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.

The application of photoacoustic (PA) phenomena to medical imaging has been investigated for more than a decade. To implement this modality, one may choose between two types of laser sources, pulsed or continuous wave (CW). This selection affects all features of the imaging technique. Nowadays pulsed lasers are the state-of-the-art technique in the PA research. In this work, various features of the alternative frequency-domain (FD) PA were investigated. An axially symmetric transfer function model of PA wave generation and a Krimholtz-Leedom-Matthaei (KLM) transducer model were developed and used to analyze the experimental results. The controllable parameters of the FD-PA were optimized to improve the signal-to-noise ratio (SNR), contrast, axial resolution and depth detectivity. For example, it was shown that employing the optimal chirp bandwidth can enhance the SNR by more than 10 dB. In addition to the image produced by the cross-correlation amplitude, the phase of the correlation signal was used as a separate channel. A statistical method was introduced to generate an image from this phase channel, and also to filter the PA amplitude channel. A study was also performed to compare FD PA and the prevalent pulsed method. Various features of both methods were examined experimentally using a dual-mode PA system and under the condition of maximum permissible exposure (MPE). The SNRs of both methods were evaluated theoretically and experimentally. It was shown that at low frequencies, both modalities generate comparable SNRs, and at high frequencies pulsed PA produces superior SNRs and depth detetivity. However, by increasing the laser power and decreasing the chirp duration within the safety limits, the SNR and depth detectivity of the FD-PA method are enhanced considerably. The main cause to achieve lower experimental SNRs than theoretical predictions for pulsed PA response was shown to be the oscillating baseline, which can be partially eliminated by filtering.

The application of photoacoustic (PA) phenomena to medical imaging has been investigated for more than a decade. To implement this modality, one may choose between two types of laser sources, pulsed or continuous wave (CW). This selection affects all features of the imaging technique. Nowadays pulsed lasers are the state-of-the-art technique in the PA research. In this work, various features of the alternative frequency-domain (FD) PA were investigated. An axially symmetric transfer function model of PA wave generation and a Krimholtz-Leedom-Matthaei (KLM) transducer model were developed and used to analyze the experimental results. The controllable parameters of the FD-PA were optimized to improve the signal-to-noise ratio (SNR), contrast, axial resolution and depth detectivity. For example, it was shown that employing the optimal chirp bandwidth can enhance the SNR by more than 10 dB. In addition to the image produced by the cross-correlation amplitude, the phase of the correlation signal was used as a separate channel. A statistical method was introduced to generate an image from this phase channel, and also to filter the PA amplitude channel. A study was also performed to compare FD PA and the prevalent pulsed method. Various features of both methods were examined experimentally using a dual-mode PA system and under the condition of maximum permissible exposure (MPE). The SNRs of both methods were evaluated theoretically and experimentally. It was shown that at low frequencies, both modalities generate comparable SNRs, and at high frequencies pulsed PA produces superior SNRs and depth detetivity. However, by increasing the laser power and decreasing the chirp duration within the safety limits, the SNR and depth detectivity of the FD-PA method are enhanced considerably. The main cause to achieve lower experimental SNRs than theoretical predictions for pulsed PA response was shown to be the oscillating baseline, which can be partially eliminated by filtering.

The recent increase in survival rates of most cancers is due to early detection greatly aided by medical imaging modalities. Combined ultrasound and photoacoustic imaging provide both morphological and functional/molecular information which can help to detect and diagnose cancer in its earliest stages. However, both modalities can benefit from the use of contrast agents. The objective of this thesis was to design, synthesize, and test a nano-sized, dual contrast agent for combined ultrasound and photoacoustic imaging named Photoacoustic nanoDroplets. This agent consists of liquid perfluorocarbon nanodroplets with encapsulated plasmonic nanoparticles. These dual contrast agents utilize optically triggered vaporization for photoacoustic signal generation, providing significantly higher signal amplitude than that from the traditionally used mechanism, thermal expansion. Upon pulsed laser irradiation, liquid perfluorocarbon undergoes a liquid-to-gas phase transition generating giant photoacoustic transients from these dwarf nanoparticles. Once triggered, the gaseous phase provides ultrasound contrast enhancement. Demonstrated in this work are the design, synthesis, characterization, and testing of Photoacoustic nanoDroplets in phantom and animal studies, and preliminary work into adapting these agents into targeted, drug delivery vehicles for simultaneous detection, diagnosis, and treatment of diseases. / text

Cancer has become one of the leading causes of death today. The early detection of cancer may lead to desired therapeutic management of cancer and to decrease the mortality rate through effective therapeutic strategies. Advances in materials science have enabled the use of nanoparticles for added contrast in various imaging techniques. More recently there has been much interest in the use of gold nanoparticles as optical contrast agents because of their strong absorption and scattering properties at visible and near-infrared wavelengths. Highly proliferative cancer cells overexpress molecular markers such as epidermal growth factor receptor (EGFR). When specifically targeted gold nanoparticles bind to EGFR they tend to cluster thus leading to an optical red-shift of the plasmon resonances and an increase in absorption in the red region. These changes in optical properties provide the foundation for photoacoustic imaging technique to differentiate cancer cells from surrounding benign cells. In photoacoustic imaging, contrast mechanism is based on the optical absorption properties of the tissue constituents. Studies were performed on tissue phantoms, ex-vivo and in-vivo tumor models to evaluate molecular specific photoacoustic imaging technique. The results indicate that highly sensitive and selective detection of cancer cells can be achieved by utilizing the plasmon resonance coupling effect of EGFR targeted gold nanoparticles and photoacoustic imaging. In conclusion, the combined ultrasound and photoacoustic imaging technique has the ability to image molecular signature of cancer using bioconjugated gold nanoparticles. / text

Molecular imaging is an emerging imaging principle which can visually represent the biological processes both spatially and temporally down to the sub-cellular level in vivo. The outcome of this research is expected to have a profound impact on facilitating the early diagnosis of diseases, accelerating the development of new drugs, and improving the efficacy of therapy. In general, molecular imaging highly relies on probes to sense the occurrence of molecular biological events, and to generate signals which could be picked up by diagnostic imaging modalities. The advances in the design of molecular probes not only have equipped traditional anatomical medical imaging with new capabilities but also, in some cases, stimulated developments of new imaging modalities and renaissance of existing medical imaging modalities. One of these is photoacoustic imaging, which as an emerging medical imaging modality, unites the merits from both optical imaging and ultrasound imaging. It shares with optical imaging, that it uses non-ionizing radiation and provides higher contrast and higher sensitivity than ultrasound imaging. Unlike optical imaging, which requires ballistic photons for imaging, photoacoustic imaging requires only diffusive photons to excite the ultrasound signal from the imaging target; therefore, it is capable of imaging much deeper into the tissue. In combination with molecular probes, photoacoustic molecular imaging has been demonstrated by several research groups using various photoacoustic molecular probes. However, the molecular probes used for most of these studies were contrast agents simply adopted from other optical imaging modalities. Our research on photoacoustic contrast agents indicated that the mechanism of photoacoustic signal generation from nanometer-sized contrast agents is distinct from that of optically homogeneous materials, such as tissue. We have discovered that, the amplitude of the photoacoustic signal generated from nano-contrast agents depends not only on the optical absorption of the particles, but more importantly, on the dynamic process of the heat conduction from the nanoparticles to the ambient, and the thermal properties of the surrounding materials. Based on our finding, we explored and further improved the photoacoustic response of the nanoparticles by exploiting the heat conduction process between the nanoparticle and its surrounding materials and by manipulating the excitations. This research allows to create optimized molecular specific contrast enhanced photothermal stable probes which can aid photoacoustic imaging and image guided photothermal cancer therapy. / text

Current methods to diagnose and treat cancer often involve expensive, time-consuming equipment and materials that may lead to unwanted side effects and may not even increase a patient’s chance of survival. Thus, for a while now, a large part of the research community has focused on developing improved methods to detect, diagnose, and treat cancer on the molecular scale. One of the most recently discovered methods of cancer therapy is targeted therapy. These targeted therapies have potential to provide a patient with a form of personalized medicine because these therapies are biological molecules that specifically target other molecules involved with a cancer’s growth. Past trials using these therapeutic molecules, however, have led to controversial results, where certain patients responded better than others to the therapy for unknown reasons. Elucidating the reason behind these mixed results can be accomplished using metal nanoparticle technologies which could provide a bright signal to monitor the path that these therapeutic molecules take in vivo as well as enhance the molecule’s efficacy. Literature has shown that presenting targeting molecules in a dense manner to their target will increase these molecules’ binding affinity. This concept has been explored here to increase binding affinity of therapeutic molecules by attaching these molecules in a dense manner on the surface of gold nanoparticles, and correlating this increased affinity with therapeutic efficacy. Additionally, gold nanoparticles provide an easy surface for molecules to be functionalized on and have shown to be effective imaging, x-ray, and photothermal therapy agents. A major roadblock to using these gold nanoparticles clinically is their non-degradability and thus potential to cause long-term negative side effects in vivo. A platform for developing biodegradable gold nanoparticles is also explored here to take advantage of the gold nanoparticles’ excellent imaging and drug delivery capabilities while still allowing them to be used safely in the long term. / text

Accurate detection of the spread of cancer is critical for planning the best treatment strategy for a patient. Currently, an invasive sentinel lymph node biopsy is commonly used to detect metastases after a primary tumor is detected. This procedure results in patient morbidity, requires weeks of waiting, and is prone to sampling error. This dissertation presents new developments in an emerging biomedical imaging modality – photoacoustic imaging – and their application to improving the detection of metastases in the lymphatic system in a metastatic mouse model of squamous cell carcinoma of the oral cavity. Label-free spectroscopic photoacoustic imaging is demonstrated to detect hypoxia that results from the development of sub-millimeter cancer foci in the lymph node. In order to improve the sensitivity to micrometastases, molecularly-activated plasmonic nanosensers which are targeted to the epidermal growth factor receptor are introduced. The nanosensors are demonstrated to detect metastases consisting of only a few tens of cells. Improvements to spectroscopic photoacoustic imaging are then demonstrated by selecting imaging wavelengths based on the spectral properties of the optical absorbers. Finally, a new contrast agent – silica-coated gold nanoplates – are used to map the sentinel lymph node with high contrast. The final result is a set of tools that can be used to noninvasively detect micrometastases and improve molecular photoacoustic imaging. / text

Gold nanoparticles attain an intense focus in biomedical imaging applications due to their unique optical properties, facile conjugation with biomolecules, and biocompatibility. Although a considerable amount of work towards the development of gold nanoparticles has been completed, these promising contrast agents have not yet reached the clinic due to several challenges including efficient accumulation at the diseased site, sensitivity of detection in vivo, potential adverse effects, and clearance from the body. High signal-to-background ratio is required to enhance sensitivity of detection. Because near infrared (near-IR) light has the best tissue penetration, contrast agents designed to work in this range can significantly increase imaging sensitivity. Moreover, efficient targeting of the molecular biomarkers on diseased cells can decrease the required dosage, increase the site-specific accumulation, and enhance the imaging sensitivity. Molecular-specific contrast agents developed in this project use directional attachment of antibody molecules to the nanoparticle surface, enhancing the targeting efficacy. Additionally, cell-based delivery of diagnostic and therapeutic agents is gaining much interest due to the immune cells’ special access to the avascular, diseased regions. The contrast agents developed in this project enable detection of just a few cells per unit of imaging volume, enable multiplex imaging, and open up a possibility for tracking different cell populations with noninvasive photoacoustic and ultrasound imaging. Finally, the clearance of nanoparticles from the body dictates their clinical translation. The in vivo pharmacokinetics study along with the proposed in vitro model explored in this project will enable fast, reliable, and cost-efficient screening of promising agents and facilitate quick optimization of nanoparticles for their potential use in the clinic. / text

Rupture of atherosclerotic plaque is closely related to plaque composition. Currently, plaque composition cannot be clinically characterized by any imaging modality. The objective of this dissertation is to use a recently developed imaging modality – ultrasound-guided intravascular photoacoustic (IVPA) imaging – to detect the distribution of two critical components in atherosclerotic plaques: lipid and phagocytically active macrophages. Under the guidance of intravascular ultrasound imaging, spectroscopic IVPA imaging is capable of detecting the spatially resolving optical absorption property inside a vessel wall. In this study, contrast in spectroscopic IVPA imaging was provided by either the endogenous optical property of lipid or optically absorbing contrast agent such as gold nanoparticles (Au NPs). Using a rabbit model of atherosclerosis, this dissertation demonstrated that ultrasound guided spectroscopic IVPA imaging could simultaneously image lipid deposits as well as macrophages labeled in vivo with Au NPs. Information of macrophage activity around lipid rich plaques may help to identify rupture-prone or vulnerable plaques. The results show that ultrasound guided IVPA imaging is promising for detecting plaque composition in vivo. Clinical use of ultrasound guided IVPA imaging may significantly improve the accuracy of diagnosis and lead to more effective treatments of atherosclerosis. / text