Aldred, N.

Arpa Sancet, M.P.

Bauer, S.

Callow, J.A.

Callow, M.E.

Clare, A.S.

FInlay, J.

Grunze, M.

Rosenhahn, A.

English

Arpa Sancet, M. P.

Callow, M. E.

Callow, J. A.

Clare, A. S.

KITopen

KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association Amphiphilic Polysaccharides  as Inert Surface Coatings S.Bauer,1,2* M.P. Arpa Sancet,1,2 J.Finlay,3 M.E.Callow,3 J.A.Callow,3 N.Aldred,4 A.S.Clare,4  M.Grunze1,2 and A.Rosenhahn1,2  1 Applied Physical Chemistry, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany 2 Institute of Functional Interfaces, IFG, Karlsruhe Institute of Technology, PO Box 3640, 76021 Karlsruhe, Germany  3 School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom 4 School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom  Institute of Functional Interfaces      * Email: stella.bauer@pci.uni-heidelberg.de Motivation and Hypothesis • Polysaccharides are biocompatible and non-toxic • Capping of free carboxyl-groups prevents complexation of bivalent cations like Ca2+ and hence syneresis of  the films • A hydrophobic protection molecule (fluorinated amine) establishes amphiphilic properties in the highly hydrophilic polysaccharide network • Additionally, the contact angle is shifted towards the minimum in the Baier curve  • Standard assays with a range of different foulers to correlate the change in surface chemistry with the different settlement preferences: biofilm building microorganisms, hard and soft macrofoulers Surface Preparation Polysaccharide coupling procedure:  APTMS - 3-Aminopropyltrimethoxy silane NHS – N-Hydroxysuccinimid EDC – N-(3-Dimethyl amino propyl)- 3-ethyl carbodiimid TFEA – Trifluoroethylamine Introduction • Polysaccharides are resistant to the adhesion of proteins, mammalian cells and bacteria[1] • Against marine biofoulers, the coatings perform worse than expected due to the complexation of bivalent ions[2]   • Glycosaminoglycans, found in the extracellular matrix, highly hydrophilic, differ in sulfatation of CS, both have only one carboxylic-acid moiety per disaccharide unit. The use of CS is bioinspired due to its presence in fish mucus and its potential contribution to protection of the skin of fish[3] • Amphiphilic surface-coatings have a higher anti-fouling performance than those which are only hydrophilic or hydrophobic[4] Hyaluronic Acid (HA) Chondroitin-6-Sulfate (CS) Coating APTMS HA CS HA + TFEA CS + TFEA Contact angle [°] 35 ± 5 < 10 11 ± 3 25 ± 5 28 ± 3 Ellipsometric thickness [Å] 11.4 ± 4.0 26.6 ± 6.6 26.5 ± 7.1 28.2 ± 5.1 28.6 ± 4.0 Thickness from XPS [Å]* n.a. 23.3 ± 2.5 18.6 ± 5.6 24.8 ± 4.2 23.2 ± 7.7 Biological Evaluations Protein adhesion assay[5] Adhesion strength of Cobetia marina Attachment of Navicula perminuta Settlement of Ulva linza zoospores A - Comparison of diatom density before and after shear stress (32Pa) B  - Percentage removal  • TFEA capping weakens attachment for HA • Attachment on CS surfaces is in range of glass standard, modification shows no effect  Adhesion behavior of Balanus amphitrite Spore density before and after shear stress (52Pa)  • Settlement is very low on both HA coatings • Modification makes CS films more attractive and adhesion strength increases  A - Initial settlement after incubation B - Critical shear stress to remove 50% of adherent bacteria  • Initial settlement is not influenced by different surface chemistries • Adhesion strength is weakened on TFEA modified HA surfaces • Native CS performs very well, behaviour after TFEA capping is reversed  [1] Morra, M.; Cassineli, C. J. Biomater. Sci.-Polym. Ed. 1999, 10, 1107. [2] Cao, X. Y.; Pettit, M. E.; Conlan, S. L.; Wagner, W.; Ho, A. D.; Clare, A. S.; Callow, J. A.; Callow,  M.E.; Grunze, M.; Rosenhahn, A. Biomacromolecules 2009, 10, 907. [3] Shephard, K. L. Rev. Fish. Biol. Fish. 1994, 4, 401. [4] Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Hexemer, A.; Sohn, K. E.; Kramer, E. J.; Fischer, D. A. Biomacromolecules 2006, 7, 1449. [5] Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. Literature Acknowledgements Conclusions • Amide modification does not worsen good protein resistance • Modification with TFEA has a positive impact on HA coatings: decreased adhesion strength of C.marina, lower settlement and decreased adhesion strength of N.perminuta, U.linza and B.amphitrite • Opposite behavior or no change in settlement of the different species is observed for CS coatings (likely related to the negative charge carrying sulfate-group) The work was funded by the Office of Naval Research (Grant number N00014-08-1-1116). Characterization: • All polysaccharide coatings are resistant against both negatively charged proteins • Positively charged proteins adhere more readily compared to the negative ones, but in most cases weakly compared to the not-resistant C12–SAM standard Lab adhesion experiments: Proteins and biofoulers • Protein affinity test:[5] Determination of protein film thickness by spectral ellipsometry • Bacteria: Microfluidic Cobetia marina assay (M.P. Arpa Sancet) • Algea: Navicula perminuta settlement and detachment assay (J. Finlay, University of Birmingham) Ulva linza settlement and detachment assay                 (J. Finlay, University of Birmingham) • Barnacles: Balanus amphitrite settlement assay (N.Aldred, University of Newcastle)  POEGMA – Poly(ethylenglykolmethacrylat) AA – Alginic Acid  Field tests in Melbourne, Florida (USA) • Settlement after 48h incubation in sea water • Quantification of organisms bigger than 10µm by optical microscopy  Proof of amine coupling with simiplified SAM-System: XPS analysis:  Conditions are suitable for intended modification of free carboxyl groups  Settlement after 96 h  • Settlement is strongly reduced on modified HA • Settlement comparable on both CS surfaces  Settlement on glass standard was unusually low and is not shown • Mainly diatom settlement • All sugar coatings reduce settlement significantly • Strongest effect of TFEA coupling is observed for AA, which contains two carboxyl-groups per disaccharide unit XP-detail spectrum of F1s region * Thickness determined by attentuation of the Si2p signal of a representative APTMS layer 1. Silanization 3. Polysaccharide modification 2. Polysaccharide coupling 

Amphiphilic polysaccharides as inert surface coatings

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Amphiphilic polysaccharides as inert surface coatings

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