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Parallel control over surface charge and wettability using polyelectrolyte architecture: effect on protein adsorption and cell adhesion Shanshan Guo, Xiaoying Zhu, Min Li, Liya Shi, June Lay Ting Ong, Dominik Ja#czewski, and Koon Gee Neoh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09481 • Publication Date (Web): 20 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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ACS Applied Materials & Interfaces

Parallel control over surface charge and wettability using polyelectrolyte architecture: effect on protein adsorption and cell adhesion Shanshan Guo†, Xiaoying Zhu*,‡,#, Min Liǁ, Liya Shiǁ, June Lay Ting Ong‡, Dominik Jańczewski*,§, Koon Gee Neoh*,†,ǁ

† NUS Graduate School for Integrative Science and Engineering, National University of Singapore, Kent Ridge, Singapore, 117576 ‡ Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research, 2 Fusionopolis Way, Singapore, 138634. Email: [email protected]; Tel: +65 6319 4819; Fax: +65 6872 0785 § Laboratory of Technological Processes, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664, Warsaw, Poland. E-mail: [email protected]; Tel: +48 22 234 5583; Fax: +48 22 234 5504 ǁ Department of Chemical & Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore, 119260. E-mail: [email protected]; Tel: +65 6516 2176; Fax: +65 6779 1936

# Department of Environmental Science, Zhejiang University, Hangzhou, China, 310058. E-mail: [email protected]; Tel: +86 571 88982651

KEYWORDS: layer-by-layer assembly, polyelectrolyte multilayer, surface charge, surface wettability, protein adsorption, cell adhesion

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Abstract Surface charge and wettability, the two prominent physical factors governing protein adsorption and cell adhesion, have been extensively investigated in the literature. However, a comparison between these driving forces in terms of their independent and cooperative effects in affecting adhesion is rarely explored on a systematic and quantitative level. Herein, we formulate a protocol which features two-dimensional control over both surface charge and wettability with limited cross parameter influence. This strategy is implemented by controlling both the polyion charge density in the layer-by-layer (LbL) assembly process and the polyion side chain chemical structures. The 2D property matrix spans surface isoelectric point ranging from 5 to 9 and water contact angle from 35° to 70°, with other interferential factors (e.g. roughness) eliminated. The interplay between these two surface variables influences protein (bovine serum albumin, lysozyme) adsorption and 3T3 fibroblast cell adhesion. For proteins, we observe the presence of thresholds for surface wettability and electrostatic driving forces necessary to affect adhesion. Beyond these thresholds, the individual effects of electrostatic forces and wettability are observed. For fibroblast, both surface charge and wettability have an effect on its adhesion. The combined effects of positive charge and hydrophilicity lead to the highest cell adhesion whereas negative charge and hydrophobicity lead to the lowest cell adhesion. Our design strategy can potentially form the basis for studying the distinct behaviors of electrostatic force or wettability driven interfacial phenomena and serving as a reference in future studies assessing protein adsorption and cell adhesion to surfaces with known charge and wettability within the property range studied here.

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1. Introduction Managing protein adsorption and cell adhesion on interfaces is important for a wide range of applications in analytical, biomedical and environmental fields. Investigations on the correlation between surface properties and protein/cell interaction are numerous in the literature. Physical cues such as surface charge,1-4 surface free energy5 and surface wettability6 have been identified as prominent factors governing physical interactions with biological entities. However, correlating protein or cell responses to a combination of surface parameters is challenging as modifying one surface property typically affects the others.4,7,8 Particularly, surface charge and wettability, both of which depend on the chemical attributes of surface structures, are strongly interdependent properties.9,10 Typical modifications in the electric charge of the surface will affect its surface free energy, which will in turn induce changes in the surface wettability. On the other hand, the introduction of functional groups responsible for hydrophobic and hydrophilic characteristics of the interphase naturally influences charge balances due to preferential adsorption of ions, even if the group is formally neutral or even hydrophobic.11 There are existing protocols in the literature for probing surface properties and protein/cell interaction that allow a systematic control over either surface charge or wettability. Such protocols include self-assembled monolayers (SAMs) presenting different head groups (e.g. alkyl, glucose and ethylene glycol) with different degrees of hydrophobicity,12 mixed SAMs,4,13,14 mixed-charge copolymer brushes2,15 and layer-by-layer (LbL) films with serially varied surface zeta (ζ) potential.3,16 This single level of control has been applied to the investigation of the mechanisms of protein adsorption,2,12,13,15 mammalian cell adhesion4 and stem cell fate.14 Other studies focused on specific materials or functional groups to probe their effects on protein adsorption,8,17 mammalian cell adhesion,8,17-20 cell skeletal organization7 and migration.21 Frequently investigations report that behaviors such as cell adhesion were dependent on individual functional groups and cannot be clearly attributed to macroscopic surface properties such as wettability.7,8,17,20 As a result, it 3



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may be difficult to translate the conclusions to other systems or complex ecosystems that often occur in biologically and technologically relevant structures. So far, no satisfactory protocol has been proposed to address the issue of independent control over wettability and surface charge. To fully capture how these two variables influence protein adsorption and cell adhesion in independent or cooperative manners necessitates a two-dimensional (2D) independent control over both charge and wettability spanning sufficient and relevant ranges. The LbL technique, comprising self-assembled, sequential adsorption of oppositely charged polymers, offers opportunities to form coatings for various applications such as drug delivery and anti-biofouling surfaces.22-26 The benefits of adopting the LbL technique include coating on a generic substrate, growing films which are structure-controlled and molecularly well-blended,27,28 and tailoring surface composition, morphology, as well as physicochemical properties readily via assembling routines.29-31 Therefore, using the LbL approach to develop a strategy to control both wettability and charge in a 2D matrix, could form the basis for fine tuning adhesion to optimize molecular recognition processes or serving as a reference in future studies assessing protein adsorption and cell adhesion to surfaces with known charge and wettability. Herein, following previous work on LbL fabrication with serially varied surface ζ potential,3 we developed a new model system with a parallel control over both surface ζ potential and surface wettability (or surface free energy). Using commercial polyethylenimine (PEI) and custom-synthesized derivatives of poly(isobutylene-alt-maleic anhydride) (PIAMAn), we created a library of substrates covering a 2D matrix of surface parameters with surface isoelectric point (pI) in the range of 5-9 and water contact angle (CA) of 35°-70°. Poly(ethylene glycol) (PEG) and short alkyl chains were attached to polyanionic building blocks to create the polyanions with different hydrophilicity, which were subsequently used to assemble LbL films with cationic PEI. The two properties were tuned independently by the judicious adjustment of both the polyion charge density in the LbL assembly process and the polyion side chain chemical structures. We used 4


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biological entities including bovine serum albumin (BSA), lysozyme (LZY) and 3T3 fibroblast cells to demonstrate the strategy of forming a 2D property matrix to delineate the individual and cooperative effects of these two parameters on interfacial interaction, with new features and possible mechanisms highlighted. 2. Materials and methods 2.1

Materials

Poly(isobutylene-alt-maleic anhydride) (PIAMAn, Mw 60 kD), 6-aminocaproic acid (Mw 131.17 D), polyethylenimine (PEI, Mw 25 kD, branched), (3-aminopropyl)trimethoxysilane (APTMS, 97%), folate-free Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, L-glutamine, penicillin, bovine serum albumin (BSA, MW 66 kDa) lyophilized powder (≥96%), BSA−fluorescein isothiocyanate conjugate (BSA−FITC), lysozyme from chicken egg white (LYZ,

MW 14.3 kDa), Trypan Blue solution (0.4%) and

4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO). Lysozyme conjugated with Rhodamine (LYZ−RH) was purchased from Nanocs. Amine-PEG-carboxylic acid (NH2-PEG-CM, MW 2,000) was purchased from Laysan Bio Inc. All solvents used including methanol, ethanol and toluene were purchased from Tedia. Silicon wafers were purchased from Latech Scientific Supply, Pte., Ltd. Ultrapure water (18.2 MΩ cm) produced by a Millipore Milli-Q integral water purification system was used to prepare aqueous solutions. 3T3 mouse fibroblast cells were purchased from American Type Culture Collection (Manassas, VA). 2.2

Synthesis of polyanions (Figure 1)

Synthesis of PIAMA-C5 and PIAMA-PEG: 6-Aminocaproic acid (0.25 g, 1.9 mmol, synthesis of PIAMA-C5) or NH2-PEG-COOH (0.75 g, 0.38 mmol, synthesis of PIAMA-PEG) was dissolved in 5 mL dimethyl sulfoxide (DMSO). PIAMAn (0.5 g, 3.8 mmol of monomeric repeats) was added to the above solution. The reaction proceeded for 24 h at a temperature of 40 °C for 6-aminocaproic acid and room 5



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temperature for NH2-PEG-COOH, after which the resultant polymer solution was slowly poured into 50 mL NaOH aqueous solution (10 g L-1) containing a small excess of NaOH with respect to the COOH groups in the polymer backbone. The polymer solution was stirred and then dialyzed using dialysis membrane (FisherBrand, 6000-8000 MWCO) against ultrapure water for 3 days. The product was then concentrated using a rotary evaporator and finally freeze-dried. PIAMA-C5 (0.45 g, yield 76.1%) was stored at room temperature and PIAMA-PEG (1.09 g, yield 88.5%) was stored at -20 °C. Synthesis of poly(isobutylene-alt-maleic acid) (PIAMAc): PIAMAn (1 g, 7.6 mmol of monomeric repeats) was dissolved in 100 mL NaOH aqueous solution (10 g L-1). The polymer solution was stirred at room temperature for 5 h and purified the same way as described for the above polymers resulting in a white solid product (0.95 g, yield 81.2%). The polymers were analysed using 1H-NMR spectrometer (Bruker Avance, 400 MHz) and Fourier transform infrared (FTIR) spectrometer (Bruker, Vertex 80v) at a resolution of 4 cm-1 across a 400-2400 cm-1 range.

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Figure 1: Synthetic scheme for PIAMA-C5, PIAMA-PEG and PIAMAc. The degree of substitution was obtained by 1H-NMR analysis and discussed in detail in Section 3.1. 2.3

Preparation of LbL films

Freshly cut silicon substrates with dimensions of 1 cm × 2 cm were cleaned via ultrasonication in water and ethanol respectively for 10 min, after which they were dried with a nitrogen stream and treated with oxygen plasma at 200 W for 2 min. A layer of APTMS in toluene (10 mM) was deposited on these substrates for 4 h and used as a positively charged layer precursor to improve initial adhesion prior to the assembly of the main LbL films. LbL assembly was conducted by alternate immersion of the substrates in polycation and polyanion solutions for 5 min, with rinsing in ultrapure water between these immersions for 1 min for all the samples. All assembly procedures were performed at room temperature of 25°C. The polyanion could be any of those three prepared polyanions mentioned above, and the polycation was PEI. Polyelectrolyte solutions of 1.0 mg mL-1 in ultrapure water were prepared. The charge density of polyelectrolytes was controlled by adjusting the solution pH and adding either 0.1 M HCl or 0.1 M NaOH aqueous solution. The polyelectrolyte layers were deposited alternately up to 6.5 or 7 bilayers. One bilayer refers to one polycationic layer on top of one polyanionic layer, and 0.5 layer refers to one polyanionic layer. After the desired number of layers was reached, the films were dried under a nitrogen stream and further dried at room temperature for 24 h before characterization measurement. 2.4

Characterization of LbL films

The ζ potential of the surfaces was measured by an electrokinetic analyzer (SurPass, Anton Paar) containing an adjustable-gap cell. Two sample films with dimensions 1 cm × 2 cm were fitted into the cell facing each other in parallel with a micro slit of 100 µm. Streaming current measurements were performed 7



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by pumping 0.01 M KCl solution through the slit. The pH of the KCl solution was adjusted via auto pH titration from 10 to 4 by adding dilute HCl. The ζ potential of each sample was determined from the streaming current data according to the Helmholtz–Smoluchowski equation32 and the Fairbrother–Mastin approach.33 Four measurements were conducted for each run and the average was used to represent the ζ potential value at a given pH. Measurement of contact angles was carried out with a goniometer (250-F1 from Ramé-Hart Instrument Co.) using the static sessile drop method. By measuring the static contact angles on LbL films with water and methylene iodide, surface free energy was calculated according to the Owens’ method.34 Measurements were performed at room temperature. The average value of four measurements on different locations was reported. Atomic Force Microscopy (AFM) experiments were performed using a NanoWizard® 3 NanoOptics Atomic Force Microscope (JPK Instruments AG). Surface morphology was visualized in the tapping mode on three 5 × 5 µm spots using silicon probes (k ≈ 40 N m-1, Tap 300AL-G, Budget sensors) in the dried state. The root-mean-square (RMS) roughness was analyzed by the installed software (JPK Data Processing, 4.3.25). Surface chemical composition was measured by X-ray photoelectron spectroscopy (XPS) (Thermo Fisher Scientific Theta Probe, monochromatic Al Kα X-ray source). Constitutive elements were identified from survey scans (0-1000 eV) at a resolution of 1 eV, and atomic concentrations corresponding to the elements were determined from high-resolution scans at a resolution of 0.05 eV. Scans were made at a take-off angle of 90°. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) spectra were obtained using an FTIR spectrometer (Bruker, Vertex 80v) with a ZnSe crystal and an MCT detector cooled with

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liquid nitrogen. Spectra were recorded at a resolution of 4 cm-1 across the 800-4000 cm-1 range and over at least 2000 scans for each sample surface. All FTIR measurements were conducted under vacuum conditions. 2.5

Monitoring of LbL assembly by quartz crystal microbalance (QCM)

In situ QCM measurements were conducted with a Qsense E4 multichannel instrument. Silicon dioxide-coated crystal surface were first coated with a layer of APTMS according to the procedure described in Section 2.3, after which the crystals were inserted into the chamber and sealed with a rubber O-ring. Then DI water was first pumped through the chamber until the frequency reading was stabilized. Subsequently, alternate injection of polyelectrolyte solutions (1 mg mL-1) into the chamber was carried out at a flow rate of 250 mL min-1 for 30 min to 1 h to achieve saturated adsorption, with DI water washing in between to remove loosely adhered polyelectrolytes until the frequency was stabilized. The procedure was repeated until 6.5 bilayers were reached. The frequency change of the crystals was recorded throughout the adsorption process. The adsorbed mass of protein on the sensor was calculated using the Sauerbrey equation: ∆ = −

∆

where C (sensor-specific proportionality constant) = 17.7 ng cm-2 Hz-1 and n = overtone number. 2.6

Protein adsorption and cell adhesion assays

For protein adsorption assays, lysozyme-RH or BSA-FITC was first dissolved in PBS (pH 7.4) to prepare stock solutions at a concentration of 1 mg mL-1 and stored at 4 °C before use. The sample surfaces with dimension 1 cm × 1 cm were incubated in PBS for 30 min, after which they were removed from PBS and covered with 100 µL of 12 µg mL-1 protein solutions diluted from the stock solutions and equilibrated at room temperature. Following 3 h of adsorption in the dark, 50 µL of the protein solution from each of the sample surfaces was withdrawn for absorbance measurement using a microplate reader (Tecan Infinite M200 monochromator). The excitation/emission wavelength of BSA−FITC and LYZ−RH were at 9



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490/520 nm and 544/576 nm, respectively. A standard calibration curve was established to estimate the amount of adsorbed proteins on surfaces by dissolving protein solution in PBS at five concentrations (50, 25, 12.5, 6.25, and 3.125 µg mL-1) from the same stock solution, followed by carrying out the same absorbance measurement procedure as described above. For fibroblast cell adhesion assay, 3T3 fibroblast cells were grown to 80% confluence, trypsinized and washed with PBS, after which they were resuspended in DMEM supplemented with 10% fetal bovine serum (v/v), 1 mM L-glutamine, and 100 IU mL-1 penicillin. Films were cut into 1 cm × 1 cm pieces and placed in a 24-well plate. Next, 1 mL of medium with 3T3 fibroblasts at a concentration of 2 × 105 cells mL-1 was placed in the well with the films and incubated in 5% CO2 at 37 °C for 24 h. Afterwards, the films were carefully rinsed with culture medium three times to remove loosely adhered cells. Cell fixation on the films was conducted with 10% formalin in PBS for 30 min. For cell counting, the cells were stained with DAPI, imaged using a Leica DM IL LED microscope and counted using ImageJ. The reported value represents the average over ten microscopy images taken at different locations for each surface. 2.7

Cytotoxicity assay

The effect of the polymer films on cell viability was tested using the MTT assay according to the standard protocol illustrated in ISO 10993−5 for evaluation of in vitro cytotoxicity by direct contact. 3T3 fibroblast cells were grown to 80% confluence, trypsinized and washed with PBS, after which they were resuspended in DMEM supplemented with 10% fetal bovine serum, 1 mM L-glutamine, and 100 IU mL-1 penicillin. 1 mL of medium with 3T3 fibroblasts at a concentration of 105 cells mL-1 was placed in a 24-well plate and incubated in 5% CO2 at 37 °C for 24 h. The growth of a cell layer in each well was confirmed using a Leica DM IL LED microscope. Afterwards, the medium in each well was replaced with a fresh one. Bare and polymer film coated silicon substrates (1 cm × 1 cm) were gently placed on top of the cell layer in

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each well. Nontoxic control experiments on cell growth without substrates were also carried out. The cells were incubated in 5% CO2 at 37 °C for another 24 h. The medium in each well was then removed and the substrates were gently taken out without disrupting the cell layer. No obvious cell attachment on the substrates was confirmed using a Leica DM IL LED microscope. 900 µL of medium and 100 µL of MTT solution (5 mg mL-1 in PBS) were then added to each well and incubated at 37 °C for 4 h. The medium in each well was then removed and the formazan crystals formed were dissolved in 1 mL of DMSO for 15 min. The optical absorbance of solutions in each well was then measured at 570 nm on a BIO-TEK microplate reader (Model Powerwave XS). 2.8

Statistical analysis

Statistical analysis of the results was conducted by one-way analysis of variance (ANOVA) with Tukey post hoc test. Data with p < 0.05 were reported as statistically significant. Numerical results were presented as mean ± standard deviation (SD). Synergy was tested using multiple regression analysis 3. Results and discussion 3.1

Surface design and fabrication

To construct the matrix of model polymer thin films with independent two-dimensional control over surface charge and wettability, three characteristics are essential: (i) independent control of wettability and surfaces charge should have minimal cross parameter influence; (ii) molecular smoothness of the films should be maintained to exclude surface topological or roughness effects;35,36 (iii) chemical structures with similar compositions should be utilized to avoid drastic alterations in chemical environment when the surface properties are varied. As demonstrated by previous reports,4,7,8 a precise and independent control over surface wettability and charge is still a challenge due to the difficulties in variation of the cross-interacting effects in relation to the 11



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chemical structures of the surface. To address this issue, we propose the use of a LbL fabrication protocol, which allows surface charge modification by varying the polyion charge density in the film buildup process, and surface wettability modification by a small change in the polyion side chain structures. We avoid the drastic alterations in chemical environments by employing materials with minimal structural changes. Confining the fabrication conditions to pH 4-10 (avoiding the extreme pH ranges), we kept the film roughness within the nanoscale range to exclude surface topological (or roughness) effects. Following this approach, eight surfaces characterized by both surface pI and water CA, forming a 2D property matrix as illustrated schematically in Figure 2 were fabricated. The water CAs of these surfaces were ~35°, ~55° and ~70° which were denoted as ‘low CA’ (LCA), ‘medium CA’ (MCA) and ‘high CA’ (HCA), respectively. The surface pIs were at pH ~5, ~6.5 and ~9 which were denoted as ‘low pI’ (LpI), ‘medium pI’ (MpI) and ‘high pI’ (HpI), respectively. The average surface pI and CA were calculated from at least four experiments for each type of surface. The main feature of the 2D matrix is that surfaces in each row or column exhibit a major change in one property without affecting the other property in a significant way. The following sections present detailed characterization of the surfaces and also demonstrate stability of the films through retention of the surface constituents after exposure to PBS (Figure S1, SI). On the basis of these investigations, we can identify the most effective combination of water CA and pI for promoting or inhibiting adhesion of different biological entities e.g. proteins and cells, and compare the individual and combined effects of these two properties.

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Figure 2: Schematic diagram showing the rationale for the surface designs developed in this work. Three polyanionic building blocks were synthesized using the same PIAMAn precursor and the anhydride groups present in the polymer backbone as grafting points.37,38 Grafted side chains of 6-aminocaproic acid (NH2-(CH2)5-COOH) or amine-PEG-carboxylic acid (NH2-PEG-COOH) were used to form polymers with enhanced hydrophilic (PIAMA-PEG) or hydrophobic (PIAMA-C5) characteristics, respectively (Figure 1). All side chain modifying agents were terminated with COOH groups to maintain the constant number of carboxylates across the three polyanions. This feature limits influence of side chain variation on the total charge density of the polymers, which is essential for surface charge modifications. Adding charged groups to the grafted side chains also facilitates the LbL assembly process, particularly for PIAMA-PEG as the grafting of PEG chains may pose steric hindrance to LbL film growth.29

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The choice of 6-aminocaproic acid containing a pentyl group to impart hydrophobicity to the polyanion was motivated by the solubility of the polymer in an aqueous solution. The selection of 45 ethylene oxide units for the PEG side chain was influenced by sufficiently strong hydration reported for this group.39,40 Evidence for the successful synthesis of the desired polyanions was provided by the complementary use of FTIR and 1H-NMR techniques. From the IR spectrum in Figure S2, 100% conversion of the poly(maleic anhydride) to its poly(maleic acid) form was confirmed by the disappearance of the peaks at 1860 cm-1 and 1780 cm-1 (anhydride groups) and the appearance of the peak at 1550-1560 cm-1 (COO-) in all three synthesized polymers. The incorporation of the PEG units was confirmed by the increasing absorbance at 1000-1200 cm-1 (-C-O-C- of PEG). A strong peak between 3.50 and 3.80 ppm (methylene hydrogen, PEG) and a peak at 3.21 ppm (pentyl-amide hydrogen) in the 1H-NMR spectra of PIAMA-PEG and PIAMA-C5 spectra, respectively (Figure S2) also confirmed the successful incorporation of PEG and C5 moieties. To estimate the degree of substitution by PEG and C5 groups, the integral areas of the peaks from protons of these groups were compared with the areas from the protons of the dimethyl groups of the main chain (peaks at 0.9 ppm). The degree of substitution was estimated to be 10 % for PIAMA-PEG and 16 % for PIAMA-C5 side chains. The synthesized polyanions have the same elemental constituents, but the grafting of side chains could elicit different molecular architectures, which may further affect the LbL film architecture. Thus, the degree of substitution was kept low to reduce such effects. Branched PEI was selected as a typical polycationic building block and was used without further modification in the LbL assembly to pair with the respective polyanions (Figure S3). To achieve desirable chemical architectures of surfaces, the LbL protocol was applied using the following polyelectrolyte pairs namely: PIAMA-PEG/PEI, PIAMA-C5/PEI and PIAMAc/PEI as schematically illustrated in Figure 3. To modulate surface charge, we altered the deposited amount of polyanion and polycation, by manipulating polymer ionization degrees through adjusting the assembly pH. The use of low 14


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pH solution, during polyanion deposition, leads to larger mass of polymers attached, compared to the high pH solution. The opposite rule guides polycation deposition.3,41,42 To modulate surface wettability, default polyacid (PIAMAc) was modified with polar (PIAMA-PEG) and non-polar (PIAMA-C5) components. Thus, a layer of polyanions brought dual functionality: generating negative charge and increasing predominance of hydrophilicity/hydrophobicity associated with the polyanion side chain structures. The control over chemical structures and the deposited amount of polyanion allowed us to fine tune the proportion of ionic and nonionic functional groups on the surface. To do so, we deposited 6.5 or 7 bilayers of the polyelectrolyte pairs PIAMA-PEG/PEI, PIAMA-C5/PEI and PIAMAc/PEI respectively, in low pH or high pH solutions as summarized in Table 1. The resulting films have tunable surface pI from pH ~5 to ~9 and tunable wettability (water CA) from ~35° to ~70°.

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Figure 3: The surface modification concept and protocol.

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Table 1: Fabrication conditions and surface properties of the LbL systems.

a

Sample

LbL(LpI/LCA) LbL(LpI/MCA) LbL(LpI/HCA) LbL(MpI/LCA) LbL(MpI/MCA) LbL(MpI/HCA)

LbL(HpI/LCA)

LbL(HpI/HCA)

Fabrication conditionb

(PIAMA-PEG,7 /PEI, 7)6.5 (PIAMAc, 6/PEI, 6)6.5 (PIAMA-C5, 6/PEI, 4)6.5 (PIAMA-PEG, 10/PEI, 10)6.5 (PIAMAc, 7/PEI, 7)6.5 (PIAMA-C5, 7/PEI, 7)6.5 (PIAMA-C5, 10/PEI, 10)6.5/(PEI, 10) (PIAMA-C5, 8/PEI, 8)6.5/(PEI, 10)

pH range and ionization of the polymer PIAMAXc

PEI

6-7: Low to medium charge density

4-7: High to medium charge density

7-10: Medium to high charge density

8-10: High charge density

Surface properties Thickness (nm)e

Water CA(°)

Rq (nm)d

5.20±0.57

32.4±1.7

0.77±0.03

47.9±5.4

5.30±0.71

57.1±2.2

0.70±0.03

32.3±5.5

4.95±0.21

67.8±3.0

4.00±0.05

39.8±5.3

7-10: Medium to low charge density

6.55±0.07

39.9±1.2

1.10±0.03

30.3±2.0

7.00±0.28

55.8±1.1

0.80±0.03

35.0±5.7

6.45±0.07

69.3±4.5

0.97±0.04

18.0±1.3

8-10: Low charge density

9.15±0.49

38.3±2.9

0.77±0.03

9.0±5.6

9.45±0.07

72.1±2.9

0.80±0.05

39.0±3.7

pI

a

in the sample name, each surface is labeled by both properties (e.g. LbL(LpI/LCA) indicates the surface with low pI and low CA)

b

in the notation (polyanion, x/PEI, x)n/(PEI, x), x refers to the deposition pH and n the number of bilayers

c

PIAMA-X denotes all three polyanions PIAMA-PEG, PIAMAc and PIAMA-C5

d

RMS roughness (Rq) measured by AFM

e

film thickness measured by the AFM scratch method

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Figure 4: Properties of films. Top: CA of surfaces characterized by: (A) low pI; (B) medium pI; (C) high pI. Bottom: representative surface ζ potential as a function of pH. The average value was calculated based on four experimental runs for each data point. In the sample name notation, each surface was labeled by both properties (e.g. LbL(LpI/LCA) indicates the surface with low pI and low CA). The pH at which PEI is 50% ionized (defined as ‘pKa’ in this study) is reported to be ~543, ~644, and 7.445. The reported pKa value for polymaleic acid is ~646, and the value for poly(isobutylene-alt-maleic acid) is 5-7.547. These reported pKa values were used to guide the pH selection as shown in Table 1. It should be noted that weak polyelectrolytes’ pKa may be a complex function of polymer charge density,48,49 therefore, the exact pH values used for the film fabrication were selected from a preliminary screening. As shown in Table 1, to fine tune surface wettability and to maintain low pI (negative surface charge), we deposited 6.5 bilayers of polyanions (PIAMA-C5, PIAMAc or PIAMA-PEG) with low charge density (low ionization) paired with PEI with high charge density. While employing polyanions as the terminating layer, polar (PEG) or non-polar (alkyl) side chain structures were exposed at the surface, manifesting variation in 18


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wettability from ~35° to ~70° (Figure 4A). To fabricate films with tunable wettability and medium pI, we increased the deposition pH for each polyelectrolyte pair. This was motivated by previously reported behaviors of weak polyelectrolytes and changes in their ionization degree with pH41,42,48 as illustrated in Figure 3. To achieve variation in wettability and to maintain high pI, we increased the deposition pH further and terminated the film with PEI at pH 10 (low ionization). The extra layer of PEI brought dual effect, increasing both surface potential and hydrophilicity. Taking advantage of these effects, we fabricated LbL(HpI/LCA) using the PIAMA-C5 (high charge density) and PEI (low charge density) as the terminating layer. However, the intrinsic hydrophilicity of PEI chains made control over the wettability parameter more challenging since the terminating layer had a stronger impact on the overall surface properties.42,50 Nonetheless, lower wettability at CA ~70° was still attainable by keeping a relatively high concentration of PIAMA-C5 in the bulk, accomplished by depositing PIAMA-C5 layers at pH 8. The composition of the films was verified by XPS and ATR-FTIR (Section 4, SI). In summary, the method presented herein allowed us to fabricate LbL films with surface charge and wettability distributed across pI from ~5 to ~9 and water CA from ~35° to ~70° in the form of a 2D property matrix with values falling under each of eight regions as illustrated in Figure 2. The 2D matrix exhibits a major change in one property without affecting the other property in a significant way. This allows a comparison of the independent and cooperative effects of surface charge and wettability. 3.2

Surface characterization

In addition to water CA measurements, surface free energy was investigated to assess the balance of polar

(γ ) and dispersive (γ ) components51 across the whole matrix of samples. As expected, surface wettability correlates well with changes in the polar component of the surface free energy (Table S2, SI). Surface roughness is also an important parameter for consideration. The root-mean-square roughness (Rq)

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and morphologies of the LbL films were assessed by AFM and shown in Table 1 and Figure 5 respectively. The as-synthesized LbL films showed only nanoscale roughness. Rq of all the films was less than 1 nm across a 5 µm × 5 µm area except for the LbL(LpI/HCA) surface with a Rq of 4 nm. The low roughness of the PIAMA-PEG/PEI and PIAMAc/PEI systems can be linked to the relatively high hydrophilic nature of both polyelectrolytes used which translated into high solubility in the depositing medium.41,52 The slightly higher roughness observed for LbL(LpI/HCA) built from PIAMA-C5 and PEI and assembled at a low pH was likely caused by the higher polymer hydrophobicity and increased short-range interaction between hydrophobic side chains,53,54 which may result in small islands distributed on a smooth underlying film (Panel 6, Figure 5). The nanoscale roughness of all the films was maintained and hence, negligible differences on adhesion of biological entities can be expected.55 This was achieved by confining the assembly pH between the values of 4 and 10. Exceeding this range led to significantly increased film roughness (Table S3, SI), which was not compliant with the design requirements. The corresponding 3D morphology images and height histograms were included in SI (Figure S6). The dry thickness of the films was assessed by the AFM scratch method and shown in Table 1. Most of the films had a similar thickness (~30-40 nm), with the exception of LbL(MpI/HCA) and LbL(HpI/LCA) which comprise PIAMA-C5/PEI couples with a decreased thickness in the range of 10 nm and 20 nm. The lower thickness can be attributed to the reduced amount of PIAMA-C5, highly ionized at pH 7-10, and as a consequence deposited in lower volume. The results confirmed that the dry thickness of the films was also kept within a relatively narrow range, which would not interfere with the subsequent protein adsorption or cell adhesion results as demonstrated in a previous report.3

20


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Figure 5: Representative AFM topography images of different LbL films: (1) LbL(HpI/HCA); (2) LbL(HpI/LCA); (3) LbL(MpI/HCA); (4) LbL(MpI/MCA); (5) LbL(MpI/LCA); (6) LbL(LpI/HCA); (7) LbL(LpI/MCA); (8) LbL(LpI/LCA). The scan area for all images is 5 µm × 5 µm. The grafting of side chains may affect LbL film growth due to steric interactions29 and/or secondary interactions such as hydrophobic interactions and hydrogen bonding.56,57 Thus, to compare the LbL growth mechanism for the PIAMA-PEG and PIAMA-C5 films, QCM experiments were conducted for two typical pH scenarios (pH 10/10 for PIAMA-PEG/PEI, pH 7/7 for PIAMA-C5/PEI) at which the same surface pI was obtained for both systems. The assembly of polymers during each adsorption step on APTES coated sensor chips was recorded as frequency changes of QCM signals in Figure 6. The results show that for both systems, the adsorption of layers generally conformed to a stepwise decrease in frequency typical for LbL assembly, thus the grafting of side chains did not hinder the film growth. For PIAMA-C5/PEI films, a slight 21



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increase in frequency appeared after passage of the polycationic PEI solution whereas for PIAMA-PEG/PEI films, the increase in frequency occurred upon DI water rinse and passage of the PEI layer. The increase in frequency upon DI water rinse can be attributed to the removal of loosely attached PIAMA-PEG chains on the surface. The increase in frequency upon passage of PEI solution was indicative of some dissolution of surface polyelectrolytes into solution for both systems. Previous studies suggest that for the assembly of LbL films based on electrostatic attraction, the charge density of the species in the solution and on the interface must be sufficient, otherwise desorption of the adsorbed species may occur upon adsorption of an incoming layer.58,59 Such desorption of surface polyelectrolytes may account for the observed increase in frequency in these two systems.

Figure 6: In situ frequency changes monitored by QCM for (A) PIAMA-C5/PEI films assembled at pH 7/7; (B) PIAMA-PEG/PEI films assembled at pH 10/10. The assembly of 6.5 bilayers was monitored for both films During adsorption of mass sensed by QCM, a variation in surface hydrophilicity can result in significant changes in observed frequency. Hydrophilic surfaces can entrap solvent molecules contributing to a hydrodynamic mass (adsorbed polyelectrolyte and trapped water molecules) increase. On the other hand, hydrophobic surfaces can entrap air in the cavities, resulting in a decrease of the measured mass.60 The hydrodynamic mass of the PIAMA-PEG and PIAMA-C5 layers in the films is 920.4 ng cm-2 and 22


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88.5 ng cm-2 respectively (calculated from the 5th bilayer to exclude the effect of the substrate). The greater hydrodynamic mass increase for the PIAMA-PEG adsorption than PIAMA-C5 remains consistent with the typically variation observed between polyions with different degrees of hydrophilicity.61 3.3

Evaluation of surface interactions with model biomolecules and cells

The fabricated LbL films library was evaluated in bio-relevant adhesion tests using proteins and cells. Particular attention was devoted to interplay between surface charge and wettability that influences protein adsorption and cell adhesion in an independent or cooperative manner. In this way, we featured a more complex but encompassing system which simulates a more realistic situation that often occurs in biologically and technologically relevant structures, but has not previously been analyzed systematically. Two fluorescent-labeled proteins, BSA as an anionic (pI = 4.8), large (Mw = 66 kDa) and soft62 protein and LYZ as a cationic (pI = 11.1), small (Mw = 14.3 kDa) and rigid63 protein, were used in adsorption studies with the current matrix of model surfaces. Proteins were deposited from PBS buffer with an ionic strength of 162.7 mM and pH 7.4 which simulates typical physiological conditions relevant to biological applications. The BSA adsorption data (Figure 7A, Table S6) shows that with increase in surface hydrophobicity, BSA adsorption was enhanced on both negatively (pI ≤ 6.5) and positively charged (pI ~ 9) films. Moreover, across the whole contact angle range, positively charged surfaces were more favorable for BSA adsorption compared to the negatively charged ones. This is consistent with previous reports and can be linked to the role of electrostatic interaction in BSA adsorption.2,64,65 These results show that both surface charge and wettability had an effect on BSA adsorption. BSA adsorption was not responsive to CA and pI changes in the region from ~55° to ~35° for CA and from 5 to 6.5 for pI (four cyan bars with a similar low adsorption). The highest BSA adsorption was observed on the surface which was both hydrophobic and positively charged (blue bar) suggesting cooperation between variables. To investigate possible synergistic

23



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effect, data was analyzed using multiple regression approach (Tables S4 and S5). The obtained polynomial equation exhibited positive coefficient (CA*pI). However, its value did not reach significant level. We may conclude, there is no significant synergistic effect between contact angle and surface charge on adsorption of BSA in the investigated range. Examination of the results of LYZ adsorption (Figure 7B) revealed that the overall adsorption of LYZ remained at a relatively high level across the whole matrix of investigated variables. A substantially higher adsorption was observed for the cluster of four hydrophobic surfaces (CA ≥ 55°) with negative charge (pI ≤ 6.5) (blue and grey bars). On the other hand, on hydrophilic surfaces (CA ~ 35°), its adsorption was not responsive to surface charge; similarly, on positively charged surfaces (pI ~ 9), its adsorption was not responsive to surface wettability (four cyan bars).

Figure 7: Protein adsorption on different LbL films: (A) BSA; (B) LYZ. Highest and lowest adsorption was represented by blue and cyan respectively. Blue: significantly different compared to the bars in the same row and column (one-way ANOVA, p < 0.05); Cyan: not significantly different within the group (one-way ANOVA, p > 0.05). For both proteins, we observed certain thresholds of surface hydrophobicity and surface charge necessary to affect their adsorption. Importantly, the manipulation of a single surface parameter within this envelope 24


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does not affect the adsorption process. Such thresholds have been reported for wettability values for generating hydrophobic effects to increase protein adsorption66 or charge loading for retaining proteins in a flow cell.67 In particular, BSA showed greater responsiveness to surface variables than LYZ. This difference is likely a result of the size, shape, packing and conformational arrangement upon adsorption.68 BSA is a relatively soft protein with structural flexibility, which can give rise to structural changes favoring adsorption onto a surface.10,62,69,70 LZY is less responsive to surface charge or wettability due to its rigidity.63 Therefore, our data show that protein structural characteristics can override certain physicochemical aspects of surfaces, which might contribute to the insignificant effects of single parameter manipulation. Mouse 3T3 fibroblast cells, cultured for 24 h, used in a variety of biological studies, were employed to evaluate cell adhesion on the model surfaces (Figure 8). The general trends in the cell attachment revealed that the highest number of attached cells was present on substrates with high pI values. The observed effect is in agreement with previous reports describing a direct correlation between cell adhesion and surface ζ potential.4,71 The driving force for such behavior might be the electrostatic attraction between positively charged surface and the cell surface/serum protein surface which are predominantly negatively charged.71 Interestingly however, for both positively and negatively charged surfaces, more cells attached to the relatively hydrophilic surfaces (CA ≤ 55°) than the hydrophobic ones (~70°). Due to the expected antifouling effect of the hydration layer, this observation is not typical. Because the cell adhesion experiments were performed using culture media containing 10% serum, the amount of cells adhered may be dictated to some extent by adsorbed serum proteins, for example, fibronectin (pI = 5.7) and vitronectin (pI = 5.4) which are predominantly negatively charged under the present experimental conditions.72 BSA adsorption results reported in the previous section showed that the protein adsorbed more on the hydrophobic films than on the hydrophilic ones. Thus, it is likely that the observed cell adhesion with respect to surface wettability may be a consequence of proteins adsorbed in different states (e.g. 25



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conformation and orientation) that direct cell interaction.73 For example, serum proteins may adsorb onto moderately hydrophilic surfaces in a conformation which favors cell adhesion compared to a denatured and rigid state on hydrophobic surfaces.7 The combined effects of positive charge (pI ~9) and hydrophilicity (CA ~35°) generated the greatest amount of cell attachment whereas negative charge (pI ~ 5) and hydrophobicity (CA ~70°) led to the lowest amount of cell attachment.

Figure 8: Fluorescence microscopy images of cell adhesion on different LbL films and the summary of cell adhesion results. Scale bar is 100 µm. Highest and lowest degree of cell adhesion was represented by blue and cyan bars respectively. Blue & cyan: significantly different compared to the bars in the same row and column (one-way ANOVA, p < 0.05). Moving along the pI axis, adhesion increased continuously from LpI to HpI for HCA and LCA cases at a statistically significant level; moving along the CA axis, adhesion increased from HCA to MCA/LCA for all pI cases at a statistically significant level. The same cell line (3T3) was used in the cell viability MTT assay to understand the effects of film 26


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structures on cell growth (Figure S7, SI). On surfaces with high pI, cell viability was lower which reflects the cytotoxicity of polycation.74 On surfaces with high wettability, cell viability was higher, and consistent with previous reports demonstrating that the grafting of PEG reduced the cytotoxicity of polycation.27,29 Overall, the combination of negative charge and high wettability yielded the highest cell viability. Taking into account that negative charge is unfavorable for cell adhesion, the implication of these results is that surfaces with an optimal combination of positive charge (that promotes cell adhesion but can be cytotoxic) and wettability (for reducing cytotoxicity) needs to be identified for enhancing cell adhesion. The above mentioned results on interaction of the surfaces with BSA, LYZ, and 3T3 fibroblast cells highlight the presence of a complex interplay that exists between the surface variables in influencing the interaction. For proteins, the individual effects of both surface charge and wettability were evident. In general, electrostatic attraction and surface hydrophobicity were more favorable for protein adsorption. However, we observed the presence of a low adhesion region where there was little or no effect upon enlarging either electrostatic attraction or hydrophobicity within the time frame and surface property range examined here. For fibroblast, both surface charge and wettability had an effect on its adhesion. The combined effects of positive charge and hydrophilicity led to the highest cell adhesion whereas negative charge and hydrophobicity led to the lowest cell adhesion. 4. Conclusion In this report, we exploited the LbL technique to create surfaces with independent control over surface ζ potential and surface wettability for investigation of the interplay between these two surface variables in influencing biological entity-interface interaction. Contrary to the conventional approaches which can lead to simultaneous variation in multiple surface properties, our protocol featured two-dimensional control over both surface charge and wettability. Thus, their effects on interfacial interaction can be compared. PIAMAn

27



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were grafted with PEG or hydrocarbon chains and were used as the polyanionic building blocks with PEI to fabricate LbL films. We found a set of guiding principles to fabricate films that allowed the control over surface charge and surface wettability in the range of 5-9 for surface pI and 35°-70° for surface water CA. The films were used to study a spectrum of biological entity-interface interactions and the results revealed the complex interplay that exists between the two surface variables in influencing the degree of interaction. Their individual, combined and synergistic effects on protein adsorption and cell adhesion could be systematically evaluated using presented protocol. The design strategy and results provided new insights into the understanding and manipulation of multi-surface variables to direct protein and cell interaction with interfaces that are not easily achievable by other methods. The protocol also featured a model for predicting protein adsorption and cell adhesion. Associated Content Supporting Information Film stability (Figure S1); polyanion characterization (Figure S2); chemical structures of film constituents (Figure S3); relative atomic percentage of surface carbon bonds present on LBL films (Table S1); XPS C-1c core level and ATR-FTIR spectra of the films (Figures S4 and S5); methylene iodide contact angle of surfaces and surface free energy (Table S2); effects of low assembly pH in LbL fabrication using the PIAMA-C5/PEI pair (Table S3); AFM 3D morphology and height histograms (Figure S6); assessment of synergistic influence of wettability and surface charge on BSA adhesion (Tables S4 and S5); MTT assays for assessing cell viability (Figure S7) and tabularized results for protein adsorption, cell adhesion and MTT assay (Table S6).

28


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Author Information Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interest Acknowledgment The authors are grateful to the Agency for Science, Technology, and Research (A*STAR) for providing financial support and the A*STAR Graduate Academy for the PhD scholarship of Shanshan Guo. References (1) Sze, A.; Erickson, D.; Ren, L. Q.; Li, D. Q. Zeta-Potential Measurement using the Smoluchowski Equation and the Slope of the Current-Time Relationship in Electroosmotic Flow. J. Colloid Interface Sci. 2003, 261, 402-410. (2) Guo, S. S.; Janczewski, D.; Zhu, X. Y.; Quintana, R.; He, T.; Neoh, K. G. Surface Charge Control for Zwitterionic Polymer Brushes: Tailoring Surface Properties to Antifouling Applications. J. Colloid Interface Sci. 2015, 452, 43-53. (3) Zhu, X. Y.; Janczewski, D.; Guo, S. F.; Lee, S. S. C.; Velandia, F. J. P.; Teo, S. L. M.; He, T.; Puniredd, S. R.; Vancso, G. J. Polyion Multilayers with Precise Surface Charge Control for Antifouling. ACS Appl. Mater. Interfaces 2015, 7, 852-861. (4) Chang, H. Y.; Huang, C. C.; Lin, K. Y.; Kao, W. L.; Liao, H. Y.; You, Y. W.; Lin, J. H.; Kuo, Y. T.; Kuo, D. Y.; Shyue, J. J. Effect of Surface Potential on NIH3T3 Cell Adhesion and Proliferation. J. Phys. Chem. C 2014, 118, 14464-14470. (5) Andrade, J. D.; Hlady, V. Protein Adsorption and Materials Biocompatibility - a Tutorial Review and Suggested Hypothesis. Adv. Polym. Sci. 1986, 79, 1-63. 29



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Parallel Control over Surface Charge and ... - ACS Publications

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