Covalent immobilization of a sulfated polysaccharide via GraftFast® technology on a surface acoustic wave (SAW) sensor: toward a tool to measure interactions with vascular biomolecules
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1
INSERM, University Paris 13, University Paris Diderot, Sorbonne Paris Cité University, Laboratory for Vascular Translational Science, INSERM U1148, France
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2
CEA Saclay, Laboratory of Innovation in surface Chemistry and Nanosciences, NIMBE UMR CNRS 3685, France
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3
CNRS, Paris-Saclay University, ISMO, CNRS, France
P-, L- and E-selectins, glycoproteins mainly expressed by platelets, leucocytes and endothelial cells respectively, are relevant biomarkers of vascular pathologies such as atherothrombosis and endothelial ischemia[1]. Fucoidans, sulfated polysaccharides extracted from brown algae, have been used to perform molecular imaging of aneurysms, endocarditis and heart ischemia[2][3]. Although a strong interaction between fucoidan and a Fc chimera P-selectin was evidenced[4][5] using Surface Plasmonic Resonance (SPR), the role of the other selectins remains to be clarified. The covalent immobilization of a low molecular weight fucoidan (LMWF) by the GraftFast® technology on SAW sensor6 has been performed to quantify the interactions with native P-, L- and E-selectins
. The kinetic parameters of the affinity of LMWF for P-, E- and L-selectins and stability constants have been evaluated.
Materials and Methods: The LMWF was obtained from Ascophyscient®, a fucoidan produced from the French company Algues et Mer[3]. The molecular weight was determined using HPSEC-MALLS-dRI. Fucose, uronic acid, sulfate and primary amines contents were determined with colorimetric assays[7][8][9][10][11]. Fucoidan amination at its reductive end was performed using Kondo et al.[10] and Seo et al.[11] methods. Methacrylated fucoidans were immobilized onto the gold surface of a SAW sensor (SAW Instruments GmbH) by the quick and easy to handle wet technology GraftFast® based on diazonium salts as described by Mévellec et al.[6]. The resulting functionalized SAW chip was introduced in a sam®5 blue SAW instrument (SAW Instruments GmbH and HBS-EP was used as running buffer with a flow rate of 30 μL/min. After equilibration of the baseline, selectin solutions (P, L and E) (Recombinant Human selectin/CD62P/CD62L/CD62E, CF-R&D systems ADP1, ADP2, ADP3), at different concentrations (10, 50 and 100 nM) were injected in order to detect association and dissociation steps as phase shifts indicating binding and unbinding events. Corresponding binding kinetic parameters were calculated via non-linear curve fitting using GraphPad Prism® 5.3.
Results and Discussion: Synthesis of methacrylated fucoidan and its immobilization on SAW chips have been successful. Binding events were characterized for P-selectin. Resulting binding kinetics for P-selectin were estimated in a range from 10 to 30 nM. After a non-linear curve fitting, the Kd of the P-selectin was estimated to 19 nM. For E and L-selectin, no binding events were observed with our functionalized SAW chip.
Conclusion: These results highlight for the first time the interaction between native P-selectin and immobilized fucoidan and demonstrate its selectivity for P-selectin against L or E-selectin. Moreover, SAW binding data demonstrate also that the grafting process used does not alter the biological activity of fucoidan. This versatile wet grafting process offers the ability in the future to produce a powerful tool to screen biological activity of polysaccharides.
ANR LabCom; Large-scale integrated project NanoAthero (NMP-2012-309820); ANRT for CIFRE fellowship n°2014/0337 (Lucas Chollet)
References:
[1] Bachelet, L.; Bertholon, I.; Lavigne, D.; Vassy, R.; Jandrot-Perrus, M.; Chaubet, F.; Letourneur, D. Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets. Biochimica et Biophysica Acta 1790. 2009, 141–146.
[2] Rouzet, F.; Bachelet-Violette, L.; Alsac, J.M.; Suzuki, M.; Meulemans, A.; Louedec, L.; Petiet, A.; Jandrot-Perrus, M.; Chaubet, F.; Michel, J.B.; Le Guludec, D.; Letourneur, D. Radiolabeled Fucoidan as a P-Selectin Targeting Agent forIn Vivo Imaging of Platelet-Rich Thrombus and Endothelial Activation. J Nucl Med. 2011, 52 (9), 1433-1440.
[3] Saboural, P.; Chaubet, F.; Rouzet, F.; Al-Shoukr, F.; Azzouna, R. B.; Bouchemal, N.; Picton, L.; Louedec, L.; Maire, M.; Rolland, L.; Potier, G.; Le Guludec, D.; Letourneur, D.; Chauvierre, C. Purification of a Low Molecular Weight Fucoidan for SPECT Molecular Imaging of Myocardial Infarction. Mar. Drugs. 2014, 12, 4851-4867.
[4] Bachelet, L.; Bertholon, I.; Lavigne, D.; Vassy, R.; Jandrot-Perrus, M.; Chaubet, F.; Letourneur, D. Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets. Biochim Biophys Acta 1790. 2009, 141–146.
[5] Suzuki, M.; Bachelet-Violette, L.; Rouzet, F.; Beilvert, A.; Autret, G.; Maire, M.; Menager, C.; Louedec, L.; Choqueux, C.; Saboural, P.; Haddad, O.; Chauvierre, C.; Chaubet, F.; Michel, J.B.; Serfaty, J.M.; Letourneur, D. Ultrasmall superparamagnetic iron oxide nanoparticles coated with fucoidan for molecular MRI of intraluminal thrombus. Nanomedicine. 2014, doi:10.2217/nnm.14.51.
[6] Mévellec, V.; Roussel, S.; Tessier, L.; Chancolon, J.; Mayne-L’Hermite, M.; Deniau, G.; Viel, P.; Palacin, S. Grafting Polymers on Surfaces: A New Powerful and Versatile Diazonium Salt-Based One-Step Process in Aqueous Media. Chem. Mater. 2007, 19, 6323–6330.
[7] Dische, Z. New Color Reactions for Determination of Sugars in Polysaccharides. In Methods of Biochemical Analysis; Glick, D., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 1955, 2, 313–358.
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[11] Seo, J. H.; Adachi, K.; Lee, B. K.; Kang, D. G.; Kim, Y. K.; Kim, K. R.; Lee, H. Y.; Kawai, T.; Cha, H. J. Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates. Bioconjug Chem. 2007, 18, 2197-201.
Keywords:
screening,
biosensing,
Surface modification,
biomacromolecule
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
Poster
Topic:
Biosensors
Citation:
Chollet
L,
Berthelot
TT,
Greff
RR,
Letourneur
DD and
Chaubet
FF
(2016). Covalent immobilization of a sulfated polysaccharide via GraftFast® technology on a surface acoustic wave (SAW) sensor: toward a tool to measure interactions with vascular biomolecules.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.02056
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.