Engineering the Biointerface of Electrospun 3D Scaffolds with

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Engineering the biointerface of electrospun 3D scaffolds with functionalized polymer brushes for enhanced cell binding Lina Duque Sánchez, Narelle Brack, Almar Postma, Laurence Meagher, and Paul J. Pigram Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01427 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 6, 2019

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Biomacromolecules

Engineering the biointerface of electrospun 3D scaffolds with functionalized polymer brushes for enhanced cell binding Lina Duque-Sanchez †‡, Narelle Brack †, Almar Postma ‡, Laurence Meagher ˩, Paul J. Pigram †*

†

Centre for Materials and Surface Science and Department of Chemistry and Physics, La Trobe University, Melbourne, Victoria 3086, Australia. ‡

˩

CSIRO Manufacturing, Bayview Avenue, Clayton, Vic 3168, Australia.

Monash Institute of Medical Engineering and Department of Materials Science and Engineering, Monash University, Clayton, Vic 3800, Australia.

KEYWORDS biomolecule coupling, electrospun fibres, surface functionalization, SI-Cu(0) polymerization, cell binding.

ACS Paragon Plus Environment

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ABSTRACT

Electrospun ultrafine fibres prepared using a blend of poly(lactide-co-glycolide) (PLGA) and bromine terminated poly(ʟ-lactide) (PLA-Br), were surface modified using surface-initiated (SI) Cu(0) mediated polymerization. Copolymers based on N-acryloxysuccinimide (NAS) and a low fouling monomer (either N,N-dimethylacrylamide (DMA), N-(2-hydroxypropyl)acrylamide (HPA) or N-acryloylmorpholine (NAM)) were grafted from the fibre surface to impart surface functionality and to reduce non-specific protein adsorption. Inclusion of the functional NAS monomer facilitated the conjugation of a non-bioactive cyclic RAD peptide and a bioactive cyclic RGD peptide, the latter expected to facilitate cell adhesion through its affinity for the αvβ3 integrin receptor. A detailed analysis of the surface of the electrospun fibre scaffolds in non-grafted form compared to the surface functionalized state is presented. Characteristic amino acid peaks are observed for both conjugated RGD and RAD peptides. Cell culture experiments confirmed cell specific attachment mediated through the presence of the bioactive RGD peptide mainly at high surface density. 1. INTRODUCTION The design and engineering of biomaterials requires not only the capacity to mimic the physical and topographical structure of the extracellular matrix (ECM) but also the presentation of an instructive background to support cell attachment and growth and control their behaviour.1 Electrospun scaffolds resemble the three-dimensional environment of the ECM. However, the lack of binding domains and cellular cues within the scaffold has been a shortcoming in their ability to support cell adhesion, growth and integration. In addition, non-specific protein adsorption mediated by the native surface chemistry of the scaffold may lead to foreign body and


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inflammatory responses after implantation.2-4 Two crucial characteristics are required to improve the biological response of electrospun scaffolds: the addition of bioactive functionality and the reduction of non-specific protein adsorption. Reduction of non-specific protein adsorption can be achieved by the surface grafting of low fouling polymers to the scaffold.5-9 Low-fouling materials typically exhibit similar structural and chemical properties including hydrogen-bond acceptor characteristics, hydrophilic nature and overall electrical neutrality.2, 10 Low fouling surfaces may reduce the likelihood of thrombus or scar tissue formation due to non-specific protein adsorption after biomaterials implantation.11, 12 The addition of functionality may be accomplished by the chemical immobilization of biomacromolecules. This approach is preferred over physical immobilization strategies as the covalent attachment of bioactive molecules delivers enhanced chemical stability over time.13 The presence of functional polymers as part of the tethered chains favors the conjugation of biomolecules that may stimulate cell attachment and subsequent cell signaling via interaction with specific cell surface receptors.14 The effectiveness of the modified surfaces relies on the chemical and physical properties of the grafted polymer chains and the degree of control over the grafted chain molecular weight and molecular weight distributions (MWDs). Reversible-deactivation radical polymerization techniques (RDRP) including surface-initiated (SI) atom transfer radical polymerization (SI-ATRP) and Cu(0) mediated radical polymerization (SICu(0)) represent an effective strategy for the surface modification of biomaterials. Xu et al.15, 16, employed SI-ATRP for the surface grafting of acrylamides and methacrylates from hydrolyzed polycaprolactone (PCL) films. Subsequent surface functionalization using biomolecules significantly increased the density of cell attachment mostly when collagen was employed.16 Similarly, semicrystalline poly(ε- caprolactone) (PCL) films with varied topologies were surface


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modified using SI-ATRP.17 Despite the demonstrated effect of the surface topologies on the attachment and spreading of human mesenchymal stem cells (hMSCs), grafting of thick brushes of poly(oligo(ethylene glycol) methacrylate (POEGMA) inhibited the influence of the underlying characteristics over the hMSCs behavior. The cell behavior on the PCL-brush samples was demonstrated to be substrate topology independent. Surface functionalization of two-dimensional (2D) samples has also been reported using SI-ATRP. Tugulu et al.18, studied polymers with both low-fouling and functional characteristics to enhance cell adhesion. RGD-based peptide ligands were employed for the functionalization of the surfaces where adhesion and spreading of human umbilical vascular endothelial cells (HUVECs) were tested. After being subjected to shear stress experiments, HUVECs remained attached to the polymer brushes, representing an attractive strategy for the fabrication of biologically active interfaces. Brush functionalization of flat surfaces through SI-ATRP has been reported with varied biomolecules including RGD peptides19 and combination of RGD peptide and the PHSRN sequence.20 The surface modification of two-dimensional samples using SI-Cu(0) mediated polymerization has been reported at room temperature. Jordan et al.21-23, introduced the use of copper plates for the surface grafting of a variety of polymers including but not limited to poly(Nisopropylacrylamide) (PNIPAAM), POEGMA, poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(4-vinylpyridine) (PMMA) from initiator-functionalized substrates. The authors modified the polymer film thickness by controlling the distance between the copper plate and the substrate obtaining well-controlled grafted chains. In addition, using the same technique, the authors reported the fabrication of block copolymer and patterned brushes.21 RDRP techniques have been employed for the fabrication of brushes with polymers containing reactive functional groups for subsequent functionalization.24-26 The presence of those functional groups for coupling to


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functional groups present on bioactive molecules (e.g. amino or thiol groups) or with synthetic reactive groups incorporated into the biological molecules (e.g. azido functional group) is critical for subsequent immobilization. Amines are often the preferred functional group targeted for bioconjugation as they are present in most proteins and peptides. One common strategy is the use of these amine groups to react with activated esters forming stable amide linkages,27 with Nhydroxysuccinimide (NHS) being the most widely reported approach.28 The NHS ester is formed by the reaction of NHS with carboxylate groups in the presence of carbodiimide. Coupling between NHS and proteins occurs principally via the α-amines at the N-terminals and the ε-amines of lysine side chains.29 NHS chemistry was employed by Gunnewiek et al.30, for the surface functionalization of microporous samples with diverse protein gradients through diffusion into the matrix. The author reported completely cell covered of functionalized scaffolds without selected areas to settle. In another study of 3D samples functionalization, additive manufacturing was employed for the fabrication of scaffolds with interconnected pore size. The surface was functionalized with growth factors which when present homogenously, stimulated the differentiation of human mesenchymal stromal cells (hMSCs).31 In this study, the surface of electrospun ultrafine fibres prepared from a blend of poly(D,L-lactideco-glycolide) (PLGA) and 2-bromoisobutyryl terminated poly(L-lactide) (PLA-Br), which acts as the surface initiator, was modified via Cu(0) mediated radical polymerization. Copolymers comprising N-acryloxysuccinimide (NAS) and a low fouling polymer; either N,N-dimethyl acrylamide (DMA) p(DMA-co-NAS), N-(2-hydroxypropyl) acrylamide (HPA) p(HPA-co-NAS) or 4-acryloylmorpholine (NAM) p(NAM-co-NAS) were grafted from the surface of the electrospun ultrafine fibre scaffolds. The presence of the copolymer at the surface of the electrospun fibres was confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight


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secondary ion mass spectrometry (ToF-SIMS). A detailed evaluation of the surface functionalization process was completed using ToF-SIMS and principal component analysis (PCA). Cell culture studies were used for the evaluation of both protein adsorption resistance and cell specific stimulation of the modified surfaces, representing a versatile strategy to improve the interfacial interactions between electrospun fibre scaffolds and cells. 2. EXPERIMENTAL SECTION 2.1 Materials Poly(ʟ-lactide), 2-bromoisobutyryl terminated (PLA-Br, Mn 10,000), poly(ᴅ,ʟ-lactide-coglycolide (Resomer® RG 756 S) (PLGA, MW 76,000-115,000), 1-amino-2-propanol, Tris[2(dimethylamino)ethyl]amine (Me6TREN, 97%), acetonitrile (≥99.9%) (MeCN), 1% penicillinstreptomycin solution (Pen-Strep), N-acryloxysuccinimide (NAS), 1,3,5-trioxane N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and paraformaldehyde (PFA) were purchased from Sigma-Aldrich and used without further purification. Methanol (99.8%) (MeOH), acryloyl chloride, ethyl acetate, chloroform (≥99%) (CHCl3) and N-hydroxysuccinimide (NHS), were obtained from Merck. N,N-dimethylacrylamide (99%) (DMA) and 4acryloylmorpholine (97%) (NAM), obtained from Sigma-Aldrich, were de-inhibited by passing through a column of inhibitor remover. N-(2-hydroxypropyl) acrylamide (HPA) was synthesized following the procedure reported by Fairbanks et al.32 Copper(I) bromide (Cu(I)Br, 98%) was washed with acetic acid, ethanol and anhydrous ether to remove Cu(II)Br2 impurities, then dried in an oven at 90 °C.33 Ethyl 2-bromoisobutyrate (≥97%) (EBiB) was employed as the sacrificial initiator for the surface-initiated polymerization. All water used was purified through a MilliQ™ water purification system (Millipore) and had an initial resistivity of 18.2 MΩ cm-1. Dulbecco's phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle medium (DMEM), trypLE


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express, and antibiotic-antimycotic (anti-anti (100X)) were obtained from Gibco. Triton-X-100, trihydrochloride, trihydrate - 10 mg/mL solution in water (Hoechst 33342), and InvitrogenActinRedTM 555 were obtained from Thermo Fisher Scientific. Fetal bovine serum (FBS) was obtained from Scientifix. The peptides, cyclo(Arg-Gly-Asp-D-Phe-Lys) c(RGDfK) and cyclo(Arg-Ala-Asp-D-Phe-Lys) c(RADfK) were obtained from Peptides International. 2.2 Fabrication of electrospun fibre scaffolds PLA-Br and PLGA in weight proportions of 1:1 were dissolved in CHCl3:MeOH (3:1), to prepare a 16%. (w/w) solution. The electrospinning equipment consisted of a syringe pump (Model NE-1000 from New Era Pump Systems, Inc.), a high voltage power supply (Spellman SL150 high voltage DC) and a 5 mL syringe (TERUMO) attached to an 18 G needle. The voltage applied was 15 kV, the distance between the tip of the needle and the drum collector was set at 15 cm and the flow rate was adjusted to 1 mL h-1. After four hours of deposition, membranes of ~100 ± 0.5 m thickness and an average fibre diameter of 1.24 ± 0.18 had been created. The samples were stored in a vacuum desiccator until required. 2.3 SI-Cu(0) mediated radical polymerization SI-Cu(0) mediated polymerizations were performed in a nitrogen-filled glove box. A molar ratio of initiators in the fibre to sacrificial initiator in solution of 95:5 was employed. The molar proportions of [monomer]:[initiator]:[Cu(I)Br]:[Me6TREN] used were varied [200]:[1]:[X]:[X] with monomer concentrations of 0.7 M unless otherwise specified. MilliQ™ water was deoxygenated before use. Low fouling monomer (either DMA, NAM or HPA), organic solvent (MeCN) and sacrificial initiator (EBiB) were purged with N2 gas individually for 15 min before use. A solution of Cu(I)Br, Me6TREN and MilliQ™ water was prepared and deoxygenated for 15 min. Disproportionation of Cu(I)Br in this study was completed in water prior to the addition of


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both organic solvent, monomer and initiator. Disproportionation of [Cu(Me6TREN)]Br was confirmed by the in situ formation of brown Cu(0) particles and the appearance of a green/blue solution colouration. The disproportionated dispersion was placed in an ice bath until use. Low fouling monomer, N-acryloxysuccinimide (NAS) monomer, MeCN, 1,3,5-trioxane as internal standard and EBiB were mixed separately (monomer system) and stored in an ice bath prior to use. The disproportionated dispersion was added to the electrospun ultrafine fibres. This was followed by the addition of the monomer system with rapid stirring. SI-Cu(0) mediated polymerization was completed in 1 hour. The reactions were carried out at 0 °C and were quenched by removing the samples from the glove box and exposing them to air. Samples of ~ 0.2 mL were taken from polymerization processes for further analysis of polymer grown in solution. Copolymerized grafted samples p(DMA-co-NAS), p(HPA-co-NAS) and p(NAM-co-NAS) were rinsed with a solution of the same water/organic solvent media used in the polymerization, followed by a large amount of MilliQ™ water. Samples were allowed to dry under vacuum for subsequent characterization. 2.4 Post-polymerization activation Potential hydrolysis of the NHS ester after the SI-Cu(0) mediated polymerization was evaluated by a post-polymerization activation reaction. Immediately after the SI-Cu(0) mediated modification, samples were activated by incubation for 30 min in an aqueous solution containing 125 mM EDC and 125 mM NHS. These samples and non-activated samples were analyzed by XPS and the relative nitrogen atomic concentrations were compared. 2.5 Surface functionalization of electrospun fibre scaffolds Functionalization of the surface of the fibres was achieved by conjugation of a bioactive c(RGDfK) and non-bioactive c(RADfK) peptide. An amide linkage was created between the


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amino group present in both cyclic peptides and the activated esters on the surface of the fibres. A wad punch was employed to cut 9 mm disc samples of either p(DMA-co-NAS) p(HPA-co-NAS) or p(NAM-co-NAS) grafted electrospun material, which were placed in a 48 well plate and incubated for 24 hours under 200 µL of peptide solution at varying concentrations (200 µM. 400 µM, 800 µM). Peptide conjugated fibres were washed 10 times in PBS (10 minutes per wash) and immediately incubated in antibiotic-antimycotic solution for subsequent cell culture. 2.6 Cell culture experiments For cell culture experiments, non-functionalized (no peptide) grafted samples were employed as control samples. In order to prevent bacterial and fungal contamination during cell culture, functionalized and non-functionalized electrospun fibre samples were incubated overnight in a 200 µL of 2× antibiotic-antimycotic solution. The L929 fibroblast cell line was employed. DMEM media was supplemented with 1% Pen-Strep and 10% FBS to obtain complete media. Cells were incubated at 37 ºC, 5% CO2 for 24 hours. 2.6.1 Cell seeding onto electrospun fibres After incubation with antibiotic-antimycotic solution, samples were rinsed once with DPBS. Complete media was then added to the wells and the samples were incubated overnight. After overnight incubation, the medium was replaced by seeding 100,000 cells/well in 0.4 mL of complete media onto the samples. Cell counts were undertaken using a hemocytometer and a 10 µL aliquot of the cell suspension. 2.6.2

Cell fixation and staining

After incubation, the cell culture medium was removed, and samples were gently rinsed with DPBS. A pre-determined amount of 4% PFA in DPBS, sufficient to cover the sample, was added to each well and allowed to react for 15 minutes at room temperature. Samples were immediately


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transferred to a fume hood where the PFA was removed and the samples were rinsed twice with DPBS. Permeabilization of the samples was achieved by adding 0.1% Triton-X-100 in DPBS solution to the samples and leaving for 15 min at room temperature. Subsequently, samples were incubated in 3% BSA for 30 minutes at room temperature. For staining, ActinRed (2 drops for each mL of 3% BSA) and trihydrochloride, trihydrate (Hoechst 33342) (1 µL for each mL of 3% BSA) were added to each well. The well plate was covered with aluminum foil and left in the dark for 1 hour. Samples were then rinsed five times with DPBS and stored in the dark at 4 °C. For imaging, samples were placed on a glass slide and imaged using a Nikon A1+confocal microscope. 2.7 Characterization of free polymer in solution Surface initiated polymerization was carried out in the presence of fibres. The resulting free polymer grown in solution was analyzed via 1H NMR and SEC. 2.7.1 Nuclear magnetic resonance spectroscopy (NMR) Monomer conversion was determined by 1H-NMR analysis with a Bruker AV400 MHz NMR spectrometer using a relaxation delay, d1 of 10 seconds. All spectra were measured, baseline corrected and analyzed using TopSpin™. The conversion of monomer to polymer was calculated based on the reduction of the integrated areas of the peaks associated with vinyl protons and compared to the integration of the 1,3,5-trioxane internal standard. 2.7.2 Size-exclusion chromatography (SEC) Size-exclusion chromatography (SEC) was performed on a Shimadzu system equipped with a CMB-20A controller system, an SIL-20A HT autosampler, an LC-20AT tandem pump system, a DGU-20A degasser unit, a CTO-20AC column oven, an RDI-10A refractive index detector, and 4× Waters Styragel columns (HT2, HT3, HT4, and HT5, each 300 mm × 7.8 mm2, providing an effective molar mass range of 1004 × 106). N,N-Dimethylacetamide (DMAc) (containing 4.34 g


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L-1 lithium bromide (LiBr)) was used as an eluent with a flow rate of 1 mL/min at 80 °C. Number (Mn) and weight average (Mw) molar masses were evaluated using Shimadzu LC Solution software. The SEC columns were calibrated with low dispersity polystyrene (PS) standards. Therefore, the molecular weight in this manuscript are reported in terms of the effective MW of the PS standards. 2.8 Surface characterization of the electrospun fibre scaffolds 2.8.1 Scanning electron microscopy (SEM) The samples were iridium coated before being imaged using a Zeiss Merlin FESEM (Field Emission Scanning Electron Microscope) operated in the secondary electron (SE) mode to highlight topographical features. An accelerating voltage of 1 kV was used for imaging using the In-Lens detector. The magnifications used are indicated by the scale bars shown in the images. The average fibre diameter was obtained by measuring the diameter of 30 fibres from the SEM images taken at 1 kV. 2.8.2 X-ray photoelectron spectroscopy (XPS) Fibre surface composition was analyzed by X-ray photoelectron spectroscopy (XPS) using either an AXIS Ultra DLD or an AXIS Nova spectrometer (Kratos Analytical Inc., Manchester, UK) with a monochromated Al Kα source at a power of 180 W (15 kV × 12 mA). Data processing was performed using CasaXPS processing software version 2.3.15 (Casa Software Ltd., Teignmouth, UK). All elements present were identified from survey spectra acquired at a pass energy of 160 eV. High-resolution spectra were acquired using a pass energy of 20 eV for the copolymer system. The atomic concentrations of the detected elements were calculated using integral peak intensities and the sensitivity factors supplied by the manufacturer. Photoelectron components resulting from the base material (electrospun fibres) were labelled “b” while those from the grafted copolymer were labelled “c”. The binding energies were


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referenced to the C5 ester functional group peak at 289.0 eV. Peak fitting of the C 1s spectrum was assigned as follows, C1b (CC and CH), C1c (C-C and C-C-Br) (284.6 – 284.8eV), C2c assigned to secondary shifted aliphatic carbon bound to electronegative elements (CCBr) or amide functional groups (C-(NC=O)) (285.1-285.3 eV), C3b assigned to C-(O=CO) (287.0 – 287.2 eV) for electrospun fibres or C3c assigned to (CO or CN) (285.6 – 285.8 eV) alcohol and amine carbons for copolymer grafted samples, C4c (NC=O) (287.2 – 287.4 eV) amides and C5 labelled as “b or c” as it could be related to the ester (O=COC) (289.0 eV) functional group present in both the copolymer (C5c) and the electrospun fibre surface (C5b). 2.8.3 Time of flight secondary ion mass spectroscopy (ToF-SIMS) Time of flight secondary ion mass spectroscopy (ToF-SIMS) was carried out using a ToF-SIMS 5 instrument (ION-TOF GmbH, Münster, Germany). Spectra were collected in positive polarity using Bi3+ as the primary ion source at 30 keV. The beam was pulsed at 0.7 ns period and each pulse was bunched to maximize mass resolution. Spectra were acquired from three separate 100 µm × 100 µm regions on the surface using a random raster and 128 × 128 pixels. Twenty scans were completed at an operating current of 0.6 pA. Charge compensation was achieved using pulsed low-energy electron flooding of the sample surface. The average pressure in the analysis chamber during data acquisition was 2 × 10-9 mbar. The mass scale of each spectrum was calibrated against the flight times of the CH+, CH2+, CH3+, C2H3+, C3H5+, C4H3+, C5H5+, C6H5+, C7H7+, secondary ions. PCA was conducted using a peak list containing 500 mass spectral peaks selected between 1350 u and with intensity greater than 102 counts. The peak list was created using SurfaceLab 6 (ION-TOF GmBH, Münster, Germany; version 6.5). Peak areas were manually assigned for each mass spectral peak. Pre-processing prior to PCA comprised normalization by the total ion intensity


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per spectrum and mean-centering of parameters. Cross-validation methods were not applied. PCA was undertaken using PLS_Toolbox (Version 8.1) (Eigenvector Research, Manson, WA) via MATLAB R2015b (Version 8.6) (The MathWorks Inc., USA). 2.9 Statistical analysis The student’s t-test was used for comparison of two groups where p ≤ 0.05 was considered significant. 3. RESULTS AND DISCUSSION Electrospun fibre samples were functionalized via a SI-Cu(0) mediated polymerization process, as described in Scheme 1. The polymerization was carried out using N-acryloxysuccinimide (NAS) as the co-monomer for reaction with either N,N-dimethylacrylamide (DMA), N-(2hydroxypropyl)acrylamide (HPA) or 4-acryloylmorpholine (NAM). Scheme 1. Schematic illustrating functionalization of the electrospun fibre samples. A) Starting from the 1:1 16% (w/w) PLGA:PLA-Br base fibres, followed by, B) SI-Cu(0) mediated copolymerization of either p(DMA-co-NAS), p(HPA-co-NAS) or p(NAM-co-NAS) systems and concluding with C) amide linkage between the NHS activated ester from PNAS and the amine group on the K amino acid of the cyclic RGD peptide.


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3.1 Optimization of the SI-Cu(0) mediated copolymerization The three copolymer systems employed and the associated reaction conditions are shown in Table 1. The solution remaining after the polymerization was evaluated via SEC to obtain an estimation of the MWDs and average molecular weight of the grafted polymer chains (Table 1). Table 1. Comparison of the free polymer from the analysis of the solution remaining from the SICu(0) mediated copolymerization of p(DMA-co-NAS), p(HPA-co-NAS) and p(NAM-co-NAS) grafted electrospun fibres. SI-Cu(0) conditions [M]:[I]:[200]:[1], H2O:MeCN (97:3) solvent solution, at 0 °C for 1 hour.

Sample

[Cu(I)Br]:[Me6TREN ]

p(DMA-co-NAS)

Conv. (%) Mn(theo)

Mn(SEC)

Ð

14100

74600

1.35

1.2:1

95 [M] 65

5 [NAS] 100

p(HPA-co-NAS)

1.6:1.2

48

100

13700

78500

1.30

p(NAM-co-NAS)

1.8:1.4

35

100

11300

64200

1.37

Faster consumption of the acrylate (NAS) monomer over acrylamide (DMA, HPA, NAM) species was observed in this study following the trend observed in previously reported studies.34,


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35

We assume that the reactivity ratios of the copolymers under Cu(0) polymerization conditions

promote the faster consumption of the NAS monomer. Increased consumption of the acrylate monomer during the polymerization may be additionally affected by other factors such as the ligand to copper concentration36 and the reported efficiency of the EBiB initiator for the polymerization of acrylates.37 Another explanation for the limited conversion of the acrylamide monomers may be the high levels of copper required for obtaining monomodal and symmetrical MWDs of the copolymers. A high concentration of deactivation species is expected and this may compromise the rate of polymerization.38 In addition, the complex geometry and curvature of the fibre substrate were found to affect the overall polymerization kinetics significantly by limiting reagent availability. Specifically, reagent diffusion may be restricted by local monomer consumption, confinement and crowding effects. Overall, higher molecular weights than the theoretically expected values were obtained when the solution from the SI-Cu(0) mediated polymerization was analyzed. This discrepancy in the molecular weight can be attributed to the effect of the physical structure of the fibres and the density and location of the surface initiators over the polymerisation kinetics.9 The molecular weight and MWDs obtained from the analysis of the solution remaining after the SI-Cu(0) polymerization represented an acceptable result, given the challenges presented by the complex system employed in this study. 3.1.2 Post- polymerization activation The reaction of the activated NHS esters with the proteins nucleophilic groups has been observed to be limited by NHS ester hydrolysis when aqueous media at basic pH was employed.39 The predominantly aqueous media employed for the SI-Cu(0) mediated reaction may induce hydrolysis of the NHS-activated ester in this study, reducing the number of reactive sites for subsequent peptide immobilization. The rate of hydrolysis of the NHS ester in aqueous media has been


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observed to increase with increasing pH. When aqueous solutions at pH 7 were employed, the halflife of hydrolysis of NHS esters at 0 ºC has been shown to be 4-5 hours.40 Although the reaction time used for the SI-Cu(0) mediated copolymerization in this study was less than 4 hours, an activation experiment was carried out in order to ensure that NHS esters were present after the polymerization. Samples obtained after the SI-Cu(0) mediated polymerization were incubated in EDC and NHS solution to promote re-activation of the NHS ester. Activated and non-activated copolymerized grafted samples were analyzed by XPS and the relative concentrations of nitrogen compared. Presented in Figure 1 are the nitrogen concentration measured at the surface of nonactivated and activated samples for each copolymer graft layer.

Figure 1. XPS nitrogen abundance of samples activated and non-activated after the SI-Cu(0) mediated modification. No statistically significant differences between the nitrogen content of the surface of activated and non-activated copolymerized grafted electrospun samples were observed (Fig. 1) confirming the stability (minimum hydrolysis) of the NHS esters during the SI-Cu(0) polymerization process. Therefore, a re-activation of any carboxylic acid groups formed as a results of hydrolysis during the SI-Cu(0) mediated process was not carried out. Previous work by Ameringer et al.41, has shown


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that NHS esters on polymer backbones have slower rates of hydrolysis than those not conjugated to polymers, supporting this approach. 3.2 Surface characterization of copolymerized samples Surface characterization of the copolymerized grafted samples was carried out using SEM, XPS and ToF-SIMS. Surface functionalization was assessed using ToF-SIMS in conjunction with PCA in order to confirm biomolecule surface immobilization and to identify specific molecular differences between surfaces with coupled bioactive RGD and non-bioactive RAD peptides. Despite the smaller sample depth of ToF-SIMS compared to XPS,42 ToF-SIMS was selected as the principal surface characterization method to evaluate the functionalization process due to its sensitivity and the precision to identify specific fragments. Particularly in this study where nitrogen containing molecule signals were expected from most of the polymers and biomolecules employed. Table 2 shows the relative atomic concentration of the elements on the surface of the copolymerized grafted samples. Table 2. Relative atomic percentage concentrations at the surface of the p(DMA-co-NAS), p(HPA-co-NAS) and p(NAM-co-NAS) copolymer samples after the SI-Cu(0) process.

Sample

% Relative atomic concentrations C 1s

O 1s

N 1s

Al 2p

p(DMA-co-NAS)

69.9

19.5

10.7

0.0

p(HPA-co-NAS)

68.2

23.8

8.0

0.0

p(NAM-co-NAS)

65.4

28.3

6.1

0.15

Relatively similar carbon concentrations were observed on the surface for all samples. A trace amount of aluminum contamination was detected in the p(NAM-co-NAS) sample. The highest


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nitrogen concentration after the surface-initiated polymerization reaction was observed for the p(DMA-co-NAS) sample. A low nitrogen content at the surface of the p(NAM-co-NAS) sample was expected due to the low rate of conversion of the NAM and NAS monomers in the SI-Cu(0) mediated polymerization and anticipated thinner surface grafted coating. SEM images and XPS high-resolution C 1s spectra from the analysis of the surface of the non-grafted electrospun fibres and copolymerized grafted samples fabricated via SI-Cu(0) mediated polymerization are presented in Figure 2.

Figure 2. XPS high-resolution C 1s spectra from the analysis of the surface of the SI-Cu(0) mediated copolymerization of A) non-grafted electrospun fibre sample. C) p(DMA-co-NAS) sample. E) p(HPA-co-NAS) sample. G) p(NAM-co-NAS) sample. The XPS spectra of the copolymer samples were fitted with: C1b (CC and CH), C1c (C-C and C-C-Br), C2c (CCBr) (C-(NC=O)), C3b C-(O=CO), C3c (CO or CN), C4c (NC=O) C5b/c (O=COC)


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components. SEM images of B) non-grafted electrospun fibre sample. D) p(DMA-co-NAS) sample. F) p(HPA-co-NAS) sample. H) p(NAM-co-NAS) sample. The carbon C 1s components associated with the chemical composition of the PLGA and PLABr, including C1b (CC/CH), C3b C-(O=CO) and C5b (O=COC) (Fig. 2A) were fitted for the non-grafted electrospun fibres with an increase of approximately 10% in carbon atomic concentration, which may be attributed to the presence of adventitious hydrocarbon. The presence of nitrogen was supported by the appearance of the C3c (CO or CN) and C4c (NC=O) components in all copolymerized grafted-electrospun fibres (Figs. 2C, 2E, 2G) indicating successful surface modification. For the NHS ester, a peak at 289 eV (C5c component) corresponding to the ester functional group, the amide functional group peak (C4c) at 288 eV and the secondary shift peak (C2c), corresponding to the carbon adjacent to the amide groups (285.4 eV), were expected.43 The C5 component at ~ 289.0 eV was detected in the high-resolution C 1s spectra for all copolymer surface coatings, consistent with surface grafting. It should be noted that this component can also be related to the ester (O=COC) (C5b) functional group from the electrospun fibre surface. A detailed evaluation of the source of this component will be presented in the following section. In addition, signals arising from the C1b, C3b carbon components of the polyester fibre substrate surface were observed in all graft copolymer coated samples, suggesting that the coatings were less than 10 nm in thickness. The low conversion rate of the p(NAM-coNAS) sample was validated in the high-resolution C 1s analysis where the intensities of both C1b and C3b peaks were the highest of all graft copolymer coated samples compared. The presence of adventitious hydrocarbon was also more evident in this sample due to the greater fibre signal as a result of the thinness of the coating (Fig. 2G). SEM was employed to characterize the samples after surface modification. As observed in Figures 2B-H, no significant changes between the non-


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grafted and copolymerized grafted electrospun fibre samples were observed. The surfaces of the fibres were smooth after the polymerization process without any visible surface variation. Small defects at the surface of the p(NAM-co-NAS) sample were observed (Fig. 2H). However, these do not significantly affect the morphology of the fibres. No significant differences between the average diameter of the copolymerized grafted samples and the non-grafted samples were observed (Fig. S1). This result confirms the surface modification process does not negatively impact the fibre morphology. 3.2.1 Step by step surface functionalization analysis via ToF-SIMS and PCA Surface modification of the grafted samples was confirmed with XPS. It was unclear whether the surface nitrogen observed resulted only from the presence of the amides in the polymer brush coating or from contributions from both the polymer brush and NAS activated functional groups in the polymer brush. ToF-SIMS and PCA were employed to confirm the presence of the NAS polymer at the surface of the copolymerized grafted electrospun fibre samples. Figure 3 shows the scores and loadings for PC1 for each individual homopolymer brush compared with its related NAS copolymer.


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Figure 3. PC1 of ToF-SIMS data where each copolymer was analyzed against the low fouling homopolymers. A) The scores plot of PC1 of p(DMA) against p(DMA-co-NAS), encompassing 99.51% of the variance. B) PC1 loadings plot where positive loadings are related to p(DMA-co-


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NAS) copolymerized grafted sample and negative loading corresponded to p(DMA) grafted homopolymer. C) The scores plot of PC1 of p(HPA) against p(HPA-co-NAS), encompassing 99.06% of the variance. D) PC1 loadings plot where positive loadings are related to p(HPA) grafted homopolymer and negative loading corresponded to p(HPA-co-NAS) copolymerized grafted sample. E) The scores plot of PC1 of p(NAM) against p(NAM-co-NAS), encompassing 97.90% of the variance. F) PC1 loadings plot where positive loadings are related to p(NAM) grafted homopolymer and negative loading corresponded to p(NAM-co-NAS) copolymerized grafted sample. PC1 accounted for a large fraction of the variance (above 97%) for all samples. A strong differentiation between homopolymer and copolymer samples is shown (Figs. 3A, 3C, 3E) in all cases. The peak assignment for each fragment is presented in Table S1. Several nitrogencontaining fragments were observed in the set of PC1 loadings for all copolymers. The C4H4O2N+ molecular ion was absent, attributed in the literature to (succinimide-H)+.44, 45 However, other NHS-related peaks were observed including the m/z = 89 (C3H7NO2+), m/z = 99 (C4H5NO2+) and the m/z = 128 (C5H6NO3+). Confirmation of the presence of the NHS-activated residues in all of the copolymer coatings provided a foundation for the subsequent surface peptide functionalization. Peptides cyclic-RGDfK and cyclic-RADfK were selected for this study with the latter acting as the negative control due to its lack of bioactivity.41, 46 Cyclic RGD peptides were selected due to their higher activity and stability compared to their linear analogues.47 The conformational stability of cyclic peptides may be attributed to the rigidity structure, which represents higher resistance to proteolysis as well as higher binding affinity towards integrin receptors.48, 49 As the reaction of the NHS ester with the amino group from other residues including arginine or histidine is not commonly observed,40, 50 peptide conjugation was expected to occur via the reaction of the NHS


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ester and the N-terminal ε amino group of the lysine side chain (primary amine), followed by the release of N-hydroxysuccinimide.40 Surface peptide conjugation on the surface of p(DMA-coNAS), p(HPA-co-NAS) and p(NAM-co-NAS) grafted samples was confirmed by significant changes observed in the analysis of samples before and after immobilization from a solution containing 800 µM peptide, using ToF-SIMS and PCA (Fig. 4).


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Figure 4. PC1 ToF-SIMS data where 800 µM RGD and 800 µM RAD functionalized copolymers were evaluated against non-functionalized copolymerized grafted samples. A) The scores plot of PC1 of peptide conjugated p(DMA-co-NAS) against p(DMA-co-NAS), encompassing 69.20% of


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Biomacromolecules

the variance. B) PC1 loadings plot where positive loadings are related to no peptide conjugated and negative loading corresponded to peptide conjugated samples. C) The scores plot of PC1 of peptide conjugated p(HPA-co-NAS) against p(HPA-co-NAS), encompassing 93.03% of the variance D) PC1 loadings plot where positive loadings are related to peptide conjugated samples and negative loading corresponded to no peptide conjugated samples. E) The scores plot of PC1 of peptide conjugated p(NAM-co-NAS) against p(NAM-co-NAS), encompassing 64.14% of the variance. F) PC1 loadings plot where positive loadings are related to RGD peptide conjugated samples and negative loading corresponded to no peptide conjugated and RAD peptide conjugated samples. It was expected that successful peptide immobilization would be characterized by the absence of NHS-related ToF-SIMS fragments after the conjugation process. Although clear separation between functionalized and non-functionalized samples for both p(DMA-co-NAS) and p(HPAco-NAS) by PC1 was obtained, NHS related fragments such as C4H5NO2+ and C5H6NO3+ were still present in the positive loadings plot (peptide conjugated samples) of the p(HPA-co-NAS) sample. Limited accessibility of water to NHS groups located within high density brushes has been reported.41 This observation and the relatively slow hydrolysis of the NHS esters previously reported suggests that hidden NHS groups did not react and may still be present after conjugation. The presence of NHS fragments from the analysis of the functionalized samples then was not considered as evidence of ineffective functionalization. In the case of p(NAM-co-NAS) copolymers, the analysis of the scores and loadings plots for PC1 of the functionalized and nonfunctionalized samples, accounted for 64.14% of the variance in the dataset (Fig. 4E-F). PC1 revealed the presence of non-functionalized and RAD functionalized samples in the negative loadings and RGD functionalized samples in the positive loadings. This unexpected sample


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separation may be attributed to a thin layer of the p(NAM-co-NAS) copolymer coating where specific differences between base materials, coating and peptide may be difficult to resolve. PC1 (64.14%) was then plotted against PC2 (28.32%) showing the expected separation between the samples (Fig. S2). Both RGD and RAD functionalized p(NAM-co-NAS) samples were most strongly loaded in PC2 where NHS-related peaks m/z = 99 and m/z = 128 fragments were present following the trend observed for p(DMA-co-NAS) and p(HPA-co-NAS) samples. The peak assignments of fragments from Figures 4 and S2 are presented in Tables S2 and S3. Despite the presence of the NHS-related fragments after the functionalization, the clear separation between the fragments before and after peptide functionalization was the first indication of the success of the functionalization process. Further confirmation was sought through cell culture experiments. ToFSIMS and PCA were then employed to identify the main differences between RGD and RAD peptides. ToF-SIMS and PCA comparison between RGD and RAD functionalized samples for each copolymer is presented in Figure 5. The single amino acid difference per sequence, including alanine from the RAD and glycine from the RGD peptide is expected to favor fragment separation.


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Biomacromolecules

Figure 5. PC1 ToF-SIMS data where RGD was evaluated against RAD functionalized copolymers. Positive loadings are related to RGD conjugated and negative loading corresponded RAD conjugated for all copolymer samples. A) The scores plot of PC1 of RGD functionalized


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p(DMA-co-NAS) against RAD functionalized p(DMA-co-NAS), encompassing 87.77% of the variance. B) PC1 loadings plot C) The scores plot of PC1 of RGD functionalized p(HPA-co-NAS) against RAD functionalized p(HPA-co-NAS), encompassing 85.19% of the variance D) PC1 loadings plot. E) The scores plot of PC1 of RGD functionalized p(NAM-co-NAS) against RAD functionalized p(NAM-co-NAS), encompassing 96.60% of the variance. F) PC1 loadings plot. All PC1 instances accounted for variance values greater than 80% with positive loadings corresponding to the characteristic fragments associated with amino acids including arginine m/z = 87 (C3H9N3+), m/z = 100 (C5H10NO+) and aspartic acid m/z = 127 (C6H9NO2+) attributed to the RGD peptide. Despite the presence of the glycine ion m/z = 30 (C2H6+) in the negative loading of some samples, most of the negative loadings were associated with the alanine amino acid (RAD peptide), including m/z = 29 (CH3N2 / C2H5 ), m/z = 57 (C3H5O ), m/z = 86 (C4H8NO+). Notwithstanding the evident fragment separation when the RGD and RAD peptides were compared, the similar chemical composition of both peptide sequences and the polymer coatings where carbon, oxygen and nitrogen are prevalent, may hinder data interpretation. In addition, inconsistent fragment grouping was observed and may also limit RGD and RAD peptide sample differentiation. Some of the ion fragments obtained from the PCA analysis of RAD and RGD peptide samples were selected and their potential amino acid and assignments are presented in Table 3. The potential assignments of all fragments in Figure 5 are presented in Table S4.


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Biomacromolecules

Table 3. Potential assignments of the characteristic peaks obtained from the PCA analysis. Where R represents arginine, f represents phenylalanine, K represents lysine, D represents aspartic acid and A represents alanine. m/z

Potential assignments

Potential amino acid

CH3N2 / C2H5

29.04

R/A

42.04

C3H6+

f

45.00

CH3NO+

D

57.03

C3H5O+

A

68.05

C5H8+/C4H6N+

R

71.05

C4H7O

R

86.06

C4H8NO+

A

87.05

C3H9N3+

R

100.07

C5H10NO+/C4H10N3+

R/K

101.08

C5H11NO+

G

127.04

C6H9NO2+

D

142.10 C7H12NO2+/ C7H14N2O2 148.08

C9H1ONO+

A f

3.3 Cell culture experiments Cell culture experiments were carried out to confirm the surface functionalization of the electrospun samples and to illustrate that the peptide surface concentrations obtained were suitable for cell attachment. The presence of non-specific proteins adsorbed on the surface of biomaterials may result in reduced interaction between synthetic materials and cells or biological tissue, for example by limiting access of cell receptors to ligands, reducing the effectiveness of the functionalized scaffold.14 At interfaces cell attachment occurring via non-specific protein


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adsorption is expected to be minimized or inhibited through the grafting of low fouling polymer brush layers. The low-fouling properties of the pDMA, pHPA and pNAM employed in the copolymer system here, has been previously confirmed via both cell culture and protein adsorption tests.9 Biomacromolecule immobilization is expected to facilitate cell attachment via specific cell material interactions, resulting in specific signaling cascades. When bioactive, biospecific peptides are presented on the surface of electrospun scaffolds, cell attachment facilitated by these peptides is expected to be observed. Evaluation of the number of cells attached at the surface of functionalized and non-functionalized samples is presented in Figure 6.

Figure 6. Cells per 0.5 mm2 on the surface of functionalized (RGD 200-800µM, RAD-800 µM) and non-functionalized copolymerized grafted samples. (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p≤ 0.001, n ≥ 3, error bars represent standard error of the mean). The number of cells present on the surface of the both p(DMA-co-NAS) and p(HPA-co-NAS) was higher compared with the p(NAM-co-NAS) copolymer. This was most likely, as a result of the thin coating fabricated using NAM/NAS co-monomer combination. A higher number of cells was observed to attach when the peptide concentration was increased, in a dose specific manner,


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except in the case of p(NAM-co-NAS) samples. Significant differences between the number of cells on the non-functionalized and the samples prepared at 400 and 800 µM peptide concentrations, were obtained for p(DMA-co-NAS) and p(HPA-co-NAS) samples. The nonbioactive RAD (800 µM) sequence did not support higher cell attachment in any of the copolymers coating systems tested. Therefore, peptide immobilization at concentrations of 800 µM was successfully achieved for all samples, especially for p(DMA-co-NAS) and p(HPA-co-NAS) modified samples. Figures 7 shows images of the cells present on the surface of each copolymer coating system prepared. The presence of low fouling coatings may facilitate the analysis of the RGD coupled samples as the influence of uncontrolled variables including protein adsorption, integrin-independent cell attachment and the material topographical characteristics, is expected to be minimized.51

Figure 7. Fluorescent images of fibroblast stained for F-actin (red) and nucleus (blue), cultured on the surface of A) p(DMA-co-NAS). B) p(HPA-co-NAS) and C) p(NAM-co-NAS) grafted


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electrospun fibre samples. Bioactive cRGDfK and peptide non-bioactive cRADfK peptides were coupled from an 800 µM peptide solution. In general, cells seeded onto non-functionalized samples and samples with coupled cRADfK peptide presented a rounded morphology which is assumed to be due to the lack of surface specific adhesion. Further immunofluorescence characterization may assist in understanding cell focal adhesion on these 3D scaffolds. The presence of the low fouling polymer brush layer resists nonspecific protein adsorption. Cell attachment is not facilitated in this case by the presence of the cRADfK peptide, as illustrated by the aggregated morphology of the cells (i.e. they form cell clusters very weakly attached or not attached to the coated surface). The coupling of the cRGDfK peptide on the surface of the fibres promotes higher attachment density and a more spread morphology of the cells. These results confirm successful peptide coupling and the higher affinity of interactions with cell surface integrins of the cyclic-RGDfK peptides. This is consistent with the expectation that cyclic peptides will support more specific interactions and higher affinity, in comparison with linear peptide sequences.52-54 4. CONCLUSIONS Functionalization of the surface of PLGA:PLA:Br electrospun fibre samples was successfully achieved through grafting of copolymer brush coatings comprised of a low fouling monomer, either DMA, HPA and NAM and copolymerized with the activated ester functional monomer, NAS. In order to obtain relatively controlled chains, the catalyst ratio in the SI-Cu(0) mediated polymerisation was significantly increased, especially for the copolymerization of p(DMA-coNAS) and p(NAM-co-NAS). The reactivity ratios of the copolymer systems under the Cu(0) mediated polymerization conditions coupled with high levels of copper employed, were assumed to negatively affect the degree of conversion of the acrylamide monomers, with the p(NAM-co-


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NAS) copolymer being more drastically affected. XPS and ToF-SIMS analysis of samples confirmed the presence of the copolymer brush coating on the surface of the electrospun samples. Additionally, the use of PCA facilitated a detailed analysis of the samples where the main fragments related to the NAS monomer were identified. Despite the observation of the NHS group in the already functionalized samples, confirmation of the peptide immobilization via ToF-SIMS was successfully achieved. Fractions of NHS residues were attributed to sterically hidden NHS groups within the brush that may not have been available for peptide immobilization. Peptide coupling to the surface of the electrospun fibre samples was validated by ToF-SIMS and confirmed via the observation of cell attachment. This peptide coupling could only be achieved through the presence of specific cell attachment motifs, delivered by the cRGDfK bioactive sequence, since the presence of low fouling polymer brush coatings result in low cell adhesion. In addition, the absence of cell attachment onto the negative control cRADfK also supported peptide coupling. Higher number of cells attached per mm2 were obtained with p(DMA-co-NAS) and p(HPA-coNAS) compared with the p(NAM-co-NAS) copolymer system, in which effective surface-initiated polymerisation was limited. Increasing the peptide concentration favoured higher cell attachment as might be expected. Overall, surface grafting of copolymers comprising of a combination of low fouling and functional monomers significantly reduced the adsorption of non-specific proteins and facilitated the addition of bio-functionality for effective cell attachment. Supporting information Average fibre diameter before and after surface modification, PC2 comparison of the RGD and RAD functionalized p(NAM-co-NAS) copolymers against non-functionalized p(NAM-co-NAS) and the potential peak assignment for each fragment from ToF-SIMS and PCA analysis Corresponding Author


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*E-mail: [email protected] ORCID Almar Postma 0000-0001-7343-7236 Laurence Meagher 0000-0002-2055-6159 Paul Pigram: 0000-0002-7972-492X ACKNOWLEDGMENTS This work was performed in part at the Australian National Fabrication Facility (ANFF), a company established under the National Collaborative Research Infrastructure Strategy, through the La Trobe University Centre for Materials and Surface Science. The authors gratefully acknowledge Dr Robert Jones and Mr Robert Madiona for their assistance in undertaking ToFSIMS measurements and for surface analysis support. REFERENCES 1. Stevens, M. M.; George, J. H., Exploring and engineering the cell surface interface. Science 2005, 310, 1135-1138. 2. Chen, S.; Li, L.; Zhao, C.; Zheng, J., Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials. Polymer 2010, 51, 5283-5293. 3. Ratner, B. D.; Bryant, S. J., Biomaterials: Where we have been and where we are going. Annu. Rev. Biomed. Eng 2004, 6, 41-75. 4. Duque-Sánchez, L.; Brack, N.; Postma, A.; Pigram, P. J.; Meagher, L., Surface modification of electrospun fibres for biomedical applications: A focus on radical polymerization methods. Biomaterials 2016, 106, 24-45. 5. Tugulu, S.; Klok, H. A., Stability and nonfouling properties of poly(poly(ethylene glycol) methacrylate) brushes under cell culture conditions. Biomacromolecules 2008, 9, 906-12. 6. Coad, B. R.; Lu, Y.; Meagher, L., A substrate-independent method for surface grafting polymer layers by atom transfer radical polymerization: Reduction of protein adsorption. Acta Biomater 2012, 8, 608-618. 7. Ameringer, T.; Ercole, F.; Tsang, K. M.; Coad, B. R.; Hou, X.; Rodda, A.; Nisbet, D. R.; Thissen, H.; Evans, R. A.; Meagher, L., Surface grafting of electrospun fibers using ATRP and RAFT for the control of biointerfacial interactions. Biointerphases 2013, 8, 1-11. 8. Lin, S. S.; Li, Y.; Zhang, L.; Chen, S. F.; Hou, L., Zwitterion-like, charge-balanced ultrathin layers on polymeric membranes for antifouling property. Environ. Sci. Technol 2018, 52, 4457-4463.


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9. Duque Sanchez, L. M.; Brack, N.; Postma, A.; Pigram, P. J.; Meagher, L., Optimisation of grafting of low fouling polymers from three-dimensional scaffolds via surface-initiated Cu(0) mediated polymerisation. J. Mater. Chem. B 2018, 5896-5909 10. Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M., A Survey of Structure−Property Relationships of Surfaces that Resist the Adsorption of Protein. Langmuir 2001, 17, 5605-5620. 11. Shen, M. C.; Martinson, L.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A., PEO-like plasma polymerized tetraglyme surface interactions with leukocytes and proteins: in vitro and in vivo studies. Journal of Biomaterials Science-Polymer Edition 2002, 13, 367-390. 12. Hucknall, A.; Rangarajan, S.; Chilkoti, A., In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins. Adv. Mater. 2009, 21, 2441-2446. 13. Yoo, H. S.; Kim, T. G.; Park, T. G., Surface-functionalized electrospun nanofibers for tissue engineering and drug delivery. Adv. Drug Delivery Rev 2009, 61, 1033-1042. 14. Rodda, A. E.; Meagher, L.; Nisbet, D. R.; Forsythe, J. S., Specific control of cell-material interactions: Targeting cell receptors using ligand-functionalized polymer substrates. Prog. Polym. Sci 2014, 39, 1312-1347. 15. Xu, F. J.; Zheng, Y. Q.; Zhen, W. J.; Yang, W. T., Thermoresponsive poly(N-isopropyl acrylamide)-grafted polycaprolactone films with surface immobilization of collagen. Colloids Surf., B. 2011, 85, 40-47. 16. Xu, F. J.; Wang, Z. H.; Yang, W. T., Surface functionalization of polycaprolactone films via surface-initiated atom transfer radical polymerization for covalently coupling cell-adhesive biomolecules. Biomaterials 2010, 31, 3139-47. 17. Gunnewiek, M. K.; Benetti, E. M.; Di Luca, A.; van Blitterswijk, C. A.; Moroni, L.; Vancso, G. J., Thin Polymer Brush Decouples Biomaterial's Micro-/Nanotopology and Stem Cell Adhesion. Langmuir 2013, 29, 13843-13852. 18. Tugulu, S.; Silacci, P.; Stergiopulos, N.; Klok, H. A., RGD-Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells. Biomaterials 2007, 28, 2536-46. 19. Paripovic, D.; Hall-Bozic, H.; Klok, H. A., Osteoconductive surfaces generated from peptide functionalized poly(2-hydroxyethyl methacrylate-co-2-(methacryloyloxy)ethyl phosphate) brushes. J. Mater. Chem. 2012, 22, 19570-19578. 20. Desseaux, S.; Klok, H. A., Fibroblast adhesion on ECM-derived peptide modified poly(2hydroxyethyl methacrylate) brushes: Ligand co-presentation and 3D-localization. Biomaterials 2015, 44, 24-35. 21. Zhang, T.; Du, Y. H.; Muller, F.; Amin, I.; Jordan, R., Surface-initiated Cu(0) mediated controlled radical polymerization (SI-CuCRP) using a copper plate. Polym. Chem 2015, 6, 27262733. 22. Zhang, T.; Du, Y. H.; Kalbacova, J.; Schubel, R.; Rodriguez, R. D.; Chen, T.; Zahn, D. R. T.; Jordan, R., Wafer-scale synthesis of defined polymer brushes under ambient conditions. Polym. Chem 2015, 6, 8176-8183. 23. Dehghani, E. S.; Du, Y.; Zhang, T.; Ramakrishna, S. N.; Spencer, N. D.; Jordan, R.; Benetti, E. M., Fabrication and Interfacial Properties of Polymer Brush Gradients by SurfaceInitiated Cu(0)-Mediated Controlled Radical Polymerization. Macromolecules 2017, 50, 24362446.


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Engineering the Biointerface of Electrospun 3D Scaffolds with

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