New ampholytic microgels based on N-isopropylacrylamide and α-amino acid: changes in swelling behavior as a function of temperature, pH and divalent cation concentration

Abstract

Several new microgels based on N-isopropylacrylamide and the amino acid L-ornithine were synthesized by means of surfactant free emulsion polymerization. N-δ-Acryloyl ornithine was copolymerized with N-isopropylacrylamide and N,N′-methylenebisacrylamide, leading to the synthesis of a new ampholytic microgel. The swelling behavior of the obtained microgels with respect to the amount of amino acid incorporated in the polymer network, temperature, concentration of ions and pH was investigated. The pH dependence of the microgel size, measured at a constant temperature, was found to exhibit a minimum; the pH range where the minimum appeared corresponded well with the pH range where zwitterions dominated. The temperature dependence of the swelling process obtained for different pH values showed that for the pH region where zwitterions dominated, the polymer networks collapsed more efficiently. The presence of free α-amino acid groups attached to the polymeric network of the microgels also enabled the complexation of some metal cations. The influence of the presence of two metal ions (Cu²⁺ and Ca²⁺), which differ significantly in their ability to form complexes with the α-amino-acid, on the swelling behavior was investigated. It was found that the presence of copper ions that form stable complexes with the α-amino acid strongly influenced the swelling behavior of the investigated microgels.

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1. Introduction

Microgels are chemically cross-linked polymers, with a network structure, that are colloidal in size and swollen in a suitable solvent.^1–3 With their low viscosities, very high surface areas and a rapid and large-magnitude volume phase transition in response to such external stimuli as a change in temperature, pH and ionic strength, microgels have great potential in controlled and regulated applications and therefore have great potential in such areas as controlled drug delivery,^4–6 catalysis⁷ and sensors.^8,9

The environmentally sensitive gels that are most often investigated are thermo sensitive gels based on N-isopropylacrylamide (NIPA). Cross-linked poly(N-isopropylacrylamide) (pNIPA) hydrogels are known to exhibit a drastic volume phase transition at temperatures above 32 °C; this critical temperature increases with increasing quantity of crosslinking agent.¹⁰ At temperatures below 32 °C these gels are swollen, whereas above this temperature they dehydrate to the collapsed state. When groups able to change their charge in response to a change in pH (weak acid, base, or their salts) are introduced to the polymeric chains, the pNIPA microgel acquires new properties, such as sensitivity to a change in pH, enhancement of the size change during the volume phase transition, increase in the temperature at which the phenomenon starts, and the possibility of switching from discontinuous to continuous volume phase transition.

Microgels based on N-isopropylacrylamide are traditionally copolymerized with pH sensitive carboxylic acid comonomers like itaconic acid, vinylacetic acid or most commonly acrylic acid (AA), and with cationic comonomers like 4-vinylpyridine and 2-(aminoethyl)-methacrylatehydrochloride.¹¹ Previous studies on temperature and pH sensitive microgels have also investigated polymers prepared from monomers that were either acidic or basic.¹² For instance, Nayak and Lyon showed that microgels synthesized by copolymerization of NIPA, AA and N-(3-aminopropyl)methacrylamide exhibited zwitterionic behavior.¹³ Ogawa et al. used AA and 1-vinylimidazole as anionic and cationic monomers, respectively; both monomers were incorporated into the network of pNIPA cross-linked with N,N′-methylenebisacrylamide (BIS).¹⁴ Tan et al.^15,16 studied pH-responsive polyampholyte microgels consisting of poly(methacrylic acid) and poly-(2-(diethylamino)ethyl methacrylate) (PMAA–PDEA). These microgel were more hydrophilic at low and high pH values and became compact between pH 4.0 and 6.0, near the isoelectric point.

The introduction of amino acid moieties may result in new microgel properties such as ampholytic behavior, catalytic activity, sorption properties, sensitivity to pH and ionic strength. The ability to complex metal ions makes polymeric microgels very attractive as metal adsorbents. Amino acid moieties also may serve as a means to facilitate further chemical reactions to incorporate molecules for improved targeting.

We recently reported the synthesis of new polymeric gels with free α-amino-acid groups based on modified L-ornithine and L-lysine, and optionally N-isopropylacrylamide.^17–20 The presence of α-amino acid in those polymers made it possible to control the charge sign and the excess of charge in the polymeric network by changing pH. The synthesized gels showed an interesting swelling behavior in response to changes in temperature, pH and concentration of divalent metal ions.

In the present study, we modified the synthesis of the gels based on N-isopropylacrylamide and N-δ-acryloyl ornithine to obtain polymeric microparticles not exceeding 1 μm in size. This investigation focused on how the swelling behavior of the synthesized microgels is influenced by the amount of α-amino acid groups incorporated into the polymer network, temperature, pH and the presence of divalent ions with differing ability to complex with the polymer network.

2. Experimental

2.1. Chemicals

N-Isopropylacrylamide (NIPA, 97%), N,N′-methylenebisacrylamide (BIS, 99%), potassium persulfate (KPS, 99.99%) and N,N,N′,N′-tetramethylethylenediamine (TEMED, 99.5%) were purchased from Aldrich. Sodium hydroxide (NaOH, 99%), hydrochloric acid (HCl, 35–38%), copper(II) sulfate pentahydrate (CuSO₄·5H₂O, 98%) and calcium(II) sulfate (CaSO₄·2H₂O, 99%) were purchased from POCh. N-δ-Acryloyl ornithine was synthesized according to the procedure described previously.²⁰

All chemicals were used as provided by manufacturer except for NIPA, which was recrystallized twice from benzene–hexane mixture (9 : 1). All solutions were prepared using high purity water obtained from a Hydrolab/HLP purification system (water conductivity: 0.056 μS cm⁻¹).

2.2. Synthesis of poly(N-isopropylacrylamide-co-N-δ-acryloyl ornithine) NIPA-AcOrn microgel

Microgels were obtained by surfactant free emulsion polymerization.²¹ Various amounts of NIPA monomer, BIS and AcOrn were dissolved in 95 mL of deionized water in a three-neck flask equipped with a magnetic stirrer (set at 500 rpm during the entire polymerization), reflux condenser, inlet and outlet of inert gas. The total concentration of NIPA, amino acid derivative and BIS was kept constant at 150 mM. The microgels were synthesized with the mole fraction of the amino acid derivative equal to Y_solution in the pre-gel solution, defined as:

In this study, Y_solution = 0, 2, 8, 20 and 25%. The mole fraction of BIS was kept constant at 1%. However, for 25% NIPA-AcOrn a lack of well-formed particles according to SEM and TEM, and no reliable measurements by DLS were observed.

The monomer solution was purged with argon for 0.5 h to remove any dissolved oxygen and heated up in an oil bath thermostated at 70 °C. Then KPS (0.1 g dissolved in 5 mL of deionized and degassed water) and 50 μL of TEMED were added to initiate the polymerization. The reaction continued for 8 h under an argon blanket, after which the solution was cooled down to room temperature. The schemes of the synthesis and the structure of the polymeric network are shown in Fig. 1. The microgels so obtained were purified by placing them into a dialysis tube with a 10 000 Da molecular weight cutoff (Spectra/Por® 7 Dialysis Membrane), thus removing the oligomers and unreacted substrates. The microgels were dialyzed against 5 L of water for three weeks at room temperature, with the water changed daily. The final conductivity of the water used was close to the initial value (conductivity measurements were carried out using a Radiometer, model CDM230 conductometer). Before further proceeding the microgel solution was filtered via a syringe inline glass filter with pore size 1–2 μm.

Fig. 1 Scheme of synthesis and structure of microgel based on N-isopropylacrylamide-co-δ-acrylic ornithine polymer crosslinked with N,N′-methylenebisacrylamide, and ¹H NMR spectrum of microgel.

2.3. Determining the amount of N-δ-acryloyl ornithine incorporated into the polymeric network of NIPA-AcOrn microgels

The amount of amino acid incorporated into the polymeric network of the gels was estimated from ¹H NMR spectra, obtained at 500 MHz using a Varian Unity Plus spectrometer. The gel samples for the NMR experiments were prepared by swelling the dry microgels in D₂O in NMR tubes. The ¹H NMR spectra were recorded one day after the swelling. A typical 20% NIPA-AcOrn microgel spectrum is shown in Fig. 1. All signals in the spectrum are assigned to the appropriate protons, except protons G from the cross-linker. The signal from protons G is expected at 4.2–4.5 ppm,¹⁸ the absence of a signal in this region is caused by the low level of mol fraction of the linker (1%). Signal C (representing the α-protons of ornithine) and signal B (representing the protons from the isopropyl group) were used for estimating the amino acid loading content into the network:
where 〈B〉 and 〈C〉 denote the areas of the corresponding signals. Fortunately, the error is very small, as the mol fraction of the linker was kept at a low level. The results so obtained are shown in Table 1 and compared with the molar fraction of acryloyl ornithine used in the polymerization.

Table 1 Content of amino acid in pre-gel solutions and of amino acid incorporated into the polymeric network of microgels (estimated from ¹H NMR spectra)^a

	2% NIPA-AcOrn	8% NIPA-AcOrn	20% NIPA-AcOrn
a Value below the detection limit.
Ysolution	2.0%	8.0%	20.0%
Ygel	—	4.8%	13.0%

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2.4. Instrumental examinations

Dynamic light scattering. The hydrodynamic diameter of the dilute aqueous microgel particles dispersions was determined using a Malvern Zetasizer instrument (Nano ZS, UK) fitted with a 4 mV He–Ne laser (λ = 632.8 nm) as the light source at the scattering angle of 173°. The solutions of microgel were cleaned through a 1–2 μm glass-fiber membrane filter just before measurement. The solutions were equilibrated at selected temperatures for 10 min before measurement.

Measurement of pH and ion impact on swelling behavior of NIPA-AcOrn microgels. Dialysis-purified NIPA-AcOrn microgel was centrifuged (1400 rpm, 30 min, 20 °C, Eppendorf Centrifuge 5430 R with a FA-45-30-11 rotor) and the solution (supernatant) was removed. The NIPA-AcOrn gel was then soaked in excess an aqueous solution of differing pH or ion concentration (Cu²⁺, Ca²⁺). The pH was changed by adding HCl or NaOH, and was monitored with a pH/ion meter (Mettler Toledo, model SevenGo-SG2). The final pH value was measured just before measurements were taken. In all experiments, the ionic strength was kept at a constant level (0.01 M) by adding NaCl. This eliminated the effect of ionic strength on the swelling equilibrium. Additionally, in all experiments where copper(II) and calcium(II) were used, pH was kept close to 6.0 to be sure that the observed effects were not caused by changing pH. Changes in the hydrodynamic diameter of NIPA-AcOrn microgel were determined from the DLS data.

Transmission electron microscopy (TEM). The samples for TEM were prepared by placing a drop of aqueous NIPA-AcOrn microgel on a formvar-coated copper grid and allowing them to dry in air. All samples were examined using a Libra 120 microscope (Zeiss).

Scanning electron microscopy (SEM). The SEM images were taken with a Merlin (Zeiss) microscope at 3 kV. The samples were first dried completely in a hot-air oven at 50 °C and next covered with a thin layer of sputtered Au–Pd alloy to a depth of approximately 3 nm using a Polaron SC7620 Mini Sputter Coater.

3. Results and discussion

Selected SEM micrographs of microgels containing various amounts of AcOrn are shown in Fig. 2a. As can be seen, all the microgels formed spherical particles. However, the higher the content of amino acid in the polymeric network (8 and 20%), the more the spherical shape of the particles was deformed. The micrographs obtained with a transmission electron microscope (see Fig. 2b) confirmed that an increase in the amount of AcOrn in the microgels led to a greater deformation in the spherical shape of the particles, probably due to increasing self-adhesion of microgels with increasing amino acid content. During the drying process the microgel particles became strongly deformed. Moreover, analysis of the SEM and TEM micrograms leads to a conclusion that microgel size decreases with increasing AcOrn content.

Fig. 2 (A) SEM and (B) TEM images of microgels with various amount of AcOrn.

The influence of temperature on the swelling behavior of the gels containing different amounts of amino acid is shown in Fig. 3. The temperature dependencies of the equilibrium swelling ratio for the NIPA-AcOrn microgels were constructed using the hydrodynamic diameters of the microgels. The hydrodynamic diameters were determined using the dynamic light scattering method (DLS). Assuming that the particles exhibit random Brownian motion, their diffusion coefficient could be obtained from the decay of the autocorrelation function. Then the hydrodynamic diameter (D_h) of the microgel particles can be calculated using the Stokes–Einstein equation^21,22

where D_h – hydrodynamic diameter, k – Boltzmann constant, T – temperature, η – solvent viscosity and D – diffusion coefficient.

Fig. 3 Change in hydrodynamic diameter of microgels with various amount of amino acid as a function of temperature.

Based on the swelling behavior of the microgels, two general trends were observed. An increase in the content of amino acid leads to: (a) a decrease in microgel size (D_h values at 25 °C were 1086, 1010, 895 and 605 nm for NIPA, 2% NIPA-AcOrn, 8% NIPA-AcOrn and 20% NIPA-AcOrn, respectively), this is in good agreement with the data obtained by SEM and TEM, and (b) an increase in the volume phase transition temperature (VPTT). pNIPA microgel was shown to have a VPTT consistent with the existing literature, i.e. around 32 °C,²³ whereas for 20% NIPA-AcOrn microgel the VPTT was ca. 35 °C.

For further investigation, we selected 20% NIPA-AcOrn microgels, which demonstrate the greatest sensitivity to a change in pH. Fig. 4B shows the size of 20% NIPA-AcOrn microgel as a function of pH. The measurements were taken at 25 °C, a temperature much lower than the phase transition temperature. The hydrodynamic diameter decreased rapidly in the pH region from 1.5 to 3. Then, in the region 3 < pH < 8, the dependence was fairly stable. The microgel size again started to grow considerably at pH 8. The changes seen in Fig. 4B may be related to the acid–base equilibrium of the amino acids built into the polymer chains. Three amino acid species exist in the microgels: I (cation, protonated amino group), II (neutral form, dissociated carboxylic groups and protonated amino group, zwitterions), and III (anion, dissociated carboxylic group). The molar fractions (X_I, X_II, and X_III) of each form of the amino acid can be calculated using the following equations:

The values of K_a1 and K_a2 needed for the calculations were taken as those for free aliphatic amino acid (leucine): pK_a1 = 2.36 and pK_a2 = 9.60.²⁴ The plots shown in Fig. 4B exhibit a minimum, characteristic for ampholytic polymer networks. The pH range over which the minimum in Fig. 4B is spread corresponds well to the pH distribution of the X_II form shown in Fig. 4A. In this range, almost all amino acid groups are in the form of zwitterions. Here, the van der Waals and hydrophobic interactions contribute significantly to the collapse of the ampholytic polymer networks. Additionally, in this region there exist both electrostatic repulsions between the groups similarly charged and Coulombic attractions between the positive and negative charges, which may lead to collapse as well. At low and high pH, on the other hand, the behavior of the ampholytic networks is determined by the ionized forms (X_I or X_III), which create an osmotic pressure in the network and therefore prompt the swelling process. Also, the charges on the ionized groups generate electrostatic repulsive forces between the polymer chains, which leads to further swelling of the network.

Fig. 4 Calculated distribution of amino acid forms as a function of pH, pK_a1 = 2.36 and pK_a2 = 9.60 (A). pH dependence of hydrodynamic diameter for 20% NIPA-AcOrn microgel measured at 25 °C (B).

To further characterize the swelling behavior of the microgels at a given excess charge, the temperature dependence of the swelling ratio was examined at eight selected values of pH and is represented in a three-dimensional diagram in Fig. 5. As can be seen, temperature and pH strongly influence the microgel size and the swelling behavior. In the pH region where zwitterions dominate, the microgel size is the smallest both before and after the phase transition. The microgel achieves a completely collapsed state at temperatures higher than 35 °C. At the pH extremes, the microgels exhibited a larger diameter before and after phase transition and the discontinuous volume phase transition can be interpreted in terms of an incomplete collapse. For temperatures higher than 35 °C, in the pH region where zwitterions dominate, the surface is nearly flat and the gel achieves a completely collapsed state. At the pH extremes, the surface is distinctly curved.

Fig. 5 Three-dimensional diagrams of hydrodynamic diameter as functions of pH and temperature for 20% NIPA-AcOrn microgels.

Next, we investigated the influence of the presence of metal ions on the swelling behavior of 20% NIPA-AcOrn microgels. For this purpose two kinds of ions were selected: calcium(II) and copper(II). The temperature dependencies of the swelling ratio were examined for several selected values of metal-ion concentration and are presented as three-dimensional diagrams in Fig. 6. In the case of calcium ions (Fig. 6A), at constant ionic strength, the temperature of phase transition does not change significantly over a wide range of calcium ion concentration. Only sensitivity to changes in temperature is exhibited. Microgels achieved completely shrunken state at temperatures higher than 35 °C and at any pCa value.

Fig. 6 Three-dimensional diagrams of hydrodynamic diameters as functions of concentration of calcium (A) and copper ions (B) and temperature for 20% NIPA-AcOrn microgels.

In the case of copper ions (Fig. 6B) the microgels exhibited the sensitivity to both temperature and ion concentration. A decrease in the swelling ratio with an increase in the concentration of Cu²⁺ was observed. For smaller concentrations of copper ions the 20% NIPA-AcOrn microgel underwent the volume phase transition at about 35 °C, while for higher Cu²⁺ concentrations we observed the transition at lower temperature, at approx. 33 °C. The temperature induced change in the swelling ratio was not very significant when pCu was smaller than 3. A more significant change in the swelling ratio was observed for pCu higher than 4. A drastic drop in the swelling ratio was observed when pCu was changed from 5 to 3 at temperature below volume phase transition.

The observed behavior of the microgels could be explained in terms of the formation of complexes between metal ions and amino acid groups attached to the polymeric chains of the microgel networks. It is known that amino acids can form stable complexes with some metal cations. Usually, two complexes of stoichiometry 1 : 1 and 1 : 2 and can be formed.^25–28

The presence of such complexes in the gel may influence its swelling ratio. The first complex (1 : 1) should expand the polymer network by introducing an excessive positive charge to the polymeric chains, which leads to an increase in the osmotic pressure between the solution and the gel. The second (1 : 2) complex increases the overall cross-link density of the gels and leads to the shrinking of the polymer network. Copper ions form complexes of relatively high stability constants. The values of the stability constants for the complexes with glycine (unbound to the gel) are log β_ML = 8.1 and log β_ML2 = 15.3. The weak influence of the calcium ions on the swelling ratio could be explained in terms of the formation of very weak complexes with only one stoichiometry: 1 : 1 (log β_ML = 1.4). The mean values of the stability constants between the metal ions and glycine were taken from ref. 26.

4. Conclusions

New ampholytic microgels based on N-isopropylacrylamide and modified natural amino acid L-ornithine were synthesized by surfactant free emulsion polymerization. The microgels with 20% content of amino acid showed an interesting swelling behavior in response to changes in temperature, pH and concentration of metal ions. The amino acids made it possible to control the excess of either positive or negative charge on the polymeric network of that microgel. The temperature dependencies of the swelling process obtained for different pH values showed that in the pH region where zwitterions dominate, the polymer networks collapse more efficiently. Metal ions that have the ability to form complexes with α-amino acid caused the microgels to shrink.

The metal ion sorption ability of the presented microgels and the temperature dependence of their swelling process make the synthesized microgels interesting materials in terms of the temperature triggered swinging of their heavy-metal binding strength. Additionally, the presence of free α-amino acid groups attached to the polymer network offers the possibility of further modification of the microgel, i.e. through binding to these α-amino acid groups. The bound compounds could be easily released from the microgel by the appropriate change in pH and temperature; the swelling state, the hydrophilicity/hydrophobicity balance and the stability of the peptide/ester bonds will be affected. Thus microgels with free amino acid groups are also interesting from the standpoint of drug delivery systems.

Acknowledgements

This work was supported by Iuventus Grant no. IP2012 015272 from the Polish Ministry of Science and Higher Education.

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