Molecularly imprinted polymers for protein capture, sequestration, and delivery

Abstract

Event Abstract Back to Event Molecularly imprinted polymers for protein capture, sequestration, and delivery John Clegg1, 2, Justin Zhong3, Heidi R. Culver1, 2 and Nicholas A. Peppas1, 2, 3, 4 1 University of Texas at Austin, Department of Biomedical Engineering, United States 2 University of Texas at Austin, Institute for Biomaterials, Drug Delivery, and Regenerative Medicine, United States 3 University of Texas at Austin, McKetta Department of Chemical Engineering, United States 4 University of Texas at Austin, College of Pharmacy, United States Introduction: Molecularly imprinted polymers (MIPs) for protein detection have been limited to a few model protein templates[1]. MIPs entrap protein within their pores during polymerization, requiring denaturing or even destructive purification techniques to purify and extract templates[2]. The cost of many therapeutic proteins is therefore prohibitive to their use as imprinting templates. Herein, we hypothesized that rationally-selected templates, possessing similar geometry, molecular weight and isoelectric point to disease-relevant biomarkers could be used as low-cost alternatives. We demonstrate a MIP system, using Trypsin as a low-cost template, to capture and sequester low molecular weight, positively charged biomolecules while excluding larger, negative proteins. The ability to displace and release a model therapeutic protein through template binding is also investigated. Materials and Methods: Trypsin was allowed to pre-assemble with methacrylate monomers possessing hydrophilic, positive, or negative moieties, as well as hydrophobic core nanoparticles and crosslinker in phosphate buffer under a nitrogen atmosphere. Following polymerization, entrapped trypsin and unreacted monomers were extracted with 10 washes in 10% acetic acid (Fig. 1). Non-imprinted control polymers (NIPs) were prepared in the same manner, excluding Trypsin. Hydrogel microparticles were subjected to binding assays, with proteins of varying charge and molecular weight (Fig. 2A). MIP sequestration and delivery of cytochrome c was demonstrated through loading cytochrome c within MIPs and NIPs, and monitoring payload release over 24 hours in the presence and absence of trypsin. Results and Discussion: MIPs bound more trypsin than NIPs (Fig. 2B) and similar quantities of cytochrome c and lysozyme, while binding small quantities of hemoglobin (Fig. 2C). Normalization of equilibrium rebinding for MIPs to NIPs (Imprinting Factor) revealed no difference in relative protein binding between trypsin, lysozyme, cytochrome c, which were all significantly greater than bovine hemoglobin (Fig 2D). All imprinting factors were significantly different from 1, (p< .05), demonstrating a measurable impact of molecular imprinting on protein binding. MIPs exhibited 84.9% loading efficiency of cytochrome c, as compared to 52.3% for NIPs. MIPs were capable of sequestering cytochrome c in 0.1x PBS, releasing only 5.7% of payload released in 24h, while subsequently releasing 41.5% during 24h incubation with trypsin. Conclusion: Trypsin imprinting impacts cytochrome c, lysozyme, trypsin, and hemoglobin binding through exclusion on the basis of size and electric potential. Cytochrome c and lysozyme bind in similar capacity to Trypsin, likely as their similar charge and smaller size permit entry and sequestration into trypsin-MIP pores. These proteins can be efficiently loaded, excluded, sequestered, and delivered. Future studies, using rationally-selected imprinting templates with structural similarity to clinical biomarkers will determine if this fully-synthetic, low cost system can be employed in medical diagnostics and drug delivery. NSF Graduate Research Fellowship; Pratt Foundation; UT-Portugal Collaborative Research Program