Effect of Urea on Phase Transition of Poly(N-isopropylacrylamide) and

Article pubs.acs.org/Macromolecules

Effect of Urea on Phase Transition of Poly(N‑isopropylacrylamide) and Poly(N,N‑diethylacrylamide) Hydrogels: A Clue for Urea-Induced Denaturation Jian Wang,†,‡ Biaolan Liu,†,‡ Geying Ru,† Jia Bai,†,‡ and Jiwen Feng*,† †

State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: 1H MAS NMR spectroscopy has been applied to study the urea effect on phase transition of two similar thermosensitive polymer hydrogels: poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA). It is found that urea influences the phase transition of the hydrogels in opposite ways: lowering the lower critical solution temperature (LCST) of PNIPAM and hence stabilizing its globular structure, whereas raising the LCST of PDEA and destabilizing the globular structure. The selfdiffusion coefficient and urea−polymer nuclear Overhauser effect (NOE) measurement reveal that urea has a stronger interaction with PNIPAM than with PDEA. Moreover, the enhanced positive water−PNIPAM NOE suggests that urea not only interacts directly with PNIPAM via hydrogen bond but also intensifies the hydrogen bonding interaction between water and PNIPAM. We suggest that different urea−polymer hydrogen bonding interaction due to the presence or absence of amide hydrogen is correlated with the distinct LCST variation of PNIPAM and PDEA.

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INTRODUCTION Urea is the simplest compound consisting of the most four major elements in life substances and is well-known as a protein denaturant.1−7 Despite extensive theoretical and experimental studies, the molecular mechanism of urea-induced denaturation is still obscure. The main challenges originate from the physiochemical complexity of proteins and small free energy change during denaturation. Both direct and indirect mechanisms have been proposed. The direct mechanism postulates that urea interacts directly with the protein via hydrogen bonds, etc.8−10 Contrastively, the indirect mechanism suggests that urea alters the tetrahedral network structure of water and makes hydrophobic parts of protein solvated more favorably.3,11,12 However, the latest result showed that urea had a negligible effect on the hydrogen-bond dynamics of water.13 Anyhow, the competing interactions of macromolecule− macromolecule, macromolecule−solvent, and solvent−solvent are believed to be the key to the denaturation process. Poly(N-isopropylacrylamide) (PNIPAM) has received great attention for its potential applications in biomedicine and separations.14−20 When the temperature is increased upon ca. 32 °C, thermal-induced phase transition of PNIPAM occurs in water for both linear chain and hydrogel system, and such critical temperature is termed as the lower critical solution temperature (LCST). The existence of both hydrophilic (amide) and hydrophobic (isopropyl) groups on the side © XXXX American Chemical Society

chain of PNIPAM accounts for its fascinating thermosensitivity, and the dainty balance of interactions with solvents determines its LCST.21,22 Below the LCST, the PNIPAM hydrogels are well hydrated, and the hydration shell is stabilized through extensive hydrogen bonding interactions between the amide groups and water. Once above the LCST, the translucent hydrogel collapses into a hardened opaque one, and the solvent is expelled from network accompanying volume phase transition. This shrinking process involves both intra- and interchain hydrogen bonding interactions. Because of the same amide moieties and similar thermo-induced phase behavior, PNIPAM is usually regarded as a simplified model system for the cold denaturation of proteins,23,24 and some meaningful results have been achieved already.25−29 Cremer et al.26 detected the direct hydrogen bonding interaction between urea and PNIPAM by FTIR and suggested that two NH2 groups of urea molecule simultaneously link to the different carbonyl groups of PNIPAM through hydrogen bonds, making two side chains into closer proximity and stabilizing the globular state. However, less attention was paid to the NH group of PNIPAM in that research. As is well-known, the NH group is a good hydrogen bond donor, and it can form Received: September 5, 2015 Revised: December 17, 2015

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DOI: 10.1021/acs.macromol.5b01949 Macromolecules XXXX, XXX, XXX−XXX

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immersed in aqueous urea solution with a gel/solution weight ratio of 1:2 (w/w), and the swollen samples were left overnight at 4 °C for equilibration. The samples with urea concentration of 0, 1, 2, 3, 5, and 8 M were prepared for NMR measurements. All materials were purchased from Sigma-Aldrich and used as received. NMR Measurements. All the NMR experiments were carried out on the Bruker Avance III spectrometer equipped with a 4 mm HRMAS probe. It is operated at a proton frequency of 600.13 MHz and uses a Bruker Cooling Unit (BCU) for temperature control. The sample temperature in the spinning rotor was calibrated by methanol before the experiments.56 The spin rate was set to 3 kHz for preventing gel from phase separation at higher speed.57 The π/2 pulse length was typically 7.5 μs, and the recycle time was as long as 5 times the longitudinal relaxation for quantitative recording. All variabletemperature NMR experiments were performed at increasing temperature, and the sample was equilibrated for more than 15 min at each temperature before the measurement. The PGSE diffusion measurements were made under MAS conditions with the gradient coil along the magic angle axis. The diffusion time (Δ) and the duration of gradient pulse (δ/2) were optimized to 100 and 1.5 ms, respectively. The gradient strength was linearly ranging from 0.96 to 45.74 G cm−1 in 16 increments. Phase-sensitive NOESY experiments were conducted with mixing time of 500 ms at 20 °C. No spectral symmetrization was applied before analysis.

hydrogen bond with urea or water molecules. In addition, strong hydrogen bonding interaction of N−H group or CO group with water was observed experimentally for the biomolecule.30,31 For PNIPAM, it has been proved that the NH group plays an important role in its conformation interconversion as the temperature across its LCST.32,33 We are concerned whether and how urea interacts with the NH group of PNIPAM. Poly(N,N-diethylacrylamide) (PDEA) is another N-substituted thermosensitive polymer which has nearly the same LCST as PNIPAM in water (Figure 4). PNIPAM and PDEA share the same backbone structure, except for the N-substituted groups, two ethyl groups for PDEA, and one isopropyl group for PNIPAM (Figure 1). Owing to the absence of NH in the

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RESULTS High-Resolution 1H NMR Spectra of PNIPAM and PDEA Hydrogels. The PNIPAM and PDEA gels swollen in aqueous urea solution with different urea concentration were analyzed using 1H HRMAS NMR spectroscopy. Figures 2 and 3 exhibit 1H NMR spectra in both hydrated and collapsed states for PNIPAM and PDEA hydrogels with 3 M urea, respectively. Below the LCST (Figures 2a,b and 3a,b), the polymer chains are surrounded by solvents, and the chains are flexible enough that the chemical shift anisotropy and residual

Figure 1. Chemical structures of (a) PNIPAM, (b) PDEA, and (c) urea.

amide group, PDEA can only act as hydrogen bond acceptor and thus disable the formation of intra- or interchain hydrogen bonds. Therefore, PDEA is a suitable candidate for comparative study with PNIPAM to check the role of NH moiety.33−39 In the present work, PNIPAM and PDEA gels swollen in aqueous urea solution were studied with high-resolution magic angle spinning (HRMAS) NMR spectroscopy. Compared to other analytical techniques, such as dynamic light scattering (DLS),40 attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR),41 and differential scanning calorimetry (DSC),42 NMR is a powerful tool to detect both the chemical structure and dynamics at atomic level in qualitative or quantitative way. The HRMAS technology can sufficiently reduce the line broadening caused by the residual dipolar coupling (RDC) or chemical shift anisotropy (CSA) and produce high resolution NMR spectra in the heterogeneous system.43,44 Thus, it is possible to conduct the sophisticated 1D and 2D NMR experiments for samples like tissues45,46 and polymer hydrogels.47−49 In addition, the nuclear Overhauser effect (NOE) is developed to study the weak intermolecular interaction based on the cross-relaxation rate, which depends on the proximity and correlation time of the interacting molecules.50−52 In this paper, we have analyzed the solvent composition in hydration shell and investigated the interaction between the solvent and the polymers. By comparison, our study reveals that the NH group in the side chain of the polymer plays a critical role in forming and stabilizing the hydrogen bond between PNIPAM and urea.

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Figure 2. 1H MAS NMR spectra of the PNIPAM gel immersed in (a) water, (b) 3 M aqueous urea solution at 20 °C in swollen state, and (c) 3 M urea aqueous solution at 40 °C in collapsed state. The inset is an enlarged view of the methyl region at 40 °C; the narrow peak and the very broad bump-like peak indicate the end methyl groups outside and inside the gel network, respectively. The detailed peak assignments are listed as follows: (1) methyl proton of the N-isopropyl group, −CH3; (2) methylene proton of the backbone, −CH2; (3) methyne proton of the backbone, −CH; (4) lone proton of the N-isopropyl group, −CH⟨; (5) H2O; (6) amide proton of urea. Asterisk corresponds to the rotational sidebands.

EXPERIMENTAL SECTION

Materials. The PNIPAM and PDEA gels used in this study were synthesized by free radical emulsion polymerization of the respective monomer (NIPAM and DEAAm). Details for the synthesis can be found elsewhere.53−55 The cross-linking ratios for each gel were kept constant at 5 mol %. Molecular biology grade urea and double-distilled water were used for solvent preparation. The freeze-dried gel was B


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Figure 3. 1H MAS NMR spectra of the PDEA gel immersed in (a) water, (b) 3 M aqueous urea solution at 20 °C in swollen state, and (c) 3 M aqueous urea solution at 55 °C in collapsed state. The inset is an enlarged view of methyl region at 55 °C. The detailed peak assignments are listed as follows: (1) methyl proton of the N-diethyl group, −CH3; (2) methylene proton of the backbone, −CH2; (3) methyne proton of the backbone, −CH; (4) methylene proton of the N-diethyl group, −CH2; (5) H2O; (6) amide proton of urea.

Figure 4. Temperature variation of normalized integration of methyl protons for (a) PNIPAM and (b) PDEA gels at 0, 1, 2, 3, 5, and 8 M urea concentration. The solid line is a fit to eq 1.

the phase transition region, and then the integration continuously decreases as temperature further rises. The phase transition ends when the integration hits a plateau again. This full process can be fitted with the phenomenological temperature function of the integration intensity I:60 Imax − Imin I= + Imin T − LCST 1 + exp (1) k

dipole−dipole coupling can be removed by the MAS method effectively. In this case, well-resolved proton peaks of PNIPAM and PDEA are observed and assigned according to the published literatures.58,59 The methyl signal of PDEA around 1 ppm is much broader than that of PNIPAM at 20 °C (Figures 2b and 3b), and the broadened methyl signal of PDEA originates from the peak overlapping of two magnetically nonequivalent methyl groups on side chain. It is known that both urea and water have labile hydrogens, and they can exchange with each other. In Figures 2 and 3, water and urea exhibit their own resonance peaks around 4.79 and 5.80 ppm, respectively. Therefore, the exchange rate is lower than their chemical shift difference (∼618 Hz), which belongs to the slow exchange region in NMR time scale. In this slow exchange region, spectral lines broaden as exchange rate increases. We also find that the line width (full width at half-maximum) of urea (∼10 Hz) is significantly reduced in swollen gels compared with urea in polymer-free aqueous solution (∼42.6 Hz), suggesting the proton exchange rate between urea and water is slowed down by the existence of gel network. When the temperature rises above the LCST, the gel network shrinks and becomes a solid-like globule. In the NMR spectra of shrunk PNIPAM and PDEA networks (Figures 2c and 3c), the polymer signals dramatically broaden and are almost undetectable because the intensive dipole−dipole interaction cannot be eliminated effectively. Meanwhile, water proton signals are split into two peaks, and the line shapes of urea are changed. Urea Effect on the Phase Transition of PNIPAM and PDEA. The variable temperature one-pulse NMR experiments have been applied to study the urea effect on the phase transition of two different thermosensitive hydrogels: PNIPAM and PDEA. The phase transition is characterized by the methyl signal integration with increasing temperature. As shown in Figure 4, the integration nearly keeps constant until reaching

(

)

where Imax and Imin are the maximum and minimum integration intensity, respectively. LCST is the phase transition temperature, which is defined at the point where the integrated intensity is decreased by 50%. The parameter k is related to the width of the transition, and a small value of k means a relatively narrow transition interval. The phase transition of PNIPAM and PDEA hydrogels with different urea concentration can be well fitted by eq 1, and the corresponding values are listed in Table 1. It indicates that the Table 1. Detailed Parameter Values of Phase Transition Fitted According to Eq 1 sample PNIPAM

LCST (°C)

k

sample PDEA

LCST (°C)

k

0-UREA 1-UREA 2-UREA 3-UREA 5-UREA 8-UREA

32.6 31.7 31.0 30.6 27.6 22.4

0.775 0.814 0.765 0.764 0.851 1.170

0-UREA 1-UREA 2-UREA 3-UREA 5-UREA 8-UREA

34.7 38.0 40.0 42.3 46.3 52.4

2.166 2.630 2.820 2.533 2.643 2.887

LCST of PNIPAM decreases gradually with urea concentration increasing, suggesting a stabilizing effect for the aggregated (or globular) state of PNIPAM, consistent with the results of Cremer et al.26 The width of PNIPAM transition does not change much after adding a certain amount of urea, except for a high concentration of 8 M. In contrast to PNIPAM, the LCST C


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Macromolecules of PDEA hydrogel exhibits an opposite trend with increasing urea concentration. When the concentration of urea is increased from 0 to 8 M, the LCST of PDEA rises markedly from 35 to 55 °C, accompanied by a sharp increase in transition width. In Figure 4, we also notice that after the phase transition the integration of PDEA methyl signals is substantially higher than that of PNIPAM, and such difference becomes larger with increasing urea concentration. According to the theory of dynamic NMR, slower motion results in larger line broadening and larger loss of integration in the fixed integral interval (here 0.2−1.4 ppm). Therefore, compared to PDEA, lower integral intensity or larger integral loss of methyl signals for shrunk PNIPAM networks at high temperature is due to the broader peak caused by its slower motion (the insets of Figures 2 and 3). The lower mobility of PNIPAM side groups can be attributed to the low solvent content inside the shrunk network (see below), and intra (or inter) macromolecular hydrogen bond, as well as urea-bridged hydrogen bonds between two side groups of PNIPAM.26,33 Solvent Component Assignment and Quantification. Different phase transition originates from specific polymer− solvent interactions. It was reported that preferential interaction or adsorption of polymer with solvents might play an important role in LCST phase transition.61,62 So it is necessary to investigate whether there exists a preferential adsorption of urea on polymer chain. Referring to our previous work,38,47 when the temperature increases across the LCST, solvent molecules expelled from the polymer network are termed as free solvents. By contrast, the restricted solvents are named as confined solvents. Therefore, we can study the preferential solvent− polymer interaction by quantitatively comparing the compositions of confined solvents inside the gel network and free solvents outside the gel network. In Figures 2c and 3c, two split signals of water are observed around 4.4 ppm when the temperature increases above the LCST (40 °C for PNIPAM, 55 °C for PDEA). The downfield narrow signal and the other broad resonance nearby are assigned to the free and confined water, respectively. This assignment is further confirmed by 1D diffusion filtered NMR experiments. Like water molecules, urea molecules are separated into two components above the LCST, but the assignments are not intuitive for their seriously overlapping signals in NMR spectra. Saturation transfer experiments were adopted to distinguish the free and confined urea signals. When free water proton signal was selectively irradiated, free urea proton signal was also completely saturated owing to the proton exchange between water and urea (Figure 5). The free solvents cannot exchange with the confined solvents freely, so only confined solvents signals are remained after irradiation. Surprisingly, in the PDEA gel system (Figure 5b), the line width of residual (confined) urea signal (∼10 Hz) is much narrower than that of free urea signal (∼66 Hz). While in the PNIPAM gel system (Figure 5a), the line width of residual (confined) urea signal (∼54 Hz) is as broad as that of the free one (∼51 Hz), and the position of confined urea has a slight downfield shift relative to free urea peak. The peak deconvolution has been executed for the quantitative analysis of solvent component in collapsed hydrogel spectra (take the gels swollen in 3 M urea aqueous solution for instance) by DMFIT software (Figure 6).63 To avoid some uncertainty caused by peak overlapping, the chemical shifts and line widths of confined solvents are fixed

Figure 5. Saturation transfer experiment for both (a) PNIPAM and (b) PDEA hydrogels with 3 M urea. The irradiated resonance is on free water, and the irradiated time is 10 s. The top (blue) and bottom (red) spectra correspond to off- and on-resonance irradiation, respectively.

during the fitting and obtained the corresponding values by saturation transfer experiment. The multiple peak fitting error is controlled less than 1%, and the detailed results are listed in Table 2. The ratio of confined water to free water in PNIPAM (0.281) is much lower than the corresponding ratio in PDEA (0.840). This difference may be associated with the presence or absence of the NH group. It is expected that the water molecules in high-temperature collapsed polymer network interact with polymer mainly via hydrogen bonding. PNIPAM contains both hydrogen bond donor (N−H) and acceptor (CO), and it can form intra- and interpolymer hydrogen bonds directly or indirectly through urea bridging in hightemperature collapsed state,26,33 reducing the number of solvent−polymer hydrogen bonds and giving rise to a tight globule structure. Whereas for PDEA, intra- and interpolymer hydrogen bonds are lacking in the network; thus, more water molecules can form the hydrogen bonds with the CO group of PDEA and remain inside the shrunk network. It was reported that the single-chain globule of PDEA in water is less compact than that of PNIPAM above their LCST, which was also interpreted in terms of the absence or presence of the intrachain hydrogen bonds.33 The area ratios of urea to water peak are the same in the confined and free environment for either PNIPAM or PDEA D


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Macromolecules Table 3. Line Width and Self-Diffusion Coefficient Variations with Urea Content line width (Hz)

solvents diffusion (10−9 m2 s−1)

PNIPAM

PDEA

PNIPAM gel system

urea molarity (M)

−CH3

−CH3

water

0 1 2 3

35.2 39.7 44.5 47.8

126.1 125.8 126.2 126.4

1.00 0.84 0.76 0.59

PDEA gel system

urea

water

urea

0.44 0.38 0.32

1.12 1.01 0.95 0.90

0.73 0.67 0.63

broadened, and the line width varied from 35 to 48 Hz with increasing urea concentration (from 0 to 3 M urea), suggesting the presence of urea reduces mobility of PNIPAM side group. However, the line width of PDEA methyl proton is very large for seriously overlapped signals of two magnetically nonequivalent methyl groups, and it almost keeps unchanged with increasing urea concentration. What is more, the self-diffusion coefficient of urea in PNIPAM gel is significantly smaller than that in PDEA gel (Table 3), indicating the stronger urea− PNIPAM interactions. Meanwhile, the self-diffusion coefficient of water decreases with increasing urea concentration for both PNIPAM and PDEA gels, but it drops more rapidly in PNIPAM gel than in PDEA gel. Solvent self-diffusion coefficient reflects average interactions (hydrogen bonding and non-hydrogen bonding) between the solvent and polymer. The direct hydrogen bonding between polymer and solvents was assessed by measuring their intermolecular 1H nuclear Overhauser effect (NOE). Two general mechanisms are used to account for the intermolecular NOE: hydrogen bonding model and diffusion model. In the diffusion model, the correlation time τD for intermolecular 1 H−1H dipolar interaction depends on the solvent translational diffusion coefficient D and is described by the Einstein− Smoluchowski relationship τD = x2/6D, where x is solvent displacement. From diffusion data (Table 3), D > 3 × 10−10 m2 s−1 for both water and urea, and the diffusion correlation time is estimated (τD < 0.2 ns) by taking x = 5 Å. Because of the short diffusion correlation time, a negative cross-peak in two-dimensional NOESY is expected for all polymer water/ urea gels. This expected negative NOE basing on diffusion model is in contrast to the experimental observation, exhibiting positive solvent−polymer NOE effects for urea as well as water. According to the NOE theory, there exists a critical correlation time τc = 1.12/ω ≈ 0.3 ns (at proton resonance of 600 MHz). When the correlation time is shorter than the critical value, NOE is negative and relatively weak (due to low crossrelaxation rate), but once the correlation time is longer than the critical value, positive NOE appears, and NOE intensity enhances rapidly with increasing correlation time.64 The correlation time of intermolecular 1H−1H dipolar interaction is regarded as the transient hydrogen-bond residence time (or

Figure 6. Component analysis of confined and free solvents in hydrogel’s collapsed state under 3 M urea by using DMFIT: (a) PNIPAM; (b) PDEA. The confined and free solvents are indicated in red and blue, respectively.

hydrogel (Table 2), suggesting that no preferential urea adsorption occurs inside the two shrunk gel networks. This result is further evidenced by our equilibrium swelling experiments. The dried polymer gels were immersed in excessive urea−water solution for more than 1 day, and then the swollen gels were taken out. The compositions of the solvents inside the swollen gel and in residual polymer-free aqueous urea solution were analyzed by NMR subsequently. No obvious enrichment or dilution of urea was observed (data not shown here). Intermolecular Interactions Detected by PGSE Diffusion and NOESY Experiment. According to the results shown above, the different phase transition behaviors of PNIPAM and PDEA swollen in urea and water mixtures are not directly associated with the preferential adsorption of solvents on polymers. In order to probe the possible solvent− polymer interaction, the measurements of line width, PFG diffusion, and the intermolecular 1H nuclear Overhauser effect (NOE) were taken. Table 3 lists the line width and self-diffusion coefficient variations with different urea concentration at 20 °C. When adding urea, the methyl proton signal of PNIPAM is markedly

Table 2. Deconvolution of Solvent Peaks above LCST and Their Distribution Ratios chemical shift (ppm)

width (Hz)

area (au)

solvents

confined

free

confined

free

confined

free

c/f

PNIPAM−urea PNIPAM−water PDEA−urea PDEA−water

5.72 4.48 5.68 4.47

5.69 4.55 5.67 4.34

53.5 17.9 9.7 14.7

50.9 7.1 65.8 9

0.222 1.710 0.459 3.582

0.778 6.082 0.541 4.264

0.285 0.281 0.848 0.840

E


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at the base, impeding spectral analysis. Fortunately, the horizontal solvent streaks are less intense, and we can clearly see the polymer−solvent cross peaks in the F1 dimension along the polymers methyl resonance (∼1 ppm). It should be noted that proton polarization transfer within the polymer (less than 100 ms) is markedly more efficient than that between the polymer and solvent. Therefore, the NOE here does not show any site-specific polymer−solvent interaction. In order to investigate the possible relationship between urea−polymer binding and phase behavior, we measured a series of 2D NOESY spectra of PNIPAM and PDEA hydrogels with different urea concentrations. The one-dimensional column slices (along the ω1 dimension) at the methyl resonance were extracted from the corresponding 2D NOESY spectra for the direct comparison. No water−polymer NOE peaks are observed in the urea-free PNIPAM hydrogel (Figure 8a), which may be attributed to the short intermolecular 1H−1H dipolar correlation time or short hydrogen-bond residence time between water and PNIPAM (τh < 0.3 ns). When urea is added, both positive water− PNIPAM and urea−PNIPAM NOE appear, and their intensities are enhanced with increasing urea concentration. The presence of urea significantly enhances the positive water− PNIPAM NOE, implying urea prolongs hydrogen-bond interaction between water and PNIPAM (τh > 0.3 ns), consistent with the previous result that urea reduces the mobility of the PNIPAM side group. To clarify the possible proton exchange effect on NOE result, we compare the intensities of urea diagonal peak and urea−water cross peak and find that the urea−urea diagonal peak is much stronger than the urea−water cross peak (Figure 7, marked in the red box), indicating the relatively slow proton exchange between water and urea in the hydrogel system. Hence, the indirect water− PNIPAM cross peak, originating from urea−PNIPAM NOE via water−urea proton exchange, should be much weaker than the direct urea−PNIPAM NOE peak compared with the experimental result that water−PNIPAM cross peak is stronger than urea−PNIPAM one at low urea concentration (0.3 ns). Figure 7 shows 2D 1H−1H NOESY spectra for PNIPAM and PDEA gels swollen in 8 M urea solution. Positive solvent−

Figure 7. 2D NOESY spectra of (a) PNIPAM and (b) PDEA swollen in 8 M urea solution with a mixing time of 500 ms at 20 °C. All the positive peaks are observed. Inevitable vertical streaks appear on the resonances of water and urea. The relatively slower proton exchange between water and urea is marked by a red box.

polymer cross-peaks (with the same sign of diagonal peaks) are clearly observed. Severe vertical streaks characteristic of “t1 noise” at the solvent resonances (especially at the water resonance of 4.7 ppm) are inevitable since the fid in the F1 dimension is not fully sampled. 256 points were generally used in our experiments, and the cutoff solvent signals get “wiggles” F


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Figure 9. NOEs in PNIPAM hydrogel with 1 M urea at two different temperatures (10 and 20 °C) below the LCST. The peaks highlighted in gray indicate urea and water.

from the first solvation shell of polymer chains, which shrinks the chains (or decreases the LCST as seen with PNIPAM) by minimizing the area of the water−polymer interface.65 However, recently experimental26 and theoretical27,29 studies suggest that collapse of polymer may also occur in the situation of either direct hydrogen bonding or attractive dispersion interactions between urea and polymer (see below). In general, urea interacts with either hydrophobic groups (van der Waals dispersion interactions) or hydrophilic groups (hydrogen bonding) of polymer. In view of the molecular structure of PNIPAM and PDEA (Figure 1), the difference of two polymers lies in the presence or absence of the hydrogen bond donor (i.e., the NH group) and different size of hydrophobic end groups (one isopropyl group or two ethyl groups) of side chains. Apparently, the amide NH group and hydrophobic group size of thermosensitive polymers are two relevant factors responsible for the observed opposite effects of urea on the phase transitions of PNIPAM and PDEA. For PNIPAM, each side chain provides two hydrogen binding sites either as hydrogen bond donor (N−H) or acceptor (CO). From the FTIR results, Cremer et al. proposed that urea bridges two different amide groups of PNIPAM via bivalent hydrogen bonds and stabilizes the globular state.26 This is in line with the observed strong positive intermolecular NOEs and the reduction of segmental mobility with the attendance of urea in present investigation. Moreover, the transient bivalent interaction is expected to have long residence time; thereby, a positive NOE cross peak is observed in our experiment. Meanwhile, urea-induced folding or crosslinking of PNIPAM segments provides steric hindrance to the exchange of bonded water with free water and thus leads to a longer hydrogen bonding correlation time and positive water− PNIPAM NOE as well. While for PDEA hydrogel, when urea is added, the NOE between the urea and PDEA is weaker and even undetectable compared with PNIPAM. It seems that the urea interacts with PDEA in a more “free” way. Comparing with PNIPAM, we suppose that urea interacts with PDEA in monovalent way rather than bivalent way since a single side group of PDEA only bears one hydrogen binding site CO. A recent molecular dynamics simulation reveals that the number of urea bridges between N−H and CO group of PNIPAM is several times larger than that between two CO groups.27 Since the monovalent interaction is substantially weaker than its bivalent counterpart, monovalent interaction may have

Figure 8. Water and urea NOE correlated with (a) PNIPAM and (b) PDEA at different urea concentration (0, 1, 2, 3, 5, and 8 M); each column slice is extracted from its corresponding 2D NOESY spectrum with diagonal (methyl) resonances being positive. The peaks highlighted in gray indicate urea and water, respectively. The peak at 7.6 ppm is the amide proton of PNIPAM. Asterisk corresponds to the rotational sidebands.

shorter than that of urea−PNIPAM and water−PNIPAM hydrogen bonds, respectively.

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DISCUSSION According to the traditional viewpoint, the macromolecular unfolded (or coiled) state is stabilized by attractive urea− polymer interactions, which allow the urea accumulation in the first solvation shell of the polymer chains and thus swell the chains (or increase the LCST as observed with PDEA). Whereas the folded (or globule) state is driven by entropic mechanism. That is, urea molecules are preferentially excluded G


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interaction induces the formation of preferential urea cloud around hydrophobic hydrocarbon groups of PNIPAM or PDEA. In this case, the preferential urea adsorption should occur inside the gel networks relative to outside of the gel work. However, we do not observe any preferential urea enrichment inside two polymer networks by NMR measurements. More work should be done to determine the relative importance of hydrogen bonding and dispersion interactions between urea and polymers to the distinct LCST variations of PNIPAM and PDEA hydrogels. As is well-known, protein consists of a series of amino acids linked by peptide bonds, and different types and sequences of amino acids result in the specificity of each protein. People usually take protein as an abstract concept but pay less attention to its particular amino acid unit when they discuss the denaturation of protein. The side chains of all the 20 proteinogenic amino acids have a great variety of chemical structures and properties different from each other, especially for the proline, containing a ring structure to the N-end amine group and no amide hydrogen like PDEA. It has been proved that proline plays a vital part in biological structure and always acts as the α-helix breaker. The amide hydrogen is crucial to the conformations and functions of proteins undoubtedly. With the comparison of PNIPAM and PDEA, different hydrogen bond strength or even different hydrogen bond patterns are involved. Regarding urea-induced protein denaturation, we can legitimately believe that urea molecules interact with different amino acid residues in different ways, either stabilizing or destabilizing the protein native structure.

shorter hydrogen bond lifetime which leads to weaker urea− PDEA NOE. The above-mentioned distinct hydrogen bonding patterns of urea with polymers PNIPAM and PDEA, due to the presence or absence of the amide hydrogen, may be correlated with the opposite LCST trends of PNIPAM and PDEA. For PDEA, the urea−PDEA monovalent interaction dominates, and adding urea should swell the PDEA chains or shift the LCST to higher temperature. While for PNIPAM, bridging-type hydrogen bonding interaction is operative which folds the PNIPAM chain and favors globular state (or decreases the LCST) of PNIPAM.26 A recently comparative study on PNIPAM and PDEA gels in water/methanol mixture reveals the same effect: methanol increasing LCST of PDEA but decreasing LCST of PNIPAM.39 The different effects of methanol on LCST of PNIPAM and PDEA are attributed to the presence or absence of the amide NH group. The presence of the amide NH group and hydrogen bonding interaction with urea also change the hysteresis behaviors of heating and cooling phase transitions. PDEA does not show any heating−cooling hysteresis in both pure water and urea aqueous solutions due to lacking of amide NH group and thus intra- or interpolymer hydrogen bonds. The hysteresis is found in PNIPAM in the present urea-free aqueous solution owing to the intra- and interpolymer hydrogen bonds,66 but such a hysteresis becomes less obvious in the water/urea solution (Figure S1 in Supporting Information). The urea-reduced hysteresis in PNIPAM can be attributed to that the urea destroys the direct intra- and interpolymer hydrogen bonds due to the formation of urea− polymer hydrogen bonds (including bridge-type bonding). Besides the above-mentioned urea−polymer hydrogenbonding patterns, however, there also exist the van der Waals dispersion interactions between urea and hydrophobic groups of polymers, which may also play an important role in chain folding or unfolding.27,29 On the basis of the molecular dynamics simulations, van der Vegt et al.27 found that the average number of bridging urea molecules in PNIPAM system is very low, and urea preferentially interacts with hydrophobic ends of PNIPAM side groups. They suggested that urea stabilizes the globular state by entropic driving force rather than attractive dispersion interactions between urea and PNIPAM, i.e., entropy penalizing the solvation of the unfolded state over the folded state. Also, on the basis of the molecular dynamics simulations, Zangi et al. observed that hydrophobic interactions between the nonpolar groups of polymer are weakened by preferential dispersion interactions of urea with the hydrophobic group, and urea either unfolds or folds a polymer chain of purely hydrophobic hydrocarbon groups depending on the size of hydrophobic groups.67 For the smaller hydrocarbons entropy dominates, and urea folds the polymer chains; but for the larger hydrophobic groups, van der Waals interactions dominate, and urea destabilizes the folded (or globular) chains. Obviously, the attractive van der Waals interactions together with entropic effects could also entirely explain the differences in the phase transitions of PNIPAM and PDEA. For the PNIPAM bearing smaller nonpolar side group (isopropyl), the entropy dominates, and urea induces polymer collapse. For the PDEA that contains larger nonpolar side group than PNIPAM, van der Waals interactions between PDEA and urea are expected to be stronger than those between PNIPAM and urea, and stronger PDEA−urea van der Waals interactions drive unfolding (swelling) of PDEA. The above mechanism predicates that the urea−polymer attractive dispersion

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CONCLUSION We have investigated the effect of urea on LCST transition of two similar thermosensitive polymer hydrogels PNIPAM and PDEA by 1H MAS NMR spectroscopy. Urea lowers the LCST of PNIPAM hydrogel and serves as a protector of the globule structure, but for the PDEA, urea increases the LCST of the PDEA hydrogel and serves as a destructor of the globule structure. Through comparing the compositions of confined solvents and free solvents inside and outside polymer network respectively, we find that the urea molecules homogeneously distribute inside and outside polymer network, and no obvious urea enrichment occurs inside the PNIPAM or PDEA network. The PGSE diffusion and NOESY experiments reveal that the urea molecules can form the direct hydrogen bond with two polymers, but the urea molecules are more strongly hydrogen bonded to the PNIPAM than to the PDEA. Therefore, the direct hydrogen binding between urea and polymer can either stabilize or destabilize the globular structure of polymers, depending on the moiety of the amide groups of the polymers. With the comparative work, we can draw the conclusion that the moiety of the amide groups and their hydrogen-bond interaction with urea of thermosensitive polymer are crucial for determining stability of the globular polymer structure.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available ACS Publications website at DOI: mol.5b01949. Data for hysteresis of heating transitions and phase transition and NMR measurements (PDF) H

free of charge on the 10.1021/acs.macroand cooling phase probed by turbidity


Article

Macromolecules

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel 86-27-87197343; Fax 86-2787199291 (J.W.F.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Nos. 21204099, 11274347, and 21221064) for grant support. REFERENCES

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