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Thermoresponsive Behavior of Poly(N-Isopropylacrylamide) Hydrogels Containing Gold Nanostructures Frances Y. Pong, Michelle Lee, Jessica R. Bell, and Nolan T. Flynn* Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, Massachusetts 02481-8203 ReceiVed December 16, 2005. In Final Form: February 21, 2006 We report the changes in the structure and thermoresponsive behavior of poly(N-isopropylacrylamide) (PNIPAm) hydrogels when gold nanostructures are synthesized in situ within the hydrogel matrix. Cross-linked PNIPAm hydrogels were synthesized using NIPAm and 0.00-3.50% (w/w versus NIPAm) of N,N′-methylenebisacrylamide (MBAm) and/or N,N′-cystaminebisacrylamide (CBAm) as cross-linking agents. The hydrogels were soaked in potassium tetrachloroaurate to introduce gold ions. The hydrogels containing Au3+ were then immersed in a sodium borohydride solution to reduce the gold ions. Infrared spectroscopy, UV-visible spectroscopy, and equilibrium swelling were used to examine the structural/physical differences between gels of different compositions; UV-visible spectroscopy and mass measurements were used to observe the kinetics and thermodynamics of the hydrogel volume phase transition. These studies revealed several differences in the physical characteristics and thermoresponsive behavior of hydrogels based on cross-linker identity and the presence or absence of gold nanostructures. Hydrogels with gold nanostructures and high CBAm and low MBAm content have equilibrium swelling masses 3-20 times their native analogues. In comparison, gold-containing hydrogels with high MBAm and low CBAm content have swelling masses that are equal to their native analogues. Additionally, the gold-containing PNIPAm hydrogels cross-linked with only CBAm have a deswelling temperature of ∼40 °C, ∼8 °C above the samples cross-linked with only MBAm. Varying the CBAm content and introducing gold enables tuning of the deswelling temperature.
Introduction Stimuli-responsive hydrogels undergo a substantial volume change when an environmental factor such as temperature, pH, or ionic strength is altered. The volume phase transition is caused by a change in the hydrogel’s water content. Hydrogels have attracted attention because of both potential and demonstrated utility of this behavior in a variety of applications ranging from flow controllers in microfluidic devices to sorbents.1-3 Biomedical applications have been the focus of much of this research.4-6 Poly(N-isopropylacrylamide) (PNIPAm) is one hydrogel material that undergoes a dramatic volume phase transition from a swollen, hydrated state below 33 °C to a shrunken, dehydrated state above 33 °C.7,8 The expulsion of its contents near physiological temperatures has resulted in extensive investigations of the drug delivery potential for PNIPAm. Investigating and understanding both the thermodynamics and kinetics of the phase transition of PNIPAm hydrogels have long been of interest.7,9-11 In the first reported study of the phasetransition behavior of PNIPAm hydrogels, Tanaka and co-workers observed a sharp, discontinuous transition from the hydrated to dehydrated state near 33 °C for PNIPAm.7 This behavior of PNIPAm hydrogels varied from other hydrogel systems in which * To whom correspondence should be addressed. Phone: 781-283-3097. Fax: 781-283-3642. E-mail:
[email protected]. (1) Bromberg, L.; Temchenko, M.; Alakhov, V.; Hatton, T. A. Langmuir 2005, 21, 1590. (2) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B.-H. Nature (London) 2000, 404, 588. (3) Peppas, N. A.; Huang, Y.; Torres-Lugo, M.; Ward, J. H.; Zhang, J. Annu. ReV. Biomed. Eng. 2000, 2, 9. (4) Hoffman, A. S. AdV. Drug DeliVery ReV. 2002, 54, 3. (5) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869. (6) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliVery ReV. 2002, 54, 37. (7) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379. (8) Li, Y.; Tanaka, T. Annu. ReV. Mater. Sci. 1992, 22, 243. (9) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311. (10) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (11) Tanaka, T.; Sato, E.; Hirokawa, Y.; Hirotsu, S.; Peetermans, J. Phys. ReV. Lett. 1985, 55, 2455.
a discontinuous transition was seen only when the hydrogel possessed ionized groups. The authors attributed this behavior to the high stiffness of the polymer chains in PNIPAm relative to other hydrogel systems. However, subsequent work revealed the important role that the polymerization initiator may play in imparting charge to the PNIPAm hydrogel.10,12 The changeover from a discontinuous to an abrupt but continuous phase transition was found to be slight and to depend strongly on the solution conditions such as ionic strength.12 The kinetics of phase transition of the PNIPAm hydrogel have also been studied extensively. Here, synthesis parameters can be varied to achieve “fast” response from the hydrogels.10 For example, adjusting temperature during the gelation process results in a PNIPAm hydrogel with rapid phase-transition kinetics.13 Similarly, introducing porosity into the PNIPAm hydrogel by synthesizing the hydrogel around silica spheres then removing the spheres results in a much more rapid deswelling with temperature increase.14,15 Of particular significance to the use of hydrogels in myriad applications is the ability to control both the rate and temperature at which deswelling occurs. Polymerization of NIPAm with a comonomer is one of the most common means to alter the phasetransition temperature.10 The nature of the comonomer, specifically its hydrophobicity or hydrophilicity, generally dictates the direction of temperature shift. Comonomers that are more hydrophobic than NIPAm result in a lower phase-transition temperature. Recently, researchers have begun exploring alternative ways to adjust properties of PNIPAm-based hydrogels, including the creation of nano- or microcomposite materials. Composite materials consisting of the hydrogel and a second phase with at least one dimension on the micrometer or nanometer scale, enable (12) Hirotsu, S.; Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1987, 87, 1392. (13) Kabra, B. G.; Gehrke, S. H. Polym. Commun. 1991, 32, 322. (14) Serizawa, T.; Uemura, M.; Kaneko, T.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 3542. (15) Serizawa, T.; Wakita, K.; Akashi, M. Macromolecules 2002, 35, 10.
10.1021/la0534165 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/21/2006
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Figure 1. Structures of (a) N-isopropylacrylamide, (b) N,N′methylenebisacrylamide, and (c) N,N′-cystaminebisacrylamide.
the tuning or creation of new properties of the composite relative to the individual components. Composites of PNIPAm with silica microparticles14,15 and gold nanoparticles,16-18 nanoshells,19,20 and nanorods21 have been studied. Inclusion of nanoscale gold in the hydrogel matrix dramatically alters the properties of the composites relative to the individual components. The alteration is a result of the properties possessed by gold nanostructures.22 The PNIPAm-gold nanocomposites often have interesting electronic and optical properties.16,17,19,20 The composites may exhibit a weak color change as they pass through the volume phase transition.16 Alternatively, introducing gold nanoshells into the hydrogel allows the absorption of nearinfrared (NIR) radiation to heat the composite and drive a volume phase transition. The ability to use NIR is noteworthy because this type of radiation can penetrate human skin, making the technique useful for in vivo applications.19,20 Here, we report the characterization and UV-visible spectroscopic and mass-based volume phase-transition studies of PNIPAm hydrogels containing gold nanostructures synthesized in situ. The PNIPAm hydrogels are cross-linked with either N,N′methylenebisacrylamide (MBAm), N,N′-cystaminebisacrylamide (CBAm), or both. Figure 1 contains the chemical structures for NIPAm, MBAm, and CBAm. The presence of the disulfide bridge in CBAm makes it a reactive or cleavable cross-linking agent, whereas MBAm is relatively unreactive.1,23,24 The well-known affinity of thiols, thiolates, and disulfides for metallic gold surfaces creates the potential for interaction between the CBAm crosslinker and gold nanostructure.25 Previously, researchers observed novel optical and physical properties resulted when gold nanostructures were grown within high-CBAm-content PNIPAm hydrogels.18 Here, we report the full characterization of the kinetics and thermodynamics of the phase transition for a variety of hydrogel compositions with and without gold nanostructures. The characterization allows for evaluation of the drug delivery potential of these nanocomposite materials. We also explore the origins of the alteration in phase-transition behavior imparted to the hydrogel by the introduction of gold into the matrix. Experimental Section Materials. CBAm (Sigma), MBAm (Fluka), methanol (MeOH, Aldrich), ammonium persulfate (APS, Aldrich), N,N,N′N′-tetram(16) Sheeney-Haj-Ichia, L.; Sharabi, G.; Willner, I. AdV. Funct. Mater. 2002, 12, 27. (17) Pardo-Yissar, V.; Gabai, R.; Shipway, A. N.; Bourenko, T.; Willner, I. AdV. Mater. 2001, 13, 1320. (18) Wang, C.; Flynn, N. T.; Langer, R. AdV. Mater. 2004, 16, 1074. (19) Sershen, S. R.; Westcott, S. L.; Halas, N. J.; West, J. L. J. Biomed. Mater. Res. 2000, 51, 293. (20) Sershen, S. R.; Westcott, S. L.; West, J. L.; Halas, N. J. Appl. Phys. B: Lasers Opt. 2001, 73, 379. (21) Gorelikov, I.; Field, L. M.; Kumacheva, E. J. Am. Chem. Soc. 2004, 126, 15938. (22) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293.
Pong et al. ethylenediamine (TEMED, Sigma), potassium tetrachloroaureate (KAuCl4, Aldrich), and sodium borohydride (NaBH4, Aldrich) were used as received. NIPAm (Aldrich) was recrystallized from hexanes prior to use. Gold nanoparticles (10 nm) were purchased from Ted Pella, Inc. and were used as received. Water was purified using a Nanopure system from Barnstead. The gel mold (35 × 100 × 0.75 mm3) was purchased from Bio-Rad. Stock solutions of 10wt%/wt APS were prepared and stored at -10 °C. Synthesis. For each hydrogel composition, 5.0 mL of pre-gel solution was prepared by combining 0.50 g of NIPAm and the appropriate amounts (0.00-3.50 wt%/wt of monomer) of MBAm and/or CBAm. The NIPAm and MBAm were dissolved in 4.8 mL of water. CBAm was first dissolved in MeOH because of its low solubility in water; 0.2 mL of the CBAm-MeOH solution, containing the appropriate amount of CBAm, was then added to the NIPAmMBAm solution bringing the total pre-gel solution volume to 5.0 mL. After 25 µL of 10% APS solution was added, the pre-gel solution was purged with N2(g) for at least 5 min on ice to removed dissolved O2. Then, 10 µL of the accelerant TEMED was added to the pre-gel solution. The pre-gel solution was then transferred to the gel mold and was allowed to polymerize at 4 °C for at least 24 h. After polymerization, 8.5 mm diameter hydrogel disks were cut from the hydrogel. The disks were then thoroughly washed to remove remaining unpolymerized monomer, initiator, and accelerator. The hydrogels were placed in Nanopure water for a minimum of 72 h, during which the water was changed at least once every 24 h. For long-term storage, hydrogels were placed in a 10% v/v ethanol/ Nanopure water solution. Hydrogel composition is denoted with the nomenclature X.XXM/Y.YYC where X.XX and Y.YY refer to the percentage of MBAm and CBAm, respectively, as wt/wt percentages relative to NIPAm in the hydrogel. Gold nanostructures were synthesized within hydrogels of all compositions using a method adapted from Wang et al.18 Gold ions were introduced into the hydrogel matrix by placing gels in 10.0 mL of 5.0 mM Au3+ (KAuCl4) solution for 24 h. The solutions were placed in the dark to prevent photoinduced gold reduction. Gold nanostructures were then formed within these gels by reducing the Au3+ soaked gels in 50 mM NaBH4 for 15-20 min. Upon reduction, gels turned colors ranging from black to red-brown. Gels with low CBAm content tended to turn black or dark brown, while gels with high CBAm content typically became red-brown. The gold-containing hydrogels were washed in Nanopure water for a minimum of 72 h, and the water was changed at least once every 24 h. For long-term storage, gold-containing hydrogels were placed in a 10% v/v ethanol/ Nanopure water solution. After three months, regardless of use, in situ gold gels were discarded because of possible changes in gold nanostructure size and shape.18 Two types of control samples were used in these studies. The first were non-gold-containing hydrogels, which are subsequently referred to as “native” hydrogels. The second control samples were made by introducing presynthesized Au nanoparticles into native hydrogels. These samples were prepared using the “breathing” method of Willner and co-workers.16,17 Specifically, following the washing step, hydrogels were dried overnight. The dried hydrogels were then immersed in a solution of 10 nm gold nanoparticles (concentration 5.7 × 1012 particles/mL) and allowed to swell to equilibrium volume. The process was repeated until additional cycling did not increase the concentration of gold nanoparticles, as indicated by stabilization in the UV-visible absorption band at ∼520 nm. Generally, four to five breathing cycles of the hydrogels were needed to achieve saturation with gold nanoparticles. Characterization of Nanocomposite Materials. IR Spectroscopy. The structural properties of the hydrogels were examined using a Perkin-Elmer Instruments Spectrum One FT-IR spectrophotometer fitted with a Perkin-Elmer Universal ATR sampling accessory. Hydrogel disks were cut into small pieces and allowed to dry for a minimum of 24 h. Spectra were recorded by placing a dried gel piece over the attenuated total reflectance crystal and acquiring 64 scans with a resolution of 1.00 cm-1. UV-Visible Spectroscopy. The spectra of the hydrogels were examined and recorded using a Cary Scan 500 UV-vis-NIR
ThermoresponsiVe BehaVior of PNIPAm Hydrogels spectrophotometer. Spectra were recorded over the 400-800 nm range with 1.0 nm resolution in the double-beam mode using baseline correction. Cuvette temperature was controlled using the CARY Varian PCB 150 Water Peltier System, which had an uncertainty of (0.3 °C. Baseline spectra were recorded with a cuvette containing Nanopure water at 25.0 °C. To eliminate the effects of changing hydrogel volume, brass plates with 3 mm diameter holes were placed between the light source and the sample. The plates served to normalize beam path through the hydrogel to an area smaller than the minimum diameter of the hydrogel. Room-temperature hydrogel samples were then prepared by placing a hydrogel against the side of a cuvette and filling the cuvette with Nanopure water. The phase transition for all hydrogels was examined in a temperature range of ∼25.0 to ∼50.0 °C, over which the temperature was increased stepwise. Spectra were recorded after samples had been kept at a constant temperature for a minimum of 20 min in order to ensure that the maximum absorbance of the hydrogel at that temperature had been reached. Phase-Transition Studies. The thermodynamics of the volumetric phase transition of the hydrogels were examined. Room-temperature gels, placed in Nanopure water-filled vials, were placed in a FischerScientific IsoTemp 205 temperature-adjustable water bath. Mass changes were recorded over a temperature range of 25.0-60.0 °C. Native gels were held at one temperature for a minimum of 2 h; gold-containing gels were held for a minimum of 6 h prior to mass measurement. Gels were removed from solution and had excess surface water removed with a damp Kimwipe. The gel was then placed on a glass slide, and the mass measured using an analytical balance. All reported masses represent the average from at least three separate samples. The kinetics of the phase transition of the hydrogels were examined using a procedure adapted from Makino et al.26 Hydrogels in Nanopure water-filled vials were placed in a water bath and allowed to equilibrate at 25.0 °C for an hour. The 25.0 °C-equilibrated gels were then transferred to a 30.0 °C or 40.0 °C bath where they were kept for 8 h. The mass of the hydrogels was measured every 15 min for the first 3 h and then once every hour for the next 5 h.
Results and Discussion Equilibrium Swelling. Figure 2 contains the equilibrium swelling masses for both native (a) and gold-containing (b) hydrogels. Only slight variation is seen among the native hydrogels regardless of the identity or concentration of the crosslinking agent. Generally, the hydrogels with the smallest total cross-linking agent, CBAm and MBAm, are the most massive. The heaviest native gel is the 0.00M/3.50C sample, which has a mass of 0.068 ( 0.003 g. The 3.50M/0.00C hydrogel has a mass of 0.042 ( 0.001 g. The 3.50M/3.50C hydrogel has the smallest mass of 0.034 ( 0.001 g. We believe the decrease in total cross-linking simply enables more structural flexibility within the hydrogel matrix. This behavior enables hydrogels with a small about of CBAm or MBAm to swell more readily than highly cross-linked hydrogels in which stiff mechanical forces balance the osmotic force for swelling. Additionally, we believe the reduction of a small fraction of the disulfide bond in the CBAm cross-linked hydrogels is the likely origin of the mass difference between the native 3.50M/0.00C and 0.00M/3.50C hydrogels. This reduction of the disulfide is possible because the hydrogels are synthesized in a decreased dissolved oxygen environment as described in the Experimental Section. The equilibrium swelling of gold-containing hydrogels is shown in Figure 2b. Here, the variation in swelling differs dramatically (23) Chiu, H.-C.; Wang, C.-H. Polym. J. (Tokyo) 2000, 32, 574. (24) Plunkett, K. N.; Kraft, M. L.; Yu, Q.; Moore, J. S. Macromolecules 2003, 36, 3960. (25) Dubois, L. H.; Nuzzo, R. G. Annu. ReV. Phys. Chem. 1992, 43, 437. (26) Makino, K.; Hiyoshi, J.; Ohshima, H. Colloids Surf., B 2000, 19, 197.
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Figure 2. Equilibrium swelling mass of (a) native and (b) goldcontaining hydrogels at 25 °C.
depending on the CBAm content in the hydrogel. This behavior is in stark contrast to the slight differences observed with the native hydrogels. The 3.50M/0.00C hydrogel is similar in mass to its native analogue, 0.039 ( 0.001 and 0.042 ( 0.001 g, respectively. In contrast, all hydrogels containing CBAm are substantially more massive following the in situ gold nanostructure synthesis. The 3.50M/3.50C hydrogel is 2.5 times as massive as the native hydrogel. The increase in mass following gold introduction becomes more pronounced as the CBAm content increases, and MBAm content decreases, as is illustrated by looking at all the X.XXM/3.50C masses in Figure 2. The 0.00M/ 3.50C hydrogel’s mass increased an average of 8-fold, from 0.068 ( 0.003 to 0.5 ( 0.1 g, with the in situ generation of gold nanostructures. The 0.00M/3.50C hydrogel’s mass may increase from 5- to as much as 16-fold with the in situ generation of gold nanostructures. The mass range for this composition extends up to more than 1.0 g. This wide mass range may indicate that there is variation between synthesis batches of hydrogels of the same composition and/or that the number of gold nanoparticles formed in each gel varies. Phase-Transition Behavior. The effect of gold nanostructure generation within the hydrogel matrix on the thermoresponsive behavior was investigated. As the hydrogels were heated, the optical and equilibrium swelling properties of the hydrogels were monitored. The results are interpreted on the basis of the aforementioned characterization of the hydrogel system. UV-Visible Spectroscopy. UV-visible spectroscopy is often used to characterize the optical properties of the hydrogels. Because of changes in transparency (turbidity) associated with PNIPAm’s phase transition, temperature-dependent spectra can also be used to monitor the phase-transition behavior of these hydrogels.10 Figure 3 shows the UV-visible spectra of native
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Figure 4. Fractional mass change of 3.50M/0.00C native (9) and gold-containing (O) and 0.00M/3.50C native (2) and gold-containing (×) hydrogels following a temperature step from 25 to 40 °C. Error bars on data represent the standard deviation from a minimum of three separate hydrogel samples.
Figure 3. Temperature-dependent UV-visible spectra of 3.50M/ 3.50C hydrogels. Spectra of (a) native hydrogels were recorded at 25, 30, 32, 34, 36, 38, 40, and 50 °C and (b) gold-containing hydrogels were recorded at 25, 30, 35, 37, 39, 41, 43, 45, 47, 49, and 51 °C. Absorbance values for each hydrogel type increase with temperature, and several spectra have been labeled.
(a) and gold-containing (b) 3.50M/3.50C PNIPAm hydrogels. At temperatures of 34.0 °C and below, the native hydrogels possess a featureless, low absorbance over the visible range. At temperatures above 34.0 °C, a featureless, high absorbance is observed. This characteristic response is well-known for PNIPAmbased hydrogels.10,27 The change results from the collapse of the polymer segments within the hydrogels followed by aggregation. These aggregated structures lead to wavelength-independent scattering across the entire visible region of the spectrum. The dramatic increase in absorbance is very abrupt, occurring between 34.0 and 36.0 °C, as seen in Figure 3a. The rapid increase in absorption by 36.0 °C is observed in all native hydrogels regardless of cross-linking agent or concentration. The temperature-dependent optical behavior of gold-containing PNIPAm hydrogels differs substantially from the native hydrogels. As Figure 3b indicates, a pronounced peak is observed at ∼520 nm for the 3.50M/3.50C hydrogel. A peak at 520 nm is characteristic of the surface plasmon resonance of gold nanostructures.28 This surface plasmon band for gold particles is known to be very sensitive to gold nanoparticle size and aggregation.29 The 520 nm peak position and the peak’s moderate tailing at longer wavelengths is characteristic of well-separated, small gold nanostructures. The absorbance at longer wavelength increases substantially as the CBAm content decreases (see Figure 2 in ref 18), indicating that nanoparticles are larger or more highly aggregated at lower CBAm content. This theory is supported by (27) Matsuo, E. S.; Tanaka, T. J. Chem. Phys. 1988, 89, 1695. (28) Brown, K. R.; Walter, D. G.; Natan, M. J. Chem. Mater. 2000, 12, 306. (29) Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci. 1982, 120, 435.
previous electron microscopy studies with these types of hydrogels.18 As the temperature is increased to 51.0 °C, the absorbance increases across the entire visible range. However, the increase in absorbance is rather gradual compared to the abrupt change exhibited by native hydrogel systems (Figure 3a). With our samples, the shape of the absorbance-temperature profile is strongly dependent on the CBAm content in the hydrogels. The 3.50M/0.00C gold-containing hydrogel exhibits a dramatic increase in absorbance between 34.0 and 35.0 °C, as shown in Figure A in the Supporting Information. The optically observed phase transition for the 3.50M/0.00C gold-containing hydrogel is identical to the native analogue. This behavior is found for all non-CBAm-containing hydrogels after the introduction of gold (data not shown). Figure 3b also shows that no change in the wavelength for peak absorbance is observed as the temperature increases from 25.0 to 50.0 °C. The lack of a shift to longer wavelength indicates that the gold nanostructures are not forming aggregated structures as the hydrogel collapses. This may result from the large average separation between gold nanostructures or the relative stability of the particles within the hydrogel matrix. This result differs from those observed with introduction of presynthesized nanoparticles using the breathing method developed by Willner and co-workers.16 The authors in that study observed a blue-shift in the color of the hydrogel containing presynthesized gold nanoparticles after the phase transition. We have observed similar shifts for hydrogels containing gold nanoparticles made using the breathing method regardless of the identity or concentration of the cross-linking agent. Kinetics. The rate at which hydrogels collapse was investigated using mass measurements.26 Hydrogels were allowed to thermally equilibrate at 25 °C and were transferred to a bath at 40.0 °C, and the mass was monitored over a period of 8 h. The fractional change in hydrogel mass, defined as Fm ) (mtime - mdry)/(m0 mdry), where mtime is the mass at a given time, mdry is the mass in the dry state, and m0 is the mass after equilibration at 25 °C, is shown in Figure 4 for native and gold-containing 3.50M/ 0.00C and 0.00M/3.50C hydrogels. Gold-containing 0.00M/3.50C and 3.50M/0.00C gels exhibit similar deswelling rates/characteristics to those of native gels. The deswelling characteristics of the gold-containing 0.00M/3.50C and 3.50M/0.00C hydrogels are remarkably similar, despite the large difference in equilibrium mass at 25 °C. Native gels, of the composition 0.00M/3.50C, 3.50M/0.00C, 1.75M/1.75C, and 3.50M/3.50C, all exhibit similar deswelling
ThermoresponsiVe BehaVior of PNIPAm Hydrogels
Figure 5. Fractional mass change of (a) native and (b) goldcontaining 3.50M/0.00C (9), 1.75M/1.75C (O), 0.00M/3.50C (2), and 3.50M/3.50C (×) hydrogels over the temperature range of 2560 °C. Error bars on data represent the standard deviation from a minimum of three separate hydrogel samples.
characteristics, in which a majority of the deswelling during the 8-h period takes place within the first 2.5-3 h. It appears that native hydrogels containing both CBAm and MBAm, regardless of the total cross-linking amount, undergo significantly faster deswelling initially. We attempted to perform a true rate determination for the phase-transition process as has been done with other hydrogel systems.11,14,15,27 Analysis using several different rate order processes (first, ln Fm vs time; second, 1/Fm vs time) resulted in possible linear behavior for native hydrogels with secondorder rates. Analysis did not reveal strong linear behavior for either first- or second-order rates for any of the gold-containing hydrogels. This phenomenon is not uncommon for the deswelling process, which typically exhibits much more complex kinetics than the hydrogel swelling.27 However, understanding the deswelling process is more vital to intended applications in drug delivery. Thermodynamics. Figure 5 shows the fractional mass change for four compositions of both native (a) and gold-containing (b) hydrogels as temperature is increased from 25.0 to 60.0 °C. All native hydrogels exhibit the same fractional mass change with temperature. Fm drops quickly with increases from 25.0 °C passing through Fm ) 0.50 at 32.0 °C. By the time the hydrogels have equilibrated at 34.0 °C, all have deswelled to 10% of the total fractional change. All native hydrogel compositions synthesized (see Figure 2) have been studied with these methods and exhibit behavior identical to the four compositions shown in Figure 5a. The similarity in deswelling behavior of native hydrogels is striking for several reasons. First, the total cross-linking agent concentration (CBAm + MBAm) varies from 3.50% to 7.00%. The variation in concentration of cross-linking agent might be
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expected to cause differences in mechanical properties of the hydrogel. However, these differences are not observed in the thermal deswelling behavior of the hydrogels. Second, a large disparity in equilibrium swelling exists between the 3.50M/0.00C (0.042 ( 0.001 g) and the 0.00M/3.50C (0.068 ( 0.003 g). Despite a large difference in the mass of water contained in the hydrogels, both compositions deswell over the same temperature range. As Figure 5b indicates, the thermal deswelling changes dramatically following the growth of gold nanostructures within some hydrogels. The 3.50M/0.00C hydrogel sample (solid squares in Figure 5b) possesses a fractional mass behavior that is nearly identical to all the native hydrogels. Specifically, the 3.50M/ 0.00C mass quickly drops to Fm ) 0.50 by 32.5 °C and to 0.10 by 35.0 °C. With the 1.75M/1.75C gold-containing hydrogel, a significant shift to higher phase-transition temperature is observed, as seen in Figure 5b. The 1.75M/1.75C gold sample drops through Fm ) 0.50 at 37.5 °C and does not reach 0.10 until 42.5 °C. These temperatures represent approximately 5 and 8 °C shifts, respectively, from the same Fm values for the 1.75M/1.75M native hydrogel. At higher CBAm cross-linking levels, a continued increase in phase-transition temperature is observed. Both the 3.50M/3.50C and the 0.00M/3.50C gold-containing hydrogels pass through Fm ) 0.50 near 40 °C and do not reach 0.10 until the solution temperature is above 45 °C. Slight differences between the phase-transition behavior of the two 3.50% CBAm hydrogels are evident in Figure 5b. The 0.00M/3.50C hydrogel exhibits an earlier onset of deswelling than the 3.50M/3.50C counterpart. Through the steepest portion of the deswelling curves, however, the two samples possess similar mass response to increased temperature. Above the midpoint of the fractional mass change, the 0.00M/3.50C hydrogel again deswells more significantly than the 3.50M/3.50C hydrogel. The differences between the two hydrogels containing 3.50% CBAm may be a result of the higher total cross-linking in the 3.50M/ 3.50C hydrogel leading to a stronger mechanical resistance to the deswelling. The higher cross-linking density has been demonstrated to affect mechanical properties of other hydrogel types.30 In combination, the alteration in CBAm content in the hydrogel followed by in situ synthesis of gold nanostructures creates the ability to tune the transition temperature of the hydrogel materials from ∼34 to 40 °C. This control over phase-transition temperature in the physiologically relevant range has long been recognized as an important goal for numerous applications involving PNIPAm-based hydrogels.6,31-33 Origins of Alteration in Behavior for High-CBAm Hydrogels Containing Gold. The alteration in equilibrium swelling and phase-transition behavior for PNIPAm hydrogels with high CBAm content after introduction of gold is readily evident in Figures 2, 3, and 5. Several possible factors may contribute to this alteration in behavior. We believe the two most likely origins are either chemical changes caused to the hydrogel itself by the gold nanostructure synthesis process or a property inherent to the gold nanostructures themselves. Chemical alteration of the PNIPAm hydrogel could result either from interactions with the Au3+ ions or the NaBH4 reducing agent. The gold nanostructures within the hydrogel have been shown to be significantly different in size and morphology, which has previously been postulated (30) Kong, H. J.; Lee, K. Y.; Mooney, D. J. Macromolecules 2003, 36, 7887. (31) Tanaka, T. Phys. ReV. Lett. 1978, 40, 820. (32) Tanaka, T.; Fillmore, D.; Sun, S.-T.; Nishio, I.; Swislow, G.; Shah, A. Phys. ReV. Lett. 1980, 45, 1636. (33) Coughlan, D. C.; Quilty, F. P.; Corrigan, O. I. J. Controlled Release 2004, 98, 97.
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as the origin of the differences in equilibrium swelling among various composition hydrogels.18 Below we describe the results from a series of experiments to test the contributions of each of these factors to modifying the properties of the high-CBAm cross-linked PNIPAm hydrogels containing gold nanostructures. Chemical Alteration of the PNIPAm Hydrogel. To test the origin of the increased equilibrium swelling for CBAm crosslinked hydrogels after gold nanostructure introduction, native hydrogels were immersed in a 50 mM solution of NaBH4, the reducing agent used for the formation of gold nanostructures. If the increased volume of the hydrogels results simply from cleavage of the disulfide linkage by the reducing agent, these CBAm cross-linked hydrogels should swell to the equilibrium size of the hydrogels containing gold nanostructures. The reduction of the disulfide bond in the 0.00M/3.50C results in an increase in mass to 0.20 ( 0.04 g. This mass is larger than that of the native hydrogel (0.068 ( 0.003 g) but significantly less than the mass of the gold-containing 0.00M/3.50C hydrogel (0.5 ( 0.1 g). Other reducing agents such as dithiothreitol and tris[2-carboxyethyl]phosphine hydrochloride, which are commonly used for disulfide reduction, also failed to induce as significant a mass change in the hydrogels as the in situ gold nanoparticle synthesis. Therefore, the reduction of the disulfide bond in CBAm alone does not lead to the observed changes in equilibrium swelling volume. The affect of exposure to Au3+ ions on the equilibrium swelling was also investigated. Here, a 0.00M/3.50C hydrogel was immersed in a 5.0 mM solution of KAuCl4 for 24 h in the dark. The treated hydrogel was then removed from the KAuCl4 solution and transferred to water without first reducing the Au3+ ions in NaBH4. After equilibrating in water for a minimum of 24 h, these Au3+-exposed 0.00M/3.50C hydrogels were characterized. Two properties were readily apparent with these hydrogels. First, the hydrogels were observed to have a pale red-pink color. Second, the hydrogels’ equilibrium swelling was significantly large than the native hydrogels. UV-visible spectroscopy on the hydrogel indicated a small peak near 520 nm, characteristic of the formation of gold nanoparticles. Equilibrium mass measurements on these hydrogels showed a mass that was 89% of the mass of the 0.00M/ 3.50C hydrogel after in situ synthesis of gold nanostructures. In other words, exposure to Au3+ ions without subsequent reduction seems to lead to some of the same behaviors observed with the Au3+ loading and reduction process. As a control, the same process of soaking in Au3+ solution without subsequent reduction in borohydride was performed with the 3.50M/0.00C hydrogels. The 3.50M/0.00C hydrogels treated with Au3+ did not exhibit any change in equilibrium swelling from the native 3.50M/0.00C hydrogels. The gels did turn pale yellow following loading with the gold salt. This color, however, is characteristic of the Au3+ solution and not indicative of formation of Au nanostructures within the hydrogel matrix. FT-IR spectroscopy was used to look for structural differences among the native, gold containing, and gold ion-treated hydrogels. For reference, raw materials spectra are shown in Figure B, Supporting Information. The raw material spectra showed the peaks expected for each compound; NIPAm, MBAm, and CBAm all possess peaks for secondary amines (-NH stretches) near 3310-3251 cm-1, the amide I (-CdO) and amide II (CNH) stretches near 1650 cm-1 and 1550 cm-1, respectively, and vinyl stretches between 995 and 905 cm-1. The spectra all have rich fingerprint regions at frequencies less than ∼1500 cm-1.34,35 (34) Kim, S. J.; Lee, C. K.; Lee, Y. M.; Kim, S. I. J. Appl. Polym. Sci. 2003, 90, 3032. (35) Petrovic, S. C.; Zhang, W.; Ciszkowska, M. Anal. Chem. 2000, 72, 3449.
Pong et al.
Figure 6. FT-IR spectra of 3.50M/0.00C and 0.00M/3.50C native and gold-containing hydrogels and the 0.00M/3.50C hydrogel treated with Au3+. Spectra have been offset along the y axis for clarity. The arrow indicates the 1040 cm-1 peak present only with CBAm crosslinked gold-containing hydrogels.
Figure 6 is the IR spectra of both native and gold-containing 3.50M/0.00C and 0.00M/3.50C hydrogels along with the Au3+treated, unreduced 0.00M/3.50C hydrogel. In comparison with the raw materials, the IR spectra of native and gold-containing, dried hydrogel possessed fewer and broader peaks below 1600 cm-1.34,35 The 3600-3100 cm-1 region has also changed significantly. All the hydrogels have a broad absorption throughout the region as is characteristic of polymerized NIPAm.34-37 The spectral differences between the reactants and product reveal that polymerization had taken place. Specifically, in comparison with the reactants, the vinyl peaks (995-905 cm-1) are nearly absent in the spectra of dried hydrogels, indicating that polymerization has occurred across the double bond. Figure 6 indicates little spectral difference between the two native hydrogels regardless of composition. This similarity holds true for all native hydrogels regardless of composition (data not shown). The -OH stretches of bound water appear as a large, broad peak at 3297 cm-1; alkane stretches at 1935 and 2971 cm-1, a strong CdO stretch at 1639 cm-1, and a strong amide stretch at 1530 cm-1 are associated with the PNIPAm polymer backbone.36 However, with gold-containing samples (Figure 6) a new peak is observed at ∼1040 cm-1 in the CBAm crosslinked hydrogels. We believe this 1040 cm-1 peak likely results from sulfonate.38,39 We postulate that a small fraction of Au3+ ions are reduced to Au0 and in the process oxidize the disulfide or free thiolates in CBAm to a sulfonate. The large standard reduction potential for Au3+ ions (1.41 V for Au3+ or 1.04 V for AuCl4-) may serve as the energetic driving force for the oxidation of the disulfide or thiolate to the sulfonate. To investigate this hypothesis, FT-IR spectra of CBAm cross-linked gels loaded with Au3+ were also recorded. The 1040 cm-1 peak is also observed in the IR spectrum of 0.00M/3.50C hydrogels treated with Au3+ but not subsequently reduced, as seen in Figure 6. Treatment of 0.00M/3.50C hydrogels with a second oxidizing agent, potassium permanganate (KMnO4), also results in the appearance of this 1040 cm-1 peak in the FT-IR spectrum. Although FT-IR spectroscopy indicates that either KMnO4 or Au3+ treatment result in the formation of a sulfonate within (36) Liang, L.; Rieke, P. C.; Liu, J.; Fryxell, G. E.; Young, J. S.; Engelhard, M. H.; Alford, K. L. Langmuir 2000, 16, 8016. (37) Percot, A.; Zhu, X. X.; Lafleur, M. J. Polym. Sci., Part B: Polym. Phys. 2000, 38, 907. (38) Park, S.-H.; Lee, C. E. J. Phys. Chem. B 2005, 109, 1118. (39) Bonazzola, C.; Calvo, E. J.; Nart, F. C. Langmuir 2003, 19, 5279.
ThermoresponsiVe BehaVior of PNIPAm Hydrogels
0.00M/3.50C hydrogels, the equilibrium swelling behavior of these two samples is significantly different. KMnO4 only induces swelling to 62% of the final mass of the 0.00M/3.50C hydrogels containing gold nanostructures. This value is much less than the 89% observed with the Au3+-treated 0.00M/3.50C hydrogels. The conversion of disulfide in CBAm to sulfonates introduces charge into the hydrogel. The presence of increased charge may account for the much of the change in equilibrium swelling and phase-transition behavior for the 0.00M/3.50C hydrogels containing gold since charge is known to play a critical role in hydrogel properties and behavior.10 For example, copolymerization of NIPAm with a variety of ionizable species such as sodium acrylate, 2-(dimethylamino)ethyl methacrylate, and methacrylamidopropyltrimethlylammonium chloride, leads to an increase in the phase-transition temperature of the hydrogel.40-42 The simplest explanation for this phenomenon is that the ionized (charged) comonomer results in a more-hydrophilic hydrogel. The increased hydrophilicity necessitates a higher temperature to overcome the increased attraction by the hydrogel for water. Affect of Gold Nanostructures on Hydrogel Properties. To test the role of gold nanostructure introduction on the behavior of the hydrogels, the breathing method was used to introduce presynthesized 10 nm Au nanoparticles into all compositions of hydrogels. These samples were then characterized using the same methods described for the hydrogels containing gold nanostructures synthesized in situ. Equilibrium swelling mass of hydrogels containing presynthesized gold nanostructures were measured. None of these samples, regardless of CBAm content, exhibited a mass dramatically different from its native counterpart (data not shown). Thus, the large equilibrium swelling is only achieved through the in situ synthesis of gold nanostructures in highCBAm-content hydrogels. The UV-visible spectra of 3.50M/0.00C, 1.75M/1.75C, 0.00M/3.50C, and 3.50M/3.50C hydrogels containing presynthesized Au nanoparticles are shown in Figure C in the Supporting Information. In contrast to the in situ synthesis samples,18 only slight differences among hydrogels are observed as a result of differences in cross-linker identity and concentration. The Au nanoparticle uptake appears highest in the 0.00M/3.50C hydrogel on the basis of the absorbance near 520 nm, which is attributed to the nanoparticles. Also of note, the absorbance value for all the hydrogels is reduced more than 50% from that observed with the in situ synthesized hydrogels (see Figure 3). The lower absorbance indicates significantly less gold is introduced into the hydrogel using the breathing method than is incorporated using the in situ synthesis method. Phase-transition studies were also performed on the control hydrogels containing the presynthesized nanoparticles introduced using the breathing method.16,17 Here, all hydrogel compositions shown in Figure 2 were investigated. Results from the 3.50M/ 0.00C, 1.75M/1.75C, 0.00M/3.50C, and 3.50M/3.50C hydrogels are shown in Figure D in the Supporting Information. As with the native samples, the hydrogels with presynthesized nanoparticles reach Fm ) 0.50 by 32 °C and are near Fm ) 0.10 by 34 °C. This fractional mass change is dramatically different than the mass change of the hydrogels containing gold nanostructures synthesized in situ. The thermodynamic fractional mass change of the hydrogels is identical to that of the native hydrogels as shown in Figure 5a, strongly suggesting that introducing the (40) Tokuhiro, T.; Amiya, T.; Mamada, A.; Tanaka, T. Macromolecules 1991, 24, 2936. (41) Beltran, S.; Hooper, H. H.; Blanch, H. W.; Prausnitz, J. M. J. Chem. Phys. 1990, 92, 2061. (42) Beltran, S.; Baker, J. P.; Hooper, H. H.; Blanch, H. W.; Prausnitz, J. M. Macromolecules 1991, 24, 549.
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gold nanostructures into the hydrogel matrix through means other than in situ synthesis does not affect the physical behavior of the hydrogel. The difference between samples is most evident with the 0.00M/3.50C gold-containing hydrogels, where the in situ generation results in a mass phase transition that is 8 °C higher than the same composition hydrogel with presynthesized Au nanoparticles. Although the PNIPAm hydrogels containing presynthesized Au nanoparticles do not exhibit the same behavior as the in situ synthesis samples, we do not believe this eliminates any role for the nanostructures in modified hydrogel properties. Rather, the equilibrium swelling of the hydrogels treated with Au3+ may induce the growth of some gold nanostructures within the hydrogel matrix. The fact that neither the hydrogels treated with Au3+ nor those treated with KMnO4 exhibit the same increase in mass as the in situ gold synthesis hydrogels indicates the importance of the in situ synthesis process on alteration of hydrogel properties. Previous work demonstrated that a high concentration of small nanostructures is formed within the high-CBAm-content hydrogels.18 The smaller gold nanostructures were believed to possess a large surface charge, leading to increased equilibrium swelling and higher phase-transition temperatures for these samples. We now postulate that this effect coupled with some oxidation of disulfide to sulfonate with the CBAm in the hydrogel leads to the alteration in behavior following gold nanoparticle formation.
Conclusions Gold nanostructures were synthesized within PNIPAm hydrogels containing either or both an inert (MBAm) or reactive/ cleavable (CBAm) cross-linking agent. The samples exhibit properties that are strongly dependent on the amount of reactive cross-linker present. The two samples which best illustrate these effects are the 3.50M/0.00C and 0.00M/3.50C in situ goldcontaining hydrogels. Optical, equilibrium swelling, and phasetransition behavior are dramatically different between 3.50M/ 0.00C and 0.00M/3.50C gold-containing hydrogels. The presence of CBAm changes the fractional mass loss observed with increases in temperature after thermal equilibrium is reached. The 3.50% CBAm hydrogels exhibit an 8 °C increase in the temperature associated with a fractional mass change of 50%. Despite this increase in temperature needed to induce deswelling in highCBAm hydrogels, little significant change is observed in the kinetics of the deswelling process for the 3.50M/0.00C and 0.00M/ 3.50C hydrogels with gold. The likely origin of the change in behavior is through charge introduction into the hydrogel by both the oxidation of disulfide to sulfonate in CBAm and the large surface area of the gold nanostructures. The ability to increase the equilibrium swelling mass (loading capacity) and tune the volume phase-transition temperature upward from ∼32 °C without substantial alteration of deswelling rate has important implications for applications most notably use as drug-delivery vehicles. Acknowledgment. The authors thank Ms. Ellane J. Park for her work elucidating the gold-sulfonate chemistry with CBAm cross-linked gold-containing hydrogels and Prof. Chun Wang for helpful discussions. This work was partially supported by the Donors of The American Chemical Society Petroleum Research Fund through Grant No. PRF#42899-GB10 and by the Department of Chemistry, Wellesley College. Supporting Information Available: Figures A-D referred to in the text (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. LA0534165
