4650
Langmuir 1998, 14, 4650-4656
Wilhelmy Plate Measurements on Poly(N-isopropylacrylamide)-Grafted Surfaces Claudio Della Volpe,*,† Clara Cassinelli,‡ and Marco Morra‡ Nobil Bio Ricerche, Str. S. Rocco 32, Villafranca d’Asti, Italy, and Materials Engineering Department, University of Trento, V. Mesiano 77, Trento, Italy Received November 14, 1997. In Final Form: April 8, 1998 Polystyrene surfaces have been modified by poly(N-isopropylacrylamide) (PIPAAm) grafting. Beside observing the adhesion and the effect of low-temperature treatment on anchorage-dependent cells, the wetting behavior of PIPAAm-grafted surfaces was studied by dynamic contact angle measurement by Wilhelmy microbalances. Measurements were performed in water at different temperatures and at different speeds of immersion. Contact angle behavior has been explained in terms of heterogeneity of the surfaces, using a slightly modified hysteresis graph. The apparent inversion of advancing and receding contact angles shown at high speeds of immersion has been analyzed in terms of kinetic effect, and it is underlined the lack of a similar critical approach in existing literature data.
Introduction The modification of polymer surfaces by a top layer whose properties depend on external stimuli (i.e., temperature, pH, etc.) is a topic of great theoretical and practical interest. In principle, this approach allows the production of polymer surfaces that can respond to changes in some environmental parameter. Surface modification by poly(N-isopropylacrylamide) (PIPAAm) is a typical example. PIPAAm in water shows
a fully expanded chain conformation below the lower critical solution temperature (LCST) of 32 °C and a collapsed, compact conformation at temperatures above the LCST.1 Because of this temperature effect, the nature of surfaces covered by a layer of PIPAAm can be switched from hydrophilic to more hydrophobic and vice versa by a temperature change. Surface coating by thermally reversible polymers (TRP) such as PIPAAm has been recently suggested as a new † ‡
University of Trento. Nobil Bio Ricerche.
(1) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci., B, Polym. Phys. 1990, 28, 923.
system for the recovery of cells from tissue culture substrata without the need for proteolytic enzymes (e.g., trypsin) to digest the matrix responsible for cell attachment.2-5 In these systems, cells can be recovered from tissue culture substrata simply by lowering the temperature below a critical threshold. In fact, while at the normal cell culture temperature of 37 °C, above the LCST, the comparatively hydrophobic, rigid surface structure of PIPAAm can support cell attachment and growth, the high hydrophilicity and water content of the PIPAAm below LCST prevent cell attachment.6 As reported in several papers, cells grown on a PIPAAmcoated culture dish can be recovered by leaving the dish below the LCST for a time that depends on the cell type, the growth state (i.e., confluent vs subconfluent cells), and the fine structure of the PIPAAm surface layer.4,5,7,8 Several methods have been suggested in the literature to produce PIPAAm-coated surfaces, from electron beam4 and UV grafting8 to physical coating.7 All published studies indicate that irrespective of the surface modification method used, the temperature-dependent properties of PIPAAm in solution are maintained when the polymer is grafted to a substrate surface. Contact angle measurement, performed at temperatures above and below the LCST, is among the most direct techniques to evaluate the environment-dependent properties of PIPAAm coated surfaces. Among contact angle techniques, the Wilhelmy plate experiment9,10 is the most suitable, because one can closely monitor the temperature of the probe liquid. Wilhelmy plate contact angle measurement on several PIPAAm-coated surfaces has been described in a recent (2) Takezawa, T.; Mori, Y.; Yoshizato, K. Biotechnology 1990, 8, 854. (3) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11, 571. (4) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biome. Mater. Res. 1993, 27, 1243. (5) Rollason, G.; Davies, J. E.; Sefton, M. V. Biomaterials 1993, 14, 153. (6) Lydon, M. T.; Minett, W.; Tighe, B. J. Biomaterials 1985, 6, 396. (7) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16, 297. (8) Morra, M.; Cassinelli, C. Polym. Prepr. 1995, 36, 55. (9) Andrade, J. D.; Smith, L. M.; Gregonis, D. E. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, chapter 7. (10) Garbassi, F.; Morra, M.; Occhiello, E. Polymer Surfaces, from Physics to Technology; Wiley: Chichester, 1994.
S0743-7463(97)01243-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/11/1998
Wetting Behavior of PIPAAm-Grafted Surfaces
paper by Okana and co-workers.11 While their results nicely show the expected temperature dependence of the advancing water contact angle, reported receding angles are, surprisingly, higher than advancing ones, and no explanation is given for this rather uncommon finding. Apparent receding angles higher than advancing ones have been qualitatively described by Andrade, as a result of the interplay of thermodynamic and kinetic phenomena at the three-phase boundary.12 It must be underlined that, as most hydrophilic or hydrogel-like surfaces, PIPAAm coatings are quite different from the ideal surface required by the Young equation, which should be rigid, non-deformable, and unable to interact with the liquid. In these systems, beside the purely thermodynamic interaction described by the Young equation, it is likely that kinetic phenomena contribute to the shape of the hysteresis loop and, ultimately, to the experimental output.9,10,13 These considerations prompted us to undertake a systematic study of the different contributions to the overall shape of the hysteresis loops and the measured contact angles obtained by Wilhelmy plate measurements on PIPAAm-coated surfaces. To this end, we performed temperature- (above and below LCST) and velocitydependent Wilhelmy plate measurements on PIPAAmcoated polystyrene (PS). Contrary to the quoted paper of Okano and co-workers,11 where pre-polymerized PIPAAm was linked to the substrate, we used the Ce(IV)-induced polymerization and grafting of aqueous solution of Nisopropylacrylamide (NIPAAm) monomer to produce PIPAAm-coated PS surfaces. This method, based on the redox chemistry of Ce(IV) ions, has been frequently used to graft acrylic monomers to corona- or plasma-treated polymer surfaces.14-16 While the main goal of this paper is to describe and discuss the hysteresis loops arising from Wilhelmy plate experiments on PIPAAm-grafted surfaces, cell behavior and, in particular, the effect of low-temperature treatment on cells are reported as well. In this way, it is possible to show that, indeed, the surface modification process used imparts the expected temperature-dependent properties to cell culture substrate surfaces. However, other parameters more directly relevant to cell culturing, that is, growth rate or the behavior of different cell lines, are outside the scope of this paper and will not be discussed here. As a rough indication, it can be said that L-929 fibroblasts used in the present experiment, albeit reaching confluence, grow on surfaces grafted with PIPAAm according to the present method at a much lower rate as compared to conventional tissue culture polystyrene (TCPS). The doubling time is about 60% longer in the former case. Experimental Section Materials. NIPAAm (98% pure) was purchased from Kodak. Surface modification was performed on PS Petri dishes (5 cm diameter) and 24-well PS plates, both non-tissue-culture treated, from Corning. Samples for the Wilhelmy plate experiment were (11) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Macromolecules 1994, 27, 6163. (12) Andrade, J. D.; Smith, L. M.; Gregonis, D. E. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, p 280. (13) Morra, M.; Occhiello, E.; Garbassi, F. J. Colloid Interface Sci. 1992, 84, 149. (14) Hoffman, A. S. Adv. Polym. Sci. 1984, 57, 142. (15) Yun, Y. K.; DeFife, K.; Colton, E.; Stack, S.; Azeez, A.; Cahalan, L.; Verhoeven, M.; Cahalan, P.; Anderson, J. M. J. Biomed. Mater. Res. 1995, 29, 257. (16) Morra, M.; Cassinelli, C. J. Biomed. Mater. Res., in press.
Langmuir, Vol. 14, No. 16, 1998 4651 obtained by cutting 2 × 1 cm samples from bacteriological grade PS Petri dishes. Surface Modification. PIPAAm grafting to PS surfaces was performed using a two-step procedure. In the first step, PS samples were air plasma treated in a stainless steel, parallel plate plasma reactor. Treatment conditions were as follows: 2 Pa air pressure, 20 sccm (standard cm3/min) flow rate, and 15 s treatment time. Treated samples were dropped in a 10% NIPAAm solution in water, containing 0.2 wt % ammonium cerium nitrate, 98.5% pure (Aldrich). The reaction was carried on for 2 h at room temperature. After treatment, samples were extensively rinsed with cold water, to remove unreacted NIPAAm and homopolymer PIPAAm. Petri dishes and plates were then filled with water and stored in a refrigerator overnight. Surface Characterization. Surface composition was evaluated by electron spectroscopy for chemical analysis (ESCA), using a Perkin-Elmer PHI 5500 ESCA system. The instrument is equipped with a monochromatic X-ray source (Al K anode), operating at 14 kV and 250 W. The diameter of the analyzed spot is 400 µm. The base pressure was 10-8 Pa. Peak deconvolution and quantification of the elements was accomplished using the software and sensitivity factors supplied by the manufacturer. Curves were fitted through a best-fit procedure, assuming a Gaussian peak shape. The angle between the electron analyzer and the sample surface was 45°. Survey spectra were acquired using a pass energy of 58.7 eV. In the high-resolution mode, the pass energy was 5.850 eV. All binding energies were referenced by setting the CHx peak maximum in the resolved C(1s) spectra to 285.0 eV. Wilhelmy Plate Experiments. Dynamic contact angles have been measured at controlled temperature using a Cahn 322 and a KSV Sigma 70 microbalances. All measurements done at an immersion speed higher than 250 µm/s have been performed on Sigma 70, while measurements below 250 µm/s have been performed on a Cahn 322. This is due to the speed limit of the Cahn 322. Temperature of the immersion liquid has been controlled on both microbalances with a precision of (1 °C; no control was performed on the air temperature in contact with the test liquid. Five subsequent immersion cycles have been accomplished for each sample; runs were performed at two temperatures, 20 and 37 °C, and at four speeds, 40, 166, 250, and 666 µm/s. Water HPLC grade by Merck has been used through all measurements as a test liquid, and its surface tension was controlled and found in good agreement with literature data. Contact angle values are the mean of five independent runs. The significant standard deviation of contact angles is partly due to the difficulty of obtaining good parallelepiped samples from the PS Petri dishes. Cell Culturing. The continous mouse fibroblasts cell line L-929 was used in cell culturing. Experimental cell culture medium (BIOCHROM KG, Berlin) consisted of Minimum Eagle’s Medium without L-glutamine, 10% fetal bovine serum, streptomycin (100 g/L), penicillin (100 U/mL), and 2 mmol/L Lglutamine in 250-mL plastic culture flask Corning). Cells were cultured at 37 °C in a humidified incubator equilibrated with 5% CO2. Fibroblasts were harvested prior to confluence by means of a sterile trypsin-EDTA solution (0.05 trypsin, 0.02 EDTA in normal phosphate buffered saline, pH 7.4) from the culture flasks, resuspended in the experimental cell culture medium, and diluted to 1 × 105 cells/mL. 5 mL of the cells suspension were seeded into PIPAAm-coated and control TCPS Petri dishes, 1 mL in the 24-well plates. Experiments were performed in triplicate. Cell Recovery. Recovery by low temperature treatment was obtained by placing the cells containing PS dishes in a refrigerator, at 10 °C for a given time. The effect of the low-temperature treatment on the number of cells remaining on the dish surface was evaluated by optical microscopy, using an inverted microscope equipped with phase-contrast optics (Leica). Scanning Electron Microscopy of Cells. The cell behavior on control and PIPAAm-coated PS was also observed by scanning electron microscopy (SEM), using a LEO 420 SEM. PIPAAmcoated and control uncoated 24-well plates were used for these experiments. Briefly, after a given time in the incubator, cells in three different wells for each kind of surface (i.e., three control and three PIPPAm-coated PS) were fixed with a 2% solution of
4652 Langmuir, Vol. 14, No. 16, 1998
Della Volpe et al.
Table 1. Surface Composition (atom %) As Detected by ESCA Analysis, of Untreated, Plasma Treated, and PIPAAm-grafted PS sample
O
C
N
PS‘ plasma-treated PS PIPAAm-grafted PS PIPAAm, theoretical
13.6 11.9 12.5
100.0 86.4 76.9 75.0
11.2 12.5
glutharaldehyde. The plate was then put into the refrigerator and, after a 30-min period, cells in three more wells were fixed. The same procedure was repeated several times, for a total follow up of 2 h in the refrigerator. Fixed cells were then dehydrated by several passages in ethanol-water solutions 30 min each, with decreasing water content. After the last passage in absolute ethanol, dehydration was completed by a 30 min immersion in hexamethyldisiloxane (Aldrich). The fixed and dehydrated cell layer was further sputter coated by gold (Agar sputter-coater) before SEM observation.
Results Surface Composition. The surface composition of the PIPAAm-grafted samples, together with the composition of PS and plasma-treated PS are reported in Table 1. As expected, the surface of untreated PS contains only carbon, and no traces of contaminants or extraneous elements are detected. Air plasma treatment introduces oxygen-containing functionalities on the PS surface. The C(1s) peak shows the well-known broadening, due to chemical moieties containing multiple carbon to oxygen bonds. After PIPAAm grafting, the surface composition is close to the composition expected for PIPAAm homopolymer. Actually, the oxygen to carbon and nitrogen to carbon ratios are a little lower than the theoretical value, probably because of some slight contamination from ubiquitous hydrocarbons; we remember here that the depth analysis of the used vacuum technique is about 8 nm, using the experimental conditions described in the relevant section, so this eliminates different interpretations, for example, internal molecular reorientation, which involves shallower layers. Anyway, data of Table 1 indicate that a PIPAAm layer, whose thickness is similar to or greater than the ESCA sampling depth, covers the PS surface and successfully withstands the cold water extraction routine described in the Experimental Section. Wilhelmy Plate Measurements. Numerical results are shown in Tables 2 and 3 and Figure 2. The results obtained during the first immersion are reported separately from the mean values of the immersions 2∧ to 5∧, which appear substantially constant. Untreated PS plates show contact angle values substantially independent of speed, temperature, and immersion cycles, except at the highest speed, where some differences are present. Plasma-coated PS plates show some differences between the first immersion and the subsequent ones and also an increasing trend with speeds particularly at 250 and 666 µm/s. However the effect of temperature is evident only on receding values at the highest speed; in this case the receding angle is higher at 37 °C than at 20 °C. In the case of PIPAAm-grafted surfaces the situation is more complex, because there is always a difference in contact angle values between the first immersion (1° cycle) and the subsequent ones (2-5 cycles); moreover during these immersions the force value changed abruptly with the immersion depth (Figure 2a). This is particularly evident and prolonged at higher immersion speeds (Figure 2b). During the reimmersion of the lower part of the sample, which was exposed to the air for the shortest time,
Table 2 cycles
40 µm/s
166 µm/s
250 µm/s
666 µm/s
(a) Advancing and Receding Dynamic Contact Angles of Untreated PS Plates at Different Speeds (20 °C, Standard Deviation (SD) ) (3°) 1-θadv 96 97 98 101 69 70 70 73 1-θrec 2-5 θadv 96 94 95 95 2-5 θrec 69 69 69 73 (b) Advancing and Receding Dynamic Contact Angles of Untreated PS Plates at Different Speeds (37 °C, Standard Deviation (SD) ) (5°) 1-θadv 98 91 1-θrec 68 72 2-5 θadv 99 93 2-5 θrec 68 73 (c) Advancing and Receding Dynamic Contact Angles of Plasma-Treated PS Plates at Different Speeds (20 °C, SD ) (5°) 1-θadv 68 70 82 86 1-θrec 16 12 17 29 2-5 θadv 62 62 69 72 2-5 θrec 15 11 15 29 (d) Advancing and Receding Dynamic Contact Angles of Plasma-Treated PS Plates at Different Speeds (37 °C, SD ) (5°) 1-θadv 74 72 75 83 1-θrec 20 17 20 45 2-5 θadv 66 62 64 73 2-5 θrec 20 16 18 45 Table 3a cycles
40 µm/s
166 µm/s
250 µm/s
666 µm/s
(a) Advancing and Receding Dynamic Contact Angles of PIPAA-Grafted PS Plates at Different Speeds (20 °C, SD ) (5°) 1-θadv 84 80 83 92 1-θrec 3 15 13 11 2-5 θadv 26 (70) 17 (71) 12 (66) cos θ > 1b (na) 2-5 θrec 0 17 12 8b (b) Advancing and Receding Dynamic Contact Angles of PIPAAm-Grafted PS Plates at Different Speeds (37 °C, SD ) (5°) 1-θadv 78 76 74 78 1-θrec 10 19 28 41 2-5 θadv na(64) na(61) na(60) 35b (58) 2-5 θrec 8 13 27 41b a Data in parentheses refer to the upper part of the sample, which is reimmersed after a more prolonged exposition to air, na ) data not available. b This apparent inversion of advancing and receding contact angles is discussed in the text.
the advancing force is equal or higher than the receding one; after a few millimeters, during the immersion of the upper part, exposed to air for a more prolonged time, the value reduces, but remains higher than the first advancing. If the speed is sufficiently high and/or the immersion depth is lower, the second part of this trend may be not shown (Figure 2c). Particularly at the highest speed it is possible to obtain a force value during the subsequent advancings higher than that obtained during the recedings (Figure 2d); this result is very impressive and strange. The effect of temperature is simply resumed: the advancing values during the first cycle and the advancing values of the upper part of the samples (if available) are greater at 20 °C than at 37 °C, and all receding values (but one) and the advancing values of the lower part of the samplkes (if available) are lower at 20 °C than at 37 °C. Cell Behavior and Low-Temperature Cell Recovery. Cells seeded on PIPAAm-grafted PS Petri dishes adhere and spread on the cell culture substrate, even if
Wetting Behavior of PIPAAm-Grafted Surfaces
Langmuir, Vol. 14, No. 16, 1998 4653
Figure 1. Scanning electron microscope photographs of (a) a nearly-confluent layer of L-929 mouse fibroblasts on a PIPAAm grafted PS dish and (b) the same cells after 1 h in the refrigerator.
the time to reach confluence is longer by about 60% than that measured on conventional TCPS. Figure 1a is a SEM image of L-929 fibroblasts after 3 days in cell culture conditions in a 24-well plate. Cells exhibit a normal morphology and are well spread on the substrate surface, and the cell layer is nearly confluent. When the same plate is placed in a refrigerator a marked change in cells
shape occurs. In the case of subconfluent cells, such as those shown in Figure 1, cells become rounded and detachment begins after about 1 h. Figure 1b shows the morphology of cells in a different well of the same plate used for the photograph of Figure 1a, after 1 h in the refrigerator. Severals cells already detached, while remaining cells are rounded, showing a dramatic come
4654 Langmuir, Vol. 14, No. 16, 1998
back from the spread morphology of Figure 1a. They can be easily removed by minimal pipetting. When low-temperature recovery was performed on completely confluent monolayers, cells detached mainly as sheets. Obviously, no modification of cell shape or cell detachment was observed on control tissue culture or air plasma treated PS, even after prolonged (6 h) storage in the refrigerator. Discussion Surface modification by TRP is an interesting strategy to produce polymer surfaces that can respond to changes in the temperature of the surrounding environment. In the present case, Ce(IV) was used to graft PIPAAm to PS surfaces. The use of Ce(IV) to graft acrylate monomers to surfaces containing hydroxyl groups, or to synthetic organic surfaces plasma or corona treated to introduce oxygen-containing functionalities, has been widely exploited.14-16 From a surface chemistry point of view, it must be noted that in the present case, no direct evidence exists that the polymerized acrylate it, indeed, covalently linked (grafted) to the substrate surface. A strong physical interaction or entanglement between the plasma-treated PS surface and the PIPAAm chains could explain the observed results as well. Xue and Wilkie recently discussed chemical grafting versus physical interaction in vinyl monomer-poly(ethylene terephthalate) systems.17 Many of their observations can be extended to other monomer-substrate systems, including the present one. From a practical point of view, it is important to note that the PIPAAm surface coating successfully withstands overnight extraction in cold water and succeeds in imparting thermally responsive properties to the cell culture substrate surface. SEM images reported in Figure 1 clearly show that L-929 cells feel the change of surface properties, and round up and detach as the compact conformation of PIPAAm chains above the LCST switches to the fully expanded chain conformation and becomes unsuitable for cell adhesion. The main goal of this paper was to understand the effect of temperature and velocity on the shape of hysteresis loops and contact angles measured by the Wilhelmy plate experiment. The changes of dynamic contact angles of liquids on rigid and chemically stable surfaces with speed have been repeatedly analyzed in literature;18,19 also the effect of speed on surfaces able to respond to a changing environment is a widely discussed argument.9,18 The nature of surfaces analyzed in the present paper changes with the temperature, but this property interacts in an unexpected way with the environment through the speed at which the DCA experiment is done. The results reported in Table 2 and Figure 2 show that water contact angle on PS and plasma-treated PS surfaces is substantially stable and independent of common experimental variables; in these cases only very high speeds could have some effect on measured values; this result is in good agreement with the literature, which confirms that at low capillary numbers the difference between dynamic and static contact angles is very low. Temperature acts mainly (but not only) through the changes in viscosity of test liquid (and thus of capillary number); the joint effects of temperature and speed could (17) Xue, T. J.; Wilkie, C. A. Polym. Prepr. 1995, 36, 251. (18) Morra, M.; Occhiello, E.; Garbassi, F. J. Adhes. Sci. Technol. 1992, 6, 653. (19) Rame´, E.; Garoff, S. J. Colloid Interface Sci. 1996, 177, 234.
Della Volpe et al.
be invoked to explain the slight differences in reported contact angle values of PS and plasma-treated PS at different speeds and temperatures. It is noteworthy that the difference between the receding angles of plasmatreated PS could be explained also invoking the effect of solvent evaporation and a consequent increase of hydrophobic character of the surface, which is greater at higher temperature. The situation appears quite different in the case of PIPAAm-grafted surfaces. In this case the differences are stronger and can be appreciated at every speed value. The peculiar behavior of these surfaces during repeated immersions needs a more detailed analysis. It is evident from Figure 2 that an unexpected variation of contact angles occurs with increasing immersion depth; this variation is slow and gradual. It permits the samples to be divided in two zones, the upper and lower part of every sample; these parts of the samples are reimmersed after different time intervals; in fact the upper part is reimmersed after a more prolonged exposition to air, which allows the evaporation of a certain quantity of the adsorbed water. Notwithstanding, these parts of samples maintain a more hydrophilic character than those parts before the first water immersion. The decrease of contact angles, however, is not more pronounced than in the case of polar plasma-treated PS surfaces, and this indication of a “polar” character is more pronounced at higher temperature. The lower part of samples, on the contrary, keeps a greater quantity of adsorbed water and shows a more pronounced polar character; in these cases advancing contact angles show a dramatic decrease after the first immersion, which is more pronounced at lower temperature; in fact at higher temperature this “lower” part is very small and cannot be evaluated at the lower speed. Moreover the receding angles are lower at lower temperature than those at higher temperature, but their decrease is less than that observed on advancing angles. These two observations suggest that the presence of water induces the formation of a more polar and hydrophilic surface on PIPAAm and that this decrease is much more pronounced at lower temperature. Using a slightly modified “hysteresis graph”,20,21 one can easily model this situation in terms of the heterogeneity of the surface with increasing water content (Figure 3), simply assuming that a lower temperature allows the permanence of a greater percentage of heterogeneity for every water content; in fact the mobility of molecules is reduced at a lower temperature. Two graphs, valid at different temperatures, are thus reported: the inner curve, corresponding to a lower degree of heterogeneity and to a higher temperature; the outer curve, corresponding to a higher degree of heterogeneity at lower temperature. For each percentage of water content the advancing angle for a lower temperature is higher and the receding angle lower. Higher water content is not allowed at higher temperatures, due to the effect of evaporation. On the left we have the case of dry material; in the central part the situation of partially wetted or partially dried PIPAAm, and finally on the right the totally wetted PIPAAm. The temperature effect is in agreement with the known properties of PIPAAm molecules, which appear to be in an “extended”, water-integrated, hydrophilic structure at low temperature, and in a “coil”, hydrophobic structure at temperatures higher than its LCST. (20) Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1994, 40, 546. (21) Johnson, R. E., Jr.; Dettre, R. H. J. Phys. Chem. 1964, 68, 1744.
Wetting Behavior of PIPAAm-Grafted Surfaces
Langmuir, Vol. 14, No. 16, 1998 4655
a
b
c
d
Figure 2. (a) Force vs immersion depth in a Wilhelmy experiment at T ) 20 °C and immersion speed of 160 µm/s for PS coated by PIPAAm: ZDOI ) zero depth of immersion; five subsequent immersions are reported; the first advancing value is clearly different from the others; the receding values are constant; during the first 2 mm of immersions 2 to 5, the force value is clearly different. (b) Force vs immersion depth in a Wilhelmy experiment at T ) 20 °C and immersion speed of 250 µm/s for PS coated by PIPAAm: ZDOI ) zero depth of immersion; five subsequent immersions are reported; the first advancing value is clearly different from the others; the receding values are constant; during the first 5 mm of immersions 2 to 5, the force value is clearly different. (c) Force vs immersion depth in a Wilhelmy experiment at T ) 20 °C and immersion speed of 250 µm/s for PS coated by PIPAAm: ZDOI ) zero depth of immersion; five subsequent immersions are reported; the first advancing value is clearly different from the others; the receding values are constant; this run is equivalent to that reported in part b, but the immersion distance is reduced. The global result is an “artifact” in which advancing and receding seem fully overlapped. (d) Force vs immersion depth in a Wilhelmy experiment at T ) 20 °C and immersion speed of 666 µm/s for PS coated by PIPAAm: ZDOI ) zero depth of immersion; five subsequent immersions are reported; the first advancing value is clearly different from the others; the receding values are constant; at this speed also for an immersion length of about 10 mm the advancing force of immersions 2 to 5 appears greater than receding.
This effect has been interpreted1 as resulting from the fact that at low temperature interactions among polymer and water molecules prevail during the mixing process; above LCST “the polymer network may undergo hydrophobic interactions between isopropyl groups on the side chain, preventing a decrease in entropy in the system”. In other words at low temperature the interaction among water and polymer is prevalent and at higher temperature the entropy in the form of the “hydrophobic effect” prevails; water is “pulled-out” from the network. For a fully wetted surface, in the lower parts of the samples, the advancing and receding measured forces are equal; actually, at the highest measured speed the advancing force is higher than the receding one. If one calculates from these force values a contact angle, one obtains a very unexpected result: advancing angles appear slightly lower than the receding ones. One must consider that dynamic contact angles are not fully equivalent to static ones, particularly at higher speeds,18,19 and in fact the inversion appears only at the highest speeds. At slow immersion speeds this particular inversion phenomenon is less relevant or has not been shown; this condition is more similar to a static angle
measurement so that one could expect that static contact angles cannot show it. To explain the inversion one could consider the effect of viscosity of the solution on the meniscus, recently treated in literature,18 but unfortunately the viscous force can only enhance the receding force and diminish the advancing one, so that this effect could not explain the inversion of advancing and receding angles. To our knowledge, in literature data the indication of a similar inversion was reported by Andrade et al.12 on fully hydrated PVP coating on glass surfaces without any modeling, but correctly stressing its peculiarity; on the contrary Takei et al. has reported a similar evidence11 on PIPAAm surfaces without any critical approach or simply a mention. Our opinion is that a similar inversion, due to its peculiarity, must be absolutely stressed and it is not correct to report it without any mention,11 because this can induce in inexperienced people the incorrect idea that this phenomenon (the inversion of advancing and receding contact angles) is common and straightforward to explain. On the contrary this interpretation is not so simple; we suggest two alternatives: (a) a water film remains on
4656 Langmuir, Vol. 14, No. 16, 1998
Della Volpe et al.
immersions. Only if air is saturated with water vapor is the evaporation forbidden. However this has not been done in our experiments (nor in the Takei experiment) due to the difficulty to saturate by water vapor the microbalance environment forbidding the liquid condensation on the microbalance mechanism. The effect can be also modulated by the degree of stiffness of grafted PIPAAm network; in the case of a less rigid structure (as in Takei Ic-120)11 a greater quantity of water molecules is accommodated, in the case of a more rigid network, as in our case or in Takei IA-3, a lower number of water molecules is accommodated. This can explain the reason for which this inversion effect is more enhanced and needs a higher immersion speed in our case than in Takei PIPAAm; in fact our PIPAAm resembles, probably, much more the Takei IA-3 material.
Figure 3. Two hysteresis graphs are reported at two different temperatures for the PS coated by PIPAm: the higher temperature corresponds to the inner cycle. The numbers reported in the correspondence of line-curve intersections correspond to the values of contact angles of the first column of Table 2d, obtained at an immersion speed of 40 µm/s. The figure is purely qualitative.
fully wetted material, and its interaction with liquid bulk in advancing corresponds to a lower contact angle than previous receding; (b) a more convincing alternative is to consider that solvent evaporation has the effect to produce a lower temperature and so a greater organizing effect, which could be shown in Figure 3 with a still more outer hysteresis graph, whose advancing can be less than the receding at higher temperature. Time spent in air modulates material wetting through water evaporation; speed of immersion has an effect through this mechanism, not by the change of meniscus shape or viscosity effect, which would change the force in the opposite direction, as noted earlier. It is to be noticed that the simple control of air and liquid temperature is necessary but not sufficient to eliminate the evaporation of the liquid from the solid surface between two subsequent
Conclusions In the present paper we have analyzed the surface properties of polystyrene surfaces modified by poly(Nisopropylacrylamide) (PIPAAm) grafting; the behavior of this material has been studied by measuring dynamic contact angles by Wilhelmy microbalances in water at different temperatures and at different speeds of immersion. The cell behavior and, in particular, the effect of low-temperature treatment on cells are reported as well. Both properties confirm that at a temperature lower than LCST there is a sharp modification of surface in the direction of a more hydrophilic structure. Global contact angle behavior has been explained in terms of heterogeneity of the surfaces, using a slightly modified hysteresis graph, remembering that at lower temperature it is possible to retain a greater percentage of heterogeneity. The apparent inversion of advancing and receding contact angles shown at higher speeds has been analyzed in terms of a kinetic effect due to the presence of a liquid film and/or to its evaporation. Acknowledgment. We are grateful to Dr. Silvano Invernizzi of Synt SpA, Zola Predosa (BO)-I for the license to use the KS Sigma 70 microbalance. LA971243G
