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Engineered, robust polyelectrolyte multilayers by precise control of surface potential for designer protein, cell and bacteria adsorption Xiaoying Zhu, Shifeng Guo, Tao He, Shan Jiang, Dominik Ja#czewski, and G. Julius Vancso Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04118 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 18, 2016
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Engineered, robust polyelectrolyte multilayers by precise control of surface potential for designer protein, cell and bacteria adsorption
Xiaoying Zhu†˭*, Shifeng Guo†˭, Tao He†, Shan Jiang†, Dominik Jańczewski§*, G. Julius Vancso‡*
†
Institute of Materials Research and Engineering A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, Innovis, #08-03, Singapore, 138634
§
Laboratory of Technological Processes, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
‡
MESA+ Institute for Nanotechnology, Materials Science and Technology of Polymers, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
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ABSTRACT Cross-linked layer-by-layer (LbL) assemblies with precisely tuned surface ζ potential were fabricated to control the adsorption of proteins, mammalian cells and bacteria for different biomedical applications. Two weak polyions including a synthesized polyanion and polyethylenimine (PEI) were assembled under controlled conditions and cross-linked to prepare three robust LbL films as model surfaces with similar roughness and water affinity but displaying negative, zero and positive net charges at the physiological pH (7.4). These surfaces were tested for their abilities to adsorb proteins including Bovine Serum Albumin (BSA) and Lysozyme (LYZ). In adsorption tests, the LbL films bind more proteins with opposite charges but less with like charges, indicating that electrostatic interactions play a major role in protein adsorption. However, LYZ showed higher non-specific adsorption than BSA because of the specific behavior of LYZ molecules such as stacked multilayer formation during adsorption. To exclude such stacking effects from experiments, protein molecules were covalently immobilized on AFM colloidal probes to measure the adhesion forces against the model surfaces utilizing direct protein molecule-surface contacts. The results confirmed the dominating role of electrostatic forces in protein adhesion. In fibroblast cell and bacteria adhesion tests, similar trends (high adhesion on positively charged surfaces, but much lower on neutral and negatively charged surfaces) were observed because the fibroblast cell and bacterial surfaces studied possess negative potentials. The cross-linked LbL films with improved stability and engineered surface charge described in this study provide an excellent platform to control the behavior of different charged objects and can be utilized in practical biomedical applications.
KEYWORDS: surface charge, polyelectrolyte, layer-by-layer assembly, composite multilayers, protein, mammalian cell, bacteria
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1. Introduction Smart, bioactive surfaces fabricated from various materials and responding to target biological objects such as nucleic acids, proteins, polysaccharides, viruses, bacteria or living cells, have been subject of great interest due to a swift growth e.g. in biomedical materials and applications.1, 2 The adsorption to target substrates can be increased by enhancing surface-object interactions, while preventing adsorption is targeted for antifouling surfaces. For example, in sensitive protein analyses, a strong adsorption ability of the surface is needed to enhance the concentration of target proteins from highly diluted solutions.3,
4
Strong protein adsorption on solid surfaces is also desired in protein
purification processes.4, 5 As another example, bacteria enrichment on solid surfaces is an alternative approach for the retrieval of novel types of bacteria from complex natural communities for subsequent assessment.6 Bacterial pathogens were coordinated with surface engineered magnetic nanoparticles and subsequently such assemblies were concentrated and removed by an applied magnetic field.7 In biomedical implants, enhancement of the cell adhesion is important for tissue development during the process of repairing and regenerating damaged tissue.8 The opposite problem to adhesion enhancement is to minimize the interactions between solid surfaces and biological substances (proteins and cells) by fabricating low adhesion, antifouling, or bioinert surfaces. The undesired accumulation of biological matters and growth of microorganisms on surfaces, immersed in an aquatic environment, i.e. during marine biofouling,9 is a rapidly growing field of interest due to the legislative ban of biocides used hitherto to control surface fouling of ships and other underwater structures. Fouling is a recognized problem for biomedical applications,10 water treatment proceses,11 and in the maritime industry.12 Obviously, low adhesion strength is desirable for antifouling surfaces.
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The interactions of solid surfaces with biological objects are dramatically affected by the properties of the surface.9, 13, 14 Micro-topography (or morphology),15 roughness,16, 17 wettability18, 19 and surface charge20, 21 are parameters that need to be controlled to fabricate high or low adhesive materials for different needs. Since most of the proteins and cells are charged entities, electrostatic interactions play an important role in their adhesion onto solid surfaces, particularly during the initial stage of contact.22, 23 Several methods have been proposed to control the charge of a surface and influence the adsorption of proteins and cells, and thus to control this adhesion process. For example, surfaces were treated with oxygen plasma24,
25
or strong oxidants26 to become covered with ionic functional groups for adhesion
enhancement. The ζ potential of the treated surfaces in such cases usually becomes more negative because of oxidation. However these surface engineering methods can only render surfaces to feature more negative charge, which makes fine tuning of surface potentials difficult. Progress in surface engineering, however, may provide alternatives. For example, the surface charge of self-assembled monolayers (SAMs) can be adjusted by mixing alkane thiols terminated with positively or negatively charged functional groups in different ratios.20, 27, 28 But in SAM systems it is difficult to isolate electrostatic contributions from other effects related to variations of the head group chemical structure, when different SAMs are compared. Thus it is notoriously difficult to independently identify electrostatic contributions to adhesion without varying other parameters of the surface. As an alternative to SAMs, polymer brushes were also used to control electrostatic charge by polymerizing mixtures of cationic and anionic monomers.29,
30, 31
Oppositely charged monomers in
different ratios were reacted to grow polymer brushes from polypropylene surface to prepare grafts with and without net charge to control the interaction with proteins and bacteria.23 However these brushrelated surface engineering approaches are difficult to scale up, which limits their potential applications.
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Oppositely charged polyelectrolytes can be sequentially assembled in a layer-by-layer (LbL) fashion to form multilayer thin films.32 Surfaces obtained by LbL assembly were used to prevent, or promote, protein adsorption through electrostatic interactions because of the overcompensated surface charge of such layers.33 In one of our recent studies, LbL assembly was explored as a surface modification method to provide substrates with well-controlled surface ζ potential.34 In this initial proof of concept work, commercially available polyelectrolytes were assembled but the problem of the long term stability of the LbL films was not addressed. Such films with well controlled surface potential may have broad applications in the biomedical area, however their stability is a key factor to achieve and maintain the desired performance in practical scenarios. Thus in this study we addressed this issue. Here, precise ζ potential control over the robust LbL film surface, constructed with a cross-linkable polyanionic ester35 and polyethylenimine (PEI), was achieved to benefit various biomedical applications that require a stable polymeric interphase. The three model cross-linked LbL surfaces with positive, negative and zero ζ potentials at physiological pH = 7.4 were fabricated. All three display only very slight variations of properties, like roughness and water contact angle. The macroscopic adsorption of charged biological substances, such as proteins, cells and bacteria, were studied using those films. Finally, the adhesion force between protein molecules and the model surfaces were investigated using the AFM force spectroscopy approach at the nanoscale. 2. Experimental section 2.1
Materials The cross-linkable polyanion (P1, Mw: 84 kDa) partly bearing methyl ester groups (8% of repeating
units) in the main chain was synthesized following the previously published protocol.35 Polyethylenimine (PEI, Mw: 25 kDa, branched) and (3-Aminopropyl)trimethoxysilane (APTMS, 97%) were provided by Sigma Aldrich. Solvents including toluene, methanol and ethanol were purchased 5 ACS Paragon Plus Environment
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from Tedia (USA). Bovine Serum Albumin (BSA, MW 66 kDa) lyophilized powder, ≥ 96%, BSA fluorescein isothiocyanate conjugate (BSA-FITC) and Lysozyme (LYZ, MW 14.3 kDa) were purchased from Sigma-Aldrich. Lysozyme conjugated with Rhodamine (LYZ-RH) was provided by Nanocs (USA). Phosphate-buffered saline (PBS) was provided by GIBCO (ThermoFisher scientific). Silicon wafers were obtained from Latech Scientific Supply Pte. Ltd (Singapore). Ultrapure water produced by a Millipore Milli-Q integral water purification system was used to prepare aqueous solutions. A triple P plasma processor (Duratek, Taiwan) was used to clean the silicon wafers. 2.2
Assembly of the LbL films Silicon wafers were cut into 2 cm × 2 cm slides using a DISCO dicing machine (DAD 321). After
ultrasonic cleaning with water and ethanol for 10 min, the slides were dried over a nitrogen gas stream and treated with oxygen plasma (200 W) for 2 min. The treated silicon wafers were immersed into 3-aminopropyltrimethoxysilane toluene solutions (10 mM) for 5 h to impart positively charged amine groups on the silicon surface. The pretreated silicon wafer slides were immersed into aqueous polyanion (P1) solutions (1 mg/mL) for 10 min and then rinsed with ultrapure water for 2 min. Subsequently, slides were immersed into polycation (PEI) aqueous solutions (1 mg/mL) for 10 min, followed by another 2 min ultrapure water rinse. The cycle was repeated until the desired number of layers was reached. The pH values of the polyelectrolyte solutions were well controlled. The silicon wafers with the deposited LbL films were dried by nitrogen stream and later under vacuum at room temperature for 5 h. The cross-linking process was conducted by heating the silicon wafers with the dried LbL films to 60 °C for 5 h under vacuum. The cross-linked LbL films were stored in a desiccator prior to further use. Since the stability of the cross-linked LbL films was previously documentedn, the storage time was not included in this work as a testing parameter.35 6 ACS Paragon Plus Environment
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Characterization of the LbL assemblies Surface morphology and thickness of the deposited LbL films were measured by the JPK (Germany),
NanoWizard® 3 NanoOptics Atomic Force Microscope (AFM) system in the “AC mode” (tapping mode). Tap300AI-G cantilevers, made by Budget Sensors (Bulgaria) were used. AFM images were recorded using dried films over scan areas of 2 µm × 2 µm for morphology observations and roughness measurements. The film thickness was measured by scratching the multilayer assembly with a fresh razor blade to expose the bare substrate (silicon) and then scanning the sample by AFM over 10 µm × 10 µm to reveal a clear step obtained by the scratch.36 Five sections crossing the step of a single scratch were used to measure the film height. The mean value of the height obtained was used as the film thickness value. AFM raw data were processed by the software of the instrument provider (JPK Data Processing, 4.3.25). A goniometer (250-F1) from Ramé-Hart Instrument Co. (USA) utilizing the static sessile drop method was applied for contact angle measurements using water on the LbL films. A 5 µL droplet of water was placed onto the dry sample surface by the microsyringe of the device. The liquid droplet image was captured and analyzed by the instrument to obtain the contact angle value of the tested surface. For each sample, ten measurements of liquid contact angles at different locations on the LbL film surface were made, and the average value of the measurements was used as a representative water contact angle of the tested LbL film. ζ potentials of flat solid surfaces were measured with a SurPASS electrokinetic analyzer from Anton Paar (Austria). Silicon wafers carrying the LbL films were cut into 1 cm × 2 cm slides. Two slides were attached to the sample holders of the electrokinetic analyzer, which were inserted into the adjustable gap cell of SurPASS. Upon adjusting the gap height between the slides to 100 µm, the ζ potential measurements were carried out in 0.001 M KCl aqueous solution by adding 0.05 M NaOH solution and
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using auto pH titration in the range 5.5 to 10. Since the slides were smooth with known surface area, the streaming current mode was used.37 2.4
Protein adsorption tests The specimen surfaces were cut into 1 cm × 1 cm for the protein adsorption assays. Fluorescent
labelled BSA-FITC and LYZ-RH were dissolved in PBS to prepare stock solutions with a concentration of 1000 µg/mL. Subsequently, the stock solution was diluted to 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL and 3.125 µg/mL using PBS. 100 µL of the 12.5 µg/mL standard solution was dropped onto each 1 cm × 1 cm sample surface and spread to completely cover the substrate. After incubation for 2 h in dark, 50 µL of protein solution from each of the sample surfaces was transferred into a well of a Greiner 96 Flat Bottom Black Polystyrol plate and mixed with 50 µL of PBS. Similarly, the standard solutions (50 µL) were also transferred and mixed with PBS (50 µL). The fluorescence emission intensity of each well in the plate was measured by a microplate reader (Infinite® M200 from Tecan). The excitation/emission wavelength of BSA-FITC and LYZ-RH were at 490/520 nm and at 544/576 nm, respectively. The calibration curve was retrieved from the fluorescence intensities and concentrations of the standard solutions. The quantity of adsorbed protein by the sample surface was calculated from its fluorescence intensity. 2.5
Protein adhesion force measurements by AFM A JPK (Germany), NanoWizard® 3 NanoOptics AFM system was used to measure the adhesion
force between protein and surface. Colloidal probes with SiO2 sphere (diameter of 1 ± 0.06 µm; Novascan Technologies, Inc., USA) were employed. The proteins including BSA and LYZ were covalently immobilized onto the colloidal probes. The probe modification process was performed as described previously.38 In short, the probes were vapor deposited with 3-aminopropyltrimethoxysilane after plasma cleaning. Then, the introduced amine groups from the probes were reacted with 8 ACS Paragon Plus Environment
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glutaraldehyde which served as the binding agent to covalently immobilize proteins. Subsequently, the treated colloidal probes were immersed in protein solutions and finally rinsed with PBS to complete the protein immobilization. Before and after the experiments the spring constant of the cantilevers were calibrated using the thermal noise method (0.04 - 0.1 N/m).39 All force curve measurements between protein modified probes and surfaces were performed in liquid conditions (PBS), using a fluid cell and allowing the system to equilibrate for 20 min before measurements. All force measurements were conducted using the force mapping imaging mode (as defined by JPK) at a vertical scan rate of 0.5 Hz.38 The ‘setpoint’ of the loading force was kept constant for the measurements, and the force data were collected in a 10 x 10 array of force curves over a scan area of 10 µm x 10 µm. AFM data were analyzed by the software JPK SPM Data Processing (v. 4.3.25) from JPK. 2.6
Cell adhesion tests A human fibroblast cell line (CCD-112CoN) from American Type Culture Collection, ATCC (USA)
was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C under 5% CO2 atmosphere. 500 µL of cell suspension at a concentration of 2 × 105 cells/mL was seeded onto the sample surfaces (1 cm × 1 cm) and placed in a Greiner 24 well plate. After culturing at 37 °C under 5% CO2 atmosphere for 24 h, the substrates were washed with PBS twice and fixed by 4% paraformaldehyde for 30 min at room temperature. After the fixation, substrates were rinsed with DI water to remove the remaining paraformaldehyde and then dried at 60 °C in the oven for 24 h. The dried samples were coated with gold and then imaged with a scanning electron microscope (SEM, JEOL JSM-5600LV). The surface coverage of cells was estimated by image analysis of the SEM pictures with the ImageJ program (available as a public domain Java image processing program provided by National Institute of Health, USA). The total area covered by the cell clusters was calculated, and then divided by the total 9 ACS Paragon Plus Environment
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area of the image to give the percentage coverage of cells on the silicon wafer surface. The cell coverage for each sample was calculated based on ten images obtained at different locations. Three samples were measured for each type of surfaces to get the average cell coverage. Plasma cleaned silicon wafers were used as a reference surface for adhesion testing. 2.7
Bacteria adhesion test Two bacterial strains were used for the antibacterial tests. Escherichia coli DH5 (E. coli, ATCC#:
53868) and Staphylococcus aureus (S. aureus, ATTC#: 25923) were obtained from American Type Culture Collection, ATCC (USA). The two bacterial strains were cultivated in LB broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl) at 37 °C. The bacteria were cultured for about 16 h before harvest. The bacteria-containing broth was centrifuged at 3000 rpm for 10 min, and after the removal of the supernatant, the cells were washed twice and re-suspended with PBS for E. coli and S. aureus. After incubation with bacterial suspension for 2 h, the samples were washed three times with PBS before fixing with 4% glutaraldehyde for 5 h at 4 °C. After the fixation, substrates were rinsed with DI water to remove the remaining glutaraldehyde and then dried at 60 °C in the oven for 24 h. The dried samples were coated with gold and then imaged with a scanning electron microscope (SEM, JEOL JSM5600LV). The surface coverage of bacteria was also estimated by image analysis of the SEM images with the ImageJ program. The protein, cell and bacteria adhesion results were analyzed with One-way analysis variance (ANOVA), followed by a Tukey post-test. Data comparison was performed using GraphPad Prism 5 (GraphPad Software Inc., USA). For all comparisons p ≤ 0.05 results were considered to be statistically significant.
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3. Results and discussion 3.1
Tuning surface charge of the cross-linked LbL films To investigate the effects of surface charge on the adsorption or adhesion of various charged objects,
the substrate surfaces must be robust and possess well-defined physicochemical characteristics. One of the essential and measurable parameters of surface charge is its ζ potential, directly affecting electrostatic interactions between the surface of the substrate and the charged (or charge-polarized) species present in the solution.34, 37 It has been reported that the surface charge of polyelectrolyte multilayers can be manipulated by changing the deposition conditions such as pH value and ionic strength of the polymer solution.34, 40, 41 In this study, pH value of the weak polyelectrolyte solution during deposition was controlled to determine the quantity of the polyion assembled in each single layer contributing to the surface charge of the whole LbL system after deposition.
Figure 1. The chemical structure of polyelectrolytes and the three model cross-linked LbL films used in this study.
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Table 1. pH values of polyelectrolyte solutions and IEPs of the LbL films before and after crosslinking. LbL
IEP before IEP after
No. of
pH of P1 -
pH of PEI -
pH of P1 -
pH of PEI -
films
cross-
cross-
bilayers
the first 5
the first 5
penultimate
the last layer
linking
linking
bilayers
bilayers
layer
LbL(-)
