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.
Controlled release devices have the potential to improve therapeutic outcomes for millions of patients worldwide; however, evaluating the performance of these devices in the body using existing techniques is costly and time-consuming. This proposal aims to validate and optimize a non-invasive, fluorescence-based approach for studying device function in the body that is less expensive, faster, and more accessible then current methods. If successful, this approach could serve as a major enabling technology for preclinical device evaluation and facilitate translation to the clinic for applications ranging from cancer and diabetes to tissue engineering.
| McHugh, Kevin J; Nguyen, Thanh D; Linehan, Allison R et al. (2017) Fabrication of fillable microparticles and other complex 3D microstructures. Science 357:1138-1142 |
