Membrane-Grafted Hyaluronan Films:  A Well-Defined Model System of Glycoconjugate Cell Coats

Richter, Ralf P.; Hock, Kai K.; Burkhartsmeyer, Jeffrey Mark; Boehm, Heike; Bingen, Pit; Wang, Guoliang; Steinmetz, Nicole F.; Evans, David; Spatz, Joachim P.

doi:10.1021/ja068768s

Cited by 73 publications

(116 citation statements)

References 15 publications

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“…This value is in good agreement with results from independent analysis of the surface density and permeability of HA films. 12 We recall that the mean value of s/A F 1/3 for ≥50 mM NaCl was 49 nm ( Figure 4B). Based on the estimate of s ≈ 57 nm, we would obtain A F ≈ 1.6, which is again on the order of unity, as expected.…”

Section: Biomacromoleculesmentioning

confidence: 74%

“…48,49 Instead, HA interacts exclusively via a biotin moiety at its reducing end with the surface. 12 The grafting-to approach produces surface densities that are large enough to induce chain stretching into the brush regime, even at high ionic strengths.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Counterion condensation 44 is unlikely because the distance between chargeable groups is larger than the Bjerrum length (0.7 nm in aqueous solution), that is, α = 1. p = a/b is expected to lie between 8 and 16, based on the equivalence of a in the freely jointed chain and 2l p in the worm-like chain models, and reported persistence lengths l p between 4 and 8 nm. 15−18 In earlier work, 12 we had estimated s ≈ 56 nm by reflectometry.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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

2012

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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 ...

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Section: Biomacromoleculesmentioning

confidence: 74%

Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

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

2012

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“…Mixtures of DOPC/DOPS (3:1 molar ratio), DOPC/OEOA-bis-NTA (9:1), and pure DOPC were dissolved in chloroform, dried, resuspended in Hepes buffer, and homogenized as described earlier (29). Small unilamellar vesicles (SUVs) were obtained by sonication (30) or extrusion (50 nm final pore size) (31). SUV suspensions were stored at 4°C under nitrogen and diluted to desired concentrations before use.…”

Section: Methodsmentioning

confidence: 99%

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

Wolny

Banerji

Gounou

et al. 2010

Journal of Biological Chemistry

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193

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CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ϳ30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of <10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

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“…245 More recently, HA grafted SLBs were prepared using biotinylated lipids and HA. 246 Similarly glycosaminoglycans (GAG) and sulphated GAGs like chondroitin sulphate (CS), large unbranched polysaccharides found in the ECM, were conjugated to SLBs as well. CS plays an important role in bone and cartilage tissue and is capable of binding growth factors and ECM molecules like bone morphogenic protein 2 (BMP-2) and collagen type I, ll A10: vascular smooth muscle cells respectively.…”

Section: Mimicking Cell-ecm Contacts By Decorating Slbs With Ecm Protmentioning

confidence: 99%

Novel biomedical applications of supported lipid bilayers

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Membrane-Grafted Hyaluronan Films: A Well-Defined Model System of Glycoconjugate Cell Coats

Cited by 73 publications

References 15 publications

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

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

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

Novel biomedical applications of supported lipid bilayers

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