Integrated Experimental and Modeling Study of Enzymatic Degradation Using Novel Autofluorescent BSA Microspheres Xiaoyu Ma, †,‡ Ji-Qin Li, §,‡ Christopher O’Connell, ∥ Tai-Hsi Fan,* ,§ and Yu Lei* ,⊥ † Department of Biomedical Engineering, University of Connecticut, Storrs, Connecticut 06269-3247, United States § Department of Mechanical Engineering, University of Connecticut, Storrs, Connecticut 06269-3139, United States ∥ Biotechnology-Bioservices Center, University of Connecticut, Storrs, Connecticut 06269-3149, United States ⊥ Department of Chemical & Biomolecular Engineering, University of Connecticut, Storrs, Connecticut 06269-3222, United States ABSTRACT: Autofluorescent bovine serum albumin (BSA) hydrogel microspheres were prepared through the spray- drying of glutaraldehyde cross-linked BSA nanoparticles and then used for a proteinase K based degradation study in an aqueous solution. Experimental results and empirical models are presented to characterize the kinetics of BSA hydrogel microsphere degradation, as well as the accompanying release of synthesized fluorophore. The BSA gel degradation dynamics is primarily controlled by the concentration of proteinase K within the Tris buffer. The coupling of swelling dynamics and the transient distributions of fluorophore are traced by confocal microscopy. Models are developed based on the linear theory of elastic deformation coupled to enzyme and fluorophore transport. This study represents a fundamental investigation of the degradation and release kinetics of protein-based materials, which can potentially be applied for the dynamic and photostable tracking of relevant in vivo systems. 1. INTRODUCTION Biodegradability and biocompatibility are well-known signifi- cant functionalities of synthetic biomaterials. 1,2 Controlled material degradation further allows for sustained release design for various biomedical applications. The advantages of controlled degradation are the avoidance of fast clearance and the availability of further access to biological response along a desired period of time. Specifically in tissue engineering, materials are often intended to degrade after serving as a temporary scaffold for cell therapy or tissue regeneration. 2 Among all materials, natural polymers such as polysaccharides and many other proteins have attracted a great deal of attention because of their biocompatibility, in vivo affinity to tissues and scaffolds, and controllable degradation properties. 3−7 More specifically, natural-polymer-based hydrogels have been widely explored in biomedical applications for drug delivery, medical diagnostics, biological tracking, and as personal care prod- ucts. 8−11 Hydrogels can absorb large amounts of water and exhibit three-dimensional structures. Protein-based hydrogels are important biodegradable and biocompatible materials, primarily because of their intrinsic high affinity to tissues and enzymatic degradation properties. Hydrogels made of bovine serum albumin (BSA) have been extensively studied, because of its low cost, good solubility and stability, and excellent ligand- binding accessibility and intrinsic fluorescence emission proper- ties. 12−15 Although several attempts to quantify the degradation of the synthesized materials using fluorescence imaging have been reported, 16,17 physics-based modeling and quantification of the process are still lacking. Moreover, in our previous studies, we proposed the comparison of in vivo fluorescence imaging and phenomenological models to systematically investigate protein-based hydrogels with different sizes. 15,18,19 However, the dynamic degradation of the protein-based hydrogel matrix and the relevant transport mechanisms at the single-microsphere level are not well understood. In this study, spray-dried autofluorescent BSA microspheres were fabricated according to the method reported in our previous study 15 and used for the further investigation of their enzymatic degradation and the release of the fluorophores. The use of autofluorescent microspheres has more advantages than the use of fluorescent microspheres with embedded chemical fluorophores. This is because autofluorescent materials can avoid photobleaching and leaking problems that exist in the majority of fluorescent materials. The diminishing of the fluorescence intensity can accurately reflect the degradation of the materials, which prevents complications from the decay of fluorescence intensity due to the leaking or photobleaching of chemical fluorophores. This advantage opens up many possibilities for using autofluorescent microspheres in investigations involving degradation kinetics. Figure 1 illustrates the problem at hand: a dynamic swelling and degrading autofluorescent hydrogel microsphere triggered by proteinase K in Tris buffer solution. The microspheres are about 2−4 μm and covalently immobilized on the surface of a cover glass. The enzymatic degradation of the BSA gel results in the swelling of the microspheres and the diminishing of the Received: August 30, 2017 Revised: November 10, 2017 Article pubs.acs.org/Langmuir Cite This: Langmuir XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.langmuir.7b03057 Langmuir XXXX, XXX, XXX−XXX