Kevin D. Belfield, University of Central Florida Komatsu, Masanobu, Sanford-Burnham Medical Research Institute Development of Novel Two-photon Fluorescence Polymer Probes for High Resolution Deep Tissue Intravital Imaging
Significance Biophotonic materials and processes are fast becoming pervasive in many new and emerging technologies from telecommunications to biomedical imaging, technologies that have far reaching impact on society. In vivo imaging, coupled with efficient multifunctional nanoprobes, promises to transformationally impact the field of nanomedicine and diagnostics in a manner that may have profound effects on societal well being. Two-photon fluorescence microscopy (2PFM) for deep tissue imaging is a developing technology with unique subcellular resolution. To take full advantage of 2PFM, fluorescence probes with high two-photon absorption (2PA) cross-sections and high fluorescence quantum yields are critical all the while being nontoxic and heavy-metal free. The future of nanomedicine and diagnostics lies in the development of multifunctional nanoplatforms that combine both highly specific targeting and efficient imaging functionality, facilitating temporal and spatial site-specific imaging, not in carefully controlled cell culture but in vivo in living tissue. This highly interdisciplinary proposal aims to develop advanced polymer-based probes for in vivo 2PFM angiogenesis imaging. In vivo 2PFM may provide real-time, minimally invasive monitoring of tumor progression, a tumor's response to anti-cancer therapies, and wound healing in much greater detail than is currently known. This project constitutes an excellent platform for interdisciplinary research training and education in advanced materials and microscopy techniques. Students that are traditionally underrepresented in science and engineering will be especially encouraged to participate in this project. There will be strong international engagement through Belfield's participation in the European Commission's FP7 Marie Curie Actions program, supporting the development of an interdisciplinary-trained, diverse, globally adept, highly-skilled workforce.
Few efforts have been reported on approaches to push the limits and improve the contrast and quality of two-photon fluorescence microscopy (2PFM) imaging in tissue. In this proposal, we propose to dramatically improve probe performance and targeting selectivity in 2PFM imaging through controlled multivalency, integrating multiple highly efficient 2PA, near-IR emitting fluorophores and vectors that target specific biomarkers in a polymeric nanoplatform. This approach is expected to enhance cellular interaction through increased avidity and bioavailability due to the small size and biocompatibility of the nanoprobe. We propose a polymeric nanoplatform approach to prepare well-defined probes that provide high local fluorophore concentration to achieve high contrast imaging and multiple targeting moieties on the same polymer chain to enhance avidity, hence reducing the dose needed. This is expected to provide optical sectioning with subcellular resolution from deeper within tissue and make it possible to track biologically important events, such as sprouting of vessel tip cells, in three dimensions. Tumor angiogenesis is a complex biological process that can only be studied in living animals. However, techniques for the study of tumor angiogenesis in vivo are acutely lacking. We will push the limits of in vivo 2PFM by studying angiogenesis and vascular cell growth and movement, processes important for the pathogenesis of a number of diseases, while helping us gain a better understanding of the biology of tumor angiogenesis.
