SPcBCl (1) with the corresponding oligoethylene glycols, and characterised with various spectroscopic methods and elemental analysis. The molecular structure of one of these compounds (subphthalocyanine 4) has also been determined. As revealed by absorption spectroscopy, these compounds are essentially non-aggregated in DMF. The low aggregation tendency of these compounds results in a strong fluorescence emission and high efficiency to generate singlet oxygen. All these subphthalocyanines, being formulated with Cremophor EL, function as efficient photosensitisers and exhibit a high photocytotoxicity against HepG2 human hepatocarcinoma and HT29 human colon adenocarcinoma cells. The phenoxy analogues 4 and 5 show a relatively high photostability and are particularly potent towards these cell lines, with IC(50) values down to 0.02 microM.     

Elucidation of Binding Mechanism of Photodynamic Therapeutic Agent Toluidine Blue O with Chicken Egg White Lysozyme by Spectroscopic and Molecular Dynamics Studies

The nature of binding mechanism of toluidine blue O (TBO) with chicken egg white lysozyme was studied comprehensively by various spectroscopic and computational methods. Both steady state and time‐resolved fluorescence studies unambiguously point to the prevalence of static quenching mechanism in lysozyme–TBO system. Thermodynamic parameters revealed that the association of TBO with lysozyme was a spontaneous process in which hydrophobic and hydrogen bond interactions played a pivotal role in the binding process. The secondary and tertiary conformational changes of lysozyme in the presence of TBO were unraveled using absorption, Fourier transform infrared spectroscopy (FT‐IR) and circular dichroism (CD) techniques. Molecular docking studies of lysozyme–TBO system substantiated the findings of site marker experiment and revealed TBO adjacent to Trp‐63 and Trp‐108 residues of lysozyme. Molecular dynamics (MD) simulation studies of lysozyme–TBO system indicate a stable and effective complexation of TBO with lysozyme. It is hoped that the results presented here will enable further understanding of TBO toxicity.     

The vascular response to photodynamic therapy with ATX-S10Na(II) in the normal rat colon

The mechanism of tissue damage from photodynamic therapy (PDT) may be cellular, vascular or both, depending on the photosensitising agent and the treatment conditions. Well established photosensitisers like porfimer sodium have an optimum drug light interval of two days and may cause skin photosensitivity lasting several weeks. ATX-S10Na(II) is a new photosensitiser that remains largely in the vasculature after systemic administration and clears from the body within a few hours. The present study looks at the factors controlling the extent of PDT necrosis using ATX-S10Na(II) and correlates these with changes in the circulation after PDT. Normal Wistar rats were sensitised with ATX-S10Na(II), 2 mg/kg. At laparotomy, a laser fibre was positioned just touching the colonic mucosa and 50 J light at 670 nm delivered varying the drug light interval (0.5-24 h) and light delivery regime (100 mW continuous, 20 mW continuous or 100 mW in five fractions). Some animals were killed at three days to document the area of necrosis, others received fluorescein shortly prior to death (from a few minutes to three days after PDT) to outline the zone of PDT induced vascular shutdown. Maximum necrosis was seen with the shortest drug light interval (0.5 h), with no effect by 6 h. Fractionating the light or lowering the power did not increase the necrosis. The area of fluorescein exclusion increased over the first 2 h after PDT (in contrast to the re-perfusion seen with other photosensitisers) and correlated with the area of necrosis. PDT with ATX-S10Na(II) is most effective with a drug light interval of less than one hour. It induces irreversible vascular shutdown that extends after completion of light delivery and which is largely independent of the light delivery regime.     

Candida albicans pathogenicity: a proteomic perspective.

Candida albicans is an opportunistic fungus which causes both superficial infections and life-threatening systemic candidiasis in immunocompromised hosts such as AIDS patients, people with cancer, or other immunosuppressed individuals. Virulence factors for this fungus include the ability to adhere to host tissues, production of tissue damaging secreted enzymes, and changes in morphological form that may enhance tissue penetration and avoidance of immune surveillance. Treatment of candidiasis patients is hampered by a limited choice of antifungal agents and the appearance of clinical isolates resistant to azole drugs. Proteome analysis involves the separation and isolation of proteins by two-dimensional gel electrophoresis and their identification and characterization by mass spectrometry. The systematic application of this methodology to C. albicans is in its infancy, but is progressing rapidly. Comparing protein profiles between avirulent and virulent C. albicans strains, between drug-sensitive and -resistant strains, or between different morphological forms, could identify key control and effector proteins. There are difficulties, however, associated with the display of low abundance and cell envelope-associated proteins and the choice of conditions for obtaining suitable C. albicans cells. This article describes the potential of applying proteome analysis to C. albicans in order to better understand pathogenicity and identify new antifungal targets.     

Modified copolymers of bifunctional vinyl ethers with methyl vinyl sulfide as active matrices of solid superbases

Reactive linear and crosslinked copolymers of diethylene glycol divinyl ether and ethylene glycol vinyl glycidyl ether with methyl vinyl sulfide have been synthesized in the presence of 2,2′-azobis(isobutyronitrile) (2%, 60 °C, 45–55 h) in ∼53% yield. The hydrolyzed at the residual vinyloxy and epoxy groups and oxidized at the methylthio groups copolymers upon treatment with KOH afford alkoxide (complex) and crown-like superbases. They are capable of catalyzing the acetone ethynylation, as well as the prototropic isomerization of methyl propargyl ether to allenyl methyl ether and vinylation of ethylene and diethylene glycols with acetylene.