J. Phys. Chem. C 2007, 111, 5309-5312
5309
Collapse and Aggregation of Poly(N-isopropylacrylamide) Chains in Aqueous Solutions Crowded by Polyethylene Glycol Yanwei Ding and Guangzhao Zhang* Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemical Physics, Structure Research Laboratory, UniVersity of Science and Technology of China, Hefei, Anhui, China ReceiVed: NoVember 21, 2006; In Final Form: December 23, 2006
Collapse and aggregation of poly(N-isopropylacrylamide) (PNIPAM) chains in aqueous solutions with polyethylene glycol (PEG) as the crowding agent have been investigated by using ultra-sensitive differential scanning calorimetry (US-DSC). For any PEG with a certain molecular weight, the transition temperature of PNIPAM decreases with PEG concentration (CPEG) due to the complexation between PEG chains and water molecules. A hysteresis has been observed in one heating-and-cooling cycle. As CPEG increases, short PEG chains lead the hysteresis to enlarge, whereas longer PEG chains result in a decrease in the hysteresis. The facts indicate that longer PEG chains suppress the interchain aggregation of PNIPAM chains. When CPEG is above the overlap concentration (C*), the hysteresis is almost independent of PEG concentration, suggesting that PEG chains form a transient network which locates PNIPAM chains in a number of pores. The widening of the transition with increasing CPEG at CPEG>C* indicates that the network is inhomogeneous. The fact that the enthalpy change (∆H) decreases with CPEG further indicates that PEG chains reduce the interchain aggregation of PNIPAM chains. The same ∆H in heating and cooling processes demonstrates that PNIPAM chains form small-scale aggregates in the presence of PEG.
Introduction It is well-known that proteins in a living cell carry out their functions in an environment crowded by macromolecules. The total concentrations of macromolecules including proteins, nucleic acids, lipid membranes, and sugars are as high as 50400 mg/mL,1,2 where each macromolecular species may not participate in a particular activity of a protein but occupy a significant fraction of the volume of the medium. Due to the macromolecular crowding, proteins in a cell (in vivo) exhibit dynamics and thermodynamics quite different from those in a dilute solution (in vitro). It has long been a challenge to observe the protein behavior in a living cell. At the present time, experiments are usually conducted in a cell-mimicking environment created by the so-called crowding agents to extract the related information. One of the most commonly used crowding agents is polyethylene glycol (PEG). It has been reported that PEG can either reduce or promote the aggregation of proteins; that is, it can act as a molecular chaperone or protein precipitating agent.3,4 This should relate to the specific and nonspecific interactions between PEG and the proteins. Actually, due to the complexity of protein structures, the influence of macromolecular crowding on the dynamics of proteins even in such a mimicking environment still remains largely unknown. Considering that poly(N-isopropylacrylamide) (PNIPAM) aqueous solutions exhibit a lower critical solution temperature (LCST) at ∼32 °C, the polymer has been used as a model to study the folding and denaturation of proteins.5-13 So far, most of studies have been performed in a dilute aqueous solution. Collapse and aggregation of PNIPAM chains in a crowding environment have not been addressed in either a real experiment or a theoretical treatment because one has to deal with a manybody problem. In the present work, we prepared a series of PNIPAM solutions with monodisperse PEG as the crowding * To whom correspondence should be addressed.
agent. By using ultrasensitive differential scanning calorimetry (US-DSC), we have investigated the collapse and aggregation of PNIPAM in such a crowding environment. Our aim is to understand the behavior of polymer chains in a solution crowded by another polymer. Experimental Section Sample Preparation. PNIPAM was synthesized by radical polymerization in benzene with azobis(isobutyronitrile) (AIBN) as the initiator. The resultant PNIPAM was fractioned in an acetone/hexane mixture. The average molecular weight (Mw), the polydispersity index (Mw/Mn), and the radius of gyration 〈Rg〉 evaluated by laser light scattering are 1.6 × 106 g/mol, 1.5, and 66 nm, respectively.13 PEG samples from Shanghai Reagents Co. were used as received. Mw/Mn of PEG was measured by a gel permeation chromatography (GPC) on a Waters 150C using a series of monodisperse polystyrenes as the calibration standard and tetrahydrofuran (THF) as the eluent with a flow rate of 1.0 mL/min. The characteristic data are summarized in Table 1. The overlap concentration (C*) of a PEG was evaluated from C* ) 3M/(4πNARg3).14,15 Since the molecular weights of PEG samples used here are relatively low, their Rg values cannot be directly measured. Alternatively, Rg was estimated from the mean square end-to-end distance (r2) by using Rg2 ) r2/6, where r2 ) C∞N(lC-C2 + 2lC-O2).16 The characteristic ratio C∞ for PEG in water is 4.1, N is the number of the -CH2CH2O- unit, and the lengths of C-C (lC-C) and C-O (lC-O) bonds are 1.54 and 1.43 Å, respectively.17 Table 1 shows the C* value of each PEG with a certain molecular weight (Mw,PEG). Obviously, when Mw,PEG e 1000 g/mol, in the range we investigated, the PEG solutions are not in the semidilute regime though they are concentrated in terms of g/L. When Mw,PEG g 3750 g/mol, we can have CPEG > C*, where PEG solutions can be taken to be semidilute.
10.1021/jp067723a CCC: $37.00 © 2007 American Chemical Society Published on Web 03/20/2007
5310 J. Phys. Chem. C, Vol. 111, No. 14, 2007
Ding and Zhang
TABLE 1: Characteristic Data of PEG Samples sample
Mw (g/mol)
Mw/Mn
C* (g/mL)
PEG-200 PEG-400 PEG-1000 PEG-4000 PEG-10000
200 405 1000 3750 10 750
1.00a 1.00 1.04 1.05 1.03
764 634 387 205 121
a
Evaluated from the molecular formula.
Figure 2. Temperature dependence of the specific heat capacity (Cp) of PNIPAM chains in one heating-and-cooling cycle at different PEG concentrations (CPEG), where Mw, PEG ) 405 g/mol.
Figure 1. FTIR spectra of PNIPAM chains in D2O in the absence and presence of PEG at 20 and 36 °C, respectively.
Ultra-Sensitive Differential Scanning Calorimeter. A PNIPAM solution with PEG as the crowding agent was measured on a VP-DSC microcalorimeter from Microcal under an external pressure of 200 kPa with the corresponding PEG aqueous solution as the reference. The investigated solution and the reference solution were degassed at 25.0 °C for 30 min and equilibrated at 10 °C for 120 min before heating. In the cooling process, they were equilibrated at 60 °C for 120 min to eliminate the effect of thermal history. The concentration of PNIPAM was 1.0 mg/mL. The concentrations of PEG (CPEG) ranged from 0 to 300.0 mg/mL. The heating and cooling rates were 1.0 °C/ min. The phase transition temperature (Tp) was taken as that centered at the transition. The enthalpy change (∆H) during the transition was calculated from the area under each peak. Fourier Transform Infrared (FTIR) Spectroscopy FTIR spectra were measured on a Nicolet Magna 750 spectrometer with a resolution of 4 cm-1. D2O was used instead of H2O so that the overlapping of the amide I band of the PNIPAM moiety with the O-H bending band of water around 1640 cm-1 can be masked.18 The concentrations of PNIPAM and PEG were 20.0 and 300.0 mg/mL, respectively. The polymer solution in D2O was added to a cell between two KRS-5 crystals with a diameter of 32 mm and thickness of 3.5 mm. The space between the crystals is 20 µm. The temperature was measured continuously by an electronic thermometer with a precision of (0.1 °C. Results and Discussions It is known that PNIPAM can strongly interact with some polymers such as poly(acrylic acid) and polymethyacrylic acid in aqueous solutions.19 Such interactions have a heavy effect on the behavior of PNIPAM chains. In order to clarify the role of PEG in the present system, we first examined the interaction between PNIPAM and PEG. Figure 1 shows the FTIR spectra of PNIPAM in the absence and presence of PEG in the range 1670-1580 cm-1 at 20.0 and 36.0 °C, respectively. The band centered at 1625 cm-1 and the shoulder at 1650 cm-1 are
assigned to the CdO groups in >CdO‚‚‚D-O-D and >CdO‚‚‚H-N< species, respectively.20 In comparison with PNIPAM without PEG, the addition of PEG leads to a small blue shift of the band at 1625 cm-1 at 20 °C. Actually, other bands do not have any shifts (not shown). The facts indicate that the interaction between PNIPAM and PEG is weak; namely, they do not form a complex in water. A shoulder at 1650 cm-1 can be observed at a temperature above the LCST of PNIPAM (36 °C), whose weighting in the presence of PEG is more than that in the absence of PEG. This indicates that the presence of PEG leads to the increase of the additional hydrogen bondings or the hydrogen bonds between >CdO and H-N C*, suggesting the transition involves more than one mode. Figure 4 clearly shows that Tp linearly decreases with CPEG at Mw,PEG e 1000 g/mol in both heating and cooling processes. It has been reported that the addition of a certain amount of methanol to water can decrease the LCST of PNIPAM due to the complexation between water and methanol, which reduces the hydrogen-bonded sites in water molecules.21,22 Similarly, previous investigations suggested that PEG forms hydrogen bonding with water molecules.23-25 Thus, PEG competes with PNIPAM in complexing water molecules. In other words, although PNIPAM forms hydrogen bonds with water molecules,26 each -CH2CH2O- unit also binds a certain number of water molecules forming the hydration layer.27,28 The presence of PEG disturbs the hydration layer around PNIPAM so
Poly(N-isopropylacrylamide) Chains
J. Phys. Chem. C, Vol. 111, No. 14, 2007 5311
Figure 5. CPEG and Mw, PEG dependence of the difference of the transition temperature (∆Tp) in one heating-and-cooling cycle.
Figure 3. Temperature dependence of the specific heat capacity (Cp) of PNIPAM chains in one heating-and-cooling cycle at different PEG concentrations (CPEG), where Mw, PEG ) 10750 g/mol.
Figure 4. CPEG and Mw, PEG dependence of transition temperature (Tp) in heating and cooling processes.
that some of the hydrophobic groups are exposed, leading to the onset of collapse and aggregation of PNIPAM chains. When Mw,PEG e 1000 g/mol, no entanglement occurs in the range we investigated, one PEG molecule can bind a certain number of water molecules. Thus, Tp linearly decreases with CPEG. When Mw,PEG g 4000 g/mol, Tp linearly decreases with PEG concentration at CPEG < C*. However, the CPEG dependence of Tp is no longer linear when CPEG > C*; namely, the increase in PEG concentration causes a more drastic decrease of Tp. The difference between macromolecule behaviors in dilute and semidilute solutions has been explained by the network formed by the entangled chains in solution.29-31 Kozer and Schreiber32 demonstrate that the crowding effect of PEG on protein-protein association has a large dependence on its molecular weight. The semidilute solution created by high molecular weight PEG behaves as a porous medium where proteins in one pore can associate relatively freely. In the present case, longer PEG chains with Mw,PEG g 3750 g/mol are expected to form physical network by chain entanglement at CPEG > C*, so that PNIPAM chains are trapped in the pores. Recently, Okada and Tanaka33 show that the LCST of PNIPAM solution arises from the cooperativity between the neighboring water molecules that are hydrogen-bonded onto the polymer chain. Since the network formed by longer PEG chains can encompass PNIPAM chains, such cooperativity would be destroyed even more by longer PEG chains than shorter ones. In other words, longer PEG chains
can effectively disrupt the hydration layer around PNIPAM, leading to a larger decrease of Tp. Figure 5 shows the difference (∆Tp) of Tp in one heatingand-cooling cycle as a function of CPEG. ∆Tp directly reflects the hysteresis. It can be seen that ∆Tp gradually increases with CPEG at Mw,PEG e 400 g/mol. Previous work shows that the hysteresis of PNIPAM chains is related to the formation and dissolution of additional hydrogen bonding.13,34 Due to the small size, PEG molecules complexed with water slightly influence the interchain aggregation scale of PNIPAM chains in the heating process. In the cooling process, as CPEG increases, more water molecules are complexed with PEG, so that it is harder for the solvent to disrupt the additional hydrogen bonds among PNIPAM chains. As a result, the hysteresis is gradually enlarged with CPEG. However, when Mw,PEG g 3750 g/mol, due to the larger size, PEG chains complexed with water molecules can suppress the interchain aggregation of PNIPAM chains in the heating process. As CPEG increases, PNIPAM chains form smaller aggregates with less interchain connection by the additional hydrogen bondings. Such aggregates are dissolved more easily than the larger aggregates. This explains why the hysteresis becomes smaller with the increasing CPEG. Particularly, when CPEG > C*, the entangled PEG chains form a transient network, which effectively limits the interchain aggregation of PNIPAM chains, so ∆Tp does not change with CPEG. When Mw,PEG ) 1000 g/mol, ∆Tp slightly increases at CPEG < C* but decreases at CPEG>C*; that is, ∆Tp has a maximum at CPEG ∼160 mg/mL. This is understandable because PEG with Mw,PEG ) 1000 g/mol should exhibit a character between the longer PEG chains (Mw,PEG g 3750 g/mol) and the shorter PEG chains (Mw,PEG e 400 g/mol). Actually, considering that C* is roughly estimated, PEG-1000 solutions at CPEG > 160 mg/mL are approximately semidilute. Figure 6 shows the half-width of the peak (W0.5) slightly increases with CPEG at CPEG < C* for any PEG in both heating and cooling processes. However, for long PEG (Mw,PEG g 3750 g/mol) at CPEG > C*, W0.5 sharply increases, clearly indicating more than one aggregation modes in the system. This can be clearly viewed in Figure 3. Note that the widening of the transition is not due to the polydispersity of PNIPAM. Otherwise, W0.5 should also be wide at CPEG < C*. As suggested before,32 the entangled PEG chains form an inhomogeneous transient network; namely, the pores have a size distribution. A bigger pore is expected to trap more PNIPAM chains, whereas a smaller pore confines less PNIPAM chains. Thus, PNIPAM chains trapped in the spectrum of the pores exhibit multi-modes of collapse and aggregation. Figure 7 shows that the enthalpy change (∆H) decreases with CPEG. It is known that both intrachain collapse and interchain aggregation of PNIPAM chains contribute to ∆H in the heating process. As discussed above, the presence of PEG suppresses
5312 J. Phys. Chem. C, Vol. 111, No. 14, 2007
Ding and Zhang that Tp decreases with CPEG because PEG is competitive with PNIPAM in complexing water molecules. The hysteresis observed in one heating-and cooling cycle has much dependence on the concentration (CPEG) and molecular weight (Mw,PEG) of PEG. In a semidilute PEG solution, PEG concentration slightly influences the hysteresis, suggesting that PEG chains form a transient network where the trapped PNIPAM chains form small-scale aggregates. Moreover, the widening of the transition with increasing CPEG indicates the inhomogeneous network with pores of different size. The enthalpy change (∆H) decreases with CPEG and indicates that PEG chains suppress the interchain aggregation of PNIPAM chains. The same ∆H in the heating and cooling processes further demonstrates that PNIPAM chains form small-scale aggregates at an elevated temperature due to the restriction of PEG chains.
Figure 6. CPEG and Mw, PEG dependence of the half-width of the peak (W0.5) in heating and cooling processes.
Acknowledgment. Financial support of National Natural Science Foundation (NNSF) of China (20474060) and The Chinese Academy of Sciences (KJCX2-SW-H14) is gratefully acknowledged. References and Notes
Figure 7. CPEG and Mw, PEG dependence of enthalpy change (∆H) in heating process (solid) and cooling process (open), where Mw,PEG is 200 (9, 0), 405 (b, O), 1000 (2, ∆), 3750 (1,3), and 10 750 ([,]) g/mol, respectively.
the interchain aggregation of PNIPAM chains. The increasing CPEG would further reduce the interchain aggregation, leading to the decrease in ∆H. In the cooling process, ∆H relates to the swelling and dissolution of PNIPAM chains. As CPEG increases, the complexation between water and PEG becomes stronger, and the dissolution becomes more difficult, so ∆H decreases with CPEG. On the other hand, Mw,PEG almost does not have any effect on ∆H in either heating or cooling process. In other words, whether PEG solution is semidilute or not does not influence the ∆H of PNIPAM chains. As discussed above, high molecular weight PEG chains would trap PNIPAM chains in pores, which can reduce the interchain aggregation of PNIPAM chains in different pores. However, such trapping would increase the interchain aggregation and intrachain collapse of PNIPAM chains in the same pore. In a solution with a certain PEG concentration in terms of g/L, PNIPAM finally collapses and aggregates to the same level in the heating process. Similarly, the aggregated PNIPAM chains would swell and dissolve to the same degree in the cooling process. Thus, the energy for the transition is independent of PEG molecular weight. Figure 7 also shows that ∆H values in the heating process and cooling process are almost equal at any CPEG except that without PEG (CPEG ) 0). The fact further indicates that PEG chains restrict PNIPAM chains from a large-scale interchain aggregation, so that the aggregated PNIPAM chains can readily be dissolved in the cooling process. Conclusion The present investigations on the collapse and aggregation of PNIPAM chains in aqueous solutions crowded by PEG reveal
(1) Ellis, R. J.; Minton, A. P. Science 2003, 425, 27. Ellis, R. J. Trends Biochem. Sci. 2001, 26, 597. (2) Cheung, M. S.; Klimov, D.; Thirumalai, D. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4753. (3) Cleland, J. L.; Wang, D. I. C. Biotechnol. Prog. 1992, 8, 97. (4) Arakawa, T.; Timasheff, S. N. Biochemistry 1985, 24, 6756. (5) Wu, C.; Zhou, S. Q. Phys. ReV. Lett. 1996, 77, 3053. (6) Tiktopulo, E. I.; Bychkova, V. E.; Ricˇka, J.; Ptitsyn, O. B. Macromolecules 1994, 27, 2879. (7) Ricˇka, J.; Meewes, M.; Nyffenegger, R.; Binkert, Th. Phys. ReV. Lett. 1990, 65, 657. (8) Zhu, P. W.; Napper, D. H. Langmuir 1996, 12, 5992. (9) Graziano, G. Int. J. Biol. Macromol. 2000, 27, 89. (10) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311. (11) Grinberg, V. Y.; Dubovik, A. S.; Kuznetsov, D. V.; Grinberg, N. V.; Grosberg, A. Y.; Tanaka, T. Macromolecules 2000, 33, 8685. (12) Grinberg, N. V.; Dubovik, A. S.; Grinberg, V.Y.; Kuznetsov, D. V.; Makhaeva, E. E.; Grosberg, A. Y.; Tanaka, T. Macromolecules 1999, 32, 1471. (13) Ding, Y. W.; Ye, X. D.; Zhang, G. Z. Macromolecules 2005, 38, 904. (14) Ying, Q. C.; Chu, B. Macromolecules 1987, 20, 362. (15) Teraoka, I. Polymer Solutions; John Wiley & Sons: New York, 2002. (16) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Khokhlov, A. R.; Doi, M. Biopolymers 1994, 34, 555. (17) Brandrup, J.; Immergut, E. H. Polymer Handbook, 3rd ed.; John Wiley & Sons: New York, 1989. (18) Maeda, Y.; Nakamura, T.; Ikeda, I. Macromolecules 2001, 34, 1391. (19) Burova, T. V.; Grinberg, N. V.; Grinberg, V. Y.; Kalinina, E. V.; Lozinsky, V. I.; Aseyev, V. O.; Holappa, S.; Tenhu, H.; Khokhlov, A. R. Macromolecules 2005, 38, 1292. (20) Maeda, Y.; Higuchi, T.; Ikeda, I. Langmuir 2000, 16, 7503; 2001, 17, 7535. (21) Winnik, F. M.; Ringsdorf, H.; Venzmer, J. Macromolecules 1990, 23, 2415. (22) Zhang, G. Z.; Wu, C. J. Am. Chem. Soc. 2001, 123, 1376. (23) Verbrugghe, S.; Laukkanen, A.; Aseyev, V.; Tenhu, H.; Winnik, F. M.; Du Prez, F. E. Polymer 2003, 44, 6807. (24) van Durme, K.; Verbrugghe, S.; Du Prez, F. E.; van Mele, B. V. Macromolecules 2004, 37, 1054. (25) Yanul, N. A.; Kirsh, Y. E.; Verbrugghe, S.; Goethals, E. J.; Du Prez, F. E. Macromol. Chem. Phys. 2001, 202, 1700. (26) Ono, Y.; Shikata, T. J. Am. Chem. Soc. 2006, 128, 10030. (27) Kjellander, R.; Florin, E. J. Chem. Soc., Faraday Trans. 1981, 77, 2053. (28) Lu¨sse, S.; Arnold, K. Macromolecules 1996, 29, 4251. (29) Devanand, K.; Selser, J. C. Nature 1990, 343, 739. (30) Hammouda, B.; Ho, D.; Kline, S. Macromolecules 2002, 35, 8578. (31) Laurant, T. Biophys. Chem. 1995, 57, 7. (32) Kozer, N.; Schreiber, G. J. Mol. Biol. 2004, 336, 763. (33) Okada, Y.; Tanaka, F. Macromolecules 2005, 38, 4465. (34) Cheng, H.; Shen, L.; Wu, C. Macromolecules 2006, 39, 2325.
