Konstantin Sokolov - US grants

0119450
Richards-Kortum
This proposal brings together scientists from very diverse areas with the goal of developing new photonic probes and contrast agents for highly sensitive and selective detection of pre-cancers in vivo . Dr. Andrew Ellington will use the approaches of combinatorial chemistry to develop a library of aptamer molecules specific for biomolecular targets on the surface of cervical cancerous and pre-cancerous cells. He will use well-established cervical cell lines at different stages of cancer development provided by Dr. Lotan. Drs. Brian Korgel and Konstantin Sokolov will develop new photonic probes based on quantum dots (BK) and metal nanoparticles (KS). They will utilize both the aptamers developed by Dr. Ellington as well as well-known antibodies currently used in clinical histopathology. Dr. Rebecca Richards- Kortum will test the conjugates as molecular specific contrast agents using optical microscopy and spectroscopy. She will use cervical cancer cell lines provided by Dr. Lotan, three-dimensional tissue phantoms and fresh cervical tissue slices from Dr. Follen. Experiments with all three biological systems representing properties of normal and neoplastic cervix at different levels of complexity will be used to assess and refine the performance and detection scheme for the new contrast agents. This refinement will include preparing bioengineered aptamers with high affinity to cancer specific targets, tailoring optical properties of metal nanoparticles and quantum dots, optimizing conjugation procedures, and developing optimal imaging geometries.

DESCRIPTION (provided by applicant): Cancer is the second leading cause of death in the US. The majority of cancers are of epithelial origin. Early diagnosis of pre-invasive epithelial neoplasia can dramatically reduce the incidence and mortality of carcinoma. Thus, there is a desperate need for highly sensitive and cost-effective screening and diagnostic techniques to identify curable precancerous lesions. Epithelial pre-cancers are characterized by a variety of architectural and morphological features including increased nuclear size, increased nuclear/cytoplasmic ratio, hyperchromasia and pleomorphism. In addition, there is increasing evidence of significant changes occurring in the stromal layer at the earliest stages of carcinogenesis as a result of epithelial-stromal interactions. A major limitation of current clinical diagnosis is that morphological and molecular changes associated with early carcinogenesis can be assessed only after invasive biopsy. Optical techniques can assess morphologic and biochemical alterations in epithelial tissue, non-invasively and in real-time. The goat of this proposal is to develop a new technology based on polarized reflectance spectroscopy and imaging to enable non-invasive real-time detection and monitoring of morphological and architectural changes in epithelium and underlying connective tissue associated with carcinogenesis. The aims of this proposal are to: (1) develop theoretical models for polarized reflectance spectroscopy of epithelial tissue and stroma; (2) to test the theoretical predictions in tissue models of human epithelium at different stages of cancer progression; (3) to use the theoretical models to determine the sensitivity of polarized reflectance spectroscopy to morphology of epithelial nuclei and morphology of stroma; (4) to design, construct and test a new fiber optic endoscope for polarized reflectance spectroscopy in vivo; (5) to evaluate the endoscope in clinical studies of oral cavity mucosa. (6) to develop an imaging instrument based on the principles of polarized illumination and detection and to test this device in clinical trials. The inexpensive optical sensors proposed here can immediately benefit health care by reducing the number of unnecessary biopsies, enabling combined diagnosis and therapy, and reducing the need for clinical expertise.

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] DESCRIPTION (provided by applicant): One of the major challenges of modern medicine is the development of novel approaches for the efficient delivery of therapeutics and molecular specific treatment of pathology that can be carried out under imaging guidance and monitoring. Recent advances in nanotechnology, biochemistry and molecular biology give an opportunity to combine all these capabilities in a single entity. In this research program we will use recent achievements in nanotechnology and biochemistry to engineer a nanomaterial with both therapeutic and MRI contrast enhancing capabilities. This material will provide the optimized combination of: efficient delivery of a deactivated therapeutic compound, selective activation of the prodrug using external stimuli, molecular specific therapeutic effect upon activation and MRI monitoring and guidance. The nanomaterial will consist of a gold-coated iron oxide nanoparticle carrier with attached oligonucleotide handles that interact with fluorinated aptamer-siRNA chimera molecules through complementary nucleotides. The aptamer portion of the chimera will be specific for a cancer biomarker and the siRNA portion will be used to down-regulate expression of genes that are essential for cancer cell survival. The oligonucleotide handle will be designed to interact with and reversibly deactivate the aptamer portion of the chimera; this will ensure that the particles do not spontaneously bind to their target especially in normal tissue. These bioconjugated nanoparticles will be delivered in cancerous tissue under T2 weighted MRI monitoring of their accumulation and biodistribution. Then, near infrared (NIR) irradiation will be delivered to the treatment site that will lead to the local heating of the gold layer, melting of the double stranded helix between oligonucleotide handles and the aptamer portion of chimera molecules, and release of the chimeras which will then diffuse deep into the cancerous tissue. We hypothesize that release and diffusion of chimera molecules can be imaged by 19F MRI. The aptamer portion will refold and regain molecular specificity, delivering the therapeutic siRNA inside cancer cells thereby inducing cell death. The nanoparticle carrier will improve delivery, reduce non-specific toxicity, and enable monitoring of accumulation and activation of molecular specific cancer therapy. Initial tests with cell cultures and mouse xenograft models will demonstrate its efficacy. The main objective of this program is to develop and initially test a new, nontoxic nanomaterial that can be activated via NIR light irradiation to release a targeted molecular compound that can be selectively internalized by cancer cells and induce a therapeutic gene-silencing response. The nanoparticle carrier will improve delivery, reduce non-specific toxicity, and enable monitoring of accumulation and activation of molecular specific cancer therapy. Initial tests with mouse xenograft models will demonstrate its efficacy. Successful completion of this project will make an important advance toward realization of one of the ultimate goals of cancer medicine, a material that can be used to simultaneously detect and treat cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): It has been convincingly shown that nanotechnology can provide unique solutions to revolutionize diagnosis and treatment of many devastating diseases such as cancer. One specific area of great interest is development of nanoparticles for molecular specific imaging, therapy and combined imaging/therapy. A major roadblock in translation of inorganic nanoparticles to clinical practice for systemic targeting of cancer cells is their non-biodegradable nature. In addition, sizes of coated nanoparticles that are used in biological applications are not small enough to be easily cleared from the body. The accumulation and resulting long- term toxicity of nanoparticles is a major concern. In this research program we will create a new class of biodegradable gold nanoparticles with plasmon resonances in the NIR region. The nanoparticles will degrade to easily clearable components in the body and, therefore, will provide a crucial missing link between the enormous potential of metal nanoparticles for cancer imaging and therapy and translation into clinical practice. Our synthetic methodology is based on controlled assembly of very small (less than 5 nm) primary gold particles into nanoclusters with <100 nm overall diameter and an intense NIR absorbance. The assembly will be mediated by biodegradable polymers and small capping ligands on the primary nanoparticles. After delivery into the body the nanoclusters will biodegrade over time into sub-6 nm ligand capped primary gold nanoparticles, which will be highly favorable for rapid clearance from the body. This hybrid polymer/inorganic material will combine advantages of biodegradability of polymer nanoparticles and strong imaging contrast and therapeutic capabilities afforded by metal nanoparticles. Whereas these nanoclusters will be shown to have widespread potential in imaging/therapy, we will develop and optimize in this particular application biodegradable plasmonic nanoclusters with intense NIR absorbance for photo-acoustic imaging (PA) of cancerous cells. The nanoclusters will be evaluated in biologically relevant models of oral cancer. PUBLIC HEALTH RELEVANCE: It has been convincingly shown that nanotechnology can provide unique solutions to revolutionize diagnosis and treatment of many devastating diseases such as cancer. One specific area of great interest is development of nanoparticles for molecular specific imaging, therapy and combined imaging/therapy. A major roadblock in translation of inorganic nanoparticles to clinical practice for systemic targeting of cancer cells is their accumulation in the body and resulting long-term toxicity. In this research program we will create a new class of biodegradable gold nanoparticles with plasmon resonances in the NIR region. The nanoparticles will degrade to easily clearable components in the body and, therefore, will provide a crucial missing link between the enormous potential of metal nanoparticles for cancer imaging and therapy and translation into clinical practice.

0968038
Johnston

Intellectual Merit:

A major challenge in nanotechnology is to design stable particles smaller than 100 nm with multifunctionality, and in particular, strong optical and magnetic properties. It is difficult to achieve the required particle morphology in the common approach of atomic growth guided by surfactants. Thus, a robust and flexible alternative will be developed to assemble ~5 nm nanoparticle building blocks physically into multifunctional nanoclusters. This colloidal kinetic and interfacial assembly concept will be developed to obtain simultaneously sizes below 100 nm and extremely high loadings (>80%) of gold and iron oxide particles to produce strong optical (NIR absorbance) and magnetic (magnetic moment/volume and spin-spin relaxivity, r2) properties. In addition, these clusters will disassemble back into the original primary nanoparticles upon breakdown of biodegradable polymer stabilizers, which is very important for their translation to the biomedical field. Specifically, nanoclusters with high loadings of closely paced gold and iron oxide nanoparticles will be formed by tuning the colloidal interactions to control the cluster growth, size, and morphology. Weakly adsorbed polymer stabilizers will favor much higher inorganic particle loadings than in the case of equilibrium self-assembly, as well as cluster biodegradation. The spatial orientation of each type of nanoparticle in the cluster will be analyzed by high resolution TEM and TEM tomography at various tilt angles for a variety of nanoparticle compositions, surface coatings and stablilizing polymers. The NIR surface plasmon resonance and the spin-spin magnetic relaxivity will be measured and explained in terms of the cluster morphology. The cluster de-aggregation will be monitored in solution with spectroscopy and dynamic light scattering and in live cells with hyperspectral optical imaging and transmission electron microscopy.

Broader Impact:

This robust kinetic assembly platform for the design of biodegradable nanoclusters with high inorganic particle loadings for strong multifunctional properties will offer broad opportunities in microelectronics, sensors, imaging and optoelectronics. The simplicity and flexibility of this colloidal approach to form novel classes of nanoclusters will likely spawn numerous experimental and theoretical studies to understand the relationship between the optical/magnetic properties and nanocluster morphology. Furthermore, the optical/magnetic nanoparticles can provide solutions to one of the major challenges of modern medicine efficient delivery of therapeutics and molecular specific treatment of pathology with real-time imaging (photoacoustic imaging and MRI) for guidance and monitoring. The biodegradation of nanoclusters into primary nanoparticles can overcome the major roadblock in nanotechnology that is toxicity upon accumulation in humans and in the broader environment.

A key theme will be to show young students that engineering can play a major role in improving health care, by integrating scientific concepts in chemistry and biology to address practical problems. The PIs will develop educational material, laboratory experiments and provide student teachers for the nationally renowned UTeach Outreach program, which provides undergraduate students to serve as volunteer instructors for science lessons in the Austin Independent School District. In the UTeach Young Scientists Summer Camps, rising sixth grade students from heavily Hispanic elementary Schools will come to University of Texas for one week to participate in hands-on inquiry-based science lessons that stress academic rigor. The PIs will add an engineering component to the science lessons and the summer camp to complement current efforts in chemistry and biology.

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 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): In the last decade there has been an exponential growth in nanotechnology research in cancer demonstrating that nanotechnology could provide unique and otherwise unattainable solutions to cancer management including very early cancer detection, accurate molecular specific diagnosis and treatment that diminishes side effects. However, achieving this promise is extremely challenging because it requires overcoming multiple constraints imposed by translational barriers in clinical applications of nanomaterials that is multiplied by complexity of cancer biology. Currently, there is a growing gap between new discoveries coming at a fast pace from academic labs and their translation into clinic. Therefore, there is an urgent need in addressing this gap in cancer nanotechnology translational pipeline. To this end, we have designed a novel training program to educate future leaders in the broad field of nanotechnology with specific interests in cancer-related applications, who are keenly aware of the needs and demands of clinical environment as well as of major challenges of translational research. We believe that the only way to train cancer translation minded Ph.D. researchers is to insert them into the environment of an outstanding cancer center. Therefore, our program is based on a close collaboration between The University of Texas MD Anderson Cancer Center and Rice University. As part of our program, we have developed a comprehensive plan for recruiting trainees from underrepresented minority groups that are historically underrepresented in health-related research, including women, minority individuals, and individuals with disabilities. Our training program includes multidisciplinary mentorship of translational research projects combined with multidisciplinary, hands-on coursework and seminar experiences. All trainees will work with at least two program faculty mentors (one from Rice and one from MD Anderson) to define and carry out an independent research problem. Didactic coursework will prepare them to contribute to research projects that directly address barriers to translation of nanotechnology-based approaches and to develop the skills needed to define and lead such projects. Incoming trainees will participate in a unique 2-week-long boot camp in Cancer Management and Nanotechnology that provides an overview of current opportunities and barriers in the field. Trainees will develop foundational background in the field by taking four courses related to translational cancer or nanotechnology topics. Trainees will gain an appreciation for federal resources to assist in cancer nanotechnology research by taking a trip to the NCI Nanotechnology Characterization Lab. Finally, trainees will gain important lab management skills by participating in a short hands-on course providing an introduction to laboratory and project management. At the end of the program, fellows will have a deep understanding of translational research in cancer nanotechnology, with the most important component being the demonstrated ability to carry out independent translational research in this challenging multidisciplinary field.

? DESCRIPTION (provided by applicant): The purpose of this application is to provide basic and translational investigators from the University of Texas MD Anderson Cancer Center (MDACC) with a small-animal, full-body optoacoustic imaging capable of facilitating and furthering their cancer research. The iThera Medical Multispectral Optoacoustic Tomography (MSOT) small-animal imaging system provides multi-wavelength optoacoustic imaging data throughout the full body of a mouse. A significant number of NIH-funded investigators work collaboratively at MDACC toward their common mission of eliminating cancer. The research foci of these investigators span basic, translational, and clinical domains, and therefore many of them rely on murine models of cancer to bridge the gap between laboratory-based development and clinical implementation. Specifically, the MSOT system will provide MDACC investigators with a powerful new tool toward development of molecular probes and therapeutics, the investigation of cancer biology in orthotopic or spontaneous tumor models, and longitudinal assessment of therapeutic efficacy. Currently, MDACC does not have an optoacoustic imaging system capable of full-body or orthotopic/spontaneous tumor imaging to conduct functional and molecular imaging of the mice used in these studies. The specific aim of this application is to acquire the iThera Medical MSOT system to enable MDACC investigators to conduct research in murine models for the purpose of improving the mechanistic understanding, diagnosis, and therapy of cancer. The principle investigator, key personnel, and all other users detailed in this application are fully able and committed to ensuring that the MSOT system will be successfully incorporated into MDACC's small-animal imaging core and leveraged to its full potential. As we highlight in this application, there are currently many NIH-funded investigators at MDACC who would significantly and immediately benefit from the acquisition of an MSOT system, which affords researchers with essential molecular and functional imaging capabilities. In addition, the MSOT system would become part of the small- animal imaging and therapy resources supported by MDACC's Small Animal Imaging Facility (SAIF), which is a shared institutional resource supported, in part, by our Cancer Center Support Grant (#: P30 CA16672; PI: R. DePinho). Lastly, although there are a significant number of investigators at MDACC who will utilize the MSOT system immediately, in time the technology could also benefit NIH-funded investigators at the other seven major medical research institutions at the Texas Medical Center in Houston, TX.

ABSTRACT. Compared to normal tissues, there are well documented and functionally important changes in mechanical properties of neoplastic cells and tumor tissue, including tissue stiffening, loss of elasticity, and densification. Clinically, high tumor stiffness correlates with aggressive subtypes of epithelial cancers and overall poor prognosis. The importance of mechanical deregulation is illustrated by evidence that cancer cells increase their metastatic potential when cultured on substrates closely resembling stiffness of tumor microenvironment. Mechanistically, a self-reinforcing link between cancer cell and ECM remodeling likely results in increased stiffness and progressive malignization of tumor cells. Despite a growing appreciation in the importance of understanding mechanical signaling in tumor biology, the methods for reliable high resolution measurements of tumor mechanical properties in 3D cell models and live tumors are severely lacking. Therefore, there is an important need to overcome this technological limitation in order to advance our understanding of cancer progression and therapeutic strategies to revert this process. Here we address this limitation by developing a new high-spatial resolution method for nanobomb-Optical Coherence Elastography (nb-OCE). OCE is an emerging optical non-invasive biomechanical imaging method that, in principle, can detect tissue stiffness with micrometer resolution. However, existing excitation and detection methodologies for force measurements are limited in their ability to produce highly localized mechanical stress that is needed for high-resolution 3D elastography mapping. To solve this limitation we propose novel ?nanobomb? contrast agents for OCE that are based on lipid-coated perfluorocarbon (PFC) nanodroplets with embedded light absorbing dyes. Illumination of PFC ?nanobombs? by a pulsed laser triggers liquid to gas transition of PFC nanodroplets due to heating produced by light absorbing chromophores in the PFC core. Our preliminary data showed that this liquid-gas phase transition induces highly localized mechanical stress that can be detected by OCE with a high signal-to-noise ratio (SNR). Further, this ?bursting? of PFC nanobombs and their expansion can be effectively triggered by a femtosecond laser allowing a straightforward combination of the proposed here nb-OCE and multi-photon microscopy (MPM). This combination will provide an unprecedented opportunity to measure tissue elasticity with high resolution in the context of tissue morphology and function given by MPM. Here we assembled a team of experts in nanotechnology (Sokolov), OCE instrumentation/data analysis (Larin) and multiphoton intravital imaging/tumor biology (Friedl) to demonstrate feasibility of this combined technology in 3D spheroid cell cultures in vitro and in window tumor models in small animals. This MPM-nbOCE technology will enable longitudinal monitoring of evolution of tissue elasticity landscape during both tumor growth and invasion thus addressing a critical gap in our understanding of tumor biology.

Abstract. Pancreatic cancer is one of the most aggressive human malignancies, with a yearly incidence that equals its mortality. Radiation therapy (RT) is an integral component of modern therapy for locally advanced unresectable pancreatic cancers. However, its ultimate utility is severely limited by the fact that some cancer cells are resistant to RT. This problem is further amplified by the presence of gastrointestinal mucosa immediately adjacent to the tumor that makes dose escalation difficult and often not readily achievable. A novel approach to enhancing the radiation dose delivered to tumors is by transiently increasing the radiation-interaction probability of the target tissues using high atomic number (Z) nanomaterials. However, pancreatic cancer is characterized by hypovascularity in the setting of a dense stromal component that serves as a formidable physiological barrier to the delivery of drugs and nanoparticles. Therapeutic strategies, which can bypass the desmoplasia `fortress' and apply therapy without significantly affecting healthy cells and tissues would address the critical issues inherently presented by the pancreatic cancer. Here we propose to solve this delivery challenge by a paradigm shift from delivery of pre-made high-Z nanoparticles to an atomic size gold precursors (i.e., gold ions) for tumor radiosensitization thus achieving the ultimate reduction in size of a therapeutic agent ? an atomic scale. Our hypothesis is that small gold ions (i) will uniformly distribute throughout the tumor as their diffusion is not likely to be impeded by the stroma, and (ii) will be reduced to gold nanoparticles (GNPs) by cancer cells that (iii) will result in cancer cell radiosensitization to RT. This hypothesis is based on our compelling preliminary data demonstrating efficient synthesis of GNPs from gold ions inside pancreatic cancer cells but not normal cells. Further, the biosynthesized GNPs exhibited a high nuclear localization that is critical for efficient radiosensitization due to a higher dose delivery to nuclei by the secondary Auger electrons. In addition, a number of recent reports demonstrated intracellular synthesis of GNPs from chloroauric acid by mammalian cells with a preferential nuclear localization of the nanoparticles further supporting our hypothesis. Interestingly, this phenomenon has not been previously considered for applications in radiotherapy. We see it as a highly innovative and exciting opportunity to greatly improve radiosensitization efficiency of cancer cells in situ. We envision clinical implementation of our approach as an added boost to significantly increase efficacy of stereotactic body radiotherapy in patients with a pancreatic tumor. Recent clinical data from our group and others shows that radiation dose enhancement increases overall survival of locally advanced pancreatic cancer patients. However, the proximity of gastrointestinal mucosa to the tumor in many instances precludes this dose escalation in clinical practice. We expect that changing the current paradigm from delivery of pre-made GNPs to in situ synthesis of GNPs by cancer cells will overcame delivery barriers in pancreatic tumors and will result in a highly significant sensitization of pancreatic cancer cells to RT that can greatly improve treatment outcomes.

Abstract. Photoacoustic imaging (PAI) is a promising modality that is non-ionizing, low-cost, and offers high- contrast and high-spatiotemporal-resolution imaging in a platform that is amenable for high-throughput preclinical use and for specific clinical applications. However, widespread use of molecular PAI is severely limited by availability of validated contrast agents. Currently available contrast agents either do not have adequate photostability under the pulsed illumination that is required for PAI, lack sufficient PAI-signal-generation ability for deep imaging, or their absorbance spectra significantly overlap with those of hemoglobin, which reduces imaging sensitivity. In order to address these limitations, a new class of PAI contrast agents was proposed that is based on phase-changing perfluorocarbon (PFC) nanodroplets (NDs). These agents are based on a liquid PFC core and a light-absorbing ?fuse? in the form of a dye or a nanoparticle. Illumination of these NDs with a pulsed laser triggers liquid-to-gas transition of the PFC core heated by light-absorbing chromophores that results in a very strong PAI signal. Therefore, these agents are often referred to as Laser-Activated NDs (LANDs). Furthermore, after laser excitation PFC microbubbles can re-condense back into their liquid nanodroplet form, which can allow multiple excitations and the possibility for dynamic imaging contrast and super-resolution PAI. However, evaluation of this exciting contrast agent design by multiple research groups revealed one critical limitation ? commonly used dye molecules or nanoparticles are not soluble or mixable with perfluorocarbons. Therefore, current LANDs contain their ?fuses? (i.e., dye absorbers) in the shell with a loading efficiency and distribution of the dyes that is highly variable depending on specifics of a LAND's coating and a dye's chemical structure. Importantly, in addition to the limitations associated with irreproducibility and a shot shelf-life of LANDs due to leakage of dye molecules from a LAND's shell, recent studies of phase-changing NDs showed the advantage of heating LANDs from within the core for an effective liquid-to-gas transition. These data underline the importance of heating inside an ND's core for activation of LANDs that cannot be effectively achieved with peripherally located chromophores. Here we propose to address weaknesses of the prior research by developing fluorinated dyes with absorbance in the first and second near-infrared tissue windows (NIR-I and NIR-II). Our hypothesis is that the fluorinated dyes will be soluble inside the PFC core, thus resulting in highly reproducible, stable LAND formulations with greatly improved laser activation efficacy. To reflect these advancements in LAND formulation, we refer to PFC NDs doped with fluorinated dyes as enhanced LANDs (eLANDs). We posit that an increase in concentration of uniformly distributed fluorinated dyes inside the PFC core will dramatically improve efficacy of eLAND's activation. Our estimates show that this gain in activation efficacy could be associated with a highly significant (on the order of centimeters) increase in depth sensitivity of PAI with eLANDs; such increase in depth penetration could be a game changer in molecular PAI in pre-clinical and clinical settings.