Radiation-induced synthesis of thermo-sensitive, gradient hydrogelsbased on 2-(2-methoxyethoxy)ethyl methacrylate

Slawomir Kadlubowski a,n, Malgorzata Matusiak a, Jacek Jenczyk b,Magdalena N. Olejniczak c, Marcin Kozanecki c, Lidia Okrasa c

a Institute of Applied Radiation Chemistry, Lodz University of Technology, Wroblewskiego 15, 93-590 Lodz, Polandb NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Polandc Department of Molecular Physics, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland

H I G H L I G H T S

� Thermo-sensitive hydrogels with gradient of cross-link density.� Analysis of parameters describing network formation.� Synthesis of hydrogels with various morphologies depending on post-irradiation procedure.

a r t i c l e i n f o

Article history:Received 3 February 2014Accepted 10 March 2014Available online 19 March 2014

Keywords:Poly(2-(2-methoxyethoxy)ethylmethacrylate)Radiation polymerizationand cross-linkingGradient hydrogels

a b s t r a c t

The gradient (“transient”) systems are required for many biomedical applications, especially if thematerials with different properties are to be joined without strong internal stresses. In this paper onestep, radiation-induced synthesis of thermo-responsive hydrogels based on 2-(2-methoxyethoxy)ethylmethacrylate with gradient in crosslink density, has been presented. The influence of the monomercomposition and synthesis procedure (irradiation dose and conditioning temperature) on the generalproperties of prepared gels has been discussed. It has been found that the properties of particularsections of the gel sample do not result from the differences in chemical structure, but from significantlydifferent morphology. The explanation of the mechanism leading to gradient behavior of obtained gelshas been proposed. The chemical structure of obtained products and morphology of synthesizedhydrogels as well as general properties of products have been analyzed.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Hydrogels, three-dimensional networks of amphiphilic poly-mer chains being able to swell in water to an equilibrium state,have been systematically investigated since the 1950s (Charlesby,1953, 1954). While studies on materials based on “classical”hydrogels are still being continued, over the last 50 years theresearch interest has shifted towards stimuli-responsive systems,which are able to change their physical properties in response toexternal triggers such as temperature, pH, ionic strength, light, orvarious external fields (Park and Hoffman, 1988; Alarcon et al.2005; Roy et al. 2010; Liu and Urban, 2010; Aoshima and Kanaoka,2008). The special interest focused on this class of gels resultsmainly from their biomimetic properties, high water content and

biocompatibility so they are perfect candidates for artificial tissues(muscle and skin), membranes, dressings and other implants(Corkhill et al., 1989; Rosiak and Yoshii, 1999).

Currently, the most popular are thermo-sensitive hydrogels.They are usually synthesized with polymers exhibiting lowercritical solution temperature (LCST), for example: poly(N-isopro-pylacrylamide) (PNIPAM) (Schild, 1992; Liu et al., 2009), poly(2-(N,N-dimethylamino)ethyl methacrylate) (PDMAEMA) (Nagase et al.,2008; Yuk et al., 1997), poly(2-oxazolines) (Park and Kataoka,2007; Hoogenboom et al., 2008; Uyama and Kobayashi, 1992;Zou et al., 2008) and poly(oligo-(ethylene oxide) methacrylate)(POEOMA) (Becer et al., 2008; Yamamoto et al., 2007; Lutz, 2008;Lutz et al., 2006; Paris and Quijada-Garrido, 2009). Contrary tolinear polymers, which precipitate from the solution under LCST,in the cross-linked systems (because of the limited number offreedom degrees) the network collapse, called volume phasetransition (VPT), occurs (Hirokawa and Tanaka, 1984). The VPTresults in abrupt deswelling process and water is pulled out.

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Radiation Physics and Chemistry

http://dx.doi.org/10.1016/j.radphyschem.2014.03.0140969-806X/& 2014 Elsevier Ltd. All rights reserved.

n Corresponding author. Tel.: þ48 426313164; fax: þ48 426840043.E-mail address: slawekka@mitr.p.lodz.pl (S. Kadlubowski).

Radiation Physics and Chemistry 100 (2014) 23–31

Thermo-responsive gels have been extensively studied due to theirpotential applications as drug delivery systems (Soppimath et al.,2002; Gupta et al., 2002; Qiu and Park, 2001) regenerativemedicine (Slaughter et al., 2009), biosensors (Gota et al., 2009),responsive membranes (Ehrick et al., 2005; Schepelina and Zharov,2007; Castellanos et al., 2007), molecular machines (Huck, 2008;Wang et al., 2005; Yeghiazarian et al., 2005; Kim and Beebe, 2007),nanotemplates for nanoparticles formation (Kim and Lee, 2007;Jiang et al., 2007; Schexnailder and Schmidt, 2009), catalysis (Yanet al., 2010) and photonic crystals (Kang et al., 2008).

Hydrogels, because of their excellent biocompatibility, func-tionality, mechanical properties etc., are considered as idealmaterials for 3D tissue scaffolds that mimic the extracellularmatrix. In spite of high similarity between the natural tissue andhydrogels, for many biomedical applications the “transient” (gra-dient) systems are required, especially if the materials withdifferent properties should be joined without strong internalstresses. Materials, including hydrogels, with gradient propertiesare broadly defined as systems possessing a gradual spatiotem-poral change in at least one physical or chemical property (Genzerand Bhat, 2008). From the engineering point of view, the mostinteresting are the hydrogels combining opposite mechanicalproperties (soft and hard matter) or opposite chemical affinity(hydrophilic and hydrophobic properties).

Gradient hydrogels usually exhibit a continuous spatial changein a given property. Therefore, they could replicate in vivo physicaland chemical gradients in vitro for tissue-engineered constructions(Sant et al., 2010). Gradient materials have been used to rapidlyscreen cell–biomaterial interaction (Simon et al., 2009) and tostudy cellular processes such as migration and angiogenesisin vitro (Chung et al., 2010). Gradient materials have also foundwidespread use in drug delivery (Peppas and Khare, 1993; Leeet al., 2001) and tissue engineering (Singh et al., 2008).

Various techniques from material science, microscale engineer-ing and microfluidics have been used to synthesize biomimetichydrogels. In particular, a lot of methods exist to incorporatemicrometer to centimeter scale of chemical and physical gradientsand complex biomaterials incorporating such gradients. Thoroughreviews show various methods being able to create chemical orsurface gradients (Sant et al., 2010; Keenan and Folch, 2008).

It is necessary to underline that currently the gradient hydro-gels are generally formed by a two-step approach. Concentrationgradients of prepolymer solutions are first formed (source-sinkdiffusion, tree-like gradient generator, dynamic mixing, and con-vection), and then stabilized by the appropriate cross-linkingmethod. By the choice of input solutions and cross-linking methodgradients of soluble factors, proteins, beads, and even cells withinhydrogels with constant concentrations of other species can begenerated. In addition, combining the concentration gradientprotocols with cross-linking gradient protocols can produce gra-dient hydrogels with superposed chemical and physical gradients.

Recently the POEOMA system has been highlighted as asuccessful alternative to PNIPAM due to the advantages of tune-able in broad range LCST (Lutz and Hoth, 2006; Dong andMatyjaszewski, 2010), high biocompatibility/low cytotoxicity(Tang et al., 2011), commercially available monomers (Lutz et al.,2007; Lutz et al., 2006) and facile polymerization by free radical,anionic (Ishizone et al., 2008) and atom transfer radical (Yoon etal., 2010, 2011a, 2011b) polymerization mechanisms. As an exam-ple of POEOMA system, hydrogels obtained from 2-(2-methox-yethoxy)ethyl methacrylate (MEO2MA) may be referred to.

Several works (Yoon et al., 2010, 2011a, 2011b) have beendevoted to the systematic analysis of poly(MEO2MA) hydrogels.Materials obtained with conventional free radical polymerization(FRP) have been compared to those formed via atom transferradical polymerization (ATRP). The gels prepared by ATRP

displayed higher swelling ratio than FRP gels due to a loweramount of densely cross-linked nanogel domains. It has been alsofound that the influence of temperature on deswelling rate of FRPhydrogels is mainly controlled by network homogeneity. Synthe-sized hydrogels were also subjected to post-polymerization mod-ification. Grafting of dangling chains to the bare network allowedthe preparation of hydrogels with higher deswelling rates andcontrollable transition temperatures. The degree of modificationwas regulated by the composition of the dangling chains, chainlength and grafting density.

The main goal of this paper is to present the possibility ofradiation synthesis of thermo-responsive hydrogels based onMEO2MA with gradient type architecture. According to our bestknowledge, presented herein results constitute the first attempt ofradiation-induced synthesis of hydrogels based on this monomer.Observation of structures with gradient heterogeneities has notyet been noticed in the samples that had been prepared usingradiation-induced free radical polymerization. Additionally phaseseparation has not been observed in the investigated systemreceived by other methods, such as thermally induced FRP orATRP. The applied method should be regarded as a good alter-native to the conventional (thermally-induced) FRP. The describedprocedure of hydrogel synthesis could be interesting from theapplication point of view. It offers some key advantages inpreparation of thermo-sensitive biomaterials such as: low tem-perature of all conducted processes, lack of additional chemicals(initiators, catalysts, stabilizers, and inhibitors) and product steri-lity. Importance of this work weights mostly on the simplicity ofthe proposed method. The influence of monomer composition(presence of additional chemical cross-linker or co-monomeruseful for post-polymerization grafting) and synthesis procedure(irradiation dose and conditioning temperature) on the mainparameters describing network formation, namely gelation dose,degree of swelling at the equilibrium state, degradation vs. cross-linking ratio is discussed. The chemical structure of obtained pro-ducts was analyzed with the use of Raman and Nuclear MagneticResonance (NMR) spectroscopies. Scanning Electron Microscopy(SEM) was used to determine the morphology of synthesizedhydrogels. Contact angle measurements and gravimetric analysiswere used to characterize the general properties of products.

2. Experimental

2.1. Materials and synthesis

The mixtures of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2

MA), 2-hydroxyethyl methacrylate (HEMA) and ethylene glycoldimethacrylate (EGDMA) (molar ratios: 100:2:1 or 100:1:1) havebeen prepared according to synthetic routes proposed by Yoon et al.(2011a). Chemical structures of have been presented in Scheme 1.

Irradiation of air-saturated monomer mixture was carried outat the ambient temperature in 2 ml glass ampoules using an ELU-6linear accelerator (Eksma, Russia) delivering pulses of 6 MeVelectrons at the repetition rate of 20 Hz and pulse duration 4 μs(dose per pulse 5.3 Gy as determined by an alanine dosimeter(ISO/ASTM51607-13)). The average dose rate under these condi-tions was 107 Gy s�1.

After irradiation gels were conditioned for 24 hours at threedifferent temperatures: 7, 25 or 50 1C. Then they were equilibratedat 7 1C at least for 3 weeks with TKA Micropure-filtered water(exchanged every 2 days) until constant gel weight was obtained.Schematically processes of irradiation (I), polymerization andcross-linking (II), conditioning (III) and swelling (IV) of monomermixture (A), gel (B) (gradient and homogenous, B0 and B″ respec-tively) and swollen hydrogel (C) have been presented in Scheme 2.

S. Kadlubowski et al. / Radiation Physics and Chemistry 100 (2014) 23–3124

2.2. Sol–gel analysis

After freeze-drying of swollen hydrogels the sol fractions (s)were calculated as

s¼ 1�g ð1Þwhere g is the gel fraction, i.e. the ratio of dry gel weight afterwashing out the sol content to the initial polymer weight in thesample.

2.3. Scanning electron microscopy

SEM measurements were performed on a Hitachi TM1000Scanning Microscope. Hydrogels obtained by irradiation (30 kGy)of MEO2MA–HEMA–EGDMA monomer mixture (molar ratio100:2:1), conditioned after irradiation at 25 1C for 24 hours andthen swollen in water (7 1C) to the equilibrium state were freeze-dried, cut into three sections along the Y-axis and then coveredwith Au film (about 20 nm).

2.4. Gravimetric analysis

Samples of gels were first equilibrated in demineralized waterat 7 1C. Then they were removed from the water, cut into couplecrosswise parts and weighed. Next they were dried at 17 1C(to protect the gel against VPT) under vacuum for until the

constant weight was achieved. The degree of swelling at theequilibrium state (DSeq) has been calculated as

DSeq ¼ms�md

mdð2Þ

where md and ms are the dry and swollen gel weights respectively.

2.5. Raman Spectroscopy Studies

Raman spectra were acquired with the resolution of 2 cm�1 usingFourier-transform Raman spectrometer (Bruker) was equipped withnear infrared Nd:YAG laser (1064 nm). Samples were put to a home-made cell ensured against falling out of the sample from themeasuring chamber. Measure conditions (32 scans, scanner velocityequal to 10 kHz, excitation light power 500 mW) were adjusted toobtain high quality Raman spectra and to avoid over-heating causingwater losses or VPT. Before and after each measurement, a hydrogelsample was weighed in order to determine its degree of swelling.

2.6. Nuclear Magnetic Resonance Studies

The NMR spectra were acquired on an Agilent spectrometeroperating at Larmor frequency of 400 MHz for protons. Thesamples were placed within 4 mm diameter zirconia rotor andspun at 10 kHz frequency. The first 901 pulse applied to the protonchannel was 5 μs long. The cross-polarization contact time was setto 900 μs. Two-pulse phase-modulated decoupling was utilizedduring the acquisition period. 13C chemical shifts were referencedto the 38.3 ppm carbon signal of Adamantane.

2.7. Contact angle measurements

A non-commercial, home-made goniometer was used to mea-sure the contact angle of water on the gel surface. A small piece(0.5�0.5�0.5 cm3) of dried polymer network was cut out byusing a razor blade and placed on a microscope glass slide. Allsamples were conditioned for 24 h under vacuum at 20 1C andnext kept under the environmental conditions for 2 h. A waterdroplet of 2.7 μL was placed on the surface and a picture of thedroplet was taken with a camera. Then the droplet was removedfrom the surface of the hydrogel. The procedure was repeated 20times per one sample and the arithmetic mean of contact anglewas evaluated.

Scheme 2. Schematic representation of hydrogel formation: irradiation (I), polymerization and cross-linking (II), conditioning (III) and swelling (IV) of monomer mixture (A),gel (B) (gradient and homogenous, B0 and B″ respectively) and swollen hydrogel.

O

O

O

O

O

O

OH

O

O

O

MEO2MA HEMA EGDMA

Scheme 1. Chemical structures of 2-(2-methoxyethoxy)ethyl methacrylate(MEO2MA), 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacry-late (EGDMA).

S. Kadlubowski et al. / Radiation Physics and Chemistry 100 (2014) 23–31 25

3. Results and discussion

3.1. Synthesis

Radiation-induced synthesis has long been recognized as asuitable method for synthesis of various biomaterials includinghydrogels. Easy process control, possibility of joining hydrogelformation and sterilization in one technological step, no waste,relatively low running costs etc. make the irradiation as a methodof choice in the synthesis of hydrogels for a number of differentpractical applications.

In previous works synthesis of hydrogels based on 2-(2-methoxyethoxy)ethyl methacrylate by means of atom transferradical polymerization (ATRP) and conventional free radical poly-merization (FRP) was proposed (Ishizone et al., 2008; Yoon et al.,2010, 2011a, 2011b). As an alternative method of polymerizationinitiation to these two methods mentioned above, the irradiationof MEO2MA–HEMA–EGDMA mixture by means of electron beam(EB) from the accelerator is applied in the present work. Hydrogelswith different compositions, cross-link densities (controlledwithin single composition by absorbed dose) and conditionedafter synthesis at various temperatures were prepared.

Calculation of the gelation dose, Dg (the dose necessary toproduce the first insoluble gel fraction), and degradation vs. cross-linking ratio, p0/q0, has been done using a customized computerprogram for the sol–gel analysis (free software available at: http://mitr.p.lodz.pl/biomat/gelsol.html) with the implemented generalversion of the Charlesby–Pinner formula (also known as Char-lesby–Rosiak equation) (Olejniczak et al., 1991; Rosiak, 1998)

sþ ffiffis

p ¼ p0q0

þ 2�p0q0

� �DvþDg

DvþD

� �ð3Þ

where s is the sol fraction, D is the absorbed dose, Dv is the virtualdose, p0 is degradation density (average number of main chainscissions per the monomer unit and per unit dose), q0 is cross-linking density (fraction of monomer units cross-linked per unitdose). One of the main advantages of Eq. (3) is the possibility toanalyze the cross-linking process in samples of any initial mole-cular weight distribution, the degree of crystallinity or containingmonomers.

Table 1 presents the gelation doses and degradation vs. thecross-linking ratios as found for various monomer mixtures.The determined gelation dose depends strongly on the presenceof the cross-linking agent EGDMA in the mixture of irradiatedmonomers. The gelation dose, calculated on the basis of the gelfraction in a function of the absorbed dose (exemplary curves forMEO2MA–HEMA–EGDMA mixture (molar ratio 100:2:1) presentedin Fig. 1), is about 2 times lower for mixture with addition ofEGDMA than for neat MEO2MA as presented in Table 1. It has alsobeen found that addition of HEMA, providing functionality to theobtained hydrogel network, does not influence the gelation dose.The addition of the cross-linker leads also to decreasing of thedegradation to the cross-linking densities ratio. For MEO2MA–HEMA–EGDMA mixtures of different molar ratio p0/q0¼0, whichindicates that at given conditions the chain scission is negligible,

while for MEO2MA p0/q0 value rises to 0.44, indicating a moderatecontribution of degradation processes.

An interesting phenomenon has been observed for samplesirradiated with low (up to 45 kGy, i.e. about 2 times of Dg) doses.Within a few hours after irradiation some heterogeneity (phaseseparation) appears in samples initially containing EGDMAwhile itis not being observed for neat MEO2MA or its mixture with HEMA.This post-irradiation effect is a common phenomenon found innumber of systems irradiated in viscous or solid state. Analysis ofthis effect on the structure of the hydrogels requires annealing ofthe samples as presented for example by Darwis et al. (1999). Thatis why another series of hydrogels has been prepared under thesame conditions, but after irradiation samples were conditioned inthree precisely controlled temperatures: 7, 25 or 50 1C. The phaseseparation effect has been observed again for samples kept at 7 or25 1C, while for the samples heated up to 50 1C no phase separa-tion has been found (Fig. 2). Samples irradiated with doses higherthan 45 kGy are not heterogeneous (with phase separation effect)but cracked because of internal tension increase caused by highcross-link density.

To analyze stability of obtained heterogeneity, hydrogels withphase separation effect i.e. irradiated and conditioned at 7 or 25 1Cwere additionally heated for 24 hours at 50 1C. No change inthe appearance has been detected – phase separation was stillobserved.

Phase separation has a strong effect on the swelling degree ofsynthesized hydrogels. In Fig. 3 changes in the degree of swellingat the equilibrium state, calculated on the basis of Eq. (2), in thefunction of the absorbed dose have been presented.

One can see that for doses close to the gelation point, i.e. withthe strongest phase separation effect, the degree of swelling at theequilibrium state increases with the absorbed dose. This unusualeffect can be, on the basis of swollen hydrogel shape, related withdifference in the swelling degree of two sections of the gel. Fordoses higher than 30 kGy, the typical decrease of the degree ofswelling at the equilibrium state with the increase in the dose hasbeen observed, which can be correlated with the increase innumber of cross-links per chain and also with the decrease innetwork mesh size resulting from the decrease of average mole-cular weight between two cross-links. For these samples the decayof the phase separation effect has been observed.

Another important difference has been observed for samplesconditioned at different temperatures. The degree of swelling atthe equilibrium state is lower for samples with pronounced phaseseparation rather than for homogenous gels conditioned after

Table 1The gelation dose and degradation vs. the cross-linking ratio determined fordifferent monomer compositions.

Composition Dg [kGy] p0/q0

MEO2MA 50.4 0.44MEO2MA–HEMA (100:2) 48.1 0.29MEO2MA–EGDMA (100:1) 27.6 0.0MEO2MA–HEMA–EGDMA (100:2:1) 27.4 0.0

Fig. 1. Changes in the gel fraction as a function of dose during irradiation of air-saturated MEO2MA–HEMA–EGDMA (molar ratio 100:2:1) mixture conditioned atdifferent temperatures. Empty symbols denote the gelation dose. Inset: the samedata plotted in coordinates corresponding to Eq. (3).

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irradiation at 50 1C. For homogenous samples (doses higher than45 kGy, over 50% of gel) the degree of swelling at the equilibriumstate does not depend, within the experiment error, on condition-ing temperature.

The described above phase separation observed as the post-irradiation effect in synthesized poly(MEO2MA-co-HEMA-co-EGDMA) hydrogels may be in the first attempt explained takinginto consideration possible inhomogeneity of the monomer mix-ture. This has to be, however, rejected because of similar chemicalstructures of monomers and what is more important, it is almostidentical densities: 1.02, 1.07 and 1.051 for MEO2MA, HEMA andEGDMA respectively.

As mentioned above, phase separation is observed as a post-irradiation effect. From this one can conclude that propagationand termination (e.g. cross-linking – the most important reactionfor synthesis of hydrogels) processes also occur after irradiation.

For samples containing the cross-linker (EGDMA) irradiated withdoses Z45 kGy, crosslinking is sufficient enough to create wall-to-wall uniform network, which fulfills whole ampoules. The applica-tion of lower doses (below 45 kGy) of irradiation leads to receivingproducts containing (directly after irradiation) a significant amountof sol fraction probably consisting of the clusters (micro- ornanogels) of poly(MEO2MA-co-HEMA-co-EGDMA). Due to gravityand limited miscibility (differences in polymer–polymer interac-tions), the high molecular weight clusters precipitate and form anopaque phase at the bottom of vessels. Stability of these clusters,during its further heating, indicates that at this point of the processall radicals are terminated and no further changes in properties ofthe hydrogel can be observed.

Conditioning of the monomer mixture at 50 1C directly afterirradiation accelerates the propagation process considerably, thisresults in the fast increase of viscosity. Growing clusters of poly(MEO2MA-co-HEMA-co-EGDMA) do not have enough time toprecipitate and they are built into the gel network. Consequently,products obtained in these conditions are optically homogenous.To confirm this hypothesis, systematic studies on selected sampleshave been done.

3.2. Characterization of heterogeneous gel samples

To explain unexpected properties of obtained gradient materialsmore careful investigations have been done on the poly(MEO2MA-co-HEMA-co-EGDMA) sample cross-linked with the dose of 30 kGyand conditioned after irradiation at 25 1C for 24 h. General obser-vations point strong heterogeneity along Y axis (see Fig. 4). Theupper section of the gel (regions circled with blue and green dashedlines in Fig. 4) was solid, transparent, homogenous and stuck tothe walls of the glass vessel, while the bottom section of the gel(marked with red color frame) was opaque and had inhomo-geneous structure with white grains, which revealed no adhesionto glass.

The SEM micrographs (Fig. 5) show completely differentmorphology of particular sections of the gel. The upper section ishomogenous in the micrometer scale, while in the bottom one thecauliflower-like structures dominate. In the case of the intermedi-ate region co-existence of both structures is visible, which provesthat the material exhibits gradient nature. Moreover, the bottom

Fig. 3. The degree of swelling at the equilibrium state as the function of the dosefor hydrogels obtained by irradiation of air-saturated MEO2MA–HEMA–EGDMA(molar ratio 100:2:1) mixture and then conditioned at different temperatures.Lines have been added to guide the eye.

Fig. 2. Changes in the appearance of hydrogels obtained by irradiation of air-saturated MEO2MA–HEMA–EGDMA (molar ratio 100:5:1) mixture as the functionof conditioning temperature and dose: a – 27.5 kGy, b – 30 kGy, c – 35.5 kGy,d – 37.5 kGy, e – 40 kGy, f – 42.5 kGy, g – 45 kGy, h – 47.5 kGy, i – 50 kGy, j – 70 kGy,and k – 100 kGy.

Fig. 4. Picture of hydrogel obtained by irradiation (30 kGy) of air-saturatedMEO2MA–HEMA–EGDMA (molar ratio 100:5:1) mixture conditioned after irradia-tion in 25 1C. Sections denoted as g1, g2 and g3 indicate the upper, intermediateand bottom part of the ampoules.

S. Kadlubowski et al. / Radiation Physics and Chemistry 100 (2014) 23–31 27

section is strongly heterogeneous and its morphology suggeststhat it may be rich in the more densely cross-linked material.

According to the best of our knowledge, observation of suchgradient heterogeneities has not yet been noticed in the samplesthat had been prepared using radiation-induced free radicalpolymerization. Moreover, the phase separation has not beenobserved in the investigated system received by other methods,such as thermally induced FRP or ATRP. These different featuresalong the Y-axis have led us to performing the analysis on a

macroscopic and molecular level. A cylindrical sample was dividedinto three sections: upper, intermediate and bottom marked g1,g2 and g3, respectively, as shown in Fig. 4. Each of themwas investigated in 2 or 3 separate, independent series (a, b, c)to assess the reproducibility of measurements. Swelling degrees ofvarious sections of the gel at the equilibrium state below LCSTwere gravimetrically determined.

Together with visual heterogeneity, the gravimetric studies(Fig. 6a) show inhomogeneity in swelling degrees along theY-axis. Clearly, swelling degrees of the upper section of the gelare higher than those of the bottom ones. This can be attributed tovarious mesh sizes due to various cross-linking density along theY-axis. Moreover, for upper and bottom sections of the hydrogels astandard deviation of DSeq is relatively low, while for the intermediatesection results arewidely scattered. Such behavior suggests occurrenceof the gradient structure in investigated hydrogels.

To correlate the water content at the equilibrium state with thechemical structure of various sections of the investigated sample,the Raman spectroscopy was used. Fig. 6b shows the Raman shiftsof the O–H and C–Hx stretching vibration modes in collectedspectra for various sections of fully swollen hydrogel. The broadband appearing in range of 3100–3600 cm�1 is attributed to O–Hstretching vibration of water filling the pores of the polymernetwork, whereas narrower bands (located between 2800 and3100 cm�1) may be assigned to C–Hx stretching modes of polymerchains (Maeda et al., 2007). The ratio of intensities (h) of bothmentioned Raman bands may be used as a measure of swellingdegree. Presented results in Fig. 6 have confirmed the gravimetric

Fig. 5. Exemplary SEM micrographs acquired for upper (a) intermediate (b) andbottom (c) sections of hydrogel obtained by irradiation (30 kGy) of air-saturatedMEO2MA–HEMA–EGDMA (molar ratio 100:5:1) mixture conditioned after irradia-tion in 25 1C.

Fig. 6. The degree of swelling at the equilibrium state and the ratio of intensity ofO–H and C–H bands for particular sections of the hydrogel sample (a) (color of barscorresponds to the appropriate section of the gel – see Fig. 4) and correspondingRaman spectra (b) for hydrogel obtained by EB-irradiation (30 kGy) of air-saturatedMEO2MA–HEMA–EGDMA (molar ratio 100:5:1) mixture conditioned after irradia-tion in 25 1C.

S. Kadlubowski et al. / Radiation Physics and Chemistry 100 (2014) 23–3128

measurements. Both the upper and the bottom sections of hydro-gel are found to be entirely homogeneous in terms of DSeq, incontrast with the intermediate section. The strong variation of theintensity ratio of water and polymer bands characteristic for thissection suggests that this is a transient phase between the phasesof low (upper section) and high (bottom section) cross-linkdensities.

Taking into consideration both hypotheses, that the opaquephase could differ on chemical structure and the fact that for thesamples with high DSeq C–Hx stretching bands are not well visible,the Raman spectra of the three, discussed above, sections ofthe gel after drying under vacuum have been collected (see Fig. 7).All spectra were normalized to maximum intensity to be directlycomparable. Presented results evidence that each section of the gelhas very similar composition from the chemical point of view.All, acquired for various sections, Raman spectra contain the sameset of bands. Neither band shift nor relative intensity changes havebeen observed.

Keeping in mind that the Raman spectroscopy provides infor-mation mainly from the surface and its sensitivity is relatively lowin the case of the compounds with similar chemical structure(such as MEO2MA and EGDMA) one can conclude that thevibrational techniques may be insufficiently powerful to distin-guish the differences between various sections of the gel. There-fore, the NMR spectroscopy was applied to compare the chemicalstructure of transparent and opaque phases.

The totally dried polymer networks were examined. As it isseen in Fig. 8a, both carbon spectra recorded for transparent (g1)and opaque (g3) sections of the sample (obtained by EB-irradiation(30 kGy) of air-saturated MEO2MA–HEMA–EGDMA (molar ratio100:5:1) mixture conditioned after irradiation in 25 1C) are vir-tually indistinguishable and there are only slight differencesconcerning the amplitude of some peaks. These discrepanciesmay come from unequal polarization transfer, which might bedue to a different cross-linking level of opaque (bottom) andtransparent (upper) specimen. Nevertheless, the chemical shiftsobserved for both samples are the same. This clear similarityindicates a comparable local environment of probed carbon nuclei.Therefore one can conclude that the local molecular structure ofmeasured samples is the same. However, according to the protonNMR measurements, presented in Fig. 8b, the global architectureof studied materials seems to differ from one another. There is asubstantial broadening of 1H NMR line observed from transparent (Δν0.5¼4.6 kHz) to opaque (Δν0.5¼5.75 kHz) specimen. This sig-

nificant half-width difference is presumably the consequence ofthe various cross-linking levels present in both the samples. Theopaque section reveals broader spectrum due to a stronger dipolarcouplings present in the system. These strong couplings are relatedto the increase of networking sites population. It is also worthmentioning that the 1H NMR spectrum is multi-componential.There are at least two distinct components visible, a narrow and abroad one. The narrow part of the spectrum probably comes fromthe protons residing on the free, side chains, which are moremobile. In the case of the transparent sample, the fraction of thisnarrow component is much bigger than the one observed in theopaque specimen. It appears that in the opaque section there is alarger population of immobilized side chains, which are taking partin polymer cross-linking.

The observed different adhesive behaviors of hydrogel obtainedby irradiation (30 kGy) of air-saturated MEO2MA–HEMA–EGDMA(molar ratio 100:5:1) mixture conditioned after irradiation in25 1C in contact with glass motivated us to conduct moresystematic studies on hydrophilicity/hydrophobicity of obtainedproducts. A standard analysis of the contact angle for each sectionof the gel after drying under vacuum has been performed. Themean values of twenty measurements of contact angle done foreach section of the gel and the standard deviations are summarized

Fig. 7. Raman spectra of the polymer networks received after drying of the hydrogelobtained by EB-irradiation (30 kGy) of air-saturated MEO2MA–HEMA–EGDMA (molarratio 100:5:1) mixture conditioned after irradiation in 25 1C. Color of Raman scatteringcurves corresponds to the appropriate section of the gel – see Fig. 4.

Fig. 8. Comparison of CP-MAS 13C NMR spectra (a) and static 1H NMR (b) recordedfor the polymer networks received after drying of the upper and bottom sections ofhydrogel obtained by irradiation (30 kGy) of air-saturated MEO2MA–HEMA–EGDMA (molar ratio 100:5:1) mixture conditioned after irradiation in 25 1C.

S. Kadlubowski et al. / Radiation Physics and Chemistry 100 (2014) 23–31 29

in Table 2. The average contact angle determined for g1 and g2sections are very similar, while the significant discrepancy wasfound between the g3 region and the others.

The significantly higher value of average contact angle indicatesthat the bottom section of the gel is more hydrophobic than theintermediate and upper ones. These results are fully consistentwith gravimetric and Raman analyses showing higher waterabsorptivity of the transparent section of the gel. It is largelyattributable to the creation of attractive forces between the waterand polymer groups being able to form hydrogen bonds (ether andesters oxygens in side groups).

Values of contact angles found for the opaque section indicatethat the attractive forces between water molecules are strongerthan the water–polymer interactions, which testifies the higherhydrophobicity of this gel section. Lower wettability of the opaquegel section may result from two reasons directly related to the gelstructure: (i) the rough surface of this gel section confirmed bySEM micrographs (see Fig. 5) and/or (ii) specific conformationresulting from the higher density of crosslinking of polymer chainsat the surface resulting in lower accessibility of hydrophilic centersfor water molecules. The second explanation supports well ourpostulate that micro- or nanoclusters of poly(MEO2MA-co-HEMA-co-EGDMA) are formed during irradiation and then they precipi-tate in form of cauliflower-like forms. In this case the polymer–polymer interactions may lead to coil (micelle like) structures withhydrophilic groups hidden inside the clusters.

To analyze thermo-sensitivity of synthesized gradient hydro-gels, changes in the degree of swelling at the equilibrium state inthe function of temperature have been followed (Fig. 9). Hydrogelobtained by irradiation (32.5 kGy) of air-saturated MEO2MA–HEMA–EGDMA (molar ratio 100:5:1) mixture conditioned afterirradiation in 7 1C has been swollen to the equilibrium state at 7 1Cand cut into two pieces: upper (transparent) and bottom (opaque)one. Both sections have been weighed and then thermostated indifferent temperature conditions (7–28 1C) for 24 h. Before the

change of temperature, samples were weighed again and after thelast measurement freeze-dried and weighed. The degree of swelling atthe equilibrium state has been calculated according to Eq. (2).

One can see, that below LCST of the sample, which for bothcases is around 15 1C, degree of swelling at the equilibrium state issignificantly different for selected sections of the hydrogel. It isabout four times lower for the bottom section than for the upperone. This is in good agreement with the previous observations ofchanges in cross-link density. The increase of the temperatureresults in the decrease in DSeq, nevertheless minimum value of thisparameter is similar for both sections of the sample. Even thoughthe total change in the degree of swelling between two hydrogelsections is different (because of differences in cross-link density),the character of these changes is similar.

4. Conclusion

The presented results showed that the induced by irradiationsynthesis of thermo-sensitive hydrogels based on MEO2MA:HEMA:EGDMA monomer mixture is possible. Based on the Char-lesby–Rosiak equation, the gelation dose was estimated to beequal ca. 28 kGy. Depending on the conditioning temperature,the hydrogels with various morphology may be produced. Forthe samples obtained with the use of small doses (not higher thanits double value), stable phase separation was found as a result ofthe post-effect and gravitational rearrangement of the gel. Thiseffect leads to the formation of gradient in physical properties ofobtained hydrogels, that is why radiation-induced polymerizationand cross-linking may be a good, one-step method for synthesisof gradient gels. Physical properties (hydrophilicity and wateruptake) of such gradient gels may be changed by adjusting theapplied dose of radiation and conditioning temperature. Interest-ingly enough, in spite of similar chemical structure confirmed byNMR and Raman spectroscopies, both sections of the sampleexhibit different physical properties. The upper section is soft,more hydrophilic and transparent (it exhibits higher adhesion tothe glass and higher water absorption), contrary to the bottomone. These differences result from different degrees of cross-linking of particular sections of the gel sample. The cauliflower-like structure of the bottom section of the samples is probablyconstituted as a result of precipitation of cross-linked poly(MEO2MA-co-HEMA-co-EGDMA) clusters formed during the initialstage of irradiation.

Acknowledgments

Authors would like to acknowledge valuable discussions withProf. Janusz M. Rosiak, Prof. Jacek Ulanski and Dr. Piotr Ulanski.This work was supported by the National Centre for Research andDevelopment under research Grant no. 178479 (Contract no. PBS1/A9/13/2012), Project no. N N209200738 from the Ministry ofScience and Higher Education of Poland and IAEA’s CoordinatedResearch Project F23030 (Research Contract No. 18281/R0).

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