Microgels as a basis for affinity delivery of bioactive proteins

Microgels combining the strengths of hydrogels and microparticles have emerged as a versatile drug delivery system with controlled drug release, high loading efficiency, stability and biocompatibility, making them an ideal vehicle for delivery of proteins. Affinity-based delivery can potentially realise sustained release of proteins while maintaining their structures and functions during delivery via mild interactions. In this study, hyaluronic acid (HA)-diviynl sulfone (DVS) microgels were fabricated by both conventional stirring and novel microfluidic approaches. A coaxial flow reactor (CFR) was used as the microfluidic device to allow continuous manufacture and achieve precise control over size properties of microgels. Taguchi design was applied as a design of experiments (DoE) method to optimise seven experimental factors during the synthesis process and determine the optimal conditions for minimised particle size and polydispersity index (PdI). With the combination of CFR and DoE, the impacts of the factors on size properties were identified within specific ranges, where non-solvents and flow rates were key factors affecting size properties greatly. A desirable batch of microgels with hydrodynamic diameter of 261 nm and PdI of 0.19 was obtained after optimisation. To enable controlled affinity-based protein delivery, microgels were modified with peptide ligands that selectively bind target proteins while preserving their structures and functions. Immunoglobulin G (IgG) as a typical antibody widely used in research for multiple treatments was utilised to prove the concept of affinity delivery. The peptide ligand CRRGW, recognising the Fc region of IgG, was synthesised via solid phase peptide synthesis (SPPS) and conjugated to HA microgels using a linker molecule 2-aminoethanol (MEA). Surface plasmon resonance (SPR) confirmed strong IgG-CRRGW binding, and sustained IgG release over a month from the peptide-HA-DVS microgel system was demonstrated by dialysis via fluorescence detection. Considering the critical role of HA in the extracellular matrix (ECM) for neuronal tissues, the peptide-HA-DVS system was further adapted for potential nervous system applications. The peptide CRQRSRPGRWHKVSVRWEKNR, targeting fibroblast growth factor 2 (FGF-2), was synthesised by automated SPPS and conjugated to microgels. Similarly, affinity binding between the peptide and FGF-2 was verified by SPR, and controlled release and protection of FGF-2 by the microgel system over 8 weeks was confirmed through dialysis with an enzyme-linked immunosorbent assay (ELISA). Overall, this work demonstrated the principle that the peptide-protein interactions could be exploited to achieve affinity delivery of therapeutic proteins from HA microgels. The peptide conjugated HA-DVS microgel system provides a promising platform for affinity-based protein delivery, offering an opportunity to improve biotherapeutics. The applications can be extended from dermal fillers and wound healing to central systems such as traumatic brain injury (TBI) treatments.

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