Protein-Based Autofluorescent Hydrogel and Nano/Micro-Particles for Bio-Imaging Applications Xiaoyu Ma University of Connecticut - Storrs, [email protected]
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University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 9-7-2017 Protein-based Autofluorescent Hydrogel and Nano/Micro-Particles for Bio-imaging Applications Xiaoyu Ma University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Ma, Xiaoyu, "Protein-based Autofluorescent Hydrogel and Nano/Micro-Particles for Bio-imaging Applications" (2017). Doctoral Dissertations. 1620. https://opencommons.uconn.edu/dissertations/1620 Protein-based Autofluorescent Hydrogel and Nano/Micro- Particles for Bio-imaging Applications Xiaoyu Ma, PhD University of Connecticut, 2017 Fluorescent polymeric materials such as hydrogels and polymeric particles have been attracting attention in many biomedical applications including bio-imaging, optical sensing, tissue engineering and therapy, due to their good biocompatibility, biodegradability, and advanced optical property. This PhD project aims at developing novel autofluorescent protein materials in different configurations with good biocompatibility and biodegradability for bio-imaging applications. Early research focused on the development of autofluorescent protein hydrogels for in vivo bio- imaging application. Glutaraldehyde cross-linked Bovine Serum Albumin (BSA) hydrogel were facilely prepared. Various advanced techniques were employed to characterize the as-prepared material. SEM study clearly revealed its 3-dimentional pore structure, while UV-vis spectra studies, in conjunction with the fluorescence spectra studies including emission, excitation and synchronous scans, indicated that three classes of fluorescent compounds are presumably formed during the gelation process. The autofluorescent hydrogel exhibited high toughness according to the compression and tensile tests. Finally its biocompatibility and biodegradability were demonstrated through extensive in vitro and in vivo studies. More interestingly, the in vivo degradation of autofluorescent hydrogel can be non-invasively tracked using fluorescence images, which provided a convenient way to model in vivo biodegradation of the protein hydrogel from a new perspective. The degradation/diffusion trends predicted by the proposed mathematical model were in good agreement with the time-dependent fluorescence images of mice. Xiaoyu Ma – University of Connecticut, 2017 Based on the aforementioned autofluorescent concept for BSA system, BSA autofluorescent nanoparticles dispersion and spray-dried microspheres were further fabricated, respectively. Their physical, optical, and biocompatible properties were extensively characterized and evaluated using SEM, FTIR, UV-vis spectra, fluorescence spectra, in vitro cytotoxicity assay, in vivo histological study, and/or Dynamic Light Scattering. The as-synthesized green and red fluorescent nanoparticles dispersion and microspheres were both applied for cell imaging, ascribing to their unique size properties. Also, in vivo degradation processes of these nanoparticles and microspheres in mouse model were also tracked via non-invasive fluorescence imaging and concurrently interpreted by the proposed mathematical model. To further study the degradation mechanism, in vitro degradation of the microspheres by proteinase K were recorded and tracked via Confocal Laser Scanning Microscopy (CLSM), which exhibited two degradation trends based on different concentrations of active enzyme. Microspheres exhibited the swelling of the micro-spherical matrix, accompanying with the decrease of the fluorescent intensity. This phenomena was ascribed to the relatively higher diffusing rate of the enzyme into microspheres matrix than that of the accompanying enzyme- based matrix degradation. A mathematical model was proposed to demonstrate the complexing of microsphere swelling, enzyme diffusion, and diffusion of the liberated fluorophores from enzyme-degraded BSA microspheres matrixes. As another application of the developed autofluorescent protein materials, the as-synthesized autofluorescent BSA nanoparticles have been applied for sensitive heme/hemin detection, and the ultrasensitive sensing performance is ascribed to Photo-induced Electron Transfer (PET) as well as specific interaction between hemin and the fluorescent protein nanoparticles. Xiaoyu Ma – University of Connecticut, 2017 Overall, this dissertation expands the cutting edge in the design and synthesis of autofluorescent materials with good biocompatibility and biodegradability for various biomedical applications. Protein-based Autofluorescent Hydrogel and Nano/Micro- Particles for Bio-imaging Applications Xiaoyu Ma B.S. Northwest A&F University, 2012 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2017 Copyright by Xiaoyu Ma 2017 ii APPROVAL PAGE Doctor of Philosophy Dissertation Protein-based Autofluorescent Hydrogel and Nano/Micro-Particles for Bio-imaging Applications Presented by Xiaoyu Ma Major Advisor_________________________________________________________________ Dr. Yu Lei Associate Advisor_______________________________________________________________ Dr. Christian Brückner Associate Advisor_______________________________________________________________ Dr. Mu-Ping Nieh Associate Advisor_______________________________________________________________ Dr. Xiuling Lu Associate Advisor_______________________________________________________________ Dr. Tai-Hsi Fan Associate Advisor_______________________________________________________________ Dr. Guoan Zheng University of Connecticut 2017 iii Acknowledgements First of all, I would like to express my sincere gratitude to my major advisor Prof. Yu Lei for his guidance throughout my graduate study. This work is impossible without his encouragement and support. Also, I wish to thank my advisory committee (Pro. Christian Bruckner, Pro. Mu-Ping Nieh, Pro. Xiuling Lu, Pro. Tai-Hsi Fan, and Pro. Guoan Zheng) for their valuable guidance, support, and feedback during my Ph.D. research. In addition, for the excellent group and cross-departmental collaboration, I acknowledge my labmates, visiting scholars and students in collaboration labs such as Xiangcheng Sun, Jun Chen, Qiuchen Dong, Donghui Song, Jing Bao, Swayandipta Dey, Derek Hargrove, and Jiqin Li, etc.. Most of all, I would like to express my love to my parents, boyfriend and family for their support and care. iv Table of Contents Chapter 1 Introduction ................................................................................................................ 1 1.1 Background of fluorescent natural polymer or natural-synthetic hybrid polymer based hydrogels and particles for bio-imaging. ....................................................................................................................1 1.2 Synthesis of natural polymer or natural-synthetic hybrid polymer based fluorescent hydrogels and particles ......................................................................................................................................................3 1.2.1 Polysaccharides based fluorescent polymeric materials ...............................................................3 1.2.2 Protein Based Fluorescent Polymer Blocks ..................................................................................8 1.3 Fluorescent Natural Polymer Hydrogels/Particles for Bio-imaging ..................................................14 1.3.1 In vivo bio-imaging .....................................................................................................................14 1.3.2 Cell imaging ................................................................................................................................22 Chapter 2 A Biocompatible and Biodegradable Protein Hydrogel with Green and Red Autofluorescence: Preparation, Characterization and In Vivo Biodegradation Tracking and Modeling ............................................................................................................................... 36 2.1 Introduction ........................................................................................................................................37 2.2 Experimental ......................................................................................................................................39 2.2.1 Cross-linked BSA or HSA Hydrogel ..........................................................................................39 2.2.2 Freeze-Dry BSA Hydrogel .........................................................................................................40 2.2.3 Characterization of the BSA hydrogel ........................................................................................40 2.2.4 In vitro cytotoxicity test ..............................................................................................................41 2.2.5 In vitro enzymatic biodegradation test ........................................................................................42 2.2.6 In vivo biodegradability and biocompatibility study ..................................................................42 2.3 Results and Discussions .....................................................................................................................43 2.3.1 UV-vis study of cross-linked BSA hydrogel. .............................................................................44 2.3.2 Fluorescent