Films of End-Grafted Hyaluronan Are a Prototype of a Brush of a Strongly Charged, Semiflexible Polyelectrolyte with Intrinsic Excluded Volume

Abstract

Hyaluronan (HA) is a naturally occurring linear, negatively charged polysaccharide that plays a vital role in the organization and function of pericellular coats and extracellular matrices in vertebrates, and that is becoming increasingly popular in biomedical applications. To gain insight into the physical phenomena that govern the mechanical behavior of HA assemblies, we have studied the response of films of end-grafted HA to compression over a large range of ionic strength. Compression forces were measured as a function of the absolute distance between a colloidal probe and the planar surface on which the HA film was constructed, using a combined atomic force microscopy and reflection interference contrast microscopy setup. The HA films were well-defined in the sense that they are made of chains with a narrow size distribution that are grafted at controlled density to a solid support. Detailed comparison of the experimental data with analytical expressions derived from polymer and polyelectrolyte brush theory reveals that films of end-grafted HA behave as strongly charged polyelectrolyte brushes. To quantitatively reproduce the experimental data, intrinsic excluded volume interactions and chain stiffness of the polymer backbone must be taken into account. At low ionic strength, chains become almost fully stretched. In our experimental system, several micrometer thick films are formed that reach a hydration of up to 99.98%, and the brush thickness decreases by more than 5-fold with increasing ionic strength. More generally, the study provides quantitative theoretical predictions for the film thickness and compressive response as a function of HA length, grafting density and ionic strength. ■ INTRODUCTIONThe polysaccharide hyaluronan (HA) is ubiquitous in the pericellular and extracellular space of vertebrates. It is also becoming increasingly popular in biomedical applications as surface coating 1 and in tissue engineering. 2,3 HA is a linear polymer of disaccharides, made of glucuronic acid and Nacetylglucosamine, that are linked via alternating β-1,4 and β-1,3 glycosidic bonds. Each disaccharide has a length of 1 nm 4 and carries one chargeable group in the form of a carboxylic acid. For polymeric HA, the pK a is approximately 3, 5 and HA is, hence, negatively charged above pH ≈ 3. In vivo, HA is expressed by HA synthases at the cell membrane and extruded into the extracellular space. These HA molecules have a molecular mass of typically a few million Daltons 6 or a contour length of several micrometers. Once produced, they can stay grafted to the synthases and/or bind to other HA receptors at the cell surface, such as CD44 7 and form so-called pericellular coats (PCCs), or they can be released into solution to serve other functions in the extracellular space. Numerous biological functions of PCCs 8−11 and extracellular assemblies of HA in general (e.g., in cartilage) have been related to their mechanical properties. To better understand how the mechanical properties of PCCs are connected to their ...

Heterogeneous Morphology and Dynamics of Polyelectrolyte Brush Condensates in Trivalent Counterion Solution

“…We introduce two dimensionless parameters to describe the system. The extent of lateral overlap between neighboring polymer chains is formulated using the chain overlap parameter χ ≡ (2R g,xy /d) 2 , where R g,xy , which scales as R 2 g,xy ∼ N 1.7 ( Fig. 2), is the in-plane component of the gyration radius of a single grafted polyelectrolyte chain.…”

“…As the main component of outer membrane in Gram-negative bacteria, lipopolysaccharides, consisting of charged O-polysaccharides side chains, form a brush layer and mediate the interaction of bacteria with their environment [1]. For vertebrates, negatively charged polysaccharide hyaluronic acids play a vital role in the organization of pericellular matrix [2,3]. Double-stranded DNA brushes on a biochip [4] at a cell-like density (∼ 10 4 kb/µm 3 ) have been developed as a platform to study cell-free gene expression [5].…”

Elastohydrodynamic Lift at a Soft Wall

“…The surface of the coverslip exposed to the flow was functionalized with a layer of HA, as described in [27,28]. A Ti/Au layer (respectively 0.5 and 5 nm in thickness) was evaporated onto the glass surface.…”

“…1a). Such a procedure yields HA films stably bound to the substrate in a polymer brush conformation [27]. To investigate the role of brush thickness and softness, we have studied three different HA surfaces made of chains of well-defined molecular weight (Hyalose, USA): two substrates obtained by incubating chains of 840±60 kDa for two different times, yielding high (HA840-h) and low (HA840-l) grafting density samples, and a third one (HA58) bearing a brush made of chains of 58±3 kDa.…”

“…This corresponds to an interaction energy per unit area F g /R ≃ 2.5 × 10 −7 N.m −1 at which we anticipate the brushes to be essentially uncompressed [27]. Compared to F g , van der Waals forces between the beads and the glass substrate are negligible at distances h > 10 nm [30], and repulsive forces of electrostatic origin are screened at the ionic strength used (Debye length < 1 nm) [31].…”