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Bio-interactions and Biocompatibility

The effect of functional groups of self-assembled monolayers on protein adsorption and initial cell adhesion Lalit M Pandey, Abshar Hasan, and Sudip K. Pattanayek ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00795 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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ACS Biomaterials Science & Engineering

The effect of functional groups of self-assembled monolayers on protein adsorption and initial cell adhesion

Abshar Hasan1, Sudip K. Pattanayek2, Lalit M. Pandey1* 1

Bio-Interface & Environmental Engineering Lab, Department of Biosciences and Bioengineering,

Indian Institute of Technology Guwahati, Assam, 781039, India 2

Macromolecules and Interfaces Laboratory Department of Chemical Engineering Indian Institute of Technology Delhi Hauz Khas New Delhi 110 016, India

*Corresponding author: Tel. +91-361-258-3201; Fax +91-361-258-2249 Email addresses: [email protected], [email protected], [email protected]

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Abstract Surface modification plays vital role in regulating protein adsorption and subsequently cell adhesion. In the present work, we prepared nanoscaled modified surfaces using silanization and characterized them using Fourier-transform infrared spectroscopy (FTIR), water contact angle (WCA) and Atomic Force microscopy (AFM). Five different (amine, octyl, mixed, hybrid and COOH) surfaces were prepared based on their functionality and varying wettability and their effect on protein adsorption and initial cell adhesion was investigated. AFM analysis revealed nanoscale roughness on all modified surfaces. Fetal bovine serum (FBS) was used for protein adsorption experiment and effect of FBS was analyzed on initial cell adhesion kinetics (upto 6 h) under three different experimental conditions: (a) with FBS in media, (b) with pre-adsorbed FBS on surfaces and (c) incomplete media, i.e., without FBS. Various cell features such as cell morphology/circularity, cell area and nuclei size were also studied for above stated conditions at different time intervals. The cell adhesion rate as well as cell spreaded area were highest in case of surfaces with pre-adsorbed FBS. We observed higher surface coverage rate by adhering cells on hybrid (rate, 0.073 h-1) and amine (0.072 h-1) surfaces followed by COOH (0.062 h-1) and other surfaces under pre-adsorbed FBS condition. Surface treated with cells in incomplete media exhibited least adhesion rate, poor cell spreading and improper morphology. Furthermore, we found that initial cell adhesion rate and ∆ adhered cells (%) linearly increased with the change in α-helix content of adsorbed FBS on surfaces. Amongst all the modified surfaces and under all three experimental conditions, hybrid surface exhibited excellent properties for supporting cell adhesion and growth and hence can be potentially used as surface modifiers in biomedical applications to design biocompatible surfaces.

Keywords: Silanization; Protein adsorption; Cell adhesion; Kinetics; Wettability



1. Introduction Interdisciplinary research in the field of tissue engineering amalgamates basic biological sciences, material science, engineering and medical technology to overcome the damage or failure of tissue/organ due to injuries, diseases or trauma and for many other applications.1-2 Bone implant, heart stents and artificial tooth are the most commonly used biomaterial implants nowadays.3 Protein adsorption and cell adhesion are the primary events that take place on biomaterial surfaces as soon as they come in contact with the body fluids. Nonspecific protein adsorption from the protein rich plasma may cause deterioration of biomaterial due to platelet adhesion and activation resulting in thrombosis.4 The implant’s surface interactions with biological system play an important role in deciding its fate as these interactions describe the response of proteins, cells, tissue and organs.5 In this direction, biomimetic surfaces are designed by various surface modification techniques to minimize the foreign body response (FBR) and maximize the purpose of implant.6-8 The tendency of modified biomimetic surfaces to imitate the extracellular matrix (ECM) provides platform to support cell adhesion and proliferation. Surface modifications using various physical, chemical and biological processes had been reported to circumvent such unwanted processes.8-10 Surfaces of ceramics, polymers and metals have been modified via physical (for ex. plasma treatment) and chemical methods (for ex. functionalization via Self Assembled Monolayers [SAMs], immobilized ligands etc.) to carefully modulate protein adsorption, cell adhesion and differentiation.11-23 In a recent study, it was found that surface functional groups tune protein adsorption, which in turn regulate cell adhesion and spreading.24 Dynamic behavior of adsorbed cell-adhesive protein (eg. vitronectin)

due to adhering cells on surface regulate cell adhesion at cell-material

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interface. Garcia et al.26 reported the enhanced expression of integrins receptors on hydrophilic surface indicating better cell adhesion on them as compared to hydrophobic surfaces. Arima and Iwata further elaborated that adhesion of HUVECs cells was best responded by CH3/OH surface (θ=40º) whereas HeLa cells adhered best on CH3/OH and CH3/COOH surfaces (θ=50º).27 Their results agrees well with the literature which states that cell adhesion and spreading takes place optimally on surfaces with moderate wettability in the range 50°-80º. On contrary, the attachment and proliferation of osteoblastic cells were found to increase with an increase in surface wettability, which was correlated to fibronectin adsorption.28-29 FTIR and XPS studies


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revealed that interactions between the internal hydrophobic domains of protein and surfaces lead to the change in the secondary structure of proteins and may expose the cell adhesive sites resulting in enhanced cell adhesion and spreading.13, 30-31 Moreover, Inoue et al.32 emphasized that surface ζ potentials of polymeric brush substrates play major role in regulating protein adsorption and cell adhesion rather than surface wettability. Therefore it highlights the need to investigate this very important yet complicated phenomenon on model surfaces with wide range of wettability. SAMs of silanes and thiols form highly organized covalently attached organic molecules with nano-thick coatings which in turn provide tunable surface properties.33-34 Desirable surface properties can be easily generated by choosing various functionalized molecules from the huge available library. Although SAMs with different functionalities such as -NH2, -COOH, -Cl, -OH, -CH3 and their mixed combinations have been explored for protein adsorption and cell adhesion31, 35-37 but their effect on initial cell adhesion kinetics and spreading is less explored. This inspired use to undertake this challenge to explore the underlying aspects of initial cell adhesion on surfaces exhibiting different surface functionalities (wettability) in the presence and absence of serum proteins. In the present work, we prepared surfaces with different wettability by varying the surface functionalities using various mono, mixed and hybrid SAMs.13,

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The adsorption of serum

proteins was examined on these modified surfaces in terms of adsorbed amounts using bicinchoninic acid (BCA) assay and change in secondary structures using FTIR analysis. The kinetics of initial L929 mouse fibroblast cell adhesion was investigated in the presence of modified surfaces (a) without FBS proteins in media, (b) with FBS in media, and (c) with FBS pre-adsorbed on surfaces. With the change in the protein behavior at differently functionalized surfaces, the behavior of the adhering cells also changes. Along with kinetics studies under all three experimental conditions stated above, we also determined the effect of surface modifications on cell adhesion and spreading, their morphology and nuclei size.



2. Material and Methods 2.1. Materials Aminopropyl triethoxysilane (APTES, Cat. No. 440140), triethoxy(octyl)silane (TEOS, Cat. No. 440213), phalloidin- fluorescein isothiocyanate (FITC) labeled (Cat. No. P5282), propidium iodide (PI, Cat. No. P4170), anhydrous toluene, dibutyltin dilaurate, and p-tolyl isocyanate were purchased from Sigma Aldrich, India. Fetal bovine serum, vinculin primary antibody (Cat no. 700062) and secondary antibody-Alexa Fluor 350 (Cat. No. A11046) were purchased from ThermoFisher Scientific, India. Methanol, toluene, circular glass coverslip (14 mm diameter), sodium chloride (NaCl), potassium chloride (KCl), monobasic potassium phosphate (KH2PO4), dibasic sodium phosphate (Na2HPO4), sulfuric acid (H2SO4), and hydrogen peroxide (H2O2) and diiodomethane (DI) were procured from Himedia, India. Double distilled water (Mili-Q, 18 MΩ) was used throughout the work. 2.2. Surface silanization/ modification Five different surfaces with varying wettability and functionality were prepared using aforementioned silane groups based on our previously reports.13, 31, 38-40 Briefly, glass coverslips were washed with piranha (H2SO4:H2O2=7:3 v/v), ammonia (water:H2O2:NH3=5:1:1 v/v) and HCl (water:H2O2:HCl=3:1:1 v/v) solutions and dried overnight at 50ºC prior to surface modification. For monotype amine and octyl SAMs surfaces, cleaned substrates were dipped in 1% (v/v) solution of APTES and TEOS in anhydrous toluene and incubated for 24 h under inert conditions at room temperature. Mixed (amine-octyl) SAM was prepared by mixing APTES and TEOS in 1:1 ratio (1% v/v) under similar experimental conditions. Carboxylic (COOH) modified surfaces were synthesized by oxidizing octyl modified surfaces with 5% acidified KMnO4 solution for 30 min at room temperature.41 Similarly, hybrid SAM was prepared by treating amine modified surface with p-tolyl isocyanate solution (1%, v/v) for 4 h in the presence of catalyst dibutyltin dilaurate. The reaction between amine (NH2) head groups at surface and isocyanate (NCO) forms urea linkage at surface.13, 38-40


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2.3. Characterization of modified surfaces The modified surfaces were characterized in terms of functional groups, wettability and morphology using FTIR, contact angle Goniometer and AFM, respectively. Surface silanization were confirmed using FTIR (Spectrum TWO, Perkin Elmer) instrument at a scanning rate of 15 scans per second with resolution 1cm−1, taking unmodified surface as background. Modified silicon surfaces were characterized for surface topology and roughness. Surface silanization resulted in nanoscale roughness as evidenced by AFM analysis and was compared with unmodified surfaces. Innova AFM system (Bruker) fitted with silicon nitride tip of

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