Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models.

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.

Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. Methods: The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Results: Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. Discussion: To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

Organic Nanodelivery Systems as a New Platform in the Management of Breast Cancer: A Comprehensive Review from Preclinical to Clinical Studies.

Breast cancer accounts for approximately 25% of cancer cases and 16.5% of cancer deaths in women, and the World Health Organization predicts that the number of new cases will increase by almost 70% over the next two decades, mainly due to an ageing population. Effective diagnostic and treatment strategies are, therefore, urgently required for improving cure rates among patients since current therapeutic modalities have many limitations and side effects. Nanomedicine is evolving as a promising approach for cancer management, including breast cancer, and various types of organic and inorganic nanomaterials have been investigated for their role in breast cancer diagnosis and treatment. Following an overview on breast cancer characteristics and pathogenesis and challenges of the current treatment strategies, the therapeutic potential of biocompatible organic-based nanoparticles such as liposomes and polymeric micelles that have been tested in breast cancer models are reviewed. The efficacies of different drug delivery and targeting strategies are documented, ranging from synthetic to cell-derived nanoformulations together with a summary of the interaction of nanoparticles with externally applied energy such as radiotherapy. The clinical translation of nanoformulations for breast cancer treatment is summarized including those undergoing clinical trials.

Treatment of 3D In Vitro Tumoroids of Ovarian Cancer Using Photochemical Internalisation as a Drug Delivery Method.

Photochemical internalisation (PCI) is a means of achieving spatio-temporal control of cytosolic drug delivery using sub-lethal photodynamic therapy (PDT), with a photosensitiser that can be activated by non-ionising visible light. Various 3D models including those developed at our laboratory, where spheroids are grown in a compressed collagen matrix, have been used for studying anti-cancer drug effects. However, the use of a more biomimetic tumouroid model which consists of a relatively hypoxic central cancer mass surrounded by its microenvironment (stroma) has not yet been explored in either toxicity or phototoxicity studies involving PCI. Here, we examined the efficacy of PCI using a porphyrin photosensitiser and a cytotoxin (Saporin) on ovarian cancer tumouroids, with HEY ovarian cancer cells in the central cancer compartment, and HDF fibroblast cells and HUVEC endothelial cells in the surrounding stromal compartment. The efficacy was compared to tumouroids treated with either Saporin or PDT alone, or no treatment. PCI treatment was shown to be effective in the tumouroids (determined through viability assays and imaging) and caused a considerable decrease in the viability of cancer cells both within the central cancer mass and those which had migrated into the stroma, as well as a reduction in the cell density of surrounding HUVEC and HDFs. Post-treatment, the mean distance of stromal invasion by cancer cells from the original cancer mass following treatment with Saporin alone was 730 µm vs. 125 µm for PCI. PDT was also effective at reducing viability in the central cancer mass and stroma but required a higher photosensitiser dose and light dose than PCI. Tumouroids, as tissue mimics, are suitable models for interrogating multicellular events following pharmacological assault.

Nanotechnology-augmented sonodynamic therapy and associated immune-mediated effects for the treatment of pancreatic ductal adenocarcinoma.

PURPOSE: Sonodynamic therapy (SDT) is emerging as a cancer treatment alternative with significant advantages over conventional therapies, including its minimally invasive and site-specific nature, its radical antitumour efficacy with minimal side effects, and its capacity to raise an antitumour immune response. The study explores the efficacy of SDT in combination with nanotechnology against pancreatic ductal adenocarcinoma. METHODS: A nanoparticulate formulation (HPNP) based on a cathepsin B-degradable glutamate-tyrosine co-polymer that carries hematoporphyrin was used in this study for the SDT-based treatment of PDAC. Cathepsin B levels in BxPC-3 and PANC-1 cells were correlated to cellular uptake of HPNP. The HPNP efficiency to induce a sonodynamic effect at varying ultrasound parameters, and at different oxygenation and pH conditions, was investigated. The biodistribution, tumour accumulation profile, and antitumour efficacy of HPNP in SDT were examined in immunocompetent mice carrying bilateral ectopic murine pancreatic tumours. The immune response profile of excised tumour tissues was also examined. RESULTS: The HPNP formulation significantly improved cellular uptake of hematoporphyrin for both BxPC-3 and PANC-1 cells, while increase of cellular uptake was positively correlated in PANC-1 cells. There was a clear SDT-induced cytotoxicity at the ultrasound conditions tested, and the treatment impaired the capacity of both BxPC-3 and PANC-1 cells to form colonies. The overall acoustic energy and pulse length, rather than the power density, were key in eliciting the effects observed in vitro. The SDT treatment in combination with HPNP resulted in 21% and 27% reduction of the target and off-target tumour volumes, respectively, within 24 h. A single SDT treatment elicited an antitumour effect that was characterized by an SDT-induced decrease in immunosuppressive T cell phenotypes. CONCLUSION: SDT has significant potential to serve as a monotherapy or adjunctive treatment for inoperable or borderline resectable PDAC.

Transparent, Robust, and Photochemical Antibacterial Surface Based on Hydrogen Bonding between a Si-Al and Cationic Dye.

Healthcare-associated infections can occur and spread through direct contact with contaminated fomites in a hospital, such as mobile phones, tablets, computer keyboards, doorknobs, and other surfaces. Herein, this study shows a transparent, robust, and visible light-activated antibacterial surface based on hydrogen bonds between a transparent silica-alumina (Si-Al) sol-gel and a visible light-activated photosensitizer, such as crystal violet (CV). The study of the bonding mechanisms revealed that hydrogen bonding predominantly occurs between the N of CV and Al-OH. Apart from CV, Si-Al can be combined with a variety of dyes, highlighting its potential for wide application. The Si-Al@CV film selectively generates singlet oxygen using ambient visible light, triggering potent photochemical antibacterial performance against Gram-positive and Gram-negative bacteria. Additionally, the Si-Al@CV film is stable even after mechanical stability tests such as tape adhesion, scratch, bending, and water immersion. In vitro cytotoxicity tests using C2C12 myoblast cells showed that the Si-Al@CV film is a biocompatible material. This work suggests a new approach for designing a transparent and robust touchscreen surface with photochemical antibacterial capability against healthcare-associated infections.

Photobiocidal-triboelectric nanolayer coating of photosensitizer/silica-alumina for reusable and visible-light-driven antibacterial/antiviral air filters.

Outbreaks of airborne pathogens pose a major threat to public health. Here we present a single-step nanocoating process to endow commercial face mask filters with photobiocidal activity, triboelectric filtration capability, and washability. These functions were successfully achieved with a composite nanolayer of silica-alumina (Si-Al) sol-gel, crystal violet (CV) photosensitizer, and hydrophobic electronegative molecules of 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (PFOTES). The transparent Si-Al matrix strongly immobilized the photosensitizer molecules while dispersing them spatially, thus suppressing self-quenching. During nanolayer formation, PFOTES was anisotropically rearranged on the Si-Al matrix, promoting moisture resistance and triboelectric charging of the Si-Al/PFOTES-CV (SAPC)-coated filter. The SAPC nanolayer stabilized the photoexcited state of the photosensitizer and promoted redox reaction. Compared to pure-photosensitizer-coated filters, the SAPC filter showed substantially higher photobiocidal efficiency (∼99.99 % for bacteria and a virus) and photodurability (∼83 % reduction in bactericidal efficiency for the pure-photosensitizer filter but ∼0.34 % for the SAPC filter after 72 h of light irradiation). Moreover, after five washes with detergent, the SAPC filter maintained its photobiocidal and filtration performance, proving its reusability potential. Therefore, this SAPC nanolayer coating provides a practical strategy for manufacturing an antimicrobial and reusable mask filter for use during the ongoing COVID-19 pandemic.

One-Step Aqueous Synthesis of Anionic and Cationic AgInS2 Quantum Dots and Their Utility in Improving the Efficacy of ALA-Based Photodynamic Therapy.

Silver-indium-sulfide quantum dots (AIS QDs) have potential applications in many areas, including biomedicine. Their lack of regulated heavy metals, unlike many commercialized QDs, stands out as an advantage, but the necessity for alloyed or core-shell structures and related costly and sophisticated processes for the production of stable and high quantum yield aqueous AIS QDs are the current challenges. The present study demonstrates the one-step aqueous synthesis of simple AgInS2 QD compositions utilizing for the first time either a polyethyleneimine/2-mercaptopropionic acid (AIS-PEI/2MPA) mixture or only 2-mercaptopropionic acid (AIS-2MPA) as the stabilizing molecules, providing a AgInS2 portfolio consisting of cationic and anionic AIS QDs, respectively, and tuneable emission. Small AIS QDs with long-term stability and high quantum yields (19-23%) were achieved at a molar ratio of Ag/In/S 1/10/10 in water without any dopant or a semiconductor shell. The theranostic potential of these cationic and anionic AIS QDs was also evaluated in vitro. Non-toxic doses were determined, and fluorescence imaging potential was demonstrated. More importantly, these QDs were electrostatically loaded with zwitterionic 5-aminolevulinic acid (ALA) as a prodrug to enhance the tumor availability of ALA and to improve ALA-induced porphyrin photodynamic therapy (PDT). This is the first study investigating the influence of nanoparticle charge on ALA binding, release, and therapeutic efficacy. Surface charge was found to be more critical in cellular internalization and dark toxicity rather than drug loading and release. Both QDs provided enhanced ALA release at acidic pH but protected the prodrug at physiological pH, which is critical for tumor delivery of ALA, which suffers from low bioavailability. The PDT efficacy of the ALA-loaded AIS QDs was tested in 2D monolayers and 3D constructs of HT29 and SW480 human colon adenocarcinoma cancer cell lines. The incorporation of ALA delivery by the AIS QDs, which on their own do not cause phototoxicity, elicited significant cell death due to enhanced light-induced ROS generation and apoptotic/necrotic cell death, reducing the IC50 for ALA dramatically to about 0.1 and 0.01 mM in anionic and cationic AIS QDs, respectively. Combined with simple synthetic methods, the strong intracellular photoluminescence of AIS QDs, good biocompatibility of especially the anionic AIS QDs, and the ability to act as drug carriers for effective PDT signify that the AIS QDs, in particular AIS-2MPA, are highly promising theranostic QDs.

Cetuximab-Ag2S quantum dots for fluorescence imaging and highly effective combination of ALA-based photodynamic/chemo-therapy of colorectal cancer cells.

Colorectal cancer (CRC) has a poor prognosis and urgently needs better therapeutic approaches. 5-Aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) based photodynamic therapy (PDT) is already used in the clinic for several cancers but not yet well investigated for CRC. Currently, systemic administration of ALA offers a limited degree of tumour selectivity, except for intracranial tumours, limiting its wider use in the clinic. The combination of effective ALA-PDT and chemotherapy may provide a promising alternative approach for CRC treatment. Herein, theranostic Ag2S quantum dots (AS-2MPA) optically trackable in near-infrared (NIR), conjugated with endothelial growth factor receptor (EGFR) targeting Cetuximab (Cet) and loaded with ALA for PDT monotherapy or ALA/5-fluorouracil (5FU) for the combination therapy are proposed for enhanced treatment of EGFR(+) CRC. AS-2MPA-Cet exhibited excellent targeting of the high EGFR expressing cells and showed a strong intracellular signal for NIR optical detection in a comparative study performed on SW480, HCT116, and HT29 cells, which exhibit high, medium and low EGFR expression, respectively. Targeting provided enhanced uptake of the ALA loaded nanoparticles by strong EGFR expressing cells and formation of higher levels of PpIX. Cells also differ in their efficiency to convert ALA to PpIX, and SW480 was the best, followed by HT29, while HCT116 was determined as unsuitable for ALA-PDT. The therapeutic efficacy was evaluated in 2D cell cultures and 3D spheroids of SW480 and HT29 cells using AS-2MPA with either electrostatically loaded, hydrazone or amide linked ALA to achieve different levels of pH or enzyme sensitive release. Most effective phototoxicity was observed in SW480 cells using AS-2MPA-ALA-electrostatic-Cet due to enhanced uptake of the particles, fast ALA release and effective ALA-to-PpIX conversion. Targeted delivery reduced the effective ALA concentration significantly which was further reduced with codelivery of 5FU. Delivery of ALA via covalent linkages was also effective for PDT, but required a longer incubation time for the release of ALA in therapeutic doses. Phototoxicity was correlated with high levels of reactive oxygen species (ROS) and apoptotic/necrotic cell death. Hence, both AS-2MPA-ALA-Cet based PDT and AS-2MPA-ALA-Cet-5FU based chemo/PDT combination therapy coupled with strong NIR tracking of the nanoparticles demonstrate an exceptional therapeutic effect on CRC cells and excellent potential for synergistic multistage tumour targeting therapy.

Photodynamic therapy of cutaneous T-cell lymphoma cell lines mediated by 5-aminolevulinic acid and derivatives.

The delta-amino acid 5-aminolevulinic acid (ALA), is the precursor of the endogenous photosensitiser Protoporphyrin IX (PpIX), and is currently approved for Photodynamic Therapy (PDT) of certain superficial cancers. However, ALA-PDT is not very effective in diseases in which T-cells play a significant role. Cutaneous T-cell lymphomas (CTCL) is a group of non-Hodgkin malignant diseases, which includes mycosis fungoides (MF) and Sézary syndrome (SS). In previous work, we have designed new ALA esters synthesised by three-component Passerini reactions, and some of them showed higher performance as compared to ALA. This work aimed to determine the efficacy as pro-photosensitisers of five new ALA esters of 2-hydroxy-N-arylacetamides (1f, 1 g, 1 h, 1i and 1 k) of higher lipophilicity than ALA in Myla cells of MF and HuT-78 cells of SS. We have also tested its effectiveness against ALA and the already marketed ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA). Both cell Myla and SS cells were effectively and equally photoinactivated by ALA-PDT. Besides, the concentration of ALA required to induce half the maximal porphyrin synthesis was 209 µM for Myla and 169 µM for HuT-78 cells. As a criterion of efficacy, we calculated the concentration of the ALA derivatives necessary to induce half the plateau porphyrin values obtained from ALA. These values were achieved at concentrations 4 and 12 times lower compared to ALA, according to the derivative used. For He-ALA, concentrations were 24 to 25 times lower than required for ALA for inducing comparable porphyrin synthesis in both CTCL cells. The light doses for inducing 50% of cell death (LD50) for He-ALA, 1f, 1 g, 1 h and 1i were around 18 and 25 J/cm2 for Myla and HuT-78 cells respectively, after exposure to 0.05 mM concentrations of the compounds. On the other hand, the LD50s for the compound 1 k were 40 and 57 J/cm2 for Myla and HuT-78, respectively. In contrast, 0.05 mM of ALA and Me-ALA did not provoke photokilling since the concentration employed was far below the porphyrin saturation point for these compounds. Our results suggest the potential use of ALA derivatives for topical application in PDT treatment of MF and extracorporeal PDT for the depletion of activated T-cells in SS.

Apoptotic cell death induced by dendritic derivatives of aminolevulinic acid in endothelial and foam cells co-cultures.

Photodynamic therapy (PDT) is an effective procedure for the treatment of lesions diseases based on the selectivity of a photosensitising compound with the ability to accumulate in the target cell. Atherosclerotic plaque is a suitable target for PDT because of the preferential accumulation of photosensitisers in atherosclerotic plaques. Dendrimers are hyperbranched polymers conjugated to drugs. The dendrimers of ALA hold ester bonds that inside the cells are cleaved and release ALA, yielding PpIX production. The dendrimer 6m-ALA was chosen to perform this study since in previous studies it induced the highest porphyrin macrophage: endothelial cell ratio (Rodriguez et al. in Photochem Photobiol Sci 14:1617-1627, 2015). We transformed Raw 264.7 macrophages to foam cells by exposure to oxidised LDLs, and we employed a co-culture model of HMEC-1 endothelial cells and foam cells to study the affinity of ALA dendrimers for the foam cells. In this work it was proposed an in vitro model of atheromatous plaque, the aim was to study the selectivity of an ALA dendrimer for the foam cells as compared to the endothelial cells in a co-culture system and the type of cell death triggered by the photodynamic treatment. The ALA dendrimer 6m-ALA showed selectivity PDT response for foam cells against endothelial cells. A light dose of 1 J/cm2 eliminate foam cells, whereas less than 50% of HMEC-1 is killed, and apoptosis cell death is involved in this process, and no necrosis is present. We propose the use of ALA dendrimers as pro-photosensitisers to be employed in photoangioplasty to aid in the treatment of obstructive cardiovascular diseases, and these molecules can also be employed as a theranostic agent.

Population pharmacokinetic modelling of indium-based quantum dot nanoparticles: preclinical in vivo studies.

There is considerable interest in biomedical applications of quantum dot (QD) nanoparticles, in particular their use as imaging agents for diagnostic applications. In order to investigate the in vivo biodistribution and the potential toxicity of quantum dots (QDs), it is crucial to develop pharmacokinetic (PK) models as basis for prediction of QDs exposure profiles over time. Here, we investigated the in vivo biodistribution of novel indium-based QDs in mice for up to three months after intravenous administration and subsequently developed a translational population PK model to scale findings to humans. This evaluation was complemented by a comprehensive overview of the in vivo toxicology of QDs in rats. The QDs were primarily taken up by the liver and spleen and were excreted via hepatobiliary and urinary pathways. A non-linear mixed effects modelling approach was used to describe blood and organ disposition characteristics of QDs using a multi-compartment PK model. The observed blood and tissue exposure to QDs was characterised with an acceptable level of accuracy at short and long-term. Of note is the fast distribution of QDs from blood into liver and spleen in the first 24 h post-injection (half-life of 28 min) followed by a long elimination profile (half-life range: 47-90 days). This is the first study to assess the PK properties of QDs using a population pharmacokinetic approach to analyse in vivo preclinical data. No organ damage was observed following systemic administration of QDs at doses as high as 48 mg/kg at 24 h, 1 week and 5 weeks post-injection. In conjunction with the data arising from the toxicology experiments, PK parameter estimates provide insight into the potential PK properties of QDs in humans, which ultimately allow prediction of their disposition and enable optimisation of the design of first-in-human QDs studies.

Synergy between Photodynamic Therapy and Dactinomycin Chemotherapy in 2D and 3D Ovarian Cancer Cell Cultures.

In this study we explored the efficacy of combining low dose photodynamic therapy using a porphyrin photosensitiser and dactinomycin, a commonly used chemotherapeutic agent. The studies were carried out on compressed collagen 3D constructs of two human ovarian cancer cell lines (SKOV3 and HEY) versus their monolayer counterparts. An amphiphilc photosensitiser was employed, disulfonated tetraphenylporphine, which is not a substrate for ABC efflux transporters that can mediate drug resistance. The combination treatment was shown to be effective in both monolayer and 3D constructs of both cell lines, causing a significant and synergistic reduction in cell viability. Compared to dactinomycin alone or PDT alone, higher cell kill was found using 2D monolayer culture vs. 3D culture for the same doses. In 3D culture, the combination therapy resulted in 10 and 22 times higher cell kill in SKOV3 and HEY cells at the highest light dose compared to dactinomycin monotherapy, and 2.2 and 5.5 times higher cell kill than PDT alone. The combination of low dose PDT and dactinomycin appears to be a promising way to repurpose dactinomycin and widen its therapeutic applications.

Synergistic interactions of cadmium-free quantum dots embedded in a photosensitised polymer surface: efficient killing of multidrug-resistant strains at low ambient light levels.

Cadmium-free quantum dots (QD) were combined with crystal violet photosensitising dye and incorporated into medical grade polyurethane via a non-covalent dipping process known as 'swell-encapsulation-shrink'. The antibacterial efficacy of the prepared quantum dot-crystal violet polyurethane substrates (QD + CV PU) was investigated under low power visible light illumination at similar intensities (500 lux) to those present in clinical settings. The antibacterial performance of QD + CV PU was superior to the constituent polymer substrates, eliminating ∼99.9% of an environmental P. aeruginosa strain, a clinical P. aeruginosa strain from a cystic fibrosis patient and a clinical E. coli strain. The nature of the reactive oxygen species (ROS) involved in antibacterial activity of the QD + CV PU surface was investigated using ROS inhibitors and time-resolved optical spectroscopy. The photo-physical interactions of the green-emitting QDs with CV lead to a combination of Type I and II electron transfer and energy transfer processes, with the highly potent ROS singlet oxygen playing a dominant role. This study is the first to demonstrate highly efficient synergistic killing of clinical and environmental strains of intrinsically resistant and multi-drug resistant Gram-negative bacteria using light-activated surfaces containing biocompatible cadmium-free QDs and crystal violet dye at ambient light levels.

Enhanced photodynamic therapy and fluorescence imaging using gold nanorods for porphyrin delivery in a novel in vitro squamous cell carcinoma 3D model.

Nanocomposites of gold nanorods (Au NRs) with the cationic porphyrin TMPyP (5,10,15,20-tetrakis(1- methyl 4-pyridinio)porphyrin tetra(p-toluenesulfonate)) were investigated as a nanocarrier system for photodynamic therapy (PDT) and fluorescence imaging. To confer biocompatibility and facilitate the cellular uptake, the NRs were encapsulated with polyacrylic acid (PAA) and efficiently loaded with the cationic porphyrin by electrostatic interaction. The nanocomposites were tested with and without light exposure following incubation in 2D monolayer cultures and a 3D compressed collagen construct of head and neck squamous cell carcinoma (HNSCC). The results showed that Au NRs enhance the absorption and emission intensity of TMPyP and improve its photodynamic efficiency and fluorescence imaging capability in both 2D cultures and 3D cancer constructs. Au NRs are promising theranostic agents for delivery of photosensitisers for HNSCC treatment and imaging.

Antibacterial Surfaces with Activity against Antimicrobial Resistant Bacterial Pathogens and Endospores.

Hospital-acquired bacterial infections are a significant burden on healthcare systems worldwide causing an increased duration of hospital stays and prolonged patient suffering. We show that polyurethane containing crystal violet (CV) and 3-4 nm zinc oxide nanoparticles (ZnO NPs) possesses excellent bactericidal activity against hospital-acquired pathogens including multidrug resistant Escherichia coli (E. coli), Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and even highly resistant endospores of Clostridioides (Clostridium) difficile. Importantly, we used clinical isolates of bacterial strains, a protocol to mimic the environmental conditions of a real exposure in the healthcare setting, and low light intensity equivalent to that encountered in UK hospitals (∼500 lux). Our data shows that ZnO NPs enhance the photobactericidal activity of CV under low intensity light even with short exposure times, and we show that this involves both Type I and Type II photochemical pathways. Interestingly, polyurethane containing ZnO NPs alone showed significant bactericidal activity in the dark against one strain of E. coli, indicating that the NPs possess both light-activated synergistic activity with CV and inherent bactericidal activity that is independent of light. These new antibacterial polymers are potentially useful in healthcare facilties to reduce the transmission of pathogens between people and the environment.

Photobactericidal activity activated by thiolated gold nanoclusters at low flux levels of white light.

The emergence of antibiotic resistant bacteria is a major threat to the practice of modern medicine. Photobactericidal agents have obtained significant attention as promising candidates to kill bacteria, and they have been extensively studied. However, to obtain photobactericidal activity, an intense white light source or UV-activation is usually required. Here we report a photobactericidal polymer containing crystal violet (CV) and thiolated gold nanocluster ([Au25(Cys)18]) activated at a low flux levels of white light. It was shown that the polymer encapsulated with CV do not have photobactericidal activity under white light illumination of an average 312 lux. However, encapsulation of [Au25(Cys)18] and CV into the polymer activates potent photobactericidal activity. The study of the photobactericidal mechanism shows that additional encapsulation of [Au25(Cys)18] into the CV treated polymer promotes redox reactions through generation of alternative electron transfer pathways, while it reduces photochemical reaction type-ІІ pathways resulting in promotion of hydrogen peroxide (H2O2) production.

Photodynamic Therapy in Primary Breast Cancer.

Photodynamic therapy (PDT) is a technique for producing localized necrosis with light after prior administration of a photosensitizing agent. This study investigates the nature, safety, and efficacy of PDT for image-guided treatment of primary breast cancer. We performed a phase I/IIa dose escalation study in 12 female patients with a new diagnosis of invasive ductal breast cancer and scheduled to undergo mastectomy as a first treatment. The photosensitizer verteporfin (0.4 mg/kg) was administered intravenously followed by exposure to escalating light doses (20, 30, 40, 50 J; 3 patients per dose) delivered via a laser fiber positioned interstitially under ultrasound guidance. MRI (magnetic resonance imaging) scans were performed prior to and 4 days after PDT. Histological examination of the excised tissue was performed. PDT was well tolerated, with no adverse events. PDT effects were detected by MRI in 7 patients and histology in 8 patients, increasing in extent with the delivered light dose, with good correlation between the 2 modalities. Histologically, there were distinctive features of PDT necrosis, in contrast to spontaneous necrosis. Apoptosis was detected in adjacent normal tissue. Median follow-up of 50 months revealed no adverse effects and outcomes no worse than a comparable control population. This study confirms a potential role for PDT in the management of early breast cancer.

Photoactivable Polymers Embedded with Cadmium-Free Quantum Dots and Crystal Violet: Efficient Bactericidal Activity against Clinical Strains of Antibiotic-Resistant Bacteria.

The rising incidence of antibiotic-resistant infections from contaminated surfaces in hospitals or implanted medical devices has led to increasing interest in new antibacterial surfaces. Photoactivatable surfaces that can generate cytotoxic reactive oxygen species under exposure to ambient light is a promising approach to inactivation of surface-borne microorganisms. There is growing interest in the use of quantum dots (QDs) as light-harvesting agents for photobactericidal applications, but the cadmium in commonly used QDs will restrict clinical application. Herein, the photobactericidal activity of novel polyurethane substrates containing cadmium-free QDs was tested against clinical multidrug-resistant Gram-positive and Gram-negative bacterial strains: methicillin-resistant Staphylococcus aureus (MRSA) and a carbapenemase-producing strain of Escherichia coli ( E. coli). To enhance the capacity for reactive oxygen species generation, QDs were incorporated into the polymer with a photosensitizing dye, crystal violet. Close proximity between the QD and dye enables electron and energy transfer processes leading to generation of cytotoxic singlet oxygen and superoxide radicals. A QD solution in cyclohexane was premixed with a solution of CV in the more polar solvent, dichloromethane, to promote the formation of QD-CV nanocomposite complexes via CV adsorption. This solution was then used to embed the QDs and crystal violet into medical grade polyurethane via swell-encapsulation. The combination of QD and CV elicited significant synergistic antibacterial activity under visible light against MRSA within 1 h (99.98% reduction) and E. coli within 4 h (99.96% reduction). Photoluminescence lifetime and singlet oxygen phosphorescence measurements demonstrated that interaction between the QDs and the crystal violet occurs within the polymer and leads to enhanced generation of reactive oxygen species. Strong inhibition of kill was observed using the superoxide scavenger, superoxide dismutase. The efficacy of these QD-CV polymer substrates, that can harvest light across the visible spectrum, against multidrug-resistant bacteria demonstrates the feasibility of this approach.