Protein therapeutics hold high efficacy in treatment for various diseases including cancer and diabetes. The cost of treatment is generally higher than other types of medicine due to <i>in vivo</i> protein degradation caused by delivery issues. This creates a demand for more efficient delivery methods to preserve the function and quality of protein therapeutics. Thermoresponsive polymer systems are being researched for use in protein therapeutics. Core cross-linked micelles, nanogels, have been developed to respond to pH and temperature stimuli. The nanogel core can undergo a hydrophobic and hydrophilic phase change at the lower critical solution temperature (LCST). Coacervate-forming polymers are another thermoresponsive system used in protein therapeutic research. Coacervate refers to a phase-separated solution in which a dense polymer phase separates from the aqueous phase, forming a nanodroplet within solution that can capture bioactive molecules. Thermoresponsive polyesters (TR-PEs) have pendent groups built into the monomer units that can nucleate binding to certain biomolecules. Furthermore, the development of biodegradable TR-PEs have been reported for medicinal research, demonstrating that TR-PEs have relatively low cytotoxicity. Biodegradable TR-PEs encapsulate and preserve the activity of smaller proteins such as insulin, 6 kDa, and bovine serum albumin, 66.5 kDa. There is limited research demonstrating if TR-PEs can encapsulate and preserve the structure and activity of larger proteins such as large enzymes. It will be determined if biodegradable TR-PEs can efficiently encapsulate large enzymes such as beta-galactosidase, which forms a 465.4 kDa homotetramer, and preserve its specific enzymatic activity upon release. The specific activity of heterologously expressed and purified beta-galactosidase has been determined using activity assays with Chlorophenol Red-β-D-galactopyranoside (CPRG). The TR-PEs have been demonstrated to capture beta-galactosidase via Bradford protein assay. The results of studies using fluorescein isothiocyanate (FITC) to track the direct encapsulation and provide images of captured protein will also be presented. Comparison of specific activity before capture and after release will determine if enzymatic activity is preserved in TR-PEs coacervates. The demonstration that TR-PEs capture and release an unaltered, larger complex protein molecule exemplifies the viability of using TR-PEs for future therapeutic delivery.

Preserving β-Galactosidase specific activity through encapsulation by thermoresponsive coacervate-forming biodegradable polyesters

ACS Fall 2021

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