Collaborative Research: Development of Novel Two-photon Fluorescence Polymer Probes for High Resolution Deep Tissue Intravital Imaging

1403525/1403535 Kevin D.Belfield，中央佛罗里达大学小松，Masanobu，Sanford-Burnham医学研究所新型双光子荧光聚合物探针的开发用于高分辨率深部组织生命内成像意义生物光子材料和工艺正在迅速普及到许多新兴技术中，从电信到生物医学成像，这些技术对社会产生了深远的影响。活体成像与高效的多功能纳米探针相结合，有望以一种可能对社会福祉产生深远影响的方式，对纳米医学和诊断领域产生革命性的影响。用于深部组织成像的双光子荧光显微镜(2PFM)是一种新兴的技术，具有独特的亚细胞分辨率。为了充分发挥2PFM的优势，具有高双光子吸收截面和高荧光量子产率的荧光探针一直是无毒和无重金属的关键。纳米医学和诊断学的未来在于多功能纳米平台的开发，这种平台结合了高度特异的靶向和高效的成像功能，促进了时间和空间部位的特定成像，不是在精心控制的细胞培养中，而是在活体组织中。这项高度跨学科的提案旨在开发先进的基于聚合物的探针，用于体内2PFM血管生成成像。在体内，2PFM可以提供实时的、微创的监测肿瘤进展、肿瘤对抗癌治疗的反应以及伤口愈合的细节，比目前已知的要详细得多。该项目为先进材料和显微技术的跨学科研究、培训和教育提供了一个极好的平台。我们将特别鼓励那些在科学和工程领域传统上代表性不足的学生参与这一项目。通过Belfield参与欧盟委员会的FP7玛丽·居里行动计划，将有强大的国际参与，支持发展一支受过跨学科培训的、多样化的、全球熟练的、高技能的劳动力队伍。技术说明关于如何突破极限并提高组织中双光子荧光显微镜(2PFM)成像的对比度和质量的方法，已有一些报道。在这项建议中，我们建议通过控制多价性，将多个高效的2PA、近红外发射荧光团和靶向特定生物标志物的载体整合在一个聚合物纳米平台上，从而显著提高2PFM成像中的探针性能和靶向选择性。由于纳米探针的小尺寸和生物兼容性，这种方法有望通过提高亲和力和生物利用度来增强细胞相互作用。我们提出了一种聚合物纳米平台的方法来制备定义良好的探针，提供高局部荧光团浓度，以实现高对比度成像和同一聚合物链上的多个靶向部分，以增强亲和力，从而减少所需的剂量。这有望提供从组织内部更深层次的亚细胞分辨率的光学切片，并使其能够以三维方式跟踪重要的生物学事件，如血管尖端细胞的萌发。肿瘤血管生成是一个复杂的生物学过程，只能在活体动物中进行研究。然而，在体内研究肿瘤血管生成的技术严重缺乏。我们将通过研究血管生成和血管细胞的生长和运动来推动体内2PFM的极限，这些过程对许多疾病的发病机制至关重要，同时帮助我们更好地了解肿瘤血管生成的生物学。

1403525/1403535Kevin D. Belfield, University of Central FloridaKomatsu, Masanobu, Sanford-Burnham Medical Research Institute Development of Novel Two-photon Fluorescence Polymer Probes for High Resolution Deep Tissue Intravital ImagingSignificanceBiophotonic 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.Technical DescriptionFew 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.