Gold Nanoparticles Induce Surface Morphological Transformation in

Corresponding author. E-mail: [email protected]. Telephone: (886) 4-2284-0510, extension 711. Fax: (886) 4-2285-4734. Address: Professor ...
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Biomacromolecules 2008, 9, 241–248

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Gold Nanoparticles Induce Surface Morphological Transformation in Polyurethane and Affect the Cellular Response Shan-hui Hsu,*,†,‡ Cheng-Ming Tang,† and Hsiang-Jung Tseng† Department of Chemical Engineering, Institute of Biomedical Engineering, National Chung Hsing University, Taichung, Taiwan, R.O.C. Received April 30, 2007; Revised Manuscript Received October 23, 2007

Nanocomposites from a hexamethylene diisocyanate (HDI)-based polyester-type waterborne polyurethane (PU) containing different amounts (17.4–174 ppm) of gold (Au) nanoparticles (∼5 nm) were prepared. The microstructure and physiochemical properties of the nanocomposites were characterized. The cell attachment and proliferation, platelet activation, and bacterial adhesion on the nanocomposites were evaluated. Gold nanoparticles in small amounts induced significant changes in surface morphology and domain structures, from hard segment lamellae to soft segment micelles. These changes resembled the morphological transformation among different mesophases occurred in diblock copolymers. Better cellular proliferation, lower platelet activation, and reduced bacterial adhesion were demonstrated for the PU nanocomposite with 43.5 or 65 ppm of Au than the pure PU or the nanocomposite containing a different amount of Au. The different cellular response on PU-Au nanocomposites was attributed to the extensively modified surface morphology and phase separation in the presence of a small amount of Au nanoparticles.

Introduction Polyurethane (PU) is one of the most interesting synthetic elastomers. Because of the unique properties arising from the microphase separation between hard and soft segments, much attention has been paid to the synthesis, morphology, chemical, and mechanical properties of the polymer. PU is also widely used in biomedical applications because of good biocompatibility and mechanical properties.1,2 Driven by the reduction in costs and the control of organic solvents, the development of waterborne PU formulations has dramatically increased. The waterborne materials present many of the features related to the conventional organic solvent-borne PU with the advantages of low viscosity at high molecular weight, nontoxicity, and good applicability.3 Many solvent-borne PUs have nanometer-sized surface features in the form of hard-segment short cylinders4 or thin lamellae (5–10 nm thick and 40–100 nm long)5 embedded in soft-segment-rich domains. A few PU nanocomposites have also been developed. The addition of 1–5% intercalated silicate layers in a polytetramethylene oxide (PTMO)-based waterborne PU (PU-silicate nanocomposite) has been shown to improve thermal and mechanical properties.6 Gold (Au) is regarded as one of the noble metals with high biocompatibility. Gold nanoparticles are not cytotoxic and do not elicit secretion of proinflammatory cytokines such as TNF-R and IL1-β.7 In our previous studies, the thermal and mechanical properties as well as the biostability of a PTMO-based waterborne PU have been significantly improved when a small amount of Au nanoparticles (∼5 nm, 43.5 ppm) was added.8,9 The surface morphology of * Corresponding author. E-mail: [email protected]. Telephone: (886) 4-2284-0510,extension 711. Fax: (886) 4-2285-4734. Address: Professor Shan-hui Hsu, Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan. † Department of Chemical Engineering, Institute of Biomedical Engineering, National Chung Hsing University. ‡ Institute of Biomedical Engineering, National Chung Hsing University.

the mentioned PU and PU-Au nanocomposites showed predominantly hard segment micelles (instead of the more common lamellae) embedded in a soft-segment-rich matrix. The change in the characteristic diameter (from 93 to 22 nm) of the hard segment micelles upon addition of the gold nanoparticles actually played an important role in the cellular response to these nanocomposites.10 In the current study, another series of PU-Au nanocomposites based on Au nanoparticles and the PU of an entirely different formula (the ester-type PU) were established. The new types of nanocomposites were characterized. The response of fibroblasts, platelets, and bacteria to the nanocomposites and their dependence on Au concentrations were examined. Quite surprisingly, the surface morphological change of the ester-type PU upon addition of Au was completely unlike that which occurred in the earlier system (ether-type PU). Instead of a mere change in the characteristic size of hard domains as in the previous system, for the first time, a phenomenon of surface morphological “transformation” was observed upon addition of a small amount of gold nanoparticles in the polyurethane. The morphological transformation is defined as the transition from one morphological arrangement to another (e.g., from lamellae to micelles). This study revealed that nanoparticles could induce morphological transformation in polyurethane, similar to the “mesophase transition” (the arrangement of two phases of different atomic species11) in diblock copolymers under the appropriate compositions. The cellular response to the polyurethane was also modified as a result of the morphological transformation induced by the Au nanoparticles.

Experimental Section Materials. The polyurethane dispersion and the diisocyanate salt were obtained from Great Eastern Resins Industrial Co., Taiwan. The polyurethane dispersion (with 50% solid content in distilled water, catalogue no. 6608) was synthesized using hexamethylene diisocyanate

10.1021/bm700471k CCC: $40.75  2008 American Chemical Society Published on Web 12/29/2007

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Hsu et al.

Figure 1. Chemical composition of the polyurethane used in the study.

(HDI) and the macrodiol poly(butadiene adipate) (average molecular weight 2000) at a molecular ratio 3:1 and chain-extended by ethylene diamine sulfonate sodium salt and ethylene diamine. The diisocyanate salt (catalogue no. 368) was a mixture of the isocyanurate trimer of hexamethylene diisocyanate (HDI trimer) and 6% Bayer hardener (made from the HDI trimer and polyethylene glycol). The chemical composition of the polymer used in this study is illustrated in Figure 1. The hard-segment weight fraction of the polymer was about 34.6%. Au nanoparticles were manufactured by Global NanoTech, Taiwan, and supplied in solution state. The solution was a dispersion of pure gold fine particles in distilled water (50 ppm/ml, which was determined by ICP-AA). The diameter of the Au nanoparticles was in the range of 4–7 nm and about 5 nm in average, confirmed by transmission electron microscopy.8 Preparation of Polyurethane-Gold (PU-Au) Nanocomposite Films. The mentioned polyurethane dispersion was diluted by distilled water or the Au solution to 10 wt % solid content. The diisocyanate salt was added to the polyurethane dispersion (the concentration of the diisocyanate salt being 1 wt %), and the mixture was then stirred for 30 min to obtain the suspension of plain polyurethane (abbreviated “PU”) or the suspensions of polyurethane-gold (abbreviated “PU-Au”) nanocomposites used in this study. The PU-Au suspensions were prepared to contain 17.4-174 ppm of Au in the final nanocomposites after water removal by adding a calculated amount of gold nanoparticles into the system. Thin films (∼0.02 mm) were cast from the PU or PU-Au suspensions on 15 mm round glass cover slips by a spin-coater (PM-490, Synrex, Taiwan), dried at 60 °C for 48 h, and further dried in a vacuum oven at 60 °C for 72 h to remove any residual solvent. The thicker films (∼0.2 mm) used in most physicochemical characterization and in the bacterial adhesion test were prepared by casting the above suspensions in Teflon molds to facilitate the release of dried samples. Characterization of the PU-Au Nanocomposites. For transmission electron microscopy (TEM), thin sections from PU-Au nanocomposites were microtomed using an ultramicrotome apparatus (Reichert-Jung Ultracut E, Leica Microsystems, Germany) equipped with a diamond knife and subsequently deposited on copper grids. The specimens were examined under a JEOL JEM-1200 microscope operated at 110 kV. The surface morphology of spin-coated films was examined under an atomic force microscope (AFM) (CP-II, Veeco) equipped with a 100 µm piezoelectric scanner. The images were obtained in the tapping

mode in air with a cantilever (force constant of 5–37 N/m) supporting an integrated pyramidal tip of silicon (PPP-SEIHR-50, Nanosensors, Switzerland). Topography and phase images were recorded simultaneously. Comparisons of the phase images among different materials were made on those images obtained with a similar tapping condition (with a free oscillation amplitude A0 ) 108 nm and a set point ratio Asp/A0 ) 0.3). Phase images provide a sharp contrast of fine structural features and emphasize the differences in mechanical properties of different components. Tapping mode AFM generates phase images with dark features corresponding to the regions of lower modulus (soft domains) and bright features corresponding to the regions of high modulus (hard domains).5 The root-mean-square (rms) average of the surface roughness values was calculated as the standard deviation of all the height values within the given area. The average size of hard or soft domains from the phase images was obtained by the image analysis using the Image Pro Plus 4.5 software (Media Cybernetics). Each sample was scanned on at least three different locations. The reproducibility of AFM images was also confirmed for samples cast from three different batches. The typical AFM images were shown. The surface contact angle (θ) was measured in the air by a static contact angle meter (CA-D, Kyowa Interface Science, Japan) at 25 °C and 70% relative humidity. Twenty µL of distilled–deionized water droplets were used. At least six readings were made on different parts of the films and were averaged. The thermogravimetric analysis (TGA) was carried out with a thermogravimetric analyzer (TGA2050, TA Instruments). The samples (5 mg) in a platinum crucible were heated at a rate of 10 °C/min under nitrogen. The pyrolytic temperatures (Tonset and Tp) were obtained from the TGA curves. The glass transition temperature (Tg) and the hardsegment crystallization temperature (Tc) as well as the associated enthalpies (∆Hf) of the PU and PU-Au nanocomposites were further determined with a dynamic scanning calorimeter (DSC) (SSC/5200, Seiko instruments, Japan). The DSC was calibrated using indium as a standard. Each of the samples was sealed into an aluminum pan with an empty pan as the reference. The measurements were performed in the air at a constant heating rate of 10 °C/min in the temperature range from -100 to 200 °C. The dynamic mechanical analysis (DMA) was performed in a dynamic mechanical analyzer (DMA 2980, TA Instruments). The samples were cooled down to -100 °C and then heated at a rate of 5 °C /min to 60 °C. The Tg of the sample was defined as the temperature

Au Nanoparticles Morphological Transformation in PU where the loss tangent (tan δ) reached a maximum. The dynamic moduli, including the storage modulus (E′) and the loss modulus (E′′), were recorded simultaneously. PU and PU-Au nanocomposites were chemically analyzed using a Fourier transform infrared spectrometer (Spectrum one FT-IR, PerkinElmer). Each sample was scanned eight times in the spectral region of 4000–400 cm-1 with a resolution setting of 2 cm-1 and averaged to produce each spectrum. The surface of the samples was analyzed by the attenuated total reflectance infrared spectroscopy (ATR-IR) in the spectral region of 4000–600 cm-1. Cell Attachment and Proliferation. Human gingival fibroblasts were obtained from a healthy human donor in a dental clinic by following the ethical guidelines. The gingival tissue of the graft recipient was removed by partial thickness flap, and the connective tissue was harvested. The tissue was minced into 0.5 mm3 pieces and explanted in 60 mm culture dishes.10 Tissue explants were cultured in alpha minimum essential medium (R-MEM) supplemented with 10% fetal bovine serum and antibiotics (penicillin G 100 U/mL, streptomycin 100 µg/mL, and gentamicin 50 µg/ml). The culture was incubated in a 37 °C/5% CO2 incubator. After 10–14 days, the nearly confluent primarily cultured cells were trypsinized using 0.05% trypsin-EDTA solution. Cells were expanded by standard cell culture techniques in 25 cm2 tissue culture flasks containing 5 mL of 10% serum supplemented medium. Cells of the eighth passage were used. PU and PU-Au nanocomposites on cover slips were sterilized by 70% ethanol, washed by the sterilized phosphate buffered saline (PBS), and placed into the bottom of 24-well tissue culture plates. One mL of cell suspension with a density of 5 × 104 cells/mL was injected into each well of the plates. After 24 and 48 h of incubation, the adherent cells were trypsinized, centrifuged, and resuspended for cell counting by a hemacytometer combined with an inverted phase contrast microscope (TE-300, Nikon, Japan). Platelet Activation Test. For platelet adhesion and activation, substrates were placed in a 24-well culture plate, and 0.5 mL of the platelet-rich plasma (∼2 × 106 platelets/µL, obtained from the Chinese Blood Foundation, Taiwan) were added to each well. After incubation for 1 h, samples were taken out and gently rinsed with the HEPES buffered saline. In one duplicate, the adherent platelets were detached by trypsin and counted by a cell counter. In another duplicate, samples were fixed by the HEPES buffered glutaraldehyde, dehydrated in the ethanol solutions of increasing concentrations, critical point dried, sputter-coated with gold, and examined by a scanning electron microscope (SEM) (S-3000, Hitachi, Japan). The common morphological change (including five stages) during platelet activation was used as a tool routinely2 to define the degree of activation of a platelet quantitatively in this study: 0 ) round (unactivated), 1/4 ) dendritic (pseudopodial but no flattening), 1/2 ) spread-dendritic (flattened pseudopodia), 3/4 ) spreading (late pseudopodial with hyaloplasm spreading), and 1 ) fully spread (totally activated). The average degree of platelet activation (0.0–1.0) was calculated based on ∼50 adherent platelets observed under the SEM. Bacterial Culture and Adhesion on Polymeric Films. Bacillus subtilis (BCRC 10447) was used for the bacterial adhesion experiment.12 Cultures were grown overnight in a shaker incubator (100 rpm) at 37 °C. An overnight culture of Bacillus subtilis was washed in PBS and centrifuged at 6500 rpm for 5 min. The cell pellet was resuspended in 1 mL of PBS and diluted to 2 × 105-3 × 106 colony forming units (CFU)/mL. The samples (7.5 mm in diameter, 1 mm in thickness) were sterilized by 70% ethanol and then washed by PBS. Each sample was placed into a glass tube. One mL of the bacterial suspension and 9 mL nutrient media (Bacto, France) were added into each tube. The tubes were incubated in the shaker incubator. After 12 h, samples were rinsed twice by sterile water and then placed into another tube. Two mL of PBS were added into each tube, and the bacteria were detached from the sample surface by an ultrasonic cleaner. One mL of the bacterial suspension was diluted to 10-, 100-, and 1000-fold with PBS. Diluted

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Figure 2. TEM micrographs for PU-Au nanocomposites containing (a) 17.4 ppm, (b) 43.5 ppm, (c) 65 ppm, and (d) 174 ppm of Au nanoparticles.

suspension (0.1 mL) was then spread in an agar dish and incubated for 24 h at 37 °C. The bacteria were then counted manually per dish. Statistical Analysis. Multiple samples were collected in each measurement (n ) 6) and expressed as mean ( standard deviation. The method of single factor analysis of variance (ANOVA) was used to assess the statistical significance of the results. p values less than 0.05 were considered significant.

Results Characterization of the Nanocomposites. The distribution of Au nanoparticles in the PU matrix was visualized by TEM and the images are shown in Figure 2. The nanoparticles (∼5 nm in the average size) at the concentration of 17.4 ppm were well dispersed in the PU matrix. At 43.5 ppm, some nanoparticles were drawn together but the size remained near 5 nm. At 65 ppm, very slight aggregation occurred and the average size of the clusters increased to ∼7 nm. At 174 ppm, some nanoparticles formed large aggregates that could reach ∼50 nm in size. An overloading of Au nanoparticles in the PU matrix started at a concentration of about 65 ppm. The surface AFM images of PU and nanocomposites are shown in Figure 3. The rms roughness of PU and PU-Au obtained from the AFM topography was in a narrow range of 2.0–2.8 nm, indicating that the surface was similarly flat for all nanocomposites. The topography diagrams provided little information about the surface, while the phase diagrams revealed more details. Still, by comparing the topography with phase diagrams, the darker area in topography corresponded to the brighter area in phase, i.e., the hard segments slightly descended and the soft segments slightly protruded. Judging from the phase diagram, the original PU surface was dominated by thin hard segment lamellae (14.3 ( 2.4 nm in width). PU-Au 17.4 ppm demonstrated fewer hard segment lamellae. Instead, the lamellae were mixed with a network of hard domains. At this time, some soft micelles also showed up. On the surface of PU-Au 43.5 ppm, the lamellae were completely absent and replaced with

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Hsu et al. Table 1. Surface Characterization of the PU and PU-Au Nanocomposite Films

materials

rms roughness (nm)

main surface morphology (and characteristic size)

2.84 ( 0.01 hard segment lamellae (width 14.3 ( 2.4 nm) PU-Au 17.4 ppm 2.47 ( 0.18 hard segment lamellae (width 16.4 ( 2.9 nm) mixed with a network of hard domains PU-Au 43.5 ppm 2.01 ( 0.05 soft segment micelles (diameter 27.3 ( 2.8 nm) PU-Au 65 ppm 2.13 ( 0.05 hard segment micelles/lamellae (dotted) (diameter 24.9 ( 3.7 nm /width 18.4 ( 3.5 nm) PU-Au 174 ppm 2.48 ( 0.13 hard lamellae mixed with a network of hard domains, some aggregates (width 22.6 ( 2.5 nm) PU

Figure 3. AFM images of topography (left) and phase (right) for (a) pure PU, and PU-Au nanocomposites containing (b) 17.4 ppm, (c) 43.5 ppm, (d) 65 ppm, and (e) 174 ppm of Au nanoparticles.

plenty of soft segment micelles (27.3 ( 2.8 nm in diameter). When Au was further increased (as in PU-Au 65 ppm), the soft micelles were still seen but started to deviate from circles.

Meanwhile, thin lamellae were mixed with micelle-like irregular hard domains, both of which had a dotted/granular appearance. On the surface of PU-Au 174 ppm, the dotted irregular hard domains expanded in size with areas of aggregation. The lamellae became thicker (22.6 ( 2.5 nm) while the soft micelles also existed. The changes of surface morphology in PU upon addition of Au are summarized in Table 1. A comparison among the phase images revealed that the surface morphology of the original PU and that of PU-Au 43.5 ppm were very distinct, each dominated by hard lamellae and soft micelles. The morphology of PU-Au 17.4 ppm appeared to be an intermediate of the lamellae in PU and the micelles in PU-Au 43.5 ppm. PU-Au 65 ppm on the other hand demonstrated more hard micelles (with a dotted/granular look). PU-Au 174 ppm resembled PU-Au 17.4 ppm in a manner that they both seemed to be an intermediate between PU and PU-Au 43.5 ppm, except that aggregation of hard domains was evident on PU-Au 174 ppm. In spite of the different surface morphology, the contact angle of PU-Au nanocomposites fell into a very narrow range (71–73°) and was higher than that of the original PU (∼62°). The PU nanocomposites were therefore a little more hydrophobic than the original PU. The pyrolytic temperatures (Tonset and Tp) were defined from the TGA curves. The transition temperatures (Tg and Tc) and the associated heat of fusion (∆Hf) were obtained from the DSC curves. The data are summarized in Table 2. When Au nanoparticles were added, the pyrolytic temperatures shifted to higher values and reached to the maximum at 43.5 ppm of Au. After that, the pyrolytic temperatures decreased with the increased Au concentrations. The change of Tc upon addition of Au followed a similar tendency. On the other hand, the dosedependent variation of Tg showed an opposite trend, i.e., Tg decreased upon the initial addition of Au (17.4–43.5 ppm) but increased thereafter (65–174 ppm). Because the lower Tg or the higher Tc indicates more complete microphase separation in PU,1 the degree of phase separation was ranked in the order of PU-Au 43.5 ppm ∼ PU-Au 65 ppm > PU-Au 17.4 ppm > PU-Au 174 ppm> PU. The heat of fusion associated with Tc was connected to the degree of crystallinity in the nanocomposite. The PU-Au nanocomposites had a slightly higher heat of fusion compared to the original PU. Therefore, the degree of crystallinity was slightly increased upon addition of Au. The degree of crystallinity was approximately in the order of PU-Au 43.5 ppm > PU-Au 17.4 ppm ∼ PU-Au 65 ppm > PU-Au 174 ppm > PU, with large standard deviation.

Au Nanoparticles Morphological Transformation in PU

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Table 2. Pyrolytic Temperatures (Tonset and Tp), Transition Temperatures (Tg and Tc), and Heat of Fusion from the Thermal Analysis of Pure PU and PU-Au Nanocompositesa materials PU PU-Au PU-Au PU-Au PU-Au

17.4 ppm 43.5 ppm 65 ppm 174 ppm

Tonset (°C)

Tp (°C)

Tg (°C)

Tc (°C)

∆Hf (J/g)

323.20 ( 0.50 326.82 ( 0.49 329.19 ( 0.33 327.43 ( 0.33 323.84 ( 0.76

395.60 ( 0.11 400.82 ( 0.33 403.33 ( 0.28 403.40 ( 0.02 394.42 ( 0.06

-41.22 ( 0.72 -51.17 ( 0.96 -53.54 ( 0.54 -52.66 ( 0.59 -46.74 ( 0.78

46.96 ( 0.21 47.56 ( 0.08 48.15 ( 0.22 48.18 ( 0.05 47.53 ( 0.11

60.0 ( 2.5 63.8 ( 4.3 65.1 ( 5.0 63.6 ( 3.3 61.4 ( 3.1

a Tonset: onset temperature of pyrolysis, obtained from TGA curves at 95% weight. Tp: peak pyrolytic temperature, obtained from TGA curves at 50% weight. Tg: glass transition temperature, obtained from the first derivative of DSC curves. Tc: hard-segment crystallization temperature, obtained from the first derivative of DSC curves. ∆Hf: heat of fusion associated with Tc, obtained from the area of the melting peaks in DSC curves.

Table 3. Mechanical Properties and the Glass Transition Temperature (Tg) from the Dynamic Mechanical Analysis of Pure PU and PU-Au Nanocomposites materials

Tg (°C)

E′ (MPa) at 37 °C

E′′ (MPa) at 37 °C

tan δ at 37 °C

PU PU-Au 17.4 ppm PU-Au 43.5 ppm PU-Au 65 ppm PU-Au 174 ppm

-29.30 ( 0.01 -30.20 ( 0.05 -31.42 ( 0.02 -30.62 ( 0.01 -30.11 ( 0.01

139.9 ( 0.45 220.1 ( 0.50 228.2 ( 0.30 219.1 ( 2.56 197.2 ( 1.38

15.8 ( 0.05 18.2 ( 0.02 19.3 ( 0.12 19.2 ( 0.27 17.4 ( 0.15

0.113 ( 0.001 0.083 ( 0.001 0.084 ( 0.001 0.088 ( 0.001 0.088 ( 0.001

The results from DMA are listed in Table 3. The Tg values were higher than those obtained from DSC, but the Au dosedependent variation remained in a similar fashion. E′ values increased in all nanocomposites. PU-Au 43.5 ppm showed the highest E′, followed by PU-Au 17.4 ppm and PU-Au 65 ppm. The data of E′′ and tan δ were also included in Table 3 as supporting information. E′′ was higher for the nanocomposites containing 43.5–65 ppm of Au where Tg was lower (i.e., rubbery and energy-dissipating). Lower tan δ occurred in the nanocomposites containing 17.4–43.5 ppm of Au, consistent with the higher crystallinity (i.e., solid-like). One percent of nanoparticles such as TiO2, nanoclay, or carbon nanotubes were reported to cause a 7–17 °C increase in the pyrolytic temperature,13 while a much smaller amount (43.5–65 ppm) of gold nanoparticles caused ∼8 °C of increase in this study. It has been suggested that nanoparticles tended to distribute at the interface of a twopolymer blend and stabilize the interface.14 Therefore, the change in thermal transition of the PU-Au nanocomposites could be due to those associated with the interface between hard and soft domains, e.g., the change of interfacial energy. The exact mechanism for the change in thermal transition, however, was not fully understood. The addition of a small amount of Au did not cause a remarkable change in the transmission IR spectra of PU. However, a close examination of the carbonyl bands for the nanocomposites in Figure 4a revealed that only one band at 1733 cm-1 (the free carbonyl band in the hard segment) existed for the pure PU, while two bands each at 1733 cm-1 (the free carbonyl band) and 1686 cm-1 (the hydrogen-bonded carbonyl band) were observed in PU-Au 43.5 ppm and PU-Au 65 ppm. The band at 1686 cm-1 was not evident in either PU-Au 17.4 ppm or PU-Au 174 ppm. These results indicated that the hydrogen bonding was enhanced upon addition of a suitable amount of Au (43.5–65 ppm). ATR-IR spectra had a smaller -NH band (around 3373 cm-1) than the transmission IR spectra for all samples, indicating that the surface was more enriched with the soft segment compared to the bulk. The difference in carbonyl bands among the samples was not as evident as that observed in the transmission IR spectra. In contrast, the ATRIR spectra of all samples had a large 1726 cm-1 peak and a tiny 1689 cm-1 peak, i.e., no obvious change in the free or hydrogen-bonded carbonyl band. Nevertheless, there was a shift in the peak location of the -NH band from 3373 to 3306 cm-1 upon addition of Au, as shown in Figure 4b, suggesting

hydrogen bonding with the Au nanoparticles.15 The shift of -NH peak location was greatest (3373 to 3306 cm-1) at 43.5 ppm of Au and least (3373 to 3361 cm-1) at 174 ppm of Au. Cell Attachment and Proliferation. The number of cells on PU and PU-Au nanocomposites is shown in Figure 5. At 24 h, there were more cells on the nanocomposites than on the

Figure 4. Infrared spectra of PU and PU-Au nanocomposites: (a) transmission IR spectra, (b) ATR-IR spectra.

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Figure 5. Attachment and proliferation of human gingival fibroblasts on PU and PU-Au nanocomposites. Significance (p < 0.05): *greater than PU, **greater than all the other samples. TCP: tissue culture polystyrene. Table 4. Platelet Adhesion and Activation on the Surface of PU and PU-Au Nanocompositesa

materials PU PU-Au PU-Au PU-Au PU-Au

17.4 ppm 43.5 ppm 65 ppm 174 ppm

number of adhered platelets ( × 103)

average degree of activation (0.0–1.0)

22.3 ( 0.6 22.0 ( 1.4 17.3 ( 0.6*,** 19.7 ( 1.5* 22.3 ( 0.6

0.79 ( 0.09 0.68 ( 0.11* 0.43 ( 0.12*,** 0.33 ( 0.12*,** 0.69 ( 0.11*

a Significance (p < 0.05): * smaller than PU, ** smaller than all the other samples.

original PU, but there was no statistically significant difference in cell number among the different nanocomposites. At 48 h, the number of cells on PU-Au 43.5 ppm and PU-Au 65 ppm was greatest, followed by PU-Au 17.4 ppm and PU-Au 174 ppm. Platelet Activation. The response of human blood platelets to the surface is summarized in Table 4. The amount of platelet adherence on PU-Au 43.5 ppm and PU-Au 65 ppm was significantly lower than that on the other samples. The platelet activation was highest on the original PU and lowest on the samples of PU-Au 43.5 ppm and PU-Au 65 ppm. Bacterial Adhesion. All PU-Au nanocomposites showed much lower bacterial adhesion than the original PU, as shown in Figure 6. The bacterial adhesion on PU-Au 43.5 ppm was the lowest, followed by PU-Au 65 ppm. PU-Au 17.4 ppm appeared to present a smaller number of bacteria than PU-Au 174 ppm; however, the difference was not statistically significant (p ∼ 0.4).

Discussion It was observed in this study that the surface morphology of the HDI-based PU was significantly modified by the existence of a small amount of gold nanoparticles. The change in surface morphology was previously reported but was less complicated in another system of PU-Au nanocomposites, where the hard segment micelles changed their size upon addition of Au nanoparticles.9 In the current system, the surface of the original PU showed hard segment lamellae (∼14 nm thick), which was the common surface morphology of PU when the hard segment content was relatively higher.4 The surface of PU-Au 17.4 ppm possessed lamellae as well as the micelle-like hard and soft domains. It seemed that the presence of gold nanoparticles on

Hsu et al.

Figure 6. Bacterial adhesion on PU and PU-Au nanocomposites. Significance (p < 0.05): *smaller than PU, **smaller than all the other samples, + smaller than PU-Au 17.4 ppm and PU-Au 174 ppm.

the surface facilitated micelle formation and interfered with the ability of the hard segments to form lamellae. On the surface of PU-Au 43.5 ppm, the soft segments formed isolated micelles, and the extent of phase separation was greatest at this time. As will be discussed later, the phase separation was probably stabilized by hydrogen bonds brought about by the Au nanoparticles. When the Au contents were further increased, the regular soft micelles started to lose the battle for the surface, possibly due to an overload of Au nanoparticles. A related and interesting phenomenon has been demonstrated recently in various hybrids of nanoparticles/block copolymer. Generally, the original diblock copolymers were found in a variety of mesophasic structures such as spheres in bodycentered lattices (BCC) and cylinders packed in hexagonal lattices (hex), depending on the relative fraction of the two blocks (Figure 7a).16 Nanoparticles in the hybrids could be sequestered into one domain to form ordered hybrids. For examples, CdS nanoparticles (3.5 nm in diameter with mercaptoacetic acid as the surfactant) in polystyrene-block-poly(4vinylpyridine) (PS-b-P4VP, the volume fraction of P4VP being 0.3) were segregated in the P4VP microdomains, possibly through the mediation of dipole–dipole interaction between the carboxylic acid groups of mercaptoacetic acid on the CdS nanoparticle surface and the P4VP chains. The morphology of the diblock copolymer at 7% CdS (with respect to the PV4P block) was transformed from a hexagonally packed cylinder structure (hex) in the absence of CdS into a lamellar structure (lam) by hydrogen bonding.17 In another example, CdSe nanoparticles (4 nm in diameter coated with the low surface energy hydrocarbon γhydrocarbon ) 30–33 mN/m) in polystyreneblock-poly(2-vinylpyridine) (PS-b-P2VP) copolymer were observed to concentrate on the surface of the P2VP microdomains and orient the domains normal to the surface.18 The above observations demonstrated that nanoparticles had the ability to induce significant morphological transformation in diblock coplymers. Analogous to the above observations, the surface morphology of PU in this study was significantly modified by introducing nanoparticles that were preferentially segregated on the surface of hard segment. Au nanoparticles and the hard segment had a dipole–dipole interaction (hydrogen bonding), as evident by the IR spectra. The interaction acted as a driving force for morphological transformation. As illustrated in Figure 7, the transformation of PU induced by Au nanoparticles resembled that originally described for diblock polymers upon

Au Nanoparticles Morphological Transformation in PU

Figure 7. (a) Schematics showing the transformation of morphology in diblock copolymers upon increase in the volume fraction of one component and the continuous morphological changes of PU surface upon increase in the weight fraction of the hard segment. (b) Surface morphological changes of PU-Au nanocomposites and their corresponding states.

an increase in the volume fraction of one of the blocks (lam-hex-BCC).16 PU, PU-Au 17.4 ppm and PU-Au 43.5 ppm each adopted lam-like, hex-like, and BCC morphology. The transformation from “hex” to “lam” upon addition of semiconductor nanoparticles has been reported in literature as described earlier.17 The continuous transformation of BCChex-lam-hex-BCC in PU with increasing hard segment contents has also been observed.1,19 Therefore, it sounded logical that morphological transformation of PU could be induced by gold nanoparticles. Beyond the concentration of 43.5 ppm, Au nanoparticles appeared to cause a revert from BCC morphology to hex-like (in PU-Au 65 ppm) and lam-like (in PU-Au 174 ppm) morphology except for the emergence of dotted hard micelles in the former and the aggregation of hard domains in the latter. It has been deduced by theoretical modeling that variations in the particle interaction energies or sizes could induce transitions between the different mesophases formed by the system.20 The Au nanoparticles in PU-Au 65 or 174 ppm were greater in size based on the TEM pictures. It has also been reported that an excess of nanoparticles at the interface could destroy the lamellar morphology.17 An overloading of Au nanoparticles in the hard domains thus could cause the dotted curvature of the hard lamellae (PU-Au 65 ppm) and structural destruction as a result of Au aggregation (PU-Au 174 ppm). The extent of phase separation predicted from Tg and Tc agreed with that estimated from the surface AFM images (i.e., PU-Au 43.5 ppm ∼ PU-Au 65 ppm > PU-Au 17.4 ppm > PU-Au 174 ppm > PU). In addition, our DSC study demonstrated that the melting peaks in PU-Au composites were narrower than those in the pure PU (data not shown). This result was in concord with the finding that the melting peak in 1%

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polypropylene (PP)/single-walled carbon nanotubes (SWNTs) composite was narrower compared to that in the pure PP.21 The observation suggested that Au nanoparticles improved the homogeneity of crystallization for PU probably through nucleation, by which the crystallites became more uniform in size. The values of heat of fusion reflecting the relative amount of crystallinity also supported the Au-induced crystallization behavior of the nanocomposites. When the surface lamellae of PU disappeared at 43.5 ppm of Au, the degree of crystallinity slightly increased. Au nanoparticles in our earlier work functioned as the nucleating agent for crystallization, enhanced hydrogen bond formation in the PTMO-based PU matrix, and improved the biostability and biocompatibility.7,8 As in the previous PU system, Au nanoparticles may have played a similar role as the nucleating agent for crystallization, enhanced the hydrogen bonding and stabilized the phase separation in the current HDI-based poly(ester urethane) matrix. Transmission IR spectra confirmed that an appropriately small amount of Au nanoparticles could enhance the formation of hydrogen bonds at the carbonyl site of the hard segment. In addition, the nanocomposite at 174 ppm of Au had large-size aggregation of Au particles that may have destroyed the hydrogen bonding between soft and hard segments (i.e., disappearance of the hydrogen-bonded carbonyl band in the spectrum). ATR-IR spectra showed that there was interaction between Au nanoparticles and the nitrogen of the -NH bond of the original PU (3373 cm-1) on the surface. A shift of the -NH peak in PU occurred upon addition of Au. The shift was largest (67 cm-1) at 43.5 ppm of Au. It has been reported that when silver nanoparticles were coated on the surface of PU, a significant shift (63 cm-1) in the -NH peak was observed.15 This interaction at the interface was presumed to serve as an inducing force for the morphological transformation.20 Solvent-casting and spin-coating were both used to prepare the samples. It was noticed that the bulk properties were not influenced by the casting method. The surface properties only slightly varied with the casting method. Because AFM generated more clear images for spin-coated films, all the measurements of surface properties were performed on the spin-coated films. The cellular response to PU was improved upon addition of a small amount of Au in the current system. A comparison with the previous PTMO-based PU system revealed that, in spite of the completely different PU formulas and the distinct surface morphology,9 the performance of PU was improved by Au nanoparticles in both systems. And most interestingly, the Au concentration for achieving the best performance in the two distinct PU systems was coincidentally in a similar range (∼43.5 ppm). At 43.5 ppm, the nanoparticles were well dispersed (not aggregated) in both systems. Under this circumstance, the two PU-Au nanocomposites demonstrated the best physical and biological properties. The performance of these nanocomposites was thus closely associated with the dispersity of the nanoparticles. Shi et al. reported that the addition of 0.05% SWNTs into poly(propylene fumarate) at low concentrations increased the rheological and mechanical properties.22,23 These effects decreased at higher concentrations due to aggregation of SWNTs. The observations suggested that the dispersion of nanomaterials be a critical factor to determine the final properties of the nanocomposites. In addition, the coincidence in concentration suggested that the limitation of dispersibility for the metal nanoparticles in polyurethane may have a stoichiometric nature. On the basis of our calculation, at 43.5 ppm, one 5 nm gold nanoparticle on average had to interact with 99.8 µmol of -NH in the previous PTMO-based waterborne PU. In the current PU

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system, one 5 nm gold nanoparticle (43.5–65 ppm) had to interact with 119.6–80.0 µmol, or on average 99.8 µmol, of -NH. These two values were interestingly identical. The limitation of dispersibility for the system may account for the low concentration to achieve the regionally best performance. For the same reason, improving the dispersibility of Au at higher concentrations may further increase the performance of PU-Au nanocomposites. The surface contact angle increased a little in the nanocomposites (∼10° higher than that of the orginal PU). This could be attributed to the slight increase of the C/O atomic ratio on the nanocomposite surface. The latter was confirmed by the data from X-ray photoelectron spectroscopy (XPS), where the atomic ratio of C/O increased from ∼0.49 for PU to ∼0.55 for the nanocomposites. XPS data showed no Au on the surface (i.e., no peak around 84.4–88.0 eV)24 and only slight change of the atomic ratio in each nanocomposite. The fibroblast response to PU-Au had no general relation with the contact angle but was in good accordance with the enhancement in the microphase separation and the changes in surface morphology and so were platelet activation (negative correlation) and bacterial adhesion (negative correlation). The unified positive change in the biological performance of PU with microphase separation has been observed earlier.9,25,26 The favorable biological response of PU-Au 43.5–65 ppm was attributed to the distinct phase separation and surface morphology in the presence of Au. The synergistic interactions between nanoparticles and a phaseseparated material can lead to a significant alteration of the surface under very small amount of gold nanoparticles. Many studies have attempted to correlate the microphase separation with the biocompatibility of PU while difficulty remains in separating the chemical influence.25 Because the tiny amount of Au added should not determine the chemical composition, this PU-Au system served as a good model to investigate the effect and identify the role of microphase separation of PU in cell behavior. On the basis of the results from the current and the previous PU systems, gold nanoparticles with a size of 5 nm that was smaller than the characteristic size of hard segment could participate in the microphase separation process of PU, leading to either “morphological transformations” or “featuredsize changes”. It is thus possible to manipulate the properties and the performance of PU materials by using gold nanoparticles of the appropriate sizes and concentrations.

Conclusions The effect of Au nanoparticles on the surface, thermal, and mechanical properties of HDI-based waterborne poly(ester urethane)-Au nanocomposites, as well as the impact on the cellular response, was investigated. All properties and performances were improved by the addition of Au nanoparticles,

Hsu et al.

especially when the Au concentrations were in an appropriate weight range (43.5–65 ppm). Au nanoparticles induced surface morphological transformations in PU, which played an important role in determining the cellular responses to the current polyurethane-Au nanocomposites. Acknowledgments. This work was supported by the National Health Research Institutes and the National Science Council, Taiwan, and conducted in the Center of Tissue Engineering and Stem Cells Research, National Chung Hsing University. The corresponding author is jointly appointed to the Center of Nanoscience and Nanotechnology of the university.

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