Aminated Thermoresponsive Microgels Prepared from the Hofmann

Article pubs.acs.org/Langmuir

Aminated Thermoresponsive Microgels Prepared from the Hofmann Rearrangement of Amides without Side Reactions Zuohe Wang and Robert Pelton* Department of Chemical Engineering, McMaster University, 1280 Main Street, West Hamilton, Ontario, Canada, L8S 4L7 S Supporting Information *

ABSTRACT: Thermoresponsive microgels bearing primary amine groups were prepared by the Hofmann rearrangement of methacrylamide groups present in cross-linked NIPMAM (N-isopropylmethacrylamide) microgels. Most thermoresponsive microgels are based on NIPAM (N-isopropylacrylamide). By substituting NIPMAM for NIPAM, and methacrylamide for acrylamide, side reactions and the generation of carboxyl groups were prevented during the Hofmann reaction. The Hofmann rearrangement is sufficiently slow under our conditions (1 h for a 51% conversion) to permit fine control of the primary amine contents in the microgels. When starting with PNIPMAM microgels containing both methacrylamide and acrylic acid residues, we prepared a series of amphoteric microgels spanning a range of amine contents, all from a common parent microgel. Therefore, every microgel in the series had the same microstructure, cross-link density, and molecular weight.

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INTRODUCTION Since the first report in 1986, hundreds of publications have described modifications and applications of thermoresponsive microgels based on poly(N-isopropylacrylamide) (PNIPAM) and related polymers. Microgels bearing primary amine groups are particularly desirable as bioconjugation platforms. Kawaguchi, one of the microgel pioneers, was the first to describe protein conjugation to aminated PNIPAM microgels. 1 Kawaguchi’s aminated microgels were prepared by the Hofmann rearrangement of poly(NIPAM-co-acrylamide) microgels cross-linked with methylenebis(acrylamide) (MBA). The Hofmann rearrangement converted the acrylamide residues to the corresponding ethylamine moieties with dilute bleach (NaClO) at high pH. This is an attractive approach because acrylamide has favorable copolymerization characteristics with NIPAM (N-isopropylacrylamide) and is easily incorporated into microgels. However, the Hofmann rearrangement of acrylamide residues also generates carboxyl groups, giving amphoteric particles. For example, Kawaguchi showed, by measuring electrophoretic mobility as a function of pH, that carboxyl groups formed in the Hofmann rearrangement of polystyrene latex with surface acrylamide groups.2 The amphoteric nature of the particle surfaces was shown by the presence of an isoelectric point and high negative mobility at pH 10. In a more recent example, aminated PNIPAM-coacrylamide microgels prepared by the Hofmann rearrangement had an isoelectric point of 8.5 and a large negative zeta potential at pH 10 (see Figure 1 in ref3). This again indicated that the Hofmann conditions produced a significant content of carboxylated side products. Finally, that PNIPAM homopolymer reacts with bleach4 and MBA hydrolyzes5 under Hofmann © 2014 American Chemical Society

conditions suggest that the Hofmann rearrangement of PNIPAM-co-acrylamide microgels is not a robust procedure. Herein we demonstrate the preparation of thermoresponsive microgels bearing primary amines based on the Hofmann rearrangement giving no detectable cross-linker hydrolysis or carboxyl group formation. Our approach (see Scheme 1) includes three important variations of Kawaguchi’s original approach: (1) we used N-isopropylmethacrylamide (NIPMAM)6 instead of NIPAM, (2) we used methacrylamide instead of acrylamide as the Hofmann target, and (3) we used ethylene glycol dimethacrylate (EGDM) instead of MBA as the cross-linking monomer. There are, of course, other approaches to generating thermoresponsive microgels bearing primary amine groups, including copolymerization of cationic monomers [2-aminoethyl methacrylate hydrochloride, 7 N-(3-aminopropyl)methacrylamide hydrochloride,8 or allylamine9], and postpolymerization grafting of polyvinylamine. Perhaps the biggest advantage of the approach described herein is the ability to prepare a series of microgels with varying amine contents while the molecular weight and cross-link density are kept constant.

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EXPERIMENTAL SECTION

Materials. All chemicals were purchased from Sigma-Aldrich and most were used without further purification. N-Isopropylacrylamide (NIPAM) (97%) and N-isopropylmethacrylamide (NIPMAM) (97%) were purified by recrystallization with a mixture of toluene and hexane (60:40) prior to use. Fluorescein isothiocyanate (FITC, ≥90%) was Received: April 14, 2014 Revised: May 26, 2014 Published: May 26, 2014 6763

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Scheme 1. Hofmann Rearrangement of AMI-0 Microgela

a

Hofmann reaction conditions for polyacrylamide.10−13 In our procedure, to a beaker containing 10 mL of water kept in an ice bath was added 0.286 mL of NaClO (1.4 M) followed by 1 mL of NaOH (1 M), which was added dropwise with vigorous stirring. Finally, 113 mg of dried microgel was dispersed in 10 mL of water, and the dispersion was added to the beaker. The beaker was then warmed to 25 °C and mixed with a magnetic stirrer. The reaction was quenched by the addition of 60 mg of sodium thiosulfate (98%) and the reaction product was purified by serum replacement and then lyophilized for storage. For PNIPAM-AM, the lyophilized microgels (100 mg) were dispersed in 4 mL of water and the suspension was stirred at 4 °C. The reaction was started with the addition of 0.02 mL of cooled NaClO (1.4 M) and 0.6 mL of cooled NaOH (1 M). After 1 h the mixture was quenched and the product was purified by serum replacement for mobility measurements. Fluorescence Labeling. The presence of primary amines in AMI microgels after the Hofmann rearrangement was confirmed by FITC labeling. AMI-0.4 microgels with an amine content of 0.4 mmol/g were added to 10 mL of borate buffer (pH 9) to give a 1 mg/mL suspension, 0.1 mL of FITC solution in DMSO (1 mg/mL) was added, and the mixture was mixed for 12 h at 4 °C. The reaction was quenched by adding 0.26 mL of NH4Cl (2 M), and the products were purified by serum replacement. Microgel AMI-0 was also treated with FITC as control. Dynamic Light Scattering (DLS). Microgels were dispersed in 1 mM NaCl, and the pH was adjusted with 1 mM HCl and NaOH. Hydrodynamic diameters of the microgels were measured by DLS with a detection angle of 90° using a BI-9000AT autocorrelator (Brookhaven Instrument Corp.). The laser source was a Melles Griot HeNe laser with a wavelength of 633 nm. The scattering intensity was between 100 and 250 kcps for all measurements. Each sample was measured for three runs with 2 min for each run. The results were analyzed with the cumulants method using Brookhaven software 9kdlsw32, version 3.34. Electrophoretic Mobility. Electrophoretic mobility was measured with a ZetaPlus analyzer (Brookhaven Instruments Corp.), operating in phase analysis light scattering (PALS) mode. Microgels were dissolved in 1 mM NaCl, and the microgel concentration was 1 g/L for all measurements. Each measurement consisted of 10 runs (15 cycles each). Conductometric Titration. Microgel amine contents were measured by conductometric titration using a Burivar-I2 Buret Module (ManTech Associates) with PC-Titrate software (version 2.0.0.79). Lyophilized microgels (25 mg) in 50 mL of NaCl (1 mM) were added to a thermostated titration cell fitted with a pH electrode, a conductivity electrode, and a temperature probe. The titration cell was purged with nitrogen for 30 min to remove dissolved CO2, and the initial pH was then adjusted to 3 before starting the titration. NaOH (0.1 M, LabChem Inc.) was titrated into the cell with 300 s between additions. Nuclear Magnetic Resonance (NMR). The content of amide groups in the microgels before the Hofmann rearrangement was estimated from 1H NMR. Microgels, after being freeze-dried, were dissolved in deuterated methanol to obtain a suspension with a concentration of about 20 g/L. The spectrum was acquired in a Bruker AV200 NMR spectrometer (200 MHz). UV−Vis Absorbance. FITC-labeled microgels and control samples were dispersed in phosphate-buffered saline at pH 7.4. Spectra were recorded at 25 °C with a Beckman Coulter DU800.

Some methacrylamide residues are converted to amines.

used directly as received. A Barnstead Nanopure Diamond Type I water system was used in all experiments. The concentration of sodium hypochlorite (bleach, NaClO, 10−15%) was determined by iodometric titration prior to use.5 Microgel Synthesis. PNIPMAM microgel polymerization recipes are shown in Table 1. To a 250 mL three-neck flask reactor equipped with a mechanical stirrer, a nitrogen inlet/outlet, and a condenser were added 1.56 g of NIPMAM, 0.13 g of ethylene glycol dimethacrylate (EGDM, 98%), 0.3 g of methacrylamide (MAM, 98%), 0.1 g of sodium dodecyl sulfate (SDS, 99%), and 150 mL of water. The mixture was deoxygenated with nitrogen for 30 min at 70 °C, followed by the addition of 0.025 g of ammonium persulfate (APS, 98%) dissolved in 5 mL of water. The preparations were clean with little evidence of coagulum. After 6 h of reaction, the microgels were purified by serum replacement. Specifically, the microgels were centrifuged (Beckman L-80 XP ultracentrifuge), decanted, and suspended in water for several cycles until the conductivity of the supernatant was less than 5 μS/cm. Microgels were then lyophilized for storage. PNIPAM-AM microgels were prepared using Kawaguchi’s recipe,1 with minor modifications. To a 250 mL three-neck flask reactor equipped with a mechanical stirrer, a nitrogen inlet/outlet, and a condenser were added 1.35 g of NIPAM, 0.15 g of N,N′methylenebis(acrylamide) (MBA, 99.5%), 0.15 g of acrylamide (AM, 99%), 0.049 g of sodium dodecyl sulfate (SDS, 99%), and 150 mL of water. The mixture was deoxygenated with nitrogen for 30 min at 70 °C, followed by the addition of 0.04 g of APS dissolved in 5 mL of water. After 6 h of reaction, the microgels were purified by serum replacement. Hofmann Rearrangement. The Hofmann rearrangement was used to convert methacrylamide moieties in the microgels to the corresponding primary amines. The literature has many examples of

Table 1. PNIPMAM Microgel Recipes designation

NIPMAM (g)

MAM (g)

AA (g)

EGDM (g)

SDS (g)

APS (g)

water (mL)

CON-0 AMI-0 AMP-0

1.56 1.56 1.56

0 0.3 0.3

0 0 0.03

0.13 0.13 0.13

0.1 0.1 0.1

0.025 0.025 0.025

150 150 150

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RESULTS AND DISCUSSION Poly(N-isopropylmethacrylamide) (PNIPMAM) is a methylated analogue of PNIPAM with a lower critical solution temperature of 38−44 °C.14 Pichot’s group were the first to prepare microgels based on NIPMAM.15 We prepared three NIPMAM microgels cross-linked with EGDM; see Table 1. Microgel CON-0 was made with only NIPMAM monomer and cross-linker, and AMI-0 also included methacrylamide monomer, the Hofmann active component. Finally, AMP-0 included both methacrylamide and acrylic acid monomers, yielding amphoteric microgels after the Hofmann rearrangement. The proton NMR spectrum for AMI-0 is given in the Supporting Information (Figure SI 1). Finally, for comparison purposes, we prepared PNIPAM-AM, a version of the Kawaguchi’s original microgel. In summary, AMI-0 was based on N-isopropylmethacrylamide, methacrylamide Hofmann target, and EGDM cross-linker, whereas PNIPAM-AM was based on N-isopropylacrylamide, acrylamide, and methylenebis(acrylamide). Three Hofmann rearrangement reactions (see Scheme 1) of AMI-0 microgels were conducted with a mixture of NaClO and NaOH at 25 °C for 10, 30, and 60 min. The amine contents of the microgels were measured by conductometric titrations. The titration curves are shown in Figure SI 2 of the Supporting Information. Figure 1 shows that the amine content was nearly

Figure 2. Comparing traditional PNIPAM-co-AM microgels to PNIPMAM-co-MAM microgels after Hofmann rearrangement treatments.

about 8.5, whereas AMI-0.4 was positively charged to pH 10. The starting microgel properties are also shown in Figure 2. The low, negative, pH-insensitive mobility values reflect the presence of sulfate end groups from the persulfate initiator. The absence of significant carboxylation of the AMI series is more apparent from the properties of microgels from lower reaction times and thus lower extents of Hofmann rearrangement. Figure 3 shows both the electrophoresis and swelling behaviors of the AMI series as functions of pH. AMI-0.4 and AMI-0.2 microgels were positively charged at pH 10, whereas the mobility of AMI-0.1 was zero at pH. None of the microgels showed a minimum in the diameter versus pH plots typical of

Figure 1. Amine contents in AMI microgels after Hofmann rearrangement determined by conductometric titration. The error bars were calculated from duplicate titrations.

a linear function of the hydrolysis time. The maximum amine content (1 h reaction time) was 0.4 mmol/g (AMI-0.4 microgels), which corresponds to 51% conversion of methacrylamide moieties in the starting microgels (AMI-0). The distribution of amine groups within the microgel particles has not been determined. The distribution should be influenced by both the distribution of acrylamide groups and by possible topochemical effects. We believe that methacrylamide gives random polymers with PNIPMAM, whereas little is known about the EGDM cross-linker distribution. The initial and the product microgels were swollen under the Hofmann reaction conditions, suggesting minimal topochemical effects. Electrophoretic mobility values, plotted as a function of pH, can distinguish between microgels bearing only amine charge groups, versus amphoteric microgels with both amine and carboxyl groups. Figure 2 compares AMI-0.4 with the Hofmann product PNIPAM-AM-Hof. Like the publications mentioned previously,2,3 PNIPAM-AM-Hof had an isoelectric point of

Figure 3. Effect of pH on (A) electrophoretic mobility and (B) hydrodynamic diameter of AMI microgels before and after Hofmann rearrangement. The measurements were conducted in 1 mM NaCl at 25 °C. The labels on the curves denote microgel amine contents and the error bars were calculated from triplicate measurements. 6765

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essentially the same molecular weight and cross-link density but varying contents of amine groups. Each amphoteric microgel had a minimum diameter (i.e., minimum swelling) at the pH values corresponding to zero mobility, the isoelectric point. As expected, the isoelectric point shifted to higher pH with increasing amine content. It would be difficult to prepare a series of microgels such as those shown in Figure 4 by copolymerization of cationic monomers. As the monomer feed composition varies in the polymerization, particle size, cross-linking, and microstructure would all change in directions dictated by the copolymerization kinetics. Our post-polymerization Hofmann rearrangement eliminates this complication. Beyond speculation, we have no explanation for the remarkable stability gain by replacing main chain protons with methyl groups (i.e., NIPMAM versus NIPAM and methacrylamide versus acrylamide). We speculate that abstraction of the hydrogen radical, α to the carbonyl, is involved in carboxyl group formation under Hofmann rearrangement conditions.

amphoteric microgels. In summary, both the swelling (diameter) and mobility versus pH behaviors in Figure 3 are consistent with the absence of carboxyl groups. The Supporting Information (Figure SI 4) confirms that aminated microgels could be conjugated with FITC, an amine reactive fluorscent dye. Also mobility and diameter values as functions of temperature confirm that the AMI series of microgels is thermoresponsive; see Figure SI 5 of the Supporting Information. The absence of carboxyl groups in the AMI series suggests that the NIPMAM or the EGDM moieties are stable under Hofmann conditions. Further support for our claimed lack of side reactions was obtained by exposing microgel CON-0 to NaClO at high pH for 2 h. Microgel diameter and electrophoretic mobility were completely unchanged over the pH range; see Figure SI 3 in the Supporting Information. Finally, to illustrate the flexibility of our approach, AMP-0 microgels (copolymers of NIPMAM, methacrylamide, and acrylic acid) were reacted for various times with NaClO at high pH to give a series of amphoteric microgels. Figure 4 shows the

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CONCLUSIONS Thermoresponsive microgels bearing primary amine groups were prepared by the Hofmann rearrangement of methacrylamide groups present in cross-linked NIPMAM (N-isopropylmethacrylamide) microgels. Most thermoresponsive microgels are based on NIPAM. By substituting NIPMAM for NIPAM, and methacrylamide for acrylamide, side reactions and the generation of carboxyl groups were prevented during the Hofmann reaction. The Hofmann rearrangement is sufficiently slow under our conditions (1 h for a 51% conversion) to permit fine control of the primary amine contents in the microgels. When starting with PNIPMAM microgels containing both methacrylamide and acrylic acid residues, we prepared a series of amphoteric microgels spanning a range of amine contents, all from a common parent microgel. Therefore, every microgel in the series had the same microstructure, cross-link density, and molecular weight.

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra for AMI-0, titration curves for the AMI series, diameters and electrophoretic mobility values for CON-0 before and after reaction, and UV−vis spectra and swelling and mobility values for the AMI series as functions of temperature. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 4. Properties of amphoteric microgels prepared by the Hofmann rearrangement of PNIPMAM-co-MAM-co-AA microgels. A homologous microgel series was obtained by varying the Hofmann rearrangement reaction time for AMP-0 microgel (see Table 1).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. mobility and diameters of four microgels derived from AMP-0 as functions of pH. As before, the extent of amide to amine conversion was controlled by the exposure time to NaClO. The resulting microgel compositions are expressed as the ratio of total concentration of titratable nitrogen ([NH2]T) to the total carboxyl concentration ([COOH]T = 0.32 mmol/g for AMP0). The ratios are based upon conductometric titrations assuming the Hofmann rearrangement did not introduce any new carboxyls. The results in Figure 4 show four microgels with

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge BASF Canada and NSERC for research funding. R.P. holds a Tier 1 Canada Research Chair in Interfacial Technologies. We thank Miles Montgomery, Wing Yan Lam, and Kyle Lefebvre for experimental help. 6766

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