Controlling the Cell-Adhesion Properties of Poly(acrylic acid

Jul 17, 2012 - A programmable slide stainer (HMS Slide Stainer, Zeiss) is used to assemble 20 bilayers of a PAA/PAAm film on a PEI-primed substrate...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Controlling the Cell-Adhesion Properties of Poly(acrylic acid)/ Polyacrylamide Hydrogen-Bonded Multilayers Sang-Wook Lee,† Kwadwo E. Tettey,† Iris L. Kim,‡ Jason A. Burdick,‡ and Daeyeon Lee*,† †

Department of Chemical and Biomolecular Engineering and ‡Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States S Supporting Information *

ABSTRACT: A thorough understanding of thermal effects on the physicochemical properties of layer-by-layer (LbL) assembled multilayers is crucial in utilizing these films in a variety of applications. In this work, we investigate the effect of thermal treatment on cross-linking and swelling of a hydrogenbonded multilayer film made of poly(acrylic acid) (PAA) and polyacrylamide (PAAm), which has been shown to exhibit excellent long-term cell adhesion resistance. We observe that the apparent swelling of PAA/PAAm multilayers treated at 90 and 180 °C in a physiologically relevant condition is similar; however, these two multilayers with different thermal history exhibit completely different cell adhesion properties when assessed with human mesenchymal stem cells (hMSCs). While the 90 °C treated samples show excellent cell adhesion resistance, those treated at 180 °C are highly cell adhesive. A combination of characterization techniques including thermogravimetric analysis (TGA) and Fourier-transform infrared (FT-IR) spectroscopy reveals that complete cross-linking between PAA and PAAm chains occurs above 150 °C. PAA/PAAm multilayer films incubated at temperatures below 150 °C have a very low degree of crosslinking. Thus, when these less-cross-linked films are exposed to aqueous solutions of pH 4 or higher, a significant loss of the constituent materials is observed. This lightly cross-linked hydrogel-like network in phosphate buffered saline (PBS) has a water content of ∼94 vol %, making the surface cell-adhesion resistant. In constrast, PAA/PAAm films incubated at 180 °C consist of ∼72 vol % water in PBS and exhibit cell-adhesive properties. The shear modulus of 180 °C treated films measured by quartz crystal microbalance with dissipation (QCM-D) monitoring is 6.0 MPa, which is 2 orders of magnitude higher than that of lightly cross-linked 90 °C treated films (0.02 MPa), suggesting that mechanical compliance plays a significant role in influencing the cell adhesion behaviors. This work emphasizes the importance of thermal treatment conditions, which potentially can be used as a postassembly route to gradually modify and control the properties of LbL multilayers over a wide range for specific applications.

■

INTRODUCTION Layer-by-layer (LbL) assembly via alternate adsorption of oppositely charged species provides a powerful tool for fabricating versatile nanocomposite thin films with tunable architecture and properties.1−5 LbL films have been utilized in a variety of advanced applications such as drug delivery,6,7 iontransport membranes,8,9 tissue engineering,10,11 and functional coatings.12−15 The LbL assembly technique has been extended to sequential deposition of hydrogen donors and acceptors,16−23 which offer unique opportunities in generating functional films owing to the dramatic responsiveness of these hydrogen-bonded (HB) multilayers to environmental pH24−26 and temperature.27 For example, pH-controlled softening28 and permeability change29 of HB multilayer capsules made of poly(methacrylic acid) (PMAA) and poly(N-vinylpyrrolidone) (PVPON) were demonstrated for applications in pH-triggered drug release. The size of theses capsules can be drastically changed by increasing the local pH above a critical value (pH ∼ 6), which ionizes the carboxylic acid groups of PMAA and, thus, disrupts hydrogen bonding between the two. The thickness and permeability of HB © XXXX American Chemical Society

multilayer shells can also be regulated by the type of polymers incorporated and their molecular weights.30 In addition, HB multilayer films are promising for biomedical applications due to their excellent biocompatibility. HB multilayer films using poly(acrylic acid) (PAA) and polyacrylamide (PAAm), for example, have been shown to exhibit high resistance to the adhesion of mammalian cells for an extended period of time.31,32 For many practical applications, it is imperative to maintain the structural integrity as well as the functionality of HB multilayers in the physiologically relevant pH range (pH ∼ 7). However, many HB multilayers that are composed of poly(carboxylic acid)s, such as poly(acrylic acid) and poly(methacrylic acid), disassemble at neutral or high pH due to the ionization of the carboxylic acid groups and the disruption of hydrogen bonds. Therefore, cross-linking between polymer Received: May 19, 2012 Revised: July 3, 2012

A

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

■

chains has been employed to prevent disintegration of HB films. To introduce cross-linking in HB multilayer films, thermal,32,33 chemical,19,34,35 and photo23,25,32 treatments have been utilized. Among these approaches, thermal treatment provides a convenient way to induce cross-linking without additional chemical treatments or labeling with photoreactive species. Heat-induced cross-linking was first demonstrated in electrostatic multilayers made of poly(allylamine hydrochloride) (PAH) and PAA, in which amide formation was observed after thermal treatment.36 The thermochemical properties of PAH/PAA films including cross-linking and glass transition were further studied by using films isolated from low surface energy substrates.37 In HB multilayers, PAA-based films underwent cross-linking through esterification38 or imidization32 between the carboxylic acid groups of PAA and the hydroxyl groups of poly(vinyl alcohol) (PVA) or the amide groups of PAAm, respectively. In most of these studies, the thermal effects on cross-linking and corresponding changes in film properties were largely understood by comparing films before and after heat treatment in which complete cross-linking was achieved. Although thermal approaches provide flexibility in tuning the incubation temperature and time, the possibility of modulating the structure and properties of HB films by controlling thermal treatment conditions has not been fully explored. In this paper, we study the effect of thermal treatment on cross-linking and swelling of a HB multilayer made of poly(acrylic acid) (PAA) and polyacrylamide (PAAm). We chose this HB multilayer because PAA and PAAm in LbL multilayers have been reported to undergo imidization after heat treatment at 175 °C.32 The hydrogen-bonding nature of these films enables micropatterning using water as a rinsing agent, which locally disrupts hydrogen-bonding and disassembles the film. In addition, when incubated at 90 °C, the PAA/ PAAm multilayer exhibits excellent cell-adhesion resistance, while remaining noncytotoxic.31 Among numerous parameters tested such as protein adhesion resistance and the effect of outermost layer, the cell adhesion resistance of cross-linked PAA/PAAm HB multilayers was attributed to the high degree of swelling and, in turn, high mechanical compliance under physiologically relevant conditions.31,39 Inspired by these prior studies, we test the adhesion of human mesenchymal stem cells (hMSCs) on PAA/PAAm multilayers incubated at two different temperatures, 90 and 180 °C. The film treated at 90 °C shows excellent cell-adhesion resistance, whereas the film treated at 180 °C is highly celladhesive. Unexpectedly, these two films have approximately the same degree of apparent swelling in a phosphate buffered saline (PBS) solution when the ratio of the thickness of the swollen film in PBS to the thickness of the as-prepared film is compared. The objective of the present study is to understand the physical mechanism behind the seemingly counterintuitive cell adhesion and swelling behaviors. We show that the thermal treatment condition has significant impact on the structure and properties of these PAA/PAAm HB multilayers. Our results suggest that controlling the thermal treatment conditions such as temperature is a versatile method to gradually tailor the properties and functionality of layer-by-layer assemblies, which will be important in their utilization in a variety of applications including biomedicine and energy conversion/storage.

Article

MATERIALS AND METHODS

Materials. Poly(acrylic acid) (PAA) (Mw ∼ 50 000, 25% aqueous solution) is purchased from Polysciences. Polyacrylamide (PAAm) (Mw ∼ 5 000 000−6 000 000), poly(ethylenimine) (PEI) (Mw ∼ 750 000, 50% aqueous solution), and gold-coated silicon wafers (layer thickness ∼ 100 nm) are purchased from Sigma-Aldrich. All chemicals are used without any further purification. Layer-by-Layer Assembly of Multilayer Films. The multilayer films are fabricated on gold-coated silicon wafers that are first cleaned in H2O/H2O2/NH4OH mixture (5:1:1 v/v/v) at 75 °C for 30 min, then rinsed with deionized (DI) water thoroughly, and finally blown dry with compressed air. PEI solution of 10 mM with 10 mM NaCl and 10 mM solutions of PAA and PAAm are prepared in DI water (18.2 MΩ cm), based on the repeat unit molecular weight. The pH values of all solutions are adjusted to 3.0 using HCl. A programmable slide stainer (HMS Slide Stainer, Zeiss) is used to assemble 20 bilayers of a PAA/PAAm film on a PEI-primed substrate. The multilayers are deposited by first immersing the substrate into the PEI solution (for 10 min) and rinsing in pH 3 water for 2 min to form a primer layer for PAA/PAAm multilayers. We found that PAA/PAAm multilayers, even with a low degree of cross-linking, adhere very strongly to PEI-primed gold-coated Si substrates.40 Subsequently, the PEI-modified substrates are alternately immersed into the PAA solution and the PAAm solution (for 10 min each) with a rinse step in pH 3 water for 2 min between the two polymer adsorption steps. After LbL assembly, the film is dried in ambient atmosphere. PAA/PAAm is treated in a vacuum oven at different temperatures (90, 120, 150, 180, and 200 °C) for 8 h. Film Thickness, Refractive Index, and Water Content Determination. The thickness and refractive index of multilayer films deposited onto gold-coated Si wafers are measured using a spectroscopic ellipsometer, Alpha-SE, and the Complete EASE software package (J.A. Woollam). Measurements are taken at an incident angle of 70° and at wavelengths from 380 to 900 nm, and the data are fitted using a Cauchy model. Refractive index values are obtained at the wavelength of 632.8 nm. All measurements are made between 30 and 60 min after the films are thermally treated at different temperatures for 8 h. To measure the degree of swelling of thermally treated PAA/PAAm multilayers in water, a home-built fluid cell based on a design described in the literature is used.41 Assuming that the swelling of films occurs in a direction perpendicular to the substrate, the content of water in a swollen film can be calculated using the following equation: (Ts − Td,r)/Ts, where Ts is the thickness of the swollen film and Td,r is the thickness of the dried film remaining after a treatment in a neutral pH condition. Thermogravimetric Analysis. The dynamic weight changes of films are monitored using a thermogravimetric analyzer (TGA, TA Instruments model SDT 2960). 100 bilayer PAA/PAAm multilayers are assembled on glass slides and then scraped off into a platinum TGA pan. The temperature is ramped at 5 °C/min to 300 °C. Fourier-Transform Infrared (FT-IR) Spectroscopy. A Nicolet 8700 FT-IR spectrophotometer (Thermo Scientific) is used to obtain absorbance spectra of 20 bilayer PAA/PAAm films on ZnSe windows (Phoenix Infrared, MA). Spectra are taken by averaging 256 scans at 4 cm−1 wavenumber resolution. Samples are purged with nitrogen until the carbon dioxide peak disappears prior to spectra collection. Cell Culture. Human mesenchymal stem cells (hMSCs, Lonza) are cultured on tissue culture polystyrene (TCPS) in complete growth medium (GM): α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum, 1% (v/v) L-glutamine (200 mM), 1% (v/ v) penicillin−streptomycin (Sigma). The thermally treated multilayers on gold-coated silicon wafers are sterilized in 70% ethanol prior to cell seeding. For attachment and proliferation assays, hMSCs are seeded onto the sterilized multilayers at a density of 5000 cells/cm2. After 1 and 5 days of culture in GM, samples are fixed in 4% formalin, stained with rhodamine phalloidin and DAPI (Invitrogen), and imaged with an upright fluorescence microscope. As a measure of proliferation, ImageJ is utilized to quantify the number of nuclei in each field of view (2.3 mm2). B

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

Figure 1. Fluorescence images of human mesenchymal stem cells (hMSCs) (5 days after seeding, 5000 cells/cm2) on PAA/PAAm films thermally treated (a) at 90 °C for 8 h and (b) at 180 °C for 8 h. (c) The apparent swelling ratio of each film is defined as the ratio of the thickness of the swollen film in PBS to the thickness of the as-prepared film. hMSCs are stained with rhodamine phalloidin for actin and DAPI for nuclei. Quartz Crystal Microbalance (QCM). An E4 QCM-D unit (QSense Inc.) is used to obtain shear modulus of PAA/PAAm films in PBS by measuring shifts in the frequency and dissipation of multilayercoated quartz crystals. The baseline frequency and dissipation are first recorded with a blank crystal under PBS, and then 20-bilayer films are formed on the crystals. The film on the back side of crystals is carefully but thoroughly removed using DI water. Then, the film is thermally treated at 90 or 180 °C for 8 h, immersed in pH 7.4 PBS for 5 min followed by gentle rinsing in DI water, and dried in ambient atmosphere. After the prepared crystals are loaded in QCM chamber, the frequency and dissipation are monitored by flowing PBS at 100 μL/min at a fixed chamber temperature (25 °C). The measured frequency and dissipation shifts from multiple overtones are then fitted using the Voigt viscoelastic model incorporated in Q-Sense analysis software (QTools) to obtain the shear modulus of films.

Figure 2. Relative film thickness (black square) and refractive index (blue circle) after thermal treatment at different temperatures for 8 h. The refractive indices are measured at 632.8 nm.

■

RESULTS AND DISCUSSION The cell adhesion resistance of thermally treated PAA/PAAm multilayers is highly dependent on the cross-linking temperature. While PAA/PAAm multilayers treated at 90 °C show excellent cell adhesion resistance, consistent with the previous reports,39,42,43 human mesenchymal stem cells (hMSCs) attach and spread aggressively on PAA/PAAm HB multilayers treated at 180 °C (Figure 1). A few cells observed on the 90 °C treated samples have a round shape, whereas cells on the 180 °C treated samples exhibit a highly noncircular and stretched morphology (Figure 1). Although it is expected that 180 °C treated multilayers would swell to a lesser degree and thus facilitate better cell adhesion, the apparent degree of swelling for the two thermally treated samples under physiologically relevant conditions (pH 7.4 PBS) is approximately the same. It should be noted that previous studies have shown a strong correlation between the swelling of multilayers and their cell adhesion properties; that is, highly swelling multilayers tend to resist cell adhesion.39,42,43 While our results indicate that thermal cross-linking can provide a facile method to drastically vary the properties (i.e., cell adhesion) of layer-by-layer films, the apparent swelling and cell adhesion results seem contradictory. To understand the effect of cross-linking temperature on the structure of PAA/PAAm multilayers, we begin by measuring the relative thickness change of dry PAA/PAAm LbL films before and after thermal treatment as a function of temperature. The thickness of PAA/PAAm HB multilayers decreases after thermal treatment and, correspondingly, the refractive index of the films (blue circles) increases as seen in Figure 2. Interestingly, the thickness is reduced by about 10% after thermal treatment at 90, 120, and 150 °C, whereas it decreases by ∼20% at 180 and 200 °C. In addition, the refractive indices

of the films show an abrupt jump between 150 and 180 °C, indicating that the film treated at 180 °C is denser than that treated at 150 °C. It has been previously shown that bound water in poly(allylamine hydrochloride)/PAA multilayers37 and PEI and PAA homopolymers44 are released in the temperature range between 40 and 150 °C. Similarly, the thickness drop observed at 90, 120, and 150 °C is most likely due to the release of physisorbed water from the PAA/PAAm multilayers. In this regard, we hypothesize that the additional decrease in film thickness and the corresponding increase in film density at 180 °C may indicate major thermochemical reactions that could lead to further dehydration in these films. Thermogravimetric analysis (TGA) is performed to obtain detailed insights into the thermochemical behavior of PAA/ PAAm multilayers as shown in Figure 3. The derivative of the mass loss as a function of temperature (blue curve in Figure 3) clearly shows two regions of major mass loss. There is a mass loss of 8.6% between 25 and 150 °C and an additional 14.1% mass loss from 150 to 250 °C. As indicated above, we believe the first mass loss is dominantly due to the release of physisorbed water from the PAA/PAAm multilayers. On the basis of prior studies,32,37 there are two chemical reactions that can further induce dehydration of PAA/PAAm multilayers in the temperature range of the second weight loss. One reaction is cross-linking through imidization between carboxylic acid groups of PAA and amide groups of PAAm, and the other reaction involves anhydride formation between carboxylic acid groups of PAA. Both of these condensation reactions release water, resulting in further mass loss. Molecular scale chemical changes due to thermal treatment are monitored using Fourier-transform infrared (FT-IR) spectroscopy. The spectrum of a PAA/PAAm multilayer with C

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

not sufficient to prevent the partial release of constituent polymers due to the ionization of PAA and the disruption of hydrogen bonding between the two polymers.32 Therefore, these results indicate that it is crucial to consider partial dissolution of the films when they are incubated at temperatures below which major cross-linking reactions are observed.

Figure 3. Thermogravimetric analysis (TGA) of a PAA/PAAm film showing weight percent (black curve) and derivative of weight percent (blue curve) as a function of temperature. The heating rate is 5 °C/ min.

no thermal treatment (black curve in Figure 4) exhibits acid groups in their nonionized form as well as the amide I peak at Figure 5. pH-triggered disintegration of PAA/PAAm films crosslinked at different temperatures. pH-adjusted DI water and pH 7.4 PBS are used.

Loss in PAA/PAAm multilayers in different pH solutions as a function of thermal treatment condition (i.e., temperature) is further investigated. Thermally treated films are immersed in pH-adjusted DI water for 5 min and then dried before their thickness is determined using ellipsometry. The total fraction remaining in each film is determined by the ratio of the thicknesses before and after immersion in pH adjusted water. A significant loss is observed in films with no treatment and thermal treatments at 90 and 120 °C after immersion in pH 4 or higher water. These results indicate that the critical pH at which partially cross-linked PAA/PAAm multilayers undergo dissolution is between pH 3 and 4. In contrast, there is essentially no loss in the films that were thermally treated at 150, 180, and 200 °C for 8 h. This observation suggests a small amount of imidization (as confirmed by FT-IR in Figure 4) induced at 150 °C, which is below the dominant cross-linking temperature detected by TGA (Figure 3), is sufficient to stabilize the film against dissolution. In essence, a few crosslinks per polymer chain are sufficient to convert the LbL multilayers into a cross-linked hydrogel film. We note that although cross-linking occurs dominantly above 150 °C, a small number of imidization reactions can take place at a lower temperature (as low as 90 °C), which has been observed in a similar imidization reaction between amide and carboxylic acid groups in poly(amic acid).45 We summarize the relative thickness change of PAA/PAAm films cross-linked at different temperatures after they have been exposed to different solution conditions. The thickness of the dry films after LbL assembly is set as the reference state of each film (step 1). After thermal treatment for 8 h (step 2) at different temperatures, the film thickness decreases by about 10−20% depending on incubation temperature as described earlier. Subsequently, the films are immersed in pH 7.4 PBS for 5 min. After a gentle rinse in DI water, each film is dried in ambient atmosphere (step 3). About 80% and 65% mass loss is observed in the films treated at 90 and 120 °C, respectively. We observe similar mass loss in these films even if thermal treatments are performed under vacuum (see Figure S1). In contrast, PAA/PAAm films treated at 150, 180, and 200 °C show a slight increase in thickness from step 2. We believe such

Figure 4. FT-IR spectroscopy of PAA/PAAm films as-assembled (black curve) and after thermal treatment at 150 °C for 8 h (red curve), at 180 °C for 8 h (green curve), and at 90 °C for 8 h followed by immersion in PBS for 5 min and subsequent drying at room temperature (blue curve).

1659 cm−1 (CO stretching) and the amide II peak at 1604 cm−1 (N−H mixed mode). After thermal treatment at 150 °C (red curve in Figure 4), small increases in the intensity of the spectrum are observed at 1700 and 1214 cm−1, which are attributed to the formation of imide bonds. Despite fairly long thermal treatment (8 h), cross-linking is not significant at this temperature. In contrast, the film thermally treated at 180 °C for 8 h shows substantial increases in the imide peaks (green curve). Moreover, two peaks at 1804 and 1042 cm −1 responsible for anhydride formation appear in the same film. These results indicate that PAA/PAAm films treated below 150 °C contain a very low degree of cross-linking, although the apparent swelling of 90 and 180 °C treated films is similar. We further investigate this contradiction by monitoring the structural changes in a PAA/PAAm film treated at 90 °C after it has been exposed to a physiological pH condition (pH 7.4 PBS solution) using FT-IR (blue curve in Figure 4). A PAA/PAAm multilayer is heated at 90 °C for 8 h, immersed in PBS for 5 min, and then dried at room temperature before FTIR characterization. Interestingly, the two amide peaks are substantially reduced, indicating a significant loss of PAAm chains in the film. Moreover, a significant loss of PAA chains is also observed. A new peak indicating ionization (COO−) of the remaining carboxylic acid groups appears after PBS (pH 7.4) treatment. The loss in the film can be understood in terms of a very low level of cross-linking in the polymer matrix, which is D

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

an increase in the thickness of the films could be due to the ionization of carboxylic acid groups in the films, which replaces protons with sodium ions from PBS, as well as the enhanced moisture uptake from ambient due to the ionization of carboxylic acid groups. When these films are immersed in PBS (step 4), all of the films swell significantly. The extent of swelling in PBS increases if the films are treated in 0.1 M NaOH solution prior to PBS immersion (step 5) for thickness measurements. We believe the exposure of thermally crosslinked films in the basic solution catalyzes the hydrolysis of anhydrides,46 leading to greater swelling. Amazingly, in PBS, all films swell to ∼300% of their original thickness (step 1). Finally, when films are dried in ambient atmosphere (step 6), the thickness of the films incubated at 150, 180, and 200 °C returns to the thickness in step 3, whereas an additional loss is observed in the films treated at 90 and 120 °C. These results clarify the seemingly contradictory results observed in Figure 1. Even though the apparent swelling of PAA/PAAm multilayers incubated at 90 and 180 °C is approximately the same, the film treated at 90 °C has lost substantial amount of polymers due to partial dissolution and, thus, have much higher water content within the polymer network compared to the film cross-linked at 180 °C. It is an interesting coincidence that the apparent swelling of the two cross-linked PAA/PAAm multilayers (i.e., 90 and 180 °C thermal treatments) is approximately the same although the physical origin of the observed constant apparent swelling is not clearly understood. It should be noted that partial dissolution of HB multilayers has been previously achieved by selectively cross-linking one component in HB multilayers using carbodiimide chemistry and completely removing the other component to generate highly pH-responsive and amphoteric microcapsules.24,47,48 Although a detailed property characterization was not provided, a recent study also has shown that a hydrogen-bonded multilayer made of a weak polyacid (e.g., PAA) and poly(vinyl alcohol) undergoes partial dissolution in a neutral pH condition if the thermal treatment was performed at a low temperature for a short period of time.33 Our study shows that a similar effect can be achieved in PAA/PAAm HB multilayers by varying the extent of crosslinking via thermal treatment. One potential advantage of thermal cross-linking over chemical cross-linking of one polymer is that both polymers remain in the film after dissolution of un-cross-linked polymers, which could be advantageous when the presence of both polymers is required for final applications. On the basis of these results shown in Figure 6, the actual swelling ratio of thermally treated PAA/PAAm films in PBS (step 4) is calculated with the thickness in step 3 as the reference state for each sample. The swelling ratio of the PAA/ PAAm film incubated at 90 °C is as high as 15 when the loss of the film is taken into consideration as shown in Figure 7. As incubation temperature increases, the swelling ratio is eventually decreased to about 3 in the film treated at 150 °C and stays around this range up to the multilayer film incubated at 200 °C. The corresponding water content in swollen films as a function of cross-linking temperature is shown in Figure S2. Previously, the swelling ratio43 and mechanical compliance42,49−51 of multilayer films, rather than protein adsorption and hydrophobicity,52 were identified as the key factors for controlling cell adhesion properties of LbL films. In this regard, if we return to Figure 1, the difference in the adhesion, spreading, and proliferation behavior of hMSCs can be largely

Figure 6. Relative thickness change of PAA/PAAm films in a series of experimental steps, in which the films are thermally treated at different temperatures at step 2.

Figure 7. Swelling ratio of PAA/PAAm films by using the relative film thickness in PBS (step 4 in Figure 6) to film thickness after dried from PBS (step 3 in Figure 6).

understood by the large difference in the actual swelling ratios of the films, which are inversely proportional to cross-linking temperature. Since the swollen film incubated at 90 °C has a very low degree of cross-linking and mostly consists of water (about 94%), once immersed in a physiologically relevant medium, the film is expected to present a high mechanical compliance. The shape of hMSCs on 90 °C treated films indeed resembles the morphologies of cells that are not fully spreading on films with a low Young’s modulus ∼105 Pa.39,51 In contrast, the cells on the film incubated at 180 °C show stretched cell morphologies as previously seen in films with a relatively higher Young’s modulus.39 To confirm the capability of modulating mechanical properties of the swollen multilayers by thermal treatments and subsequent partial dissolution of un-cross-linked polymers, the shear modulus (G) of these films in PBS is measured using a quartz crystal microbalance with dissipation (QCM-D) monitoring. As shown in Figure 8, the shear modulus of a 180 °C treated film (G180 °C ∼ 6.0 × 106 Pa) is 2 orders of magnitude higher than that of a 90 °C treated film (G90 °C ∼ 2.3 × 104 Pa). Previously, Young’s modulus (E) of chemically functionalized PAA/PAAm films has been shown to strongly correlate with cell adhesion. Cell-adhesive films were shown to have E ∼ 107 Pa, whereas cell-resistant films have E ∼ 105 Pa.39 These values are in good agreement with our estimates by converting shear moduli to Young’s moduli (E180 °C ∼ 1.8 × 107 Pa and E90 °C ∼ 6.9 × 104 Pa) using E = 2G(1 + ν), where Poisson’s ratio ν = 0.5 is used. The cross-linking density, determined using the theory of rubber elasticity,53 also shows that the cross-linking density of 180 °C treated films is ∼2 E

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

imidization and anhydride formation) of these multilayer films occurs above 150 °C. When PAA/PAAm films are incubated below this temperature, only a small degree of cross-linking is achieved, and such films undergo partial dissolution when they are immersed in pH 4 or higher water. When the mass loss is taken into consideration, the actual swelling ratio and Young’s modulus of the film treated at 90 °C are approximately 15 and 6.9 × 104 Pa in PBS, respectively, and these films become highly cell-adhesion resistant. In contrast, the film incubated at 180 °C swells about 3.5 times its dry thickness with a high Young’s modulus (1.8 × 107 Pa) in PBS and exhibits celladhesive properties. Our work emphasizes the importance of understanding the effect of thermal treatment on the structure and properties of one particular layer-by-layer film in depth. We believe controlling the thermal treatment conditions such as temperature and treatment duration provides a simple, powerful, perhaps underutilized method to gradually modify the properties of LbL films after their assembly. It can be envisioned, for example, that partial cross-linking of LbL films can lead to mechanically robust membranes that retain high mobility of constituent polymers, which can be useful for energy storage and conversion applications.8 Also, a new class of hydrogel films for biomedical applications can potentially be generated by partially cross-linking LbL films below their complete crosslinking temperature and inducing partial dissolution of uncrosslinked polymers from the multilayers.

Figure 8. Shear modulus of 90 and 180 °C treated PAA/PAAm films measured under PBS.

orders of magnitude greater than that of 90 °C treated films (see Figure S3). We further investigate how the difference in the mechanical compliance of PAA/PAAm films affects the proliferation of hMSCs. At day 1 after cell seeding, the number of cells on 180 °C treated film is equivalent to TCPS control, whereas the cell density on the 90 °C treated film is about half of that on the 180 °C treated film (Figure 9). At day 5, the cell number on 90

■

ASSOCIATED CONTENT

S Supporting Information *

Relative thickness change of films treated at 90 °C with and without applying vacuum, water content in films as a function of incubation temperature, and cross-linking density of 90 and 180 °C treated films. This material is available free of charge via the Internet at http://pubs.acs.org.

■

Figure 9. Proliferation of cells on 90 and 180 °C treated PAA/PAAm films and TCPS as a control (field of view = 2.3 mm2).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

°C treated film is decreased substantially, indicating that hMSCs are not able to proliferate on 90 °C treated film, and some may even detach from the film. In contrast, cell numbers on 180 °C treated film and TCPS control have doubled and quadrupled, respectively. These observations indicate that the mechanical compliance of substrates strongly affects the proliferation of cells as well as cell adhesion to the films. Our results are consistent with previous studies that demonstrated mechanical compliance is a dominant factor that determines the cell adhesion properties of LbL films;39,50,54 we note, however, that the interplay between mechanical compliance and other properties cannot be completely ruled out. Taken together, our results demonstrate that thermal treatment on LbL multilayer films can provide an effective way of modulating cross-linking, swelling, and mechanical compliances of films and thereby controlling the cell-adhesion properties of PAA/PAAm multilayers.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported primarily by an NSF CAREER Award (DMR-1055594) and partly by the PENN MRSEC (DMR1120901). We thank S. Szewczyk for measurements with differential scanning calorimeter. D.L. also acknowledges the support of the Korean-American Scientists and Engineers Association (KSEA), and I.L.K. acknowledges support from a National Science Foundation Graduate Research Fellowship.

■

REFERENCES

(1) Decher, G. Science 1997, 277, 1232−1237. (2) Decher, G.; Eckle, M.; Schmitt, J.; Struth, B. Curr. Opin. Colloid Interface Sci. 1998, 3, 32−39. (3) Decher, G.; Schlenoff, J. B. Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials, 1st ed.; Wiley-VCH: Weinheim, 2003. (4) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396−5399. (5) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117−6123.

■

CONCLUSIONS We have studied the effect of thermal treatment on the crosslinking, dissolution, and swelling of a hydrogen-bonded LbL film containing poly(acrylic acid) (PAA) and polyacrylamide (PAAm). We have revealed that complete cross-linking (i.e., F

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

(42) Thompson, M. T.; Berg, M. C.; Tobias, I. S.; Rubner, M. F.; Van Vliet, K. J. Biomaterials 2005, 26, 6836−6845. (43) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96−106. (44) Yoo, P. J.; Zacharia, N. S.; Doh, J.; Nam, K. T.; Belcher, A. M.; Hammond, P. T. Acs Nano 2008, 2, 561−571. (45) Anthamatten, M.; Letts, S. A.; Day, K.; Cook, R. C.; Gies, A. P.; Hamilton, T. P.; Nonidez, W. K. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 5999−6010. (46) Petersen, J. C.; Barbour, F. A.; Dorrence, S. M. Anal. Chem. 1975, 47, 107−111. (47) Kharlampieva, E.; Erel-Unal, I.; Sukhishvili, S. A. Langmuir 2007, 23, 175−181. (48) Kozlovskaya, V.; Kharlampieva, E.; Erel, I.; Sukhishvili, S. A. Soft Matter 2009, 5, 4077−4087. (49) Richert, L.; Engler, A. J.; Discher, D. E.; Picart, C. Biomacromolecules 2004, 5, 1908−1916. (50) Schneider, A.; Francius, G.; Obeid, R.; Schwinte, P.; Hemmerle, J.; Frisch, B.; Schaaf, P.; Voegel, J. C.; Senger, B.; Picart, C. Langmuir 2006, 22, 1193−1200. (51) Schmidt, S.; Zeiser, M.; Hellweg, T.; Duschl, C.; Fery, A.; Mohwald, H. Adv. Funct. Mater. 2010, 20, 3235−3243. (52) Salloum, D. S.; Olenych, S. G.; Keller, T. C. S.; Schlenoff, J. B. Biomacromolecules 2005, 6, 161−167. (53) Blacklock, J.; Sievers, T. K.; Handa, H.; You, Y. Z.; Oupicky, D.; Mao, G. Z.; Mohwald, H. J. Phys. Chem. B 2010, 114, 5283−5291. (54) Schmidt, S.; Madaboosi, N.; Uhlig, K.; Kohler, D.; Skirtach, A.; Duschl, C.; Mohwald, H.; Volodkin, D. V. Langmuir 2012, 28, 7249− 7257.

(6) Wood, K. C.; Boedicker, J. Q.; Lynn, D. M.; Hammond, P. T. Langmuir 2005, 21, 1603−1609. (7) Becker, A. L.; Johnston, A. P. R.; Caruso, F. Small 2010, 6, 1836− 1852. (8) Lutkenhaus, J. L.; Hammond, P. T. Soft Matter 2007, 3, 804−816. (9) Taylor, A. D.; Michel, M.; Sekol, R. C.; Kizuka, J. M.; Kotov, N. A.; Thompson, L. T. Adv. Funct. Mater. 2008, 18, 3003−3009. (10) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203−3224. (11) Gribova, V.; Auzely-Velty, R.; Picart, C. Chem. Mater. 2012, 24, 854−869. (12) Zhai, L.; Cebeci, F. C.; Cohen, R. E.; Rubner, M. F. Nano Lett. 2004, 4, 1349−1353. (13) Podsiadlo, P.; Paternel, S.; Rouillard, J. M.; Zhang, Z. F.; Lee, J.; Lee, J. W.; Gulari, L.; Kotov, N. A. Langmuir 2005, 21, 11915−11921. (14) Mertz, D.; Vogt, C.; Hemmerle, J.; Mutterer, J.; Ball, V.; Voegel, J. C.; Schaaf, P.; Lavalle, P. Nat. Mater. 2009, 8, 731−735. (15) Shchukin, D. G.; Zheludkevich, M.; Yasakau, K.; Lamaka, S.; Ferreira, M. G. S.; Mohwald, H. Adv. Mater. 2006, 18, 1672−1678. (16) Seo, J.; Lutkenhaus, J. L.; Kim, J.; Hammond, P. T.; Char, K. Macromolecules 2007, 40, 4028−4036. (17) Kharlampieva, E.; Kozlovskaya, V.; Ankner, J. F.; Sukhishvili, S. A. Langmuir 2008, 24, 11346−11349. (18) Kharlampieva, E.; Kozlovskaya, V.; Sukhishvili, S. A. Adv. Mater. 2009, 21, 3053−3065. (19) Yang, S. Y.; Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2004, 20, 5978−5981. (20) Lutkenhaus, J. L.; Hrabak, K. D.; McEnnis, K.; Hammond, P. T. J. Am. Chem. Soc. 2005, 127, 17228−17234. (21) Lutkenhaus, J. L.; McEnnis, K.; Hammond, P. T. Macromolecules 2007, 40, 8367−8373. (22) Sukhishvili, S. A.; Granick, S. J. Am. Chem. Soc. 2000, 122, 9550−9551. (23) Liang, Z.; Cabarcos, O. M.; Allara, D. L.; Wang, Q. Adv. Mater. 2004, 16, 823−827. (24) Kozlovskaya, V.; Kharlampieva, E.; Mansfield, M. L.; Sukhishvili, S. A. Chem. Mater. 2006, 18, 328−336. (25) Zeng, G.; Gao, J.; Chen, S.; Chen, H.; Wang, Z.; Zhang, X. Langmuir 2007, 23, 11631−11636. (26) Ono, S. S.; Decher, G. Nano Lett. 2006, 6, 592−598. (27) Swiston, A. J.; Cheng, C.; Um, S. H.; Irvine, D. J.; Cohen, R. E.; Rubner, M. F. Nano Lett. 2008, 8, 4446−4453. (28) Elsner, N.; Kozlovskaya, V.; Sukhishvili, S. A.; Fery, A. Soft Matter 2006, 2, 966−972. (29) Kozlovskaya, V.; Sukhishvili, S. A. Macromolecules 2006, 39, 5569−5572. (30) Kozlovskaya, V.; Kharlampieva, E.; Drachuk, I.; Cheng, D.; Tsukruk, V. V. Soft Matter 2010, 6, 3596−3608. (31) Yang, S. Y.; Mendelsohn, J. D.; Rubner, M. F. Biomacromolecules 2003, 4, 987−994. (32) Yang, S. Y.; Rubner, M. F. J. Am. Chem. Soc. 2002, 124, 2100− 2101. (33) Lee, H.; Mensire, R.; Cohen, R. E.; Rubner, M. F. Macromolecules 2012, 45, 347−355. (34) Lee, D.; Rubner, M. F.; Cohen, R. E. Chem. Mater. 2005, 17, 1099−1105. (35) Li, Q.; Quinn, J. F.; Caruso, F. Adv. Mater. 2005, 17, 2058− 2062. (36) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978−1979. (37) Shao, L.; Lutkenhaus, J. L. Soft Matter 2010, 6, 3363−3369. (38) Jin, X.; Hsieh, Y. L. Polymer 2005, 46, 5149−5160. (39) Thompson, M. T.; Berg, M. C.; Tobias, I. S.; Lichter, J. A.; Rubner, M. F.; Van Vliet, K. J. Biomacromolecules 2006, 7, 1990−1995. (40) Cebrian, V.; Martin-Saavedra, F.; Yague, C.; Arruebo, M.; Santamaria, J.; Vilaboa, N. Acta Biomater. 2011, 7, 3645−3655. (41) Brunner, H.; Vallant, T.; Mayer, U.; Hoffmann, H. J. Colloid Interface Sci. 1999, 212, 545−552. G

dx.doi.org/10.1021/ma301025a | Macromolecules XXXX, XXX, XXX−XXX