Peptide-targeted dendrimeric prodrugs of 5-aminolevulinic acid: A novel approach towards enhanced accumulation of protoporphyrin IX for photodynamic therapy.

Photodynamic therapy (PDT) is a promising approach for the targeted treatment of cancer and various other human disorders. An effective, clinically approved approach in PDT involves the administration of 5-aminolevulinic acid (ALA) to generate elevated levels of the natural photosensitiser protoporphyrin IX (PpIX). The development of prodrugs of ALA is of considerable interest as a means to enhance the efficiency and cell selectivity of PpIX accumulation for PDT applications. In this work a novel peptide-targeted dendrimeric prodrug of 5-aminolevulinic acid (ALA) 13 was synthesised which displays nine copies of ALA on a core structure that is linked to a homing peptide for targeted delivery to a specific cancer cell type. The synthesis was accomplished effectively via a flexible, modular solid phase and solution phase route, using a combination of solid phase peptide synthesis and copper-catalysed azide-alkyne cycloaddition chemistry. The prodrug system shows a sustained and enhanced production of protoporphyrin IX (PpIX) in the MDA-MB-231 cell line that over-expresses the epidernal growth factor receptor (EGFR+) in comparison to equimolar ALA and the corresponding non-targeted ALA dendrimer (nine copies of ALA). This study provides a proof of concept for the development of a new generation of prodrugs for ALA-based photodynamic therapy that can deliver an enhanced ALA payload to specific tissue types.

5-Aminolevulinic acid for fluorescence-guided surgery in pancreatic cancer: Cellular transport and fluorescence quantification studies.

5-Aminolevulinic acid (ALA) is a potential contrast agent for fluorescence-guided surgery in pancreatic ductal adenocarcinoma (PDAC). However, factors influencing ALA uptake in PDAC have not been adequately assessed. We investigated ALA-induced porphyrin fluorescence in PDAC cell lines CFPAC-1 and PANC-1 and pancreatic ductal cell line H6c7 following incubation with 0.25-1.0 mM ALA for 4-48 h. Fluorescence was assessed qualitatively by microscopy and quantitatively by plate reader and flow cytometry. Haem biosynthesis enzymes and transporters were measured by quantitative polymerase chain reaction (qPCR). CFPAC-1 cells exhibited intense fluorescence under microscopy at low concentrations whereas PANC-1 cells and pancreatic ductal cell line H6c7 showed much lower fluorescence. Quantitative fluorescence studies demonstrated fluorescence saturation in the two PDAC cell lines at 0.5 mM ALA, whereas H6c7 cells showed increasing fluorescence with increasing ALA. Based on the PDAC:H6c7 fluorescence ratio studies, lower ALA concentrations provide better contrast between PDAC and benign pancreatic cells. Studies with qPCR showed upregulation of ALA influx transporter PEPT1 in CFPAC-1, whereas PANC-1 upregulated the efflux transporter ABCG2. We conclude that PEPT1 and ABCG2 expression may be key contributory factors for variability in ALA-induced fluorescence in PDAC.

Photodynamic therapy: Inception to application in breast cancer.

Photodynamic therapy (PDT) is already being used in the treatment of many cancers. This review examines its components and the new developments in our understanding of its immunological effects as well as pre-clinical and clinical studies, which have investigated its potential use in the treatment of breast cancer.

Flexible synthesis of cationic peptide-porphyrin derivatives for light-triggered drug delivery and photodynamic therapy.

Efficient syntheses of cell-penetrating peptide-porphyrin conjugates are described using a variety of bioconjugation chemistries. This provides a flexible means to convert essentially hydrophobic tetrapyrolle photosensitisers into amphiphilic derivatives which are well-suited for use in light-triggered drug delivery by photochemical internalisation (PCI) and targeted photodynamic therapy (PDT).

Aminolevulinic acid dendrimers in photodynamic treatment of cancer and atheromatous disease.

The use of endogenous protoporphyrin IX after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). We have previously reported that the conjugation of ALA dendrimers enhances porphyrin synthesis. The first aim of this work was to evaluate the ability of ALA dendrimers carrying 6 and 9 ALA residues (6m-ALA and 9m-ALA) to photosensitise cancer cells. For this aim, we employed LM3 mammary carcinoma cells. In these tumour cells, at low concentrations porphyrin synthesis from dendrimers was higher compared to ALA, whereas at high concentrations, porphyrin synthesis was similar from both compounds. Topical application of ALA dendrimers on the skin overlying a subcutaneous LM3 implanted tumour showed no diffusion of the molecules either to distant skin sites or to the adjacent tumour, suggesting a promising use of the ALA macromolecules in superficial cancer models. As a second objective, we proposed the use of ALA-dendrimers in vascular PDT for the treatment of atherosclerosis. Thus, we focused our studies on ALA-dendrimer's selectivity towards macrophages in comparison with endothelial cells. For this aim we employed Raw 264.7 macrophages and HMEC-1 microvasculature cells. Porphyrin synthesis induced in macrophages by 6m-ALA and 9m-ALA (3 h, 0.025 mM) was 6 and 4.6 times higher respectively compared to the endothelial cell line, demonstrating the high affinity of ALA dendrimers for macrophages. On the other hand, ALA employed at low concentrations was slightly selective (1.7-fold) for macrophages. Inhibition studies suggested that ALA dendrimer uptake in macrophages is mainly mediated by caveloae-mediated endocytosis. Our main conclusion is that in addition to being promising molecules in PDT of superficial cancer, ALA dendrimers may also find applications in vascular PDT, since in vitro they showed selectivity to the macrophage component of the atheromatous plaque, as compared to the vascular endothelium.

Conjugatable water-soluble Pt(II) and Pd(II) porphyrin complexes: novel nano- and molecular probes for optical oxygen tension measurement in tissue engineering.

Measurement of oxygen tension in compressed collagen sheets was performed using matrix-embedded optical oxygen sensors based on platinum(II) and palladium(II) porphyrins supported on polyacrylamide nanoparticles. Bespoke, fully water-soluble, mono-functionalised Pt(II) and Pd(II) porphyrin complexes designed for conjugation under mild conditions were obtained using microwave-assisted metallation. The new sensors display a linear response (1/τ vs. O2) to varying oxygen tension over a biologically relevant range (7.0 × 10(-4) to 2.7 × 10(-1) mM) in aqueous solutions; a behaviour that is maintained following conjugation to polyacrylamide nanoparticles, and following embedding of the nanosensors in compressed collagen sheets, paving the way to innovative approaches for real-time resolution of oxygen gradients throughout 3D matrices useful for tissue regeneration.

Photochemical internalisation: the journey from basic scientific concept to the threshold of clinical application.

Efficient delivery of therapeutic agents to subcellular targets is a major challenge in pharmacology. Physical properties including size and charge may adversely affect the cellular uptake of molecules, and consequently reduce the accessibility of intracellular targets. For example macromolecules, which do not pass freely through the phospholipid membrane, are internalised via endocytosis and subsequently retained in endosomes or lysosomes before enzymatic degradation or cell efflux. Photochemical internalisation (PCI) is a novel drug delivery technology based on light-activated release of biologically active compounds retained within endosomes/lysosomes. PCI is founded upon the principle of photodynamic therapy (PDT), which uses light to activate photosensitisers to ultimately produce reactive oxygen species (ROS) and cause local damage/cell death. In PCI, photosensitisers are selectively localised in endosomal/lysosomal membranes. PCI triggers membrane rupture facilitating release and delivery of endocytosed molecules to intracellular targets.

Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model.

BACKGROUND: 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). METHODS: Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0-10 microg 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. RESULTS: MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 microg 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 microg ml(-1)), which caused neuron death equivalent to other cell types. CONCLUSION: Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.

Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism.

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namely serine protease inhibitors. Using siRNA knockdown of acylpeptide hydrolase (ACPH) protein expression, we showed the involvement of ACPH, a member of the prolyl oligopeptidase family of serine peptidases, in the hydrolytic cleavage of ALA from the peptide derivatives. In conclusion, ALA peptide derivatives are capable of delivering ALA efficiently to cells and enhancing porphyrin synthesis and photocytotoxicity; however, the N-terminus state, whether free or substituted, plays an important role in determining the biological efficacy of ALA peptide derivatives.

Spatially defined oxygen gradients and vascular endothelial growth factor expression in an engineered 3D cell model.

Tissue hypoxia results in rapid angiogenesis in vivo, triggered by angiogenic proteins, including vascular endothelial growth factor (VEGF). Current views of tissue viability are founded on whether 'deeper-lying' cells receive sufficient nutrients and oxygen for normal activity and ultimately survival. For intact tissues, levels of such essential nutrients are governed by micro-vascular perfusion. However, there have been few effective quantitatively defined 3D models, which enable testing of the interplay or interdependence of matrix and cell density, and path diffusion on oxygen consumption in vitro. As a result, concepts on cell vulnerability to low oxygen levels, together with the nature of cellular responses are ill defined. The present study has adapted a novel, optical fibre-based system for in situ, real-time oxygen monitoring within three-dimensionally-spiralled cellular collagen constructs, which were then unfurled to enable quantitative, spatial measurements of VEGF production in different parts of the same construct exposed to different oxygen levels. A VEGF response was elicited by cells exposed to low oxygen levels (20 mmHg), primarily within the construct core.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells.

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1-0.2 microg ml(-1)) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6-28%). Hypericin (0.1-0.2 microM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy.

Photodynamic therapy using verteporfin photosensitization in the pancreas and surrounding tissues in the Syrian golden hamster.

BACKGROUND/AIM: Photodynamic therapy (PDT) is a potential treatment for locally advanced pancreatic cancer. We aimed to assess the safety of interstitial PDT using verteporfin (benzoporphyrin derivative monoacid A - a novel photosensitizer with a short drug-light interval and limited cutaneous photosensitivity) in the Syrian golden hamster, and compare it to meso-tetrahydroxyphenylchlorin (mTHPC) which we have previously evaluated in preclinical and clinical studies. METHODS: Verteporfin (2 mg/kg) was administered at laparotomy by inferior vena caval injection (n = 57), with plasma levels quantified at 5, 15, 30, 60 and 240 min, and 24 h. 15 min after photosensitization, tissues (liver, pancreas, duodenum, colon, aorta) were illuminated with 690 nm red laser light (150 mW), at a range of light doses (1-100 J/cm(2)). The PDT effects on the targeted organ and adjacent structures were assessed at post-mortem on days 3 and 21, or at the time of death. RESULTS: The elimination half-life of verteporfin was 4-5 h. Light doses of 10, 25 and 50 J/cm(2) were safe in the hamster pancreas, liver and colon, respectively, and produced coagulative necrotic lesions of 3 (range 3-4), 10 (9-10) and 7 (7-8) mm diameter. Collagen was resistant to damage and lesions healed mainly by regeneration of normal tissue. At higher light doses, necrosis extended to the edge of organs, sometimes causing sealed duodenal perforations as seen with mTHPC. CONCLUSION: The safety profile of verteporfin is very similar to mTHPC, with the advantages of a shorter drug-light interval and drug elimination time. Phase I clinical studies using this photosensitizer in pancreatic cancer should be feasible.

Photodynamic therapy down-regulates the invasion promoting factors in human oral cancer.

Squamous cell carcinomas of the head and neck are characterized by their high tendency for invasion and metastasis. Several studies have identified the roles of matrix metalloproteinases (MMPs), vascular endothelial growth factors (VEGF) and urokinase plasminogen activators (uPA) in this process. Photodynamic Therapy (PDT) is an emerging treatment currently in clinical practice for the treatment of early cancer. Here we evaluate, in vitro, the influence of PDT on the expression of these molecules. A series of human keratinocyte cell lines derived from human oral squamous cell carcinomas (OSCC) were used as the PDT 'targets' in this study. Each cell line was subjected to sublethal dose of PDT. Activity of MMP-2, MMP-9, MMP-13, uPA and VEGF were evaluated at protein levels using zymography and ELISA on culture medium. For uPA, a chromogenic assay was performed. Gelatin zymography results revealed that, in control medium, MMP-9 and MMP-2 were secreted in proform. MMP-2 was highly expressed by H376 cells while VB6 and UP cells relatively show similar MMP-2 with comparatively low expression. For MMP-9, the latent type was highly expressed by VB6 cells and only slightly by H376, while active-MMP-9 was expressed by VB6 cell line only. Following PDT, both active and latent MMP-2 and MMP-9 were down regulated by UP and VB6 cells (p<0.001), while H376 showed an increase in active-MMP-2. These observations were supported by ELISA. This study has demonstrated that, PDT causes the suppression of factors responsible for tumour invasion which may be of therapeutic value.

Fluorescence spectroscopy combined with 5-aminolevulinic acid-induced protoporphyrin IX fluorescence in detecting oral premalignancy.

BACKGROUND: Early detection of premalignant/malignant lesions in the oral cavity can certainly improve the patient's prognosis. This study presents fluorescence imaging with the topical application of 5-aminolevulinic as a way to improve detection of various oral tissue pathologies. This procedure depends mainly on comparing the intensity of red and green fluorescence emitted from tissues during examination. MATERIALS AND METHODS: Seventy-one patients who presented with clinically suspicious oral leukoplakia were recruited for this study. Each of the patients was required to have 5-aminolevulinic acid in the form of mouth rinse prior to fluorescence imaging. Following this a surgical biopsy was acquired from the exact examination site. The results of the fluorescence spectroscopy have been compared with histopathology. RESULTS: A Student's t-test was applied to test the viability of the ratio between red and green fluorescence. The red-to-green ratio was found to increase significantly when the lesion was identified as dysplastic or carcinoma in situ. By applying a threshold line to discriminate between normal and dysplastic lesions; a sensitivity of 83-90% and specificity of 79-89% were obtained. CONCLUSION: Fluorescence spectroscopy combined with 5-aminolevulinic acid-induced protoporphyrin IX was found as a valuable tool in the diagnosis of oral premalignancy. This technique offers the potential to be advantageous over other non-optical techniques in terms of providing real-time diagnosis, in situ monitoring, cost effectiveness and more tolerated by patient compared to surgical biopsy.

Correlation of real-time haemoglobin oxygen saturation monitoring during photodynamic therapy with microvascular effects and tissue necrosis in normal rat liver.

Photodynamic therapy (PDT) requires a photosensitising drug, light and oxygen. While it is known that the haemoglobin oxygen saturation (HbSat) can be altered by PDT, little has been done to correlate this with microvascular changes and the final biological effect. This report describes such studies on the normal liver of rats sensitised with aluminium disulphonated phthalocyanine. In total, 50 J of light at 670 nm, continuous or fractionated at 25 or 100 mW, was applied with a single laser fibre touching the liver surface. HbSat was monitored continuously 1.5-5.0 mm from the laser fibre using visible light reflectance spectroscopy (VLRS). Vascular shutdown was assessed by fluorescein angiography 2-40 min after light delivery. Necrosis was measured at post mortem 3 days after PDT. In all treatment groups at a 1.5 mm separation, HbSat fell to zero with little recovery after light delivery. At 2.5 mm, HbSat also decreased during light delivery, except with fractionated light, but then recovered. The greatest recovery of fluorescein perfusion after PDT was seen using 25 mW, suggesting an ischaemia/reperfusion injury. Necrosis was more extensive after low power and fractionated light than with 100 mW, continuous illumination. We conclude that VLRS is a useful technique for monitoring HbSat, although the correlation between HbSat, fluorescein exclusion and necrosis varied markedly with the light delivery regimen used.

Porphyrin synthesis from ALA derivatives for photodynamic therapy. In vitro and in vivo studies.

The aim of this work was to test in vitro and in vivo the efficacy of the derivatives of 5-aminolevulinic acid (ALA): hexyl-ALA (He-ALA), undecanoyl-ALA and R,S-2-(hydroximethyl)tetrahydropyranyl-ALA (THP-ALA) as pro-photosensitising agents. The compounds were assayed in a cell line derived from a murine mammary tumour, in tumour explants and after injection of the cells into mice. In vitro, undecanoyl-ALA and THP-ALA did not improve ALA efficacy in terms of porphyrin synthesis. On the other hand, half of the amount of ALA is required to obtain the same plateau amount of photosensitiser from He-ALA. However, this plateau value cannot be surpassed in spite of the four-times higher accumulation of ALA/He-ALA from the ALA derivative. This shows that He-ALA conversion to porphyrins but not He-ALA entry to the cells is limiting. Employing ionic exchange chromatography, we found that 80% of total uptake was He-ALA whereas only 20% was ALA. This suggests that the esterases, probably themselves regulated by the heme pathway, are limiting the conversion of ALA derivatives into porphyrins. A similar situation occurs with THP-ALA. Tumour explant porphyrin results correlate well with cell line data. However, i.p. injection of ALA derivatives to mice resulted in a lower porphyrin concentration in the tumour when compared to the administration of equimolar amounts of ALA, indicating that there should be retention of ALA derivatives either within the blood vessels in the initial phase of distribution and/or within the capillaries of the tumour.

In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitization in an animal model.

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.

Optical measurement of three-dimensional collagen gel constructs by elastic scattering spectroscopy.

Analysis of the formation and organization of new connective tissue formed in tissue-engineered constructs is a major requirement for tissue bioreactor technology. We have analyzed early-stage responses in collagen lattices, using elastic scattering spectroscopy to assess its potential to monitor tissue structural changes in structures up to 3 mm thick, under normal culture conditions. The method is based on an optical system in which an optical fiber delivers white light onto the tissue and the back-scattered light is collected for spectroscopy by another optical fiber. Results show correlation between changes in the spectral signatures with changes in the collagen gel contraction or internal organization in all three models of collagen construct analyzed. Therefore elastic scattering spectroscopy is a promising tool to monitor tissue-engineered constructs or early repair in collagenous tissues.

Fluorescence biodistribution and photosensitising activity of toluidine blue o on rat buccal mucosa.

The antimicrobial activity of toluidine blue O (TBO) in the presence of red light has been demonstrated for a wide range of microorganisms. The response of tissues to TBO-induced photosensitisation is an important factor in assessing the clinical usefulness of this technique for the treatment of infectious diseases. The aims of this study were to determine the effect of TBO-mediated photosensitisation on rat buccal mucosa and the biodistribution of the photosensitiser in this tissue. An aqueous solution of TBO was applied to one side of the buccal mucosa of the animals. A 6 mm diameter area was then exposed to light (633 nm) from a copper vapour pumped-dye laser. The opposite, untreated, side of the buccal mucosa served as a control. TBO concentrations of 25, 50 and 200 microg/ml, laser light doses of 110, 170 and 340 J/cm(2) were assessed. Control groups of animals were subjected to 340 J/cm(2) laser light alone or to 200 microg/ml TBO alone. Serial sacrifices were performed after 72 h to obtain mucosal tissue samples for histological examination. For the determination of TBO biodistribution, additional groups received the same TBO doses and were sacrificed after 1 min or 10 min. Specimens were removed and frozen immediately for digital fluorescence imaging. No necrotic or inflammatory changes were found in the buccal mucosa of the animals with any of the treatments (using up to 200 microg/ml TBO and 340 J/cm(2) laser light). A high TBO fluorescence in the epithelium, particularly in the keratinised layer, with almost no fluorescence in the underlying connective tissue was demonstrated by the digital imaging. The results of this study suggest that TBO-mediated PDT (within the concentrations and light doses tested) could be a safe antimicrobial approach for the oral infections without damaging the adjacent normal tissue.