Nanopartículas de ouro têm mostrado enorme potencial de aplicação em modalidades diagnósticas e terapêuticas fotoativadas. Em especial, nanoestruturas de ouro anisotrópicas ramificadas apresentam excelente desempenho atuando tanto como contrastes de imagens fotoacústicas, quanto como agentes ativos para terapias fototérmicas de câncer. Apesar dos avanços nas suas rotas de síntese, o desenvolvimento dessas nanoestruturas de forma simples e reprodutível ainda é desafiador. O presente trabalho visou o desenvolvimento de nanopartículas de ouro anisotrópicas ramificadas, ou nanoflores, que sejam fotoativas no infravermelho-próximo para a terapia e diagnóstico de câncer. Em particular, buscou-se o desenvolvimento de uma síntese simples para sua obtenção, assim como a verificação de sua atuação como agente de contraste fotoacústico e como agente ativo para hipertermia de tumores. Para tanto, desenvolveu-se uma síntese in situ que permitiu a obtenção de nanoflores monodispersas com tamanho e propriedades ópticas controláveis. Através da variação de aspectos da síntese, como a temperatura e a concentração de ouro, foi possível sintonizar a atividade óptica das partículas entre 590 e 960 nm. Sua formação foi confirmada por microscopia eletrônica de varredura, espalhamento de luz dinâmico e espectroscopia UV-visível. As partículas apresentaram boa estabilidade de suas características físico-químicas por dois meses e meio. Ainda, as nanoflores se mostraram estáveis, também, quando suspensas em meio de cultura, sob irradiação de lasers, e quando mantidas a temperatura corpórea por longos intervalos. Sua resposta fotoacústica foi caracterizada, apresentando sinais significativos e permitindo a obtenção de imagens claras de sua localização, mesmo em baixas concentrações. Testes realizados em cultura de células mostraram que as nanoflores foram eficazes na hipertermia de uma linhagem de hepatocarcinoma de rato (HTC), ao mesmo tempo que não apresentaram sinais de toxicidade a uma linhagem de fibroblastos de camundongos (FC3H). Esses resultados revelam uma possibilidade simples de fabricação de nanoestruturas de ouro anisotrópicas ramificadas, que podem servir como uma plataforma promissora para o diagnóstico e terapia do câncer. / Gold nanoparticles have shown enormous potential of application in photodiagnostic and in phototherapeutic procedures. Notably, branched anisotropic gold nanostructures present distinguished performance acting as contrast agents of photoacoustic images and as active agents for photothermal therapies for cancer. Despite advances in their synthesis routes, the growth of these nanostructures in a simple and reproducible way is still challenging. The present study was aimed at developing branched anisotropic gold nanoparticles, coined nanoflowers, that are photoactive in the near-infrared for therapy and diagnosis of cancer. In particular, we sought to develop a simple synthesis route, as well as to verify its application for both, as photoacoustic contrast agents and as active agents for tumor hyperthermia. An in situ synthesis was developed which allowed the development of monodisperse nanoflowers with controllable size and optical properties. Through variations of certain aspects of this procedure, such as temperature and gold ions concentration, it was possible to tune the optical activity of the particles between 590 and 960 nm. The nanostructure morphology was confirmed by scanning electron microscopy, dynamic light scattering and UV-visible spectroscopy. The particles exhibited consistent physicochemical characteristics and good stability for two and a half months. Furthermore, the nanoflowers were also stable when suspended in cell culture medium, under laser irradiation and when maintained at body temperature for long intervals. Its photoacoustic response was characterized, presenting significant responses and generating clear images of its location, even at low concentrations. In vitro tests revealed that these nanoflowers were effective therapeutic agents for photothermal therapy of a rat hepatocarcinoma (HTC) lineage, while showing no signs of toxicity to mouse fibroblast (FC3H) cell line. These results reveal a simple procedure of synthesizing branched anisotropic gold nanostructures, which can serve as a promising platform for cancer diagnosis and therapy.

Ce travail de thèse s'inscrit dans le domaine de l'imagerie photoacoustique en tant que modalité d'imagerie prometteuse pour la médecine. Il a consisté en l'expérimentation du guidage photoacoustique de la thérapie par ultrasons focalisés de haute intensité (HIFU), et en l'étude de la génération par des agents de contraste spécifiques que sont les nanoparticules d'or. Dans un premier temps lors d'expériences in vitro, une sonde conçue à la fois pour l'imagerie photoacoustique et la thérapie ultrasonore a été utilisée pour démontrer la faisabilité du guidage photoacoustique pour traiter par HIFU une zone tissulaire cible. L'usage de la photoacoustique pour le contrôle des HIFU révèle ainsi son caractère prometteur. Dans un second temps la modélisation physique de la génération photoacoustique par des nanoparticules d'or permet de quantifier les mécanismes non-linéaires thermoélastiques impactant le signal émis. Dans le cas d'une nanosphère d'or unique, nous décrivons en fonction des paramètres du problème l'importance de la contribution de ces phénomènes non-linéaires, qui sont causés par la dépendance en température de la dilatation thermique de l'eau. Nous en tirons un ensemble de prédictions quantitatives quant à l'influence et le poids de ces non-linéarités sur le signal photoacoustique. Puis nous généralisons ces résultats théoriques à une collection de nanosphères d'or, et les confrontons à nos résultats expérimentaux. Nous mettons ainsi en évidence une nouvelle forme prometteuse de contraste en imagerie photoacoustique: le contraste de non-linéarité thermoélastique. / This work falls within the field of photoacoustic imaging as a promising modality for biomedical applications. It consists in experimenting photoacoustic guidance of high intensity focused ultrasound (HIFU) for therapy, and in studying photoacoustic generation by gold nanoparticles as contrast agents. First of all, a probe designed for both photoacoustic imaging and ultrasound therapy was employed for \textit{in vitro} experiments. We demonstrate the feasability of photoacoustic guidance to treat a target embedded in biological tissue, as a promising tool for HIFU control. In a second part we model the physical mechanisms of photoacoustic generation by gold nanoparticles. This allows to quantify thermoelastic nonlinearities impacting the emitted signal, which origins derive from the temperature dependence of the thermal expansion coefficient of water. In the case of a single gold nanosphere, we describe the nonlinear contribution to the signal according to the different parameters of the problem. We infer a set of quantitative predictions concerning the weight of nonlinearities on photoacoustic signals. Then we generalize these theoretical results to a collection of gold nanospheres, and confront them to our experimental results. Thermoelastic nonlinearity thereby offers a promising new type of contrast for photoacoustic imaging.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21713号 / 薬科博第104号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 小野 正博, 教授 山下 富義, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23127号 / 人健博第89号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 杉本 直三, 教授 黒木 裕士, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

<p><a>Molecular imaging revolutionized the way researchers and clinicians visualize and investigate complex biochemical phenomena, and it is beneficial </a><a>for disease diagnosis, drug design and therapy assessment</a>. Among a variety of different imaging techniques, the non-ionizing and non-invasive photoacoustic (PA) imaging is attracting increased attentions, owing to its high spatial and temporal resolutions with reasonable penetration depth in tissue. Parallel efforts have been the preparation of PA imaging agents which has high PA efficacy and can specifically label the targets at cellular or molecular level. Particularly, there is exponentially growing interest in imaging in the second near-infrared (NIR-II) window (1000–1700 nm), where offers reduced tissue background and improved penetration depth. However, study of PA imaging in the NIR-II window is incomplete, partly due to the lack of suitable materials. Therefore, in my dissertation work I studied NIR-II PA imaging through semiconducting polymer. </p> <p>Firstly, the performance of PA imaging in the NIR-II window is explored by using a semiconducting polymer nanoparticle (SPN) which has strong absorption in the NIR-II window. Compared with lipid, blood and water, such SPN shows outstanding PA contrast in the NIR-II window <i>in situ</i> and <i>in vivo</i>, and an imaging depth of more than 5 cm at 1064 nm excitation is achieved in chicken-breast tissue. These results suggest that SPN as a PA contrast in the NIR-II window opens new opportunities for biomedical imaging with improved imaging contrast and centimeter-deep imaging depth.</p> <p>Next, targeted PA imaging of prostate cancer is achieved by functionalizing a NIR-II absorbing SPN with prostate-specific membrane antigen (PSMA)-targeted ligands. Insights into the interaction of the imaging probes with the biological targets are obtained from single-cell to whole-organ by transient absorption (TA) microscopy and PA imaging. TA microscopy reveals the targeting efficiency, kinetics, and specificity of the functionalized SPN to PSMA-positive prostate cancer at cellular level. Meanwhile, the functionalized SPN demonstrates selective accumulation and retention in the PSMA-positive tumor after intravenous administration <i>in vivo</i>. Taken together, it is demonstrated that BTII-DUPA SPN is a promising targeted probe for prostate cancer diagnosis by PA imaging. </p> <p>Lastly, PA imaging in the NIR-II window is also achieved water-soluble semiconducting polymer, which is prepared by oxygen-doping. After doping, it shows broadband absorption in the entire NIR-II window, with great chemical stability, photostability and biocompatibility. Owing to its merit of broadband absorption, the imaging depth comparison among different NIR-II wavelengths is also achieved. Moreover, this doped semiconducting polymer is readily soluble in normal physiological pH by virtue of carboxyl groups on side chains and tends to aggregate at an acidic environment which results in a 7.6-fold PA enhancement at pH 5.0. Importantly, a 3.4±1.0-fold greater signal in tumor tissue than that in muscle is revealed <i>in vivo</i>. This study provides a more attainable yet effective platform to the field for achieving water-soluble NIR-II absorbing contrast agents for activatable PA imaging. </p>

[pt] A maioria dos métodos de avaliação de ondas vibratórias é realizada através do contato entre o sensor e o objeto a ser analisado. Esse requisito de contato impõe algumas limitações de quais objetos podem ser avaliados, como por exemplo, o tamanho do mesmo. Uma técnica que expandiu a capacidade de medição se comparado aos sensores tradicionais de vibração é a vibrometria laser doppler. Ela possibilita medições não intrusivas com alta resolução espacial e tempo de teste reduzido. Dentre as várias aplicações que se beneficiam das aquisições com o vibrômetro laser doppler, o imageamento fotoacústico se encontra no centro do objetivo deste trabalho. O imageamento fotoacústico é uma tecnologia emergente que supera o alto espalhamento óptico em amostras biológicas utilizando o efeito fotoacústico. Ele combina aspectos de imageamento óptico e ultrassom e, consequentemente, algumas de suas vantagens: imagear certos componentes biológicos em profundidades de alguns centímetros e com alto contraste. A técnica mais direta de obter imagens em um sistema fotoacústico é a excitação pontual da amostra e a detecção por varredura. O foco deste trabalho é analisar e implementar uma técnica óptica para detecção das vibrações ultrassonoras em um sistema fotoacústico. Foram avaliados diferentes sistemas interferométricos experimentais, tanto por mapeamento pontual quanto por campo pleno. O princípio da interferometria e a teoria das medições quantitativas são apresentados para explicar as técnicas experimentais utilizadas para aquisição das ondas vibratórias. Igualmente, a técnica de processamento do sinal vibratório é discutida. Dentre as técnicas testadas experimentalmente, duas foram implementadas, analisadas e discutidas em detalhe e seus resultados foram apresentados e comparados entre si. A primeira técnica discutida é o Laser Optical Feedback Imaging (LOFI), um interferômetro heteródino que utiliza a dinâmica do laser e a sua grande sensibilidade ao fenômeno de reinjeção óptica para realizar medições de alta precisão do sinal retroespalhado da amostra. Essa técnica realiza suas aquisições ponto a ponto, através do do escaneamento do feixe do laser na superfície da amostra. Esse modo de aquisição possui um tempo de teste e processamento elevado, mas com alta resolução espacial e com a possibilidade de medir a região transitória. A segunda técnica utilizada para a aquisição de medidas de vibrações ultrassonoras é o estroboscópio Mach-Zehnder, um interferômetro de campo pleno que possibilita a aquisição do padrão de speckle de toda a região de interesse da amostra com baixo tempo de aquisição e processamento. Dessa forma, essa técnica tem um grande potencial para aplicações em tempo real. Os resultados experimentais de ambos os sistemas são apresentados e discutidos em detalhe. Um transdutor piezoelétrico é utilizado para as medições com os dois sistemas, com as mesmas características de teste. Adicionalmente, são apresentados os resultados com a técnica LOFI quando o ganho não-linear é compensado. Esse tipo de teste não é possível no sistema estroboscópico Mach-Zehnder campo pleno, considerando que apenas o LOFI é capaz de realizar medidas transitórias. Para concluir o trabalho, são discutidos e comparados os principais parâmetros para as técnicas testadas: ruído, potência do laser, resolução espacial, resolução temporal, e tempo de aquisição. / [en] Photoacoustic imaging combines aspects of optical and ultrasound imaging and, consequently, combines some of their particular advantages: imaging of specific tissue components in a depth up to several centimeters becomes possible with a high image contrast. The most straightforward strategy to produce photoacoustic images is point-wise excitation and detection in a scanning mode. The main focus of this work is to analyze and implement optical techniques to detect ultrasound vibrations in a photoacoustic system. We evaluate different interferometric systems, both point scanning and full field. Two techniques that were implemented and further discussed are the Laser Optical Feedback Imaging (LOFI), which is a sensitive, point scanning, heterodyne interferometer that uses the dynamics of the laser, and a full field, stroboscopic Mach-Zehnder to acquire ultrasound vibration measurements.We discuss the results from each system and its advantages with the intended application.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult type of cancer to treat, due to late diagnosis which is a result of vague symptoms and lack of biomarkers, as well as refractory behavior toward current treatment protocols. Imaging of the disease progression therefore plays a crucial role in identifying potentially curable PDAC patients at an early stage. Nanoparticle-based contrast agents have shown multimodal capabilities and potential to enhance the contrast of previously undetectable pathological changes, including PDAC. In this master’s thesis study, an iron oxide nanoparticle (IONP) was evaluated as a potential multimodal contrast agent for both photoacoustic imaging (PAI) and magnetic resonance imaging (MRI). The investigated particle was composed of Fe3O4 with a hydrodynamic size of 418.5 nm and a zeta potential of -27.7 mV. In the agarose suspended IONP phantom studies, the IONP demonstrated a two-fold higher T2 contrast compared to commercial IONP VivoTrax (Magnetic Insight), as well as generating strong and stable photoacoustic signal throughout the first near-infrared window (700 to 1000 nm). Based on this thesis’ proof of concept study, Fe3O4 IONP showed good potential as multimodal contrast agent for MRI and PAI. Future work consists of modification of the particle composition and in vivo imaging on animals to evaluate the application in PDAC diagnostics.

Although the drug-eluting stent (DES) has dramatically reduced the rate of coronary restenosis, it still occurs in up to 20% of patients with a DES. Monitoring drug delivery could be one way to decrease restenosis rates. We demonstrate real-time photoacoustic imaging and spectroscopy (PAIS) using a wavelength-tunable visible laser and clinical ultrasound scanner to track cardiac drug delivery. The photoacoustic signal was initially calibrated using porcine myocardial samples soaked with a known concentration of a drug surrogate (Dil). Next, an in situ coronary artery was perfused with DiI for 20 min and imaged to monitor dye transport in the tissue. Finally, a partially DiI-coated stent was inserted into the porcine brachiocephalic trunk for imaging. The photoacoustic signal was proportional to the DiI concentration between 2.4 and 120 mu g/ml, and the dye was detected over 1.5 mm from the targeted coronary vessel. Photoacoustic imaging was also able to differentiate the DiI-coated portion of the stent from the uncoated region. These results suggest that PAIS can track drug delivery to cardiac tissue and detect drugs loaded onto a stent with sub-mm precision. Future work using PAIS may help improve DES design and reduce the probability of restenosis. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE)

En microscopie, savoir focaliser la lumière à l’échelle micrométrique est déterminant. Dans les tissus biologiques néanmoins, les inhomogénéités du milieu diffusent la lumière, empêchant toute focalisation au-delà d’une profondeur de l’ordre du millimètre. Des techniques de façonnage de front d’onde ont été développées afin de pré-compenser la distorsion du faisceau lumineux induite par la propagation à travers un milieu diffusant. Pour parvenir à focaliser la lumière à l’intérieur même du milieu diffusant, l’enjeu est de mesurer l’intensité lumineuse en profondeur de manière non invasive. Nous proposons d’utiliser l’effet photoacoustique pour sonder cette intensité. Une structure optiquement absorbante éclairée par une impulsion lumineuse émet en effet un signal ultrasonore, dont l’amplitude est proportionnelle à l’intensité lumineuse. Ces ultrasons se propagent de façon quasi-balistique dans les tissus mous et peuvent donc être détectés à l’aide d’un transducteur acoustique externe. Cette mesure permet donc de déterminer l’intensité lumineuse éclairant l’absorbeur. Nous avons montré qu’il était possible d’utiliser l'imagerie photoacoustique pour mesurer la matrice de transmission d’un échantillon diffusant. Cette caractérisation nous permet de focaliser la lumière sur des structures absorbantes et de sonder des propriétés mésoscopiques du milieu diffusant. Nous avons montré que la large bande spectrale des signaux photoacoustiques permet d’améliorer la focalisation. Enfin, nous avons montré que l’utilisation d’une source de lumière cohérente permet de pallier certains artefacts de l’imagerie photoacoustique, ainsi que de franchir la limite de résolution acoustique. / Light focusing is a crucial requirement for high resolution optical imaging. In biological tissue though, refractive index inhomogeneities scatter light, preventing any focusing beyond one millimeter. Wavefront shaping techniques have been recently developed to partially compensate for light scattering after propagation through a scattering medium. These techniques require a measurement of the light intensity at the target point. These techniques hold much promise for performing wavefront correction in order to focus light deep inside scattering media. This would require a non-invasive measure of the light intensity at depth. In this PhD study, we propose to use the photoacoustic effect for such task. An optically absorbing structure under pulsed illumination indeed generates ultrasonic waves, whose amplitude is proportional to the absorbed light intensity. These ultrasounds mostly propagate in a ballistic way, and can therefore be detected with an external transducer. We have shown that photoacoustic imaging could be used to measure the transmission matrix of a scattering sample, enabling to focus light on absorbing structures as well as to retrieve mesoscopic properties of the medium. We have shown that the broadband spectral content of the photoacoustic signals can be harnessed to improve the focusing performances. Finally, we demonstrated that coherent illumination could be used to remove fundamentals artefacts, as well as to break the acoustic resolution limit of conventional deep tissue photoacoustic imaging.

L'imagerie optique des tissus biologiques est un défi du fait de la diffusion de la lumière. Pour sonder les propriétés optiques à quelques cm de profondeur, on peut coupler l'information optique des ultrasons. De cette idée sont nées les imageries acousto-optique et photoacoustique. La première repose sur la modulation de la lumière par des ultrasons balistiques. La seconde se base sur la génération d'ultrasons lors de l'absorption de lumière par un objet. Que ce soit pour l'une ou pour l'autre, l'enregistrement du signal nécessite la mesure de très faibles modulations de phase dans une figure de speckle. L'holographie dynamique est une bonne solution. En effet, les techniques interférométriques sont suffisamment sensibles pour mesurer de telles modulations et l'holographie permet de corriger la nature speckle de la lumière. Dans cette thèse nous démontrons la faisabilité de fabriquer un système d'holographie adaptative basé sur un milieu laser (Nd :YVO4). Un des grands avantages de ce type de milieu est le temps de réponse. On montrera que le rafraîchissement d'un hologramme dans notre cristal peut se faire en moins de 100 ?s, bien inférieur au temps de décorrélation du speckle (? 1ms) qui pourrait grandement perturber les techniques de détection plus lentes lors d'expériences in vivo. Trois montages sont présentés ici, le premier pour la détection acousto-optique par conjugaison de phase, le deuxième pour la détection acousto-optique par adaptation de front d'onde et enfin le troisième pour détection photoacoustique. Dans les trois cas on mesure un temps de réponse entre 15 ?s et 50 ?s, et on utilise le montage imager un échantillon. / Strong scattering properties of biological media make their optical imaging in depth a challenge. A solution to probe the local optical properties is to couple the optical information with ultrasound. Two imaging techniques were born from this idea, acousto-optic imaging and photoacoustic imaging. The first technique is based on the local modulation of light by ballistic ultrasound. The latter relies on the emission of ultrasound following the absorption of light by an object. Whether it is acousto-optic imaging or photoacoustic imaging, the recording of the the signal requires a detection system sensitive to weak phase modulation. In addition, the detection system must be compatible with a speckle pattern. Dynamic holography is a good solution. Indeed, as it is based on interferometry, it is very sensitive to small phase variations and holography can be used to correct the speckle nature of light. In this manuscript, we show the use of an holographic detection system based on a laser medium (Nd:YVO4). One of the main advantage of this type of material is the very fast response time. It will be highlighted that the recording of a hologram inside our crystal can be done in less than 100 μs, much faster than the speckle decorrelation time (≈ 1ms), which is one of the major obstacle towards in vivo imaging. Three optical setups will be presented in this manuscript. The first one is a phase conjugation setup for acousto-optic detection. The second one is a wavefront adaption setup, also for acousto-optic detection. Finally, the third setup is an adaptive vibrometry setup for photoacoustic detection. In each setups the measured response time is between 15 μs and 50 μs.