ABSTRACT Porphyromonas gingivalis is one of the major causative organisms of periodontitis and has been shown to be susceptible to toluidine blue-mediated photosensitization in vitro. The aims of the present study were to determine whether this technique could be used to kill the organism in the oral cavities of rats and whether this would result in a reduction in the alveolar bone loss characteristic of periodontitis. The maxillary molars of rats were inoculated with P. gingivalis and exposed to up to 48 J of 630-nm laser light in the presence of toluidine blue. The number of surviving bacteria was then determined, and the periodontal structures were examined for evidence of any damage. When toluidine blue was used together with laser light there was a significant reduction in the number of viable P. gingivalis organisms. No viable bacteria could be detected when 1 mg of toluidine blue per ml was used in conjunction with all light doses used. On histological examination, no adverse effect of photosensitization on the adjacent tissues was observed. In a further group of animals, after time was allowed for the disease to develop in controls, the rats were killed and the level of maxillary molar alveolar bone was assessed. The bone loss in the animals treated with light and toluidine blue was found to be significantly less than that in the control groups. The results of this study show that toluidine blue-mediated lethal photosensitization of P. gingivalis is possible in vivo and that this results in decreased bone loss. These findings suggest that photodynamic therapy may be useful as an alternative approach for the antimicrobial treatment of periodontitis.
While histopathology of excised tissue remains the gold standard for diagnosis, several new, non-invasive diagnostic techniques are being developed. They rely on physical and biochemical changes that precede and mirror malignant change within tissue. The basic principle involves simple optical techniques of tissue interrogation. Their accuracy, expressed as sensitivity and specificity, are reported in a number of studies suggests that they have a potential for cost effective, real-time, in situ diagnosis.We review the Third Scientific Meeting of the Head and Neck Optical Diagnostics Society held in Congress Innsbruck, Innsbruck, Austria on the 11th May 2011. For the first time the HNODS Annual Scientific Meeting was held in association with the International Photodynamic Association (IPA) and the European Platform for Photodynamic Medicine (EPPM). The aim was to enhance the interdisciplinary aspects of optical diagnostics and other photodynamic applications. The meeting included 2 sections: oral communication sessions running in parallel to the IPA programme and poster presentation sessions combined with the IPA and EPPM posters sessions.
Introduction: 5-aminolevulinic acid (ALA) generates protoporphyrin IX (PpIX)-induced fluorescence by acting as a substrate for the haem biosynthesis pathway. Despite suggestions that ALA could be used for pancreatic cancer photodiagnostics, the pancreatic cancer cell line PANC-1 only shows weak fluorescence following ALA administration. A possible explanation was that the haem biosynthesis pathway varies between cancers. Methods: We compared the mRNA expression of the haem biosynthesis pathway of PANC-1 (weak fluorescence) with the pancreatic cancer cell line CFPAC-1 (strong fluorescence) and the pancreatic ductal cell line H6c7 (control) with or without 24 hours ALA incubation. Cells were seeded on day one, fresh media with or without ALA (0.5mM) added on day two, and RNA extracted on day three. Quantitative real-time polymerase chain reaction was performed to assess the relative mRNA expression of four membrane transporters and eight enzymes responsible for haem biosynthesis. Results: Post-ALA incubation, CFPAC-1 demonstrated significant downregulation of cell membrane ALA influx transporter PEPT1, downregulation of ALA synthase and upregulation of the mitochondrial membrane transporter ABCB6. PANC-1, whilst showing similar changes to ALA synthase and ABCB6, showed significant upregulation of the PpIX efflux transporter ABCG2. PANC-1 also had minimal PEPT1 expression pre- and post-ALA. H6c7 demonstrated significant up- or downregulation of three transporters and five enzymes. Conclusion: Poor PpIX-induced fluorescence in PANC-1 is likely to be secondary to decreased ALA influx from low PEPT1 expression and increased ABCG2 expression. The use of nanocarriers to deliver ALA and/or ABCG2 inhibitors may improve ALA-induced fluorescence in PANC-1 and other ALA-resistant cancers.
The conjugation of tetrapyrroles and related photosensitisers with proteins or targeting peptides is now a well-established approach for enhancing the solubility and tissue selectivity of such molecules in the photodynamic therapy (PDT) of cancer. We are interested in developing chemical strategies for the generation of novel porphyrin-related molecules for targeted PDT, and also porphyrin-based systems whose structures are tailored for use in the light-triggered delivery of therapeutic agents by photochemical internalisation (PCI) [1]. PCI is a light-based approach that exploits the technique of PDT to enhance the delivery of nano-sized biotherapeutics that would normally be prevented from reaching their intended intracellular targets due to sequestration in certain sub-units or organelles within the cell. Effective PCI requires the use of photosensitisers that have the appropriate physical properties to localize in the membranes of the organelles (endosomes or lysosomes) where a therapeutic agent may be trapped. Upon irradiation, reactive oxygen species that are generated may then induce a highly selective damaging effect and cause partial rupture of the organelles, allowing entrapped molecules to escape to their targets within the cell. In this communication, we will describe some of our studies on the synthesis and biological evaluation of porphyrin and chlorin derivatives that are targeted with cationic cell-penetrating peptides (CPPs) via biorthogonal ligation chemistries [1, 2], and the application of these conjugates for PCI of saporin, a nano-sized protein toxin. We will also describe the extension of this principle to the CPP-targeting of liposomal nanocarriers [3] to produce molecular assemblies that allow the codelivery of a porphyrin photosensitizer and a macromolecular therapeutic in order to produce an optimal PCI effect. [1] R. Dondi, E. Yaghini, K. M. Tewari, L. Wang, F. Giuntini, M. Loizidou, A. J. MacRobert and I. M. Eggleston, Org. Biomol. Chem. 2016, 14 , 11488-11501. [2] E. Yaghini, R. Dondi, K. M. Tewari, M. Loizidou, I. M. Eggleston and A. J. MacRobert, Sci. Rep. 2017, 7 , 6059. [3] E. Yaghini, R. Dondi, K. J. Edler, M. Loizidou, A. J. MacRobert and I. M. Eggleston, Nanoscale 2108, 10 , 20366-20376.
The existence of a threshold photodynamic dose has been postulated because of the sharp demarcation between an area of PDT necrosis and normal undamaged tissue 1. An experiment was designed to measure the light intensity at various distances from the source of irradiation during PDT. Experiments were performed on normal rodent colon using a standard intravenously injected dose of the photosensitiser aluminium sulphonated phthalocyanine (A1SPc). Irradiation was performed 1 hour after injection of 5mgkg-1 using an argon pumped dye laser delivering 100mW from the end of the fibre. The time of irradiation was varied and the amount of necrosis measured 72 hours after treatment. The light intensity at specific points could be correlated with the amount of necrosis. Threshold photodynamic effects are important, if a total photodynamic dose (light fluence x photosensitiser concentration) is considered, since selective tumour destruction may be possible if the dosimetry is carefully adjusted to allow a photodynamic thresholdto be reached in the tumour and not in the normal colon. Adjustment of the light or photosensitiser dose can be used to manipulate the effect. Under apprpriate circumstances A1SPc can be photodegraded so that a total threshold photodynamic dose may never be reached in normal colon.
The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7). Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0–10 μg ml–1) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture. MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 μg ml–1 mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0–3 μg ml–1), which caused neuron death equivalent to other cell types. Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.
What's known on the subject? and What does the study add? Fluorescence cystoscopy with hexylaminolevulinate (h-ALA, Hexvix®) is known to improve tumour detection in non-muscle-invasive bladder cancer. However, specificity is relatively low and the intensity of the observed fluorescence signal decreases over time due to protoporphyrin IX (PpIX) efflux. This study evaluates in an in vivo model the use of a dendritic 5-aminolevulinic acid compound for fluorescence diagnosis. Fluorescence ratios between tumour and urothelium as well as muscle were significantly better as compared with h-ALA. Sustained synthesis of PpIX accounts for preservation of fluorescence for >24 h.• To overcome the relative lack of tumour selectivity of fluorescence-guided cystoscopy using 5-aminolevulinic acid (ALA) or its ester derivative (e.g. hexylaminolevulinate, h-ALA; Hexvix®), we evaluated the use of dendrimers bearing different ALA loads in rats bearing orthotopic bladder tumours.• Rat bladders were instilled with h-ALA or ALA dendrimers and fluorescence ratio between tumour and normal urothelium, as well as tumour and muscle and depth of fluorescence were determined with Image J software. • Quantification of ALA and/or esters systemic reabsorption was evaluated by high-performance liquid chromatography.• Slow hydrolysis of ALA from dendrimers as observed in vitro implies a higher initial ALA load and longer resting times in vivo. Sustained synthesis of protoporphyrin IX (PpIX) explains persistence of fluorescence for >24 h. • There were significantly better fluorescence ratios with dendrimers, as well as higher penetration depths and absence of systemic reabsorption.• The prolonged and sustained PpIX synthesis, the improved tumour selectivity with a deeper penetration and the absence of systemic reabsorption are primary indicators that ALA dendrimers could be an alternative to h-ALA in fluorescence-guided cystoscopy.
A quantitative technique based on fluorescence microscopic detection has been developed for measurements of photosensitizer distributions in sections of tissue and cultured cells. Imaging of photosensitizer fluorescence was achieved with negligible interference from sensitizer photodegradation and tissue autofluorescence using laser excitation and detection with a highly sensitive CCD (charge-coupled device) camera system. Techniques for the measurement of sensitizer fluorescence decay parameters in living biological cells are also described.
Silver thin films were deposited on SiO2-barrier-coated float glass, fluorine-doped tin oxide (FTO) glass, Activ glass, and TiO2-coated float glass via AACVD using silver nitrate at 350 °C. The films were annealed at 600 °C and analyzed by X-ray powder diffraction, X-ray photoelectron spectroscopy, UV/vis/near-IR spectroscopy, and scanning electron microscopy. All the films were crystalline, and the silver was present in its elemental form and of nanometer dimension. The antibacterial activity of these samples was tested against Escherichia coli and Staphylococcus aureus in the dark and under UV light (365 nm). All Ag-deposited films reduced the numbers of E. coli by 99.9% within 6 h and the numbers of S. aureus by 99.9% within only 2 h. FTO/Ag reduced bacterial numbers of E. coli to below the detection limit after 60 min and caused a 99.9% reduction of S. aureus within only 15 min of UV irradiation. Activ/Ag reduced the numbers of S. aureus by 66.6% after 60 min and TiO2/Ag killed 99.9% of S. aureus within 60 min of UV exposure. More remarkably, we observed a 99.9% reduction in the numbers of E. coli within 6 h and the numbers of S. aureus within 4 h in the dark using our novel TiO2/Ag system.
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