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Biomaterials

Biomaterials - Poster Session E

Poster N12 - Development of Bioorthogonal PEG-based microhydrogels for enhanced protein delivery

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Protein therapeutics have revolutionized the treatment of numerous diseases, including cancer, inflammatory disease, and diabetes. Despite their clinical success, protein therapies are subject to enzymatic degradation, off-target effects, and nonspecific activity in the body. To overcome these challenges, polymeric nanoparticle protein delivery vehicles have been explored that diminish off target effects and enhance protection of protein therapeutics. However, these platforms typically have low encapsulation efficiencies and can denature proteins during formulation and upon degradation-mediated release of the particles. Here, we present a bioorthogonal microhydrogel protein delivery vehicle that can efficiently encapsulate proteins and limit protein exposure to hydrophobic denaturing surfaces during formulation. Our microhydrogels consist of a 2-component hydrogel that cross-links via copper-free click reaction between azides and ring-strained alkynes, and thus is unreactive to amino acid functional groups. Our microhydrogels are formed when two buffer solutions each containing one of our two gel components are introduced into a rapidly stirred surfactant/mineral oil solution, forming an inverse emulsion. Through the addition of protein into one of the solutions containing one of our gel components, we can formulate protein-loaded microgels with nearly 100% protein encapsulation. In this work, we describe the formulation of our microhydrogels and demonstrate the efficient encapsulation of the model vaccine antigen ovalbumin (OVA). Given their ease of formulation and efficient encapsulation, our microhydrogel has the potential to improve the efficacy of numerous protein therapeutics as well as subunit vaccines.

Materials and

Methods:  The microgels are formed upon the reaction with an azide-functionalized linear co-polymer “backbone” poly [tetraethylene glycol methacrylate)-co-(azidotetraethylene glycol methacrylate)] (1) and a 4-dibenzocyclooctynol (DBCO)- modified poly(ethylene glycol) (PEG) “crosslinker” (2). To synthesize microgels, our backbone and crosslinker polymers are suspended in PBS and these solutions are mixed and added to a rapidly agitated solution of 2%SPAN 80 in mineral oil to form inverse microemulsion, within which the backbone and crosslinker react via a Huisgen cycloaddition. The emulsion is vortexed at high speeds for 30 seconds then the microgels are shaken for 30 minutes. Finally, the excess crosslinker is capped using Azido-PEG4-Amine. The aqueous phase is extended via the addition of PBS, separated and washed with hexanes to remove mineral oil prior to dynamic light scattering (DLS) measurement. Three concentrations of the PEG-DBCO (25% w/v, 12.5% w/v, and 6.25%w/v) and two concentrations of PEG-N3 (50% w/v and 30%w/v) were tested to evaluate their effects on size. To load the particles, AlexaFluor 647-labeled OVA was added to the crosslinker solution, and OVA-loaded particles were synthesized via the procedure described above. OVA loading was verified via fluorescent imaging of the gels.

Results, Conclusions, and Discussions: NMR was used to characterize the synthesized backbone and crosslinker (Fig. 1). Dynamic light scattering measurements showed that the particles were in the µm range with a polydispersity of ~0.6 (Fig. 3). Other methods of agitation, such as sonication and homogenization, will be explored and may result in more consistent sizes. As crosslinker density was increased, the particle size decreased slightly but without statistical significance, so more crosslinker and polymer densities will be tested to optimize the crosslinking rate and stability of the formulation. We also hypothesize that the release profiles of the hydrogels could be controlled by altering the crosslinker density. The microgels were successfully loaded with OVA linked to AlexaFluor 647 and visualized using fluorescence microscopy (Fig.2). The encapsulation efficiency and release profiles of the microgels will be calculated by the detection of fluorescence using a plate reader. The OVA-loaded particles will be injected into mice to assess anti-OVA antibody production along with antigen-specific T-cell responses in vivo. In addition to protein cargo, hydrophobic drugs may also be loaded into the particle and evaluated similarly.

Acknowledgements (Optional):