Thermo-Reversible Swelling of Thin Hydrogel ... - ACS Publications

Oct 6, 2004 - Welsh School of Pharmacy, Redwood Building, King Edward VII Avenue, Cardiff ... responsive polymers(SRP).7 The polymers exhibit a volume...
0 downloads 0 Views 185KB Size
Download PDF
Langmuir 2004, 20, 10107-10114

10107

Thermo-Reversible Swelling of Thin Hydrogel Films Immobilized by Low-Pressure Plasma Dirk Schmaljohann,*,† Detlev Beyerlein,‡,§ Mirko Nitschke,‡ and Carsten Werner*,‡ Welsh School of Pharmacy, Redwood Building, King Edward VII Avenue, Cardiff University, Cardiff, CF10 3XF, Wales, UK, and Institute of Polymer Research Dresden and Max Bergmann Center of Biomaterials Dresden, Hohe Strasse 6, 01069 Dresden, Germany Received April 16, 2003. In Final Form: August 17, 2004 Thin films of graft copolymers consisting of poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,Ndiethylacrylamide) (PDEAAm) as polymer backbone and poly(ethyleneglycol) as side chains were crosslinked on fluoropolymer substrates by low-pressure plasma treatment. All immobilized polymers exhibit a lower critical solution temperature between 34 and 40 °C. The swelling and collapsing of the hydrogels was examined with temperature-dependent spectroscopic ellipsometry. Two time ranges of swelling were observed: a fast ‘dynamic’ and a slow ‘equilibrium’ swelling. The dynamic swelling occurs within minutes or less, whereas the equilibrium swelling needs several days to complete. The surface-bound hydrogels show a shift in the transition temperature toward lower temperatures compared with the behavior in solution. Full reversibility of the dynamic swelling/collapsing was found, but the temperature scan exhibits a hysteresis between heating and cooling cycles. The PNiPAAm-containing hydrogels show a sharper transition compared to the PDEAAm-containing hydrogels, which is almost linear over a wide temperature range.

Introduction Thin films of synthetic hydrogels with stimuli-responsive behavior receive more and more attention in the development of advanced in vitro cell carriers for regenerative medicine.1,2 Hydrogels in general exhibit several advantages for this application as compared to conventional cell culture substrates: Swelling in aqueous media makes them sufficiently soft to allow cellular matrix reorganization. On the other hand, hydrogels provide enough mechanical strength and elasticity to permit the anchorage or entrapment of cells. Also, delivery of nutrients and growth factors is possible and can be controlled through the meshwork characteristics of the hydrogels. In addition, certain hydrogel substrates offer the most valuable advantages for cell harvest without enzymatic treatment since they allow for switching of cell adhesion and detachment under the action of environmental physical signals.3-6 Those hydrogels, exposing or hiding surface functionalities and changing integral characteristics (hydrophilicity, charge) related to varied degrees of swelling, can be achieved with so-called stimuli* Authors to whom correspondence should be addressed. E-mail: [email protected] (D.S.). † Welsh School of Pharmacy. ‡ Institute of Polymer Research Dresden and Max Bergmann Center of Biomaterials Dresden. § Current address: Thu ¨ ringisches Institut fu¨r Textil- und Kunststoff-Forschung e.V., Breitscheidstrasse 97, 07407 Rudolstadt, Germany. (1) Okano, T., Ed. Biorelated Polymers and Gels; Academic Press: San Diego, 1998. (2) Kwon, O. H.; Kikuchi, A.; Yamato, M.; Okano, T. Biomaterials 2003, 24, 1223-1232. (3) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Macromol. Rap. Comm. 1990, 11, 571-576. (4) Ebara, M.; Yamato, M.; Hirose, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano T. Biomacromolecules 2003, 4, 344-349. (5) Ratner, B. D.; Cheng, X.; Wang, Y.; Hanein, Y.; Bo¨hringer, K. F. Polym. Prepr. 2003, 44, 198-198. (6) Yamato, M.; Konno, C.; Kushida, A.; Hirose, M.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2000, 21, 981-986.

responsive polymers (SRP).7 The polymers exhibit a volume phase transition upon change in the environmental pH, temperature, or illumination due to an altered balance of competing interactions (i.e., electrostatic forces, hydrophobic dehydration) determining the polymer structure. For use in cell-culture technologies, the layered molecules have to undergo sufficiently strong structural transitions upon variation of the environmental conditions within a physiological range of settings. While pH- and irradiationsensitive polymers could so far hardly fulfill this requirement, poly(N-isopropyacrylamide) (PNiPAAm) represents a very successful and widespread example of a thermoreversible hydrogel polymer out of a selection of various other polymers showing transitions between 20 and 40 °C.8 To enhance thermoresponsive cell-culture carriers by implementation of matrix biopolymers and growth factors,2,9-11 we recently prepared a series of graft copolymers consisting of PNiPAAm or poly(N,N-diethylacrylamide) (PDEAAm) as polymer backbone and poly(ethyleneglycol) (PEG) as side chains.12,13 In view of numerous studies demonstrating the very weak interactions between proteins and PEGs,14 our strategy anticipated that this PEGylation of the PNiPAAm and PDEAAm polymers may facilitate the structural and functional conservation as well as the release performance of (7) McCormick, C. L., Ed. Stimuli-responsive Water-soluble and Amphiphilic Polymers; Symposium Series 780; American Chemical Society: Washington, DC, 1999. (8) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163-249. (9) Van Recum, H.; Okano, T.; Wan Kim, S. J. Contr. Release 1998, 55, 121-130. (10) Shimizu, T.; Yamato, M.; Akutsu, T.; Shibata, T.; Isoi, Y.; Kikuchi, A.; Umezu, M.; Okano, T. J. Biomed. Mater. Res. 2002, 60, 110-117. (11) Harimoto, M.; Yamato, M.; Hirose, M.; Takahashi, C.; Isoi, Y.; Kikuchi, A.; Okano, T. J. Biomed. Mater. Res. 2002, 62, 464-470. (12) Schmaljohann, D.; Gramm, S. Polym. Prepr. 2002, 43, 758759. (13) Gramm, S.; Komber, H.; Ha¨ussler, L.; Tarek El Tahan, N.; Schmaljohann, D. Macromolecules, submitted for publication. (14) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436.

10.1021/la034653f CCC: $27.50 © 2004 American Chemical Society Published on Web 10/06/2004

10108

Langmuir, Vol. 20, No. 23, 2004

Figure 1. Synthetic scheme for the polymer synthesis.

biomacromolecules embedded in the hydrogels. As a first criterion of the feasibility of the approach, PEGylationinduced variations of the thermoresponsive characteristics of the polymers have been carefully monitored. The N-alkylacrylamide-containing polymers exhibit a lower critical solution temperature (LCST). This temperaturetriggered phase transition is usually accompanied by a transition from soluble to insoluble, whereas the PEGcontaining graft copolymers show a transition from coil structure to micelles. This is due to the fact that the volume phase transition of, e.g., PNiPAAm goes along with a transition from hydrophilic to hydrophobic, which finally converts an all-hydrophilic graft copolymer at low temperatures into an amphiphilic polymer above the LCST. The corresponding hydrogels have a so-called lower gel transition temperature (LGTT) in the same region as the LCST. We further found that the LCSTs and LGTTs of poly(N-alkylacrylamide)s can be fine-tuned around 37 °C by adding small amounts of a comonomer, e.g., the LCST of PNiPAAm homopolymer is 32 °C and the addition of PEG shifts the LCST to higher temperatures.12,13 In this way, the LCST can be adjusted between 32 and 83 °C, depending on the composition and the polymer architecture. With this setup, we were able to synthesize a series of graft copolymers with different PEG chain lengths and different thermoresponsive monomers, which all show an LCST around 37 °C. To further analyze the obtained variety of thermoresponsive polymers as cell-culture substrates, a thin-film technology was required for the well-defined immobilization of the polymers on macroscopic surfaces. Low-pressure plasma treatment of the preadsorbed polymer films was selected for that purpose sincesgiven the appropriate settingssthis method provides a very versatile and powerful means for the cross-linking of thin organic layers on top of a wide variety of carrier materials.15-18 We report in the following details of the plasma immobilization of poly(N-isopropylacrylamide-gethylenglycol) or poly(N,N-diethylacrylamide-g-ethylenglycol) on top of poly(tetrafluorethylene) (PTFE)-like fluorocarbon surfaces and give results of the analysis of the obtained coatings with respect to their thermoreversible swelling behavior. Data of protein entrapment and cell-culture experiments with similar substrates will be provided in a companion article. Experimental Section Materials. The graft copolymers P1-P4 (Figure 1) were synthesized from N-isopropylacrylamide (NiPAAm) or N,N(15) Nitschke, M.; Menning, A.; Werner, C. J. Biomed. Mater. Res. 2000, 50, 340-343. (16) Terlingen, J. G. A.; Feijen, J.; Hoffman, A. S. J. Colloid Interface Sci. 1993, 155, 55-65. (17) Terlingen, J. G. A.; Brenneisen, L. M.; Super, H. T. J.; Pijpers, A. P.; Hoffman, A. S.; Feijen, J. J. Biomater. Sci. Polymer Ed. 1993, 4, 165. (18) Sheu, M. S.; Hoffman, A. S.; Feijen, J. J. Adhesion Sci. Technol. 1992, 6, 995.

Schmaljohann et al. diethylacrylamide (DEAAm) and poly(ethyleneglycol)monomethyl ether monomethacrylate via free-radical copolymerization. Experimental details are given elsewhere.12,13 Low-Pressure Plasma Immobilization. Nonbranched fluorocarbon films with a structure close to PTFE were prepared on silicon substrates with an oxide layer of 50 nm, which allows the ellipsometric investigation of the hydrogel preparation. The fluorocarbon films, kindly provided by the Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences (Chernogolovka, Russia), were deposited by plasma polymerization. Tetrafluoroethylene (C2F4) was introduced downstream into a low-pressure argon discharge. Silicon wafers were placed further downstream relative to the discharge. The thickness of the obtained fluorocarbon films was about 50 nm.19 The fluorocarbon surfaces were treated in argon plasma, as described below, for 120 s to obtain an appropriate wetting behavior for spin coating. Thin films of the polymers P1-P4 were prepared on the fluorocarbon surfaces by spin coating from a 0.5% wt/wt solution in CHCl3. A spin coater RC5 by Karl Suss, France, was operated at a velocity of 5000 rpm and an acceleration rate of 5000 rpm/s ()83.3 s-2). The films of P1-P4 were immobilized on the fluorocarbon surface using low-pressure argon plasma. The plasma treatment was carried out in a computer-controlled MicroSys apparatus by Roth and Rau, Germany. The cylindrical vacuum chamber, made of stainless steel, has a diameter of 350 mm and a height of 350 mm. The base pressure obtained with a turbomolecular pump is 44 °C. Thus, the system has a uniform thermal history after heating the first time above the LCST. The data for the three characteristic points of this plot (initial swelling, below, and above the LCST after one heating/cooling cycle) are given for P1-P4 in Table 3. Another observation from Figure 4 is that the heating and the cooling curve exhibit a hysteresis. This hysteresis is in the range of 2-4 K, which also causes a change in the determined LGTT (Table 3). A comparison of film thickness, water content, and degree of swelling for P1 is shown in Figure 5. The hydrogels based on polymers P3 and P4 show very broad transitions (Figure 6b). Therefore, the evaluation of the LGTT as a point in the film thickness-temperature plot is misleading, but the data of Table 3 was given to indicate the hysteresis. In both cases, the transitions span more than 15 K. Film thickness and degree of swelling show a similar curve, whereas the water content underestimates changes at high swelling ratios. This is due to the fact that DS ≈ 1/WC or dswollen ≈ 1/WC, respectively (compare eqs 1 and 2). Both film thickness and degree of swelling can be analyzed for the determination of the volume phase transition. The degree of swelling will be later discussed to compare the different hydrogel films. Two Time Scales of Swelling. Figures 4 and 5 represent data taken at heating rates of ca. 0.25 K/min (and cooling rates of -0.25 K/min, respectively). The results do not vary much when the heating and cooling rates are varied

Thermo-Reversible Swelling of Thin Hydrogel Films

Langmuir, Vol. 20, No. 23, 2004 10111

Table 3. Characterization of the Hydrogel Films after Initial Swelling and after One Heating/Cooling Cycle initial swelling

second heating/cooling cycle

polymer solution

no.

film thickness

water content

degree of swelling

thickness at 25 °C

thickness at 42 °C

LGTT at heatinga

LGTT at coolinga

LCST at coolingb

P1 P2 P3 P4

253 nm 158 nm 87 nm 91 nm

92.8% 91.2% 79.5% 83.0%

13.9 11.3 4.9 5.9

92 nm 77 nm 61 nm 83 nm

30 nm 39 nm 35 nm 52 nm

37 °C 35 °C ∼35 °C ∼34 °C

35 °C 33 °C ∼32 °C ∼31 °C

40.6 °C 35.1 °C 38.8 °C 37.3 °C

a LGTT is determined as the inflection point of the film thickness-temperature plot; the LGTTs for P3 and P4 are only estimates due to the very broad and gradual transition. b LCST is determined as the inflection point of the UV/vis transmission-temperature plot.

Figure 5. Collapsing of the P1 film after initial swelling, comparison of film thickness (bottom), water content (middle), and degree of swelling (top).

within certain limits ((0.1-1 K/min). However, when the film thickness is followed over a longer time period at constant temperature, one can measure a slow increase. Thus, the ‘regular’ heating/cooling cycle reflects a ‘dynamic’ swelling, but it does not represent an equilibrium state (Figure 2). These two time scales of swelling (dynamic swelling and equilibrium swelling) are investigated in the following sections. Dynamic Swelling. The dynamic swelling can be shown by the second heating/cooling cycle because the hydrogels have a well-defined thermal history after the first heating above the LGTT. Figure 6 gives the film thickness vs temperature plots of the second cycle for all investigated hydrogel films. The PNiPAAm-containing films (P1, P2, Figure 6a) have a sharp transition with a clearly detectable transition point. In contrast, the change in film thickness of the PDEAAm-containing films (P3, P4, Figure 6b) is more gradual, almost linear. Thus, the LGTT of P3 and P4, respectively, can only be given with an error of (2 °C (Table 3). If the intended application requires a sharp transition, the PNiPAAm-containing films will be advantageous. However, the gradual change of P3 and P4, respectively, might be of interest for sensing applications. Figure 6 also shows the hysteresis of the dynamic swelling, which is in the range of 2-3 K.

Figure 6. Film thickness vs temperature during the second heating/cooling cycle for (a) P1, P2, and (b) P3, P4.

Figure 7. Different hydrogen bonding properties of the NiPAAm units (left) and the DEAAm unit (right).

The reason the DEAAm-containing films exhibit a much broader transition is not yet clear. One possible explanation is that the swelling-and-collapsing process is influenced by the capability of the hydrogel to form hydrogen bonds to water. The amide group of the NiPAAm unit is a hydrogen-bond donor and acceptor, whereas the amide group of the DEAAm unit can only act as a hydrogenbond acceptor (Figure 7). Thus, the NiPAAm-containing hydrogels should transport water much faster in and out of the film, which then would yield differences in the dynamic swelling. Furthermore, the NiPAAm unit can also form hydrogen bonds to the ether-oxygen group of the ethyleneglycol units, which is only a hydrogen-bond

10112

Langmuir, Vol. 20, No. 23, 2004

Schmaljohann et al.

Figure 8. Degree of swelling of P1-P4 vs temperature during the second heating.

acceptor. This gives the potential for an internal stabilization of the system through either interaction with the PEG units or trapping water molecules. So far, this is only a hypothesis; a more detailed study on the differences between the two kinds of polymer is currently underway. Discussion of the Degree of Swelling. The degree of swelling DS (eq 2) gives an indication of how efficient the cross-linking process was. A temperature-dependent plot of the swelling shows how complete the collapsing process above the LGTT is. Figure 8 gives a plot of DS vs temperature during the second heating scan. Again, the gradual change of P3 and P4, is observed, compared to a sharp transition of P1 and P2, respectively. In addition, the comparison of P1 and P3 gives further information. Both films have almost the same film thickness as dry films (Table 2). P1 has not only a larger degree of swelling below the LGTT but also a smaller value above the LGTT in the dynamic swelling. This means that the swelling is favored, which can be explained by the differences in hydrogen bonding to water (Figure 7), where NiPAAm can also act as hydrogen bonding donor. For the same reason, the NiPAAm units can also be more efficient in the collapsing process above the LGTT because in this state internal hydrogen bonds stabilize the system, which works for NiPAAm as hydrogen-bond donor and acceptor similar to internal stabilization of proteins. Figure 8 also demonstrates that the films are partially swollen even above the volume phase transition. Fifty percent or more of the so-called collapsed films is still water. This is important for the further application because that preserves a certain hydrogel character above the transition temperature, which is favored against standard cell-cultivation carriers. It is advantageous compared to the similar hydrogels based on the PNiPAAm homopolymer, which are almost completely collapsed above the transition temperature.26 Comparison of the Hydrogel Swelling to the LCST Behavior of the Polymer Solutions. Table 3 also compares the LCST of the polymer solution with the LGTT of the surface-immobilized hydrogel films. The LGTT of the hydrogels appears at a slightly lower value compared to the LCST of the polymer solutions. This is also demonstrated in Figure 9, where the full symbols represent the polymer solution and the open symbols represent the hydrogel films. All four polymer solutions show a sharp transition at the LCST. On the other hand, the situation of the hydrogel films is more complicated. The NiPAAm-containing films (26) Kuckling, D.; Harmon, M. E.; Frank, C. W. Macromolecules 2002, 35, 6377-6383.

Figure 9. Comparison of the transition in polymer solution (UV transmittance, filled symbols) and in the hydrogel film (film thickness, open symbols) for (a) P1, P2 and (b) P3, P4.

(P1, P2, Figure 9a) have only a slightly broader transition, whereas the DEAAm-containing films (P3, P4, Figure 9b) have an almost linear change in film thickness. Concluding from this results, in addition to the equilibrium swelling experiments, the DEAAm-containing films might be suitable for sensing applications. On the other hand, the NiPAAm-containing films have a great potential to be used in biomedical applications. It is noteworthy that the hydrogel films also show a change in the surface polarity, which could be demonstrated by inverse contact-angle measurements. However, the change is much less pronounced as compared to the corresponding hydrogel prepared from PNiPAAm homopolymer. This is due to presence of the PEG units giving the collapsed state a more-hydrophilic character.27 It should be noted that not only the immobilization and cross-linking step causes a shift in the transition temperature toward lower values but also electrolytes, as present in cell cultivation medium, are shifting the transition temperature of both the soluble polymers and the hydrogels toward lower values (∼2-4 K).28 Thus, all hydrogel films discussed in this paper are in the collapsed state at 37 °C. A detailed study on the effect of addition of electrolytes will be given in a forthcoming paper. Equilibrium Swelling. The equilibrium swelling of the hydrogel films was followed with a different experiment. First, the films were annealed in water at 55 °C for 24 h. Then, the wafers were immersed into water at 23 °C (27) Schmaljohann, D.; Beyerlein, D.; Nitschke, M.; Zschoche, S.; Werner, C. PMSE Prepr. 2003, 88, 551-552. (28) Schmaljohann, D.; Nitschke, M.; Beyerlein, D.; Werner, C. Polym. Prepr. 2003, 44, 196-197.

Thermo-Reversible Swelling of Thin Hydrogel Films

Langmuir, Vol. 20, No. 23, 2004 10113

Figure 10. Equilibrium swelling experiment at 23 °C, film thickness of P2 and P4 vs time; inset, first 5 h.

followed by ellipsometry measurements as function of time. These experiments were performed similarly to the measurement of the swelling kinetics of PNiPAAm gels T-jumped across the volume phase-transition temperature by Shibayama et al.29 It is a dynamic measurement of the film thickness, which leads to the swelling equilibrium as the final value. Figure 10 shows a plot of the film thickness of P1 and P4 as function of time. The point when the sample is brought into contact with the cold water is taken as t ) 0. Consequently, the film thickness at 55 °C is the starting value. The ellipsometry measurements were evaluated starting from t ) 10 min because the fluid cell needed that time to equilibrate the temperature at 23 °C. Figure 10 shows that the swelling equilibrium is reached after 5-10 days. Another observation is that there are differences between the first hours of swelling and the following time period. The inset in Figure 10 shows the first 5 h, where the largest swelling rate is observable. Tanaka et al.30 and Shibayama et al.29,31 described the time variation of the gel size by a single-exponential function.

|d(t) - d(∞)| 6 ≈ e- t/τ |d(0) - d(∞)| π2 τ)

d2(∞) 4π2D

wD)

d2(∞) 4π2τ

(3)

(4)

with d(t) ) film thickness, t ) time, τ ) characteristic swelling time, and D ) diffusion coefficient Equation 3 can be linearized by plotting ln{(d(t) - d(∞))/ (d(0) - d(∞))} vs t. The slope of this graph is -τ-1. The resulting τ was plugged into eq 4 to estimate the cooperative diffusion coefficient, D. The evaluation of a graph of ln{(d(t) - d(∞))/(d(0) - d(∞))} vs t yields τ ) 1.6 × 102 min and D ) 6.9 × 10-16 cm2/s for P1 and τ ) 1.5 × 102 min and D ) 5.7 × 10-16 cm2/s for P4. It is noteworthy that this plot does not give a linear correlation for the whole time period. For this reason, only the data of the first 30 min were evaluated. The data at 40-50 min already show the deviation from linearity. This means the diffusion coefficient is also a function of time. The characteristic time of swelling, τ, is 2 orders of magnitude larger than those values for the PNiPAAm (29) Shibayama, M.; Nagai, K. Macromolecules 1999, 32, 7461-7468. (30) Tanaka, T.; Fillmore, D. J. J. Phys. Chem. 1979, 70, 1214-1218. (31) Shibayama, M.; Uesaka, M.; Shiwa, Y. J. Phys. Chem. 1996, 100, 4350.

bulk gels.29 Even though the data evaluation was not carried out for the dynamic swelling due to the experimental setup, one can estimate that the dynamic swelling occurs with τ values that are 1-2 orders of magnitude lower. This supports that there are two different time scales of swelling, which is also indicated by the fact that the linearized plots of Figure 10 do not go through the origin. In addition, we observed further differences in the dynamic swelling for t > 30 h. This is probably due to physical cross-links caused by entanglements, which are later rearranged because of the increasing mobility. Interestingly, the NiPAAm-containing hydrogel film P1 does not reach the film thickness of the initial swelling anymore, whereas the DEAAm-containing film P4 exceeds the original value. The equilibrium swelling experiment was conducted below the LGTT because the largest differences can be observed below the phase transition. Yet another question is whether there are also changes over time above the LGTT. To answer this question, two points can be compared: the film thickness of the dynamic swelling above the LGTT and the value after 24 h equilibration at 55 °C. Even though it is not clear whether the deswelling equilibrium has been reached yet, it will be denoted as equilibrium. In any case, a comparison of the two points indicates changes of the film thickness as a function of time. P1 has a film thickness of 22 nm (dynamic, at 55 °C) compared with 47 nm (equilibrium, at 55 °C). P4 has a film thickness of 52 nm (dynamic, at 42 °C) compared with 49 nm (equilibrium, at 55 °C). Again, differences are evident between P1 and P4. P1 increases its swelling ratio during equilibration at 55 °C, whereas P4 exhibits almost no changes after 24 h at 55 °C. Concluding from that, the equilibrium swelling of the NiPAAm-containing films is above the dynamic swelling curve for the entire temperature range. On the other hand, the DEAAm-containing films only show differences between swelling equilibrium and dynamic swelling below the phase-transition temperature, and the overall changes are not as pronounced as those observed for the NiPAAm-containing films. It could still be possible that the swelling equilibrium of the DEAAM-containing film needs more time at elevated temperatures. As already shown in Figure 7, PNiPAAm and PDEAAm have different hydrogen-bonding capabilities, which might be responsible for the differences between those two groups of hydrogel film and also for the difference in hydrogels based on the PNiPAAm homopolymer. Usually, the swelling rate is inversely proportional to the square of the characteristic dimension of the gel.30 This would mean that a thin hydrogel of the dimensions of some nanometers would have a very fast response time, and this is in contrast to our observed results. Hence, the same polymers also exhibit differences in solution compared to the ‘regular’ behavior of thermoresponsive copolymers. It was found that for both thermoresponsive monomers (NiPAAm and DEAAm, respectively) the graft copolymers with PEG side chains exhibit a dependence of the LCST from the molar ratio of ethyleneglycol units rather than the molar ratio of monomer units along the main chain. This already suggests that there is an interaction of the PEG chains with the thermoresponsive backbone due to the graft copolymer architecture, and NMR studies give further evidence for this behavior.13 On the basis of these results, we have proposed that the two-stage mechanism of volume phase transition (1. intramolecular collapse and 2. intermolecular aggregation) has to be replaced in this particular case by a three-stage mechanism: 1. intramo-

10114

Langmuir, Vol. 20, No. 23, 2004

lecular stabilization through hydrogen bonding to the PEG, 2. intramolecular collapse, and 3. intermolecular aggregation. A similar effect could take place in the corresponding hydrogels, where the PEG can also interact with the thermoresponsive backbone, leading to a completely different swelling and collapsing kinetics besides shifting the transition temperature. In general, the volume phase transition is a result of a delicate balance between enthalpic and entropic effects. At low temperature, the solubilization is favored due to a reduction in the free energy caused by hydrogen bonding of water molecules, which exceeds the loss in entropy due to structured water around the NiPAAm units. This effect reverses above the transition temperature, which is accompanied by a release of the water molecules. For PEG-containing copolymers, the intramolecular stabilization through hydrogen bonding to PEG is not accompanied by a large entropy penalty because only some degrees of orientational freedom are reduced, which causes an entrapment of water in the polymer or hydrogel even above the transition temperature.13 So far, a complete discussion of the observed effect is not possible, and it must be accompanied by a further analysis of the soluble polymers. Conclusions Four graft copolymers of poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as polymer backbone and poly(ethyleneglycol) (PEG) as side chains were immobilized and cross-linked on a solid substrate using low-pressure Ar plasma. All immobilized polymers have a LCST between 34 and 40 °C. The crosslinked polymers absorb water from humidity to raise the film thickness by the factor 1.3-3.2 even before swelling in water, which is due to the hygroscopic character of the material. The swelling and collapsing of the hydrogels

Schmaljohann et al.

was examined in aqueous solutions with temperaturedependent spectroscopic ellipsometry. Two time ranges of swelling were observed: a fast ‘dynamic’ swelling, which occurred within minutes or less, and a slow ‘equilibrium’ swelling, which took several days. The formation of the hydrogels resulted in a shift of the transition temperature toward lower values compared with those in solution, which leads to final transition temperatures between 32 and 37 °C. This temperature range allows for study of the material in biological experiments, where, e.g., cells can be cultivation in the collapsed state at 37 °C and can be detached upon lowering the temperature.32 Full reversibility of the dynamic swelling/collapsing was found, but the temperature scan exhibits a hysteresis between the heating and cooling cycle. In the dynamic range, the PNiPAAm-containing hydrogels P1 and P2 have a degree of swelling of 6-7 at low temperatures and of 2-3.5 at elevated temperatures, whereas the PDEAAm-containing hydrogels P3 and P4 exhibit variations to higher and lower values, probably due to a changing cross-linking density. The differences in the swelling dynamics of P1 and P2 compared to P3 and P4, respectively, were attributed to the different hydrogen bonding capabilities of NiPAAm compared with DEAAm. Acknowledgment. The authors thank Prof. B. Voit, Dr. K.-J. Eichhorn, and Dr. S. Richter for valuable discussion and support of this work. We are also grateful to N. Tarek El Tahan for help with the synthesis of the polymers. LA034653F (32) Schmaljohann, D.; Oswald, J.; Jørgensen, B.; Nitschke, M.; Beyerlein, D.; Werner, C. Biomacromolecules 2003, 4, 1733-1739.