Structural and Viscoelastic Properties of Layer-by-Layer Extracellular

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ACS Biomaterials Science & Engineering

Structural and Viscoelastic Properties of Layer-byLayer ECM Nanofilms and Their Interactions with Living Cells Akihiro Nishiguchi, Michiya Matsusaki, and Mitsuru Akashi*

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

1 Environment ACS Paragon Plus


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ABSTRACT

Modulation of living cell surfaces by chemical and biological engineering and the control of cellular functions has enormous potential for immunotherapy, transplantation, and drug delivery. However, traditional detection techniques have limitations in the identification of physical properties of viscoelastic films and interaction with living cells in real time. Here, we present the structural analysis of extracellular matrix (ECM) based nanofilms and their interaction with living cells using a quartz crystal microbalance (QCM) with dissipation (QCM-D), multiple parameter surface plasmon resonance (SPR), and flow cytometry measurements.

QCM-D

measurements according to the Voigt-based viscoelastic model allowed for the evaluation of the kinetic adsorption of extracellular matrix (ECM) proteins and physical parameters of viscoelastic ECM-nanofilms in a swelled state. These results reflected the characteristics of viscoelastic films as compared to Sauerbrey’s equation. Moreover, we found that gelatin molecules played crucial role as a binder to build up layered films and control their properties. Using the multiple parameter SPR approach, we confirmed the interaction between FN-G nanofilms and living cells from signal response in real time which was different from gold substrate-protein signal. Moreover, flow cytometry analysis supported the importance of the domain interaction between the RGD sequence in FN and integrin as a driving force to form the films on cell surfaces. The use of three different analyses supported to clarify the contribution of protein-protein interaction and viscoelastic properties of ECM films, and investigate the interaction between films and living cells. The knowledge about protein-protein and protein-cell interaction in real time would make a contribution to biomaterial design using protein interactions for modulating the living cell surfaces in biomedical applications. Keywords: Surface science, Layer-by-layer assembly, Extracellular matrix, Nanofilm, QCM-D 2 Environment ACS Paragon Plus

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INTRODUCTION

In vitro functionalization of living cells is one of the most promising approaches to control their activity for biomedical applications including immunotherapy, transplantation, and drug delivery.1-6 For example, clinical treatments using immunocompetent cells that are artificially activated in vitro have been applied for immunotherapy against viruses and cancer.7,8 On the cell surface, a number of proteins, sugars, and lipids accumulated with fluid and organized membrane structures.9 Thus, it is necessary to modify the properties of the living cells on the surface without impairing the original function of each component.

Until now, various types of

methodologies including metabolic engineering,10,11 chemical modification using covalent bonding,12

hydrophobic

interaction,13,14

antigen-antibody

reaction,15

and

electrostatic

interaction16 have been attempted. However, almost all methodologies have drawbacks such as low cytocompatibility, low stability, and dysfunction by undesired chemical reactions and nonspecific adsorption of compounds.

Layer-by-layer (LbL) assembly has the potential to

functionalize living cell surfaces by preparing micro- to nano-meter sized thin films onto a substrate by alternate immersion into interactive polymer solutions.17,18 Thin films formed could be promising materials to modify interfacial properties including electric property, roughness, charge, and surface free energy, leading to the use for electronic devices and biomedical field.19,20 However, it is known that cationic polymers as a component of the electrostatic interaction-driven films may induce serious cytotoxicity due to the disruption of the cell membrane and non-specific adsorption.21 Therefore, a functionalization method that can control cellular properties under mild conditions would be required for practical use.



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Previously, we reported the preparation of several to hundreds of nanometer-sized thin films based on extracellular matrix (ECM) components by LbL assembly.22-25 We prepared celladhesive ECM nanofilms comprising fibronectin (FN) and gelatin (G) nanofilms (FN-G nanofilms) on the cell surface, which allowed for enhancement of cell adhesive property and construction of three dimensional tissues in vitro.

FN is a flexible and multifunctional

glycoprotein and plays an important role in cell adhesion, migration, and differentiation.26 Even though FN and G have a negative charge under physiological conditions, they can interact with each other through collagen binding domains (Kd = 0.6 ~ 5 x 10-6 M) in FN,27 which allows for the preparation of LbL films without the use of cationic polymers. Moreover, FN interacts not only with a variety of ECM proteins but also with the α5β1 integrin receptor on the cell surface through the RGD sequence.28 FN-G nanofilms fixed onto cell membranes through domain interactions could support cell adhesion with each other to construct 3D-engineered tissues.24 We also found interesting protection properties against physical stress in spite of the nanometersized thin films.25 However, the physical properties of ECM-nanofilms during the LbL process and the interaction between ECM proteins and living cells have not been clarified yet.

To characterize the physical properties of formed films, quartz crystal microbalance (QCM) measurement according to Sauerbrey’s equation, which can estimate the mass of deposited polymers (∆m) from the frequency decrease of the QCM (∆f), was previously used.23,29 QCM detects the change in mass from the frequency shift at high sensitivity (in the ng/cm2 range). However, it was hard to estimate actual thickness, viscoelasticity, and the structure of viscoelastic materials like hydrogels by QCM measurement according to Sauerbrey’s equation. The propagation of the oscillatory motion of the crystal into the viscoelastic materials should be considered to evaluate the actual mass because viscoelastic materials behave as a coupled


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oscillator and the frequency shift is not proportional to the change in mass.30 To evaluate the physical properties of the viscoelastic materials, QCM with energy dissipation (D) measurements (QCM-D) can be used. QCM-D analysis evaluates the elastic components of the shear wave propagation into the materials from the measurements of the change of frequency and energy dissipation30, which allows us to evaluate the thickness and viscoelasticity of the films. On the other hand, to assess the interaction between the ECM protein films and living cells, optical techniques including surface plasmon resonance (SPR) for adsorption kinetics and flow cytometry for end-point measurement of fluorescence have potential as compared to QCM-D because the measurement based on mass change is possibly affected by morphological changes in the cells at high mass.

Here, we report the structural analysis of ECM-based nanofilms and their interaction with living cells using three types of measurements including QCM with dissipation (QCM-D), multiple parameter surface plasmon resonance (SPR), and flow cytometry measurements (Scheme 1).

QCM-D analysis allows for kinetic measurement of the change of not only

thickness but also viscoelasticity for viscoelastic materials like FN-G nanofilms.

We also

attempted to directly monitor of the formation process of FN-G nanofilms onto living cells and their interaction with living cells using SPR and flow cytometry measurements.



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MATERIALS AND METHODS

QCM-D analysis. The kinetics of the adsorption of proteins and polymers were measured by QCM-D analysis (Q-Sense E1, Q-Sense, Sweden). The QCM-D monitors the frequency shifts (∆f) and the energy dissipation change (∆D) by switching off the driving force power periodically. The measurement of up to seven harmonics (fundamental frequency and 5, 15, 25, 35, 45, 55, and 65 MHz, corresponding to the overtones n = 1, 3, 5, 7, 9, 11, and 13, respectively) of the crystal with 5 MHz fundamental resonance frequency (Q-Sense, Sweden) was performed. The four types of films were prepared: FN (Mw = 460 000, Sigma-Aldrich) / G (Mw = 100 000, Wako Pure Chemical Industries) nanofilm (FN-G nanofilm), FN nanofilm, poly(diallyldimethylammonium

chloride)

(PDDA,

Mw

=

240

000,

Polyscience)

/

poly(styrenesulfate sodium salt) (PSS, Mw = 200 000, Sigma-Aldrich) nanofilm (PDDDA-PSS nanofilm), and laminin (LN, Mw = 850 000, Sigma-Aldrich) / type IV-collagen (Col, Mw = 540 000, Sigma-Aldrich) nanofilm (LN-Col nanofilm). Gold-coated AT-cut quartz crystals were exposed to UV-ozone cleaner for 10 min and washed with ethanol. For the preparation of polymer nanofilms, 0.2 mg/mL of protein or polyelectrolyte solution (50 mM Tris-HCl buffer (pH =7.4)) was alternately flowed with a peristaltic pump at 0.1 mL/min for approximately 5 minutes at room temperature. The washing process was performed by flowing 50 mM Tris-HCl buffer between each step to remove unabsorbed polymers. After repeating these processes, the four types of LbL nanofilms were prepared on the crystals in a flow cell ((FN/G)4FN, FN5, (PDDA/PSS)3PDDA, (LN/Col)4LN). The obtained ∆f and ∆D were fit to the mass change as a function of time using the Voight model with Q-tools software (Q-Sense, Sweden). Based on this viscoelasticity analysis, the thickness (dV), shear elasticity (µ) and viscosity (η) of each LbL


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nanofilm was analyzed (polymer density: 1.3 g/cm3).

As a comparison, the mass change

(∆mQCM-D-S) and thickness (dS) were calculated according to Sauerbrey’s equation.

SPR measurement. SPR monitors the kinetics of molecular adsorption and can estimate the thickness, association constant, and dissociation constant. The measurement is based on surface plasmon resonance, which occurs on a thin gold substrate. The measurement can be used to characterize layer properties and adsorption kinetics of forming layers of molecular interactions from the change in plasmon angle and intensity.31 The formation processes of FN-G nanofilms on gold substrate and FN-G nanofilms on confluently-cultured human colon carcinoma cell line (Caco-2) on gold substrate were monitored using a multi-parameter SPR device (MP-SPR Navi 200, BioNavis Ltd, Finland). The gold-coated SPR sensor slides (Bionavis Ltd. (Tampere, Finland) were rinsed with ethanol and dried with nitrogen. The 1 x 105 cells/cm2 Caco-2 were then seeded onto on gold surface in an SPR sensor chip which was placed in culture dish with a cell growth area of 8.8 cm2. After the 3 days of culture, a confluent monolayer of Caco-2 were obtained. The cultured cells were washed with PBS before use to remove detached cells and cell debris. The sensor chip without or with cells was inserted into the SPR device and then 50 mM Tris-HCl buffer (pH=7.4) was flowed at 30 µL/min at room temperature followed by incubation for 2 minutes to stabilize the cells. Then, FN and G at 0.2 mg/mL were alternately flowed for 3 minutes in 50 mM Tris-HCl buffer during the washing step for 3 minutes. All experiments were done at 20 oC using the angular scan mode that provided a full SPR angular spectrum every four seconds. The obtained SPR curves were fitted using SPR Navi LayerSolver v. 0.9 software (BioNavis Ltd, Finland). After finishing the measurements, the morphology and viability of Caco-2 was checked with trypan-blue exclusion assay using phase microscopy.



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Cell coating with FN-G nanofilms and fluorescent observation. The normal human dermal fibroblasts (NHDFs) after trypsinization were alternately immersed in 0.2 mg / mL of Rhodamine-labeled FN (Rh-FN) and G dissolved in 50 mM of Tris-HCl buffer solution (pH = 7.4) for 1 minute using a Micro-tube Rotator (MTR-103, AS ONE, Japan) with a washing step. The centrifugation was performed at 400 xg for 1 minute at each step to separate cells. After 9 cycles of FN-G coating, FN-G nanofilms were formed onto the individual cell surfaces. To confirm the formation of FN-G nanofilms onto the cell surfaces, the cells were observed by confocal laser scanning microscopy (CLSM) (FLUOVIEW FV10i, Olympus, Japan).

Flow cytometry. For more quantitative analysis, flow cytometry analysis was performed on cells coated with FN-G nanofilms in 0, 3, or 9 steps. The cells without FN-G nanofilms and with (FN/G)FN and (FN/G)4FN nanofilms were obtained in the same manner described above. The cells were immunostained with an anti-human fibronectin antibody conjugated with FITC (BD falcon). Briefly, after the Fc blocking treatment for 5 min, the cells were incubated with an antihuman fibronectin antibody (1:50) for 1 hour at 4 oC. The cells were washed with PBS three times and the number of cells was adjusted to more than 5 x 105 cells / mL for the measurements. The flow cytometry was performed using a guava easyCyte HT sampling flow cytometer (Millipore). The cell suspensions were flowed into a capillary with stirring and 5000 cells were counted. The percentage of FITC-FN positive cells was measured using the InCyte software (Millipore).

Immunofluorescent staining. To evaluate the morphological changes of FN-G nanofilms prepared on individual cell surfaces, NHDFs coated with nanofilms of FN derived from bovine and G were cultured on a glass bottom dish for 24 hours and immunostained with a mouse anti-


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bovine FN monoclonal antibody (Sigma-Aldrich) and a rabbit anti-integrin β1 monoclonal antibody (Millipore). Briefly, the cells were fixed with 4% PFA for 15 minutes and blocking was performed with 1% bovine serum albumin (BSA)/PBS for 1 hour. The cells were then incubated with the primary antibodies (1:100) for 1 hour. After washing with PBS, the secondary antibodies (Life Technologies, 1:200) were added and incubated for 1 hour. After washing with PBS, nuclei were stained with DAPI.

Statistical analysis. All data were expressed as means ± SD unless otherwise specified. The values represent the mean ± SD from three independent experiments. Statistical comparisons between groups were analyzed by Student’s t-test. A p value < 0.05 was considered to be statistically significant.



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RESULTS AND DISCUSSION

Evaluation of structure and viscoelastic properties of ECM-nanofilms by QCM-D. To investigate the formation process of FN-G nanofilms during LbL assembly, the change in frequency and energy dissipation were monitored by QCM-D measurement (Figure 1). QCM-D that allows for monitoring of the adsorption kinetics can be applied to interaction analysis of various biomolecules such as proteins and DNA and cells.31-35 The real-time measurement of ∆f and ∆D from the seventh overtone was used to monitor the adsorption kinetics of FN and G because the values obtained at lower overtones (n =1, 3) were much higher due to viscoelastic property of FN-G films.30 In the initial step for the FN-G nanofilms, FN was adsorbed onto the gold substrate via non-specific interactions. Then, the alternate adsorption of G and FN through the domain interaction including electrostatic, hydrophobic, or Van der Waals force interaction sequentially increased ∆f and ∆D as repeated an increase and decrease (Figure 1a). On the other hand, nanofilms that were formed by interaction with FN repeatedly five times (without G) reached saturation on the initial step and ∆f and ∆D did not sequentially increase (Figure 1b). This result shows the differences of adsorption behavior and film characteristics between FN-G and FN films. As a qualitative evaluation to understand the adsorption process, a plot of ∆D-∆f was used to estimate the growth rate and viscosity of the films from the obtained ∆D and ∆f values (Figure 2).34 The plot of ∆D-∆f plot for FN-G nanofilm showed a slope in the ∆D-∆f plot that drastically increased when FN was absorbed at the third step (Figure 2a). This behavior indicated that domain interactions between FN and G overcame electrostatic repulsion and formed layered structures of (FN/G)FN nanofilms during LbL assembly. There was no change in the slope of the ∆D-∆f plots for FN films without G even after 5 step assembly (Figure 2b).


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To evaluate the formation process and physical properties of ECM films, we used the Voight model which explains the propagation of shear acoustic waves into viscoelastic materials in the presence of a semi-infinite Newtonian liquid.36 According to Voight model, the obtained ∆f and ∆D were fit and analyzed to the thickness (dV), shear elasticity (µ) and viscosity (η) (polymer density: 1.3 g/cm3) as a function of time using the grid fit module of the Q software tool.

As a comparison, the mass change (∆mQCM-D-S) and thickness (dS) were calculated

according to Sauerbrey’s equation. The thickness (dV), shear elasticity (µ) and viscosity (η) were estimated by the Voight model using ∆D and ∆f values from the fifth and seventh overtones (Figure 3). The thickness and shear modulus of the FN-G nanofilms sequentially increased in a manner that was similar to the tendency for the ∆f and ∆D values (Figure 3a). After 9 step assembly, the thickness of the FN-G films was 26 nm and four times thicker than that of dried FN-G nanofilms measured by QCM measurement using Sauerbrey’s equation23 which is valid only when ∆D is less than 10-6 per 5 Hz of ∆f.21 On the other hand, FN films did not show a sequential increase in thickness or shear modulus (Figure 3b). The nanofilm structure can be predicted from the change in thickness and shear modulus at each step as follows (Scheme 2). First, FN is adsorbed onto the gold substrate via non-specific interaction. Then, G molecules are adsorbed loosely and the complexation of G with FN occurs due to the domain interaction, which indicated by the decrease in thickness and increase in shear modulus. Sequentially, the increase in thickness and decrease in shear modulus indicated that FN molecules adsorbed loosely to form layered structures with soft properties. During the washing process, complexation with G caused a decrease in thickness and increase in shear modulus again. In addition, it is thought that other factors including the dissociation of water and protein molecules were involved in the formation process of FN-G film. Repeating these processes led to the fabrication FN-G films 26mm thick



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with a shear modulus of 120 kPa. These FN-G films were thicker and stiffer than FN films. The results obtained from QCM-D analysis of FN-G and FN-only nanofilms clearly suggested that G molecules played an important role in building up LbL films and functioned as a binder to connect FN molecules. In addition, the importance of LbL assembly using G molecules to control the physical properties including the thickness and shear modulus was shown.

As compared to FN-G nanofilm formation that were driven by domain interactions, electrostatic interaction-driven LbL films that have been widely used display different adsorption behaviors (Figure 4a,b). The film comprising PDDA and PSS (PDDA-PSS films) showed clear step-by-step increases in ∆f and ∆D (Figure 4a and Figure S1a). The thickness, elastic modulus, and viscosity of PDDA-PSS films after 7 step assembly were estimated to be 8.2 nm, 5200 kPa, and 18.1 mPa•s, respectively, from viscoelastic analysis (Figure 4b), resulting in the formation of thinner and stiffer films compared to the protein films as shown in Figure 3. The obtained result was in agreement with the value estimated according to Sauerbrey’s equation since the films were stiff and less able to swell. The combination of polyelectrolytes formed solid-like films by complexation based on strong cation-anion interactions between polymers. We also evaluated LbL films using LN and Col (LN-Col films) as other types of ECM proteins (Figure 4c,d and Figure S1b). The adsorption behavior of LN and Col was complicated, which may be attributed to the soft gel-like structure and strong complexation between macromolecular LN and Col. There were no large differences in film viscosity except PDDA-PSS film (Figure S2). The results from QCM-D analysis and QCM data23 are summarized in Table 1. Compared to the results for QCM (8.9 nm), which were calculated from dried polymer films according to Sauerbrey’s equation, QCM-D analysis using Voight’s model displayed a four-fold increase in thickness (25.6 nm). This finding supports the QCM-D method’s use in estimating the actual


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thickness of soft gel-like ECM nanofilms in a swelled state with consideration of the viscoelasticity.

The method was a useful tool to investigate the adsorption kinetics and

ultrastructure of various films.

Measurement of cell response to FN-G nanofilm formation by SPR. As we mentioned before, for the investigation of the interaction between living cells and polymers, the measurement based on mass change such as QCM-D measurement have limitation because morphological changes in the cells at high mass strongly affect the detection. To directly detect the interaction between FN-G nanofilms and living cells, we attempted to monitor signal responses of confluently-cultured monolayer cells during LbL assembly using SPR measurement. SPR is label-free optical detection technique and can be used to evaluate kinetics for biosensing.37 However, since the evanescent wave by the surface plasmon excited at a certain incident light angle propagates into the medium in the range of half wavelength of incident light, the detection range is limited to less than 300-500 nm depth with an exponential decay of the sensitivity as a function of distance.38 Therefore, it is difficult to monitor the whole signal response of cultured cells greater than 3000 nm in thickness using a traditional SPR system, although information on the interface between the metal and the cells was probed. To overcome the limitation, a multiple parameter SPR approach has been established for sensing living cells.39 This SPR technique, based on a tunable angle of incident light (50~80o), measures a full SPR angular spectra and provides important parameters including SPR peak angular position (PAP), SPR peak minimum intensity (PMI), and the changes in the total internal reflection (TIR) region, which enables the investigation of cell-drug interaction. The detection is not direct detection of molecular interaction as in traditional molecule-molecule SPR assays, but indirect detection of the interaction through the cell response to the stimulus (molecules or other).



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Using the multiple parameter SPR approach, we first investigated FN-G film formation onto a bare gold substrate (Figure 5). The changes in SPR PAP and SPR PMI were monitored in real time using incident lights sources with wavelengths of 670 nm and 785 nm. The alternate injection of FN and G solutions sequentially increased the changes in SPR PAP and SPR PMI, mainly after FN injection, which was in agreement with QCM-D behavior. After 15 step assembly, FN-G film approximately 10 nm thick with a refractive index of 1.42 was formed onto the bare gold substrate. To extend the SPR measurement for living cells and to investigate cell responses, human epithelial cells (colon carcinoma cell line, Caco-2) were cultured on the gold substrate because epithelial cell line Caco-2 can cover almost all the substrate area but not fibroblast cells like NHDFs. The 1 x 105 cells/cm2 Caco-2 were seeded and adhered to the gold substrate and after the SPR measurements the cultured Caco-2 did not change their tightly adhered morphology and showed high viability (>80%, which was estimated by trypan blue exclusion assay). It was confirmed by phase contrast microscopy that the gold surface was not exposed into the flow chamber, which indicated that the interaction between the injected protein and gold may be negligible.

The 5 step assembly of FN-G nanofilm was performed to

confluently-cultured Caco-2 and the signal responses of SPR PAP and SPR PMI were monitored (Figure 6). As compared to bare gold substrate, the main SPR PAP in the full SPR spectrum increased from 69 to 71

o

and the shape of the TIR region changed (Figure S3). The obtained

angular spectrum supported the existence of cultured cells on the gold substrate and the spectrum was stable before the injection of FN and G. During LbL assembly of FN and G, the signal response for both SPR PAP and SPR PMI was confirmed when FN and G were flowed. After the dissociation process of adsorbed polymers occurred during the washing step, these values showed a stepwise increase during the 6 steps of assembly. Interestingly, we found that there


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was larger response time lag between polymer injection and signal changes as compared to the case with substrate without cells. This indicated that the changes in SPR PAP and SPR PMI originated from cell responses caused by the interaction between FN/G and living cells and not by the adsorption of FN/G onto the gold substrate. When the culture media containing 10% FBS were injected to Caco-2 on gold substrate as a control experiment, there were no changes in the signal, indicating that FN may interact with cells during SPR measurement and protein component and concentration were important to interact with cells (data not shown). Moreover, the substrate with cultured cells showed a much higher increase in SPR PAP and SPR PMI when 785 nm of incident light was used and a behavior opposite to what was seen for the bare substrate was observed. When the changes in SPR PAP versus SPR PMI at 785 nm of incident light during the assembly were plotted, the large difference in the changes between the substrate with and without living cells was confirmed (Figure 6c) and cell responses such as morphological change and polymer adsorption were monitored. When combining the MP-SPR experimental results of FN-G nanofilm formation onto living cells with the experimental results using bare gold sensor in both MP-SPR and QCM-D, these results indicated that the interaction between FN-G nanofilms and living cells during LbL assembly could be evaluated in real time using the multiple parameter SPR approach.

Interaction analysis between FN and integrin by flow cytometry. Finally, we evaluated the formation of FN-G nanofilms on living cell surfaces by CLSM observation and flow cytometry. The use of antigen-antibody reaction in flow cytometry allowed for interaction analysis at the protein level. Flow cytometry analysis using an anti-human fibronectin antibody conjugated with FITC was performed. The cell population with higher fluorescent intensity increased with an increasing step number of LbL assembly and there were more than 80 percent FN positive



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cells after 9 steps of assembly (Figure 7a). Importantly, the adsorption of FN onto living cells drastically decreased after blocking with an anti-integrin α5 and β1 antibody. This result clearly suggested that the domain interaction between the RGD sequence in FN and integrin α5β1 works as a driving force for nanofilm formation (Figure 7b). As an additional analysis, NHDFs soon after coating with Rhodamine-labeled FN (Rh-FN) and G at the 9 steps displayed the adsorption of FN by CLSM observation (Figure 7c). After 1 day of incubation, it was found that the coated FN transformed the morphology to a fibrous structure (Figure 7d,e). These FN fibers, similar to natural fibrous ECMs,40,41 can interact with integrin molecules and function as a nano-meshwork scaffold to provide a suitable platform for cellular adhesion.


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CONCLUSIONS

Here we presented the structural evaluation of ECM-based nanofilms and their interaction with living cells using QCM-D, the multiple parameter SPR technique, and flow cytometry measurements. QCM-D measurement, employing the Voigt-based model, was used to evaluate adsorption kinetics and physical properties including thickness, viscosity, and shear modulus of viscoelastic ECM-nanofilms in a swelled state. As compared to previous QCM methods using Sauerbrey’s equation, QCM-D analysis provided new insights on the characteristics of ECMnanofilms under conditions similar to that of the physiological environment. We found that FNG films showed a stepwise increase in adsorption amounts during LbL assembly and grew to 26 nm thick with a shear modulus of 120 kPa after 9 steps. These FN-G films were thicker and stiffer than FN films, suggesting the importance of G molecules as a binder. Using the multiple parameter SPR approach, the full SPR angular spectra of cultured cells onto gold substrate were monitored in real time to investigate the interaction between FN/G and living cells.

We

confirmed the changes in SPR PAP and SPR PMI of the gold substrate with cultured cells in response to the flow of FN and G during LbL assembly and the different behavior in the PAPPMI plot as compared to results for bare gold substrate. These findings indicated that the use of the multiple parameter SPR approach enabled interaction analysis between FN-G nanofilms and living cells. Flow cytometry analysis also supported the formation of FN-G nanofilms onto living cell surfaces and the importance of the domain interaction between the RGD sequence in FN and integrin α5β1 as a driving force. The combination of these three useful analyses can be used to clarify the detailed structures and formation process of polymer films and will be essential tools in investigating the interaction between functional materials and living cells.



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ASSOCIATED CONTENT

Supporting Information

The following file is available free of charge via the Internet at http://pubs.acs.org. ∆D-∆f plots versus time during LbL assembly of (PDDA/PSS)PDDA and (LN/Col) nanofilms; Viscosity versus time during LbL assembly of (FN/G)4FN, (FN)5, (PDDA/PSS)3PDDA, and (LN/Col)4LN nanofilms; Full SPR angular spectra of a bare gold and monolayer-cultured Caco-2 on the gold substrate.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]

Present Addresses Akihiro Nishiguchi; DWI Leibniz Institute for Interactive Materials and Institute of Technical and Macromolecular Chemistry of RWTH Aachen University, Forckenbeckstr 50, D-52056, Aachen, Germany Mitsuru Akashi; Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, 565-0871, Osaka, Japan.


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Author Contributions A.N. designed and carried out the studies, and wrote the paper. M.M. organized the project. M.A. supervised the project. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources

This work was supported by the NEXT Program (LR026), a Grant-in-Aid for Scientific Research (S) (A232250040), the SENTAN-JST Program (13A1204), and Grand-in-Aid for JSPS Fellows (24・622).

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

We thank S. Nishimura at MEIWAFOSIS Co. Ltd. for QCM-D analysis and N. Granqvist at BioNavis Ltd. for SPR analysis.



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Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


QCM-D

SPR

gold substrate FN

G

Water molecule

Flow cytometry

gold substrate cell integrinα5β 1

lipid bilayer

Scheme 1. Schematic illustration of analysis procedure for the interaction between FN-G and FN-cells and nanofilm structures using QCM-D, SPR, and flow cytometry measurements which can be used to detect FN-G interactions, cell response to FN, and FN-cell membrane proteins interaction, respectively.



a

0

50

(FN/G)4FN

40

∆f (Hz)

-50 -100

30

F

20

-150

D

-200

10

-250 0

∆D (x 10-6)

3000

0 9000

6000

Incubation time (min)

b

0

20

(FN)5

∆f (Hz)

-50

15

F

-100

10 -150

D

∆D (x 10-6)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 35

5

-200

0

-250 0

1000

2000

3000


Figure 1. ∆f and ∆D from seventh overtone versus time during LbL assembly of (a) (FN/G)4FN and (b) (FN)5 nanofilms. 0.2 mg/mL of protein solutions were alternately flowed with washing step at room temperature. F and D denote the axes of frequency and dissipation shifts.


Page 27 of 35

a ∆D (x 10-6)

12

(FN/G)FN

10 8 6 4 2 0 0

-20

-40

-60

-80

-100

-120

∆f (Hz)

b 7

∆D (x 10-6)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(FN)5

6 5 4 3 2 1 0 0

-20

-40

-60

-80

-100

∆f (Hz)

Figure 2. ∆D-∆f plots from the seventh overtone versus time during LbL assembly of (a) (FN/G)FN and (b) (FN)5 nanofilms.



a

FN 40

FN

G

FN

wash

G G

FN G FN G FN

Thickness (nm)

35

350 300

30

250

25

200

20 15

150

10

100

5

50 0 8000

0 0

2000

4000

Shear modulus (kPa)

6000


b

FN

40

FN

FN

FN

wash FN

FN

Thickness (nm)

35

350 300

30

250

25

200

20

150

15 10

100

5

50

0 0

500

1000

1500

2000

2500

Shear modulus (kPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 35

0 3000


Figure 3. Viscoelastic analysis from fifth and seventh overtone versus time during LbL assembly of (a) (FN/G)4FN and (b) (FN)5 nanofilms. The results of real-time measurements of thickness and shear modulus of nanofilms were shown.


Page 29 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FN

G

Water molecule

Adsorption of FN (1st)

Adsorption of G (2nd)

Complexation of G (2nd)

26 nm

Adsorption of FN (3rd)

Complexation of FN (3rd)

(FN/G)4FN assembly

Scheme 2. Schematic illustration of the mechanism of the formation of FN-G nanofilms during LbL assembly.



a

(PDDA/PSS)3PDDA

0

b

PDDA

5

PSS

wash

10

10000

∆f (Hz)

-30

4 3

-60 2

D

-90

1

-120 0

1000

0 3000

2000

8

8000

6

6000

4

4000

2

2000 0

0 0

500


180

-200

120

F

-300

90

-400

60

D

30

-500 -600 0

2000

4000

6000

8000


2000

2500

0 10000

LN Col

LN

wash

LN Col LN Col LN Col LN

1200

100

1000

80

800

60

600

40

400

20

200

0 0

2000

4000

6000

8000

Shear modulus (kPa)

-100

150

Col 120

∆D (x 10-6)

∆f (Hz)

d

LN-Col

0

1500


Thickness (nm)

c

1000

Shear modulus (kPa)

F

Thickness (nm)

PDDA PSSPDDAPSSPDDAPSS PDDA

∆D (x 10-6)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

0 10000


Figure 4. ∆f and ∆D from seventh overtone and viscoelastic analysis from fifth and seventh overtone versus time during LbL assembly of (a,b) (PDDA/PSS)3PDDA and (c,d) (LN/Col)4LN nanofilms. 0.2 mg/mL of protein and polymer solutions were alternately flowed with washing step at room temperature.


Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


n

FN-G

FN

QCM

∆mQCM (ng cm-2)

1160 ± 117 -a)

QCM-D

thickness (nm) ∆mQCM-D-S (ng cm-2)

8.9 ± 0.9 2080 1970 12 12 16 15 26 2.2 120

∆D dS (nm) dV (nm) mPa s µ (kPa)

5 7 5 7 5 7

-a) 1540 1490 4.7 4.9 12 11 20 2.1 30

PDDA-PSS Col-LN 2720 ± 300 -a) 22.6 ± 2.5 1070 1040 0.7 1.4 8 8 9 18 5200

-a) 5280 4760 36 44 41 37 43 2.2 710

Table 1. Summary of the results from QCM and QCM-D analysis. The results of FN-G films after 13 step assembly and PDDA-PSS films after 16 step assembly estimated from QCM according to Sauerbrey’s equation were shown.23 a) It was not measured in previous paper. n means overtone. ∆mQCM-D-S and dS were calculated according to Sauerbrey’s equation. dV was calculated according to Voigt’s model.



a 0.8

FN

FN G

G FN

wash G FN

SPR PAP (o)

0.7 670 nm 785 nm

0.6 0.5 0.4 0.3 0.2 0.1 0 0

500

1000

1500

2000

2500

Incubation time (s)

b SPR PMI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.01

FN

FN G

G FN

wash G FN

670 nm 785 nm

0.008 0.006 0.004 0.002 0 0

500

1000

1500

2000

2500

Incubation time (s)

Figure 5. (a) The changes in the angular position of the SPR peak minimum and (b) the changes in the SPR minimum intensity during LbL assembly of FN-G nanofilms onto a bare gold substrate.


Page 32 of 35

Page 33 of 35

SPR PAP (o)

a

1.5

FN G

FN

wash G FN G FN

670 nm 785 nm

1.2 0.9 0.6 0.3 0 0

500

1000

1500

2000

2500

Incubation time (s)

b 0.16

SPR PMI

FN G

FN

0.14

G

wash FN

1500

2000

G FN

670 nm 785 nm

0.12 0.1 0.08 0.06 0.04 0.02 0 0

c

500

1000

2500

Incubation time (s) 0.14 Bare gold substrate

0.12

Caco-2 monolayer

SPR PMI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.1 0.08 0.06 0.04 0.02 0 0

0.2

0.4

0.6

0.8

SPR PAP

1

1.2

(o)

Figure 6. (a) The changes in the angular position of the SPR peak minimum and (b) the changes in the SPR minimum intensity during LbL assembly of FN-G nanofilms onto monolayer-cultured Caco-2 cells on the gold substrate. (c) Change in SPR PAP versus SPR PMI of a bare gold during 9 steps of assembly and monolayer-cultured Caco-2 during 3 steps of assembly with FN and G when 785 nm of incident light was used.



a Count

150

: Control : 0 step : 3 step : 9 step

100 50 0 1

10

100

Fluorescence intensity

b FN+ cells (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

**

100

**

**

80 60 40

**

20 0

0 step

c

xy xz

3 step

Rh-FN

20 µm

9 step anti-integrin α5β1 ab

d

Rh-FN Integrin β1 Nucleus

50 µm

e

10 µm

Figure 7. (a,b) Flow cytometry analysis of FN-G coated cells. An anti-integrin α5 and β1 antibody were used for blocking. FN positive cells were determined as cells with more than 10 in fluorescent intensity. (c) CLSM image of NHDFs coated with Rh-FN-G nanofilms. (d) CLSM image and (e) magnified image of Rh-FN-G coated NHDFs after 1 day. Integrin β1 was immunostained with an anti-integrin β1 antibody.


Page 35 of 35

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Table of Contents Graphic Modification of living cell FN G

FN-G nanofilms (26 nm) Domain interaction between FN and G Domain interaction between FN and integrin

Cell coated with FN-G nanofilms

Integrinα5β 1

Structural and Viscoelastic Properties of Layer-by-Layer ECM Nanofilms and Their Interactions with Living Cells Akihiro Nishiguchi, Michiya Matsusaki, and Mitsuru Akashi*


Structural and Viscoelastic Properties of Layer-by-Layer Extracellular

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