Photodynamic therapy (PDT) is an emerging therapy for the treatment of cancer and various other human disorders. 5-Aminolaevulinic acid (ALA) is a simple natural product that is of great interest for PDT because it can be converted within cells via the haem biosynthetic pathway to the photosensitiser, protoporphyrin IX (PpIX). ALA-PDT has become a first line clinical approach for the treatment of cancerous and precancerous skin lesions (e.g Bowen’s disease, basal skin carcinomas, and actinic keratosis) that would otherwise require significant conventional surgery. However, ALA being a zwitterion suffers from poor lipid solubility and at the same time has stability issues at physiological or alkaline pH. The work herein describes some novel strategies to enhance the delivery of ALA to specific cell types using targeted ALA dendrimeric prodrugs. Specifically, it describes the synthesis of molecules consisting of branched units with 3 or more copies of ALA attached to a central core structure (e.g. gallic acid) using copper-catalysed azide-alkyne click chemistry (CuAAC). Selective delivery of the dendrimeric ALA cargo was achieved by attachment of a homing peptide to an independently addressable functional group on the prodrug core. As proof of concept of this approach, systems were prepared containing a peptide that allows selective targeting of the epidermal growth factor receptor (EGFR) which is overexpressed in a variety of tumours. Targeted ALA delivery and PpIX production was studied with these prodrugs in EGFR-expressing breast adenocarcinoma cells (MDA-MB-231 cells) and a peptide-targeted derivative with 9 ALA units was found to have enhanced PDT efficacy compared to an equimolar dose of ALA. Other targeting units that have been attached to these dendrimeric ALA prodrugs include biomolecules such as vitamin E, thymidine (a nucleoside) and a glucose derivative. Additionally, strain-promoted azide-alkyne cycloadditions (SPAAC) of the same EGFR-targeting peptide with some classical photosensitisers were also investigated and biological studies in EGFR-overexpressing cell lines were carried out. Lastly, a group of cell penetrating peptide-ALA conjugates have been synthesised via CuAAC as a novel approach for targeted ALA delivery.

Background: We report here early attempts of adapting a prototype fluorescence imaging system (PRODIGI™) to be used as a surgical guidance tool to improve margin-detection in breast cancer. Methods: 36 patients were recruited to study the autofluorescence characteristics of ex vivo specimens. 5-ALA (20 mg/kg) was used as a contrast agent in human breast cancer cell lines and xenograft tumour models to detect PpIX fluorescence. Results: Administrative approvals were obtained and a surgical drape was used for sterilization. PRODIGITM could differentiate between normal and tumour tissues based on autofluorescence alone in ex vivo samples. PpIX signal was detected in experimental mice, and absent in control mice. The threshold of detection was on the order of 10 nM. Conclusions: Autofluorescence alone with PRODIGI™ was not sufficient for margin assessment of ex vivo breast tumour surgical specimens. 5-ALA at an optimal dosage may be adopted as a contrast agent to enhance tumour signal.

Background: We report here early attempts of adapting a prototype fluorescence imaging system (PRODIGI™) to be used as a surgical guidance tool to improve margin-detection in breast cancer. Methods: 36 patients were recruited to study the autofluorescence characteristics of ex vivo specimens. 5-ALA (20 mg/kg) was used as a contrast agent in human breast cancer cell lines and xenograft tumour models to detect PpIX fluorescence. Results: Administrative approvals were obtained and a surgical drape was used for sterilization. PRODIGITM could differentiate between normal and tumour tissues based on autofluorescence alone in ex vivo samples. PpIX signal was detected in experimental mice, and absent in control mice. The threshold of detection was on the order of 10 nM. Conclusions: Autofluorescence alone with PRODIGI™ was not sufficient for margin assessment of ex vivo breast tumour surgical specimens. 5-ALA at an optimal dosage may be adopted as a contrast agent to enhance tumour signal.

Neste trabalho foi investigada a ação fotodinâmica dos pró-fármacos 5-ALA e 8-ALA incorporados em nanopartículas de quitosana, através da incidência de luz em células de melanoma, utilizando sinergicamente a Eletroquimioterapia e a Terapia Fotodinâmica. O ácido 5-aminolevulínico (5-ALA) e seus ésteres derivados são precursores metabólicos da protoporfirina IX (PpIX) e apresentam uma penetração limitada no estrato córneo. Portanto, o objetivo desse trabalho foi desenvolver, caracterizar e avaliar o efeito fotodinâmico das nanopartículas de quitosana contendo os pró-fármacos 5-ALA e seu éster derivado 8-ALA e investigar a produção efetiva de PpIX. Concomitantemente, o efeito sinérgico da Eletroquimioterapia (ECT) com a TFD também foi estudado com a finalidade de aumentar a permeação dos fármacos fotossensibilizantes através da pele, aumentando a eficiência da TFD no tratamento de neoplasias cutâneas. As nanopartículas de quitosana foram obtidas pelo método de gelificação ionotrópica, produzindo nanopartículas em tamanho nanométrico (636,0 nm ± 6,5), com uma distribuição de tamanho homogênea (0,37), além de apresentar um potencial zeta significantemente positivo + 35,3 mV (± 2,21). Seu perfil de liberação e capacidade de incorporação de 80% dos pró-fármacos também foi determinado. Os estudos de citotoxicidade na ausência de luz demonstraram o caráter biocompatível das nanopartículas de quitosana desenvolvidas, assim como a produção significativa de PpIX e foi possível comprovar o excelente efeito fototóxico da formulação desenvolvida. Os estudos de sinergismo da ECT com a TFD comprovaram que a combinação das nanopartículas de quitosana contendo os pró-fármacos, com aplicação de microcorrentes de 400 ?A e dose de luz de 3 J/cm2, nestas condições apresentou uma morte celular significativa. / This study investigated the photodynamic action of the prodrug 5-ALA and 8-ALA into chitosan nanoparticles, through the light incidence in melanoma cells, using synergistically the Electrochemotherapy and Photodynamic Therapy. The 5- aminolevulinic acid (5-ALA) and its ester derivatives are metabolic precursors of protoporphyrin IX (PpIX) and have a limited penetration in the stratum corneum. Therefore, the objective of this study was to develop, characterize and evaluate the photodynamic effect of chitosan nanoparticles containing the prodrug 5-ALA and its ester derivative 8-ALA. Besides, was investigated the effective production of PpIX. Concomitantly, the synergistic effect of Electrochemotherapy (ECT) with PDT was also studied for the purpose of increasing the permeation of drugs through the skin, enhancing the efficiency of the PDT in the treatment of skin cancers. Chitosan nanoparticles were obtained by the ionotropic gelation producing nanoparticles in nanosize (636.0 ± 6.5 nm), with a homogenous size distribution (0.37), and present a significantly positive zeta potential + 35.3 mV (± 2.21). Release profile and ability to incorporate 80% of the prodrug was also determined. The cytotoxicity studies demonstrated in the absence of light the biocompatible nature of chitosan nanoparticles developed, as well as the significant production of PpIX and it was possible to demonstrate the excellent effect of phototoxic formulation developed. The synergism studies of ECT with PDT have shown that the combination of chitosan nanoparticles containing prodrugs, applying microcurrents 400 ?A and light dose of 3 J/cm2 under these conditions showed a significant cell death.

Neste trabalho foi investigada a ação fotodinâmica dos pró-fármacos 5-ALA e 8-ALA incorporados em nanopartículas de quitosana, através da incidência de luz em células de melanoma, utilizando sinergicamente a Eletroquimioterapia e a Terapia Fotodinâmica. O ácido 5-aminolevulínico (5-ALA) e seus ésteres derivados são precursores metabólicos da protoporfirina IX (PpIX) e apresentam uma penetração limitada no estrato córneo. Portanto, o objetivo desse trabalho foi desenvolver, caracterizar e avaliar o efeito fotodinâmico das nanopartículas de quitosana contendo os pró-fármacos 5-ALA e seu éster derivado 8-ALA e investigar a produção efetiva de PpIX. Concomitantemente, o efeito sinérgico da Eletroquimioterapia (ECT) com a TFD também foi estudado com a finalidade de aumentar a permeação dos fármacos fotossensibilizantes através da pele, aumentando a eficiência da TFD no tratamento de neoplasias cutâneas. As nanopartículas de quitosana foram obtidas pelo método de gelificação ionotrópica, produzindo nanopartículas em tamanho nanométrico (636,0 nm ± 6,5), com uma distribuição de tamanho homogênea (0,37), além de apresentar um potencial zeta significantemente positivo + 35,3 mV (± 2,21). Seu perfil de liberação e capacidade de incorporação de 80% dos pró-fármacos também foi determinado. Os estudos de citotoxicidade na ausência de luz demonstraram o caráter biocompatível das nanopartículas de quitosana desenvolvidas, assim como a produção significativa de PpIX e foi possível comprovar o excelente efeito fototóxico da formulação desenvolvida. Os estudos de sinergismo da ECT com a TFD comprovaram que a combinação das nanopartículas de quitosana contendo os pró-fármacos, com aplicação de microcorrentes de 400 ?A e dose de luz de 3 J/cm2, nestas condições apresentou uma morte celular significativa. / This study investigated the photodynamic action of the prodrug 5-ALA and 8-ALA into chitosan nanoparticles, through the light incidence in melanoma cells, using synergistically the Electrochemotherapy and Photodynamic Therapy. The 5- aminolevulinic acid (5-ALA) and its ester derivatives are metabolic precursors of protoporphyrin IX (PpIX) and have a limited penetration in the stratum corneum. Therefore, the objective of this study was to develop, characterize and evaluate the photodynamic effect of chitosan nanoparticles containing the prodrug 5-ALA and its ester derivative 8-ALA. Besides, was investigated the effective production of PpIX. Concomitantly, the synergistic effect of Electrochemotherapy (ECT) with PDT was also studied for the purpose of increasing the permeation of drugs through the skin, enhancing the efficiency of the PDT in the treatment of skin cancers. Chitosan nanoparticles were obtained by the ionotropic gelation producing nanoparticles in nanosize (636.0 ± 6.5 nm), with a homogenous size distribution (0.37), and present a significantly positive zeta potential + 35.3 mV (± 2.21). Release profile and ability to incorporate 80% of the prodrug was also determined. The cytotoxicity studies demonstrated in the absence of light the biocompatible nature of chitosan nanoparticles developed, as well as the significant production of PpIX and it was possible to demonstrate the excellent effect of phototoxic formulation developed. The synergism studies of ECT with PDT have shown that the combination of chitosan nanoparticles containing prodrugs, applying microcurrents 400 ?A and light dose of 3 J/cm2 under these conditions showed a significant cell death.

Siekiant pagreitinti 5-aminolevulino rūgšties (5-ALA) skvarbą į (pro) odą ir sudaryti terapines fotodinaminiam gydymui tinkamas jos koncentracijas, įvertinta 5-ALA ir naujo provaisto skvarba į (pro) odą iš įvairių vietinio poveikio vaisto formų. Atlikta naujo provaisto 2-(dimetilamino)-etil-5-amino-4-oksopentanoato (DMAE-ALA) sintezė, ištirtos jo charakteristikos. Nustatytas susintetinto provaisto stabilumas įvairių pH verčių buferiniuose vandeniniuose tirpaluose ir odos esterazių poveikyje, įrodytas provaisto tinkamumas pernašai į (pro) odą. Atlikti 5-ALA ir du teigiamus krūvius vaisto formoje įgaunančio provaisto jontoforetinės ir pasyvios pernašos iš buferinių tirpalų ir celiuliozinės bei polivinilkarboksilinės kilmės polimerinių gelių į (pro) odą tyrimai in vitro. Patvirtintas reikšmingas skatinamas jontoforezės poveikis krūvinių molekulių pernašai į (pro) odą ir krūvio dydžio reikšmė elektros srovės skatinamos pernašos greičiui. Ištirta vaisto formų, pagamintų iš polivilinkarboksilinės kilmės ir celiuliozės polimerų, kokybė ir jų pasyvioji bei jontoforetinė pernaša. Įrodyta, kad provaisto įterpimui naudojant celiuliozės polimerų pagrindu pagamintus gelius, pasiekiama didžiausia 5-ALA pernaša. Nustatyta, kad du krūvius turintis 5-ALA provaistas į (pro) odą skverbėsi geriau negu vieną krūvį įgaunantys arba elektriškai neutralūs 5-ALA dariniai. Sudarytos rekomendacijos, skirtos pasiekti didžiausiai 5-ALA pernašai į (pro) odą. / Passive and iontophoretic delivery of 5-aminolevulinic acid and newly synthesized prodrug into (through) skin from various topical formulations was evaluated, while trying to accelerate the penetration of 5-ALA and its derivative into (through) skin and to generate the therapeutic concentration suitable for photodynamic therapy. A new prodrug 2-(dimethylamino)-ethyl-5-amino-4-oxopentanoate (DMAE-ALA) was synthesized, its characteristics have been evaluated. Stability of the prodrug in aqueous solutions of various pH values and at exposure with skin esterases (dermal and epidermal stability) has been evaluated. Suitability of the prodrug for topical delivery was confirmed. Studies for the evaluation of 5-ALA and DMAE-ALA passive and active delivery from cutaneous solutions, cellulose and polyvinyl carboxy polymer gels into (through) skin in vitro have been performed. Significant effect of iontophoresis on delivery of charged molecules into (through) skin as well as the significance of the charge of the molecule on the delivery rate has been confirmed. It has been shown that the insertion of the prodrug into cellulose-based polymer gels produces the highest amount of 5-ALA delivered into (through) skin. It was determined that the two-charged prodrug DMAE-ALA penetrated into (through) the skin more efficiently than single-charged or electrically neutral 5-ALA derivatives. Recommendations for achieving most efficient 5-ALA delivery into (through) the skin have been composed.

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.

Photodynamic therapy, PDT, is a method to diagnose and treat cancer. In PDT a sensitizer is administered to the patient and this sensitizer is accumulated in tumors. If the sensitizer-containing tumor is subjected to a laser of a specific wavelength the tumor is fluorescing allowing diagnostics. If other wavelengths are used a process involving reactive oxygen species and singlet oxygen is started and the tumor cells are killed. This process thus requires oxygen as well. This thesis investigates how UV-induced damage of the skin and different physiological factors of the skin influences the uptake of 5- aminolevulinic acid, ALA, and its conversion to the active sensitizer protoporphyrin IX, PpIX. It shows that UV-induced damage affects both the uptake and production of PpIX. UV-induced damage lowers the PpIX produced after ALA application both if the damage is acute and in chronically UV-affected skin. The PpIX production differs inter and intra individually. When looking how different physiological factors affect the PpIX production after topically applied ALA the thesis shows that an increase of temperature increases the production. No correlation between the formation of PpIX and the density of hair follicles was found and a weak correlation was seen comparing the epidermal and total dermal thickness and PpIX production The thesis also shows how PDT is used in treating laryngeal malignancies. It shows that it is possible to cure laryngeal tumors (both squamous cell carcinomas and sarcomas) using PDT primarily, and that the cure rate as well as outcome of voice and patient safety is comparable to the conventional treatment modalities. PDT can also be used as a function and organ sparing treatment for recurring laryngeal cancers, both squamous cell carcinomas and sarcomas.