Stanislav Y. Emelianov - US grants

[unreadable] DESCRIPTION (provided by applicant): With the great success of tissue engineering over the past decade, there is a definite and urgent need to image the engineered living tissues in a qualitative and quantitative manner. The overall goal of our research program is to develop an advanced in-vivo imaging technology; namely, combined ultrasound, photoacoustic and elasticity microscopy, capable of visualizing both the structural and functional properties of living tissue such as internal micro- and macro-architecture, surface topography, conformation, transformation, compliance, homogeneity, growth rate, biomechanics and even cell function within tissues. The underlying hypothesis of this project is that remote, non-invasive, high-frequency, high-resolution, in-vivo microscopy is possible and will provide marked advantages over existing imaging tools available for tissue engineers. The fundamental premise of our research program is to develop an advanced in-vivo microscopy based on the fusion of three complementary imaging modalities - ultrasound, photoacoustics, and elastography - and to take full advantage of the many synergistic features of these systems, thus providing a much needed quantitative imaging tool to tissue engineers. Indeed, ultrasound-based imaging on the microscopic scale offers a conceptually and technically novel imaging tool for tissue engineering. The main objective of this application is to develop a prototype of the high-resolution, multifunctional microscope for tissue engineers. To achieve our objective, we will design and build the combined ultrasound- based microscopy system based on a mechanically scanned, single element transducer interfaced with a laser source. We will also develop algorithms for ultrasound, photoacoustic and elasticity imaging to optimize the performance of the combined system. We will then test the developed microscope and corresponding signal and image processing algorithms using tissue mimicking phantoms. Finally, based on the insights gathered during the project, we will outline the design and technical specifications of an in-vivo microscopy system. The long-range goal of our research program is to develop a combined ultrasound-based microscopy system for tissue engineers. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In cancer patients, determination of whether a malignancy has spread is the single most important factor used to develop a therapeutic plan and to predict prognosis. In most cases cancer cells initially spread through regional lymph nodes. Therefore, clinical evaluation for the presence of regional lymph node metastases is of paramount importance. Unfortunately, there are no real-time, non-invasive clinical methods that can reliably detect and diagnose micrometastases in lymph node. Therefore, there is an urgent clinical need for an imaging technique that is widely available, is non-invasive and simple to perform, is safe, and can reliably detect and adequately diagnose lymph node micrometastases in real time. The overall goal of our research program is to develop an advanced, in-vivo, noninvasive, molecular specific imaging technology, i.e., integrated ultrasound and photoacoustic imaging combined with targeted plasmonic nanosensors, capable of immediate and accurate assessment of sentinel lymph node micrometastases in real time. The underlying hypothesis of this project is that photoacoustic imaging integrated with widely used clinical ultrasound imaging is possible and both ultrasound and photoacoustic imaging can be performed in real time, yielding an immediate diagnosis and allowing early implementation of treatment. A wide range of scientific and engineering, biomedical and clinical problems must be addressed to fully explore the capabilities of molecular specific ultrasound and photoacoustic lymphatic (MS-USPAL) imaging in detection and characterization of sentinel lymph node micrometastases. The central theme of the current application is threefold: to design and build a laboratory prototype of the integrated ultrasound and photoacoustic imaging system, to develop lymphatic contrast agent based on gold bioconjugated plasmonic nanosensors, and to initially test the develop imaging technology in 3D tissue phantoms, small animal model and, finally, excised cancerous tissue samples. Therefore, all theoretical and experimental studies will be conducted to evaluate applicability of the developed MS-USPAL imaging system for sentinel lymph node micrometastases. At the end of the study, we will outline the design and technical specifications of a clinical MS-USPAL imaging system. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The overall goal of our program is to develop an imaging technology to assess viscoelastic properties of tissue based on gas bubble dynamics, and, generally, to understand the gas bubble dynamics in viscoelastic medium in the context of several biomedical technologies. Indeed, microbubbles are playing an increasingly important role in biomedical and clinical applications where microbubbles are introduced not only in liquids but also in biological soft tissues. While bubble dynamics in liquid is well understood, the behavior of bubbles in tissue is not. However, such an understanding can lead to the development of novel and improvement of existing biomedical techniques based on how bubbles behave in a viscoelastic medium and how the bubble responds to internal or external excitation. In the current project, we will focus our attention on laser microsurgery of the eye tissue where the bubbles, produced as a result of laser-tissue interaction, are used to cut tissue. The remote, non-invasive, high temporal and spatial resolution, real-time measurements of gas microbubble behavior can be used to image or sense much needed mechanical properties of the eye tissues prior, during and after the surgery. Theoretical, numerical, and experimental studies of gas bubble dynamics in viscoelastic medium are proposed. We will develop a nonlinear model of radial oscillations of a gas bubble in an incompressible elastic medium. The developed model will account for internal gas pressure and the complexity of soft tissue including slight compressibility, tissue viscosity, and tissue elastic heterogeneity and anisotropy. We will also investigate passive bubble dynamics and bubble translational motion and oscillations in response to external excitation such as acoustic radiation force. Next, we will develop ultrasonic and optical techniques capable of measuring the temporal and spatial behavior of the gas bubble. Given these measurements, the algorithms to estimate elasticity (Young's or shear modulus) and viscosity of the tissue surrounding the bubble will be developed. Finally, we will perform laboratory studies to verify our model and to demonstrate the ability of our method to image or sense viscoelasticity of ocular tissues using gas bubble dynamics. Therefore, all theoretical and experimental studies will be conducted to evaluate applicability of the developed methods for ophthalmologic applications. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In the United States alone, approximately 500,000 deaths will result from rupture of plaques considered "insignificant" on an angiographic evaluation. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs. Therefore, there is a definite and urgent clinical need for an imaging technique that can identify and characterize the vulnerability of atherosclerotic plaques during coronary artery interventions. The overall goal of our research program is to develop an in-vivo imaging technology - intravascular photoacoustic imaging - capable of visualizing both structural and functional properties of atherosclerotic plaques. The underlying hypothesis of this project is that intravascular photoacoustic (IVPA) imaging combined with intravascular ultrasound (IVUS) imaging is possible and can be used to distinguish vulnerable plaques, thus assisting pre-intervention planning, the intervention itself, and improving the post-intervention outcome. Most importantly, the proposed photoacoustic imaging will not significantly change the current protocol of coronary artery intervention. A wide range of technical, scientific and clinical problems must be addressed to fully explore the capabilities of intravascular photoacoustic imaging in interventional cardiology. The central theme of the current project is to address the areas of known technical concerns so that the broader developmental efforts may proceed with minimal risk. The main objective of this R21 application is, therefore, to develop and initially test a prototype of the photoacoustic imaging system for intravascular applications. To achieve our objective, we will first design and build the intravascular photoacoustic imaging system based on a mechanically scanned, single element transducer IVUS cathetrer interfaced with a tunable laser source. We will also develop both theoretical and numerical foundations for photoacoustic and ultrasound imaging to optimize the performance of the system. Second, we will test the developed imaging system using tissue mimicking phantoms. Third, the developed imaging system will be tested using animal tissue samples where ultrasonic and photoacoustic images will be correlated with histological slides and biochemical analysis of tissue. Finally, based on the insights gathered during the project, we will outline the design and technical specifications of an intravascular photoacoustic imaging system. Atherosclerotic cardiovascular disease results in more than 19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs - in the United States alone, approximately 500,000 deaths per year will result from rupture of plaques considered "insignificant" on an angiographic evaluation. Therefore, there is a definite and urgent clinical need for an imaging technique that can identify and characterize the vulnerability of atherosclerotic plaques during coronary artery interventions including percutaneous balloon angioplasty, endovascular stenting, ablation/vaporization and brachytherapy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The incidence of malignant melanoma of the skin the most serious form of skin cancer is increasing faster than that of any other cancer in the United States, and this rising incidence is expected to continue for at least the next 20 years. The prognosis for patients with melanoma is determined by the histology of the primary tumor and by the presence and extent of metastatic disease. Accurate staging at diagnosis is important to assess the prognosis and to determine best therapeutic strategy. The physical examination of regional lymph nodes is often inaccurate. More definitive information about the status of the regional nodes can be obtained from elective lymph node dissection (ELND), lymphoscintigraphy with sentinel node biopsy (LSNB), or fine needle aspiration. However, there are several major drawbacks of these procedures. Although some sites of metastatic disease may be clinically apparent, imaging must be used to detect unsuspected metastases almost all patients who die from melanoma do so with disseminated disease. Imaging studies are, therefore, an important component of the evaluation of patients with both localized and advanced melanoma. However, current imaging techniques including computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound imaging are ineffective in majority of asymptomatic patients with stage I or II disease. Therefore, there is an urgent and definite clinical need for an objective imaging technique that is widely available, is noninvasive and simple to perform, is safe, and can reliably detect and adequately diagnose early lymph node micro-metastases in real- time. The overall goal of our research program is to develop an in-vivo, minimally noninvasive, molecular specific imaging technology magneto-motive ultrasound (MMUS) imaging capable of immediate and accurate assessment of presence and extent of metastatic disease at all stages. In MMUS imaging, the targeted magnetic iron oxide nanoparticles are injected into the tissue and the ultrasound is used both to visualize the tissue and to accurately evaluate the internal tissue motion induced by the externally applied magnetic field. The central theme of the current application is threefold: to design and build a laboratory prototype of the magneto-motive ultrasound imaging system, to develop molecularly sensitive contrast agent for MMUS imaging system, and to initially test the developed MMUS imaging technology in tissue-mimicking phantoms, 3D cell tissue constructs and, finally, small animal cancer model ex-vivo, in vitro and in vivo. The skin is the largest organ in the body, and it is not surprising that cancer of the skin is the most common of all cancers. Melanoma a cancer that begins in skin cells called melanocytes is the most deadly skin cancer, accounting for 79% of skin cancer deaths. Melanoma is currently the sixth most common cancer in American men and the seventh most common in American women. The median age at diagnosis is between 45 and 55, although 25% of cases occur in individuals before age 40. It is the second most common cancer in women between the ages of 20 and 35, and the leading cause of cancer death in women ages 25 to 30. The overall goal of our research program is to develop an advanced, noninvasive (or minimally invasive), real- time imaging technique magneto-motive ultrasound (MMUS) imaging to assess sentinel lymph node metastases thus identifying the best therapeutic intervention including immediate therapy if necessary.

DESCRIPTION (provided by applicant): There is a definite and urgent need for improved imaging techniques for early detection and accurate staging of pancreatic cancer. Furthermore, targeted therapy, consisting of image guided chemotherapeutic drug release directed at the tumor site, will reduce harmful side effects on healthy cells, dramatically improving treatment efficacy while relieving patient suffering. Several imaging modalities are currently used to diagnose and stage pancreatic cancer. Primary methods include helical computed tomography (CT) and endoscopic ultrasound (EUS). Helical CT is generally used to initially detect the presence of a pancreatic mass and any distant metastasis, while EUS is used for tumor staging and predicting vascular invasion. EUS is a fine art and only highly experienced endosonographers who have performed at least 100 EUS examinations are generally trusted to evaluate and stage pancreatic cancer. Improving these EUS devices and ultimately the imaging of pancreatic tumors is critical for clinicians to properly assess patient treatment strategies. The overall goal of our research program is two-fold: 1) to develop molecular sensitive, targeted nanocage systems (NCSs) encompassing either imaging contrast agents, therapeutic agents, or both;and 2) to develop a sophisticated in-vivo imaging technology - endoscopic photoacoustic and ultrasound (EPAUS) imaging augmented with NCSs - that will allow for diagnosis, disease characterization and more precise staging of pancreatic cancer. The current project, however, is focused on development of both an intravenously injectable targeted NCS (with contrast agents only) and endoscopic photoacoustic and ultrasound imaging for early detection and reliable staging of pancreatic cancer. Therefore, the work proposed here aims to a) design and build the targeted, molecularly sensitive NCSs for EPAUS imaging of pancreatic cancer;b) design and build a laboratory prototype of the EPAUS imaging system;and c) initially demonstrate that the NCS-augmented EPAUS imaging can be used for early detection and accurate staging of pancreatic cancer. Elaborating on these three proposed aims, the core of NCS is a biodegradable polymer matrix of poly(lactic-co-glycolic) acid (PLGA). A silver nanocage will surround the polymer core and impart contrast agent properties for photoacoustic imaging. This nanocage will be shielded from the reticuloendothelial system by attachment of poly(ethylene glycol) (PEG) chains to the exterior. Finally, the entire system will be targeted by attaching antibodies, specific to pancreatic cancer antigens, also to the exterior. The final nanocage system will provide enhanced imaging of pancreatic cancer when used in conjunction with a custom designed EPAUS imaging device. The laboratory prototype of an EPAUS imaging device will be based on an endoscopic, linear array ultrasound probe interfaced with a multi-channel ultrasound imaging system and tunable pulsed laser source. Finally, the developed NCS-augmented EPAUS system will be tested using tissue-mimicking phantoms with cancer-simulating inclusions containing various concentrations of NCS, tissue culture cell phantoms, and xenographic mouse models of pancreatic cancer. The EPAUS images will be correlated with histological slides and compared with immunohistochemical analysis of tissue samples. PUBLIC HEALTH RELEVANCE: There is a definite and urgent need for improved imaging techniques for early detection and accurate staging of pancreatic cancer. Furthermore, targeted therapy, consisting of image guided chemotherapeutic drug release directed at the tumor site, will reduce harmful side effects on healthy cells, dramatically improving treatment efficacy while relieving patient suffering. Several imaging modalities are currently used to diagnose and stage pancreatic cancer. Primary methods include helical computed tomography (CT) and endoscopic ultrasound (EUS). Helical CT is generally used to initially detect the presence of a pancreatic mass and any distant metastasis, while EUS is used for tumor staging and predicting vascular invasion.

DESCRIPTION (provided by applicant): In the United States alone, approximately 500,000 deaths will result from rupture of plaques considered "insignificant" on an angiographic evaluation. Available screening and diagnostic methods are insufficient to identify possible victims before the event occurs. Therefore, there is a definite and urgent clinical need for an imaging technique that can identify and characterize the vulnerability of atherosclerotic plaques during coronary artery interventions. The overall goal of our research program is to develop an in-vivo imaging technology - combined intravascular ultrasound and photoacoustic imaging - capable of visualizing both structural and functional properties of atherosclerotic plaques. The underlying hypothesis of this project is that intravascular photoacoustic (IVPA) imaging combined with intravascular ultrasound (IVUS) imaging is possible and can be used to distinguish vulnerable plaques, thus assisting pre-intervention planning, the intervention itself, and improving the post-intervention outcome. Most importantly, the proposed photoacoustic imaging will not significantly change the current protocol of coronary artery intervention. A wide range of scientific and engineering, biomedical and clinical problems must be addressed to fully explore the capabilities of intravascular photoacoustic imaging in interventional cardiology. The central theme of the current project is to develop and test the prototype of the combined IVUS/IVPA in- vivo imaging system prior to extensive clinical studies. Therefore, the main objective of our multi- disciplinary application is to develop an in-vivo, minimally invasive, functional and even molecular specific imaging technology - combined IVUS/IVPA imaging - capable of immediate and accurate assessment of the presence and vulnerability of atherosclerotic plaques at critical stages. To achieve our objective, first we will design and build a prototype of the IVUS/IVPA imaging system based on an available IVUS imaging systems and catheters interfaced with a tunable laser source. Furthermore, we will develop the signal/image processing algorithms and optimize the performance of the system. Second, we will develop a novel molecularly sensitive contrast agent appropriate for the IVUS/IVPA imaging system. Third, we will test the developed IVPA/IVUS imaging technology in tissue-mimicking phantoms, 3-D cell tissue constructs, small animal model of atherosclerosis, and, lastly, excised human tissue. Finally, based on the insights gathered during the project, we will design the intensive animal and clinical studies to demonstrate that the IVUS/IVPA imaging system may become a superior clinical imaging tool needed in interventional cardiology. PUBLIC HEALTH RELEVANCE: Atherosclerotic cardiovascular disease results in more than 19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify possible victims before the event occurs - in the United States alone, approximately 500,000 deaths per year will result from rupture of plaques considered "insignificant" on an angiographic evaluation. There is a definite and urgent clinical need for a technique that can a) identify the presence and location of atherosclerotic plaques, b) characterize pathologic features that predict plaque rupture including large lipid collection within the plaque, thinning of the fibrous cap, and infiltration of macrophages at the shoulders of the fibrous cap, and c) guide coronary artery interventions including percutaneous balloon angioplasty, endovascular stenting, ablation/vaporization and brachytherapy. To address this clinical need, we propose to develop an advanced, catheter-based combined ultrasound and photoacoustic imaging technique capable of visualizing functional properties of atherosclerotic plaques. Therefore, the overall goal of our research program is to develop an in-vivo, minimally invasive, functional and even molecular specific imaging technology - combined IVUS/IVPA imaging - capable of immediate and accurate assessment of presence and vulnerability of atherosclerotic plaques at critical stages. A wide range of scientific and engineering, biomedical and clinical problems must be addressed to fully test IVUS/IVPA imaging. The central theme of the current application is threefold: to design and build a prototype of the IVUS/IVPA imaging system, to develop novel molecularly sensitive contrast agent appropriate for IVUS/IVPA imaging system, and to initially test the developed IVPA/IVUS imaging technology in tissue-mimicking phantoms, 3-D cell tissue constructs, small animal model of atherosclerosis, and, finally, excised human tissue. The current study is designed to demonstrate that in-vivo IVUS/IVPA imaging is practical and feasible. At the conclusion of this study and in cooperation with industrial partners, we will be ready to build the clinical IVUS/IVPA imaging system demonstrating the application of developed technology in interventional cardiology.

DESCRIPTION (provided by applicant): Curative treatment of local and/or regional breast cancer requires surgery and adjuvant therapy such as thermotherapy. In thermal treatment of breast cancer, the tissue is exposed to high temperatures that damage and kill cancer cells with minimal injury to normal tissues. The overall goal of our research program is to develop an image-guided, molecular specific photothermal therapy of cancer using targeted metal nanoparticles. Specifically, using targeted plasmonic nanosensors and an advanced, in-vivo, noninvasive, functional, molecular specific imaging technology (i.e., integrated ultrasound, photoacoustic and elasticity imaging), photothermal therapy can be greatly improved. Indeed, before the therapeutic procedure, using ultrasound (anatomical and blood flow imaging) and elastography (biomechanical functional imaging), the tumor will be non-invasively imaged to develop an appropriate treatment plan. Furthermore, the delivery and interaction of molecular specific photoabsorbers with cancerous tissue will be imaged using photoacoustics - a technique capable of in-vivo imaging of plasmonic nanoparticles at sufficient depth. During the therapy, the real-time imaging system will be used to guide photothermal therapy by tracking the temperature rise and, therefore, monitoring cancer treatment. Finally, after the therapy, the combined imaging will be used to accurately assess the short-term and the long-term treatment outcome. The central theme of the current application is threefold: to develop multifunctional plasmonic nanoparticles acting as both photoabsorbers for photothermal therapy and contrast agent for molecular and thermal imaging;to design and build a laboratory prototype of the integrated ultrasound, photoacoustic and elasticity imaging system;and to initially test the developed nanoparticles and imaging technology in 3-D tissue phantoms and small animal cancer model ex vivo and in vivo. Therefore, all theoretical and experimental studies will be conducted to evaluate the applicability of the molecular specific, image-guided photothermal therapy to treat cancer. At the end of the study, we will outline the design and technical specifications of a clinical image- guided photothermal therapy system. PUBLIC HEALTH RELEVANCE: Cancer is a disease characterized by uncontrollable, abnormal growth of cells. The resulting tumor can invade and destroy the surrounding healthy tissue. Cancer is the second leading cause of death in the United States, exceeded only by heart disease. Breast cancer treatment often requires surgery and adjuvant therapy such as thermotherapy. In thermal treatment of breast cancer the tissue is exposed to high temperatures that damage and kill cancer cells with minimal injury to normal tissues. The primary goal of thermal treatment of cancer is to selectively heat a small volume of cancerous cells leading to tumor necrosis while protecting the surrounding healthy tissue. Thus, to successfully perform photothermal cancer therapy, an imaging technique that can help effectively plan, guide and monitor the photothermal therapy is needed. The overall goal of our research program is to develop the targeted multifunctional nanoparticles and the combined ultrasound, photoacoustic and elasticity imaging system to assist photothermal therapy. Before the therapeutic procedure, the tumor will be non-invasively imaged to develop an appropriate treatment plan. During the therapy, the real-time imaging system will be used to guide photothermal therapy by tracking the temperature rise and monitoring the cancer treatment. Finally, after the therapy, the combined imaging will be used to accurately assess the short-term and the long-term treatment outcome.

DESCRIPTION (provided by applicant): The goal of this application is to provide the investigators from the University of Texas at Austin with a small animal imaging system that will provide the necessary means and resources to further their multi- disciplinary research. The instrument, a high resolution Vevo 2100 Ultrasound Micro-Imaging System, is capable of in-vivo, real-time imaging of small animals such as mice and rats. The University of Texas at Austin is the home of a large group of NIH-funded investigators conducting fundamental studies and translational research. These investigators are associated with departments from the Cockrell School of Engineering, the College of Natural Sciences, the College of Pharmacy and the College of Liberal Arts. Currently, the University of Texas at Austin is not equipped with the state-of-the-art equipment to conduct anatomical, functional, physiological and molecular imaging of small animals widely used in our studies, despite the large number of scientists that have an overwhelming need to have access to small animal imaging facilities. Therefore, the specific aim of this application is to acquire the Vevo 2100 ultrasound imaging system to enable investigators to conduct basic science and applied research projects ranging from cancer biology to cardiovascular disease, from studying the disease to finding the cure for the pathology, from development of new imaging methods to building the devices, etc. Overall, the principal investigator and the key personnel in this application are committed to further their understanding of the science and technology. Together, these investigators cover large and complementary research territories focusing their efforts on basic science and the translation of laboratory discoveries to clinical practice. The project and, more importantly, the unique instrument bring together researchers that have different, yet complementary conceptual approaches, and thereby enrich both the fields of science and technology. PUBLIC HEALTH RELEVANCE: The anatomical, functional and molecular imaging of small animals allows scientists and engineers to understand the pathophysiological processes of human diseases using animal models. The non-invasive, realtime, high-resolution Vevo 2100 ultrasound micro-imaging system can provide researchers with a method to efficiently examine extremely small physiological structures. Furthermore, the Vevo 2100 is capable of imaging in real-time with near-microscopic resolution. Finally, morphological, physiological and molecular imaging is possible using the Vevo 2100. Therefore, acquiring the small animal imager, such as the Vevo 2100, will support multi-disciplinary biomedical research at the University of Texas at Austin.

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This project supports a cooperative research project by Dr. Stanislav Emelianov, University of Texas at Austin and Dr. Hassan Talaat, Ain Shams University, Cairo. They plan to study nanomaterials for developing devices for Improving Biomedical Imaging and Early Diagnosis of Cancer. Photothermal therapy, a targeted, non-invasive treatment of cancer where heat is used to cause tumor necrosis, is a suitable alternative to traditional surgery when surgery is impossible. The PIs plan a combined ultrasound and photoacoustic imaging system to non-invasively assist, guide and monitor photothermal cancer therapy. Metal nanoparticles will be developed as optically tuned photoabsorbers to efficiently transfer light irradiation into thermal energy to the tumor. Further, bioconjugation protocols will be designed to specifically target the photoabsorbers to cancerous cells. Ultrasound imaging will be utilized to identify the anatomy of the tumor while photoacoustic imaging will be used to ensure the presence of photoabsorbers before beginning therapy. Both ultrasound and photoacoustic imaging will be used to monitor temperature during therapy to ensure tumor necrosis and protect the surrounding healthy tissue. Finally, ultrasound imaging can track thermal damage in real-time during and after therapy to ensure cancer destruction. Ultrasound-guided molecular photoacoustic imaging and photothermal therapy will have a major impact in breast oncology.

Intellectual merits: The main objective of this proposal is to design a prototype device for biomedical imaging based on using the photoacoustic tomography technique and different nanomaterials as contrast agents. The activity does carry a potential to advance our knowledge and understanding within the photoacoustic and ultrasound imaging field as well as nanotechnology and medicine. Nanomaterials and photoacoustic biomedical imaging are both emerging technologies in its own field. Integrating these two technologies in this proposed study will form an important foundation and guidelines to design and develop a hybrid imaging/therapy system for biomedical applications such as cancer treatments. The multi-disciplinary team consists of experts in nanotechnology and photoacoustic biomedical imaging. The roles of each coordinator are well defined and the collaborating mechanisms are adequately planned. This project involves exploring original and potentially transformative concepts of combining novel imaging strategies and advancing potentially new cancer therapies.

Broader impacts: Highly beneficial education and training of students, postdoctoral fellows, and junior faculties in the wide spectrum of the research areas of nanotechnology, biomedical imaging and medicine are expected. The already established network between UT and ASU will be strengthened and the successful outcomes of this project may lead a strong industrial partnership between US and Egypt with high potential. If successful, this hybrid technology may eventually bring an important imaging/therapy tool for the cancer treatments in clinic. The activity aims to broaden the participation of under-represented groups specifically women. Successful outcomes could greatly improve the multi-modal imaging techniques and would significantly advance the field. Indeed, the developed technology will be able to identify molecular composition of cancer and functional changes during image-guided photothermal therapy. The research will impact the fields of cancer research and clinical oncology as the developed methods and approaches can be applied in wide range of applications.

This proposal is supported under the US-Egypt Joint Fund Program where NSF supports the US side and the Government of Egypt funds the Egyptian side.

ABSTRACT In cancer patients, determination of whether a malignancy has spread is the single most important factor used to develop a therapeutic plan and to predict prognosis. In most cases, cancer cells initially spread through regional lymph nodes. Therefore, clinical evaluation for the presence of regional lymph node metastases is of paramount importance. Unfortunately, there are no real-time, non-invasive clinical methods that can reliably detect and diagnose micrometastases in lymph nodes. Therefore, there is an urgent clinical need for an imaging technique that is widely available, is non-invasive and simple to perform, is safe, and can reliably detect and adequately diagnose lymph node micrometastases in real time. The overall goal of our research program is to develop an advanced, in-vivo, noninvasive, molecular specific imaging technology, i.e., integrated ultrasound and photoacoustic imaging combined with targeted plasmonic nanosensors, capable of immediate and accurate assessment of sentinel lymph node micrometastases in real time. The underlying hypothesis of this project is that photoacoustic imaging integrated with widely used clinical ultrasound imaging is possible and both ultrasound and photoacoustic imaging can be performed in real time, yielding an immediate diagnosis and allowing early implementation of treatment. A wide range of scientific and engineering, biomedical and clinical problems must be addressed to fully explore the capabilities of molecular specific ultrasound and photoacoustic lymphatic (MS-USPAL) imaging in detection and characterization of sentinel lymph node micrometastases. The current application is focused on important aspects of clinical translation of MS-USPAL imaging. We will develop and validate clinically translatable plasmonic nanosensors for MS-USPAL. We will use ultra-small gold nanoparticles to target epidermal growth factor receptor (EGFR), which is overexpressed in squamous carcinoma and in many other epithelial neoplasms. For highly sensitive detection of cancer cells, we will explore EGF receptor mediated endocytosis and the effect of plasmon resonance coupling between closely spaced molecular specific nanoparticles. The ultra-small size of nanoparticles will be highly favorable for rapid clearance from the body which will allow safe transition into clinical practice. Additionally, 5 nm ligand capped gold nanoparticles will greatly reduce nonspecific interactions and reduce the uptake of nanoparticles by immune cells such as macrophages present due to lymph node inflammation, thus diminishing false positive results. Furthermore, we will design and construct a prototype of the clinical MS-USPAL imaging system capable of imaging 5 nm nanoparticles in-vivo.

DESCRIPTION (provided by applicant): Small animal models, particularly genetically engineered mice, are increasingly recognized as powerful discovery tools in cancer research. However, the potential of animal models has not yet fully been realized since they are often sacrificed to perform tissue analysis to determine the effects of therapy. Sacrificing prevents long-term, in vivo observation of natural or perturbed processes. Therefore, there is a need for a morphologic, functional, cellular/molecular, and quantitative imaging technique capable of visualizing biochemical, genetic, or pharmacological processes in vivo and longitudinally in small animals. The overall goal of our academic-industrial partnership is to enable the molecular and cellular sensitivity of ultrasound-guided photoacoustic (USPA) imaging, furthering its development and translation into the preclinical research arena for longitudinal animal studies. Our hypothesis is that a non-invasive USPA imaging system, capable of simultaneous anatomical, functional, cellular and molecular visualization of cancer in small animals, will significantly enhance the outcome of fundamental and preclinical cancer research. The central theme of our application is to develop contrast agents and signal/image processing algorithms for USPA imaging to enable quantitative imaging of tumor angiogenesis and functional, cellular/molecular properties of tissue in small animal models of breast cancer. In this project we will specifically demonstrate our approach by developing imaging contrast agents sensitive to human epidermal growth factor receptor 2 (HER2) and the 1v23 integrin. These agents will be used to detect the molecular composition of a tumor in vivo to monitor the effect of the therapeutic small molecule tyrosine kinase inhibitor lapatinib on breast cancer cellular function and angiogenesis in longitudinal studies.

DESCRIPTION (provided by applicant): In the United States alone, approximately 500,000 deaths per year are due to ruptured vascular plaques that were considered insignificant by angiographic evaluations. Available screening and diagnostic methods are insufficient to identify possible victims before the event occurs. Therefore, there is a definite and urgent clinical need for a diagnostic imaging technique that can identify and characterize the vulnerability of atherosclerotic plaques during coronary artery interventions. The overall goal of our research program is to develop an in vivo imaging technology - combined intravascular ultrasound and photoacoustic imaging - capable of visualizing both structural properties and composition of atherosclerotic plaques. The underlying hypothesis of this project is that intravascular photoacoustic (IVPA) imaging combined with intravascular ultrasound (IVUS) imaging can be implemented clinically and used to detect and determine the vulnerability of atherosclerotic plaques. Therefore, combined IVUS/IVPA imaging can improve pre-intervention planning and assist with the intervention itself, thus improving the post-intervention outcome and reducing patient morbidity. Most importantly, the proposed IVUS/IVPA imaging will not significantly change the current clinical protocol of coronary artery intervention. A wide range of scientific, biomedical engineering, and clinical problems must be addressed to fully explore the capabilities of IVUS/IVPA imaging in interventional cardiology. The central theme of the current project is to develop and test a prototype of the combined IVUS/IVPA system for real-time in vivo imaging prior to extensive clinical studies. To achieve our objective, first we will design an build a prototype of the real-time in vivo IVUS/IVPA imaging system consisting of the custom-built IVUS/IVPA imaging catheters integrated with a motor assembly and interfaced with pulsed laser source, ultrasound transmitter/receiver, and microprocessor control unit. Second, we will develop the signal/image processing algorithms necessary to assess the anatomical features of the vessel wall and plaque to identify and quantify lipid-rich necrotic cores within atheroscleroti plaques. We will optimize the performance of the system hardware and signal/image processing algorithms by imaging ex vivo atherosclerotic arteries from animal models (rabbit and swine) and human coronary artery autopsy samples. Finally, the efficacy of the in vivo real-time IVUS/IVPA imaging system will be validated using live animal models of atherosclerosis. Based on the insights gathered during this project, we will be ready to perform further large animal and clinical studies to demonstrate that the IVUS/IVPA imaging system may become a superior clinical imaging tool needed in interventional cardiology.

ABSTRACT The development of treatments for central nervous system disorders has been limited because of difficulties in translating positive preclinical findings in small animal models to clinical studies. One major limit in preclinical studies is that it is difficult to directly correlate biomarker expression to cognitive/behavioral results in longitudinal studies. Animals are generally sacrificed to evaluate biomarker expression. Therefore, there is a need for a morphological, functional, and cellular/molecular neuroimaging technique capable of longitudinal assessment of brain tissue in vivo, non-invasively, and in real time. The goal of our research program is to develop a preclinical neuroimaging system based on ultrasound and photoacoustic (USPA) imaging and to use gas microbubble- assisted focused ultrasound (FUS) disruption of the blood brain barrier (BBB) for delivery of targeted contrast agents. We believe that this system would improve the ability of neuroscientists to study the underlying mechanisms of diseases and to develop and evaluate potential treatments. The underlying hypothesis of this project is that non-invasive, high-resolution, high-sensitivity, in vivo imaging of the brain is possible and will provide marked advantages over existing imaging tools available to neuroscientists. USPA imaging, in combination with FUS and molecular probes, is of interest because it is non-invasive, cost-effective, and portable while also capable of imaging the whole rodent brain in vivo. The main goal of this exploratory R21 application is to develop and test a laboratory prototype of the USPA neuroimaging system and address known concerns in order to proceed with broader development efforts in the future. We will develop an FUS-mediated USPA imaging system to non-invasively visualize protein biomarkers in vivo in preclinical models of Alzheimer?s disease (AD). AD is a progressive neurodegenerative disease, which causes significant morbidity and mortality, characterized by pathogenic protein plaques composed of ?-amyloid. In order to visualize plaques, ?-amyloid-targeted gold nanorods (AuNRs) will be developed. AuNRs have been shown to be strong optical absorbers and good photoacoustic contrast agents that can be tuned to absorb in the near-infrared region. Specifically, anti-?-amyloid antibodies will be conjugated to AuNRs and tested in AD tissue samples as well as via surface plasmon resonance assays. After confirming targeting of the rods, an FUS positioning system will be developed for targeting the hippocampus of mice. The ability of this system to target the hippocampus will be confirmed with Evans Blue leakage as well as delivery of non-targeted AuNRs (visualized with USPA imaging). The distribution of AuNRs, clearance mechanisms, and clearance timeline will be assessed acutely and longitudinally. Finally, targeted AuNRs will be delivered to the hippocampus of AD and wild type mice using FUS and visualized with USPA in vivo, acutely and longitudinally. The ability to visualize ?-amyloid in the hippocampus with USPA in vivo longitudinally would prove the utility of our system. If successful, these studies will demonstrate that USPA imaging is a valuable neuroimaging tool that will help to translate the result of preclinical studies and facilitate the clinical success of disease diagnosis and therapy.

Project Summary/Abstract Glaucoma is a major cause of blindness and current treatments are insufficient. A major risk factor for glaucoma, and the only treatable risk factor, is elevated intraocular pressure (IOP). Current IOP-lowering therapies fail too often, and thus there continues to be great interest in novel IOP control strategies. The trabecular meshwork (TM), the key tissue determining IOP, has reduced cellularity in glaucoma, which has led a number of groups to study stem cell-based therapies for the TM. An obstacle to such therapies is cell delivery: current approaches have low cell delivery efficiency and specificity for the TM, and cannot deliver cells to all parts of the TM. Here a novel technological solution for ?steering? stem cells to the TM is proposed, which can be additionally used to visualize stem cell delivery and to monitor stem cell apoptosis. This approach will be tested in several models of ocular hypertension. The key technology is superparamagnetic and photosensitive nanoparticles. Their superparamagnetic properties mean that cells which have taken up these nanoparticles can be rapidly steered to the TM by a magnet placed at the limbus. Their photosensitivity means that cells can be visualized by ultrasound/photoacoustic imaging in the living eye. The functionality of these nanoparticles will be further enhanced by using a photosensitive marker of active caspase-3 to monitor stem cell apoptosis. Our overall objective is to validate these technologies as a safe and effective approach for monitoring and steering of stem cells to the TM, thereby restoring intraocular pressure (IOP) homeostasis in glaucoma patients. Three specific aims towards this long-term goal are proposed, building on our significant preliminary data. In aim 1, a novel caspase-3-sensitive reporter for monitoring apoptosis will be synthesized and characterized, and magnets for steering stem cells to the TM will be optimized. In aim 2, an instrument capable of imaging of labeled stem cells in whole eyes, including longitudinal monitoring of stem cell distribution and apoptosis, will be developed. In aim 3, stem cells will be delivered to the TM in two glaucoma models, and their ability to restore IOP homeostasis will be evaluated. The ability of ultrasound/photoacoustic imaging to monitor stem cell delivery to the TM and stem cell apoptosis will also be validated; and mesenchymal stem cells (MSCs) will be compared to differentiated induced pluripotent stem cells (iPSC-TMs) for their efficacy in restoring IOP homeostasis. This project is highly innovative: it the first study to steer and visualize stem cells as part of a treatment for ocular hypertension. It is also the first to compare the efficacy of MSCs vs. iPSC-TMs for treating ocular hypertension. We expect, as suggested by our strong preliminary data, to discover that stem cells can be efficiently and selectively steered to the TM by a simple magnet placed at the limbus for as little as 15 minutes; and that it will be possible to accurately monitor the location of stem cells in the eye and stem cell apoptosis over time. Further, we expect that TM function will be improved by stem cells steered in this way, as tested in 2 glaucoma models.

ABSTRACT The goal of this research program is to develop an advanced, noninvasive, molecularly specific imaging tech- nology ? ultrasound-guided modulated photoacoustic imaging augmented by optically-activatable, targeted con- trast agents, capable of immediate, accurate, background-free assessment of pathologies in vivo. The underlying hypothesis of this project is that ultrasound-guided photoacoustic imaging of optically modulated contrast agents can be performed in real time, yielding immediate diagnostic information. The approach is based on the unique combination of an optically-activatable molecularly-targeted imaging contrast agent and corresponding laser/ul- trasound imaging device. Specifically, development of a highly-sensitive imaging contrast agent consisting of silica nanoparticles doped with optically-activatable photoabsorbers and coated by an antibody-functionalized lipid shell will be undertaken. The photoabsorbers embedded in the silica core exhibit ~50?s-lived, transient near-infrared optical absorption upon prior red laser irradiation. The contrast agent will be imaged using a clinical ultrasound (US) imaging system, interfaced with two (pump/probe) pulsed laser sources, operating in either ultrasound or photoacoustic imaging modes. Pump laser pulses will repeatedly activate the contrast agent, thus allowing for probe laser pulses to generate photoacoustic signal that can be processed to generate background- free modulated photoacoustic (mPA) images spatially co-registered with grayscale ultrasound (US) images. Fur- thermore, by probing different dark state lifetimes of optically activated contrast agents, multiple mPA ?colors? will be simultaneously imaged within tissue. These US-mPA images will display molecular and functional signa- tures of the disease within the structural content of the tissue. The specific objective of this project is to develop an ultrasound-guided modulated photoacoustic imaging approach and demonstrate the developed approach in the background-free detection of micrometastases in sentinel lymph nodes using a murine model of metastatic breast cancer. Indeed, one of the critical components of the clinical cancer management is the analysis of re- gional lymph nodes where current diagnostic methods including imaging suffer from low sensitivity and specificity. Therefore, this US-mPA system will be specifically designed for imaging micrometastases in sentinel lymph nodes. Improved sensitivity and specificity of US-mPA imaging will be demonstrated through synthesis of mo- lecularly targeted contrast agent and coordinated instrumentation development to maximize impact of these new materials and the imaging approach. The successful outcome of this study will enable design and development of a clinical imaging system and contrast agents for background-free molecular imaging of various pathologies.

ABSTRACT Spinal cord diseases and disorders such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS) are debilitating, often resulting in loss of mobility and decreased quality of life for those affected. One promising treatment involves the transplantation of neural progenitor cells (NPC) into the spinal cord, which has been shown to have neuroprotective properties. However, most NPC-based therapies fail after reaching clinical trials, and without a method to monitor the injected cells and viability, locating injected NPCs is limited to postmortem histology. The ability to track and assess the viability of transplanted cells could be crucial in understanding whether the failure is technical or biological in nature, such as whether the injected cells were delivered to and remain at the correct location or survived transplantation. We propose the development of multi-modal contrast agents and a clinical ultrasound and photoacoustic (US/PA) imaging system for image guided delivery of trans- planted cells and longitudinal monitoring. Together with clinical magnetic resonance imaging (MRI), the use of US/PA imaging can allow for pre-, intra- and post-operative visualization of the cells. The MRI and US imaging modalities are well established in the clinic, and PA can be easily integrated with existing clinical US imaging systems. We hypothesize that visualizing the location and viability of injected neural progenitor cells obtained through the labeling of NPCs with contrast agents will allow for better understanding of transplanted cell behavior and improved translation of cell based therapies. Two contrast agents will be developed: photo-magnetic nano- particles, which will allow for photoacoustic and MRI tracking, and a dye-based apoptosis reporter, which will provide photoacoustic contrast upon apoptotic activity. These will be delivered to the cytosol of the neural pro- genitor cells and assessed for labeling efficiency and toxicity. Once optimized, labeled NPCs in different ratios of live to dead will be injected into the spinal cords of rats to assess the feasibility of distinguishing live and dead cells in vivo. After injection, the NPCs will be monitored longitudinally using trimodal US/PA/MR imaging. After validating the performance of the contrast agents in vivo, a clinical US/PA imaging system will be developed to demonstrate real-time image guided-delivery, US/PA/MRI longitudinal tracking, and PA viability assessment of the labeled cells. The ability to monitor the transplanted cells at every point during and after the procedure will allow for more accurate delivery of the cells and could elucidate common issues and behaviors of injected NPCs that could lead to therapeutic improvements. Furthermore, if successful, it will validate both the contrast agents and US/PA as a valuable tool for tracking and monitoring cell-based therapies to improve clinical translation.

ABSTRACT Stem cells, including mesenchymal stem cells (MSCs) have proven to be an exciting and promising thera- peutic for the treatment of numerous diseases and disorders of the spinal cord, such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS). Positive results in preclinical studies have led to an increase in transla- tion of stem cell therapies. However, upon reaching clinical trials, many stem cell therapeutics fail. In search for answers, the clinicians and researchers are missing critical information; the fate of the cells once implanted. Therefore, there is a definite and urgent clinical need for a technique that is capable of accurate real-time guid- ance of quantitative stem cell delivery to target areas, noninvasive longitudinal in vivo tracking of stem cell loca- tion, and assessment of stem cell viability over time after the delivery. This project is based on a hypothesis that stem cells can be effectively labeled with a photoacoustic imaging contrast agent and a photoacoustic apoptosis sensor to visualize cell localization and ascertain their viability, respectively; and ultrasound-guided photoacous- tic (USPA) imaging system and approach can be developed for longitudinal, quantitative, and noninvasive track- ing of double-labeled stem cells and their fate in vivo. The overall goal of this research program is to develop a novel cell labeling system and corresponding USPA imaging approach allowing real time image guidance for precise and accurate injection of the stem cells, and longitudinal tracking both the location and viability of stem cells in vivo in the spinal cord. First, a PA sensitive tracker will be developed, which will allow for real time feedback on the location of the stem cells during and immediately after injection, as well as post-operative tracking the location of the transplanted cells over time. Second, a PA sensor of apoptosis will be developed. This sensor will provide a unique PA signal in cells which are undergoing apoptosis, allowing us to ascertain the cells viability. The combination of both PA tracker and apoptotic sensor to double label stem cells is critical for real time imaged guided delivery of cells, and tracking viable and apoptotic cells longitudinally in vivo. The USPA imaging double-labeled cells will provide highly valu- able information of the fate of stem cells in vivo, which can be used to refine and advance the field of stem cell transplantation in the spinal cord. If successful, this work will lay foundation for in vivo real-time intra-operative and then longitudinal USPA imaging of transplanted cells within the spinal cord ? the technology needed by both scientists and clinicians.