(269d) An Integrated Fabrication-Conjugation Approach for Biopolymeric-Synthetic Hybrid Polymeric Microspheres and Improved Protein Conjugation Capacity and Kinetics Via Robust Micromolding and Bioorthogonal Click Reactions

Hydrogel microparticles with controlled structures have gained increasing attention in various biomedical applications as biosensing platforms, drug carriers, and building blocks for tissue engineering.  However, it still remains challenging to fabricate such microparticles in a simple and reliable manner.  We present facile fabrication of chemically functional and monodisperse biopolymeric–synthetic hydrogel microspheres with controlled macroporous structures that can be utilized as conjugation platforms for large biomolecules.  Specifically, we exploited a simple micromolding-based approach utilizing surface tension-induced droplet formation followed by photo-induced radical polymerization in order to fabricate highly uniform poly(ethylene glycol) (PEG)-based hydrogel microspheres, in which a potent aminopolysaccharide chitosan providing chemically reactive and abundant amine groups is incorporated.  The as-prepared microspheres showed macroporous and/or intriguing core-shell structures stemming from spontaneous polymerization-induced phase separation.  Fluorescent labeling studies utilizing an amidation reaction on the chitosan’s primary amines confirmed stable incorporation of the chitosan moieties with retained chemical reactivity, showing potential of such amines as conjugation handles.  The utility of our microspheres as biomolecular conjugation platforms was thoroughly examined in terms of conjugation capacity and kinetics by conjugating model biomolecules (fluorescein-labeled single-stranded DNA and red fluorescent protein R-phycoerythrin) via two high-yield bioorthogonal reactions (strain-promoted alkyne–azide cycloaddition (SPAAC) and tetrazine–trans-cyclooctene (Tz–TCO) cycloaddition reactions).  The conjugation results showed well-defined and macroporous network structures of the microspheres, leading to enhanced protein conjugation capacity and kinetics and allowing programmable protein conjugation.  In this presentation, we will also highlight simple methods to further fine-tune the macroporous strucutres via inert PEG porogens.