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New ampholytic

Faculty of Chemistry, University of Warsaw

E-mail: karbarz@chem.uw.edu.pl; Fax: +48-

Cite this: RSC Adv., 2014, 4, 48905

Received 19th August 2014Accepted 18th September 2014

DOI: 10.1039/c4ra08924c

www.rsc.org/advances

This journal is © The Royal Society of C

microgels based onN-isopropylacrylamide and a-amino acid: changesin swelling behavior as a function of temperature,pH and divalent cation concentration

Marcin Mackiewicz, Jan Romanski and Marcin Karbarz*

Several newmicrogels based onN-isopropylacrylamide and the amino acid L-ornithine were synthesized by

means of surfactant free emulsion polymerization. N-d-Acryloyl ornithine was copolymerized with

N-isopropylacrylamide and N,N0-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 a-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

a-amino-acid, on the swelling behavior was investigated. It was found that the presence of copper ions

that form stable complexes with the a-amino acid strongly influenced the swelling behavior of the

investigated microgels.

1. Introduction

Microgels are chemically cross-linked polymers, with a networkstructure, that are colloidal in size and swollen in a suitablesolvent.1–3With their low viscosities, very high surface areas and arapid and large-magnitude volume phase transition in responseto such external stimuli as a change in temperature, pH andionic strength, microgels have great potential in controlled andregulated applications and therefore have great potential in suchareas as controlled drug delivery,4–6 catalysis7 and sensors.8,9

The environmentally sensitive gels that are most oeninvestigated are thermo sensitive gels based on N-isopro-pylacrylamide (NIPA). Cross-linked poly(N-isopropylacrylamide)(pNIPA) hydrogels are known to exhibit a drastic volume phasetransition at temperatures above 32 �C; this critical temperatureincreases with increasing quantity of crosslinking agent.10 Attemperatures below 32 �C these gels are swollen, whereas abovethis temperature they dehydrate to the collapsed state. Whengroups able to change their charge in response to a change inpH (weak acid, base, or their salts) are introduced to the poly-meric chains, the pNIPAmicrogel acquires new properties, such

, Pasteura 1, PL 02-093 Warsaw, Poland.

22-822-02; Tel: +48-22-822-02

hemistry 2014

as sensitivity to a change in pH, enhancement of the size changeduring the volume phase transition, increase in the tempera-ture at which the phenomenon starts, and the possibility ofswitching from discontinuous to continuous volume phasetransition.

Microgels based on N-isopropylacrylamide are traditionallycopolymerized with pH sensitive carboxylic acid comonomerslike itaconic acid, vinylacetic acid or most commonly acrylicacid (AA), and with cationic comonomers like 4-vinylpyridineand 2-(aminoethyl)-methacrylatehydrochloride.11 Previousstudies on temperature and pH sensitive microgels have alsoinvestigated polymers prepared from monomers that wereeither acidic or basic.12 For instance, Nayak and Lyon showedthat microgels synthesized by copolymerization of NIPA, AA andN-(3-aminopropyl)methacrylamide exhibited zwitterionicbehavior.13 Ogawa et al. used AA and 1-vinylimidazole as anionicand cationic monomers, respectively; both monomerswere incorporated into the network of pNIPA cross-linked withN,N0-methylenebisacrylamide (BIS).14 Tan et al.15,16 studiedpH-responsive polyampholyte microgels consisting of poly-(methacrylic acid) and poly-(2-(diethylamino)ethyl methacrylate)(PMAA–PDEA). These microgel were more hydrophilic at low andhigh pH values and became compact between pH 4.0 and 6.0, nearthe isoelectric point.

RSC Adv., 2014, 4, 48905–48911 | 48905

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The introduction of amino acid moieties may result in newmicrogel properties such as ampholytic behavior, catalyticactivity, sorption properties, sensitivity to pH and ionicstrength. The ability to complex metal ions makes polymericmicrogels very attractive as metal adsorbents. Amino acidmoieties also may serve as a means to facilitate further chemicalreactions to incorporate molecules for improved targeting.

We recently reported the synthesis of new polymeric gelswith free a-amino-acid groups based on modied L-ornithineand L-lysine, and optionally N-isopropylacrylamide.17–20 Thepresence of a-amino acid in those polymers made it possible tocontrol the charge sign and the excess of charge in the poly-meric network by changing pH. The synthesized gels showed aninteresting swelling behavior in response to changes intemperature, pH and concentration of divalent metal ions.

In the present study, we modied the synthesis of the gelsbased on N-isopropylacrylamide and N-d-acryloyl ornithine toobtain polymeric microparticles not exceeding 1 mm in size.This investigation focused on how the swelling behavior of thesynthesized microgels is inuenced by the amount of a-aminoacid groups incorporated into the polymer network, tempera-ture, pH and the presence of divalent ions with differing abilityto complex with the polymer network.

2. Experimental2.1. Chemicals

N-Isopropylacrylamide (NIPA, 97%), N,N0-methylenebisacrylamide(BIS, 99%), potassium persulfate (KPS, 99.99%) and N,N,N0,N0-tetramethylethylenediamine (TEMED, 99.5%) were purchasedfrom 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 purchasedfrom POCh. N-d-Acryloyl ornithine was synthesized accordingto the procedure described previously.20

All chemicals were used as provided by manufacturer exceptfor NIPA, which was recrystallized twice from benzene–hexanemixture (9 : 1). All solutions were prepared using high puritywater obtained from a Hydrolab/HLP purication system (waterconductivity: 0.056 mS cm�1).

2.2. Synthesis of poly(N-isopropylacrylamide-co-N-d-acryloylornithine) NIPA-AcOrn microgel

Microgels were obtained by surfactant free emulsion polymer-ization.21 Various amounts of NIPA monomer, BIS and AcOrnwere dissolved in 95 mL of deionized water in a three-neck askequipped with a magnetic stirrer (set at 500 rpm during theentire polymerization), reux condenser, inlet and outlet ofinert gas. The total concentration of NIPA, amino acid derivativeand BIS was kept constant at 150 mM. The microgels weresynthesized with the mole fraction of the amino acid derivativeequal to Ysolution in the pre-gel solution, dened as:

Ysolution ¼ nAcOrn

nNIPA þ nAcOrn þ nBIS100% (1)

48906 | RSC Adv., 2014, 4, 48905–48911

In this study, Ysolution ¼ 0, 2, 8, 20 and 25%. The mole frac-tion of BIS was kept constant at 1%. However, for 25% NIPA-AcOrn a lack of well-formed particles according to SEM andTEM, and no reliable measurements by DLS were observed.

The monomer solution was purged with argon for 0.5 h toremove any dissolved oxygen and heated up in an oil baththermostated at 70 �C. Then KPS (0.1 g dissolved in 5 mL ofdeionized and degassed water) and 50 mL of TEMED were addedto initiate the polymerization. The reaction continued for 8 hunder an argon blanket, aer which the solution was cooleddown to room temperature. The schemes of the synthesis andthe structure of the polymeric network are shown in Fig. 1. Themicrogels so obtained were puried by placing them into adialysis tube with a 10 000 Da molecular weight cutoff (Spectra/Por® 7 Dialysis Membrane), thus removing the oligomers andunreacted substrates. Themicrogels were dialyzed against 5 L ofwater for three weeks at room temperature, with the waterchanged daily. The nal conductivity of the water used was closeto the initial value (conductivity measurements were carried outusing a Radiometer, model CDM230 conductometer). Beforefurther proceeding the microgel solution was ltered via asyringe inline glass lter with pore size 1–2 mm.

2.3. Determining the amount of N-d-acryloyl ornithineincorporated into the polymeric network of NIPA-AcOrnmicrogels

The amount of amino acid incorporated into the polymericnetwork 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 byswelling the dry microgels in D2O in NMR tubes. The 1H NMRspectra were recorded one day aer the swelling. A typical 20%NIPA-AcOrn microgel spectrum is shown in Fig. 1. All signals inthe spectrum are assigned to the appropriate protons, exceptprotons G from the cross-linker. The signal from protons G isexpected at 4.2–4.5 ppm,18 the absence of a signal in this regionis caused by the low level of mol fraction of the linker (1%).Signal C (representing the a-protons of ornithine) and signal B(representing the protons from the isopropyl group) were usedfor estimating the amino acid loading content into the network:

Ygel ¼ hC ihC iþhBi 100%

where hBi and hCi denote the areas of the correspondingsignals. Fortunately, the error is very small, as the mol fractionof the linker was kept at a low level. The results so obtained areshown in Table 1 and compared with the molar fraction ofacryloyl ornithine used in the polymerization.

2.4. Instrumental examinations

Dynamic light scattering. The hydrodynamic diameter of thedilute aqueous microgel particles dispersions was determinedusing a Malvern Zetasizer instrument (Nano ZS, UK) tted witha 4 mV He–Ne laser (l ¼ 632.8 nm) as the light source at thescattering angle of 173�. The solutions of microgel were cleanedthrough a 1–2 mm glass-ber membrane lter just before

This journal is © The Royal Society of Chemistry 2014

Fig. 1 Scheme of synthesis and structure of microgel based on N-isopropylacrylamide-co-d-acrylic ornithine polymer crosslinked with N,N0-methylenebisacrylamide, and 1H NMR spectrum of microgel.

Table 1 Content of amino acid in pre-gel solutions and of amino acidincorporated into the polymeric network of microgels (estimated from1H NMR spectra)a

2%NIPA-AcOrn

8%NIPA-AcOrn

20%NIPA-AcOrn

Ysolution 2.0% 8.0% 20.0%Ygel — 4.8% 13.0%

a Value below the detection limit.

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measurement. The solutions were equilibrated at selectedtemperatures for 10 min before measurement.

Measurement of pH and ion impact on swelling behavior ofNIPA-AcOrn microgels. Dialysis-puried NIPA-AcOrn microgelwas centrifuged (1400 rpm, 30 min, 20 �C, Eppendorf Centri-fuge 5430 R with a FA-45-30-11 rotor) and the solution (super-natant) was removed. The NIPA-AcOrn gel was then soaked inexcess an aqueous solution of differing pH or ion concentration(Cu2+, Ca2+). The pH was changed by adding HCl or NaOH, andwas monitored with a pH/ion meter (Mettler Toledo, modelSevenGo-SG2). The nal pH value was measured just beforemeasurements were taken. In all experiments, the ionicstrength was kept at a constant level (0.01 M) by adding NaCl.This eliminated the effect of ionic strength on the swellingequilibrium. Additionally, in all experiments where copper(II)and calcium(II) were used, pH was kept close to 6.0 to be surethat the observed effects were not caused by changing pH.Changes in the hydrodynamic diameter of NIPA-AcOrnmicrogelwere determined from the DLS data.

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

Scanning electron microscopy (SEM). The SEM images weretaken with a Merlin (Zeiss) microscope at 3 kV. The sampleswere rst dried completely in a hot-air oven at 50 �C and nextcovered with a thin layer of sputtered Au–Pd alloy to a depth of

This journal is © The Royal Society of Chemistry 2014

approximately 3 nm using a Polaron SC7620 Mini SputterCoater.

3. Results and discussion

Selected SEM micrographs of microgels containing variousamounts of AcOrn are shown in Fig. 2a. As can be seen, all themicrogels formed spherical particles. However, the higher thecontent of amino acid in the polymeric network (8 and 20%),the more the spherical shape of the particles was deformed. Themicrographs obtained with a transmission electron microscope(see Fig. 2b) conrmed that an increase in the amount of AcOrnin the microgels led to a greater deformation in the sphericalshape of the particles, probably due to increasing self-adhesionof microgels with increasing amino acid content. During thedrying process the microgel particles became stronglydeformed. Moreover, analysis of the SEM and TEMmicrogramsleads to a conclusion that microgel size decreases withincreasing AcOrn content.

The inuence of temperature on the swelling behavior of thegels containing different amounts of amino acid is shown inFig. 3. The temperature dependencies of the equilibriumswelling ratio for the NIPA-AcOrn microgels were constructedusing the hydrodynamic diameters of the microgels. Thehydrodynamic diameters were determined using the dynamiclight scattering method (DLS). Assuming that the particlesexhibit random Brownian motion, their diffusion coefficientcould be obtained from the decay of the autocorrelation func-tion. Then the hydrodynamic diameter (Dh) of the microgelparticles can be calculated using the Stokes–Einsteinequation21,22

Dh ¼ kT

3phD(2)

where Dh – hydrodynamic diameter, k – Boltzmann constant,T – temperature, h – solvent viscosity and D – diffusioncoefficient.

Based on the swelling behavior of the microgels, two generaltrends were observed. An increase in the content of amino acid

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

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

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leads to: (a) a decrease in microgel size (Dh values at 25 �C were1086, 1010, 895 and 605 nm for NIPA, 2% NIPA-AcOrn, 8%NIPA-AcOrn and 20% NIPA-AcOrn, respectively), this is in goodagreement with the data obtained by SEM and TEM, and (b) anincrease in the volume phase transition temperature (VPTT).pNIPA microgel was shown to have a VPTT consistent with theexisting 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-AcOrnmicrogels, which demonstrate the greatest sensitivity to achange in pH. Fig. 4B shows the size of 20% NIPA-AcOrnmicrogel as a function of pH. The measurements were takenat 25 �C, a temperature much lower than the phase transitiontemperature. The hydrodynamic diameter decreased rapidly inthe pH region from 1.5 to 3. Then, in the region 3 < pH < 8, thedependence was fairly stable. The microgel size again started togrow considerably at pH 8. The changes seen in Fig. 4B may be

48908 | RSC Adv., 2014, 4, 48905–48911

related to the acid–base equilibrium of the amino acids builtinto the polymer chains. Three amino acid species exist in themicrogels: I (cation, protonated amino group), II (neutral form,dissociated carboxylic groups and protonated amino group,zwitterions), and III (anion, dissociated carboxylic group). Themolar fractions (XI, XII, and XIII) of each form of the amino acidcan be calculated using the following equations:

1

XI

¼ 1þ Ka1�Hþ�þ

Ka1Ka2�Hþ�2 ;

1

XII

¼�Hþ�

Ka1

þ 1þ Ka2�Hþ�;

1

XIII

¼�Hþ�2

Ka1Ka2

þ�Hþ�

Ka2

þ 1

The values of Ka1 and Ka2 needed for the calculations weretaken as those for free aliphatic amino acid (leucine): pKa1¼ 2.36and pKa2 ¼ 9.60.24 The plots shown in Fig. 4B exhibit aminimum, characteristic for ampholytic polymer networks.The pH range over which the minimum in Fig. 4B is spreadcorresponds well to the pH distribution of the XII formshown in Fig. 4A. In this range, almost all amino acid groupsare in the form of zwitterions. Here, the van der Waals andhydrophobic interactions contribute signicantly to thecollapse of the ampholytic polymer networks. Additionally, inthis region there exist both electrostatic repulsions betweenthe groups similarly charged and Coulombic attractionsbetween the positive and negative charges, which may lead tocollapse as well. At low and high pH, on the other hand, thebehavior of the ampholytic networks is determined by theionized forms (XI or XIII), which create an osmotic pressurein the network and therefore prompt the swelling process.Also, the charges on the ionized groups generate electrostaticrepulsive forces between the polymer chains, which leads tofurther swelling of the network.

To further characterize the swelling behavior of the micro-gels at a given excess charge, the temperature dependence of theswelling ratio was examined at eight selected values of pH and isrepresented in a three-dimensional diagram in Fig. 5. As can beseen, temperature and pH strongly inuence the microgel sizeand the swelling behavior. In the pH region where zwitterionsdominate, the microgel size is the smallest both before and

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Fig. 4 Calculated distribution of amino acid forms as a function of pH,pKa1 ¼ 2.36 and pKa2 ¼ 9.60 (A). pH dependence of hydrodynamicdiameter for 20% NIPA-AcOrn microgel measured at 25 �C (B).

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aer the phase transition. The microgel achieves a completelycollapsed state at temperatures higher than 35 �C. At the pHextremes, the microgels exhibited a larger diameter before andaer phase transition and the discontinuous volume phasetransition can be interpreted in terms of an incomplete

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

This journal is © The Royal Society of Chemistry 2014

collapse. For temperatures higher than 35 �C, in the pH regionwhere zwitterions dominate, the surface is nearly at and thegel achieves a completely collapsed state. At the pH extremes,the surface is distinctly curved.

Next, we investigated the inuence of the presence ofmetal ions on the swelling behavior of 20% NIPA-AcOrnmicrogels. For this purpose two kinds of ions were selected:calcium(II) and copper(II). The temperature dependencies ofthe swelling ratio were examined for several selected valuesof 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 phasetransition does not change signicantly over a wide range ofcalcium ion concentration. Only sensitivity to changes intemperature is exhibited. Microgels achieved completelyshrunken state at temperatures higher than 35 �C and at anypCa value.

In the case of copper ions (Fig. 6B) the microgels exhibitedthe sensitivity to both temperature and ion concentration. Adecrease in the swelling ratio with an increase in the concen-tration of Cu2+ was observed. For smaller concentrations ofcopper ions the 20% NIPA-AcOrn microgel underwent thevolume 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 theswelling ratio was not very signicant when pCu was smallerthan 3. A more signicant change in the swelling ratio wasobserved for pCu higher than 4. A drastic drop in the swellingratio was observed when pCu was changed from 5 to 3 attemperature below volume phase transition.

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

The presence of such complexes in the gel may inuence itsswelling ratio. The rst complex (1 : 1) should expand thepolymer network by introducing an excessive positive charge tothe polymeric chains, which leads to an increase in the osmoticpressure between the solution and the gel. The second (1 : 2)complex increases the overall cross-link density of the gels andleads to the shrinking of the polymer network. Copper ionsform complexes of relatively high stability constants. The valuesof the stability constants for the complexes with glycine(unbound to the gel) are log bML ¼ 8.1 and log bML2

¼ 15.3. Theweak inuence of the calcium ions on the swelling ratio couldbe explained in terms of the formation of very weak complexeswith only one stoichiometry: 1 : 1 (log bML ¼ 1.4). The meanvalues of the stability constants between the metal ions andglycine were taken from ref. 26.

4. Conclusions

New ampholytic microgels based on N-isopropylacrylamide andmodied natural amino acid L-ornithine were synthesized bysurfactant free emulsion polymerization. The microgels with

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

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20% content of amino acid showed an interesting swellingbehavior in response to changes in temperature, pH andconcentration of metal ions. The amino acids made it possibleto control the excess of either positive or negative charge on thepolymeric network of that microgel. The temperature depen-dencies of the swelling process obtained for different pH valuesshowed that in the pH region where zwitterions dominate, thepolymer networks collapse more efficiently. Metal ions thathave the ability to form complexes with a-amino acid caused themicrogels to shrink.

The metal ion sorption ability of the presented microgelsand the temperature dependence of their swelling process makethe synthesized microgels interesting materials in terms of the

48910 | RSC Adv., 2014, 4, 48905–48911

temperature triggered swinging of their heavy-metal bindingstrength. Additionally, the presence of free a-amino acidgroups attached to the polymer network offers the possibility offurther modication of the microgel, i.e. through binding tothese a-amino acid groups. The bound compounds could beeasily released from the microgel by the appropriate change inpH and temperature; the swelling state, the hydrophilicity/hydrophobicity balance and the stability of the peptide/esterbonds will be affected. Thus microgels with free amino acidgroups are also interesting from the standpoint of drug deliverysystems.

Acknowledgements

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

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