Enzymatic Degradation of Glycosaminoglycans and Proteoglycan-Mimetic Materials in Solution and on Polyelectrolyte Multilayer Surfaces

Abstract

Proteoglycans (PGs) play many important roles in biology, contributing to the mechanical properties of tissues, helping to organize extracellular matrix components, and participating in signaling mechanisms related to mechanotransduction, cell differentiation, immune responses, and wound healing. Our lab has designed two different types of PG mimics: polyelectrolyte complex nanoparticles (PCNs) and PG-mimetic graft copolymers (GCs), both of which are prepared using naturally occurring glycosaminoglycans. This work evaluates the enzymatic stability of these PG mimics using hyaluronidases (I-S, IV-S, and II), chondroitinase ABC, and lysozyme, for PG mimics suspended in solution and adsorbed onto surfaces. Hyaluronan (HA)-and chondroitin sulfate (CS)-containing PG mimics are degraded by the hyaluronidases. PCNs prepared with CS and GCs prepared with heparin are the only CS-and HA-containing PG mimics protected from chondroitinase ABC. None of the materials are measurably degraded by lysozyme. Adsorption to polyelectrolyte multilayer surfaces protects PG mimics from degradation, compared to when PG mimics are combined with enzymes in solution; all surfaces are still intact after 21 days of enzyme exposure. This work reveals how the stability of PG mimics is controlled by both the composition and macromolecular assembly of the PG mimic and also by the size and specificity of the enzyme. Understanding and tuning these degradation susceptibilities are essential for advancing their applications in cardiovascular materials, orthopedic materials, and growth factor delivery applications.

Modifications to Polysaccharide Polyelectrolyte Multilayers: Improved Mechanical Stability and Hemocompatibility

“…PG-mimetic graft copolymers are composed of an HA backbone with side chains consisting of GAGs (HEP or CS). The synthesis of these graft copolymers has been previously reported by our group. ,, The synthesis is a three-step process which consists of HA backbone thiolation, backbone hydrazide activation, and coupling via reductive amination. During the first step, HA (250 mg) was activated with EDC (645 mg) and NHS (976 mg) in MES buffer (50 mL, pH 6.0).…”

“…9,25 We have also recently reported studies of the stability of these surfaces with respect to enzymatic degradation. 23 However, their interactions with whole human blood and their mechanical durability under flowing fluid have not yet been characterized. For bloodcontacting applications, these surfaces may lose their functionality because of mechanical shear when in contact with flowing blood.…”

“…Additional details and spectroscopic confirmation of the synthesis are provided in our previous publications. 23,31,32 Preparation of Polyelectrolyte Nanoparticle (PCN) PG Mimics. Polyelectrolyte complex nanoparticles (PCN) were prepared as previously published by our group.…”

“…Approaches include designing bioinert, thrombolytic, drug releasing, bioactive, and bioinspired surfaces. − Bioactive surfaces using heparin (HEP) have been successful enough to be produced commercially but have limitations that prevent their long-term use . Polyelectrolyte multilayers (PEMs) have been extensively used to modify biomaterial surfaces. − The popularity of these surfaces is in part due to their ease of construction, versatility in the polymers used, and suitability for modifying surface chemistry while preserving microscale and nanoscale topographical features of the underlying substrate. PEM surfaces also provide substrates for nanoparticle adsorption or drug release, which can be used to affect biological responses. ,, PEMs therefore represent a platform technology from which blood compatibility of surfaces can be improved.…”

“…We have previously reported on PEM surfaces that mimic features of the endothelial glycocalyx and that thereby reduce platelet adhesion and activation, reduce protein adsorption and polymerization, suppress pro-inflammatory responses from leukocytes and macrophages, and reduce bacterial attachment. ,,,,, These biomimetic surfaces designed to mimic the topography and chemistry of the glycocalyx are composed of (polycationic) chitosan and (polyanionic) hyaluronan PEMs, with heparin- or chondroitin sulfate-containing proteoglycan-like nanoparticles and graft copolymers adsorbed onto their surfaces. ,, These materials have shown promise as blood-contacting materials, and their interactions with bacteria, platelets, blood proteins, and leukocytes have been evaluated. , We have also recently reported studies of the stability of these surfaces with respect to enzymatic degradation . However, their interactions with whole human blood and their mechanical durability under flowing fluid have not yet been characterized.…”