A New Method for Crosslinking Polymers in Aqueous Solutions

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Microwave-Assisted Hydrogel Synthesis: A New Method for Crosslinking Polymers in Aqueous Solutions Joseph P. Cook, Glenn W. Goodall, Olga V. Khutoryanskaya, Vitaliy V. Khutoryanskiy*

It has been found that hydrogels may be formed by microwave irradiation of aqueous solutions containing appropriate combinations of polymers. This new method of hydrogel synthesis yields sterile hydrogels without the use of monomers, eliminating the need for the removal of unreacted species from the final product. Results for two particularly successful combinations, poly(vinyl alcohol) with either poly(acrylic acid) or poly(methylvinylether-alt-maleic anhydride), are presented. Irradiation using temperatures of 100–150 °C was found to yield hydrogels with large equilibrium swelling degrees of 500–1000 g g−1. Material leached from both types of hydrogel shows little cytotoxicity towards HT29 cells.

1. Introduction Hydrogels are crosslinked polymer networks that can absorb large amounts of aqueous fluids; they are typically soft, flexible and biocompatible. The high water content, porosity and softness of hydrogels gives them very similar properties to living biological tissue, which makes them especially well suited for applications in tissue engineering,[1,2] wound dressings[3] and drug delivery systems.[4–6] Chemically crosslinked hydrogels are most commonly synthesized by the polymerization of appropriate monomers and crosslinkers. Many common monomers and crosslinkers are harmful or toxic, particularly those used in radical (addition) polymerization, despite forming inert polymers and/or gels. It is therefore typical that unreacted Dr. J. P. Cook, Dr. G. W. Goodall, Dr. O. V. Khutoryanskaya, Dr. V. V. Khutoryanskiy Reading School of Pharmacy, School of Chemistry, Food and Pharmacy, University of Reading, Whiteknights, Reading, RG6 6AD, UK E-mail: [email protected]

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species must be removed from hydrogels before they can be used for biomedical applications. Whilst an extensive range of polymers has been synthesized using microwave irradiation,[7–10] this is usually undertaken using monomer reactants. A range of step-growth,[11–13] ring-opening[14–16] and addition[17–19] polymerization reactions using microwave irradiation have been reported. Although the main benefit of microwave-assisted polymer synthesis is reduced reaction times and the associated limitation of side reactions, it has also been shown that addition polymerization can occur without the need for an initiator.[20] The use of polymers as reactants in microwave-assisted reactions is less common than the use of monomers. Here, we demonstrate that microwave irradiation could serve as a valuable method of hydrogel synthesis by using a combination of polymeric reactants. This is the first report of the use of microwaves to form hydrogels in this way. Although ionizing radiation may be used to crosslink polymers to give sterile hydrogels,[21,22] this method requires a source of γ–rays or an electron beam, which are more expensive, more hazardous and less common than microwave reactors. Microwave irradiation is not ionizing and

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DOI: 10.1002/marc.201100742

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cannot be used to form hydrogels from a single type of polymer.

2.3. Toxicity of Leachable Material

2. Experimental Section 2.1. Materials 150 kDa poly(vinyl alcohol) (PVA, 99+% hydrolyzed), 450 kDa poly(acrylic acid) (PAA), 1.08 MDa poly(methylvinylether-altmaleic anhydride) (PMVEMA), 90 kDa 2-hydroxyethylcellulose (HEC) and 6 MDa poly(acrylamide) (PAAM) were all obtained from Sigma–Aldrich. Solutions were made by manually mixing the appropriate mass of polymer and deionized water and leaving the mixture on a tumbling machine for 24 h. In general, concentrations of 1 mol dm−3 with respect to the repeat unit were used. Owing to the high molecular weight of PAAM, this is an unrealistically high concentration and so a lower concentration of 2.5 wt% was used in this case.

2.2. Hydrogel Synthesis Aliquots of 1 cm3 of two different polymer solutions (i.e., a 1:1 molar ratio with respect to the repeat units) were mixed and added to 10 cm3 glass microwave tubes (CEM) and left on a tumbling machine for at least 24 h. The mixtures were subjected to microwave irradiation (CEM Discover LabMate) with set temperatures and hold times. Processing conditions in the range 100–200 °C and 10–60 min were used with a pressure cut-off of 200 psi and a power of 200 W. After microwave irradiation, the newly formed hydrogels were placed into 200 cm3 of deionized water and washed for at least 6 days with the water changed at least once every 2 days. The final swollen hydrogels were weighed (Ws) before being freezedried (Thermo Heto PowerDry LL3000); the dry hydrogels were then weighed (Wd) and the yield and equilibrium swelling degree (ESD) calculated from the following equations, where Wi is the calculated initial mass of the reactants in 2 cm3 wd . wi

(1)

ws − wd . wd

(2)

Yield / % = 100 ×

ES D/gg − 1 =

Additional methods of polymers and hydrogels characterization are described in Supporting Information.

The cytotoxicity of material leached from the hydrogels was tested using HT29 cells (caucasian colon adenocarcinoma cells). The protocols used in these experiments can be found in Supporting Information.

3. Results and Discussion A selection of commercially available water-soluble polymers was subjected to microwave irradiation in an attempt to produce hydrogels. Aqueous solutions of all possible combinations of PVA, PAA, PMVEMA, PAAM and HEC were subjected to microwave irradiation. No gel was formed if only one polymer was used. The most successful polymers used were PVA, PAA, PMVEMA and PAAM, and results for these are shown in Table 1. Although combinations such as PAA and PAAM resulted in low yields, higher yields may be possible by optimizing the processing conditions. No hydrogel was obtained following irradiation of PMVEMA with PAA or PAAM, nor PVA with PAAM, showing that appropriate combinations of functional groups must be present in order to produce a hydrogel by this method. A number of strategies have previously been employed to synthesize bulk hydrogels, films and fibres using PAA and PVA. Physically crosslinked bulk hydrogels are often formed by freeze-thaw of a solution of PAA/PVA.[23–26] Films are commonly formed by solvent casting, with subsequent heating to induce esterification;[27–29] films have also been crosslinked by ionizing radiation.[22] Fibres are usually formed by electrospinning,[28,30] but have also been formed by extrusion into a solution of ammonium persulphate.[31] In these cases, the esterification reaction that results in chemically crosslinked PAA/PVA generally occurs in dry or partly dehydrated mixtures. Conversely, the hydrogels reported in this study are crosslinked in aqueous solutions with an excess of water. In contrast to the perhaps more familiar condensation reaction between PVA and PAA, there are very few examples of bulk hydrogels based on PMVEMA. The only

Table 1. Examples of polymer combinations that have yielded hydrogels following microwave irradiation. Aqueous solutions (1 mol dm−3 of repeat unit for all polymers except PAAM—Section 2) of the two polymers were mixed and irradiated with microwaves at the specified temperature for 20 min.

Polymer reactants PVA and PAA

Temperature [°C]

Yield [%]

Equilibrium swelling degree in deionised water [g g−1]

150

20

939

PVA and PMVEMA

100

83

569

PAA and PAAM

135

12

1146

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Figure 1. (a) A photograph of a typical PAA/PVA hydrogel produced by microwave-assisted synthesis, irradiating at 150 °C for 20 min. (b) A typical SEM image of a freeze-dried PAA/PVA hydrogel.

examples that we are aware of involve reacting PMVEMA with poly(ethylene glycol)[32] or hydroxyethylcellulose[33] to form films, or partially hydrolyzed poly(vinyl acetate)[34] in mixed solvents to form bulk hydrogels. Images of a typical PAA/PVA hydrogel produced by microwave-assisted synthesis are shown in Figure 1. The hydrogels have flexibility, transparency and water absorption that are typical of such materials. Scanning electron microscope (SEM) images of freeze-dried samples show some evidence of a fibrous microstructure, although the freezing process could contribute to its formation. A range of microwave processing conditions can produce hydrogels when a suitable combination of polymers is used (Figure 2). The wide range of successful conditions shows that this is a simple and versatile method. In the case of PAA/PVA, using temperatures below 120 °C result in yields of lower than 5%; using temperatures above 155 °C generally produces a hard, red solid, which could be attributed to oxidation. Similar burnt products are obtained for processing times of more than 40 min at 150 °C; processing times of less than 10 min gives yields of less than 5%. For intermediate times and temperatures, the yield increases with both time and temperature. The ESD increases with both processing time and temperature, reaching a maximum at approximately 145 °C (20 min) or 15 min (150 °C) and subsequently decreases. We attribute this trend to the formation of ‘hot spots’ that could result from fluctuations in polymer concentration in viscous solutions. At lower processing times and temperatures it is possible that conditions are only suitable for the esterification to occur in small regions of the reaction mixture, and so a small mass of densely crosslinked hydrogel may be formed. As the energy input increases, due to either increases in temperature or time, the yield increases as more crosslinks are formed, and the ESD may increase because these are distributed over a larger volume. Beyond a threshold temperature and time, the maximum volume may have become crosslinked, so that the formation of any further bonds may simply increase

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Figure 2. Yield and equilibrium swelling degree for PAA/PVA hydrogels produced by microwave-assisted synthesis as a function of (a) synthesis temperature (20 min) and (b) synthesis time (150 °C).

the crosslink density, limiting the network flexibility and reducing the ESD. Although other authors have been able to use spectroscopic techniques to identify newly formed ester bonds and anhydrides,[27,32] it has not been possible to do so with hydrogels produced by microwave-assisted synthesis. FTIR and Raman spectra of PAA, PVA, a freeze-dried PAA/ PVA hydrogel and a freeze–dried PAA/PVA mixture were recorded (Figure S1, Supporting Information) and no new peaks could be identified. It is likely that ester bonds and/ or anhydrides are formed between the two polymer reactants, but in insufficiently high numbers to be identified by Raman or FTIR spectroscopy. As ester groups have characteristic peaks at similar wavelengths to carboxylic acid and alcohol groups,[35] the unreacted groups, which are expected to far outnumber the new bonds, will make it difficult to resolve peaks resulting from any new bonds. Some polymers, particularly polysaccharides,[36,37] may degrade under the conditions used in this study. Whilst this is certainly the case for PMVEMA, there is no evidence of degradation for either PAA or PVA, as evidenced by size exclusion chromatography (SEC) (Figures S2 and S3, Supporting Information). There is no substantial change in the SEC trace of PAA or PVA following microwave irradiation using 150 °C and 20 min, showing that the molecular weights

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of these polymers are not significantly altered. The weight average apparent molecular weight of PMVEMA is reduced from 1 500 to 300 kDa following microwave irradiation at 120 °C for 20 min and to 110 kDa following irradiation at 150 °C for 20 min. Given the SEC data, it is likely that the only materials that may leach from PAA/PVA hydrogels produced by microwave-assisted synthesis are unreacted PAA and PVA, and possibly some partially reacted fragments containing both polymers; such hydrogels are therefore likely to be biocompatible. The hydrogels that contain PMVEMA are likely to contain some lower molecular weight fragments that may be leachable, particularly if the temperature used is 120 °C or higher. The leached material was collected and evaluated by a gravimetric analysis and a cytotoxicity assay. For PAA/PVA irradiated at 150 °C, the mass obtained was 42.35 ± 5.00 mg (≈38%). For PMVEMA/ PVA, the mass obtained was 43.83 ± 4.00 mg (≈20%) when irradiated at 100 °C and 56.75 ± 6.00 mg (≈26%) when irradiated at 120 °C. There is a greater mass of leached material when PMVEMA is subjected to a higher temperature of 120 °C because smaller fragments are expected to be present, and these are less likely to be involved in crosslinking as well as diffusing from the gel more quickly. A range of concentrations of the material leached from the hydrogels was used to assess their cytotoxicity. Sodium dodecyl sulphate (SDS) was used as a positive control and serum-free tissue culture medium was used as a negative control. Cell viability was assessed by fluorescence after 48 h using 4′ 6–diamidino–2–phenylindole (DAPI)[38] (Figure 3). The half maximal inhibitory concentration (IC50) of SDS was estimated to be 0.044 ± 0.06 mg cm−3. At the highest available concentration of leached material, mean cell viability remains above 75% for PAA/PVA, so the IC50 of the leached material is well above 0.21 mg cm−3. The mean cell viability is lower for PMVEMA/PVA at the highest concentrations, but remains above 65% in all cases and so the IC50 of the leached material is well above 0.22 mg cm−3 for material leached from gels synthesized at 100 °C and 0.28 mg cm−3 for gels synthesised at 120 °C. When 100 μL of serum-free medium was used as a negative control, the mean cell viability was 89 ± 6%. These data suggest that the material leached from hydrogels produced by microwave–assisted synthesis has little cytotoxicity towards HT29 cells. Microwave-assisted chemical reactions have received increasing interest over the past few decades, with much effort invested into debate over the existence or otherwise of a ‘non-thermal’ or ‘microwave effect’.[39] The reaction of PAA and PVA to produce a hydrogel was also carried out in the absence of microwaves, to eliminate the possibility of any specific microwave effect.

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Figure 3. HT29 cell viability with material leached from PAA/PVA and PMVEMA/PVA hydrogels produced by microwave-assisted synthesis using the specified temperatures for 20 min.

The same mixture of PAA and PVA was placed in a 35 cm3 pressure cell (ACE) and heated to 150 °C in a sand bath on a hotplate. The process is much slower under these conditions, with a yield of only 10% obtained after 1 h; microwave-assisted synthesis gave a yield of 20% in 20 min. Microwave reactors simply offer a safe and efficient means of heating reaction mixtures and they are now common in research laboratories as they provide a facile means of performing many reactions at an accelerated rate.[39]

4. Conclusion We have reported the rapid synthesis of bulk hydrogels from polymeric reactants by microwave irradiation. The hydrogels produced are sterile, have high swelling ratios and do not require purification as no monomers are used. We anticipate that this method of synthesis could be applicable to a wide range of combinations of polymers, potentially leading to new families of hydrogels. The most successful combinations of polymers found so far have been PVA with either PMVEMA or PAA.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: This work was funded by BBSRC grant BB/ FOF/289. We acknowledge the use of the Centre for Advanced Microscopy (CfAM) and the Chemical Analysis Facility (CAF) at the University of Reading.

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Received: November 4, 2011; Revised: November 25, 2011; Published online: January 18, 2012; DOI: 10.1002/marc.201100742 Keywords: biomaterials; crosslinking; hydrogels; microwaves; synthesis

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