Fluorescence-based molecular imaging of in vivo release kinetics

项目摘要/摘要 控释装置有可能通过以下方式改善治疗结果和患者的依从性 提供长期、局部的药物输送，使药物浓度维持在治疗窗口内。 这一方法已被证明对包括激素替代疗法在内的许多应用是有效的，1 避孕，2和癌症治疗，3但这些设备的开发用于更多的应用 由于无法轻松研究他们在体内的行为，继续受到阻碍。在过去的体外研究中 评估可生物降解聚合物基质或水凝胶的治疗释放简单且廉价，但 由于水合作用的不同，经常会产生不能代表体内释放动力学的结果， 缓冲、对流和酶活性。不幸的是，非侵入性的临床前成像方式 可用于研究体内释放，如磁共振成像(MRI)、计算机断层扫描 (CT)、正电子发射断层扫描(PET)和单光子发射计算机断层扫描(SPECT) 价格昂贵，吞吐量低，可能需要造影剂，其释放不能代表 4这个项目的目的是验证和优化一种高通量的 跟踪对照荧光标记蛋白的耗竭研究体内释放动力学 释放设备。这种方法比现有的方法更快，成本更低，而且可能更安全 可供选择，也允许研究蛋白质特定的动力学，这可能与模型有很大不同 蛋白质。简而言之，这项技术将使用商业上可用的荧光标记试剂盒，基于SIMPLE 马来酰亚胺或N-羟基琥珀酰亚胺酯化学，通过硫醇或伯胺标记蛋白质， 然后，荧光标记的蛋白质将被封装到聚合物装置中(例如， 微粒、支架)并植入或注射体内。一项由体内周期性研究组成的纵向研究 将进行荧光测量以评估在受控释放中剩余的蛋白质的量 随着时间的推移，设备。为了验证这种方法的准确性，PET，一种低通量但高度定量的 技术，将使用双标记荧光团的蛋白质和一个 放射性同位素。荧光强度下降与衰变校正正电子强度下降的相关性 发射将被用来证明蛋白质含量和荧光之间的定量关系 信号。基于荧光的释放的稳健性将使用各种荧光团、材料、 装置几何形状和植入位置，以减轻组织吸收、光漂白、 以及荧光团对pH的敏感性，否则可能会对这种方法的准确性产生负面影响。

PROJECT SUMMARY/ABSTRACT Controlled release devices have the potential to improve therapeutic outcomes and patient compliance by providing prolonged, localized delivery of drugs that maintain concentrations within the therapeutic window. This approach has proven effective for a number of applications including hormone replacement therapy,1 contraceptives,2 and cancer therapy,3 yet the development of these devices for additional applications continues to be hindered by an inability to easily study their behavior in the body. In vitro studies used to assess therapeutic release from biodegradable polymer matrices or hydrogels are simple and inexpensive, but frequently yield results that are not representative of in vivo release kinetics due to differences in hydration, buffering, convection, and enzyme activity. Unfortunately, non-invasive preclinical imaging modalities that could be used to study in vivo release such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) are expensive, low-throughput, and may require contrast agents whose release is not representative of the therapeutic protein of interest.4 This project aims to validate and optimize a high-throughput technique for studying in vivo release kinetics by tracking the depletion of fluorescently labeled proteins from controlled release devices. This approach is faster, considerably less expensive, and potentially safer than existing alternatives and also allows for the study of protein-specific kinetics which could differ greatly from model proteins. In brief, this technique will use commercially available fluorescent labeling kits based on simple maleimide or N-hydroxysuccinimide ester chemistry to label proteins via thiols or primary amines, respectively.5 Fluorescently labeled proteins will then be encapsulated into polymeric devices (e.g. microparticles, scaffolds) and implanted or injected in vivo. A longitudinal study consisting of periodic in vivo fluorescence measurements will be performed to assess the amount of protein remaining in controlled release devices over time. To validate the accuracy of this approach, PET, a low-throughput but highly quantitative technique, will be performed in parallel using proteins that are double labeled with fluorophores and a radioisotope. Correlation between the decrease in fluorescence and decrease in decay-corrected positron emission will be used to demonstrate the quantitative relationship between protein content and fluorescent signal. The robustness of fluorescence-based release will be evaluated using various fluorophores, materials, device geometries, and implant sites in order to mitigate the influence of tissue absorbance, photobleaching, and fluorophore pH-sensitivity that could otherwise negatively impact the accuracy of this approach.