Fluorescent Probes for Tracking Insulin Secretion in Therapeutic and Diagnostic Devices

 描述(由申请人提供)：意义：随着世界各地患者数量的激增，1型和2型糖尿病(T1D和T2D)正在成为社会经济资源的日益沉重的负担。胰岛素依赖治疗方案，如定期注射胰岛素，已经得到了很好的证实，但与患者生活质量的负面影响和各种长期并发症有关。40多年来，寻找胰岛素非依赖性治疗方案一直是一个重要的科学目标，尤其是在胰岛移植领域取得了令人振奋的结果。在这里，要么移植整个身体胰腺，要么移植被包裹的胰岛，这些胰岛由保护性胶囊中的胰岛素分泌β细胞组成，一直是研究的主要重点。然而，可利用的身体胰腺组织很难获得移植成功，主要受β细胞的细胞存活限制。另一个需要解决的问题是如何根据胰岛素释放活动来选择功能性胰岛，以实现更一致和更可预测的移植结果，并帮助患者在更长的一段时间内保持胰岛素独立。到目前为止，还没有一种简单、高效的方法可以在这一监测过程中用于量化单个胰岛的胰岛素分泌。此外，这种技术还可以用于获得诊断信息，以便在确定糖尿病进展和病因时有效地监测胰岛素分泌。假设：我们假设我们的荧光显像剂，即ZIMIR，在用于荧光激活细胞分选(FACS)或ZIMIR功能化光纤等技术时，有能力解决这些治疗和诊断问题。初步数据：我们已经证明，ZIMIR是锌敏感荧光剂的先锋，具有标记β细胞和成像胰岛素分泌的能力，这两个功能都是筛选功能性胰岛、包膜胰岛或β细胞的基本特征。此外，我们发现ZIMIR足够灵敏，可以量化门静脉内可通过荧光检测到的锌离子浓度的增加，并与胰岛素分泌直接相关，这是设计能够定量体内胰岛素分泌的设备的关键前提。具体目标：第一阶段的主要目标包括大量ZIMIR(目标1)和ZIMIR-马来酰亚胺(目标2)的合成、纯化和表征。后者将用于第二阶段，用于由二氧化硅组成的改性光学纤维的功能化。然后这些纤维将被用来构建纤维光度学 锌离子检测仪，经体外和体内验证。ZIMIR将用于该试剂的临床前测试的第一阶段，以在随后的移植实验中使用FACS进行胰岛分类。总体影响：VitalQuan开发的这些技术将共同展示ZIMIR在ISELT细胞分选应用中的效用，以提高胰岛移植成功率，并将建立史无前例的体内胰岛素跟踪研究。这些应用结合在一起，将加速ZIMIR的商业化，用于糖尿病的临床研究和治疗。

 DESCRIPTION (provided by applicant): Significance: Type 1 and Type 2 Diabetes Mellitus (T1D and T2D) are becoming an increasing burden on socioeconomic resources with surging patient numbers all over the world. Insulin-dependent treatment regimen such as regular insulin injections are well established but have been associated with a negative impact on the patients' quality of life with a variety of long-term complications. The search for insulin-independent treatment options has been an important, albeit elusive, scientific goal for over 40 years with promising results particularly in the field of pancreatic islet transplantation. Here, grafting of either entire cadaveric pancreases or encapsulated islets comprised of insulin producing beta cells in a protective capsule has been the main focus of research. However, usable cadaveric pancreatic tissue is hard to acquire with graft success mostly limited by the cellular survival of the beta cells. Another issue that needs to be addressed is how to select functional islets based on their insulin release activity, in order to achieve a more consistent and more predictable transplant outcome, and to help patients maintain insulin independence over a longer period of time. No simple method with high efficacy exists to date that could be useful in this monitoring process to quantify insulin secretion of individual islets. Furthermore, such a technique can also be adapted to gain diagnostic information in an effort to monitor insulin secretion efficiently when determining diabetes progression and etiology. Hypothesis: We hypothesize that the use of our fluorescent imaging agent, namely ZIMIR, has the ability to solve these therapeutic and diagnostic problems when used in technologies such as fluorescence-activated cell sorting (FACS) or in ZIMIR-functionalized optical fibers. Preliminary Data: We have demonstrated that ZIMIR represents the vanguard of a zinc sensitive fluorescent agent with the ability to label beta cells and image insulin secretion, both essential features for the screening of functional islets, encapsulated islets, or beta cells. Furthermore, we showed that ZIMIR is sensitive enough to quantify a concentration increase of Zn2+ that can be detected by fluorescence at the portal vein and directly correlated to insulin secretion, a crucial prerequisite for the design of a devic that can quantify insulin secretion in vivo. Specific Aims: The key objectives in Phase I entail th synthesis, purification and characterization of a large amount of ZIMIR (Aim 1) and ZIMIR-maleimide (Aim 2). The latter will be used in Phase II for the functionalization of modified optica fibers comprised of silicon dioxide. These fibers will then be used to construct a fiber photometry instrument for the detection of Zn2+, validated in vitro and in vivo. ZIMIR will be used in Phase I for preclinical testing of the agent to find use in islet sorting using FACS with subsequent transplantation experiments. Overall Impact: Together, these technologies developed by VitalQuan will demonstrate the utility of ZIMIR in iselt cell sorting applications to increase isle graft success rates and will also establish unprecedented studies designed for in vivo insulin tracking. Taken in concert, these applications will accelerate the commercialization of ZIMIR for clinical use in diabetes research and treatment.