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Affinity binding of EMR2 expressing cells by surface-grafted chondroitin sulfate B Anouck L. S. Burzava, Marek Jasieniak, Michaelia P. Cockshell, Claudine S. Bonder, Frances J. Harding, Hans Joerg Griesser, and Nicolas H. Voelcker Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 25, 2017

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Affinity binding of EMR2 expressing cells by surface-grafted chondroitin sulfate B Anouck L. S. Burzava,†,‡ Marek Jasieniak,‡ Michaelia P. Cockshell,§ Claudine S. Bonder,§,^ Frances J. Harding,†,‡ Hans J. Griesser,‡ Nicolas H. Voelcker †,‡,* † ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of South Australia, Mawson Lakes, South Australia 5095, Australia ‡ Future Industries Institute, University of South Australia, Mawson Lakes, South Australia, 5095, Australia § Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia, 5000, Australia ^ Adelaide Medical School, Faculty of Health Sciences, University of Adelaide, Adelaide, Australia

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Abstract

The propensity of glycosaminoglycans to mediate cell-cell and cell-matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS) -B is of particular interest, since it interacts with the receptor (EGF)-like modulecontaining mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells pre-incubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types.

Keywords: Chondroitin sulfate, Cell-material interactions, Dermatan sulfate, EMR2, Glycosaminoglycans, Plasma polymer, heptylamine.


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Introduction Glycosaminoglycans (GAGs), linear polysaccharides consisting of repeating disaccharide units of amino sugar (N-acetyl-D-galactosamine or N-acetyl-D-glucosamine) and uronic acid (Liduronic acid or D-glucuronic),1,

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are expressed by almost all animal cells, forming a major

component of the extracellular matrices (ECM) that provide structural support to tissues.1, 2 Not confined to physical scaffolding, GAG molecules play an extensive role in biochemical signaling, mediating cell communication, cell-matrix and cell-cell interaction, notably in events related to inflammation and tissue repair.1-3 GAGs have the capacity to selectively bind and sequester growth factors,4 and modulate their signaling activity.5 The physical and biochemical properties of GAGs have led to their incorporation into biomaterials.6 The affinity of specific growth factors, such as fibroblast growth factor (FGF2) and epidermal growth factor (EGF), to an otherwise low protein-binding surface has led to application of GAG-functionalized surface to vascular implants.7-11 While differential adhesion profiles of endothelial cells and fibroblasts to GAG libraries have been noted,12 the ability of direct affinity capture of specific cell types onto GAG-functionalized materials surfaces remains largely unexploited. Four distinct categories of GAGs have been defined: heparan sulfate and heparin, keratan sulfate, hyaluronan, and finally chondroitin (or dermatan) sulfate (CS).1 CS, which is the most abundant GAG in the body,13 can be further classified according to its sulfation patterns (CS-A, B, -C, -D and -E). Cell binding to sulfated GAG ligands may be influenced by proteins presented by GAGs,12 but is also known to be mediated through specific receptors, such as selectins, presented at the cell surface.14 Receptor affinity is sensitive to small changes in the GAG ligand structure: CS-B and CS-E chains are able to specifically bind L- and P-selectins but not any other


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GAGs, and CS-A, CS-B, CS-C, CS-D, and CS-E are able to specifically bind to CD44 but not heparan or keratan sulfate.14 The (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2, also known as CD312) is a cell surface protein receptor of the EGF-TM7 family expressed by cells of the immune system, a prompt for a wide range of biological events, including adhesion and/or migration of cells.15-21 The fourth EGF domain of EMR2 has been shown to interact with CS-B.20, 22

Interaction between those two entities is believed to play a role in the interaction between B

cells and activated T cells, macrophages or dendritic cells; thus potentiating the effects of proinflammatory response.19, 21 EMR2 expression was recently identified on a population of nonadherent endothelial progenitor cells (naEPCs) (see data deposited in the NCBI gene expression omnibus (GEO Accession number: GSE25979).23, 24 These circulating EPCs are of major interest as they are capable of in vivo vascularization, while showing low immunogenicity and rapid expansion in response to interleukin-3.23, 25 The potential of these cells for clinical application raises the question of how they can be harnessed, via novel strategies, for transplantation.25, 26 The aim of the work reported here was to develop a platform technology capable of selectively capturing EMR2-expressing cells via their affinity for CS-B; this could find applications in functionalizing scaffolds for cell isolation in vitro, and in particular the binding and release of specific cells, including naEPCs.27 To enable the technology to be applied to a wide range of solid carrier materials and biomedical devices, we selected an interlayer fabricated by the deposition of a plasma polymer coating from heptylamine (HApp)

28,29

, whose surface amine

groups enable interfacial bio-conjugation by amide formation with carboxyl groups of CS-B. While GAGs can be simply adsorbed onto a positively charged substrate via electrostatic binding,30 adsorbed GAGs can be displaced by other macromolecules; many applications require


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covalent immobilization.31 Moreover, adsorption of GAGs is feasible only on a restricted range of materials surfaces. We have explored three different covalent bio-conjugation strategies to elucidate the biologically most effective presentation of immobilized CS-B. We used X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToFSIMS) to characterize the grafted CS layers. Surface-grafted CS layers were assessed for their capacity to bind EMR2-expressing U937 cells; as serum proteins were thought likely to adsorb onto grafted CS and mask its activity, experiments were performed in PBS, which would be the required protocol for capture of EMR2-expressing cells via our technology. By using grafted CSA layers as comparative controls, we show that affinity binding of EMR2-expressing U937 cells can be achieved onto surface-bound CS-B in vitro.


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Materials and methods Chemicals and Supplies CS-A (cat# C9819), CS-B (cat# C3788), heptylamine (HA), sodium periodate, sodium cyanoborohydride

purum,

morpholino)ethanesulfonic

acid

phosphate

buffer

monohydrate

saline

(MES),

tablets

(PBS),

5-azido-2-nitrobenzoic

2-(Nacid

N-

hydrosuccinimide ester (NSANB), fluorescein diacetate (FDA), propidium iodide (PI) and methanol ACS reagent were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) and N-hydroxysulfosuccinimide (Sulfo-NHS) were obtained from ProteoChem (Rockford, III, USA). Twenty-four-well tissue culture polystyrene (TCPS) cell plates were from ThermoFisher Scientific Pty Ltd (Adelaide, SA, Australia). For surface analyses, 13 mm diameter Thermanox plastic coverslips (Th) were obtained from ProSciTech (Townsville, QLD, Australia) whilst silicon wafers (100) were supplied by MMRC Pty Ltd (Melbourne, VIC, Australia).

Plasma Polymerization Substrates were coated with an interfacial bonding layer of heptylamine plasma polymer (HApp) in a custom-built glass reactor.32, 33 Plasma deposition was carried out under application of continuous RF power of 40 W for 60 s. The monomer pressure during the treatment was 0.2 Torr. After plasma polymerization, to minimize the presence of active free radicals remaining in the surface layer,28 the monomer flow into the chamber was maintained for an additional 3 min to assist in quenching free radicals.


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TCPS 24-well plates were directly coated with HApp, and Thermanox coverslips were put inside the wells before plasma deposition. On Thermanox substrates, it was found to be advantageous to deposit a thin layer of hexamethydisiloxane plasma polymer before depositing the HApp layer. This approach avoided occasional coating delamination.

GAG Immobilization Protocols CS-A and CS-B were grafted covalently onto HApp surfaces using three different immobilization chemistries, due to the possibility that surface densities of grafted CS molecules might vary between the different protocols and translate to differences in cell binding. EDC– Sulfo-NHS, NSANB photo-crosslinking, and periodate chemistry were chosen, and immobilization conditions for each chemistry were optimized in order to obtain maximum coverage of CS on the surface.

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EDC–Sulfo-NHS

A 2 mg/mL CS solution was activated with EDC-HCl and Sulfo-NHS (at a molar ratio 1:2:5 CS: EDC-HCl: Sulfo-NHS) for 15 min in MES buffered to pH 4. The activated solution was then applied onto HApp-modified substrates to react. Samples are left to react overnight, before being washed multiple times with MilliQ water (every 15 min for 4 h) in order to remove any unbound CS molecules.

-

Periodate

CS was dissolved in 1X PBS to produce a solution at a final concentration of 2 mg/mL (pH 7.4). A 10 mg/mL solution of sodium periodate was formed in MilliQ water, and 218 μL of this


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solution was added to the CS solution (molar ratio 1:1). Oxidation was carried out at room temperature for 1 h. Then, 1 mL of glycerol was added to the activated solution to quench the reaction, and the mixture was deposited onto the HApp-modified substrates. After 2 h of reaction, 100 μL of sodium borohydride solution (20 mg/mL in PBS) was added to each well to stabilize the links, i.e. to reduce formed imines to amines. The solution was left to react overnight, before being thoroughly washed with MilliQ water (every 15 min for 4 h) in order to remove any unbound CS.

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NSANB Photo-crosslinker

The day preceding the experiment, the photo-crosslinker NSANB was dissolved in methanol to a final concentration of 1 mg/mL. The HApp-modified substrates were washed once with methanol and then incubated with the NSANB solution at room temperature for 2 h. Then, the samples were washed twice with methanol. After methanol evaporation, CS (2 mg/mL in a 10% MilliQ water / 90% methanol solution) was deposited onto the surfaces comprising immobilized NSANB. After evaporation of the solvent, substrates were exposed to UV radiation of 100 W for 30 min. The UV lamp (50% intensity, 320-500 nm), was positioned 10 cm above the sample. All the steps prior to this UV activation were performed in a dark room. After the UV radiation, the samples were left in MilliQ water overnight, before being further washed with MilliQ water (every 15 min for 4 h) in order to remove any unbound molecule.

X-ray Photoelectron Spectroscopy (XPS) XPS was performed with a Kratos AXIS Ultra DLD spectrometer, using monochromatic AlKα radiation (hν = 1486.7 eV). The system is equipped with a magnetically confined charge


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compensation system (low energy electrons are confined and transported to the sample surface by magnetic field). Spectra were recorded using an acceleration voltage of 15 keV at a power of 225 W. Survey spectra were collected with a pass energy of 160 eV and an analysis area of 300 × 700 μm2. High-resolution spectra were obtained using a 20 eV pass energy and an analysis area of 300 × 700 μm2. Data analysis was performed with CasaXPS software (Casa Software Ltd). All binding energies were referenced to the low energy C1s peak at 285.0 eV. Core level envelopes were curve-fitted with the minimum number of mixed Gaussian–Lorentzian component profiles. The Gaussian–Lorentzian mixing ratio (typically 30% Lorentzian and 70% Gaussian functions) the full width at half maximum, and the positions and intensities of peaks were left unconstrained to result in a best fit.

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) ToF-SIMS measurements were performed with a PHI TRIFT V nanoTOF instrument (PHI Electronics Ltd, Chanhassen, MN, USA). A 30 keV pulsed primary 197Au+ ion beam was used to sputter and ionize species from each sample surface. A dual-beam charge neutralization system using a combination of low energy argon ions (up to 10 eV) and electrons (up to 25 eV) was employed to improve charge neutralization performance. Positive mass axis calibration was performed with CH3+, C2H5+ and C3H7+ whilst CH-, C2H- and Cl- were used to calibrate negative mass axis. Spectra were acquired in the bunched mode for 60 s from an area of 100 x 100 μm2. The corresponding total primary ion dose was less than 1 x 1012 ions cm-2, and thus met static SIMS conditions.34 A mass resolution m/Δ > 7000 at nominal m/z = 27 amu (C2H3+) was typically achieved.


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Five positive and five negative ion mass spectra were acquired from each sample, which were collected from sample areas that did not overlap. All recognizable, clear (i.e. unobscured by overlaps) fragment ions from 2 up to 100 amu range were used in calculations. In each mode of operation (positive and negative), the peaks were normalized to the total intensity of all selected peaks. Multiple mass spectra were processed with the aid of principal component analysis (PCA).35 PCA was performed using PLS Toolbox version 3.0 (Eigenvector Research, Inc., Manson, WA) along with MATLAB software v. 6.5 (MathWorks Inc., Natick, MA). Selected normalized intensities were also subjected to Analysis of Means.

Ellipsometry The thickness of HApp films was determined by coating a silicon wafer reference substrate in parallel with Thermanox cover slips or TCPS well plates. The measurements were performed using a variable-angle spectroscopic ellipsometer (VASE, J.A. Woolam Co. Inc. NE, USA). Scans over the wavelength region 400 – 800 nm were performed at three incident angles, viz., 65°, 70° and 75°. The thicknesses of the transparent plasma polymer films were calculated by applying a Cauchy model (silicon + one Cauchy overlayer) using the WVASE32 software (J. A. Woollam Co., Inc., USA), neglecting the extremely thin (~ 1nm) native oxide layer.

Cell Adhesion to CS Coated Surfaces U937 cells are an immortalized human myeloid cell line which are maintained as a suspension cell culture (i.e. non-adherent) and are often used to study the behavior and differentiation of monocytes.36 Prior to experimentation, the U937 cells were maintained as the non-adherent cell culture in RPMI media supplemented with 10% FBS. An experimental protocol was developed


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to support U937 cell adhesion to TCPS and surface-modified TCPS which included washing and resuspending the U937 cells in PBS (+Ca, +Mg) prior to seeding at a concentration of 3 x 105 cells/mL (equivalent to 470 cells/mm2) or 105 cells/mL (equivalent to 157 cells/mm2) in the 24 well plates (Costar® TCPS) and allowed to adhere for 30 min, 2 h or 4 h in 300 μL of PBS (+Ca, +Mg) buffer at 37°C and 5% CO2.. Notably, viability of U937 cells in PBS over time was confirmed in comparator studies wherein U937 cells were also seeded at 104, and allowed to adhere in full media for 48h. In similar experiments, the inhibition of cell adhesion was examined when U937 cells were preincubated with CS-B in solution (100 μg/mL) for 20 min prior to cell seeding on CS-coated surfaces. To stain viable cells, a working stock containing 30 μL of a 5 mg/mL FDA solution and 53.4 μL of a 1.5 mM PI solution in 10 mL of PBS was prepared. The cells were rinsed twice with PBS and the FDA/PI mix was added into the wells. The well plate was incubated for 3 min in the dark at room temperature. The wells were then rinsed with PBS, mounted and visualized with appropriate excitation/emission filter immediately by means of fluorescence microscopy (Nikon TiS). Three images were taken per well, with a minimum of three samples per condition. Two independent experiments were performed (total of 18 images per condition) and images were analyzed using ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD).37 Statistical analysis was carried out using a Student test one-tailed distribution with GraphPad Prism version 7.0a for Mac OS X (GraphPad Software, La Jolla CA, USA, www.graphpad.com). A p-value < 0.05 was considered significant for all tests. * p £0.05 ** p £ 0.01; *** p £ 0.001; **** p £ 0.0001.

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Flow Cytometry


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Flow cytometry to assay EMR2 expression was performed in HUVEC medium (Media 199 (Sigma), 20% FCS, 1% 10 mM HEPES and 1% penicillin streptomycin solution (Gibco)) with cells blocked with 1 mg/ml human IgG for 10 minutes followed by incubation with either antiEMR2-APC (R & D systems, Minneapolis, MN, USA) or IgG2b isotype control (BD). 7AAD viability dye (BD) was added in the last 5 min of incubation to exclude dead cells from analysis. After washing, cells were fixed in 1% formaldehyde, 20 g/L glucose, 5 mM sodium azide in PBS, prior to analysis using an Accuri C6 Cytometer (BD). Further analysis was performed using FCS Express 4 Flow Cytometry: Research Edition software (De Novo Software, CA, USA).

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Immunostaining

An immunostaining control was performed with the same anti-EMR2-APC antibody. 100 μL of a U937 cell suspension (105 cells/mL) were immobilized onto a microscope glass slide using a Cytospin device (Thermo Scientific). Cells were then rinsed with PBS and stained with Hoechst 33342 (2 μg/mL) at 37°C for 30 min. U937 were later rinsed, and immersed in a solution of PBS + 2% FBS for 10 min at 4°C to prevent non-specific staining. Following this saturation step, cells were stained with the anti-EMR2 antibody (R&D) for 30 min at 4°C. The slide was then rinsed with PBS, mounted and imaged with appropriate excitation/emission filter immediately by means of fluorescence microscopy (Nikon TiS).


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Results and Discussion CS-A and CS-B were covalently immobilized via EDC–Sulfo-NHS 38, periodate 39-41, or NSANB photo-crosslinker

42

chemistries onto HApp-coated substrates (Scheme 1). The three chemistries

were individually optimized to maximize the surface density of grafted CS grafted, as assessed by XPS. Immobilization onto a plasma polymer interlayer was used since it is substrateindependent and highly reproducible.43,

44

Plasma polymerization with heptylamine monomer

vapor allows the formation of a thin coating that comprises free amine groups on its surface. The composition of heptylamine plasma polymers changes slowly upon exposure to air due to oxidative chain reactions

45,46

, resulting in a progressive decrease in the density of functional

amine groups on the HApp surface. To minimize the effect of functionality loss on the resultant GAG surface density, the immobilization reactions were performed on samples freshly coated with HApp. The thickness of the HApp coating was adjusted to 44 nm as measured by ellipsometry. The coatings were then characterized by means of XPS and ToF-SIMS.


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Scheme 1. (a) Structure of CS-A and CS-B. Schematic of covalent immobilization of CS onto HApp-coated substrates via: (b) EDC–Sulfo-NHS chemistry; (c) NSANB photo-crosslinker; (d) oxidation with periodate.

Table 1 shows the composition of the HApp modified substrate and the CS-functionalized surfaces produced and studied in this work.

Table 1. Atomic composition obtained from XPS survey spectra of HApp-coated Thermanox (Th) substrates. Sample

0 (%)

N (%)

C (%)

S (%)

HApp-Th

5.4

10.1

84.5

-

CS-A-HApp-Th (EDC–Sulfo-NHS)

13.7

8.2

77.2

0.9


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CS-B-HApp-Th (EDC–Sulfo-NHS)

14.7

7.7

76.6

1.0

CS-A-HApp-Th (NSANB)

16.9

7.7

74.9

0.5

CS-B-HApp-Th (NSANB)

16.1

11.1

72.2

0.6

CS-A-HApp-Th (Periodate)

10.0

9.2

80.5

0.3

CS-B-HApp-Th (Periodate)

9.0

8.6

82.1

0.3

In all cases the XPS data show a net increase in both oxygen and sulfur content compared to the HApp control, which confirmed the immobilization of CS on the surface (Table 1). Particularly the marked increase in the oxygen content provides clear evidence for successful grafting; the S signals were of low intensity but clearly above the baseline noise level. The highest CS surface density was obtained by applying the EDC–Sulfo-NHS chemistry, which resulted in a CS-B coating containing 1.0 at.% of sulfur and 14.7 at.% of oxygen. The other two chemistries showed slightly less CS being immobilized, with 0.6 and 0.3 at.% S for NSANB and periodate chemistries, respectively. In principle, one could perform an overlayer model calculation to estimate the dry thickness of the CS layers. However, we can infer that the dry thickness is in the range of 1-2 nm, which is consistent with the expectation of grafting close to a monolayer of such molecules. Judging from the at.% S, the CS graft densities obtained in this work surpass or equal those previously reported, where 0.3 at % of sulfur was measured by XPS.8 CS-A and CS-B seem to be grafted at the same densities within the experimental uncertainty of the XPS technique. XPS C1s spectra (Figure S1) are consistent with expectations that components assignable to CO and C=0/N-C-0 are of increased intensity. However, with the expected presence of a number of close-lying components (secondary-shifted C, C-N, C-O, C=O, C(=O)-N, COOH, and even secondary-shifted C-O), fitted component intensities are subject to considerable uncertainty. For


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the present purpose, it is sufficient to conclude that the C1s spectra clearly indicate increased intensity of C-O in particular, in accordance with grafting of a GAG. To collect further evidence for successful grafting of CS-A and CS-B, samples of HApp on Thermanox and CS-grafted samples were also characterized by ToF-SIMS. This technique has a much higher chemical information content than XPS, by recording molecular fragments (as ions), and a lesser depth of analysis (1-2 nm), thus providing detailed information on the chemical composition of surface layers. Compared with the HApp control, there were clear changes in the ToF-SIMS spectra recorded after grafting of CS. However, with the high mass resolution achievable with modern ToF-SIMS instruments, the large number of peaks provides an overwhelming amount of information that can be challenging to disentangle and interpret. Accordingly, Principal Component Analysis (PCA) is often used to filter and display complex ToF-SIMS data in a more accessible manner.35, 47 In short, PCA effectively selects those peaks that vary most between spectra recorded with related samples. Those peaks can then be interpreted in terms of the intended surface chemistry. Figure 1 shows a representative example of such PCA analysis, comparing negative ion spectra recorded before and after grafting of CS-B onto HApp by EDC–Sulfo-NHS chemistry. The scores plot (Figure 1a) shows that the data sets, comprising five spectra each that were recorded on separate areas of the samples, separate well into two distinct clusters, indicating clear differences in surface chemistry. The distinct clustering also indicates homogeneity of surface coverage by CS. The first Principal Component (PC) captures 97.7% of the original data variance, making it unnecessary to extend the analysis to higher PCs. The associated loadings plot (Figure 1b) displays the peaks whose relative intensity varied most between the two surfaces; these peaks speak to the differences in chemical composition. Peaks more intense for


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HApp show with a positive loading (“upwards”), whereas peaks more intense after CS-B grafting load negatively (pointing downwards). The HApp surface yields relatively intense hydrocarbon fragments, predominantly CH-, and also by CN-. After grafting of CS-B, the spectrum contained more intense oxygen containing fragments (O- and OH-); and multiple sulfurcontaining fragments, including S-, SO-, SO2-, SO3-, SO4- and HSO4-. These peaks provide clear evidence of the presence of CS-B (as opposed to an unexpected adventitious surface contaminant for example). For the other grafting chemistries employed and CS-A, virtually identical ToFSIMS spectra and PCA analyses were obtained (Figure S2). Since the CS coatings were analyzed after thorough washing with MilliQ water (every 15 min for up to 4 h), the loadings plot provides strong evidence of covalent immobilization of CSB onto the HApp-modified surface. The ToF-SIMS data are consistent with the XPS results shown above. The uniform and dense coverage of the highly water soluble CS molecules on the HApp-modified substrate indicates their covalent attachment.

Figure 1. PCA of ToF-SIMS spectra of 67 negative fragment ions, for HApp-coated Thermanox and immobilized CS-B (via EDC–Sulfo-NHS chemistry); (a) scores plot of spectra on PC1 and PC2; (b) loadings of negative fragments ions on PC1. The CN- ion, indicated in red, is diagnostic of HApp.


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Having established that CS was immobilized onto the surfaces, we verified that the GAG structure was sufficiently preserved to selectively bind cell surface receptors. The U937 cell line was chosen for its robust display of the EMR2 cell receptor, which interacts preferentially with CS-B over CS-A

19, 20

and because of the inherent non-adherent nature of this cell line. The

presence of the EMR2 receptor on the cells used was verified by means of flow cytometry and immunostaining (Figure 2a-b).

Figure 2. (a) Flow cytometry to confirm EMR2 expression on live U937 cells (solid line) compared to a non-specific isotype control (dashed line). (b) Immunostaining (Hoechst (blue) /


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EMR2 antibody (red)). Density of live U937 cells per mm2 (c) 30 min after cell seeding and (d) 2 h after cell seeding, at a cell seeding concentration of 3 × 105 cells/mL.

Given the non-adherent nature of the U937 cells onto tissue culture plastic, which mimics a cell in circulation, we developed a protocol which facilitated the binding of cells in tissue culture well plates and avoided possible contributions from serum proteins, growth factors and other molecules present in the cell culture media, by performing the experiments in PBS. This is particularly important since CS molecules are known to interact with growth factors that may be present in serum.7, 8, 48-50 And thus our study investigated and compared interactions of the EMR2 receptor with the CS molecules. Binding of U937 cells to CS-A and CS-B-grafted TCPS was assessed 30 min and 2 h after seeding. Surfaces produced via EDC–Sulfo-NHS, periodate and photoactive crosslinker mediated immobilization were examined, together with HApp-TCPS and native TCPS control substrates (Figure 2c-d). The U937 attachment profiles were almost identical for both time points (Figure 2c-d), suggesting that the interaction between the cells and the substrates occurred rapidly. The viability of the cell population bound to the CS-coated surfaces was verified by FDA-PI (live-dead) staining

51

(Figure S3). As shown in Figure 2, cell

adhesion occurred on TCPS and TCPS-HApp controls and this was significantly reduced on the CS-A grafted surfaces. For all conjugation chemistries tested, CS-B grafted surfaces bound significantly higher cell numbers than surfaces presenting CS-A. The differences in the amount of CS covalently immobilized for each chemistry, as measured by XPS and ToF-SIMS, had little bearing on the efficiency of cell binding, suggesting that the density and presentation of the CS molecules on all grafted surfaces was greater than required for robust cell attachment.


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Figure 3. Density of live U937 cells per mm2 2 h after cell seeding (a) at a cell seeding concentration of 105 cells/mL and (b) after cell seeding at the concentration of 3 x 105 cells/mL with and without preincubation with soluble CS-B on EDC-Sulfo-NHS modified surfaces.

At reduced cell densities, preferential cell binding to CS-B was even more marked, with cell capture on CS-B coated surfaces surpassing that of TCPS for periodate chemistry (Figure 3a). Biomolecular surface coverage sparser than a contiguous monolayer has been shown elsewhere to be sufficient to support cell adhesion.52 We have also investigated the hypothesis that the surface-grafted CS layers would bind serum proteins, and that then there would occur nonselective adhesion of cells onto those adsorbed proteins. Indeed, in full media U937 adhere similarly on all substrates (Figure S4). As hypothesized, the presence of serum appears to mask the preferential adhesion of this cell line for CS-B over CS-A.

To further implicate the involvement of CS-B as the cause of the trends in adhesion observed, we immersed U937 cells in a 100 μg/mL CS-B solution for 20 min to bind EMR2 receptors prior


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to seeding, thus blocking interaction with the CS-B presented on the surface. At 2 h after seeding, cell affinity for the CS-B substrate was observed (Figure 3b). Notably, the U937 cells still adhered preferentially onto CS-B-HApp-TCPS as compared to CS-A-HApp-TCPS, whether they were blocked with CS-B in solution or not. It is worth noting that the CS-A product used was specified to contain residual CS-B and CS-C by the supplier, which could explain why cell affinity was also reduced on CS-A-HApp-TCPS after blocking with CS-B. Cell affinity was significantly lower for CS-B-HApp-TCPS substrates that were pre-incubated with CS-B, as compared to the ones that were not. Although the thickness of our coatings appears sufficient for cell recognition, a thin layer of CS would not be expected to have effective anti-fouling properties. Non-specific cell adhesion pathways, different from the EMR2 one, must be at play. These results verify the involvement of the EMR2 receptor in governing U937 cell interactions with CS-B grafted surfaces.


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Conclusions We have studied the prospects of non-adherent EMR2 expressing cells preferentially bind to CS-B over CS-A-grafted substrates. Using CS-A as a control, we have shown that EMR2+ cell binding to CS-B-coated surfaces is enhanced. This was confirmed by our observations of cell adhesion being significantly reduced when cells were blocked with CS-B in solution prior to the experiment. Cell adhesion could be attributed to direct interaction between the cells and the CS molecules, as all experiments were performed in a simple buffer in the absence of serum and growth factors. CS-A and CS-B were both successfully covalently immobilized via EDC–SulfoNHS, NSANB photo-crosslinker and periodate chemistries. XPS data showed that more CS was immobilized using EDC–Sulfo-NHS chemistry compared to NSANB photo-crosslinker and periodate chemistry. These variations, however, did not significantly impact cell behavior toward the substrates. XPS and ToF-SIMS data reveal the high homogeneity and reproducibility of the coatings. The amine plasma polymer coating common to all three approaches is a highly versatile and convenient process that can be easily applied to a wide range of substrates and materials. Surfaces were shown to be non-toxic, and CS molecules preserved their functionality in all grafted layers. We therefore conclude that CS-B coated substrates are able to capture EMR2-expressing cells, even when these cells are of a typically non-adherent phenotype. Rather than solely being used for their ability to present growth factors and to improve bio-integration, our study potentially paves the way for a new use for GAG coatings. CS-B-coated substrates are therefore a viable option to capture EMR2-expressing cells in vitro, for instance in order to enrich such populations as a part of the isolation process.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. C1s core level spectra for HApp-functionalized Thermanox-HApp-CS-B immobilization via EDC–Sulfo-NHS chemistry; PCA of negative ToF-SIMS spectra for periodate and NSANB photo-crosslinker chemistry; photomicrographs of FDA-PI stained cells on test surfaces; density of live U937 cells in full media.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions All authors contributed to the writing of this manuscript and have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We would like to acknowledge the Cell Therapy Manufacturing CRC as well as the Playford Trust foundation for their financial support.


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ABBREVIATIONS 7AAD, 7-aminoactinomycin D; CS, chondroitin sulfate; ECM, extracellular matrix; EDC, 1ethyl-3-(3-dimethylaminopropyl)carbodiimide; EGF, epidermal growth factor; EMR2, EGF-like module-containing mucin-like hormone receptor-like 2; EPC, endothelial progenitor cell; FDA, fluorescein diacetate; GAG, glycosaminoglycan; HA, heptylamine; HApp, heptylamine plasma; HCl, hydrochloride; HUVEC, human umbilical vein endothelial cells; MES, 2-(Nmorpholino)ethanesulfonic acid monohydrate; naEPC, non-adherent endothelial progenitor cell; NSANB, 5-azido-2-nitrobenzoic acid N-hydrosuccinimide ester; PBS, phosphate buffer saline; PCA,

principal

component

analysis;

PI,

propidium

iodide;

Sulfo-NHS,

N-

hydroxysulfosuccinimide; TCPS, tissue culture polystyrene; Th, thermanox; ToF-SIMS, time-offlight secondary ion mass spectrometry; XPS, X-ray photoelectron spectroscopy.

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