Glycosaminoglycan-peptide and copolymer interactions

Arjun R Ishwar, Purdue University

Abstract

The extracellular matrix (ECM) is a critical mediator of cellular function. Key roles include aiding in cell communication, cell development and differentiation, structural support, and providing a physical barrier. For this reason, current research in tissue engineering has aimed to better understand and mimic ECM structure and function. The work conducted first attempted to design and implement Raman spectroscopy to study peptide-glycosaminoglycan (GAG, a critical ECM component) interactions in a rapid and efficacious method. Based on information gathered, it was aimed to incorporate important functional groups into a cost-efficient synthetic copolymer in attempts of simulating heparin-binding for inclusion into a gel-like ECM-mimicking matrix. Raman spectroscopy was used to identify chemical interactions, and conformational changes occurring upon binding between a synthetic peptide (QRRFMQYSARRF) and two GAGs, heparin and chondroitin 6-sulfate (C6S). The results identify three main chemical groups that are involved in the binding of the synthetic peptide with heparin and C6S. Tyrosine formed hydrogen bonds with the GAGs via its hydroxyl group. The amide I band demonstrated substantial shifts in Raman wavenumbers when bound to heparin and C6S (Δω = -10.2 ± 0.7 cm-1 and Δω = -11.9 ± 0.3 cm -1, respectively), suggesting that the peptide underwent planar conformational changes after binding occurred. Upon binding to the peptide, the sulfate peak of heparin displayed a substantially greater shift in the Raman wavenumber (-7.5 ± 0.5 cm-1) than that of C6S (-2.6 ± 0.5 cm-1). The greater amide I and sulfate band shifts seen during peptide-heparin interactions are indicative of a stronger association compared to that between the peptide and C6S. This observation was confirmed by capillary electrophoresis which demonstrated a lower dissociation constant (KD) between the peptide and heparin (KD of 19.2 ± 3.3 μM) than between the peptide and C6S (26.7 ± 2.5 μM). It was concluded that the shift in the Raman wavenumbers of amide I and sulfate groups can be used for high-throughput screening of interaction affinities between libraries of peptides and GAGs. Synthesis of a heparin-binding copolymer was performed with the intention of inclusion into a multifunctional ECM-like poly(ethylene glycol) gel, similar to previous work conducted by B.L. Seal and A. Panitch. The copolymers created integrate acrylamide and acrylic acid to act as backbone mediators. Styrene was included to mimic the aromatic groups, found to be important in the Raman study. Finally, agmatine sulfate was incorporated to mimic the positively charged guanidinyl groups of arginine, vital to electrostatic binding with the negatively charged GAGs. Binding was examined via heparin-affinity liquid chromatography using a sodium chloride (NaCl) gradient to elute polymer from the column. Binding strength was measured as a function of NaCl concentration at time of elution and was found to be 179, 171, and 168 mM for copolymers containing 0, 2.5, and 5% styrene addition, respectively. Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FTIR) spectroscopy, and molecular weight analysis were performed to characterize the copolymers’ structure. FTIR results showed styrene incorporation at higher levels (5 and 15%), while NMR results displayed agmatine incorporation of varied levels within all synthesized copolymers. It was concluded that the work conducted provided a positive step in the direction of creating heparin-bound, cost-efficient synthetic copolymers for inclusion into an ECM-mimicking matrix; however, further work in optimizing the chemistry and molecular structure must be performed.

Degree

M.S.B.M.E.

Advisors

Panitch, Purdue University.

Subject Area

Biomedical engineering

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