HYALURONAN
`Volume 1 - Chemical, Biochemical and Biological Aspects
`
`Editors:
`JOHN F. KENNEDY BSe, PhD, OSe, EurChem CChem FRSc, CBiol FIBiol, FCIWEM, FCMI, FIFST
`Director of Birmingham Carbohydrate and Protein Technology Group,
`SchoolofChemical Sciences,The University ofBinningham, BirminghamB15 2IT, England,UK,
`Director of Chembiotech Ltd,
`University of Birmingham Research Park, Birmingham B15 2SQ, England, UK,
`Director of Inovamed Ltd,
`Chembiotech Laboratories, University of Birmingham Research Park, Vincent Drive,
`Birmingham B15 2SQ, England, UK, and
`Professor of Applied Chemistry,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK
`
`GLYN O. PHILLIPS BSe, PhD, OSe, HODOSe, HODLIB, CChem FRSC
`Chairman of Research Transfer Ltd,
`Newtech Innovation Centre,
`Professorial Fellow,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK, and
`Professor of Chemistry,
`The University of Salford, England, UK
`
`PETER A. WILLIAMS BSe, PhD, CChem FRSC
`Director of the Centre for Water Soluble Polymers,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK,
`Director of the Centre for Advanced and Renewable Materials at
`Institute and University of Wales, Bangor,
`The North East Wales Institute of Higher Education, P1as Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK
`Professor of Polymer and Colloid Chemistry,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`. LL11 2AW, Wales, UK
`
`the North East Wales
`
`Guest Editor:
`VINCE C. HASCALL PhD
`Co-Direetor of the Orthopaedic Surgery Musculoskeletal Research Center,
`Department of Biomedical Engineering ND-20, Lerner Research Institute. Cleveland Clinic
`Foundation, Cleveland, Ohio 44195, USA
`Adjunct Professor
`Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA
`Adjunct Professor
`Department of Biochemistry, Rush Presbyterian S1. Lukes Medical Center, Chicago, Illinois,
`60612 USA
`
`WOODHEAD PUBLISHING LIMITED
`
`ALL 2018
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`Published by Woodhead Publishing Ltd, Abington Hall, Abington,
`Cambridge CB I 6AH, England
`www.woodhead-publishing.com
`
`First published 2002
`
`© 2002, Woodhead Publishing Ltd
`The authors have asserted their moral rights
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`ISBN I 85573 5709 (2 volume set)
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`Printed in Great Britain by MFK Group Ltd
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`

`

`EFFECT OF METAL IONS ON THE RHEOLOGICAL
`FLOW PROFILES OF HYALURONATE SOLUTIONS
`
`Charles J. Knill t, John F. Kennedy t, Yasmin Latif 1& Derek C. Ellwood 2
`
`J Chembiotech Laboratories, Institute a/Research & Development,
`University 0/ Birmingham Research Park, Vincent Drive,
`Edgbaston, Birmingham, Bl5 2SQ, UK.
`
`2 Department a/Microbiology, Medical School, University a/Newcastle,
`Newcastle Upon Tyne, UK
`
`ABSTRACT
`
`The effects of various metal ions on the rheological flow profiles of hyaluronate
`solutions were investigated, by controlled incubation of metal chloride salt solutions
`with sodium hyaluronate (NaHA) solution under ambient conditions. Results from
`application of the Williamson model to the flow profiles of incubated solutions showed
`a progressive decrease in Williamson zero shear viscosity (110) with increasing atomic
`number / atomic mass of the metal ion, which, with the exception of data for u', could
`be related to a power equation (y = cxb
`) . Such reductions in viscosity were not a result
`of hydrolysis (determined by GPC), but may be due to diffusing ions disrupting
`hydrogen bonding and shielding electrostatic repulsions between carboxylate groups.
`
`INTRODUCTION
`
`Hyaluronan is a biocompatible / biodegradable, linear, water-soluble, ionic polymer
`composed of repeating (1-H) linked disaccharide units consisting of ~-D-GlcpA and
`~-D-GlcpNAc, linked together by (l~3) glycosidic bonds 1.
`It has a high molecular
`weight (around 105_107 Da), depending on source, giving a DP range of - 250-25000 2.
`It is found in all vertebrates, being present in almost every tissue as a component of the
`extracellular matrix and is distributed throughout the mammalian body, especially in
`synovial fluid, loose connective tissue, umbilical cord and the vitreous body of the eye 3.
`The largest amount of hyaluronan (7-8 g per average human, 50 % of the total in the
`body) is in the skin tissues (both the dermis and epidermis) 4. The most commonly
`isolated / utilised (in vitro) forms of hyaluronan are the free acid (hyaluronic acid, HA)
`and its sodium salt (sodium hyaluronate, NaHA).
`In solution the hyaluronan backbone is stiffened by the chemical structure of linked
`disaccharide units,
`internal hydrogen bonds, mutually repelling anionic groups and
`interactions, making it a rigid and highly hydrated molecule.
`It adopts an
`solvent
`expanded random coil structure in physiological solutions, occupying a large domain.
`Small molecules, e.g. water and electrolytes, can freely diffuse through the domain,
`whilst large molecules are partially excluded due to their hydrodynamic size. At low
`concentrations,
`individual chains entangle forming a continuous network, giving
`viscoelastic and pseudoplastic properties, which is unique for a water-soluble polymer
`at low concentration. At higher concentrations entangled networks can be formed,
`which can resist rapid, short duration fluid flow,
`thus exhibiting elastic properties.
`However, short fluid flow of longer duration can partially separate and align molecules,
`allowing movement and thus exhibiting viscous properties 5.
`
`

`

`176
`
`Rheological behaviour of hyaluronan
`
`The viscoelastic properties of hyaluronan solutions are ideal for use as a biological
`shock absorber and lubricant, which is why it is present in synovial fluid, where it
`lubricates the cartilage between joints. The cartilage provides a cushion between the
`bones allowing the joint to move smoothly. However, in an arthritic joint the elasticity /
`viscosity of the joint fluid is reduced, diminishing the shock absorbing and barrier
`properties 6. Highly viscoelastic hyaluronan solutions can be injected into joints
`(viscosupplementation) in order to restore the rheological environment of the joint and
`thus improve joint function. This is used in osteoarthritic joints to provide instant
`protection and shock absorption, thus decreasing pain associated with mobility.
`The aim of this investigation was to see how the interaction of hyaluronan with a
`range of metal ions (of differing ionic size, valency, etc.) affected the rheological flow
`characterisitics (especially the zero shear viscosity, 110) and molecular weight profile.
`This is of particular interest with respect to localised in vivo applications of hyaluronan,
`since there are numerous metal ions in the body which could interact with administered
`hyaluronan resulting in significant changes in desired physical properties.
`
`MATERIALS & METHODS
`
`Preparation of boiled, nitrogen flushed, deionised water
`
`Deionised water was boiled (~ 5 minutes, under vacuum) to remove dissolved air,
`cooled to ambient temperature, and flushed with nitrogen (- 15 minutes) to displace any
`residual air, and prevent air redissolving. The resultant boiled, nitrogen flushed,
`deionised water was refridgerated (4°C) until required, and was used for the preparation
`of all subsequent solutions (the NaHA and metal salt solutions detailed below).
`Boiling / nitrogen flushing was used to exclude dissolved oxygen from the water to try
`and minimise any oxidative hyaluronan degradation effects.
`
`Preparation of sodium hyaluronate solution
`
`fermentation using
`Sodium hyaluronate (NaHA, 6 g, produced by microbial
`Streptococcus equi) was dispersed in boiled, nitrogen flushed, deionised water (600 mL)
`in a conical flask (1 L), and the flask headspace was flushed with nitrogen. The flask
`was stoppered and the NaHA allowed to dissolve slowly over a period of- 48 hours (at
`4°C), with ocasional gentle agitation / swirling to assist solubilisation, resulting in a
`viscous, homogenous NaHA solution (1 % w/v). Care was taken to avoid the use of any
`metal equipment
`in the production of the hyaluronate solution (especially items
`containing iron / stainless steel), to minimise potential degradatory effects.
`
`Preparation of metal ion solutions
`
`Metal chloride salt solutions (0.25 M) were prepared by dissolving the necessary
`amounts of anhydrous lithium chloride (LiCI, M, 42.39), sodium chloride (NaCl,
`M, 58.44), potassium chloride (KCI, M, 74.55), anhydrous calcium cloride (CaCh,
`Mw 111.0), manganese (II) chloride tetrahydrate (MnCh.4H20, Mw 197.9), cobalt (II)
`chloride hexahydrate (CoCh.6H20, M, 237.9), and cerium (ill) chloride heptahydrate
`in
`(CeCh.7H20, Mw 372.6), in boiled, nitrogen flushed, deionised water (50 mL)
`volumetric flasks (50 mL). All salt solutions were filtered (using 0.45 11m pore size,
`25 mm diameter Titan nylon membrane filters), and flushed with nitrogen (to remove
`any dissolved air / oxygen), before use in hyaluronate incubation experiments.
`
`

`

`Effect of metal ions on now profiles
`
`177
`
`Incubation of hyaluronate with metal ions
`
`Aliquots of NaHA solution (1 % w/v, 5 mL) were transferred into individual
`headspace vials (20 mL, Merck). Aliquots of the metal chloride salt solutions detailed
`above (0.25 M, 5 mL) were transferred into the individual headspace vials containing
`the NaHA solution (in duplicate) to give overall concentrations of 0.5 % w/v NaHA and
`0.125 M metal ions. The pipette tip used for individual NaHA solution dispensing was
`also utilised for subsequent metal ion solution dispensing, so that the latter ensured any
`residual NaHA was washed out ofthe pipette tip. Duplicate controls were also prepared
`using boiled, nitrogen flushed, deionised water aliquots (5 mL). The resultant solutions
`were flushed with nitrogen (to remove any dissolved air / oxygen, which also facilitated
`mixing), and the vials sealed with butyVPTFE lined (3.0 mm) plain aluminium crimp
`caps (20 mm diameter, Merck). The solutions were incubated at ambient temperature,
`and the rheological flow profiles determined exactly 1 hour after metal ion solution
`addition (as detailed below).
`[This incubation test procedure had been previously
`validated by performing replicate rheological analyses on test solutions (NaHA and
`metal salts), which resulted in Williamson infinite shear viscosity (110) values with %
`variation values of < 10 %].
`
`Determination of rheological flow profiles
`
`All rheological flow profile measurements were performed using a TA Instruments
`AR 1000 'Rheolyst' controlled stress rheometer, equipped with 'Rheology Solutions'
`software (v. 1.2.2). The software is split into two modules, the 'ARI000' module
`controls the instrument itself and enables the operator to set up experimental procedures
`and perform the actual experiments, whilst the 'Data' module manipulates and presents
`the collected data. Duplicate rheological flow profile measurements were performed
`using the test parameters detailed below. Approximately 5 mL of sample was used for
`each flow test (the remainder being stored at 4 °C until molecular weight determinations
`were performed). The 'Data' module was used to apply the Williamson model to the
`resultant flow profiles (shear rate vs viscosity) in order to determine the Williamson
`zero shear viscosity (110) values. The Williamson model describes the low shear
`viscosity behaviour and is derived from the Cross model when 11 »1100 7.
`
`Geometry:
`Geometry gap:
`Geometry inertia:
`Instrument inertia:
`Temperature:
`Pre-experimental shear stress:
`Test stress range:
`Number ofPoints:
`
`parallel plate (4 em diameter)
`500 urn
`- 1.5 J.lNms2(calibrated before each test)
`-14.2 IlNms2(calibrated daily)
`20°C (controlled by Peltier plate)
`1.768 Pa (for 10 s)
`0.5 - 500 Pa (log ramp)
`3I (max. point time 1 min)
`
`Determination of molecular weight profiles
`
`The molecular weight profiles of incubated solutions (0.5 % w/w NaHA, 0.125 M
`metal salt, & controls) detailed above were determined - 12-24 hours after incubation,
`by GPC analysis using the isocratic HPLC system detailed below.
`
`

`

`178
`
`Rheological behaviour ofhyaluronan
`
`Instrumentation:
`
`GPC Columns:
`
`Eluent:
`
`Flow rate:
`Calibration:
`
`Injection volumes:
`Data collection:
`Data manipulation:
`
`Knauer HPLC pump 64
`Waters 712 WISP autoinjector
`Knauer column oven & control unit (set to 30°C)
`Knauer differential refractometer
`Dionex ill 20 Universal interface (for PC data link)
`Progel™TSK G5000, G4000 & G2500 PW (131J.IIl,
`300 x 7.5 mm ill) columns (& guard) linked in series
`0.05 M phosphate buffer containing 0.25 M NaCl, pH 7.0
`(prepared using 18 Mil UHQ water with helium sparging)
`0.5 mL/minute
`Pullulan standards with Mp values of 853, 380, 186, 100,
`48,23.7, 12.2 & 5.8 kDa (0.1 Ilg/J.J.L in UHQ water)
`200 J.J.L (solutions diluted 1 in 10 with UHQ water)
`Dionex Chromeleon software (v. 6.11)
`PL Caliber Reanalysis software (v. 7.04)
`
`RESULTS & DISCUSSION
`
`Mean flow profiles (of duplicate analyses, shear rate vs viscosity) for incubated
`NaHA / metal ion solutions (and controls) are displayed in Figures I & 2, and show that
`incubation with metal ions results in a reduction in viscosity. GPC analysis showed that
`the control sample (NaHA + water, i.e. no additional metal ions added) had Mp and
`polydispersity (Mw/Mn) values of2.5 x 106 Da and 1.6, respectively. Incubated samples
`had Mp values in the region of 1.9-2.5 x 106 Da and polydispersity values in the region
`of 1.7-2.0, i.e. no significant degradation had taken place.
`The Williamson model was applied to the flow profiles to determine the Williamson
`zero shear viscosities (Tlo) 7. This is the region where increasing shear stress / shear rate
`has little or no effect on viscosity (i.e. Newton's law ofliquid flow is obeyed).
`
`__ control
`
`-.-Na
`__ Li
`
`-+-Mn
`
`1.00
`
`Ui'
`iiie:.
`~ 0.10
`IIIo
`II's
`
`UI
`
`0.01 ~--r"""''''''''",,"",r'I'T''"-'''''''''I""''T""'I"T'''l''I''IT"--'-T"''T...........,-......-r-I'''T'''I,,","
`10
`100
`1000
`10000
`1
`shear rate (1/5)
`Mean flow profiles (shear rate vs viscosity) for NaHA solutions
`incubated with water (control), Na+,u: and Mn2+ ions (all cr salts).
`
`Figure 1.
`
`

`

`Effect of metal ions on now profiles
`
`179
`
`_K
`-.-Ca
`_Co
`_Ce
`
`1.00
`
`'iii'
`rei
`!:!;.
`~ 0.10
`'iii
`ou
`VI.:;
`
`0.01 -l-......-'T""T""r-TT........--r-..,.....,,.....,........or----r--r............,.,.......-or--or-r..,...,.......,
`10
`100
`1000
`10000
`1
`shear rate (1/s)
`Mean flow profiles (shear rate vs viscosity) for NaHA solutions
`incubated with K+, C~+, C02+ and CeJ+ ions (all cr salts).
`
`Figure 2.
`
`The Williamson zero shear viscosities (110) were related to the atomic number and
`atomic mass of the associated metal ions used in the incubation (Figure 3). The data
`
`(with the exception ofLn could be satisfactorily modelled using the power equation
`y = cxb (Figure 3). No obvious correlation could be obtained between the Williamson
`shear viscosity (Tjo) and ionic radii /
`ionic volume of the metal ions used in the
`incubation.
`
`0.6
`
`0.5
`
`0.4
`
`-;
`cO
`2::- 0.3
`0
`!='
`
`0.2
`
`0.1
`
`\
`
`\
`
`\0
`
`\
`
`\
`
`\,
`\.,
`
`.0
`
`\ 0,
`"
`0' ••
`
`•
`
`o
`
`atomic number
`
`atomic mass
`
`y = 4.9571 X.o·
`
`oCr--------......-- .....-----------0 _
`
`7615
`
`0.0 +---.---r---.---.---r-...---r-or---.--..--.......-....-.......-....--.
`o 10
`20 30
`40 50
`60 70
`80 90 100 110 120 130 140 150
`
`Figure 3.
`
`The relationship between the Williamson zero shear viscosity (Tjo) and
`the atomic number / atomic mass of the metal ion used for incubation.
`
`

`

`180
`
`Rheological behaviour of hyaluronan
`
`These results are in agreement with previous published data, where zero shear
`viscosity (110) was found to decrease for the series Na+, K+ and Ca2+, and Na+, Lt and
`It has been suggested that these observed reductions in viscosity are a result of
`K+ 8,9.
`ions diffusing between the hyaluronan chains and shielding electrostatic repulsions
`(between nearby carboxylate groups) and disrupting hydrogen bonding interactions
`between chains 8. This would result in a collapse of expanded stiffened coil structure to
`a more compact structure where chains could slide over each other more easily
`(i.e. flow), thereby leading to a reduction in viscosity.
`
`CONCLUSIONS
`
`As the atomic number / atomic mass of the metal ion increased, a progressive
`Contact with metal
`decrease in Williamson zero shear viscosity (110) was observed.
`ions thus alters the rheological characteristics of hyaluronan, which may in tum
`adversely affect its functional application properties. The body contains an abundance
`of metal ions (especially K+, Na+, Ca2+ and Mg2+), which therefore have the potential to
`alter the viscosity of administered hyaluronan-based materials. The effect of Li+
`seemed to be more pronounced than expected (based solely upon its charge, size, etc),
`which is of particular interest since a considerable number of pharmaceutical products
`contain lithium.
`The effects of a broader range of metal ions of higher valency should be investigated
`(e.g. Al3+, Ti4+ & Sn4+) to see if any further correlations can be made. The effects of
`such metal ions on the viscoelasticity ofhyaluronan materials (using creep & oscillation
`measurements) also merits investigation.
`
`REFERENCES
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`A. Linker & K. Meyer, Production of unsaturated uronides by bacterial hyaluronidases,
`Nature (London), 1954, 174, 1192-1193.
`T. C. Laurent, Structure of hyaluronic acid, In: Chemistry and Molecular Biology of the
`Extracellular Matrix, Vol. 2, E. A. Balazs (ed.), 1970, Academic Press, New York,
`pp.703-732.
`K. Meyer & J. W. Palmer, The polysaccharide of the vitreous humor, J. Bioi. Chem.,
`1934, 107,629-634.
`J. R. E. Fraser & T. C. Laurent, Turnover and metabolism ofhyaluronan, In: The Biology
`ofHyaluronan, Ciba Foundation Symposium 143, D. Evered & J. Whelan (eds.), 1989,
`John Wiley & Sons, Chichester, pp. 41-53.
`R. E. Turner, P. Lin & M. K. Cowman, Self-association of hyaluronate segments in
`aqueous NaCl solution, Arch. Biochem. Biophys., 1988, 265, 484-495.
`M. F. McCarty, A. L. Russel & M. P. Seed, Sulphated glycosaminoglycans and
`glucosamine may synergize in promoting synovial hyaluronic acid synthesis, Med.
`Hypotheses, 2000, 54 (5), 798-802.
`H. A. Barnes, J. F. Hutton & K. Walters, An Introduction to Rheology, Rheology Series
`Vol. 3, 1989, Elsevier, Amsterdam, pp. 16-23.
`Y. Kobayashi, A. Okamoto & K. Nishinari, Viscoelasticity of hyaluronic acid with
`different molecular weights, Biorheology, 1994,11 (3), 235-244.
`K. Ogino, Viscoelastic properties of hyaluronic aqueous solutions, Kobushu Ronbunshu,
`1998, 55 (12), 736-748.
`
`