End-Grafted Low-Molecular-Weight PNIPAM Does Not Collapse

162

Langmuir 2007, 23, 162-169

End-Grafted Low-Molecular-Weight PNIPAM Does Not Collapse above the LCST† X. Zhu,‡ C. Yan,‡ F. M. Winnik,|,⊥ and D. Leckband*,‡,§ Department of Chemical and Biomolecular Engineering and Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801, Department of Chemistry and Faculty of Pharmacy, UniVersity of Montreal, Montreal, Quebec, Canada, and Department of Chemistry, McMaster UniVersity, Hamilton, Ontario, Canada ReceiVed June 1, 2006. In Final Form: August 31, 2006 The interfacial properties of end-grafted temperature-responsive poly(N-isopropylacryamide) (PNIPAM) were quantified by direct force measurements both above and below the lower critical solution temperature (LCST) of 32 °C. The forces were measured between identical, opposing PNIPAM films and between a PNIPAM film and a lipid membrane. At the grafting densities and molecular weights investigated, the polymer extension did not change significantly above the LCST, and the polymers did not adhere. Below the LCST, the force-distance profiles suggest a vertical phase separation, which results in a diluter outer layer and a dense surface proximal layer. At large separations, the force profiles agree qualitatively with simple polymer theory but deviate at small separations. Importantly, at these low grafting densities and molecular weights, the end-grafted PNIPAM does not collapse above the LCST. This finding has direct implications for triggering liposomal drug release with end-grafted PNIPAM, but it increases the temperature range where these short PNIPAM chains function as steric stabilizers.

Introduction Changes in the solubility and swelling of poly(N-isopropyl acrylamide) above its lower critical solution temperature (LCST) have been extensively studied. In water, PNIPAM is soluble below the LCST and is assumed to adopt a swollen, random configuration. Above the LCST of 32 °C,1 the solvent quality decreases, the solubility drops, and a large body of evidence indicates that the chains collapse.1-6 This thermally driven transition and associated changes in the hydrodynamic volume and polymer interactions has been exploited in many applications such as controlled drug delivery,7-11 tissue culture,12-15 and † Part of the Stimuli-Responsive Materials: Polymers, Colloids, and Multicomponent Systems special issue. * Corresponding author. E-mail: [email protected]. Tel: 217-2440793. Fax: 217-333-5052. ‡ Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign. § Department of Chemistry, University of Illinois at Urbana-Champaign. | University of Montreal. ⊥ McMaster University.

(1) Heskins, M.; Guillet, J. E. J. Macromol. Sci. Chem. 1968, A2, 1441-1455. (2) Kubota, K.; Fujishige, S.; Ando, I. J. Phys. Chem. 1990, 94, 5154-5158. (3) Plunkett, K.; Zhu, X.; Moore, J. S.; Leckband, D. E. Langmuir 2006, 22, 4259-4266. (4) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163-249. (5) Wang, X.; Qiu, X.; Wu, C. Macromolecules 1998, 31, 2972-2976. (6) Wu, C.; Zhou, S. Macromolecules 1995, 28, 8381-8387. (7) Hoffman, A.; Stayton, P. S.; Press, O.; Murthy, N.; Lackey, C. A.; Cheung, C.; Black, F.; Campbell, J.; Fausto, N.; Kyriakides, T. R.; Bornstein P. Polym. AdV. Technol. 2002, 13, 992-999. (8) Bromberg, L. E.; Ron, E. S. AdV. Drug DeliVery ReV. 1998, 31, 197-221. (9) Francis, M. F.; Dhara, G.; Winnik, F. M.; Leroux, J.-C. Biomacromolecules 2001, 2, 741-749. (10) Polozova, A.; Winnik, F. M. Biochim. Biophys. Acta 1997, 1326, 213224. (11) Hoffman, A. S. J. Controlled Release 1987, 6, 297-305. (12) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506-5511. (13) Ide, T.; Nishida, K.; Yamato, M.; Sumide, T.; Utsumi, M.; Nozaki, T.; Kikuchi, A.; Okano, T.; Tano, Y. Biomaterials 2006, 27, 607-614. (14) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16, 297-303. (15) Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 561-567.

protein adsorption.16 The proximity of its LCST (32 °C) to physiological conditions makes PNIPAM an excellent candidate for numerous biomedical applications.17 The phase behavior of PNIPAM in aqueous solutions was studied with several techniques, including turbidity,18 light scattering,2,5,6 and IR spectroscopy.19 A vast majority of studies of high-molecular-weight PNIPAM indicate that the hydrodynamic volume changes substantially above 32 °C. The associated cloud point also signals chain aggregation under the poor solvent conditions.2,5,18,19 This collapse occurs at the level of individual chains. The hydrodynamic volume of monodisperse (Mw/Mn < 1.05), high-molecular-weight (Mw ) 1.3 × 107) PNIPAM chain in very dilute solution (∼6.7 × 10-7 g/mL) decreased 7-fold above the LCST.5 Single chains attached to streptavidin similarly blocked the biotin binding site above 32 °C.20 Thus, isolated chain behavior appears to mirror the phase behavior of more concentrated solutions. Despite extensive investigations of PNIPAM changes at the LCST, an increasing body of research shows that the temperatureinduced collapse of end-grafted PNIPAM depends on the grafting density and molecular weight.3,21-24 This was predicted by both scaling and self-consistent mean field theories.25-27 The design (16) Kawaguchi, H.; Kisara, K.; Takahashi, T.; Achiha, K.; Yasui, M.; Fujimoto K. Macromol. Symp. 2000, 151, 591-598. (17) Galaev, I. Y.; Mattiasson, B. Trends Biotechnol. 1999, 17, 335-339. (18) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311-3313. (19) Katsumoto, Y.; Tanaka, T.; Sato, H.; Ozaki, Y. J. Phys. Chem. A 2002, 106, 3429-3435. (20) Stayton, P.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G. H.; Harris, J. M.; Hoffman, A. S. Nature 1995, 378, 472-474. (21) Schonhoff, M.; Larsson, A.; Welzel, P. B.; Kuckling, D. J. Phys. Chem. B 2002, 106, 7800-7808. (22) Yim, H.; Kent, M. S.; Mendez, S.; Balamurugan, S.; Balamurugan, S. S.; Lopez, G. P.; Satija, S. Macromolecules 2004, 37, 1994-1997. (23) Yim, H.; Kent, M. S.; Mendez, S.; Lopez, G. P.; Satija, S.; Seo, Y. Macromolecules 2006, 39, 3420-3426. (24) Yim, H.; Kent, M. S.; Huber, D. L. Macromolecules 2003, 2003, 52445251. (25) Baulin, V. A.; Halperin, A. Macromol. Theory Simul. 2003, 12, 549559. (26) Halperin, A. Eur. Phys. J. B 1998, 3, 359-364. (27) Mendez, S.; Curro, J. G.; McCoy, J. D.; Lopez, G. P. Macromolecules 2005, 38, 174-181.

10.1021/la061577i CCC: $37.00 © 2007 American Chemical Society Published on Web 10/31/2006

PNIPAM Does Not Collapse aboVe the LCST

rules for “smart” polymer coatings should therefore include both the polymer identity and the architecture of the polymer films. Studies show that chemical adsorption strongly perturbs the conformational statistics from those in solution.28,29 Scho¨nhoff et al. showed that the temperature-dependent transition was broadened for adsorbed PNIPAM on colloidal silica compared with that of the polymer in solution, especially at low surface coverage.21,30 Others reported that the surface tension change due to PNIPAM adsorption at the air-water interface was less sensitive to temperature for lower-molecular-weight (Mw ) 13 100) PNIPAM than for higher-molecular-weight PNIPAM (Mw ) 547 000).31 There are also several reports of unexpected stability of PNIPAM particles above the LCST.32 Grafting can also alter the chain configuration. Theory and experiment suggest that the segment density profile of end-grafted PNIPAM exhibits a dilute outer region and a dense surface proximal region rather than a simple parabolic profile.3,23,24,26,30,33-35 Experiments also showed that grafted PNIPAM brushes exhibit a two-stage transition on heating.30,34 The first occurred below 30 °C, under better than θ solvent conditions, and the particles did not flocculate. The second transition occurred at higher temperature, under worse than θ solvent conditions. This two-step transition is compatible with the predicted vertical phase separation.25,26 Neutron reflectivity measurements of endgrafted PNIPAM exhibited a dense inner phase and a dilute outer phase.23,24 The dense phase could be due to segment adsorption to the underlying substratum. The latter explanation could not be ruled out in the reflectivity studies. Nevertheless, there is increasing evidence that the PNIPAM grafting architecture influences both the polymer configuration and phase behavior. This report describes measurements of the temperaturedependent force profiles between PNIPAM brushes end-grafted to supported lipid bilayers. These studies were motivated by the potential for PNIPAM-coated liposomes to release therapeutic agents upon selective heating.9,36 The efficacy of these formulations depends on design parameters that control PNIPAM swelling and collapse. We investigated PNIPAM interactions as a function of the polymer grafting density and molecular weight. Importantly, at the molecular weights and grafting densities explored in this work, the PNIPAM chains do not collapse above the LCST. These findings agree with other recent reports of end-grafted PNIPAM films3,22,23 and have direct consequences for the use of grafted PNIPAM in liposomal drug delivery. Materials and Methods Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were purchased in powder form (purity >99%) from Avanti Polar Lipids Inc (Alabaster, AL). All inorganic salts were high purity (>99.5%) and were purchased from Aldrich (Milwaukee, WI). All aqueous solutions were prepared with water purified with a Milli-Q (28) Chakraborty, A. K.; Shaffer, J. S.; Adriani, P. M. Macromolecules 1991, 24, 5226-5229. (29) Shaffer, J. S.; Chakraborty, A. K.; Tirrell, M.; Davis, H. T. J. Chem. Phys. 1991, 95. (30) Shan, J.; Chen, J.; Nuopponen, M.; Tenhu, H. Langmuir 2004, 20, 14711476. (31) Zhang, J.; Pelton, R. Colloids Surf. 1999, 156, 111-122. (32) Temperature-Dependence of the Colloidal Stability of Neutral Amphiphilic Polymers in Water; Aseyev, V., Tenhu, H., Winnik, F. M., Ed.; Springer-Verlag: Heidelberg, 2006; Vol. 196, pp 1-85. (33) Yim, H.; Kent, M. S.; Satija, S.; Mendez, S.; Balamurugan, S S.; Balamurugan, S.; Lopez, G. P. Phys. ReV. E. Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2005, 7205, 1801. (34) Zhu, P. W.; Napper, D. H. J. Colloid Interface Sci. 1994, 164, 489-494. (35) Baulin, V. A.; Zhulina, E. B.; Halperin, A. J. Chem. Phys. 2003, 119, 10977-10988. (36) Ringsdorf, H.; Venzmer, J.; Winnik, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 315-318.

Langmuir, Vol. 23, No. 1, 2007 163 Table 1. End-Grafted NIPAM-(C18)12 Monolayers molecular mole percent grafting density surface weight NIPAM-(C18)12 (chain/µm2) × 10-5 s/2RF configuration 5

1.22 ( 0.05

10

2.56 ( 0.23

2500

0.5

0.12 ( 0.01

2

0.48 ( 0.04

4 10

0.93 2.56 ( 0.07

10 000

0.8 weakly overlapping mushroom 0.6 weak overlap/brush 1.2 nonoverlapping mushroom 0.6 weak overlap/brush 0.4 brush 0.3 brush

UV-Plus water purification system (Millipore, Bedford, MA). Water had a resistivity of 18.2 MΩ cm-1. HPLC-grade methanol and chloroform purchased from Mallinckrodt (St. Louis, MI) were used to prepare lipid solutions. High-purity silver shot (99.99%, Aldrich, Milwaukee. WI) used for the preparation of silver films on mica was from Alfa Aesar (Ward Hill, MA). Chromium chips (99.997%) for the evaporation of adhesion layers between gold and glass were purchased from Alfa Aesar (Ward Hill, MA). Gold was purchased from A-1 coin buyers (Champaign, IL). PNIPAM-(C18)2 Synthesis. The lipophilic initiator dioctadecylamide-4,4′-azobis(4-cyanovalerate)37 (0.05 g, 0.04 mmol) and the appropriate amount of NIPAM were dissolved in methanol (50 mL). The solution was degassed by bubbling with N2 for 10 min. The solution was heated rapidly to 60 °C and kept at this temperature for 24 h. The polymerization mixture was then cooled to room temperature. The solution was concentrated in vacuum. The polymer was isolated by precipitation into hexane. It was further purified by repeated precipitations from THF into ether. The molecular weight (Mn) of the polymers was determined by 1H NMR spectroscopy, using the signals at δ 0.9 (resonance of the octadecyl terminal methyl protons) and at δ 4.0 (resonance of the isopropyl methine proton) as described previously.38 The polydispersity Mn/Mw was on the order of 2 for preparations with mass average molecular weights of 2500 and 10 000. End-Grafted PNIPAM-(C18)2 Monolayers. Solutions of PNIPAM-(C18)2 and pure DSPE were prepared in 9:1 chloroform/ methanol solutions. Mixtures of various molar ratios of PNIPAM(C18)2 to DSPE were prepared by mixing solutions of pure DSPE and lipopolymer in the appropriate proportions. Surface pressure versus area isotherms were measured on a commercial LangmuirBlodgett trough (NIMA Technologies, type 611) equipped with a standard Wilhelmy microbalance. The subphase used to prepare the Langmuir films was pure water, which was maintained at 25 °C by a circulating water jacket. The bilayer was prepared by depositing the DSPE/PNIPAM(C18)2 monolayer onto a gel-phase monolayer of DPPE (43 Å2/lipid) that was transferred by Langmuir-Blodgett (LB) deposition from the vapor-water interface onto a freshly cleaved mica substrate. After the lipopolymer mixture was spread on the water surface of the Langmuir trough, it was compressed to a surface pressure of 37 mN/m at 25 °C. The monolayer was then deposited at this constant pressure onto the supported DPPE monolayer. The transfer ratio, which is the area transferred relative to the area coated by the film, was close to unity in all cases. Adjusting the PNIPAM mole percent in the monolayer thereby controls the polymer grafting density. In this study, the mole percentages of PNIPAM-(C18)2 in the lipidpolymer mixtures used were 5 and 10 for PNIPAM Mw 2500 and 0.5, 2, 4, and 10 for PNIPAM Mw 10 000. The corresponding chaingrafting densities in the resulting monolayers are in Table 1. The surface density of PNIPAM-(C18)2 is determined from the average area per lipid in the monolayer divided by the mole fraction of PNIPAM-(C18)2 in the mixture. The mixed films were all stable at (37) Kitano, H.; Akatsuta, Y.; Ise, N. Macromolecules 1991, 24, 1678-1686. (38) Winnik, F. M.; Davidson, A. R.; Hamer, G. K.; Kitano, H. Macromolecules 1992, 25, 1876-1880.

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Zhu et al.

Figure 1. Chemical structure of PNIPAM-(C18)2. Figure 3. Surface pressure-area isotherms of mixed PNIPAM(C18)2: DSPE monolayers. The subphase was water maintained at 25 °C.

Figure 2. (a) Different configurations of grafted chains: nonoverlapping mushrooms (0.5 mol % Mw 10 000), weakly overlapping mushroom (5 and 10 mol % Mw 2500, 2 mol % Mw 10 000), and dense brushes (4 and 10 mol % Mw 10 000). L is the length of the polymer chains, and s is the distance between each grafting site. (b) Sample configuration used in the direct force measurements between end-grafted PNIPAM surfaces. (c) Sample configuration used in the direct force measurements between end-grafted PNIPAM and the DSPE layer. In both b and c, the distances, D, are the separations between the bare DSPE lipid membranes beneath the grafted PNIPAM-(C18)2, as shown here. 37mN/m, which indicates that the polymers are not squeezed out of the monolayer. In addition, the ratio of the film transferred from the air/water interface to the substrate area coated was unity in all cases. Three different PNIPAM chain configurations were investigated, namely, the nonoverlapping “mushroom”, weakly overlapping “mushroom”, and “brush” regimes. These are defined according to the ratio of the Flory radius, RF, to the average distance between polymer chains, s (Figure 2a). The Flory radius is RF ) ln3/5,where l is the effective segment length, assumed to be 3 Å,39 and n is the number of segments.40 Here we assume that the chains are swollen at 25 °C. Under these conditions, at s/2RF > 1, the chains are in the nonoverlapping mushroom regime (Figure 2a). If s/2RF < 1, then the polymers are in the weak overlap regime (Figure 2a). For s/2RF , 1, the polymers form brushes (Figure 2c). The Flory radius, RF, of PNIPAM with Mw 2500 (n ) 22) is 19 Å, and it is 44 Å for PNIPAM with Mw (39) Carey, F. A. Organic Chemistry, 2nd ed.; McGraw-Hill: New York, 1992. (40) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press: San Diego, CA, 1991.

10 000 (n ) 88).40 The s/2RF ratios for the DSPE/PNIPAM-(C18)2 mixtures are given in Table 1. We note that the radius of collapsed chains in a bad solvent differs from RF as defined above. For an unperturbed, collapsed chain, the radius scales with n according to R ≈ an1/3, where a is the monomer volume.41 Because we did not see any evidence of collapse in these studies, we did not use the collapsed radius in our analyses. Force Measurements with Grafted PNIPAM. A Mark III surface force apparatus (SFA) (SurForce Co., Santa Barbara, CA) was used to quantify the force between thin films confined between two crossed cylinders as a function of the distance D between the surfaces. The reported lower critical solution temperature of PNIPAM is ∼32 °C.1 4 Measurements were carried out at 25 and at 35 °C in a temperaturecontrolled room. The intersurface distances are measured with a resolution of (1 Å by multiple beam interferometry.42 The force, normalized by the geometric average radius of the two disks R ) (R1R2)1/2, that is, F/R, is determined with a resolution of (0.1 mN/m from the deflection of a sensitive leaf spring that supports the lower disk.43 The normalized force Fc/R between these curved disks is directly proportional to the interaction energy per area between two equivalent flat plates Ef according to Fc/R ) 2πEf.40 This wellestablished Derjaguin approximation applies when the radius is much greater than the range of the measured forces, R . D. In these measurements, R is ∼0.01 m, and D < 1000 Å.

Results Monolayers at the Vapor-Water Interface. Figure 3 shows the pressure-area isotherms of DSPE/PNIPAM mixtures containing different molar ratios of DSPE and PNIPAM-(C18)2 with a molecular weight of either 2500 or 10 000. The area indicated in Figure 3 is the average area per lipid. The minimum area occupied by a phosphatidylethanolamine lipid in the gel phase is 43 Å2. The increase in surface pressure at much larger areas is due to the lateral interactions between the polymer chains. As expected, the molecular area at the onset of the increase in surface pressure increases with the PNIPAM-(C18)2 concentration in the monolayer and with the PNIPAM-(C18)2 molecular weight. This is expected because both parameters increase the volume fraction of segments in the polymer film and hence the pressure at a given average molecular area. The pressure-area isotherms are reminiscent of those for poly(ethylene oxide) conjugates with DSPE. The apparent transition and plateau region at smaller molecular areas was also observed with PEO-lipid conjugates and was attributed to chain desorption (41) Williams, D. R. M. J. Phys. II 1993, 3, 1313-1318. (42) Israelachvili, J. J. Colloid Interface Sci. 1973, 44, 259-272. (43) Israelachvili, J.; McGuiggan, P. J. Mater. Res. 1990, 5, 2223-2231.


Langmuir, Vol. 23, No. 1, 2007 165

sheet with each 1 °C increment, was 0.35 Å/°C‚µm, as determined from measurements between bare mica sheets. At 35 °C, the range of the repulsion was similar, within error, at 42 ( 5 Å, but the compressed polymer thickness decreased slightly to 16 ( 2 Å (Figure 4). The surfaces did not adhere at this temperature. To compare interactions between grafted PNIPAM layers with simple polymer behavior, we fit the data measured at 25 °C for 10 mol % PNIPAM-(C18)2 to the Alexander-de Gennes model (Figure 4, inset). The Alexander-de Gennes model (eq 1) describes the force-distance profile between polymer brushes in good solvent. At distances D < 2L, the normalized force is Figure 4. Force profiles between 10 mol % end-grafted PNIPAM Mw 2500 layers in water at 25 and 35 °C. The circles show the force curves measured at 25 °C, and the triangles show the data obtained at 35 °C. The inset shows the fit of the data at D > 19 Å to eq 1.

from the air-water interface and extension into the bulk phase.44 In the isotherms in Figure 3, we see a similar transition at ∼20 mN/m. Further compression results in a densely packed layer at molecular areas 19 Å (R2 ) 0.97) but deviates sharply at D < 19 Å (Figure 4 inset). The fitted parameters obtained with data at D > 19 Å are summarized in Table 3. We then compared the polymer extension determined from direct measurements, model calculations (eq 2), and fits to eq 1. The experimentally measured polymer thickness is half the range of the intersurface repulsion. At 25 °C, this was at 23 ( 5 Å. The calculated (eq 2) extension is 18 Å. The fitted value of L was 28 ( 6 Å. These results are summarized in Table 3. Figure 5 shows the force profile between end-grafted 5 mol % PNIPAM-(C18)2 (Mw 2500) monolayers at 25 °C. At this polymer density, the chains form weakly overlapping mushrooms. There are two significant differences relative to the data in Figure 4. First, the onset of intersurface repulsion was closer in at 29 ( 5 Å, and the hard wall was also at a slightly smaller distance D < 11 Å. Second, upon separation, the surfaces adhered and jumped out of contact at 32 ( 5 Å, with a pull-off force of -0.11 ( 0.02 mN/m. Similar to the behavior of the 10 mol % 2500 Mw brushes, fits of the data beyond the hard wall to eq 1 agreed with theory (Figure 5 inset). However, the data deviated from the fits at smaller separations where the repulsive force increased sharply. At 35 °C, the forces were similar. The range of the repulsion was slightly smaller at 22 ( 4 Å, and the hard wall was closer in at 8 ( 2 Å (Table 2). The surfaces adhered and jumped out of adhesive contact at 26 ( 6 Å with a pull-off force of -0.22 ( 0.03 mN/m. The adhesion results are summarized in Table 4. Forces between End-Grafted Mw 10 000 PNIPAM (n ) 88). Forces were measured with end-grafted PNIPAM-(C18)2 mushrooms (0.5 mol %) and brushes (2 and 10 mol %) (Table 1). The corresponding polymer grafting densities were (0.12 ( 0.05) × 105, (0.48 ( 0.04) × 105, and (2.56 ( 0.07) × 105 chains/µm2, respectively. Figure 6 shows the force-distance profiles between 2 mol % PNIPAM-(C18)2 ((0.48 ( 0.04) × 105 chains/µm2) layers in water. The intersurface forces were repulsive at all distances. The onset of the soft repulsion was at 53 ( 5 Å, and the incompressible layer thickness was 20 ( 3 Å (Table 2). The data at 25 °C were fit to eq 1 (Figure 6 inset). Again, eq 1 describes the data at large distances D > 37 Å (Table 3). However, as in the other cases,

166 Langmuir, Vol. 23, No. 1, 2007

Zhu et al. Table 2. Polymer Extension and Position of the Hard Wall PNIPAM-PNIPAM

molecular weight

extended thickness (Å) 35 °C p value

compressed thickness (Å) 25 °C 35 °C p value

surface density chains/µm2 (× 10-5)

s/2Rg

25 °C

1.22

0.8

29 ( 2

22 ( 4

0.05

11 ( 4

8(2

0.14

2.56 0.12

0.6 1.2

46 ( 2 17 ( 3

42 ( 5 15 ( 3

0.27 0.46

19 ( 2 13 ( 1

16 ( 2 12 ( 1

0.14 0.29

0.48 2.56

0.6 0.2

53 ( 4 107 ( 6

47 ( 4 96 ( 5

0.14 0.07

20 ( 5 32 ( 6

20 ( 3 37 ( 11

1 0.53

2500 10 000

PNIPAM-DSPE extended thickness (Å) 35 °C p value

compressed thickness (Å) 25 °C 35 °C p value

molecular weight

surface density chains/µm2 (× 10-5)

s/2Rg

25 °C

2500

2.56 0.12

0.6 1.2

17 ( 1 11 ( 1

16 ( 3 10 ( 1

0.61 0.29

14 ( 1 11 ( 1

16 ( 3 10 ( 1

0.18 0.29

0.93

0.4

39 ( 9

35 ( 4

0.47

19 ( 2

17 ( 2

0.48

10000

Table 3. Calculated and Experimentally Determined Brush Parameters molecular weight

molar % PNIPAM 5 10

2500

s/2RF

measured thickness (Å)

predicted thickness (Å)

thickness from fits (Å)

R2

prefactors

25 °C

0.8 0.6

15 ( 1 23 ( 1

19 19

28 ( 6

0.92

0.1 ( 0.02

35 °C

0.6

25 (3

19

32 ( 5

0.93

0.11 ( 0.03

1.2 0.6

15 ( 3 27 ( 2

44 44

35 ( 6

0.97

0.05 ( 0.01

0.2 0.2

54 ( 3 65 ( 4

65 65

80 ( 7 90 ( 6

0.97 0.97

0.11 ( 0.02 0.12 ( 0.02

0.5 2 10 000 10

25 °C 35 °C

the data deviate from the simple polymer model at small separations. With 2 mol % PNIPAM-(C18)2 monolayers, the forces measured at 25 and at 35 °C are indistinguishable. With 10 mol % PNIPAM-(C18)2 brushes, the force profiles were similar. The range of the soft repulsion was 107 ( 10 Å, and the hard wall was also farther out at 32 ( 6 Å (Table 2). The two surfaces did not adhere. The force curves measured at the two temperatures were also very similar. At 35 °C, the range of the osmotic repulsion was 96 ( 10 Å, and the hard wall was at 37 ( 11 Å. However, the Student’s t test showed that these values are not statistically different from those measured at 25 °C (Table 2). At 0.5 mol % PNIPAM-(C18)2, the grafted chains are sparsely distributed and do not overlap laterally. At 25 °C, the force profiles between these layers were featureless prior to hard contact

Figure 5. Force profiles between 5 mol % end-grafted PNIPAM Mw 2500 in water at 25 and 35 °C. The filled circles show the force profile measured during approach at 25 °C, and the open circles show the force measurements during separation. The outward-directed arrow indicates the position of the force minimum and the position at which the surfaces jumped out of adhesive contact. The inset shows the fit to the data plotted on a logarithmic scale.

at 17 ( 3 Å. The repulsion then increased steeply at D < 17 Å. These curves differed from those at higher grafting density, which exhibited a soft repulsion prior to the hard wall. Upon separation, the surfaces adhered with a normalized force of -0.53 ( 0.04 mN/m. The pull-off distance was 13 ( 3 Å at 25 °C. At 35 °C, the profile was similar, except that the hard wall was at 15 ( 3 Å. The surfaces also adhered and jumped out of contact at 14 ( 2 Å with a pull-off force of -0.5 ( 0.1 mN/m (Table 4). Forces between End-Grafted PNIPAM and DSPE Monolayers. Mw 2500 PNIPAM Brushes. In these measurements between end-grafted PNIPAM and DSPE, we sought to identify the basis of the adhesion between dilute PNIPAM films (cf. Figure 5, Table 4). Additionally, in applications using PNIPAMcoated liposomes, drug carrier uptake could depend on interactions between the PNIPAM brush and cell membrane. The adsorption of polymer segments to the lipid bilayer would also affect the chain conformation and the resulting polymer interactions. Measurements were carried out between end-grafted PNIPAM and DSPE monolayers at 10 mol % PNIPAM-(C18)2 (Mw 2500). The polymer was in the brush regime. As observed for the interactions between dilute polymer mushrooms, the two surfaces came smoothly into “hard”, repulsive contact at 17 ( 2 Å at both temperatures (Figure 7). There was no detectable attraction or soft repulsion at larger distances. Upon separation, the surfaces adhered. The disks jumped out of contact from 17 ( 2 Å with a pull-off force of -0.22 ( 0.02 mN/m. At 35 °C, the disks also jumped out of contact from 17 ( 2 Å with a pull-off force of -0.23 ( 0.03 mN/m (Table 4). Mw 10 000 PNIPAM. Force measurements were conducted between DSPE and end-grafted PNIPAM (Mw 10 000) at 0.5 and 4 mol % PNIPAM-(C18)2 at both 25 and 35 °C. With 4 mol % PNIPAM-(C18)2, the chains form brushes (Table 1). With 4 mol


Langmuir, Vol. 23, No. 1, 2007 167

Table 4. Measured Adhesion and Predicted van der Waals Attraction PNIPAM-PNIPAM adhesive force at 25 °C

systems molecular weight

adhesive force at 35 °C

molar concentration (mol %)

van der Waals force, Fth (mN/m)

pull-off force, Fpo (mN/m)

pull-off position, Dpo(Å)




5

-0.09 ( 0.06

-0.11 ( 0.02

32 (5

-0.2 ( 0.1

-0.22 ( 0.03

26 ( 6

10 0.5

-0.55 ( 0.28

0 0.53 ( 0.04

13 ( 3

-0.8 ( 0.3

0 -0.5 ( 0.1

14 ( 3

2500 10000 2 10

0 0

0 0 PNIPAM-DSPE


systems molecular weight


molar concentration (mol %)







4

-0.4 ( 0.2

-0.32 ( 0.03

16 ( 1

-0.2 ( 0.1

-0.28 ( 0.01

19 ( 3

10 0.5

-0.3 ( 0.1 -0.7 ( 0.3

-0.22 ( 0.02 0.73 ( 0.04

17 ( 2 11 ( 1

-0.4 ( 0.2 -0.8 ( 0.3

-0.23 ( 0.03 -0.73 ( 0.06

17 ( 2 12 ( 1

2500 10000 4

0

% PNIPAM-(C18)2, the advancing and receding force profiles were completely repulsive at both temperatures (Figure 8). At 25 °C, the onset of the soft repulsive force during approach was at 39 ( 9 Å, and the compressed polymer thickness was 19 ( 1 Å (Table 2). At 35 °C, the magnitude of the repulsive force was slightly higher, but the range of the force was the same, within error. The hard wall shifted in to 17 ( 4 Å. There was no adhesion in either case.

Figure 6. Force profiles between 2 mol % PNIPAM Mw 10 000 layers at 25 and 35 °C. The filled circles correspond to the force curve measured at 25 °C, and the filled triangles correspond to the advancing curves at 35 °C. The inset shows the fit of the data at D > 20 Å to eq 1.

Figure 7. Force profiles between 10 mol % end-grafted PNIPAM Mw 2500 layers and the DSPE bilayer in water at 25 and 35 °C. The filled circles correspond to the advancing curve at 25 °C, and the open circles correspond to the receding curve at 25 °C. The filled triangles correspond to the advancing curves at 35 °C, and the open triangles correspond to the receding curves at 35 °C.

0

Force profiles were also measured between DSPE and 0.5 mol % PNIPAM-(C18)2 (Mw 10 000). The polymer configuration at T < LCST should be an unperturbed mushroom. As in Figure 7, there was no attractive or repulsive force prior to the steep steric repulsion at 11 Å. The surfaces adhered at 25 °C and jumped out of contact from 11 ( 1 Å with a pull-off force of -0.73 ( 0.04 mN/m at 25 °C. At 35 °C, the pull-off force was -0.73 ( 0.06mN/m at 12 ( 1 Å (Table 4). Analysis of Attractive Intersurface Forces. We considered the origin of the measured adhesion between the surfaces (Table 4) at both 25 and 35 °C. This could be due to either the van der Waals attraction between the underlying DSPE layers or to PNIPAM attraction to the DSPE monolayer. Because of the inverse power law dependence on distance, the van der Waals attraction between membranes should decrease with increasing pull-off distance. However, segment-surface attraction would increase with the polymer segment density. The adhesion values in Table 4 decrease with increasing chain density and molecular weight. Adhesion also decreases with increasing pull-off distance, d0, which increases with the polymer molecular weight and density. To address this more quantitatively, we compared the adhesion with the predicted van der Waals attraction between DSPE layers at the measured pull-off distance d0. The calculated40,45 values

Figure 8. Force-distance profiles between 4 mol % end-grafted PNIPAM Mw 10 000 and a DSPE bilayer in water at 25 and 35 °C. The filled circles were measured at 25 °C, and the triangles are data measured at 35 °C.

168 Langmuir, Vol. 23, No. 1, 2007

Zhu et al.

of the adhesion, considering only the van der Waals force, are in Table 4. For these calculations, we determined the Hamaker constant between the bilayers in buffer. The reported Hamaker constant between lipid bilayers across a water gap is (7 ( 1) × 10-21 J.45 We also estimated the Hamaker constant between the DSPE lipid bilayers in buffer using eq 3:

A vast majority of studies of the phase behavior of PNIPAM suggest that its LCST is ∼32 °C. However, recent experimental findings show that, under some grafting conditions, end-anchored PNIPAM does not become insoluble and collapse above 32 °C.3,22,23 In particular, at low molecular weights (Mw < 75 000) the measured change in the polymer extension dropped from -18% at the highest grafting density to 0% at the lowest density.3,24 This contrasts with the -30% change observed with ∼250 000 Mw PNIPAM.3 Our findings agree with recent reflectivity and ellipsometry data of end-grafted PNIPAM at comparable molecular weights and grafting densities.3,23,24 We could not directly check the collapse of these lipopolymer films by ellipsometry because exposure to air destroys the bilayer. However, by analogy with prior work,3,23,24,46 we might anticipate that the measured force profiles at 25 and 35 °C would be similar, as we observed. Some of the data in Table 2 suggest differences in the polymer extension and hard wall positions at the two temperatures. We

used the Student’s t test to compare the polymer thickness at the two temperatures (Table 2) and to determine the statistical significance of the differences. Within experimental error, there was no detectable volume change across the LCST (p < 0.01 in all cases) (Table 2). We considered that the absence of the transition could be due to grafting-induced broadening of the transition and/or the polydispersity of the chains. We did not explicitly explore this with these lipid-grafted chains. However, PNIPAM grafted from alkanethiol monolayers confirmed that the transition, as evidenced by contact angle changes, remained at 32 ( 1 °C, independent of the molecular weight and grafting density.3 The latter films were also prepared by atom-transfer radical polymerization so that the polydispersity was low. The lipo-PNIPAM layers did not adhere to each other or to the lipid bilayers above or below the LCST. Cloud points signal polymer aggregation due to increased segment-segment attraction under poor solvent conditions. The lack of polymer adhesion in this study is attributed to the fact that the grafted chains remain swollen above the LCST. This is supported by the work of Yim and co-workers; they also did not see any conformational change with temperature for low-molecularweight (33 000), end-grafted PNIPAM-COOH at the air-water interface.24 These chains therefore remain swollen above the LCST and repel rather than attract. The measured intersurface repulsion above 32 °C agrees with reports of unexpectedly stable PNIPAM particles above the transition temperature. This is attributed, in part, to the formation of mesoglobules, which are mesoscopic aggregates of more than one PNIPAM chain.32 The basis of their colloidal stability is not understood. One proposed explanation is that aggregation is kinetically hindered by the low collision probability in dilute solutions and by the activation barrier to forming dense and compact aggregates.32 At the grafting densities used in our system, the distances between chains could kinetically hinder the coalescence of neighboring PNIPAM globules41 and hence the film collapse. Alternatively, differential scanning calorimetry suggests that the collapse of high-molecular-weight chains involves cooperative domains of ∼100 monomers.32 The degree of polymerization of both polymers considered here is less than 100, and this may frustrate the cooperative coil-to-globule transition. There are clearly many unanswered questions, and there is a need for more extensive theoretical and experimental study of this complex material. All of the measured force profiles exhibit two compressibility regimes. The steep increase in the short-range repulsion indicates a denser, less compressible inner phase. The dense phase might be attributed to segment adsorption to the underlying surface, but our analysis showed that the polymer does not adhere to the lipid bilayers. The two-layered PNIPAM structure is therefore a property of the chains and is not due to segment adsorption to the underlying surface. It is possible that the scaling relationships in eqs 1 and 2 may not be appropriate for the short chains used in this study. Equations 1 and 2 are also applicable only for athermal solvents. As an alternative, the use of Flory exponents could be more suitable because they describe a wider range of molecular weights and solvent conditions.47 However, even in the Flory analysis, the free-energy dependence on the polymer thickness predicts a power-law dependence47 and would not explain the steep increase in repulsion at small surface separations. The PNIPAM behavior at T < LCST is consistent with the

(46) Kent, M. S.; Majewski, J.; Smith, G. S..; Lee, L. T.; Satija, S. J. Chem. Phys. 1998, 108, 5635-5645.

71.

6Fpod02 A) R

(3)

Fpo/R is the normalized pull-off force, and d0 is the pull-off distance relative to the van der Waals plane, which is assumed to be at the bilayer surface. The thus-determined Hamaker constant was (6 ( 2) × 10-21 J at 25 °C and (7 ( 2) × 10-21 J at 35 °C. Within experimental error, the measured adhesion agrees with the predicted van der Waals attraction between DSPE layers at both temperatures in all systems where we measured adhesion (Table 4). When the two surfaces attract, they will spontaneously jump into contact when the gradient of the attractive force exceeds the spring constant.45 If only the van der Waals attraction operates, then the distance at which the jump-in occurs is45

Dj )

(AR 3K)

1/3

(4)

Here, A is the Hamaker constant, R is the geometric average radius (0.02 m in these measurements), and K is the spring constant (135 N/m in this study). Neglecting the PNIPAM chains, eq 4 predicts that the van der Waals attraction would cause the dilute mushroom layers to jump into contact from ∼67 Å, yet there is no force detected between the layers at D > 30 Å. The absence of a jump-in suggests the presence of a weak repulsive force between the layers that is presumably due to the osmotic repulsion between the dilute chains. This explanation would predict that the weak repulsion should increase with the polymer grafting density. In agreement, the force curves between DSPE and the denser PNIPAM brush (4 mol % PNIPAM Mw 10 000) (Figure 8) do exhibit a weak but detectable repulsion beyond the hard wall. This is sufficient to overwhelm the attraction and abolish adhesion, as expected.

Discussion

(47) Halperin, A.; Tirrell, M.; Lodge, T. P. AdV. Polym. Sci. 1992, 100, 31-


Langmuir, Vol. 23, No. 1, 2007 169

postulated one-dimensional phase separation.3,25,26,33 Reflectivity measurements showed a two-layer segment density profile for end-tethered PNIPAM chains in D2O23,24 in which PNIPAM exhibited a thin layer with a high segment density near the surface and a more dilute outer layer. This two-layer structure would generate force profiles with qualitatively similar features to those reported here and elsewhere.3 A consequence of this two-layer structure is the smaller apparent chain extension relative to the theoretical predictions. This is most apparent with isolated and weakly overlapping mushrooms. The fitted values in our system agree somewhat better than the measured range of the repulsion. Nevertheless, in several cases, the chain extensions are less than predicted, presumably because of the altered segment distribution. These and other recent findings are somewhat unexpected, given the body of literature demonstrating large changes in PNIPAM gels, plasma-polymerized coatings, and other formulations above the transition temperature. For example, in a configuration closer to that of end-grafted chains, hydrophobically modified PNIPAM attaches to liposomes through multiple hydrophobic anchors along the backbone.10,36,48,49 Conformational changes above the LCST destabilized the liposomes50 and induced changes in pyrene reporter fluorescence.36 Adsorbed chains on polystyrene spheres also collapse above the LCST.51 The

theoretical descriptions of PNIPAM qualitatively predict some of the experimental findings with grafted chains,25-27 but they still do not fully explain differences that may be linked to chain architecture. At the surface densities and molecular weights investigated in this study, PNIPAM does not exhibit the rapid, thermally driven conformational change presumed to destabilize liposomes and trigger drug release in PNIPAM formulations. As demonstrated by us and by others,3,23 end-grafted PNIPAM undergoes temperature-driven collapse on the time scale of the measurements (minutes-days) only at high molecular weight and grafting density. At low molecular weight and density, PNIPAM remains swollen above the LCST and retains its capacity to stabilize particles sterically,32 similar to the behavior of poly(ethylene oxide). Thermally responsive, grafted PNIPAM coatings require much higher densities and molecular weights, and it is unclear whether such liposomes would be stable.52 Nevertheless, the dependence of the phase behavior of grafted PNIPAM on molecular weight and density increases the versatility of this material by expanding the conditions where it sterically stabilizes drug carriers or other colloidal particles.

(48) Polozova, A.; Yamazaki, A.; Brash, J. L.; Winnik, F. M. Colloids Surf., A 1999, 147, 17-25. (49) Ringsdorf, H.; Sackmann, E.; Simon, J.; Winnik, F. M. Biochim. Biophys. Acta 1993, 1153, 335-344. (50) Franzin, C. M.; Macdonald, P. M.; Polozova, A.; Winnik, F. M. Biochim. Biophys. Acta 1998, 1415, 219-234.

LA061577I

Acknowledgment. This research was supported by NSF BES 0349915.

(51) Hu, T.; Gao, J.; Wu, C. J. Macromol. Sci., Phys. 2000, B39, 407-414. (52) Leckband, D. E.; Borisov, O. V.; Halperin, A. Macromolecules 1998, 31, 2368-2374.

End-Grafted Low-Molecular-Weight PNIPAM Does Not Collapse

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