Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (- Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ - Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center Oklahoma City OK 73126 USA

Institute of Experimental Medicine Czech Academy of Sciences Vídeňská 1083 Prague Czech Republic

LIPhy Univ Grenoble Alpes CNRS 38000 Grenoble France

School of Biomedical Sciences Faculty of Biological Sciences University of Leeds Leeds LS2 9JT UK

School of Chemistry Faculty of Engineering and Physical Sciences University of Leeds Leeds LS2 9JT UK

School of Physics and Astronomy Faculty of Engineering and Physical Sciences Astbury Centre for Structural Molecular Biology and Bragg Centre for Materials Research University of Leeds Leeds LS2 9JT UK

Zobrazit více v PubMed

Russell DL, Salustri A. Extracellular matrix of the cumulus-oocyte complex. Semin. Reprod. Med. 2006;24(4):217–227. doi: 10.1055/s-2006-948551. PubMed DOI

Toole BP. Hyaluronan in morphogenesis. Semin. Cell Dev. Biol. 2001;12:79–87. doi: 10.1006/scdb.2000.0244. PubMed DOI

Richter RP, Baranova NS, Day AJ, Kwok JC. Glycosaminoglycans in extracellular matrix organisation: Are concepts from soft matter physics key to understanding the formation of perineuronal nets? Curr. Opin. Struct. Biol. 2018;50:65–74. doi: 10.1016/j.sbi.2017.12.002. PubMed DOI

Knudson CB, Knudson W. Cartilage proteoglycans. Semin. Cell Dev. Biol. 2001;12:69–78. doi: 10.1006/scdb.2000.0243. PubMed DOI

Seror J, Zhu L, Goldberg R, Day AJ, Klein J. Supramolecular synergy in the boundary lubrication of synovial joints. Nat. Commun. 2015;6:6497. doi: 10.1038/ncomms7497. PubMed DOI PMC

Jiang D, Liang J, Noble PW. Hyaluronan as an immune regulator in human diseases. Physiol. Rev. 2011;91(1):221–264. doi: 10.1152/physrev.00052.2009. PubMed DOI PMC

Day AJ, de la Motte CA. Hyaluronan cross-linking: A protective mechanism in inflammation? Trends Immunol. 2005;26(12):637–643. doi: 10.1016/j.it.2005.09.009. PubMed DOI

Gray AL, Pun N, Ridley AJL, Dyer DP. Role of extracellular matrix proteoglycans in immune cell recruitment. Int. J. Exp. Pathol. 2022;103:34–43. doi: 10.1111/iep.12428. PubMed DOI PMC

Toole BP. Hyaluronan: From extracellular glue to pericellular cue. Nat. Rev. Cancer. 2004;4:528–539. doi: 10.1038/nrc1391. PubMed DOI

Day AJ, Sheehan JK. Hyaluronan: Polysaccharide chaos to protein organisation. Curr. Opin. Struct. Biol. 2001;11:617–622. doi: 10.1016/S0959-440X(00)00256-6. PubMed DOI

Baranova NS, Inforzato A, Briggs DC, Tilakaratna V, Thakar D, Enghild JJ, Milner CM, Day AJ, Richter RP. Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking. J. Biol. Chem. 2014;289(44):30481–30498. doi: 10.1074/jbc.M114.568154. PubMed DOI PMC

Cyphert JM, Trempus CS, Garantziotis S. Size matters: Molecular weight specificity of hyaluronan effects in cell biology. Int. J. Cell Biol. 2015;2015:563818. doi: 10.1155/2015/563818. PubMed DOI PMC

Tavianatou AG, Caon I, Franchi M, Piperigkou Z, Galesso D, Karamanos NK. Hyaluronan: Molecular size-dependent signaling and biological functions in inflammation and cancer. FEBS J. 2019;286(15):2883–2908. doi: 10.1111/febs.14777. PubMed DOI

Rivas F, Erxleben D, Smith I, Rahbar E, DeAngelis PJ, Cowman MK, Hall AR. Methods for isolating and analyzing physiological hyaluronan: A review. Am. J. Physiol. Cell Physiol. 2022;322:C674–C687. doi: 10.1152/ajpcell.00019.2022. PubMed DOI PMC

Malm L, Hellman U, Larsson G. Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis. Glycobiology. 2012;22(1):7–11. doi: 10.1093/glycob/cwr096. PubMed DOI

Cowman MK. Methods for hyaluronan molecular mass determination by agarose gel electrophoresis. Methods Mol. Biol. 2019;1952:91–102. doi: 10.1007/978-1-4939-9133-4_8. PubMed DOI

Volpi N, Maccari F, Suwan J, Linhardt RJ. Electrophoresis for the analysis of heparin purity and quality. Electrophoresis. 2012;33(11):1531–1537. doi: 10.1002/elps.201100479. PubMed DOI PMC

Rivas F, Zahid OK, Reesink HL, Peal BT, Nixon AJ, DeAngelis PL, Skardal A, Rahbar E, Hall AR. Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor. Nat. Commun. 2018;9(1):1037. doi: 10.1038/s41467-018-03439-x. PubMed DOI PMC

Rivas F, DeAngelis PL, Rahbar E, Hall AR. Optimizing the sensitivity and resolution of hyaluronan analysis with solid-state nanopores. Sci. Rep. 2022;12(1):4469. doi: 10.1038/s41598-022-08533-1. PubMed DOI PMC

Jackson DG. Immunological functions of hyaluronan and its receptors in the lymphatics. Immunol. Rev. 2009;230(1):216–231. doi: 10.1111/j.1600-065X.2009.00803.x. PubMed DOI

McDonald B, Kubes P. Interactions between CD44 and hyaluronan in leukocyte trafficking. Front. Immunol. 2015;6:68. PubMed PMC

Morra M. Engineering of biomaterials surfaces by hyaluronan. Biomacromol. 2005;6(3):1205–1223. doi: 10.1021/bm049346i. PubMed DOI

Morra M, Cassinelli C, Cascardo G, Fini M, Giavaresi G, Giardino R. Covalently-linked hyaluronan promotes bone formation around Ti implants in a rabbit model. J. Orthop. Res. 2009;27(5):657–663. doi: 10.1002/jor.20797. PubMed DOI

Korn P, et al. Chondroitin sulfate and sulfated hyaluronan-containing collagen coatings of titanium implants influence peri-implant bone formation in a minipig model. J. Biomed. Mater. Res. A. 2014;102(7):2334–2344. doi: 10.1002/jbm.a.34913. PubMed DOI

Highley CB, Prestwich GD, Burdick JA. Recent advances in hyaluronic acid hydrogels for biomedical applications. Curr. Opin. Biotechnol. 2016;40:35–40. doi: 10.1016/j.copbio.2016.02.008. PubMed DOI

Ryan CN, Sorushanova A, Lomas AJ, Mullen AM, Pandit A, Zeugolis DI. Glycosaminoglycans in tendon physiology, pathophysiology, and therapy. Bioconjug. Chem. 2015;26(7):1237–1251. doi: 10.1021/acs.bioconjchem.5b00091. PubMed DOI

Mizrahy S, et al. Hyaluronan-coated nanoparticles: The influence of the molecular weight on CD44-hyaluronan interactions and on the immune response. J. Control. Release. 2011;156(2):231–238. doi: 10.1016/j.jconrel.2011.06.031. PubMed DOI

Lee H, Lee K, Kim IK, Park TG. Synthesis, characterization, and in vivo diagnostic applications of hyaluronic acid immobilized gold nanoprobes. Biomaterials. 2008;29(35):4709–4718. doi: 10.1016/j.biomaterials.2008.08.038. PubMed DOI

Almalik A, Karimi S, Ouasti S, Donno R, Wandrey C, Day PJ, Tirelli N. Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles. Biomaterials. 2013;34(21):5369–5380. doi: 10.1016/j.biomaterials.2013.03.065. PubMed DOI

Oommen OP, Duehrkop C, Nilsson B, Hilborn J, Varghese OP. Multifunctional hyaluronic acid and chondroitin sulfate nanoparticles: Impact of glycosaminoglycan presentation on receptor mediated cellular uptake and immune activation. ACS Appl. Mater. Interfaces. 2016;8(32):20614–20624. doi: 10.1021/acsami.6b06823. PubMed DOI

Migliorini E, Thakar D, Sadir R, Pleiner T, Baleux F, Lortat-Jacob H, Coche-Guerente L, Richter RP. Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels. Biomaterials. 2014;35(32):8903–8915. doi: 10.1016/j.biomaterials.2014.07.017. PubMed DOI

Migliorini E, et al. Cytokines and growth factors cross-link heparan sulfate. Open Biol. 2015;5(8):150046. doi: 10.1098/rsob.150046. PubMed DOI PMC

Bano F, Tammi MI, Kang DW, Harris EN, Richter RP. Single-molecule unbinding forces between the polysaccharide hyaluronan and its binding proteins. Biophys. J. 2018;114(12):2910–2922. doi: 10.1016/j.bpj.2018.05.014. PubMed DOI PMC

Peerboom N, Block S, Altgärde N, Wahlsten O, Möller S, Schnabelrauch M, Trybala E, Bergström T, Bally M. Binding kinetics and lateral mobility of HSV-1 on end-grafted sulfated glycosaminoglycans. Biophys. J. 2017;113(6):1223–1234. doi: 10.1016/j.bpj.2017.06.028. PubMed DOI PMC

Cowman MK, Spagnoli C, Kudasheva D, Li M, Dyal A, Kanai S, Balazs EA. Extended, relaxed and condensed conformations of hyaluronan observed by atomic force microscopy. Biophys. J. 2005;88(1):590–602. doi: 10.1529/biophysj.104.049361. PubMed DOI PMC

Ng L, Grodzinsky AJ, Patwari P, Sandy J, Plaas A, Ortiz C. Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy. J. Struct. Biol. 2003;143(3):242–257. doi: 10.1016/j.jsb.2003.08.006. PubMed DOI

Wei W, et al. Self-regenerating giant hyaluronan polymer brushes. Nat. Commun. 2019;10(1):5527. doi: 10.1038/s41467-019-13440-7. PubMed DOI PMC

Reviakine I, Johannsmann D, Richter RP. Hearing what you cannot see and visualizing what you hear: Interpreting quartz crystal microbalance data from solvated interfaces. Anal. Chem. 2011;83:8838–8848. doi: 10.1021/ac201778h. PubMed DOI

Johannsmann D, et al. The quartz crystal microbalance in soft matter research. In: Piazza R, et al., editors. Soft and Biological Matter. Springer; 2015.

Easley AD, Ma T, Eneh CI, Yun J, Thakur RM, Lutkenhaus JL. A practical guide to quartz crystal microbalance with dissipation monitoring of thin polymer films. J. Polym. Sci. 2021;60:1–18.

Bingen P, Wang G, Steinmetz NF, Rodahl M, Richter RP. Solvation effects in the QCM-D response to biomolecular adsorption—A phenomenological approach. Anal. Chem. 2008;80(23):8880–8890. doi: 10.1021/ac8011686. PubMed DOI

Johannsmann D, Reviakine I, Richter RP. Dissipation in films of adsorbed nanospheres studied by QCM. Anal. Chem. 2009;81(19):8167–8176. doi: 10.1021/ac901381z. PubMed DOI

Eisele NB, Andersson FI, Frey S, Richter RP. Viscoelasticity of thin biomolecular films: A case study on nucleoporin phenylalanine-glycine repeats grafted to a histidine-tag capturing QCM-D sensor. Biomacromol. 2012;13(8):2322–2332. doi: 10.1021/bm300577s. PubMed DOI

Du B, Johannsmann D. Operation of the quartz crystal microbalance in liquids: Derivation of the elastic compliance of a film from the ratio of bandwidth shift and frequency shift. Langmuir. 2004;20(7):2809–2812. doi: 10.1021/la035965l. PubMed DOI

Tellechea E, Johannsmann D, Steinmetz NF, Richter RP, Reviakine I. Model-independent analysis of QCM data on colloidal particle adsorption. Langmuir. 2009;25(9):5177–5184. doi: 10.1021/la803912p. PubMed DOI

Tsortos A, Papadakis G, Gizeli E. Shear acoustic wave biosensor for detecting DNA intrinsic viscosity and conformation: A study with QCM-D. Biosens. Bioelectron. 2008;24(4):842–847. doi: 10.1016/j.bios.2008.07.006. PubMed DOI

Jing W, Haller FM, Almond A, DeAngelis PL. Defined megadalton hyaluronan polymer standards. Anal. Biochem. 2006;355(2):183–188. doi: 10.1016/j.ab.2006.06.009. PubMed DOI

Jing W, Roberts JW, Green DE, Almond A, DeAngelis PL. Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: A natural sugar-based drug delivery system to treat neutropenia. Glycobiology. 2017;27(11):1052–1061. doi: 10.1093/glycob/cwx072. PubMed DOI PMC

Jing W, DeAngelis PL. Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida. Glycobiology. 2003;13(10):661–671. doi: 10.1093/glycob/cwg085. PubMed DOI

Jing W, DeAngelis PL. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J. Biol. Chem. 2004;279(40):42345–42349. doi: 10.1074/jbc.M402744200. PubMed DOI

Tracy BS, Avci FY, Linhardt RJ, DeAngelis PL. Acceptor specificity of the Pasteurella hyaluronan and chondroitin synthases and production of chimeric glycosaminoglycans. J. Biol. Chem. 2007;282(1):337–344. doi: 10.1074/jbc.M607569200. PubMed DOI PMC

Thakar D, Migliorini E, Coche-Guerente L, Sadir R, Lortat-Jacob H, Boturyn D, Renaudet O, Labbe P, Richter RP. A quartz crystal microbalance method to study the terminal functionalization of glycosaminoglycans. Chem. Commun. (Camb.) 2014;50(96):15148–15151. doi: 10.1039/C4CC06905F. PubMed DOI

Dubacheva GV, Araya-Callis C, Geert Volbeda A, Fairhead M, Codee J, Howarth M, Richter RP. Controlling multivalent binding through surface chemistry: Model study on streptavidin. J. Am. Chem. Soc. 2017;139(11):4157–4167. doi: 10.1021/jacs.7b00540. PubMed DOI PMC

Dubacheva GV, Curk T, Mognetti BM, Auzély-Velty R, Frenkel D, Richter RP. Superselective targeting using multivalent polymers. J. Am. Chem. Soc. 2014;136(5):1722–1725. doi: 10.1021/ja411138s. PubMed DOI PMC

Jasnin M, van Eijck L, Koza MM, Peters J, Laguri C, Lortat-Jacob H, Zaccai G. Dynamics of heparan sulfate explored by neutron scattering. Phys. Chem. Chem. Phys. 2010;12(14):3360–3362. doi: 10.1039/b923878f. PubMed DOI

Richter RP, Mukhopadhyay A, Brisson A. Pathways of lipid vesicle deposition on solid surfaces: A combined QCM-D and AFM study. Biophys. J. 2003;85(5):3035–3047. doi: 10.1016/S0006-3495(03)74722-5. PubMed DOI PMC

Richter RP, Hock KK, Burkhartsmeyer J, Boehm H, Bingen P, Wang G, Steinmetz NF, Evans DJ, Spatz JP. Membrane-grafted hyaluronan films: A well-defined model system of glycoconjugate cell coats. J. Am. Chem. Soc. 2007;129(17):5306–5307. doi: 10.1021/ja068768s. PubMed DOI

Baranova NS, Nileback E, Haller FM, Briggs DC, Svedhem S, Day AJ, Richter RP. The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers. J. Biol. Chem. 2011;286(29):25675–25686. doi: 10.1074/jbc.M111.247395. PubMed DOI PMC

Bano F, Banerji S, Howarth M, Jackson DG, Richter RP. A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force. Sci. Rep. 2016;6:34176. doi: 10.1038/srep34176. PubMed DOI PMC

Takahashi R, Al-Assaf S, Williams PA, Kubota K, Okamoto A, Nishinari K. Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains. Biomacromol. 2003;4(2):404–409. doi: 10.1021/bm025706v. PubMed DOI

Alexander S. Adsorption of chains molecules with a polar head. A scaling description. J. Phys. (Paris) 1977;38(8):983–987. doi: 10.1051/jphys:01977003808098300. DOI

Rubinstein M, Colby RH. Polymer Physics. Oxford University Press; 2003. p. 440.

Chen X, Richter RP. Effect of calcium ions and pH on the morphology and mechanical properties of hyaluronan brushes. Interface Focus. 2019;9(2):20180061. doi: 10.1098/rsfs.2018.0061. PubMed DOI PMC

Richter RP, Bérat R, Brisson AR. The formation of solid-supported lipid bilayers—An integrated view. Langmuir. 2006;22(8):3497–3505. doi: 10.1021/la052687c. PubMed DOI

Ligoure C, Leibler L. Thermodynamics and kinetics of grafting end-functionalized polymers to an interface. J. Phys. 1990;51:1313–1328. doi: 10.1051/jphys:0199000510120131300. DOI

Attili S, Richter RP. Self-assembly and elasticity of hierarchical proteoglycan–hyaluronan brushes. Soft Matter. 2013;9(44):10473–10483. doi: 10.1039/c3sm51213d. DOI

Davies HS, Baranova NS, El Amri N, Coche-Guerente L, Verdier C, Bureau L, Richter RP, Debarre D. An integrated assay to probe endothelial glycocalyx–blood cell interactions under flow in mechanically and biochemically well-defined environments. Matrix Biol. 2019;78–79:47–59. doi: 10.1016/j.matbio.2018.12.002. PubMed DOI

Attili S, Borisov OV, Richter RP. Films of end-grafted hyaluronan are a prototype of a brush of a strongly charged, semi-flexible polyelectrolyte with intrinsic excluded volume. Biomacromol. 2012;13(5):1466–1477. doi: 10.1021/bm3001759. PubMed DOI

Johannsmann D, Langhoff A, Leppin C. Studying soft interfaces with shear waves: Principles and applications of the quartz crystal microbalance (QCM) Sensors (Basel) 2021;21(10):3490. doi: 10.3390/s21103490. PubMed DOI PMC

Domack A, Prucker O, Rühe J, Johannsmann D. Swelling of a polymer brush probed with a quartz crystal resonator. Phys. Rev. E. 1997;56(1):680–689. doi: 10.1103/PhysRevE.56.680. DOI

Davies HS, Debarre D, El Amri N, Verdier C, Richter RP, Bureau L. Elastohydrodynamic lift at a soft wall. Phys. Rev. Lett. 2018;120:198001. doi: 10.1103/PhysRevLett.120.198001. PubMed DOI

Competitive Specific Anchorage of Molecules onto Surfaces: Quantitative Control of Grafting Densities and Contamination by Free Anchors