J. Phys. Chem. B 2008, 112, 5327-5332
5327
Physical, Chemical, and Chemical-Physical Double Network of Zwitterionic Hydrogels Zheng Zhang, Timothy Chao, and Shaoyi Jiang* Department of Chemical Engineering, UniVersity of Washington, Seattle, Washington 98195 ReceiVed: NoVember 7, 2007; In Final Form: January 21, 2008
Zwitterionic hydrogels are very promising for biomedical applications. They are usually copolymerized with other polymers to improve their mechanical properties often at the expense of their biological properties. In this study, physically cross-linked poly(sulfobetaine methacrylate) (polySBMA) hydrogels were prepared, and their physical properties including phase behavior were investigated. Linear polySBMAs, with an average molecular weight ranging from 20.9 kDa to 316 kDa, were prepared via free radical polymerization at different KCl concentrations. The opaque-transparent phase transition of polySBMA-water mixtures were measured using a UV-vis spectrometer. Analysis from dynamic rheometry showed the formation of physically crosslinked hydrogels with mechanical ductility due to reversible charge interactions. Chemically cross-linked hydrogels were also prepared, and their swelling and mechanical properties were evaluated. It was found that the introduction of cross-linkers could lead to a decrease in the amount of physical cross-links in chemical hydrogels. In order to improve the mechanical properties of SBMA hydrogels, linear polySBMA was introduced to the network of chemically cross-linked polySBMA gels, creating a chemical-physical double network (DN) with both chemical and physical cross-links. The chemical-physical DN provides a desirable method to improve the mechanical properties of zwitterionic hydrogels without introducing other hydrophobic moieties.
1. Introduction Poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC), poly(sulfobetaine methacrylate) (polySBMA), and poly(carboxybetaine methacrylate) (polyCBMA) are zwitterionic polymers with both positive charges and negative charges in their pendant groups. All these polymers were shown to be highly protein/cell resistant and biocompatible.1-4 The biomimetic pendant groups of polyMPC were designed to be phosphorylcholine (PC), which is the head group of lipid bilayers of cell membranes. The pendant groups of polySBMA and polyCBMA are analogues of taurines and glycine betaines, respectively, both of which were found in living organisms. In this work, polySBMA with a sulfobetaine pendent group (CH2CH2N+(CH3)2CH2CH2CH2SO3-) and a methacrylate main chain was studied as an example of these zwitterionic polymers.3 The covalently cross-linked hydrogels of MPC, SBMA, and CBMA can be prepared through initiating a solution of zwitterionic monomers and cross-linkers. Different from other hydrophilic polymers, zwitterionic polymers are characterized by their anti-polyelectrolyte behavior, i.e., the polymeric chains usually expand in aqueous solution in the presence of ions.5 For a zwitterionic hydrogel such as SBMA hydrogel, the gel tends to swell with the ion concentration.6-8 Some zwitterionic hydrogels are also responsive to the changes in temperature and pH.9 For biomedical applications, dual functional carboxybetaine hydrogels can be modified with proteins to facilitate their interactions with cells.10 With these unique properties, zwitterionic hydrogels may have many potential applications ranging from tissue engineering to drug delivery. However, since the pendant groups of zwitterionic hydrogels are highly hydrophilic, their mechanical properties are very fragile and weak. In order to improve the mechanical properties of hydrogels, MPC was * To whom correspondence should be addressed. E-mail: sjiang@ u.washington.edu.
often used as a comonomer with other monomers, such as 2-hydroxyethyl methacrylate (HEMA) to form copolymeric hydrogels.11 Other methods such as interpenetrating network (IPN)12 or double network13 have also been used to improve the mechanical properties of hydrogels. However, all these methods will reduce protein-resistant properties of zwitterionic materials when the nonzwitterionic moieties were introduced. Hydrogels can be classified as chemical hydrogels and physical hydrogels based on the way how they are crosslinked.14 For zwitterionic polymers, most studies are on their chemical hydrogels, which are covalently cross-linked networks, and their cross-linking points are fixed. Taking advantage of the anti-polyelectrolyte behavior of zwitterionic polymers, we found that high molecular weight linear polySBMA can form physical hydrogels in pure water or aqueous solutions with low ionic strengths. Zwitterionic physical hydrogels are cross-linked via the charge-charge interactions among the pendant groups, which are larger than solvation interactions from the surrounding water molecules. The physical cross-linking is not permanent but can be moved when stress is applied. As a result, unlike fragile chemical hydrogels, physical hydrogels are ductile. However, the formation of zwitterionic physical gels is usually reversible and the physical gels can be dissolved in solution at higher ionic strengths. Not only sensitive to ionic strength, the morphology of physical gels may also be changed when temperature or pH is changed. For example, we found that polySBMA could change from a transparent gel into an opaque gel. Combination of the ductile physical hydrogels with the fragile chemical hydrogels may improve the mechanical properties of chemical hydrogels without losing their ionic and structural stability. In this work, linear SBMA polymers with different molecular weights were synthesized. Two transitions, opaque-transparent phase transition and sol-gel transition, were measured from polySBMA-water mixtures. PolySBMA physical hydrogels
10.1021/jp710683w CCC: $40.75 © 2008 American Chemical Society Published on Web 04/08/2008
5328 J. Phys. Chem. B, Vol. 112, No. 17, 2008 were obtained at high polySBMA concentrations. Chemical cross-linked hydrogels were polymerized, and their swelling properties were studied at different ionic strengths and crosslinker concentrations. The ductile physical networks were introduced into the chemical hydrogels to improve their mechanical properties. 2. Experimental Section 2.1. Materials. N-(3-Sulfopropyl)-N-(methacryloxyethyl)N,N-dimethylammonium betaine (SBMA, 97%), N,N′-methylenebisacryamide (MBAA), sodium metabisulfite, ammonium persulfate, and potassium persulfate were purchased from Sigma-Aldrich (Milwaukee, WI). Phosphate buffer saline (PBS, 138 mM NaCl, 2.7 mM KCl, pH 7.4, 0.15 M) was purchased from Sigma Chemical Co. Water used in experiments was purified using a Millipore water purification system with a minimum resistivity of 18.0 MΩ.cm. 2.2. Preparation of Linear PolySBMA. SBMA (0.5 M) was polymerized in 0.5, 1, and 2 M KCl solutions. The polymerization was initiated using 5 mM potassium persulfate. The reaction was stirred under nitrogen for 5 h or more at 60 °C. Then dialysis with a cellulose membrane (Sigma-Aldrich, retain MW >12 000) and ca. 1500 mL of water was performed for 1 day to remove small molecules and ions. The precipitates after dialysis were collected and dried for further measurements. 2.3. Gel Permeation Chromatography (GPC). The molecular weight of linear polySBMA was estimated using a Waters Alliance 2695 Separations Module equipped with a Waters Ultrahydrogel 1000 column and detected with a Waters 2414 Reflex Detector. The mobile phase was a buffered 0.05 M solution of tris(hydroxymethyl)aminomethane in 1.0 M NaCl aqueous solution at a flow rate of 0.5 mL/min.15 The instrument and column were calibrated with poly(ethylene oxide) standards from Polymer Laboratories. All measurements were performed at 35 °C. The molecular weight of the polymer and its distribution were calculated using Empower Pro from Waters. 2.4. Characterization of Linear PolySBMA Solutions and Physical Hydrogels. The phase transition temperatures of the precipitated linear polymers were measured on a HewlettPackard 8480A diode array UV-Visible spectrophotometer. The turbidity of the gels was monitored as a function of temperature at 500 nm and under a heating rate of 0.5 °C/min. Rheological measurements were performed on a Physica MCR300 rheometer, using a parallel geometry with a diameter of 25 mm and a gap of 0.05 mm. The samples with linear polySBMA and water were kept in an oven at 60 °C for 1 day and then kept at room temperature for more than 1 day before the measurements. 2.5. Preparation of Chemical Hydrogels and ChemicalPhysical DNs. To prepare chemical hydrogels for swelling tests, 2.5 M SBMA was dissolved in an aqueous solution of 16, 40, 81, 122, or 162 mM MBAA and initiated with ammonium persulfate (2.6 mol %) and sodium metasulfite (1.2 mol %) for polymerization. The reaction was carried out between a pair of glass substrates, separated with a polytetrafluoroethylene (PTFE) spacer with a thickness of 0.4 mm at 37 °C for 12 h. After polymerization, the gel was immersed in a large amount of water for one week, and water was changed every day to remove residual chemicals. SBMA chemical hydrogels were punched (8 mm biopsy punch, Acuderm Inc., FL) into disks with a diameter of 8 mm and stored in water before use. For the compressional test samples, an aqueous solution with monomers, cross-linkers, and initiators was added to a disposable 1 mL syringe (Becton, Dickinson and Company) with an
Zhang et al. inner diameter of 5 mm, and the inlet was then sealed. After polymerization, one end of the syringe was cut and the gel in the cylindrical form was carefully pushed out. The cylindrical gels were cut into pieces of 5 mm height. To prepare chemical hydrogel samples for mechanical tests, 2.5 M SBMA was dissolved in an aqueous solution of 81 mM MBAA. The reaction was initiated with 0.04 mM potassium persulfate at 60 °C overnight. To prepare physical-chemical DNs, the linear polySBMA of desired concentration in 2 M NaCl solution was added instead of water. 2.6. Swelling Properties of Hydrogels. Swollen hydrogel disks were equilibrated in deionized water or a solution for more than one week and weighed. Dried hydrogel disks were weighed after the disks were put in a vacuum desiccator at room temperature for 3 days. The volume fraction of polymer within a hydrogel Φ in a particular solvent is given by:
Φ ) (D0/D)3 where D0 and D are the diameters of dried and swollen disks, respectively. D0 and D were measured with a caliber. The swelling ratios (100 wt %) were determined by the ratio of the swollen gel weight to the dry gel weight. Because of the weight loss of the low-cross-linked hydrogels during soaking, the swelling ratios were only measured for the samples with a MBAA concentration higher than 40 mM. 2.7. Compressional Properties. Before the compressional tests, the samples were kept in water for more than 5 days. The compression test was performed using an Instron 4550 tester with a cross-head speed of 1 mm/min. The fracture stress σm was determined by the failure points of compressive stressstrain measurements, i.e., the peak of the stress-strain curve. The elastic modulus was determined by the average slope in a range of 0 and 0.1 of strain ratios from the stress-strain curve. Five samples were repeated for each composition, and a typical stress-strain curve was obtained from the sample with the median compressional stress of the repeated samples. 3. Results and Discussion 3.1. Synthesis of Linear PolySBMA. Linear polySBMA was polymerized at 60 °C in an aqueous solution of 0.5, 1, or 2 M KCl. After the polymerization, the solutions were poured into dialysis membranes to remove salts and other soluble molecules with molecular weights less than 12 kDa. During the dialysis, the polymers precipitated from the solution. For similar polymerization in pure water, linear polySBMA was precipitated in ethanol after the polymerization. Zwitterionic polymers such as polySBMA present a unique anti-polyelectrolyte behavior. The polymers dissolve well in the presence of salt ions but cannot dissolve well in pure water. 5,7,16 The salt ions screen the charges of polySBMA and lead to chain expansion (salting-in). When the salt ions are removed from polySBMA through dialysis, intermolecular and intramolecular association causes the polySBMA chains to collapse and then precipitate from the solution. Physically cross-linked hyrogels can be prepared from the precipitates, which keep their shape unless external force is applied. The properties of polySBMA physical gels are discussed in the following section. In order to prepare linear SBMA polymers with different molecular weights, KCl of different concentrations was added to the reaction solutions to control their polymerization rate. In this work, SBMA polymers with a molecular weight of 316 kDa, 203 kDa, 169 kDa, and 20.9 kDa were obtained with different KCl concentrations (Table 1). Their molecular weights
Double Network of Zwitterionic Hydrogels
J. Phys. Chem. B, Vol. 112, No. 17, 2008 5329
TABLE 1: Molecular Weight and Polydispersity of Linear PolySBMA Polymerized under Different KCl Concentrationsa monomer concn (M) b
0.5 0.5c 0.5c 0.5c
KCl concn (M)
Mn (kDa)
polydispersity
0 0.5 1 2
316 203 169 20.9
1.48 1.36 1.45 1.37
a The reaction was initiated using 5 mM potassium persulfate and stirred under nitrogen at 60 °C for more than 5 h. b The samples were precipitated by adding ethanol after polymerization. c The samples were dialyzed with a cellulose membrane (retaining MW >12 000).
were measured using GPC in a buffered solution with 1.0 M NaCl. As shown in Table 1, the molecular weight of polySBMA decreases with the concentration of KCl. When the concentration of KCl was increased from 0 to 2 M, the molecular weight of polySBMA decreased from 316 kDa to 20.9 kDa. It was reported previously that the addition of some alkaline metal salts such as KCl could increase the initial polymerization rate of SBMA in aqueous solution, and the innitial polymerization rate also increased with the concentration of KCl.17 The acceleration of initial polymerization can consume monomers in a shorter time, resulting in the rapid increse of viscosity, the induction of more chain transfer reactions, and the decrease of their molecular weights. 3.2. Phase Transition and Sol-Gel Transition of PolySBMA Physical Hydrogels. The white precipitates after dialysis from KCl solution can reversibly change from opaque to transparent with increasing temperature. The transition temperature increased with polySBMA concentration and molecular weight. The optical transmittance of the precipitates was measured using a UV-visible spectrophotometer with programmed temperature control. For polySBMA with a molecular weight of 203K and a concentration of 61 wt %, the phase transition temperature is 32-37 °C (Figure 1a). The precipitates or physical gels at their transparent, opaque, and transitional stages were observed under an optical microscope, respectively (Figure 1b). The opaque gels were composed of aggregated round particles, most of which with a diameter of 5-20 µm. The round particles were attributed to the polySBMA-rich phase formed during phase-separation. When temperature was increased to the phase transition temperature, the polySBMA-rich particles began to dissolve (Figure 1b). At temperatures higher than the phase transition temperature, the gel turned transparent and was macroscopically homogeneous under the microscope (Figure 1b). The reversible opaquetransparent transition is believed to be a phase transition similar to the upper critical solution temperature (UCST) found in the dilute solution of polysulfobetaines. In a dilute solution and below UCST, polySBMA is considered to exist as a collapsed coil in water due to intramolecular and/or intermolecular associations.18-21 This work shows that intramolecular and intermolecular associations can be strong enough to form selfsustained physical hydrogels, which can exhibit a phase transition with sufficient water content. Different from the UCST in a dilute solution, the phase transition of polySBMA physical gels occurs within a highly cross-linked environment, and both opaque and the transparent phases keep their properties as crosslinked gels. It should be pointed out that the transparent-opaque transition can only be observed when the water concentration is sufficient to keep the phase separation. Further dehydration from an opaque gel can make polySBMA-rich particles to merge together and turn transparent again. In this case, no transparentopaque transition was detected as temperature is changed.
In addition to the phase transition, the polySBMA-water mixture also displays a sol-gel transition. Using a dynamic rheometer, the elastic modulus G′ and the viscous modulus G′′ of polySBMA solution with a concentration of 50 wt % or 38 wt % were measured under different oscillatory frequencies (Figure 2). Both solutions were transparent at the experimental temperature (25 °C). Usually, the gel can be defined when G′ is higher than G′′. For polySBMA with a concentration of 50 wt %, there was a crossover of G′ and G′′, indicating the formation of physical gel. When the concentration of polySBMA was decreased to 38 wt %, G′ is lower than G′′ for the entire frequency range. At this concentration, the association of polySBMA was not strong enough to keep polymer chains from flowing. Thus, the mixture still behaved as a solution. Based on the results from UV-visible spectrometry and dynamic rheometry, a schematic phase diagram was drawn for water-polySBMA mixtures (Figure 3). Within a certain concentration range, two transition curves, corresponding to phase transition and sol-gel transition, are shown on this diagram as a function of polySBMA concentration and temperature. 3.3. Properties of Single-Network Chemical Hydrogels. Transparent SBMA chemical hydrogels were prepared via free radical polymerization using MBAA as a cross-linker. In our previous work, tetraethylene glycol dimethacrylate (TEGDMA) was used as a cross-linker for zwitterionic hydrogels.10 Since TEGDMA does not dissolve well in water, a mixed solvent was used to prepare transparent gels. It was found that the change in cross-linker concentration could lead to phase separation. In this work, MBAA was chosen as a cross-linker because of its high solubility in water. All the single-network hydrogels prepared using MBAA in this work are transparent, indicating that these hydrogels are homogenously cross-linked. The swelling properties of polySBMA hydrogel in water, 150 mM PBS, and 2 M NaCl as a function of cross-linker concentration were studied. The polymer volume fraction and the swelling ratio were measured (Figure 4). PolySBMA hydrogels swelled in the presence of salt ions and the swelling ratio increased with ionic strength, which is attributed to the anti-electrolyte behavior characteristic of zwitterionic hydrogels7,22 and balanced polyampholytic hydrogels.23,24 On the basis of Flory’s theory of swollen network, hydrogels usually swell less with cross-linking density, which means that the polymer volume fraction usually increase with cross-linker concentration.25,26 However, for SBMA chemical hydrogels, when the MBAA concentration is very low (
