Separation of Storage and Loss Modulus of Polyelectrolyte Multilayers

Atomic force microscopy (AFM) is used to carry out rheology measurements on the nanoscale and to determine the mechanical properties of poly(l-lysine)...
1 downloads 0 Views 902KB Size
Download PDF
Article pubs.acs.org/Langmuir

Separation of Storage and Loss Modulus of Polyelectrolyte Multilayers on a Nanoscale: A Dynamic AFM Study Johannes Hellwig, Samantha Micciulla, Julia Strebe, and Regine von Klitzing*

Langmuir 2016.32:10505-10512. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/03/19. For personal use only.

Stranski-Laboratorium, Department of Chemistry, TU Berlin, Strasse des 17. Juni 124, D-10623 Berlin, Germany ABSTRACT: Atomic force microscopy (AFM) is used to carry out rheology measurements on the nanoscale and to determine the mechanical properties of poly(L-lysine) (PLL)/hyaluronic acid (HA) multilayer films. Storage (G′) and loss modulus (G″) of the films are calculated and compared with the values obtained from quartz crystal microbalance with dissipation monitoring measurements (QCM-D). A predominant elastic behavior independently of the applied frequencies (5−100 Hz) is observed for native HA/PLL films consisting of 36 double layer. If the layers are crosslinked, the value of G′ increases by 2 orders of magnitude, while the loss modulus becomes negligible, making these films a purely elastic chemical gel. The values of G′ and G′′ extracted from QCM-D measurements on native films are much higher, due to the different frequency regime of the applied shear stress. However, the viscoelastic ratio from the two methods is the same and proves the elastic dominated response of the multilayer in both frequency regimes.

■

film.15 Therefore, a change in the stiffness of the films can be used to trigger the cell adsorption.25−27 The stiffness of these HA/PLL films can easily be changed by cross-linking the native films after the preparation.16,17 Recent studies reported that native HA/PLL films show a viscoelastic behavior.17,28,29 Viscoelastic properties were investigated by a falling sphere experiment through a HA/PLL film, showing that HA/PLL films can be described either as a viscoelastic liquid with zero equilibrium elasticity or as a viscoelastic solid with a very small equilibrium elastic modulus.30 Furthermore, measurements with different approach speeds demonstrate that the indentation modulus increases for higher indentation velocity, while relaxation measurements were used to investigate the viscoelastic behavior of these films showing a full relaxation to zero.29 Also the effect of preparation conditions and cross-linking on the mechanical properties of the films was investigated.16,28 HA/PLL films are considered as a model system for nonlinear growing due to the high mobility of PLL, which can diffuse into and out of the entire multilayer film. However, a switch from exponential to linear growth is observed at 12 double layers where the diffusion zone reaches a limited and constant thickness.31,32 The HA/PLL films used in this study consist of 36 double layers and are therefore in the linear regime. To summarize, static measurements or measurements at a very low frequency indicate that the HA/PLL PEMs are very soft with a very low elastic modulus. This is in agreement with the ability for the polyelectrolytes to diffuse within the

INTRODUCTION Nowadays the assembly of complex molecules onto a solid substrate is a widely used method to prepare thin films to be applied to numerous applications. The assembly process can be carried out by using the layer-by-layer technique introduced by Decher and co-workers.1,2 By this method thin layers are achieved by alternate adsorption of oppositely charged polyelectrolytes to produce polyelectrolyte multilayers (PEMs). A large number of studies has been published over the past decade, dealing with growth behavior, swelling, roughness, and the effect of relative humidity. Furthermore, many different PEM systems have been studied with the most common being poly(styrene sulfonate) (PSS)/poly(diallyl dimethylammonium chloride) (PDADMAC),3 PSS/poly(allylamine) (PAH),4 or poly(acrylic acid) (PAA)/PAH. Such a method is not limited to simple synthetic molecules, but also more complex materials like viruses,5,6 polysaccharides,7 or hydrogels8−10 have been adsorbed and characterized on surfaces to understand their mechanical properties. Understanding and tuning the mechanics of various coatings opens a lot of different applications like drug delivery,11−14 cell adhesion,15−17 superhydrophobic surfaces,18 cell and protein resistance coatings,19−21 or bioresponsive sensors.22 This work is focused on a biocompatible polyelectrolyte multilayer made of poly(L-lysine) (PLL) and hyaluronic acid (HA). Their bicompatibility and the possibility to load the HA/PLL films with drugs and other molecules make them suitable as drug delivery systems.23,24 Studies on HA/PLL films as template for cell growing showed that chondrosarcoma cells adhere very well on cross-linked films, while the native films are highly antiadhesive.17 Also smooth muscle cells deposited on HA/PLL films show an increase in spreading when adsorbed on a stiffer © 2016 American Chemical Society

Received: July 25, 2016 Revised: September 7, 2016 Published: September 9, 2016 10505

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir

Figure 1. Schematic example of a dynamic force measurement on (a) native and (c) cross-linked HA/PLL films shown as force over time profile. Including the approach and indentation of the cantilever into the HA/PLL film surface, the relaxation of the film and the measurement where the cantilever oscillates with a given frequency and amplitude. (b) The change in the force signal (nN) and the change in the indentation (nm) measured at an indentation depth of 100 nm. Silicon wafers, 15 × 30 mm, used as substrate were cleaned in a piranha solution (1:1 mixture of 35% hydrogen peroxide and 98% sulfuric acid) for 20 min followed by multiple rinsing with Milli-Q before deposition of the polyelectrolytes. The film build-up was achieved by alternate dipping of the silicon wafers into the poly(Llysine) and hyaluronic acid solutions (0.5 mg/mL in 10 mM Trisbuffer containing 15 mM NaCl, pH 7.4) for 10 min. Each polyelectrolyte solution was filtered through a 0.24 μm syringe filter before use. Rinsing was done between each adsorption step for 10 min in the pure Tris-buffers solutions. The dipping procedure was repeated until n = 36 with n corresponding to the number of deposited double layers of (HA/PLL). For better adhesion of the polyelectrolytes, a polyethylenimine PEI layer was adsorbed as precursor layer under the same conditions as the other polyelectrolytes. The films were stored in Tris-buffer solution containing 15 mM NaCl at 4 °C prior to measurement. The films were never dried during all preparation and measurement steps. HA/PLL Cross-Linking. Cross-linking of HA/PLL films is based on the reaction between the primary amine (PLL) and activated carboxylic groups (HA). Activation of carboxylic groups is achieved by using water-soluble 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide EDC and N-hydrosulfosuccinimide sulfo-NHS.16,17 A 1:1 EDC/sulfoNHS solution was prepared using 400 mM EDC and 100 mM sulfoNHS solutions containing 15 mM NaCl at pH 5. A volume of 1 mL of the prepared mixture of EDC/sulfo-NHS was deposited in a Petri dish containing the prepared native HA/PLL films. Cross-linking of the HA/PLL films was achieved over 12 h at 4 °C followed by multiple rinsing with Tris-buffer solution. All samples were measured and stored in Tris-buffer solution to prevent them from drying out. Atomic Force Microscopy. Force measurements on all samples were performed at room temperature with an MFP−3D BIO AFM (Asylum Research, Oxford Instruments, Santa Barbara, CA). To get a reliable statistic of the measured properties at least three different positions of each sample were measured, recording spatially resolved force maps on 90 × 90 μm2 with 5 × 5 points. The built-in force map mode of the MFP−3D AFM was used to measure force−distance and force−time curves which provide information about the local mechanical response of the PEMs. The approach velocity of the measurements was kept constant at 800 nm/s. Moreover, all measurements were done in Tris-buffer solution using a CoolerHeater cell (Asylum Research) to ensure a constant temperature of 25 °C. Cantilever Calibration and Microsphere Attachment. Two different cantilevers were used for measurements: a soft cantilever (k = 0.05 N/m, HQ:CSC38/tipless/CR−AU, MikroMasch, USA) with an attached colloidal silica particle for the native PEMs and a stiffer cantilever (k = 2.8 N/m, HQ:NSC18/CR−AU, MikroMasch, USA) with a tip (length = 12−18 μm, full tip cone angle = 40°, radius = 35 nm) for the cross-linked films. The use of different cantilevers was necessary because of the huge difference in stiffness between native and cross-linked films, making the use of the same cantilever unsuitable. The colloidal silica particles with a radius of 3.35 μm (Bangs Laboratories, Inc., USA) were manually glued on the soft

PEM. No quantitative separation into the elastic and viscous part using AFM was achieved so far. To compare the elastic and viscous behavior in more detail frequency dependent rheological measurements are needed. According to static measurements one would expect that the loss modulus G″ is much larger than the storage modulus G′. The present study addresses the quantitative separation of the storage and loss modulus for non-cross-linked and cross-linked HA/PLL films. The mechanical properties of HA/PLL films were determined by dynamic force measurements using a colloidal probe atomic force microscope33,34 to distinguish between the elastic and viscous properties of the films. Dynamic force measurements have previously been performed on biological samples such as cells.35−41 It has been shown that the exact values of G′ and G″, as well as their relation, are dependent on the frequency used during the dynamic measurements. For these biological samples G′ is higher than G″ at small frequencies (∼10 Hz) and shows a low positive relation with frequency. G″ shows also a positive relation with frequency, but of higher magnitude, resulting in G″ > G′ for higher frequencies. The crossover (G′ = G″) for theses samples occurs between 10 and 100 Hz. In this work for the first time, dynamic force measurements with an AFM were used to determine the mechanical properties of HA/PLL films and differentiate between the storage and loss modulus as a function of the frequency and indentation depth. It was observed that the mechanical properties of these films are independent of the applied frequency in the range of 5−100 Hz, as well as on the indentation depth. However, higher viscoelastic moduli and loss ratio were found from QCM-D measurements, due to higher frequency applied to produce a shear stress in the direction parallel to the substrate. The knowledge of the film mechanics over a broad frequency range is fundamental for the proper design of biomimetic films where specific pressures are involved, which is the case of many biological processes.

■

MATERIALS AND METHODS

Materials. Tris(hydroxymethyl)aminomethane (Tris buffer), poly(ethylenimine) (PEI, MW = 750 kDa), N-(3-(dimethylamino)propyl)N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxy-sulfosuccinimide (NHS), and poly-L-lysine hydrobromide (PLL, MW = 22 kDa, MW/Mn = 1.3, purity = 96%) were purchased from Sigma-Aldrich (Steinheim, Germany) and used without further purification. Sodium hyaluronate (HA, MW = 357 kDa, purity = 96β %) was purchased from Lifecore (Minnesota, US). Preparation: HA/PLL Films. Polyelectrolyte multilayer films PEI− (HA/PLL)n/HA were prepared with the layer-by-layer technique1,2 using a dip robot (Riegler & Kirstein GmbH, Berlin, Germany). 10506

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir cantilevers using a two-component epoxy adhesive (UHU plus endfest 300, UHU GmbH, Germany). Placement of the adhesive and silica particle on the cantilever was achieved by using a micromanipulator and a microscope. The used cantilevers were calibrated before each measurement. For this the optical lever sensitivity of the cantilevers was determined against a cleaned silicon wafer, followed by the calculation of the spring constants by fitting the thermal noise spectra of the cantilevers (built-in procedure in the MFP−3D). Dynamic Force Measurements. Dynamic force measurements can be used to determine the complex modulus of a sample.36,37,39,42 For these measurements the cantilever is sinusoidal excited using the Z-piezo of the AFM. The excitation of the cantilever takes place at low frequency, small amplitude, and below its resonance frequency, while the deflection and the excitation signal are detected over time. Depending on the surrounding medium of the cantilever the deflection and excitation signals of the cantilever can show different phase shifts to each other: If the cantilever is placed on a purely elastic surface, deflection and excitation signal are in phase (i.e., 0° phase shift). If the cantilever is surrounded by a purely viscous medium, excitation and deflection signals show a 90° phase shift. The measurement on a sample can show a phase shift between 0° and 90° behaving more like an elastic solid or like a viscous liquid, respectively. In a static force measurement the cantilever is pushed into a sample surface until a certain trigger point is reached (e.g., deflection, force, Z-movement, etc.) and immediately retracted afterward. An apparent indentation modulus can be determined from the force curve. In a dynamic force measurement the oscillation of the cantilever will be applied during a dwell in the sample surface after reaching the trigger point and before retracting the cantilever from the sample surface. In this work the trigger point was set to three different indentation depths, namely, 100, 150, and 200 nm. During this dwell the cantilever was kept indented in the sample until the sample relaxed into a steady state, as shown in Figure 1. After relaxation the cantilever was excited for approximately 10 s to a sinusoidal movement using the Z-piezo. Frequency and amplitude of the excitation were set to 5−100 Hz and 10 nm, respectively. After the measurement the cantilever was retracted from the sample surface. Force and indentation signals were measured for further analysis of the complex modulus of the samples. The detected signals include the amplitudes AD, AF, Aδ, AZ and the phases φD, φF, φδ, and φZ for the deflection (D), force (F), indentation (δ), and Z-piezo (Z) over time. The deflection signal in this work is used to calculate the force signal by multiplying it with the spring constant of the cantilever (Hooke’s law). The indentation signal is calculated by subtracting the deflection from the Z-piezo signal, which is set to zero at the contact point between the probe and the surface of the sample. All settings for the dynamic measurements were done by using the built-in indenter panel of the Asylum Research MFP−3D software. Quartz Crystal Microbalance with Dissipation Monitoring. The use of quartz crystal microbalance with dissipation monitoring (QCM-D) is well established to study the adsorption and viscoelastic properties of thin polymer films onto solid substrates.43−48 The working principle of the technique is based on the piezo-electric properties of quartz, which is driven to oscillate to its resonance frequency and higher overtones by the application of a driving sinusoidal potential. When the driving potential is cut off, the amplitude of the freely decaying crystal oscillation is recorded described by the relation

A(t ) = A 0 exp(− π /τ ) sin(2πft + ψ )

response to the applied shear stress. Using the Voigt model, Voinova et al.52 described the physical features of a viscoelastic film, which is characterized by a complex shear modulus, G* = G′ + iG″ = μf + i 2πf0 ηf

(2)

with shear modulus μf and shear viscosity ηf correlated to the corresponding storage (G′) and loss (G″) moduli. The viscoelastic parameters can be obtained by proper modeling of the measured Δf and ΔD on a viscoelastic system, given that some parameters for the quartz crystal and the bulk liquid are known. QCM-D measurements reported in this work were carried out on the instrument E1 from Q-Sense (Sweden). Polyelectrolyte multilayers of PEI(HA/PLL) were prepared on a silicon-coated substrate (QSX3030, Q Sense). A solution flow of 0.1 mL/min was used to mimic the conditions of the dipping process. The adsorption time was set to 10 min, each step followed by rinsing with Tris-buffer solution for 5 min. The viscoelastic modeling of the measured signals for third, fifth, and seventh overtone was performed using the software Q-Tools from Q-Sense. Density and fluid viscosity of the bulk medium were fixed to 997 kg/m3 and 0.9 mPa·s,53,54 respectively. The film density was fixed to 1400 kg/m3.

■

DATA ANALYSIS Dynamic force measurements were previously reported in the work of Alcaraz et al.36,37 and Rother et al.39 Based on the Hertzian contact mechanics model,55,56 it is possible to calculate the complex modulus of the sample by G* = G′(ω) + iG″(ω) =

⎞ 1 − ν ⎛ F(ω) − iωb(h0)⎟ ⎜ 3δ tan(ϕ) ⎝ δ(ω) ⎠ (3)

where ν is the Poisson ratio of the sample, which is set to 0.5, δ is the indentation depth, and ϕ is the half opening angle of the pyramidal tip of the cantilever. The force F(ω) and indentation signals δ(ω) as a function of the angular frequency are given by A (ω) i(φF(ω)− φδ(ω)) F(ω) = F e δ(ω) Aδ (ω)

(4)

The complex modulus consists of a real part G′ (storage modulus) and an imaginary part G″ (loss modulus). The real part gives information about the energy stored in the sample and the imaginary part about the energy that is dissipated in the sample. For the spherical indenter used for the native PEMs the prefactor of the equation is changed to38 G* = G′(ω) + iG″(ω) =

⎞ 1 − ν ⎛ F(ω) − iωb(h0)⎟ 1/2 ⎜ ⎠ 4(Rδ) ⎝ δ(ω) (5)

When applying a sinusoidal oscillation to a cantilever which is surrounded by a viscous medium, in this work Tris-buffer, the cantilever experience a hydrodynamic drag due to the viscosity of the medium.36,37 This hydrodynamic drag can be corrected by a drag coefficient b0(h0) which is already included in eqs 3 and 5. Hydrodynamic Drag Correction. The measure of the hydrodynamic drag acting on a cantilever can be done by exciting the cantilever at several frequencies and small amplitudes as a function of cantilever-sample separation. These measurements were performed in a Tris-buffer solution at room temperature against a cleaned silicon wafer. A sinusoidal oscillation at different frequencies ranging from 10 to 100 Hz with an amplitude of 10 nm was applied to the cantilever at different separations of 0.2−2 μm above the silicon

(1)

with A0 being the amplitude of oscillation at t = 0 and ψ the phase shift, f the resonance frequency and τ the decay time constant.49 The dissipation factor D is obtained from the relation D = 1/πfτ. Literature on the topic can be found in refs 43, 48−51. The frequency shift Δf n = ( f n − f n0) and the dissipation change ΔDn = (Dn − Dn0) are typically reported by QCM-D measurements, and they describe the change of adsorbed mass and the energy dissipated by the system. Since they allow to directly correlate adsorption processes and dissipative losses, QCM-D measurements can support the interpretation of the viscoelastic behavior of a film in 10507

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir

Figure 2. Fits of the Z-sensor and force signals of a cantilever oscillation at 60 Hz in liquid to determine the hydrodynamic drag acting on the cantilever.

surface. In these measurements only the interaction between the surrounding medium and the cantilever is detected. The signals detected for the calculation of the hydrodynamic drag are the Z-piezo signal, AZ and φZ, as well as the force signal, AF and φF as shown in Figure 2. Before correction of the hydrodynamic drag it is necessary to correct a phase shift between the phases φZ and φF. Water as pure viscous medium leads to a phase shift of 90° between the Z-piezo and force signal. Deviations between the measured phase shift and 90° is corrected by a phase lag φlag which is already included in following equations: H * = H′(f ) + iH″(f ) =

Hα =

kHα(f ) k − Hα(f )

AΔF i(φF(ω)− φZ(ω)− φlag(ω)) e AΔZ

Figure 4. Dependency of the drag coefficient on the probe−sample distance. Fitting of the data was done by using a scaled spherical model eq 8. The fit is extrapolated to the probe−sample distance of 0 to get the drag coefficient b(h0).

(6)

(7)

■

where k is the spring constant of the cantilever. The calculation of HD′ and HD″ using the measured signals at different separations is shown in Figure 3. The real part of the

RESULTS Force measurements were carried out, spatially resolved, on two different samples: (1) a native HA/PLL film that shows an indentation modulus lower than hundred kPa (static measurements) and (2) a HA/PLL film that is cross-linked with EDC/ sulfo-NHS, showing an indentation modulus of several MPa. Dynamic force measurements were performed at different frequencies, f = 5, 10, 20, 40, 60, 80, and 100 Hz, and three different indentation depths δ = 100, 150, and 200 nm. Native HA/PLL films show no dependency either in the storage modulus G′ nor in the loss modulus G″ on the applied frequencies, as shown in Figure 5. Furthermore, G″ show values around 23−27 kPa, nearly 3 times lower than the G′ values at 75−85 kPa. Measurements at different indentation depths 100, 150, and 200 nm show the same values for the native HA/PLL films, as shown in Figure 5. To summarize for native HA/PLL

Figure 3. Calibration of the drag coefficient by plotting the real H′ and imaginary part H″ of the hydrodynamic drag function vs oscillation frequency. H″ shows the linear dependency of the hydrodynamic drag on the oscillation frequency and is fitted with the equation: m = 2πb(h) to obtain the drag coefficient for a probe−sample distance of 2000 nm.

hydrodynamic drag HD′ shows no dependency on the increasing oscillation frequency, while H″D shows a linear increase. The drag coefficient b(h) at a fixed separation can be determined by linear fitting of the HD″ with m = 2πb(h). Fitting HD″ for each separation gives a dependency of the drag coefficient on the separation, showing a higher drag at smaller separations, as shown in Figure 4. A scaled spherical model, eq 8, can be used to fit these data and determine the drag coefficient37 at 0 separation b(h0). 6πηαeff2 b(h) = h + heff

Figure 5. Native HA/PLL films: Storage modulus G′ (filled symbols) and loss modulus G″ (open symbols) as a function of the used oscillation frequency for three different indentation depths of 100 nm (circles), 150 nm (squares), and 200 nm (triangles).

(8) 10508

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir

modulus μf, shear viscosity ηf of HA and PLL-terminated films, 15 and 16 layer, respectively, are reported. From these values and according to eq 2, storage G′, loss G″ moduli, and the corresponding loss tangent G″/G′ were calculated. First of all, minor differences were found between HA and PLL termination. This indicates that the internal structure mostly determines the viscoelastic response of the system to the shear stress. A minor influence on G′ and G″ might be given by the different water content of the outermost layer, in particular with a slightly more flexible and hydrated structure for PLL layer (lower G′ and G″). Second, the viscoelastic moduli are roughly 1 order of magnitude higher than the values measured by AFM, likely due to the higher frequency applied for the shear stress (in the range from 25 to 45 MPa for QCMD measurements). However, the loss tangent is similar to AFM, which means that the elastic and viscous components increase proportionally under the increased applied frequency, leading at the end to the same material properties as found by AFM. QCM-D measurements on cross-linked films were not carried out, due to the high film rigidity. This makes the viscoelastic models (Maxwell and Voigt models) unsuitable to the description of the mechanical properties of these systems.

films no dependence of storage and loss modulus on the frequency and indentation depth was found. Cross-linked HA/PLL films were also measured at the same frequencies and indentation depths as the native ones. Figure 6

Figure 6. Cross-linked HA/PLL films: Storage modulus G′ (filled symbols) and loss modulus G″ (open symbols) as a function of the used oscillation frequency for three different indentation depths of 100 nm (circles), 150 nm (squares), and 200 nm (triangles).

shows that the storage modulus G′ of the cross-linked films is around 2 orders of magnitude higher than the values for the native PEMs. In contrast the G″ values are just 4 times higher. Analogous to the behavior of native HA/PLL films, the moduli are independent of frequency and indentation depth, showing constant values around 3 MPa and 80 kPa, respectively. The loss tangent (η = G″/G ′) of the native HA/PLL films is higher than for the cross-linked HA/PLL films. As shown in Figure 7, for the native films G″/G′ ≈ 0.3, while the cross-linked ones show a much lower G″/G′ ratio of 0.05.

■

DISCUSSION

Usually, a rheometer works in the dimension of hundreds of micrometers up to millimeters, whereas the AFM can probe the mechanical properties in the nanoscale using a colloidal probe or a tip with a radius in nanometer scale. The AFM was so far used to determine mechanical properties of native and crosslinked HA/PLL films by static force measurements. This method is not suited for films with a viscoelastic character. The calculation of the indentation modulus usually is done by using a Hertz based model. Since Hertz based models require a pure elastic behavior of the samples, the calculated values do not fully describe the investigated native HA/PLL films. On the other hand dynamic force measurements can be used to determine the elastic and the viscous properties of the films, as it is usually done by the use of a shear rheometer. Although dynamic force measurements are already used in cell biology due to the mostly viscoelastic behavior of cells, it is rarely used for polyelectrolyte multilayers. The storage and loss moduli of the native films are shown in Figure 5. The storage modulus is with a value between 50−80 kPa about 4 times higher than the loss modulus with 15−25 kPa, in contrast to our expectation. This indicates that the film responses more like an elastic solid in the used frequencies from 5 to 100 Hz. Also no crossover was found in the investigated frequency range, unlike to the biological samples.36,38,39 Both the storage and loss moduli show no dependency on the used frequencies. Both curves are parallel to each other without giving any hint for a crossover at lower or higher frequencies. At the crossover (G′/G″ = 1), which is called sol−gel transition, the film properties changes from viscous to predominant elastic behavior, giving these films a gel like character. In order to determine if a crossover can be

Figure 7. Loss tangent for the native (filled symbols) and cross-linked (open symbols) HA/PLL films as a function of the frequency.

The modeling of QCM-D data on a Voigt viscoelastic element was performed on native HA/PLL films with a different outermost layer, containing either 15 or 16 layers. The analysis of thicker PEMs, comparable to those studied by AFM, was not possible due to the detection limits (60−250 nm depending on the overtone number) of the technique, which is determined by the penetration depth δ of the acoustic wave in the bulk liquid.57 In fact, the values of δ in aqueous media was estimated between 90 and 140 nm from third to seventh overtone, which corresponds to the thickness of a PEI(HA/ PLL)8 swollen in water. In Table 1 shear thickness df, shear

Table 1. Viscoelastic Parameters Obtained by Modeling of QCM-D Data for PEI(HA/PLL) Multilayers According to a Voigt Element termination

μf (× 105) (Pa)

ηf (× 10−3) (Pa ·s)

df(nm)

G′ (kPa)

G″ (kPa)

G″/G′

HA PLL

15.0 ± 0.1 10.6 ± 0.2

13.2 ± 0.1 8.6 ± 0.1

141.1 ± 0.2 172.3 ± 0.4

1500 ± 10 1060 ± 20

410 ± 2 269 ± 4

0.27 0.25

10509

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir

for a qualitative analysis. The properties of the HA/PLL films (AFM: η = 0.32; QCM-D: η = 0.27) show similar loss tangent values compared to PAA hydrogels60 (η = ∼0.35) or a similar PEM systems as chitosan/heparin54 (η = 0.3−0.5). PAA hydrogels show smaller values for the storage and loss modulus (G′ = 110 Pa, G″ = 35 Pa at 10 Hz) while chitosan/heparin films in contrast show a similar stiffness in QCM-D (G′ = 0.5− 1 MPa) compared to HA/PLL films (AFM: G′ = 50−80 kPa, G″ = 15−25 kPa; QCM-D: G′ = 1000−1500 kPa, G″ = 300− 400 kPa). The storage and loss modulus was also measured and calculated for different indentation depths at 100, 150, and 200 nm. The values of the measurements at the three different indentation depths are quite similar, as shown in Figure 5, proving that the film has a homogeneous structure which does not change with the indentation depth. Cross-linked films in contrast show a really high storage modulus compared to the value for the native films, as shown in Figure 6. The calculated storage modulus at all indentation depths is around 2 MPa. This is a 25-fold increase in the modulus due to the crosslinking of the films. The ratio between the storage and loss modulus is shown in Figure 7, where the loss tangent drops to values around 0.01−0.05. These low values indicate that the films show no viscoelastic behavior anymore. The films are fully cross-linked and are now a chemical gel with a pure elastic behavior. The storage and loss modulus of the cross-linked film shows no dependency on the used frequencies, 5−100 Hz, as it was also observed with the native HA/PLL films. Due to the cross-linking over 12 h after preparation of the films, it was not possible to measure the mechanical properties of the film with the QCM-D. For a pure elastic cross-linked film of HA/PLL the storage and loss moduli are not expected to show a cross over at low frequencies.

observed at higher frequencies the moduli were also determined by QCM-D. The obtained values are reported in Table 1 for input stimuli of 15, 25, and 35 MHz. The storage modulus for both HA and PLL terminated films is roughly 4 times higher than the loss modulus, meaning that the mechanical response is always elastic-dominated. The absolute values of QCM-D-moduli, 1000−1500 kPa and 300−400 kPa for G′ and G″, respectively, are about 10−20 times higher than the values measured by AFM. This significant difference can be due to the following reasons: (1) QCM-D films were subjected to shear stress, i.e., in the horizontal direction, while AFM measurements apply a compression stress, in the vertical direction. The higher moduli under an applied shear stress indicate an enhanced strength against flow, likely due to interdigitation among subsequent layers. (2) The large difference in the frequency might also be responsible for the corresponding different moduli. Since both storage and loss modulus are frequency-dependent quantities,58,59 it is likely that the viscoelastic moduli are higher at higher applied frequency and lower in the low frequency regime of AFM. To speculate about the reason, one might assume that the relaxation time at static experiments reflects the time which is needed to break the complexation sites on a submolecular scale. At higher frequencies the complexation sites have not enough time to break and rearrange, which reduces the yield and let the system appear stiffer and more elastic. (3) It is questionable if the different preparation methods might have induced structural differences, which in turn produce diverse mechanical properties in the film. While the HA/PLL films, which are measured using the AFM, are prepared by dipping, the preparation of the HA/PLL film in the QCM-D is done under a steady flow of the polyelectrolyte solution. During dipping the polyelectrolyte solution is not stirred, and the polyelectrolytes have more time to adsorb and rearrange. In contrast during adsorption under flow more polyelectrolytes reach the surface at the same time, which leads to a more quenched chain conformation without enough time for rearrangement. This leads to a different alloverall structure in both PEM types. Nevertheless, the loss tangent (η = G″/G′) of the native films shows values which are about 0.3 for both AFM and QCM-D measurements, as shown in Figure 7 and Table 1. These values are well below 1, meaning that the films behave more as an elastic system rather than a viscous one, in the observed frequency range. Considering the fact that the HA/PLL films do not show any dependence on the input frequencies, the following model can be deduced: PLL is highly mobile; it can move through the film and build complexation sites between its amino group and the carboxylic acid groups of the HA. This implies that the film behaves like a weak physical gel network which is stabilized due to the complexation sites. A similar behavior of the mechanical properties to HA/PLL films can be found in un-cross-linked polyacrylamide hydrogels (PAA). Compared to the HA/PLL films, PAA hydrogels are showing a similar behavior of its mechanical properties measured by shear rheology. At frequencies between 5 and 100 Hz the storage modulus is higher than the loss modulus, the same as HA/PLL films, and showing mostly no dependency on the frequencies. At very low frequencies at 0.04−0.05 Hz a crossover was reported by Suriano et al.60 Frequencies lower than 1 Hz cannot be measured with the used AFM without further modifications. Instabilities of the piezo signal due to electrical and temperature fluctuations or noise over long times >1−2 min makes it impossible to get enough stable oscillations

■

SUMMARY AND CONCLUSION In this work, the mechanical properties of native and crosslinked HA/PLL films were measured by dynamic force measurements using an atomic force microscope at frequencies between 5 and 100 Hz. A quartz crystal microbalance was used to study the mechanical response in the frequency range of tens of MHz and compare it with the behavior at low frequency. As obtained from AFM studies, native HA/PLL films have a much higher storage (75−85 kPa) than loss (23−27 kPa) moduli (η = G″/G′ = 0.32) giving these films a more elastic response to dynamic measurements. Both moduli were independent of the used frequencies, for AFM, showing no change in their values and furthermore no indication of a crossover. QCM-D measurements were used to determine if the film properties were showing a crossover at higher frequencies. The measured moduli were much higher values those obtained by AFM (G′ = 1000−1500 kPa and G″ = 300−400 kPa), but they confirmed the elastic response and similar loss tangent (η = G″/G′ = 0.27). The difference between the values for AFM and QCM-D measurements was explained by the difference in preparation of the films, the different frequency regime of the applied stress, and the scale of measurement from nanometer size for the AFM to micrometer size for the QCM-D. A crossover of the storage and loss moduli is expected at frequencies much lower than 1 Hz, as it is reported for polyacrylamide hydrogels. Such low frequencies are not accessible with the used AFM. Crosslinking of the HA/PLL films in comparison leads to a purely elastic response and a 25 fold higher value for the storage modulus (3 MPa). Also in this case, G′ and G″ were independent of the used frequencies, and the loss tangent 10510

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir

(15) Engler, A. J.; Richert, L.; Wong, J. Y.; Picart, C.; Discher, D. E. Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films: Correlations between substrate stiffness and cell adhesion. Surf. Sci. 2004, 570, 142−154. (16) Francius, G.; Hemmerlé, J.; Ohayon, J.; Schaaf, P.; Voegel, J.-C.; Picart, C.; Senger, B. Effect of crosslinking on the elasticity of polyelectrolyte multilayer films measured by colloidal probe AFM. Microsc. Res. Tech. 2006, 69, 84−92. (17) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking. Biomacromolecules 2004, 5, 284−94. (18) Zhai, L.; Cebeci, F. C.; Cohen, R. E.; Rubner, M. F. Stable superhydrophobic coatings from polyelectrolyte multilayers. Nano Lett. 2004, 4, 1349−1353. (19) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J. a.; Hochbaum, A. I.; Rubner, M. F. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules 2003, 4, 96− 106. (20) Yang, S. Y.; Mendelsohn, J. D.; Rubner, M. F. New class of ultrathin, highly cell-adhesion-resistant polyelectrolyte multilayers with micropatterning capabilities. Biomacromolecules 2003, 4, 987−94. (21) Richert, L.; Arntz, Y.; Schaaf, P.; Voegel, J.-C.; Picart, C. pH dependent growth of poly (L-lysine)/ poly (L-glutamic) acid multilayer films and their cell adhesion properties. Surf. Sci. 2004, 570, 13−29. (22) Ellis, D. L.; Zakin, M. R.; Bernstein, L. S.; Rubner, M. F. Conductive polymer films as ultrasensitive chemical sensors for hydrazine and monomethylhydrazine vapor. Anal. Chem. 1996, 68, 817−822. (23) Szarpak, A.; Cui, D.; Dubreuil, F.; De Geest, B. G.; De Cock, L. J.; Picart, C.; Auzély-Velty, R. Designing hyaluronic acid-based layerby-layer capsules as a carrier for intracellular drug delivery. Biomacromolecules 2010, 11, 713−720. (24) Vodouhê, C.; Le Guen, E.; Garza, J. M.; Francius, G.; Déjugnat, C.; Ogier, J.; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Control of drug accessibility on functional polyelectrolyte multilayer films. Biomaterials 2006, 27, 4149−56. (25) Vazquez, C. P.; Boudou, T.; Dulong, V.; Nicolas, C.; Picart, C.; Glinel, K. Variation of Polyelectrolyte Film Stiffness by Photo-CrossLinking: A New Way To Control Cell Adhesion. Langmuir 2009, 25, 3556−3563. (26) Seo, J.-H.; Sakai, K.; Yui, N. Adsorption state of fibronectin on poly(dimethylsiloxane) surfaces with varied stiffness can dominate adhesion density of fibroblasts. Acta Biomater. 2013, 9, 5493−5501. (27) Wang, L.-m.; Chang, H.; Zhang, H.; Ren, K.-f.; Li, H.; Hu, M.; Li, B.-c.; Martins, M. C. L.; Barbosa, M. A.; Ji, J. Dynamic stiffness of polyelectrolyte multilayer films based on disulfide bonds for in situ control of cell adhesion. J. Mater. Chem. B 2015, 3, 7546−7553. (28) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J.-C. Buildup Mechanism for Poly(L-lysine)/ Hyaluronic Acid Films onto a Solid Surface. Langmuir 2001, 17, 7414−7424. (29) Uzüm, C.; Hellwig, J.; Madaboosi, N.; Volodkin, D.; von Klitzing, R. Growth behaviour and mechanical properties of PLL/HA multilayer films studied by AFM. Beilstein J. Nanotechnol. 2012, 3, 778−88. (30) Picart, C.; Senger, B.; Sengupta, K.; Dubreuil, F.; Fery, A. Measuring mechanical properties of polyelectrolyte multilayer thin films: Novel methods based on AFM and optical techniques. Colloids Surf., A 2007, 303, 30−36. (31) Volodkin, D.; von Klitzing, R. Competing mechanisms in polyelectrolyte multilayer formation and swelling: Polycation-polyanion pairing vs. polyelectrolyte-ion pairing. Curr. Opin. Colloid Interface Sci. 2014, 19, 25−31. (32) Porcel, C.; Lavalle, P.; Ball, V.; Decher, G.; Senger, B.; Voegel, J.; Schaaf, P. From exponential to linear growth in polyelectrolyte multilayers. Langmuir 2006, 22, 4376−4383.

decreases to values around 0.01−0.05. It is shown that dynamic force measurements can be used to measure the rheological properties of HA/PLL films on a nanoscale. This method can be extended to probe the dynamics of many different systems, not only polymer thin films but also chemical cross-linked or physical stabilized gels, polymer brushes, as well as under different environments (water, organic solvent, and gases). As solvent in these measurements it is not necessary to use water based solvents, but it is also possible to use ambient air or other gases.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Decher, G.; Hong, J.; Schmitt, J. Buildup of ultrathin multilayer films by a self-assembly process: III. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces. Thin Solid Films 1992, 210−211, 831−835. (2) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277, 1232−1237. (3) Zerball, M.; Laschewsky, A.; von Klitzing, R. Swelling of Polyelectrolyte Multilayers: The Relation Between, Surface and Bulk Characteristics. J. Phys. Chem. B 2015, 119, 11879−11886. (4) Steitz, R.; Leiner, V.; Tauer, K.; Khrenov, V.; von Klitzing, R. Temperature-Induced Changes in Polyelectrolyte Films at the SolidLiquid Interface. Appl. Phys. A: Mater. Sci. Process. 2002, 74, 519−521. (5) Lvov, Y.; Haas, H.; Decher, G.; Moehwald, H.; Mikhailov, A.; Mtchedlishvily, B.; Morgunova, E.; Vainshtein, B. Successive Deposition of Alternate Layers of Polyelectrolytes and a Charged Virus. Langmuir 1994, 10, 4232−4236. (6) Dimitrova, M.; Arntz, Y.; Lavalle, P.; Meyer, F.; Wolf, M.; Schuster, C.; Haikel, Y.; Voegel, J.-C.; Ogier, J. Adenoviral gene delivery from multilayered polyelectrolyte architectures. Adv. Funct. Mater. 2007, 17, 233−245. (7) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Layer by Layer Buildup of Polysaccharide Films: Physical Chemistry and Cellular Adhesion Aspects. Langmuir 2004, 20, 448−458. (8) Burmistrova, A.; Richter, M.; Eisele, M.; Ü züm, C.; von Klitzing, R. The Effect of Co-Monomer Content on the Swelling/Shrinking and Mechanical Behaviour of Individually Adsorbed PNIPAM Microgel Particles. Polymers 2011, 3, 1575−1590. (9) Matzelle, T. R.; Ivanov, D. a.; Landwehr, D.; Heinrich, L. a.; Herkt-Bruns, C.; Reichelt, R.; Kruse, N. Micromechanical properties of “smart” gels: Studies by scanning force and scanning electron microscopy of PNIPAAm. J. Phys. Chem. B 2002, 106, 2861−2866. (10) Wellert, S.; Richter, M.; Hellweg, T.; von Klitzing, R.; Hertle, Y. Responsive Microgels at Surfaces and Interfaces. Z. Phys. Chem. 2015, 229, 10.1515/zpch-2014-0568. (11) Smith, R.; Riollano, M.; Leung, A.; Hammond, P. Layer-byLayer Platform Technology for Small-Molecule Delivery. Angew. Chem. 2009, 121, 9136−9139. (12) Chung, A. J.; Rubner, M. F. Methods of loading and releasing low molecular weight cationic molecules in weak polyelectrolyte multilayer films. Langmuir 2002, 18, 1176−1183. (13) Müller, M.; Kessler, B.; Adler, H.-J.; Lunkwitz, K. Reversible switching of protein uptake and release at polyelectrolyte multilayers detected by ATR-FTIR spectroscopy. Macromol. Symp. 2004, 210, 157−164. (14) Jiang, S.; Chen, X.; Liu, M. The pH stimulated reversible loading and release of a cationic dye in a layer-by-layer assembled DNA/PAH film. J. Colloid Interface Sci. 2004, 277, 396−403. 10511

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512

Article

Langmuir

(53) Iturri Ramos, J. J.; Stahl, S.; Richter, R. P.; Moya, S. E. Water Content and Buildup of Poly(diallyldimethylammonium chloride)/ Poly(sodium 4-styrenesulfonate) and Poly(allylamine hydrochloride)/ Poly(sodium 4-styrenesulfonate) Polyelectrolyte Multilayers Studied by an in Situ Combination of a Quartz Crystal Microb. Macromolecules 2010, 43, 9063−9070. (54) Lundin, M.; Solaqa, F.; Thormann, E.; Macakova, L.; Blomberg, E. Layer-by-Layer Assemblies of Chitosan and Heparin: Effect of Solution Ionic Strength and pH. Langmuir 2011, 27, 7537−7548. (55) Hertz, H. On the contact of elastic solids. Reine u. Angew. Math. 1882, 92, 156−171. (56) Johnson, K. L. Contact mechanics; Cambridge University Press, 1987. (57) Alves, N.; Picart, C.; Mano, J. Self assembling and crosslinking of polyelectrolyte multilayer films of chitosan and alginate studied by QCM and IR spectroscopy. Macromol. Biosci. 2009, 9, 776−785. (58) Pritz, T. Frequency dependences of complex moduli and complex Poisson’s ratio of real solid materials. J. Sound Vib. 1998, 214, 83−104. (59) Strganac, T. W.; Letton, A.; Payne, D. F.; Biskup, B. A. Characterization of nonlinear viscoelastic behavior using a dynamic mechanical approach. AIAA J. 1995, 33, 904−910. (60) Suriano, R.; Griffini, G.; Chiari, M.; Levi, M.; Turri, S. Rheological and mechanical behavior of polyacrylamide hydrogels chemically crosslinked with allyl agarose for two-dimensional gel electrophoresis. J. Mech. Behav. Biomed. Mater. 2014, 30, 339−346.

(33) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Direct measurement of colloidal forces using an atomic force microscope. Nature 1991, 353, 239−241. (34) Butt, H. J. Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope. Biophys. J. 1991, 60, 1438−1444. (35) Shroff, S. G.; Saner, D. R.; Lal, R. Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy. Am. J. Physiol. 1995, 269, C286−C292. (36) Alcaraz, J.; Buscemi, L.; Grabulosa, M.; Trepat, X.; Fabry, B.; Farré, R.; Navajas, D. Microrheology of human lung epithelial cells measured by atomic force microscopy. Biophys. J. 2003, 84, 2071− 2079. (37) Alcaraz, J.; Buscemi, L.; Puig-De-Morales, M.; Colchero, J.; Baró, a.; Navajas, D. Correction of microrheological measurements of soft samples with atomic force microscopy for the hydrodynamic drag on the cantilever. Langmuir 2002, 18, 716−721. (38) Smith, B. A.; Tolloczko, B.; Martin, J. G.; Grütter, P. Probing the viscoelastic behavior of cultured airway smooth muscle cells with atomic force microscopy: stiffening induced by contractile agonist. Biophys. J. 2005, 88, 2994−3007. (39) Rother, J.; Nöding, H.; Mey, I.; Janshoff, A. Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines. Open Biol. 2014, 4, 140046. (40) Mizuno, D.; Tardin, C.; Schmidt, C. F.; Mackintosh, F. C. Nonequilibrium mechanics of active cytoskeletal networks. Science 2007, 315, 370−373. (41) Pullarkat, P. A.; Fernández, P. A.; Ott, A. Rheological properties of the Eukaryotic cell cytoskeleton. Phys. Rep. 2007, 449, 29−53. (42) Mahaffy, R.; Shih, C.; MacKintosh, F.; Kas, J. Scanning probebased frequency-dependent microrheology of polymer gels and biological cells. Phys. Rev. Lett. 2000, 85, 880−883. (43) Marx, K. a. Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface. Biomacromolecules 2003, 4, 1099−120. (44) Salomäki, M.; Laiho, T.; Kankare, J. Counteranion-Controlled Properties of Polyelectrolyte Multilayers. Macromolecules 2004, 37, 9585−9590. (45) Ramos, J. J. I.; Moya, S. E. Water content of hydrated polymer brushes measured by an in situ combination of a quartz crystal microbalance with dissipation monitoring and spectroscopic ellipsometry. Macromol. Rapid Commun. 2011, 32, 1972−8. (46) Micciulla, S.; Dodoo, S.; Chevigny, C.; Laschewsky, A.; von Klitzing, R. Short versus long chain polyelectrolyte multilayers: a direct comparison of self-assembly and structural properties. Phys. Chem. Chem. Phys. 2014, 16, 21988−21998. (47) Wang, X.; Berger, R.; Ramos, J. I.; Wang, T.; Koynov, K.; Liu, G.; Butt, H.-J.; Wu, S. Nanopatterns of polymer brushes for understanding protein adsorption on the nanoscale. RSC Adv. 2014, 4, 45059−45064. (48) Reviakine, I.; Johannsmann, D.; Richter, R. Hearing what you cannot see and visualizing what you hear: interpreting quartz crystal microbalance data from solvated interfaces. Anal. Chem. 2011, 83, 8838−8848. (49) Rodahl, M.; Kasemo, B. On the measurement of thin liquid overlayers with the quartz-crystal microbalance. Sens. Actuators, A 1996, 54, 448−456. (50) Dixon, M. C. Quartz crystal microbalance with dissipation monitoring: enabling real-time characterization of biological materials and their interactions. J. Biomol. Technol. 2008, 19, 151−8. (51) Johannsmann, D.; Reviakine, I.; Rojas, E.; Gallego, M. Effect of sample heterogeneity on the interpretation of QCM(-D) data: comparison of combined quartz crystal microbalance/atomic force microscopy measurements with finite element method modeling. Anal. Chem. 2008, 80, 8891−9. (52) Voinova, M.; Rodahl, M.; Jonson, M.; Kasemo, B. Viscoelastic Acoustic Response of Layered Polymer Films at Fluid-Solid Interfaces: Continuum Mechanics Approach. Phys. Scr. 1999, 59, 391−396. 10512

DOI: 10.1021/acs.langmuir.6b02764 Langmuir 2016, 32, 10505−10512