Indocyanine green (ICG) is a fluorescent dye used as an indicator in medicine and surgery. The maximum absorption wavelength of ICG is at 785 nm, while the maximum emission is around 820 nm. ICG is nontoxic and is rapidly excreted into the bile. Near infrared (NIR) fluorescence imaging or spectroscopy offer new settings for seeing the blood vessels, and also in oncological applications for finding sentinel lymph nodes (SLN) to investigate if the cancer has spread from the tumor to the lymphatic system. Given the aforementioned applications, the aim of this thesis was to develop a hardware control and a user interface in LabVIEW, and to evaluate the software, as well as the instrumentation using phantom measurements.The system consisted of a spectrometer, a laser (785 ± 5 nm) for ICG excitation, optical filters, and a fiber optical probe containing five fibers for light excitation, and one for light collection. The basic LabVIEW program designed for the spectrometer was used, and additional features were added such as the recording functions, online measurements, opening of the recorded files, saving comments, and a loop was created for the laser control. Optical phantoms were prepared to model tissue for measurements using 20 % intralipid that gave μs = 298 mm−¹ at the excitation wavelength. Agar 1% w/v and ICG were added to the phantoms using different fluorophore concentrations of 2 μg/mL, 10 μg/mL, 20 μg/mL, 25 μg/mL, and 40 μg/mL. The objective was to perform controlled measurements of steady state ICG fluorescence, the dynamics of photobleaching at different concentrations, and to find the optimal ICG concentration for obtaining the maximum fluorescence intensity. The light to excite ICG fluorescence emission was provided by using a laser output power of 10.4 mW and 200 ms of integration time in the spectrometer for optimal measurements.Measurements using the different gel phantoms showed maximum fluorescence ICG concentration to be between 16 μg/mL and 20 μg/mL. Moreover, photobleaching measurements showed to be ICG concentration-dependent, where those concentrations higher than the optimal one incrementally photobleached with time after being exposed to light. Higher concentrations presented an incremental photobleaching where they first reached a maximum peak and then the intensity decayed with time. Additionally, laser reflection at 782 nm showed that the reflection increased with time ranging from 130% – 460% as the ICG photobleached to 50% of its initial value. Normalization of ICG by the laser reflection signal was investigated to compensate for the intensity variations due to the measurement parameters including the distance from the light source to the target, and the angle of inclination of the probe. The lowest ICG concentration detectable by the system was 0.05 μg/mL.In conclusion, a LabVIEW hardware control and user interface was developed for controlling the spectrometer and the laser. Several measurements were made using the different phantoms, where the optimal concentration of ICG was estimated. It was shown that ICG fluorescence intensity and photobleaching behavior were dependent on the concentration. The results gave suggestions for future experimental design. / NIRF

Cancer mortality is progression-dependent thus its treatment relies on effective therapy and monitoring of responses. Nanoparticles have long been used to improve the therapeutic index of drugs by facilitating their transit to the target site at higher concentrations than free drugs, whilst protecting healthy tissues from an often potent and cytotoxic payload. Through the EPR (enhanced permeability and retention) effect, injected, PEGylated nanoparticles preferentially accumulate in tumour tissue deeming them eminently suitable for cancer intervention for delivery of both therapeutic and contrast agents The development of theranostic liposomal systems comprising both imaging and therapeutic capabilities exploits the facets of liposomes, and forms an elegant strategy to address major problems which hinder effective cancer therapy. Liposomes can be tailored to be thermosensitive in a low hyperthermic range of ~42°C, above physiological temperature but below that which can induce tissue damage. This allows the use of heating as an external triggering modality to induce targeted drug release. Throughout the course of this work, the photoacoustic contrast agent ICG was successfully incorporated into PEGylated doxorubicin-encapsulating liposomes, marrying two FDA approved entities. The project commenced with the development of the basic liposomal-DOX. Differing lipid compositions of varying fluidities were tested against those which have been previously established. These compositions carried a range of phase transition temperatures, above which the liposomes release the encapsulated DOX. This study concluded with the generation of a library of liposomes with differing release kinetics at 42°C in simulated physiological conditions. The second section of the project investigated the methodology behind the incorporation of ICG into the liposomal bilayers. The lipid composition used for the study was based on the DOXIL® formulation, due to its robust structure and establishment in the field of cancer therapy. The protocols used varied on the basis of chronology in regards to the liposome preparation protocol. The film insertion method incorporated the ICG in initial lipid film generation. The freeze fracture protocol introduced the ICG during lipid film hydration. The post insertion protocol introduced ICG in the final stages of DOX loading. The downsizing protocol was also varied between extrusion and sonication. Through varying of the protocols and downsizing methodology, it was possible to incorporate differing ICG concentrations and attain differing levels of structural stability. The most successful liposome was then tested for its imaging potential in vivo through a photoacoustic imaging modality namely multispectral optoacoustic tomography. This validated accumulation of the liposomes at the tumour site along with co-localisation of both drug and dye. The project culminated in the combination of the two studies, producing a thermosensitive theranostic ICG labelled liposomal doxorubicin system. The system showed improved blood stability than the current clinical systems, and demonstrated imaging potential through IVIS based fluorescence imaging. Through exploitation of the photothermal effects of ICG within a thermosensitive lipid vesicle, it was also possible to induce drug release through irradiation with a non-thermal near-infrared laser. This shows promise for future therapy, allowing simultaneous imaging, optimum release induction and monitoring response to therapy, in a cheap, effective and time-efficient manner.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21625号 / 医博第4431号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂井 義治, 教授 黒田 知宏, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

- An extensive summary in French is available in Appendix E on page 189 -

Photodynamic therapy (PDT), which utilizes the combination of light and a photosensitizing drug to cause tissue damages, has emerged as a novel clinical approach for the treatment of numerous cancers, as well as some other non-malignant conditions. Although a few photosensitizers have been approved for clinical uses, the mechanism of drug action, especially the initial photochemical reactions that lead to the formation of the reactive oxygen species (ROS), is still not well understood. Moreover, the PDT efficiency of currently used drugs is limited due to the strong attenuation of light by tissues in the wavelength range of 630-690 nm, where these drugs are photo-activated. Photosensitizers which are sensitive to near infrared (NIR) light are believed to be able to overcome this limitation. In this thesis work, the molecular mechanism of action of indocyanine green (ICG), a potential NIR PDT drug, was investigated using our femtosecond time-resolved laser spectroscopy. Femtosecond time-resolved fluorescence decay profiles of ICG in water were obtained using the fluorescence up-conversion technique. The lifetime of ICG excited singlet state was determined to be about 150 ps, directly from the fluorescence decay kinetic traces. The excited triplet-state yield of ICG in water was found to be extremely low, according to the result of the ground-state bleaching recovery measurement. This observation is contrary to the conventional understanding that the ROS would be generated mainly from the excited triplet state of the photosensitizer and, therefore, suggests the existence of a new reaction pathway. Pump-probe transient absorption spectroscopy was applied to study the reaction between ICG and oxygen in more details. The results reveal that the formation of ICG and oxygen ground-state complexes ([ICG]_m:[O₂]_n) is a key step in the generation of the ROS. Electron transfer from the excited singlet state of ICG to oxygen has been proposed to be a possible pathway for the generation of ROS.

Photodynamic therapy (PDT), which utilizes the combination of light and a photosensitizing drug to cause tissue damages, has emerged as a novel clinical approach for the treatment of numerous cancers, as well as some other non-malignant conditions. Although a few photosensitizers have been approved for clinical uses, the mechanism of drug action, especially the initial photochemical reactions that lead to the formation of the reactive oxygen species (ROS), is still not well understood. Moreover, the PDT efficiency of currently used drugs is limited due to the strong attenuation of light by tissues in the wavelength range of 630-690 nm, where these drugs are photo-activated. Photosensitizers which are sensitive to near infrared (NIR) light are believed to be able to overcome this limitation. In this thesis work, the molecular mechanism of action of indocyanine green (ICG), a potential NIR PDT drug, was investigated using our femtosecond time-resolved laser spectroscopy. Femtosecond time-resolved fluorescence decay profiles of ICG in water were obtained using the fluorescence up-conversion technique. The lifetime of ICG excited singlet state was determined to be about 150 ps, directly from the fluorescence decay kinetic traces. The excited triplet-state yield of ICG in water was found to be extremely low, according to the result of the ground-state bleaching recovery measurement. This observation is contrary to the conventional understanding that the ROS would be generated mainly from the excited triplet state of the photosensitizer and, therefore, suggests the existence of a new reaction pathway. Pump-probe transient absorption spectroscopy was applied to study the reaction between ICG and oxygen in more details. The results reveal that the formation of ICG and oxygen ground-state complexes ([ICG]_m:[O₂]_n) is a key step in the generation of the ROS. Electron transfer from the excited singlet state of ICG to oxygen has been proposed to be a possible pathway for the generation of ROS.

Submitted by Ligia Souza (ligia@ufscar.br) on 2017-09-21T14:27:46Z No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-09-25T12:32:38Z (GMT) No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-09-25T12:32:44Z (GMT) No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) / Made available in DSpace on 2017-09-25T12:37:57Z (GMT). No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) Previous issue date: 2017-07-31 / Outra / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Infectious pneumonia is a major cause of morbidity/mortality, mainly due to the increasing rate of microorganisms resistant to antibiotics. Photodynamic Inactivation (PDI) is emerging as a promising approach, as effects are based on oxidative stress, preventing the emergence of resistant microorganism strains. In previous studies, the in vitro inactivation of Streptococcus pneumoniae using indocyanine green (ICG) and infrared light source was successful, and achieved reduction of 5 log10 colony-forming units (CFU/mL) with concentration as low as 10 μM ICG. In the present study, a proof-of-principle protocol was designed to treat lung infections by PDI using extracorporeal illumination with a 780 nm laser device and ICG as photosensitizer. In a first row of experiments, hairless mice were infected with S. pneumoniae and PDI was performed two days after infection. For control groups, CFU recovery ranged between 103-104 CFU/mL/mouse. For PDT group, however, no bacteria were recovered in 80% of the animals. Animal survival was evaluated separately over 50 days. No deaths occurred in PDT group, whereas 60% of the control did not survive. Lung injury analyses were performed in BALB/c mice, the bacteria reduction were 2 and 4 log10 in 2 mice (5 in total) and the wet-to-dry ratio showed that PDI did not increase the edema in lungs. The bronchoalveolar lavage data indicated a larger absolute number of cells (mononuclear and polymorphonuclear) in the PDI group in contrast to control group, meaning that the technique could increase the immune system response. In vitro results showed the irradiated ICG could generate aggregates or photoproducts that help PDI to inactivate the bacteria. Our results indicate that extracorporeal PDI has potential for pneumonia treatment, and pulmonary decontamination with PDI may be used as a single therapy or as an antibiotics adjuvant. / A pneumonia infecciosa é uma das principais causas de morbidade/mortalidade no mundo, principalmente devido à taxa crescente de micro-organismos resistentes a antibióticos. A inativação fotodinâmica (IFD) está emergindo como uma abordagem promissora, cujos efeitos são baseados no estresse oxidativo, impedindo o surgimento de cepas de micro-organismos resistentes. Em estudos anteriores, a inativação in vitro de Streptococcus pneumoniae utilizando indocianina verde (ICV) e fonte de luz de infravermelho foi efetiva, inativando 5 log10 (UFC/mL) com apenas 10 μM ICV. Neste estudo, foi avaliado protocolo de prova de princípio para tratar infecções pulmonares por IFD usando irradiação extracorpórea com dispositivo a laser emitindo em 780 nm e ICV como fotossensibilizador (FS). Em uma primeira série de experimentos, camundongos hairless SKH-1 foram infectados com S. pneumoniae e a IFD foi realizada dois dias após a infecção. Para os grupos de controles, a recuperação de UFC variou entre 103-104 UFC/mL/animal. Para o grupo IFD, no entanto, nenhuma bactéria foi recuperada em 80% dos animais. A sobrevivência animal foi avaliada durante 50 dias. Não ocorreram mortes no grupo IFD, enquanto 60% do grupo controle foi a óbito. Foram realizadas análises de lesões pulmonares em camundongos BALB/c, onde a redução bacteriana foi de 2 e 4 log10 em dois animais (total 5) em comparação com o grupo controle, e a proporção de peso úmido e seco mostrou que a IFD não aumentou o edema nos pulmões. Os dados de lavagem broncoalveolar indicaram um aumento no número absoluto de células (mononucleares e polimorfonucleares) no grupo IFD, o que indica que a técnica pode aumentar a resposta do sistema imunológico. Os resultados in vitro mostraram que a ICV irradiada pode gerar agregados ou fotoprodutos que auxiliam a IFD a inativar a bactéria. Os resultados deste estudo indicam que a IFD com irradiação extracorpórea tem potencial para o tratamento de pneumonia, e a descontaminação pulmonar com IFD pode ser usada como terapia ou como adjuvante aos antibióticos. / CAPES: 99999.003154/2015-07

A capsulotomia curvilínea contínua (CCC) é uma das etapas mais importantes da técnica de facoemulsificação. Em cataratas brancas e com reflexo de fundo de olho deficiente ou ausente, a identificação da cápsula anterior do cristalino é dificultada, e é necessária a utilização de substâncias, como os corantes vitais, para permitir a sua diferenciação. Contudo, antes da utilização de substâncias intraoculares, é necessário determinar se elas são seguras para as estruturas do globo ocular, principalmente para as células do endotélio corneano, que podem sofrer lesões irreversíveis. A indocianina verde é uma substância capaz de corar a cápsula do cristalino, tendo seu uso relatado em humanos, mas não existem dados sobre sua utilização em equinos. Objetivou-se determinar o efeito agudo da exposição do endotélio corneano de equinos à indocianina verde 0,5%. Foram estudadas 24 córneas provenientes de 12 equinos divididos em 2 grupos: 12 córneas dos bulbos oculares direitos (grupo controle) e 12 córneas dos bulbos oculares esquerdos (grupo tratamento). As córneas do grupo tratamento foram expostas à indocianina verde durante 1 minuto, e após lavadas com solução salina balanceada. Posteriormente, as córneas foram coradas pela técnica de coloração vital com alizarina vermelha e azul de tripano, visualizadas ao microscópio óptico e fotografadas. As córneas do grupo controle também foram coradas com alizarina vermelha e azul de tripano, visualizadas e fotografadas. Não foram encontradas áreas de perda celular com a coloração pela alizarina vermelha, e nem células com núcleo corado pelo azul de tripano, não havendo diferenças entre grupo controle e grupo tratamento. Baseado nos resultados do presente estudo foi possível concluir que a indocianina verde não induziu dano às células do endotélio da córnea de equinos. / The continuous curvilinear capsulotomy (CCC) is one of the most important steps on the phacoemulsification technique. In white cataract combined with poor or absent red reflex the identification of the anterior capsule is hampered, thus requiring the use of substances such as vital dyes to allow their differentiation. However, before the use of intraocular substances it is necessary to determine whether these substances are safe to the structure of the ocular globe, mainly to the corneal endothelium cells, which may suffer irreversible damage. The indocyanine green is a substance capable of staining the lens capsule with documented use in humans, but there are no data on its use in horses. This study aimed to determine the acute effect of exposure of equine corneal endothelium to indocyanine green 0.5%. The sample consisted of 24 corneas from 12 horses divided into 2 groups: 12 corneas of right eye bulbs (control group) and 12 corneas of left eye bulbs (treatment group). The corneas belonging to the treatment group were exposed to the indocyanine green for 1 minute, and then washed with balanced saline solution. Subsequently, the corneas were stained by the technique of vital staining with alizarin red and trypan blue, and so visualized and photographed under an optical microscope. The corneas of the control group were also stained with alizarin red and trypan blue, visualized and photographed. No areas of cell loss were found with alizarin red staining, and no cell nuclei stained with trypan blue was visualized, with no difference between control group and treatment group. Based on the results of this study, we concluded that the indocyanine green did not induce damage to equine corneal endothelium.

A capsulotomia curvilínea contínua (CCC) é uma das etapas mais importantes da técnica de facoemulsificação. Em cataratas brancas e com reflexo de fundo de olho deficiente ou ausente, a identificação da cápsula anterior do cristalino é dificultada, e é necessária a utilização de substâncias, como os corantes vitais, para permitir a sua diferenciação. Contudo, antes da utilização de substâncias intraoculares, é necessário determinar se elas são seguras para as estruturas do globo ocular, principalmente para as células do endotélio corneano, que podem sofrer lesões irreversíveis. A indocianina verde é uma substância capaz de corar a cápsula do cristalino, tendo seu uso relatado em humanos, mas não existem dados sobre sua utilização em equinos. Objetivou-se determinar o efeito agudo da exposição do endotélio corneano de equinos à indocianina verde 0,5%. Foram estudadas 24 córneas provenientes de 12 equinos divididos em 2 grupos: 12 córneas dos bulbos oculares direitos (grupo controle) e 12 córneas dos bulbos oculares esquerdos (grupo tratamento). As córneas do grupo tratamento foram expostas à indocianina verde durante 1 minuto, e após lavadas com solução salina balanceada. Posteriormente, as córneas foram coradas pela técnica de coloração vital com alizarina vermelha e azul de tripano, visualizadas ao microscópio óptico e fotografadas. As córneas do grupo controle também foram coradas com alizarina vermelha e azul de tripano, visualizadas e fotografadas. Não foram encontradas áreas de perda celular com a coloração pela alizarina vermelha, e nem células com núcleo corado pelo azul de tripano, não havendo diferenças entre grupo controle e grupo tratamento. Baseado nos resultados do presente estudo foi possível concluir que a indocianina verde não induziu dano às células do endotélio da córnea de equinos. / The continuous curvilinear capsulotomy (CCC) is one of the most important steps on the phacoemulsification technique. In white cataract combined with poor or absent red reflex the identification of the anterior capsule is hampered, thus requiring the use of substances such as vital dyes to allow their differentiation. However, before the use of intraocular substances it is necessary to determine whether these substances are safe to the structure of the ocular globe, mainly to the corneal endothelium cells, which may suffer irreversible damage. The indocyanine green is a substance capable of staining the lens capsule with documented use in humans, but there are no data on its use in horses. This study aimed to determine the acute effect of exposure of equine corneal endothelium to indocyanine green 0.5%. The sample consisted of 24 corneas from 12 horses divided into 2 groups: 12 corneas of right eye bulbs (control group) and 12 corneas of left eye bulbs (treatment group). The corneas belonging to the treatment group were exposed to the indocyanine green for 1 minute, and then washed with balanced saline solution. Subsequently, the corneas were stained by the technique of vital staining with alizarin red and trypan blue, and so visualized and photographed under an optical microscope. The corneas of the control group were also stained with alizarin red and trypan blue, visualized and photographed. No areas of cell loss were found with alizarin red staining, and no cell nuclei stained with trypan blue was visualized, with no difference between control group and treatment group. Based on the results of this study, we concluded that the indocyanine green did not induce damage to equine corneal endothelium.

A capsulotomia curvilínea contínua (CCC) é uma das etapas mais importantes da técnica de facoemulsificação. Em cataratas brancas e com reflexo de fundo de olho deficiente ou ausente, a identificação da cápsula anterior do cristalino é dificultada, e é necessária a utilização de substâncias, como os corantes vitais, para permitir a sua diferenciação. Contudo, antes da utilização de substâncias intraoculares, é necessário determinar se elas são seguras para as estruturas do globo ocular, principalmente para as células do endotélio corneano, que podem sofrer lesões irreversíveis. A indocianina verde é uma substância capaz de corar a cápsula do cristalino, tendo seu uso relatado em humanos, mas não existem dados sobre sua utilização em equinos. Objetivou-se determinar o efeito agudo da exposição do endotélio corneano de equinos à indocianina verde 0,5%. Foram estudadas 24 córneas provenientes de 12 equinos divididos em 2 grupos: 12 córneas dos bulbos oculares direitos (grupo controle) e 12 córneas dos bulbos oculares esquerdos (grupo tratamento). As córneas do grupo tratamento foram expostas à indocianina verde durante 1 minuto, e após lavadas com solução salina balanceada. Posteriormente, as córneas foram coradas pela técnica de coloração vital com alizarina vermelha e azul de tripano, visualizadas ao microscópio óptico e fotografadas. As córneas do grupo controle também foram coradas com alizarina vermelha e azul de tripano, visualizadas e fotografadas. Não foram encontradas áreas de perda celular com a coloração pela alizarina vermelha, e nem células com núcleo corado pelo azul de tripano, não havendo diferenças entre grupo controle e grupo tratamento. Baseado nos resultados do presente estudo foi possível concluir que a indocianina verde não induziu dano às células do endotélio da córnea de equinos. / The continuous curvilinear capsulotomy (CCC) is one of the most important steps on the phacoemulsification technique. In white cataract combined with poor or absent red reflex the identification of the anterior capsule is hampered, thus requiring the use of substances such as vital dyes to allow their differentiation. However, before the use of intraocular substances it is necessary to determine whether these substances are safe to the structure of the ocular globe, mainly to the corneal endothelium cells, which may suffer irreversible damage. The indocyanine green is a substance capable of staining the lens capsule with documented use in humans, but there are no data on its use in horses. This study aimed to determine the acute effect of exposure of equine corneal endothelium to indocyanine green 0.5%. The sample consisted of 24 corneas from 12 horses divided into 2 groups: 12 corneas of right eye bulbs (control group) and 12 corneas of left eye bulbs (treatment group). The corneas belonging to the treatment group were exposed to the indocyanine green for 1 minute, and then washed with balanced saline solution. Subsequently, the corneas were stained by the technique of vital staining with alizarin red and trypan blue, and so visualized and photographed under an optical microscope. The corneas of the control group were also stained with alizarin red and trypan blue, visualized and photographed. No areas of cell loss were found with alizarin red staining, and no cell nuclei stained with trypan blue was visualized, with no difference between control group and treatment group. Based on the results of this study, we concluded that the indocyanine green did not induce damage to equine corneal endothelium.