Synthetic copolymers for multivalent ligand synthesis and data-driven enzyme stabilization
Creators: name: Kosuri, Shashank
Publisher: name: No Publisher Supplied
Year: 2022

description: Synthetic polymers have revolutionized the field of biomedical engineering and continue to permeate the space with applications ranging from tissue engineering to drug delivery. The ability to utilize them for a wide variety of applications results from the ability to fine tune their physicochemical parameters such as chain length, composition, valency and architecture as needed. However, until recently, studying structure-function relationships utilizing polymer scaffolds remained elusive due to the oxygen intolerant nature of radical polymerization techniques which limit the number of polymers that can be made and tested in a high throughput (HTP) manner. Development of oxygen tolerant techniques such as enzyme-assisted reversible addition fragmentation chain transfer polymerization (Enz-RAFT) and photoinduced electron/energy transfer RAFT (PET-RAFT) now allow polymer chemists to synthesize complex polymer architectures on benchtops in well plates. While HTP techniques are highly desirable for studying structure-function relationships, it is also important to understand the limitations that arise out of rational design and screening approaches. This is mainly due to the large diversity of the chemical landscape available to choose the materials from as well as the complex interactions between polymers and respective bio-interfaces. Therefore, there is a need to couple HTP screening approaches with data-driven design to provide experimental feedback for designing new materials. In this regard, we hypothesize that structure-activity testing is optimized through the development of reliable and robust HTP tools and by coupling them with data-driven design to improve upon existing designs. To address these challenges, this thesis focuses on developing a platform technology for synthesis of multivalent polymer scaffolds and utilizes a “Design-Build-Test-Learn” strategy for identifying novel copolymers that stabilize enzymes under thermally challenging conditions. The first part of the dissertation focuses on developing a HTP strategy for synthesis of multivalent polymer scaffolds libraries in a simple, dual wavelength, two step polymerize and click approach. This was achieved by synthesizing a cyclopropenone-masked dibenzocyclooctyne (cp-DIBAC) acrylamide monomer that was copolymerized into the side chain of linear polymers or as an end group of star polymers using photopolymerization in 384 well plates. This functional monomer can be unmasked using ultraviolet (UV) light resulting in generation of an alkyne moiety which was then used for bioconjugation using strain promoted azide-alkyne click chemistry (SPAAC). Utilizing this technique, we were able to synthesize a large library of linear and star shaped copolymers with precise control over valency and position of ligands conducive to HTP synthesis. After copolymerization, the synthetic scaffolds were deprotected using UV light and PEG-azides were “clicked” onto the polymer backbone using SPAAC. We demonstrated remarkable control over post-polymerization modification by achieving &gt;70% conjugation efficiency which was confirmed using UV-VIS, NMR spectroscopy and size exclusion chromatography.The second part of the dissertation focuses on utilizing a data-driven combinatorial approach for synthesizing novel copolymers to stabilize enzymes from thermal stress. Four enzymes were stabilized using this approach, namely, horseradish peroxidase (HRP), glucose oxidase (GOx), lipase and chondroitinase ABC (ChABC). Particular focus is devoted to ChABC because of its application in treating spinal cord injuries. ChABC, a 115 kDa protein derived from proteus vulgaris, has shown to be a promising candidate for treating SCIs because of its ability to degrade chondroitin sulfate proteoglycan (CSPG) side chains in the scar tissue and promote tissue regeneration. However, a major drawback that limits its usage is its thermal instability at physiological temperature. The enzyme is thermally unstable and loses all activity within 24 hrs at 37°C. Therefore, in order to maintain therapeutic efficacy, it needs to be continuously administered in large doses, which is not feasible and also highly expensive. Therefore, there is an immediate need to thermostabilize ChABC to increase its therapeutic potential. Recently, polymer-enzyme complexes (PECs) are gaining a lot of attention because of their ability to stabilize enzymes and retain their activity in non-native environments. In these complexes, the enzyme is wrapped around in a synthetic copolymeric shell that protects it from denaturation by directly controlling the enzyme’s surrounding microenvironment. We hypothesized that by carefully designing a synthetic copolymer that matches the surface characteristics of ChABC, we will be able to stabilize it and retain its activity for longer durations at 37°C. We initially designed a seed library of over 500 methacrylate/methacrylamide based copolymers using automated photopolymerization and tested their ability to retain ChABC activity for 24 hrs at 37°C in simulated cerebrospinal fluid (sCSF). Polymer compositional information along with retained enzyme activity (REA) data were then utilized to train Gaussian process regressor (GPR) models coupled with Bayesian optimization (BO) to predict new copolymer designs. Using this active learning approach, three iterations of 24 copolymer candidates were proposed for experimental synthesis and testing. By the end of the iterative process, significant improvement in enzyme activity retention was observed across generations. One best polymer composition was then selected to perform long term stability study and we were able to retain 30% activity of ChABC at the end of one week at 37°C while native enzyme lost all activity within 24 hrs. Polymer cytotoxicity and inflammatory profiles were studied on cultures of astrocytes and were found to be non-cytotoxic and non-inflammatory at the concentrations tested. These encouraging results motivate further preclinical studies to test the efficacy of PECs to degrade scar tissue and promote tissue regeneration in relevant animal models. In conclusion, the results from the studies discussed in this dissertation indicate the importance of HTP tools and the need to couple them with active learning for studying structure-function relationships to design advanced polymeric materials that can elicit required biological function.