Fluorescence-based molecular imaging of in vivo release kinetics

项目概要/摘要 控释装置有可能通过以下方式改善治疗结果和患者依从性： 提供长时间、局部的药物输送，将药物浓度维持在治疗窗内。 这种方法已被证明对许多应用有效，包括激素替代疗法，1 避孕药具2 和癌症治疗3 但这些设备的其他应用的开发 由于无法轻松地研究它们在体内的行为，继续受到阻碍。体外研究用于 评估可生物降解聚合物基质或水凝胶的治疗释放既简单又便宜，但是 由于水合作用的差异，经常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，非侵入性临床前成像方式 可用于研究体内释放，例如磁共振成像 (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) 昂贵、低通量，并且可能需要其释放不能代表实际情况的造影剂 4 该项目旨在验证和优化一种高通量技术 通过跟踪受控荧光标记蛋白的消耗来研究体内释放动力学 释放装置。与现有方法相比，这种方法速度更快，成本更低，而且可能更安全 替代方案，还可以研究可能与模型有很大不同的蛋白质特异性动力学 蛋白质。简而言之，该技术将使用基于简单的市售荧光标记试剂盒 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， 5 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。一项纵向研究，包括定期体内 将进行荧光测量以评估受控释放中剩余的蛋白质量 随着时间的推移设备。为了验证该方法的准确性，PET（一种低通量但高度定量的方法） 技术，将使用双标记荧光团和 放射性同位素。荧光减少与衰变校正正电子减少之间的相关性 发射将用于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、 装置的几何形状和植入部位，以减轻组织吸光度、光漂白、 和荧光团 pH 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.