The skin acts as a protective barrier, is crucial for thermoregulation and also forms part of the sensory, immunological and endocrine system. Therefore skin preservation is paramount to preserving life. The loss of skin homeostasis, through injury, initiates the wound healing process where the final outcome is the formation of a scar. Scar treatment remains a challenge, despite a plethora of treatments, resulting in a poor outcome and sub-optimal response to existing therapies. Photodynamic therapy (PDT) has been used to treat oncologic conditions affecting the skin. Its action depends on a photosensitiser and a specific light source. Aminolevolinic acid (5ALA) and its methyl ester (MALA) are commonly used pro-drugs of the photosensitiser protoporphyrin IX (PpIX), which in combination with red light produces reactive oxygen species (ROS). ROS will cause different responses such as cell death and tissue destruction. There is limited clinical evidence emerging for the use of PDT in treating wound healing and pathological skin scarring. For this reason, further investigations are required to better understand the role of PDT in adult human skin wound healing and skin scarring. The aim of this investigation was to evaluate the accumulation of PpIX after exposure to 5ALA or MALA, phototoxicity of red light arrengment, citotoxicity, cell death inducction, ROS generation and a gene related analysis post-PDT in keloid fibroblasts compared to normal skin fibroblasts. Optimization of a wound healing organ culture (WHOC) model and evaluation of re-epithelialization, cell death, proliferation, extracellular matrix arreangment (ECM) and a related gene analysis after 5ALA-PDT ex vivo. General histology, cell death, proliferation, ECM rearrengment and a gene related analysis after PDT in skin scarring ex vivo. This investigation found PpIX accumulation higher with MALA compared to 5ALA. Phototoxicity and cytotoxicity was site specific within the lesion and increased proportionately to fluence rates. ROS generation leads to the decrease of cytoproliferation and increased apoptosis and necrotic cell death, COLI, COLIII an HSP70 were found down-regualted. Ex vivo wound geometry, system of support and growth media were optimized in a human wound healing organ culture (WHOC). WHOCs treated with 5ALA-PDT (20 J/cm2), showed an advancing re-epithelialization tongue 3.5 folds longer, which were highly proliferative, showing increased CK14 and p16 levels. The neo-epidermis was fully differentiated and neo-collagen was present. PCNA, p16, COLI, COLIII, MMP3, MMP19 and alpha-SMA were significantly more expressed in the dermis. MALA/5ALA-PDT (40 J/cm2) applied to striae alba, fine line, hypertrophic and keloid scars ex vivo coused an increased of apoptosis while proliferation decreased, matrix components were found to be re-organised, both according to the severity of the scar. COLI and COLIII genetic expression decreased while MMP3 and tropoelastin increased significantly. However, no statistically significant difference was observed between 5ALA and MALA-PDT treatments. In conclusion, this thesis shows that cytotoxicity post-PDT in KD fibroblasts is dependent on the lesional site within the scar, a precursor of intracellular photosensitiser and fluence. PDT in wound healing ex vivo shows increased re-epithelialization and ECM reconstruction and remodelling. Finally, in dermal fibrosis morphological and cellular effects of the application of PDT correlate with the degree and severity of dermal fibrosis. In view of this, PDT may be ideal for treating abnormal skin scarring and improving human cutaneous wound healing.

Orientador: Juliana Campos Junqueira / Banca: Antonio Olavo Cardoso Jorge / Banca: Aguinaldo Silva Garcez Segundo / Resumo: O objetivo do presente estudo foi avaliar os efeitos da terapia fotodinâmica, utilizando-se os fotossensibilizadores azul de metileno e azuleno associados ao laser em baixa intensidade, sobre cepas de Candida isoladas da cavidade bucal humana. Foram estudadas 20 cepas de Candida, sendo 5 C. albicans, 4 C. tropicalis, 4 C. glabrata, 2 C. parapsilosis, 2 C. kefyr, 1 C. krusei, 1 C. stellatoidea e 1 C. lipolytica. Cada cepa foi submetida a 6 condições experimentais: associação de laser em baixa intensidade com 660 nm e azul de metileno (L+AM+), associação de laser e azuleno (L+AZ+), irradiação com laser (L+F-), tratamento com azul de metileno (L-AM+), tratamento com azuleno (LAZ+) e tratamento apenas com solução fisiológica como controle (L-F-). Após o tratamento de cada cepa, foram realizadas diluições seriadas e semeaduras em ágar Sabouraud dextrose. Os dados de unidades formadoras de colônias (UFC/mL) foram submetidos à análise de variância e teste de Tukey (p<0,05). Os resultados demonstraram que todos os grupos tratados com laser (L+AM+, L+AZ+ e L+F-) apresentaram médias de UFC/mL (Log) inferiores aos grupos sem laser (L-AM+, L-AZ+ e L-F-). Os grupos com terapia fotodinâmica (L+AM+ e L+AZ+) apresentaram média de UFC/mL (Log) semelhante ou superior ao grupo L+F-. Concluiu-se que as cepas de Candida isoladas da cavidade bucal de pacientes submetidos à antibioticoterapia prolongada foram resistentes a terapia fotodinâmica com azul de metileno e azuleno nos parâmetros utilizados neste trabalho. / Abstract: The aim of this study was to evaluate the effects of the photodynamic therapy using methylene blue and azulene on isolated Candida strains of the oral cavity of patients who underwent a prolonged antibioticotherapy to treat lung tuberculosis. Twenty Candida strains were studied: 5 C. albicans, 4 C. tropicalis, 4 C. glabrata, 2 C. parapsilosis, 2 C. kefyr, 1 C. krusei, 1 C. stellatoidea and 1 C. lipolytica. Each strain underwent 6 experimental conditions: laser in combination with methylene blue (L+MB+), laser in combination with azulene (L+AZ+), irradiation with laser (L+P-), treatment with methylene blue (L+MB+), treatment with azulene (L+AZ+), and treatment with physiologic solution as a control group (L-P-). After the treatment of each strain, serial dilutions and spreading on Sabouraud dextrose agar were performed. Data from colony forming units per milliliter (CFU/ml) were analyzed by Tukey's test (p<0.05). The results showed that all the groups treated with laser (L+MB+, L+AZ+ and L+P-) presented the lowest mean CFU/ml (Log) value in relation to the groups treated without laser (L+MB+, L+AZ+ and L+F-). The groups treated with photodynamic therapy (L+MB+, L+AZ+) presented an average of CFU/ml (Log) similar or higher to the L+P- group. In summary, the results showed that isolated Candida strains of the oral cavity of patients who underwent a prolonged antibioticotherapy were resistant to the photodynamic therapy with methylene blue and azulene. / Mestre

Orientador: Renato Atílio Jorge / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-17T02:06:37Z (GMT). No. of bitstreams: 1 Alves_JulianaMachadodaSilveira_M.pdf: 2543897 bytes, checksum: cfe682f5663f70da68e8234395efa7bc (MD5) Previous issue date: 2010 / Resumo: As porfirinas 5,10,15,20(3-hidroxifenil)porfirina (m-THPP), 5-hexil-10,20-bis(3hidrofenil)porfirina (hex-m-bisHPP) e 5-hexil-10,15,20-tris(3-hidroxifenil)porfirina (hex-m-triHPP), têm o mesmo rendimento quântico de oxigênio singleto e foram encapsuladas em nanopartículas (NPs) poli(láctico-co-glicólico) (PLGA) 50:50, preparadas através do método de emulsão e evaporação. As NPs obtidas têm diâmetros médios na faixa de submicrometros (<240 nm) e pequena dispersividade. O potencial zeta medido mostrou uma pequena variação (de -21.6 mV a -19.3 mV). A porcentagem de encapsulação das porfirinas obtidas foram: 93 ± 2; 97 ± 2 and 69 ± 1 para m-THPP, hex-m-bisHPP e hex-m-triHPP respectivamente. A eficácia fotodinâmica e a internalização das porfirinas foi investigada a 37oC utilizando células humanas cancerígenas de próstata (LNCaP). Após 2 horas de incubação com as NPs contendo a porfirina as porcentagens de internalização celular foram iguais para todas as porfirinas. As três porfirinas causam morte celular e dão a mesma viabilidade quando se varia o tempo de incubação (30-120 min), concentração da porfirina (2,5 a 7,5 mmol L) e dose de luz incidente (33 a 99 J cm). Análise das células por microscopia confocal mostrou que as porfirinas encapsuladas nas NPs foram localizadas no citoplasma, sempre na região perinuclear. Estes resultados mostram que porfirinas com estruturas semelhantes e anfifilicidades diferentes, com igual rendimento quântico de rendimento singleto e internalizadas nas células em concentrações iguais, tem a mesma eficácia fotodinâmica / Abstract: Porphyrins (5,10,15,20-tetra(3-hydroxyphenyl)porphyrin (m-THPP), 5-hexyl-10,20-bis(3-hydroxyphenyl) porphyrin (hex-m-bisHPP) and 5-hexyl-10,15,20-tris(3-hydroxyphenyl)porphyrin (hex-m-triHPP)) with different amphiphicities and equal singlet oxygen quantum yield in ethanol, were encapsulated into 50:50 poly(lactide-co-glycolide,) (PLGA) nanoparticles (NPs) prepared by the emulsion/evaporation technique. The NPs obtained had submicron average diameters (<240 nm) with low polydispersity. The zeta potential measurements showed slight variations in negativity (from -21.6 mV to -19.3 mV). Particle recovery (%) was determined with results of 93 ± 2; 97 ± 2 and 69 ± 1 for m-THPP, hex-m-bisHPP and hex-m-triHPP porphiryns, respectively. The photodynamic efficacy of the porphyrin-loaded nanoparticles and their cellular uptake at 37 oC was investigated with LNCaP prostate tumour cells. After 2 h incubation with porphyrin-loaded nanoparticles the percents of intracellular uptake were the same for all porphyrins. The three porphyrins cause cell death and gave the same cell viability with variations of incubation time (30¿120 min), drug concentration (2.5 to 7.5 mmol L) and incident light dose (33 to 99 J cm). Confocal laser scanning microscopy data showed that the porphyrin-loaded nanoparticles, were localized in the cells, always in the perinuclear region. These results show that porphyrins with similar structures and equal singlet oxygen quantum yields and internalized in equal concentrations in the cells, but different amphiphilicities, have equal photodynamic efficacy / Mestrado / Físico-Química / Mestre em Química

No description available.

The phenomenon of photobleaching of a photosensitizer during photodynamic therapy (PDT) is well known. For second generation photosensitizers it may be possible to exploit this effect to enhance the volume of damaged tissue and improve the efficacy of PDT. In addition, as a consequence of photobleaching, the fluorescence emitted by the photosensitizer will decrease during PDT. Mathematical models were developed which describe fluorescence emission, photobleaching and tissue necrosis resulting from the irradiation of tissue containing photosensitizer using an appropriate light source. Diffusion theory was used to model bleaching in a semi-infinite medium caused by broad-beam irradiation, and both pencil and broad-beam fluorescence excitation of the photosensitizer. In addition, models were developed for an isotropic point source imbedded in an infinite medium. Based on the relationship between the decreasing fluorescence signal and the increasing volume of tissue damage, these models allow the extent of necrosis achieved during treatment to be monitored. By analysing spatially resolved fluorescence measurements predictions about necrosis depth that are insensitive to treatment parameters such as photosensitizer concentration, tissue optical properties and bleach rate are possible. Tissue simulating optical phantoms that allow for relatively simple and accurate alteration to optical properties were developed. Photosensitizers which still undergo fluorescence and photobleaching in the solid medium were also added. Using these phantoms, treatment was simulated and spatially resolved fluorescence was measured as a function of time for a wide range of initial treatment parameters. Photobleaching of the photosensitizers was observed to occur through a decrease in fluorescence emission. Also, spatially resolved measurements provided information about the average photosensitizer depth, which was seen to increase with time, with little knowledge of initial treatment parameters. These experimental results were then compared with predictions from the mathematical theory, illustrating the validity of the models. The value and feasibility of this technique for photodynamic therapy dosimetry are discussed, along with planned improvements. / Thesis / Master of Science (MS)

No description available.

The motivation of this study lies in the necessity for a microfocal therapy to specifically target diseased areas in vascular pathologies such as age-related macular degeneration (AMD). AMD is the most common cause of legal blindness among people over the age of 60 in developed countries. This degenerative condition affects the macula, the central region of the retina, severely impairing detailed vision and hindering everyday activities. Worldwide, 25-30 million people live with some form of AMD. Among them, ~10% suffer from the more advanced and damaging form, wet-AMD, which causes rapid and severe loss of central vision. To date, there is no cure or long-term alternative for this degenerative disease despite intensive research efforts. With recent developments in biophysical tools and experimental procedures, in this study, we demonstrate a highly-localized therapeutic option: two-photon (2-photon) photodynamic therapy (PDT) that could be advantageous for the cure of wet-AMD, either alone or in combination with recently discovered anti-angiogenic therapies. This new approach offers selective targeting of the diseased area, thus minimizing damage to the surrounding sensitive healthy eye tissues, which is a major concern with the clinically-used, standard wide-beam, one-photon (1-photon) PDT. The objective of the research was to test the feasibility of microfocal 1-photon and the inherently localized 2-photon PDT, their optimization and also to evaluate the efficacy of existing 1-photon and novel 2-photon photosensitizers. In this thesis, I illustrated the in vitro (endothelial cell monolayer) and in vivo (window chamber mouse (WCM)) models that can be used to quantitatively compare the 2-photon efficiency of photosensitizers. Using the in vitro model, I compared the 2-photon efficacy of clinically used 1-photon PDT drugs Photofrin and Visudyne, and showed that the Visudyne is an order of magnitude better 2-photon photosensitizer than Photofrin. With the WCM model, I demonstrated a novel designer 2-photon photosensitizer is 20 times more efficient than Visudyne for single vessel occlusion. I also generated the drug and light dose reciprocity curve for localized single-vessel microfocal PDT. This is a necessary step towards applying the method to the relevant ocular models of AMD, which is the next phase for this research.

Photodynamic Therapy (PDT) has proven itself in previous studies to be a successful therapeutic treatment for surface tumors, but its effectiveness is limited to only shallow depths that allow for the penetration of light. This study demonstrates that we have improved upon the conventional method of PDT and have overcome the previous depth limitation by creating the light at the location of the tumor in situ. We conjugated a bioluminescent protein, Luciferase, to a semiconductor nanoparticle, TiO2, and with a cell specific antibody, anti-EGFR monoclonal antibody C225. The nanoconjugate, TiDoL-C225, was then activated by ATP and Luciferin in a reaction that creates reactive oxygen species (ROS) and induces apoptosis in the tumor cells. We created the optimal nanoconjugate synthesis protocol to make TiDoL and TiDoL-C225 for use in the PDT treatment. The TiDoL-C225 nanoconjugate is able to bind specifically to colon caner cells as the C225 antibody recognizes EGFR expressed at the surface of the cells, and further, when activated it will react only with the tumor cells. The optimal cell staining protocols were developed to visualize the treatment process and later analyze with the laser confocal microscope. The TiDoL nanoconjugate was found to only be operational and effective at killing tumor cells after being activated by Luciferin and ATP, which then enhances the control we have over the therapy. The TiDoL-C225 nanoconjugate increases the efficacy of binding to tumor cells and the speed of the reaction in the cells to begin apoptosis, even in lower concentrations when compared to the free TiDoL nanoconjugate. Finally, our PDT technique allowed us to monitor the tumor cells as they begin to undergo apoptosis in less than five minutes after the Luciferin was added to activate the reaction. The advantage of our method of PDT with the TiDoL-C225 nanoconjugate is that it can be used for early detection as well as developed into an effective treatment for cancers in all depths of tissue.

Photodynamic Detection (PDD) is a diagnostic technique involving administration of a photosensitizer to the targeted cells that can be stimulated by short wavelength light which then leads to emission of light at a different wavelength (lower energy). The light emitted by the cells can be detected and analysed (by a spectroscope). All cells have the innate ability (due to endogenous fluorophores) to fluoresce, termed autofluorescence. Any cellular, metabolic or structural changes can alter the fluorescence intensity peaks. In this study 5-aminolevulinic acid (5-ALA) photosensitizer prodrug was used, which is metabolised in highly active cells to protoporphyrin IX (PpIX). Excitation of a cell at 405nm wavelength (light) leads to emission of autofluorescence at 500nm and PpIX at 635nm. The purpose of this investigation was to evaluate the use of compact spectroscopy together with the photosensitizer prodrug 5-ALA, in assessing clinically suspicious oral lesions. To that end the followings were assessed: • The fluorescence intensity ratio (FIR) or Red/Green ratio at 635/500nm measurements of normal anatomical sites at ten oral anatomical sites to map and create baseline readings for normal oral mucosal site fluorescence. • The effect of participants’ characteristics on the normal oral mucosal site FIR measurements. • The use the fluorescence intensity ratio (FIR) measurements to determine any differences between the lesion and the normal oral readings and whether the FIR from clinically suspicious oral lesions is associated with the histopathology grade. In addition to the sensitivity and specificity of the technique in assessing clinically suspicious (premalignant) oral lesions for potential malignant change.Prior to the trial commencing, approval were obtained from the Research Ethics Committee (REC), local NHS Research and Development (R&D), and Medicine Healthcare product Regulatory Agency (MHRA) and the University of Dundee Research Innovation Services (RIS). A total of thirty five participants with clinically suspicious oral lesions were recruited in Dundee (Dundee Dental Hospital) and Glasgow (Southern General Hospital). A Photodynamic Detection method using compact fluorescence spectroscopy and 5-ALA mouth rinse was applied. FIR measurements from ten normal anatomical sites were obtained in every patient to study the variation at different normal oral sites and the effect of the participant’s characteristics on these readings. In addition, two FIR measurements were obtained from each lesion and a further one taken from normal looking mucosa well beyond the lesion boundary (i.e. more than 5mm away) prior to biopsy. The readings were compared to study the reliability, reproducibility and efficacy of the photodynamic method in detecting mucosal abnormality. A total of 292 spectral readings obtained from normal mucosa were used to study the FIR measurements at normal oral anatomical sites. The results showed that the oral regions could be grouped into two broad categories with similar readings, the palatal and tongue readings in one group and buccal, ventral tongue, floor of the mouth, gingiva and lip mucosa on the other (essentially keratinized and non keratinized groups). The same set of readings were further analysed to study the effect of individual characteristics (age, gender, presence of oral prosthesis, metabolic diseases, smoking and alcohol consumptions) on the FIR measurements. There was no significant difference between FIR measurements within each of the groups studied, although at times sample sizes were very small.A total of 134 spectral readings obtained from 47 lesions that were biopsied from 35 patients recruited to the trial were used for the next part of the study. There were 91 spectra obtained from the lesions and 43 spectra obtained from the normal sites (more than 5mm away from the borders of the lesion) for comparison. There was a significant difference between the lesion and normal site readings. The FIR readings for the dysplastic lesions were significantly different when compared with the normal and benign hyperkeratoses. However there was no significant difference between dysplastic and inflammatory lesions (lichen planus, lichenoid lesions and candidal leukoplakia) on the one hand and inflammatory lesions and hyperkeratotic lesion on the other. Further analysis showed the sensitivity in detecting all the clinically suspicious oral lesions from the normal sites was 59.5% and specificity was 73.8%. The sensitivity in detecting dysplasia from normal sites was 100% and specificity 100%.Photodynamic Detection was able to detect a difference between the oral lesions from normal mucosa (but so is the naked eye!). However there was variation in the sensitivity and specificity in detecting a range of different pathological conditions. The technique was highly sensitive in detecting dysplasia from normal mucosa but unfortunately the technique is not able to discriminate reliably between dysplasia and inflammatory lesions whose clinical appearance can be very similar. In conclusion, the photodynamic detection method used in this study would not appear to offer a reliable screening tool for the early detection of oral dysplasia/cancer. The need to consider adjunctive tests that discriminate inflammation from dysplasia is required. Photodynamic Detection (PDD) is a diagnostic technique involving administration of a photosensitizer to the targeted cells that can be stimulated by short wavelength light which then leads to emission of light at a different wavelength (lower energy). The light emitted by the cells can be detected and analysed (by a spectroscope). All cells have the innate ability (due to endogenous fluorophores) to fluoresce, termed autofluorescence. Any cellular, metabolic or structural changes can alter the fluorescence intensity peaks. In this study 5-aminolevulinic acid (5-ALA) photosensitizer prodrug was used, which is metabolised in highly active cells to protoporphyrin IX (PpIX). Excitation of a cell at 405nm wavelength (light) leads to emission of autofluorescence at 500nm and PpIX at 635nm. The purpose of this investigation was to evaluate the use of compact fluorescence spectroscopy together with the photosensitizer prodrug 5-ALA, in assessing clinically suspicious oral lesions. To that end the followings were assessed: • The fluorescence intensity ratio (FIR) or Red/Green ratio at 635/500nm measurements of normal anatomical sites at ten oral anatomical sites to map and create baseline readings for normal oral mucosal site fluorescence. • The effect of participants’ characteristics on the normal oral mucosal site FIR measurements. • The use the fluorescence intensity ratio (FIR) measurements to determine any differences between the lesion and the normal oral readings and whether the FIR from clinically suspicious oral lesions is associated with the histopathology grade. In addition to the sensitivity and specificity of the technique in assessing clinically suspicious (premalignant) oral lesions for potential malignant change. Prior to the trial commencing, approval were obtained from the Research Ethics Committee (REC), local NHS Research and Development (R&D), and Medicine Healthcare product Regulatory Agency (MHRA) and the University of Dundee Research Innovation Services (RIS). A total of thirty five participants with clinically suspicious oral lesions were recruited in Dundee (Dundee Dental Hospital) and Glasgow (Southern General Hospital). A Photodynamic Detection method using compact fluorescence spectroscopy and 5-ALA mouth rinse was applied. FIR measurements from ten normal anatomical sites were obtained in every patient to study the variation at different normal oral sites and the effect of the participant’s characteristics on these readings.

Photodynamic Therapy (PDT) has proven to be a minimally invasive alternative treatment option for various conditions including cancer. The treatment efficacy of deep-seated tumours with PDT is variable, compared to the treatment of tissue surfaces such as the skin and esophagus. This is partly due to inadequate monitoring of the three interrelated treatment parameters: treatment light, photosensitizer and tissue oxygenation. This thesis presents the development of a system for explicit dosimetry of PDT treatment light and tissue oxygenation using multi-sensor fiber optic probes for spatially resolved parameter measurements. The system uses embedded fluorescent sensors for treatment light quantification. Tissue oxygenation measurement is accomplished using frequency domain techniques with embedded phosphorescent metalloporphyrin compounds as sensors.