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Biofunctionalised Patterned Polymer Brushes via Thiol-Ene Coupling for the Control of Cell Adhesion and the Formation of Cell Arrays Burcu Colak, Stefania Di Cio, and Julien Ezra Gautrot Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01436 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018
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Biomacromolecules
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Biofunctionalised Patterned Polymer Brushes via Thiol-
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Ene Coupling for the Control of Cell Adhesion and the
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Formation of Cell Arrays
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Burcu Colak,1,2 Stefania Di Cio1,2 and Julien E. Gautrot1,2*
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Institute of Bioengineering and 2 School of Engineering and Materials Science, Queen
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Mary, University of London, Mile End Road, London, E1 4NS, UK.
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* To whom correspondence should be addressed E-mail:
[email protected].
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Abstract
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Thiol-ene radical coupling is increasingly used for the biofunctionalisation of biomaterials.
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Thiol-ene chemistry presents interesting features that are particularly attractive for platforms
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requiring specific reactions with peptides or proteins and the patterning of cells, such as
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reactivity in physiological conditions and photo-activation. In this work, we synthesised
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alkene-functionalised (allyl and norbornene residues) anti-fouling polymer brushes (based on
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poly(oligoethyleneglycol methacrylate)) and studied thiol-ene coupling with a series of thiols
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including cell adhesive peptides RGD and REDV. The adhesion of human umbilical vein
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endothelial cells human umbilical vein endothelial cells (HUVECs) to these interfaces was
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studied and highlighted the absence of specific integrin engagement to REDV, in contrast to
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the high level of cell spreading observed on RGD-functionalised polymer brushes. This
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revealed that α4β1 integrins (binding to REDV sequences) are not sufficient on their own to
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sustain HUVEC spreading, in contrast to αvβ3 and α5β1 integrins. In addition, we
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photopatterned peptides at the surface of poly (oligoethylene glycol methacrylate) 1 ACS Paragon Plus Environment
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(POEGMA) brushes and characterised the quality of the resulting arrays by epifluorescence
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microscopy and atomic force microscopy (AFM). This allowed the formation of cell patterns
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and demonstrated the potential of thiol-ene based photopatterning for the design of cell
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microarrays.
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Introduction
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The control and manipulation of interfaces between materials and biological systems is
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particularly important for the development of biomedical applications such as tissue
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engineering and in vitro cell-based assays. Surface engineering using polymer coatings has
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proved very successful for the control of hydrophilicity, surface charge, chemical reactivity,
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protein resistance, adhesion, mechanics and topography.1-6 In particular, recent advances in
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patterning technologies have allowed the precise patterning of cells and cell colonies,
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therefore enabling unprecedented control of single cell and cell cluster shape and organisation
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or migration.7-14 Indeed extra-cellular matrix (ECM) protein micropatterns have been
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developed to control cell spreading and shape and how such geometrical cues regulate cell
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phenotype.15, 16 In the case of mesenchymal stem cells and epidermal stem cells, changes in
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cytoskeleton organisation, guided by the geometry of the ECM, regulate differentiation15, 16
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and the compartmentatlisation of stem cell clusters.10
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For such applications, polymer coatings should allow a precise control of protein adsorption
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and integrin binding. In this respect, polymer brushes are particularly well-suited as they can
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be design with a wealth of chemical functionalities and enable the attachment and
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immobilisation of biomolecules.17, 18 In particular, polymer brushes based on oligo(ethylene
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glycol methacrylate) (OEGMA), carboxybetaine methacrylamide and hydroxypropyl
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methacrylamide display exceptional protein resistance, even in the presence of complex 2 ACS Paragon Plus Environment
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Biomacromolecules
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protein solutions such as serum (relevant to cell culture), plasma and blood.9, 19-23 Therefore
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such brushes have been patterned using micro-contact printing (µCP)9, 19 and, more recently
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electrospun fibre nanolithography24 (ENL) to define nano- to micro-scale ECM protein
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patterns that can regulate cell adhesion and spreading. In this respect, the excellent prolonged
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protein resistance of polymer brushes such as POEGMA, even in tissue culture conditions, is
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particularly attractive for cell-based assays requiring several days of culture.9 In addition, the
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ease with which polymer brushes with a wide range of chemistries can be patterned is
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attractive to study the synergistic roles of ECM geometry and matrix physico-chemical
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properties on cell adhesion, spreading and phenotype.25
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The biofunctionalistion of polymer brushes has also attracted considerable interest, from the
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immobilisation of enzymes26-28 to the development of biosensors.21,
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peptides have been coupled to polymer brushes through a range of strategies,31-35 to confer
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biospecificity to antifouling polymer brushes such as POEGMA. This strategy proved very
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successful for the control of cell adhesion and the study of the synergistic binding of different
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integrin ligands as well as their 3D spatial localisation.36, 37 However, to date few polymer
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brush-based interfaces have been micropatterned with peptides to enable the simultaneous
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control of cell shape and the binding of specific integrins35 and parameters controlling
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suitable biofunctionalisation strategies and patterning fidelity remain largely unexplored.
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In the present work, we report the functionalisation of POEGMA brushes with peptides, via
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thiol-ene chemistry. Radical mediated thiol-ene coupling has attracted attention from the
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biomaterials community owing to its efficiency in mild conditions, including at physiological
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pH, buffer and ionic strength.14, 38-40 In addition, peptides are attractive candidates used for
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biofunctionalisation via thiol-ene coupling as they can be designed to present cysteine
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residues relatively readily.41-43 Maleimide-based Michael addition has been used to promote
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29, 30
In particular,
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the coupling of antibacterial peptides32, 33, 44 to polymer brushes, but few works have focused
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on the radical-based tethering of thiols to brushes14,
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antifouling properties. Previous work in this area focused on the use of allylamine-
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functionalised poly(glycidyl methacrylate) brushes to promote thiol-ene coupling35, however
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these substrates are poorly adapted for the specific control of cell adhesion as the resulting
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coatings are too hydrophobic and positively charged. We proposed to simply functionalise
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hydroxyl-terminated POEGMA brushes (POEGMA-OH) with alkenes that can sustain thiol-
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ene coupling. We characterised the efficiency of POEGMA brush functionalisation with a
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range of thiols, including peptides. We then studied cell adhesion to the resulting
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biofunctionalised brushes, with the aim to characterise the specificity of the peptides tethered
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to promote integrin binding in human umbilical vein endothelial cells (HUVECs). In addition
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we used thiol-ene coupling of labelled peptides to characterise the quality of peptide patterns
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generated via photo-irradiation through masks, and demonstrated the design of cellular
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microarrays using this approach.
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, in particular those displaying
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Materials and methods
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Materials
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Anhydrous
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(trimethoxysilyl)propyl methacrylate (98%), methanol (99.9 %), phosphate buffer saline
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(PBS) tablets, Dulbecco's PBS, poly (ethylene glycol) methacrylate average Mn 360
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(409537), bipyridine (99 %), copper (II) bromide (99.999 %), copper (I) chloride (99.995 %),
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disuccinimyl carbonate (DSC) (95 %), 4-dimethylaminopyridine (DMAP) (99 %),
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dimethylformamide (DMF) (pharmaceutical secondary standard), allylamine (98 %),
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hexamethylenediamine (98 %) triethanolamine (99 %), Eosin Y (99 %), IRG2959 (98 %), N-
toluene
(99.8
%),
triethylamine
(99
%),
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ethanol
(99.5
%),
3-
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acetyl L-cysteine (T1, 99%), L-glutathione reduced (T2, 98 %), paraformaldehyde (PFA) (95
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%), Triton-X 100, phalloidin–tetramethylrhodamine B isothiocyanate and DAPI (98 %) were
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obtained from Sigma Aldrich. Atom-transfer radical polymerisation (ATRP) silane initiator,
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3-trimethoxysilyl propyl 2-bromo-2 methylpropionate was purchased from Fluorochem.
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Medium 199, 2-{4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) was
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purchased from life technologies. Collagen rat type I was purchased from BD Biosciences.
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Custom peptides (> 95% purity, high-performance liquid chromatography (HPLC)
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GCGREDVSG (T3, REDV), GCGRVDESG (T4, RDVE), GCGYGRGDSPG (T5, RGD),
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GCGYGRGESPG (T6, RGE), GCGPHSRNSG (T7, PHSRN), GCGYGRGESPG-Lys-FITC-
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G (T8, FITC-RGE) were purchased from Proteogenix, France. Penicillin streptomycin (5000
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U/mL) and medium 199 HEPES (22340020) were obtained from life technologies. Fetal
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bovine serum (FBS), South American Origin was purchased from Labtech. (Endo-cis-5-
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norbornene-2,3- dicarboxylic anhydride) (99 %), L-glutamine (200 mM), versene and trypsin
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(0.25 %) phenol red, were obtained from Thermo Fisher Scientific.
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Instrumentation
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Dry brush thicknesses were measured via ellipsometry, using an α-SE ellipsometer from J.A.
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Woolam Co., Inc. Ellipsometry solutions. For in situ measurements, in deionised water,
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substrates were placed in chamber fitted with quartz windows normal to the incident beam
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path. The refractive index of the medium was obtained using data recorded for bare silicon
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substrate in deionised water. Measurements were carried out in triplicate.Grazing angle
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fourier-transform infrared spectroscopy (FTIR) was carried out using a Bruker Tensor 27
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spectrometer equipped with a MCT detector; results were acquired at a resolution of 16 cm-1
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and a total of 128 scans per run in the region of 600-4000 cm-1. 1H nuclear magnetic
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resonance (NMR) and
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spectrometry was carried out using an Agilent Technologies 1100 series equipped with a
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C NMR spectroscopy was produced using Bruker AV 400. Mass
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LC/MSD trap. XPS was measured using thermo K-Alpha XPS system. The ultraviolet (UV)
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light used to initiate reactions was the Omnicure series 1500. The radiometer used to measure
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the UV light intensity was from International light technologies, ILT 1400-A radiometer
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photometer. The visible light used to initiate the reaction was generated using an OSL2 fibre
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illuminator from Thorlabs. AFM was used to scan surfaces, in semicontact mode and the row
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pictures were corrected with a first order function using Nova software. Non-contact NSG01
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cantilevers from NT-MDT were used (force constant 1,45-15,1 N/m and resonant frequency
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87-230 kHz). A Leica DMI4000B epifluorescence microscope fitted with a HCX PL
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FLUOTAR 10x/0.3 PJ1 objective and a Leica DFC300 FX CCD camera was used to image
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patterned surfaces. Analysis of microscopy images was carried out using Image J; singe
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channel images were changed to grey scale then to black and white using a threshold (with
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cells appearing black), binary watershedding was performed to identify individual cells.
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Calculation of functionalisation levels from ellipsometry data
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As described in a previous report35, polymer brush funcationalisation levels were calculated
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according to: ߂ܶ = ܶ݊ − ܶ݅
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Equation 1
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Where ∆T is the difference in dry brush thickness (in nm) between Tn (new dry brush
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thickness after functionalisation) and Ti (initial dry brush thickness). The degree of
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functionalisation (in %) fn is obtained according to: ௱்
݂݊ = ቀ
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்
ெଵ
× ெଶቁ × 100
Equation 2
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Where M1 and M2 are the molecular weight of initial repeat units and that of the added
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fragments (in g/mol).
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Biomacromolecules
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Statistical analysis
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All data were analysed by Tukey's test and significance was determined by * p < 0.05, ** p
