aPDI meets PPE: photochemical decontamination in healthcare using methylene blue-where are we now, where will we go?

Personal protective equipment (PPE) reuse, first recommended in the context of the SARS-CoV-2 pandemic, can mitigate shortages in crisis situations and can greatly reduce the environmental impact of typically single-use PPE. Prior to safe reuse, PPE must be sanitized and contaminating pathogens-in current circumstances viruses in particular-must be inactivated. However, many established decontamination procedures are not equitable and remain unavailable in low-resource settings. In mid-2020, an interdisciplinary consortium of researchers first studied the potential of implementing cheap and easy-to-use antimicrobial photodynamic inactivation (aPDI) using methylene blue as photosensitizer to decontaminate face masks and filtering facepiece respirators. In this perspective piece, we describe the development of this novel method, discuss recent advances, and offer insights into how equitable PPE decontamination via methylene blue-based aPDI may be integrated into circular economy policies in the healthcare sector.

Extraordinary Control of Photosensitized Singlet Oxygen Generation by Acyclic Cucurbituril-like Containers.

Supramolecular control of singlet oxygen generation is incredibly valuable for several fields with broad applications and thus still challenging. However, macrocyclic inclusion complexes inherently restrict the interaction of photosensitizers with surrounding oxygen in the media. To circumvent this issue, we turned our attention in this work to acyclic cucurbituril-like containers and uncover their properties as supramolecular hosts for photosensitizers with extraordinary control of their photophysics, including singlet oxygen generation. Thermodynamic and photophysical studies were carried out showing that these acyclic containers compare very favorably to benchmark macrocycles such as cucurbiturils and cyclodextrins in terms of their binding affinities and supramolecular control of singlet oxygen generation. Acyclic container with terminal naphthalene walls offers a similar cavity to cucurbit[7]uril and the same carbonyl-lined portals for a tight binding of phenothiazinium dye methylene blue and stabilizing its singlet and triplet excited states. Thus, generation of singlet oxygen for this container is higher than for other macrocycles and even higher than the free photosensitizer. While the acyclic container with smaller terminal benzene walls, stacks over the dye through sulfur-π and π-π interactions deactivating the singlet and triplet excited states, thus showing the lowest generation of singlet oxygen out of all of the studied systems. Due to the great water solubility and biocompatibility of these systems, they possess great potential for novel applications in photocatalysis, synthesis, and biomedical fields, among others.

Nanocaging Rose Bengal to Inhibit Aggregation and Enhance Photo-induced Oxygen Consumption†.

Photosensitized crosslinking of proteins in tissues has many medical applications including sealing wounds, strengthening tissues, and beneficially altering tissue properties. Rose Bengal (RB) is used most frequently as the photosensitizer but is not as efficient as would be desired for broad utilization in medicine. Aggregation of RB, at the high concentrations used for medical treatments, decreases the yield of singlet oxygen, which mediates protein crosslinking. We hypothesized that nanocages that sequester RB would inhibit self-association, increasing photosensitization efficiency. We tested cucurbituril and cyclodextrin nanocages, demonstrating that hydroxypropyl-functionalized cyclodextrins are most effective in inhibiting RB aggregation. For these RB/cyclodextrin solutions, we investigated the effect of nanocaging on the photobleaching and oxygen consumption kinetics under 530 nm LED light in aqueous phosphate-buffered solutions. At 100 µm RB, the initial oxygen consumption rates increased by 58% and 80% compared with uncaged RB for the ß and γ (2-hydroxypropyl) cyclodextrins, respectively. For 1 mm RB, the enhancement in these rates was much greater, about 200% and 300%, respectively. In addition, at 1 mm RB these two cyclodextrins increased the RB photobleaching rate by ~20% and ~75%. These results suggest that nanocages can minimize RB aggregation and may lead to higher-efficiency photo-medical therapies.

Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus.

BACKGROUND: In the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure). METHODS: Here, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate. RESULTS: Methylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titers by over four orders of magnitude on surgical mask surfaces. DISCUSSION AND CONCLUSIONS: Inactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Methylene blue in combination with sunlight as a low cost and effective disinfection method for coronavirus-contaminated PPE.

Using the Murine Hepatitis Virus (MHV) A59 coronavirus as a SARS-CoV-2 animal surrogate, we validated that methylene blue (MB) in combination with sunlight exposure is a robust, fast, and low-cost decontamination method for PPE that should be added to the toolbox of practical pandemic preparedness.

Addressing personal protective equipment (PPE) decontamination: Methylene blue and light inactivates severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on N95 respirators and medical masks with maintenance of integrity and fit.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus. DESIGN: The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance. METHODS: MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. RESULTS: Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3. CONCLUSIONS: MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.

Photobleaching of Erythrosine B in Aqueous Environment Investigation Beyond pH.

In the scientific literature, the term aqueous environment is loosely employed as it encompasses a broad range of different buffering agents. While there is an increasing number of experimental evidence that point toward specific buffer effects extending far beyond pH, the impact of the chemical nature of the buffering ions is often disregarded, especially in photochemical studies. Herein, we highlighted the importance of buffer specific effects on both the photobleaching and the singlet oxygen quantum yields of a dye in aqueous environments. For this study, we chose erythrosine B (EB) as our model photosensitizer as its photochemistry and photobleaching are well documented in the literature. We followed EB's photobleaching via absorption spectroscopy in four different aqueous solvents, including pure water, phosphate, Tris and HEPES buffer. These buffer systems were selected because they are commonly used in biochemical and biological applications. Our results show that specific buffer effects cannot be neglected. Indeed, the singlet oxygen quantum yield for EB is significantly different in HEPES compared to the other solvents. Furthermore, we showed that EB's photoproduct is highly dependent on the nature of the chemical buffer being used.

Synthetic Access to Benzimidacarbocyanine Dyes to Tailor Their Aggregation Properties.

Developing structure-aggregation relationships of cyanine dyes is crucial for controlling their optical properties for various uses. This study develops a synthetic route and the structure-dependent self-assembly of a family of benzimidacarbocyanine dyes for J- or H-aggregation properties. It was found that both the presence and placement of halogen atoms play a defining role in the resulting supramolecular interactions of these compounds.

Complex Photophysical Properties of K114 Make for a Versatile Fluorescent Probe for Amyloid Detection.

Protein aggregation is a hallmark of Alzheimer's disease (AD) and many other neurodegenerative disorders. Small organic fluorophores such as Congo Red preferentially bind to cross-ß-sheet-rich deposits and have been used to label amyloid plaques and tau tangles in histological samples. However, distinguishing between different conformations of protein aggregates is not trivial. Using silkworm and spider silks (prototypical amyloids) and transgenic AD mouse (5XFAD) and human AD brain samples, we report how spectral confocal microscopy allowed for improved detection and differentiation of protein aggregates based on the unexpected photophysical behavior of the amyloid-specific dye K114. The pH and excitation power had pronounced effects on the emission spectrum and intensity of amyloid-bound K114 fluorescence. When bound to ß-sheet-rich assemblies, the emission spectrum of K114 was governed by the local pH of the binding pockets much more than by the pH of the mounting medium, likely due to ionization of titratable phenols. Unexpectedly, exposure to high excitation power caused a permanent increase in fluorescence intensity and a spectral blue-shift. These light-induced fluorescence changes were dependent in a complex manner on laser power, exposure time, pH, and amyloid type examined. The above-mentioned phenomena were observed in silk fibers and Alzheimer brain sections from mouse and human, indicating that this may be a general characteristic of K114 when bound to tightly aggregated macromolecules. Potential mechanisms are discussed, likely involving photoinduced electron transfer. Our findings illustrate how the complex photophysical behavior of amyloid-bound K114 can be exploited for improved detection and differentiation of protein aggregates.

Influence of Rose Bengal Dimerization on Photosensitization.

Protein crosslinking photosensitized by rose Bengal (RB2- ) has multiple medical applications and understanding the photosensitization mechanism can improve treatment effectiveness. To this end, we investigated the photochemical efficiencies of monomeric RB2- (RBM 2- ) and dimeric RB2- (RBD 2- ) and the optimal pH for anaerobic RB2- photosensitization in cornea. Absorption spectra and dynamic light scattering (DLS) measurements were used to estimate the fractions of RBM 2- and RBD 2- . RB2- self-photosensitized bleaching was used to evaluate the photoactivity of RBM 2- and RBD 2- . The pH dependence of anaerobic RB2- photosensitization was evaluated in ex vivo rabbit corneas. The 549 nm/515 nm absorption ratio indicated that concentrations > 0.10 mm RB contained RBD 2- . Results from DLS gave estimated mean diameters for RBM 2- and RBD 2- of 0.70 ± 0.02 nm and 1.75 ± 0.13 nm, respectively, and indicated that 1 mm RB2- contained equal fractions of RBM 2- and RBD 2- . Quantum yields for RB2- bleaching were not influenced by RBD 2- in RB2- solutions although accounting for RB2- concentration effects on the reaction kinetics demonstrated that RBD 2- is not a photosensitizer. Optimal anaerobic photosensitization occurred at pH 8.5 for solutions containing 200 mm Arg. These results suggest potential approaches to optimizing RBM 2- -photosensitized protein crosslinking in tissues.

The regulation of skin pigmentation in response to environmental light by pineal Type II opsins and skin melanophore melatonin receptors.

Coupling skin colour with the light/dark cycle helps regulate body temperature in ectotherms. In X. laevis, nocturnal release of melatonin from the pineal complex induces pigment aggregation and skin lightening. This nocturnal blanching is initiated by a sensor (type II opsin) that triggers melatonin release when light intensity falls below a minimum threshold, and an effector (melatonin receptor) in the skin which induces pigment aggregation. The sensor/s and effector/s belong to two families of G-protein coupled receptors that originated from a common ancestor, but diverged with subsequent evolution. The aim of this work was to identify candidate sensor/s and effector/s that regulate melatonin-mediated skin colour variation. In X. laevis, we identified a developmental time (stage 43/44) when skin lightening depends on pineal complex photosensitivity alone. At this stage, the pineal complex comprises the frontal organ and pineal gland. A total of 37 type II opsin (14 duplicated) and 6 melatonin receptor (3 duplicated) genes were identified through a full genome analysis of the allotetraploid, X. laevis. These genes were grouped into subfamilies based on their predicted amino acid sequences and the presence of specific amino acids essential for their function. The pineal complex expresses mainly blue light sensitive opsins [pinopsin, parietopsin, opn3, and melanopsins (opn4 and opn4b)] and UV-light sensitive opsins (opn5 and parapinopsin), while visual opsins and va-ancient opsin are absent, as determined by RT-PCR and in situ hybridization. The photoisomerase retinal G-protein coupled receptor, and an uncharacterized opn6b opsin, are also expressed. The spectral sensitivity that triggers melatonin secretion, and therefore melanophore aggregation, falls in the visible spectrum (470-650 Î·m) and peaks in the blue/green range, pointing to the involvement of opsins with sensitivities therein. The effector-melatonin receptors expressed in skin melanophores are mtnr1a and mtnr1c. Our data point to candidate proteins required in the neuroendocrine circuit that underlies the circadian regulation of skin pigmentation, and suggest that multiple initiators and effectors likely participate.

Cytotoxicity, cellular localization and photophysical properties of Re(I) tricarbonyl complexes bound to cysteine and its derivatives.

The potential chemotherapeutic properties coupled to photochemical transitions make the family of fac-[Re(CO)3(N,N)X]0/+ (N,N = a bidentate diimine such as 2,2'-bipyridine (bpy); X = halide, H2O, pyridine derivatives, PR3, etc.) complexes of special interest. We have investigated reactions of the aqua complex fac-[Re(CO)3(bpy)(H2O)](CF3SO3) (1) with potential anticancer activity with the amino acid L-cysteine (H2Cys), and its derivative N-acetyl-L-cysteine (H2NAC), as well as the tripeptide glutathione (H3A), under physiological conditions (pH 7.4, 37 °C), to model the interaction of 1 with thiol-containing proteins and enzymes, and the impact of such coordination on its photophysical properties and cytotoxicity. We report the syntheses and characterization of fac-[Re(CO)3(bpy)(HCys)]·0.5H2O (2), Na(fac-[Re(CO)3(bpy)(NAC)]) (3), and Na(fac-[Re(CO)3(bpy)(HA)])·H2O (4) using extended X-ray absorption spectroscopy, IR and NMR spectroscopy, electrospray ionization spectrometry, as well as the crystal structure of {fac-[Re(CO)3(bpy)(HCys)]}4·9H2O (2 + 1.75 H2O). The emission spectrum of 1 displays a variance in Stokes shift upon coordination of L-cysteine and N-acetyl-L-cysteine. Laser excitation at λ = 355 nm of methanol solutions of 1-3 was followed by measuring their ability to produce singlet oxygen (1O2) using direct detection methods. The cytotoxicity of 1 and its cysteine-bound complex 2 was assessed using the MDA-MB-231 breast cancer cell line, showing that the replacement of the aqua ligand on 1 with L-cysteine significantly reduced the cytotoxicity of the Re(I) tricarbonyl complex. Probing the cellular localization of 1 and 2 using X-ray fluorescence microscopy revealed an accumulation of 1 in the nuclear and/or perinuclear region, whereas the accumulation of 2 was considerably reduced, potentially explaining its reduced cytotoxicity. Replacing the aqua ligand with cysteine in the antitumor active fac-[Re(CO)3(bpy)(H2O)](CF3SO3) complex significantly reduced its cellular accumulation and cytotoxicity against the MDA-MB-213 breast cancer cell line, shifted its maximum emission to considerably higher energies, and decreased its fluorescence quantum yield.

Singlet oxygen partition between the outer-, inner- and membrane-phases of photo/chemotherapeutic liposomes.

Liposomes carrying membrane-embedded porphyrin-phospholipid (PoP) are capable of chemo- and photo-therapeutic modes of action, which make them a potential candidate material for next-generation cancer treatments. This study examines singlet oxygen (1O2) production and release by PoP liposomes carrying either no chemotherapeutic cargo (EMPTY), or those carrying either doxorubicin (DOX) or irinotecan (IRT) chemotherapy drugs. Herein, we developed a strategy to quantify the fraction of 1O2 lifetime spent in the three distinct local liposomal environments by obtaining four key pieces of information for each system: average 1O2 deactivation rate constants (kΔ) for liposome suspensions in H2O and in D2O solvents, as well as the absolute and the apparent 1O2 production quantum yields (ΦΔ). Despite the characteristic differences in their photophysical behavior, namely in ΦΔ values, all three formulations of PoP liposomes were found to carry out 1O2 release in a similar manner. It was found that >80% of all sensitized 1O2 from the ensemble of PoP liposomes deactivates within the nanostructures themselves, with the largest portion (∼50%) deactivating in the lipid membrane specifically. Based on these findings, we conclude that the current design of the PoP liposomes is well suited for light-induced chemotherapeutic drug release. Importantly, the 1O2 partition quantification approach reported herein has potential to be a tool for characterizing nanoparticulate light-activated chemo- and phototherapeutic systems.

In-Operando Mapping of pH Distribution in Electrochemical Processes.

In aqueous electrochemical processes, the pH evolves spatially and temporally, and often dictates the process performance. Herein, a new method for the in-operando monitoring of pH distribution in an electrochemical cell is demonstrated. A combination of pH-sensitive fluorescent dyes, encompassing a wide pH range from ≈1.5 to 8.5, and rapid electrochemically coupled laser scanning confocal microscopy is used to observe pH changes in the cell. Using electrocoagulation as an example process, we show that the method provides new insights into the reaction mechanisms. The pH close to the aluminium electrode surface is influenced by the applied current density, hydrolysis of aluminium cations, and gas evolution. Through quantification of the pH at the anode, along with gas analysis, we find that hydrogen is evolved at the anode due to a non-Faradaic chemical reaction. This leads to increased production of coagulant, which may open new routes to enhance the process performance. This method for in-operando dynamic visualization of pH paves the way for studies of electrochemical processes, including other water treatment, electrosynthesis, and batteries.

Synthesis of Tetrathia-Oligothiophene Macrocycles.

The synthesis of six tetrathia-oligothiophene macrocycles is described with modest ring-closing yields between 21 and 55%. Single-crystal X-ray studies of four of the macrocycles indicated that encapsulated solvent or guest molecules were possible. A variety of guest molecules were explored for inclusion complexes via NMR, absorption, emission, and X-ray techniques. The solution-phase inclusion complexes were uninformative; yet the solid-state experiments revealed that solvent exchangeable channels exist through the macrocyclic pores.

Roles of Near and Far Fields in Plasmon-Enhanced Singlet Oxygen Production.

In plasmon-enhanced singlet oxygen (1O2) production, irradiation of a hybrid photosensitizer-metal nanoparticle leads to a significant alteration of the photosensitizer's 1O2 yield. The quest for a more rational design of these nanomaterials calls for a better understanding of the enhancement mechanism that, to this day, remains largely unexplored. Herein, we introduce a new methodology to distinguish the near- and far-field contributions to the plasmon-enhanced 1O2 production using a tunable model nanoplatform, Rose Bengal-decorated silica-coated metal nanoparticles. By correlating 1O2 production to the experimental and simulated optical properties of our nanoparticles, we effectively discriminate how the near- and far-field effects contribute to the plasmonic interactions. We show that these effects work in synergy; i.e., for nanoparticles with a similar local field, the production of 1O2 correlates with maximized scattering yields. Our results expound the critical plasmonic aspects in terms of near and far fields for the design of an efficient hybrid plasmonic nanoparticle photosensitizer.

Hybrid Silver Nanocubes for Improved Plasmon-Enhanced Singlet Oxygen Production and Inactivation of Bacteria.

Plasmonic nanoparticles can strongly interact with adjacent photosensitizer molecules, resulting in a significant alteration of their singlet oxygen (1O2) production. In this work, we report the next generation of metal-enhanced 1O2 nanoplatforms exploiting the lightning rod effect, or plasmon hot spots, in anisotropic (nonspherical) metal nanoparticles. We describe the synthesis of Rose Bengal-decorated silica-coated silver nanocubes (Ag@SiO2-RB NCs) with silica shell thicknesses ranging from 5 to 50 nm based on an optimized protocol yielding highly homogeneous Ag NCs. Steady-state and time-resolved 1O2 measurements demonstrate not only the silica shell thickness dependence on the metal-enhanced 1O2 production phenomenon but also the superiority of this next generation of nanoplatforms. A maximum enhancement of 1O2 of approximately 12-fold is observed with a 10 nm silica shell, which is among the largest 1O2 production metal enhancement factors ever reported for a colloidal suspension of nanoparticles. Finally, the Ag@SiO2-RB NCs were benchmarked against the Ag@SiO2-RB nanospheres previously reported by our group, and the superior 1O2 production of Ag@SiO2-RB NCs resulted in improved antimicrobial activities in photodynamic inactivation experiments using both Gram-positive and -negative bacteria model strains.

Visualizing Oncolytic Virus-Host Interactions in Live Mice Using Intravital Microscopy.

Oncolytic virus (OV) therapy is an emerging cancer treatment that uses replicating viruses to infect and kill tumor cells and incite anticancer immunity. While the approach shows promise, it currently fails most patients, indicating strategies to improve OV activity are needed. Developing these will require greater understanding of OV biology, particularly in the context of OV delivery and clearance, the infection process within a complex tumor microenvironment, and the modulation of anticancer immunity. To help achieve this, we have established a technique for high-resolution 4D imaging of OV-host interactions within intact tissues of live mice using intravital microscopy (IVM). We show that oncolytic vesicular stomatitis virus (VSV) directly labeled with Alexa Fluor dyes is easily visualized by single- or multiphoton microscopy while retaining bioactivity in vivo. The addition of fluorophore-tagged antibodies and genetically encoded reporter proteins to image target cells and the virus infection enables real-time imaging of dynamic interactions between VSV and host cells in blood, tumor, and visceral organs of live mice. The method has sufficient in vivo resolution to observe leukocytes in blood binding to and transporting VSV particles, foci of VSV infection spreading through a tumor, and antigen-presenting cells in the spleen interacting with and being infected by VSV. Visualizing OV-host interactions by IVM represents a powerful new tool for studying OV therapy.

Assessment of encapsulated dyes' distribution in silica nanoparticles and their ability to release useful singlet oxygen.

Working with silica nanoparticle encapsulated BODIPY and xanthene photosensitizers, we have determined that singlet oxygen spends up to 78% of its lifetime inside the nanocarriers. Our systematic investigation indicates that hydrophobicity rules the photosensitizer distribution in nanoparticles, which in turn dictates the ability of these structures to release singlet oxygen.

Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy.

For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC-demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis.