solvent feed ratio on POEGDMA thermoresponsive hydrogels: Radiation-induced synthesis, swelling properties and VPTT

Radiation Physics and Chemistry 158 (2019) 37–45

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The influence of monomer/solvent feed ratio on POEGDMA thermoresponsive hydrogels: Radiation-induced synthesis, swelling properties and VPTT

T

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Edin Suljovrujic , Zorana Rogic Miladinovic, Maja Micic, Denis Suljovrujic, Dejan Milicevic Vinca Institute of Nuclear Sciences, University of Belgrade, M. Petrovica Alasa 12-14, 11001 Belgrade, Serbia

A R T I C LE I N FO

A B S T R A C T

Keywords: Hydrogel Temperature responsive Oligo(ethylene glycol) dimethacrylate VPTT

The oligo(ethylene glycol) dimethacrylate (OEGDMA), one of the most commonly utilized crosslinking monomers, was used to tailor thermoresponsive POEGDMA hydrogels by varying the monomer/solvent feed ratio (from 10 to 100 wt% of monomer). For the first time, synthesis of different POEGDMA homopolymeric networks was performed by gamma radiation, and due to the expected thermoresponsiveness their temperature-dependent properties were extensively analysed. The sol-gel study was performed as the first step after radiation-induced synthesis. The swelling properties were investigated over the wide pH (2.2–9.0) and temperature (5–80 °C) range. Characterization of structure and properties was additionally conducted by SEM, DSC, FTIR, and UV–Vis spectroscopy. The results indicated that all POEGDMA hydrogels showed a high gel content, inverse thermoresponse and volume phase transition temperature (VPTT) with no respect to initial preparation. On the other hand, by altering the monomer/solvent ratio in the prepolymerisation mixture a large diversity in the microstructure, swelling capacity and VPTT was obtained.

1. Introduction Over the years, researchers have defined hydrogels in many different ways. The most common of these is that hydrogel is a waterswollen three-dimensional network of polymer chains. The ability to swell and the extent of swelling of hydrogels are mainly governed by two factors, namely the hydrophilicity of polymer chains and the crosslinking density. Two general classes of hydrogels can be defined – physical gels (pseudo-gels), where the chains are connected by electrostatic forces, hydrogen bonds, hydrophobic interactions or chain entanglements (such gels are non-permanent and usually they can be converted to polymer solutions by heating) and chemical gels (permanent) with covalent bonds linking the chains (Benamer et al., 2006). In this paper, we assess the chemically crosslinked hydrogels obtained via high-energy radiation. Their synthesis through chemical crosslinking implies that homopolymers or copolymers are directly crosslinked with the use of a crosslinking agent. Due to the fact that difunctional structures, i.e., structures with polymerisable groups at both ends, such as di(meth)acrylates allow fast and intensive crosslinking dynamics, oligo(ethylene glycol) dimethacrylates (OEGDMAs), especially the ones with small number of EG units, i.e., with lower molecular weights, are among the most used crosslinking agents (Ismail et al., 2013). While

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dimethacrylate (DMA) part is responsible for efficient polymerisation and crosslinking, oligo(ethylene glycol) (OEG) is known for thermoresponsive behaviour and good biocompatibility (Lutz, 2008; Smeets et al., 2014a, 2014b). Polymers and hydrogels with significant PEG content are particularly interesting from a biomedical perspective, given their low toxicity/immunogenicity and aforementioned biocompatibility (Roy et al., 2013). In recent years, there has been a great deal of interest in (co)polymers with short oligo(alkylene glycol) (OAG) side chains, i.e., nonlinear PAG-analogues (Badi, 2017; Bakaic et al., 2015; Cao et al., 2018; Javadi et al., 2017; Roy et al., 2013; Son and Lee, 2016; Szweda et al., 2017). Therefore, thermoresponsive polymers and hydrogels based on acrylate, methacrylate or di(meth)acrylate with OEG side chains have been proposed as PAG analogues for biomedical applications (Lutz, 2008). In general, these structures are non-immunogenic, non-cytotoxic and protein-repellent, thus qualifying them for clinical applications (Smeets et al., 2014a, 2014b). Previous work in our laboratory has involved a variety of homopolymeric and copolymeric thermoresponsive hydrogels with OAG pedant chains synthesized by gamma radiation from different OAG(M)A monomers (Micic and Suljovrujic, 2013; Rogic Miladinovic et al., 2018, 2016; Tomic et al., 2010). Owing to the possibility to combine VPTT close to the human body

Corresponding author. E-mail address: [email protected] (E. Suljovrujic).

https://doi.org/10.1016/j.radphyschem.2018.12.034 Received 28 August 2018; Received in revised form 20 November 2018; Accepted 30 December 2018 Available online 02 January 2019 0969-806X/ © 2019 Elsevier Ltd. All rights reserved.

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properties in relation to the number of EG units was performed by Reiche et al. (2000). For potential biomedical applications, the influence of OEGDMA type and initial composition on gel content, mechanical and some other properties were investigated as well (Bäckström et al., 2012; Killion et al., 2011; Poplawska et al., 2014). Regardless of an enviable number of published papers concerning OEGDMA based hydrogels, swelling properties were investigated mainly at room temperature and in distilled water. Until now, despite the presence of thermoresponsive EG pedant chains in structure, there is no information about temperature and/or pH swelling behaviour as well as about VPTT of the OEGDMA based homopolymeric hydrogels. The goal of the current study is to synthetize and to investigate a series of POEGDMA hydrogels by gamma radiation (which offers unique advantages for the synthesis of such systems) from different OEGDMA550-water/ethanol mixtures. Expected thermoresponsiveness (due to the presence of EG units) and two methacrylate groups qualify this EG-oligomer as a good starting point for synthesis of thermo-responsive OEG-based polymer networks for biomedical applications. Different monomer-solvent mixtures were prepared and a complete screening in initial composition was elaborated from 10 to 100 wt% of monomer since the presence of a solvent should have significant impact on polymerisation/crosslinking as well as on the swelling properties. Thermoresponsive behaviour and VPTT of five different POEGDMA homopolymeric networks are investigated over a wide temperature range. In addition, despite the fact that neither OEGDMA monomer nor OEGDMA network itself possess pH sensitive groups, swelling over a wide pH range is also investigated since for some polymeric structures with OEG pedant chains a clearly visible difference in the swelling behaviour was observed for acidic and basic buffer solutions in comparison to neutral ones (Becer et al., 2008; Rogic Miladinovic et al., 2018). Along with swelling measurements some essential information are obtained by DSC, SEM, FTIR and UV–Vis spectroscopy.

temperature with good biocompatibility, new hydrogels with EGPG “block” pendant chains were proposed (Rogic Miladinovic et al., 2018). In addition, alternation of the EG pedant chain length and copolymerisation of OEG with other monomers offer a versatile platform for the design of new thermoresponsive hydrogels for various biomedical applications (Smeets et al., 2014b). Nevertheless, hydrogels formed by difunctional PEG structures with polymerisable end-groups, i.e., acrylate or methacrylate groups at both ends (PEGDA and PEGDMA, respectively) have received attention as biomaterials for wound dressing as well as for cartilage, dental and bone repair (Bryant and Anseth, 2003; Elisseeff et al., 1999; Haryanto and Mahardian, 2018; Lin-Gibson et al., 2004; Malo de Molina et al., 2015; Son and Lee, 2016; Zhang et al., 2008). Despite the fact that PEGDA has been more widely investigated as a scaffold material for bone regeneration (Guarino et al., 2015; Ma et al., 2010; Zhang et al., 2008), PEGDMA has also been proposed in recent years (Killion et al., 2011; Poplawska et al., 2014). Mechanical performance is of crucial importance for materials that will be utilized for bone regeneration application, where scaffolds are subjected to various loading conditions in vivo. Killion et al. (2011) showed that the mechanical properties of the PEGDMA hydrogels can be controlled by varying the monomer concentrations and molecular weights prior to photopolymerisation; both of these factors could be used as a means of manipulating the strength and flexibility of the hydrogels. PEGDMA homopolymeric hydrogels also showed minimal toxicological response according to Poplawska et al. (2014) and using either bioceramics such as hydroxyapatite or bioactive glasses, different bioinert or bioactive PEGDMA based composites for cartilage and bone reparation have been reported (Killion et al., 2014; Mohamad Yunos et al., 2008; Zhou et al., 2009). Furthermore, methacrylate groups coupled with hydrolysable polymers or proteins can provide a means of generating controlled biodegradable material, which could be removed from the implantation site by triggered enzymatic digestion (Poplawska et al., 2014). High-energy radiation such as gamma radiation offers unique advantages for the synthesis of new and modification of existing materials: it is a simple, additive-free process, where reactions such as polymerisation, crosslinking and grafting can be easily controlled. Hydrogels prepared by the radiation method have the potential for use in biomedical applications due to the absence of extraneous toxic additives (chemical initiators, etc.). Another advantage of this method is simultaneous sterilization along with the synthesis process (Hennink and van Nostrum, 2012; Rosiak, 2002; Safrany et al., 2010; Schmidt et al., 2003). In radiation chemistry, OEGDMA is one of the most commonly used precursors which even for very small quantities ensures efficient crosslinking and high gel content. Nevertheless, in most circumstances hydrogels with higher content of OEGDMA monomers were synthesized by UV induced free radical polymerisation in the presence of photoinitiator (Bäckström et al., 2012; Hwang et al., 2015; Reiche et al., 2000) but some other methodologies were also proposed (Wang et al., 2008); by altering type (determined by OEGDMA molecular weight, i.e., by number of EG units), initial composition (monomer/ solvent feed ratio), and other conditions prior and during synthesis (such as type of solvent, irradiation dose, etc.) POEGDMA hydrogels were fabricated. The study of thermal, mechanical and electrochemical

2. Experimental 2.1. Materials Oligo(ethylene glycol) dimethacrylate (OEGDMA) (Sigma-Aldrich, labelled as poly(ethylene glycol) dimethacrylate, Mn = 550 g mol−1, with 270–330 ppm BHT and 80–120 ppm MEHQ as inhibitors), was used as the main component for hydrogel fabrication. Demineralized water (demi-water, Millipore) and ethanol were used for the synthesis of the hydrogels and for the preparation of buffer solutions. Buffer solutions with different pH values (and constant ionic strength 0.1 mol dm−3) were prepared using hydrochloric acid (La Chema), potassium chloride, potassium mono- and di-hydrogen phosphate, sodium hydroxide, ammonium hydroxide and ammonium chloride (Fluka). All chemicals are readily commercial products and were used without any further purification. 2.2. Hydrogel preparation OEGDMA550 monomer was dissolved in a water/ethanol (1/1, by volume) mixture by stirring at room temperature for 15 min. A series of

Table 1 Feed ratio, gel content and equilibrium swelling degree (at 20 °C and 50 °C) of the investigated POEGDMA hydrogels prepared by gamma radiation. Hydrogel

POEGDMA-10 POEGDMA-25 POEGDMA-50 POEGDMA-75 POEGDMA-100

Feed ratio OEGDMA monomer (wt%)

Water/ethanol mixture 1/1, by volume (wt%)

10 25 50 75 100

90 75 50 25 0

38

Gel (%) 8 kGy

Gel (%) 25 kGy

qe (g/g) 20 °C

qe (g/g) 50 °C

90.9 91.3 91.8 92.2 92.8

99.7 99.5 99.3 99.2 99.5

5.0 2.5 1.3 0.78 0.32

2.7 1.3 0.7 0.38 0.18

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After reaching qe the weight of the sample was measured at each predetermined temperature up to 80 °C. The VPTT values were determined from the equilibrium swelling data obtained over a wide temperature range. The point at which a significant change in the slope of the equilibrium swelling ratio curve appears is taken as the VPTT; after this critical temperature, at which the hydrogel undergoes pivotal changes in swelling capacity, only minor changes in qe were observed.

OEGDMA-water/ethanol mixtures with different monomer feed ratios, from 10 to 100 wt% of OEGDMA, were prepared (Table 1). The reaction mixtures were degassed and sealed under nitrogen between two glass plates (with the distance of 4 mm between them) and irradiated in a 60 Co gamma source under ambient conditions at a dose rate of 0.5 kGy/ h to two different absorbed doses (8 and 25 kGy). After irradiation, the specimens were cut into discs (16 mm in diameter) and dried in a vacuum oven at 40 °C to a constant weight.

2.7. Calorimetric measurements 2.3. Gel content and sol-gel conversion Differential scanning calorimetry (DSC) was used to determine the VPTT of the obtained hydrogels. DSC measurements were performed using TA Instruments Q2000 Differential Scanning Calorimeter at a heating rate of 1 °C min−1 from 4 to 85 °C. The xerogels were immersed in pH 7.4 buffer solution and left to swell to equilibrium at 4 °C for 24 h before the DSC heating scan. The temperature at the onset point of the DSC endothermic peak was referred as the VPTT (Rogic Miladinovic et al., 2018; Zhang et al., 2009).

To remove unreacted components, the xerogels obtained after synthesis and drying were subjected to Soxhlet extraction at 40 °C in water/ethanol for 48 h. After that, the extracted samples were dried in a vacuum oven at the same temperature for additional 72 h, in order to obtain a constant weight. The gel fraction was determined gravimetrically according to the following equation, Gel fraction (%) = (We/ W0)×100, where Wo is the initial weight of the xerogel and We is the weight of the xerogel after extraction.

2.8. Scanning electron microscopy (SEM) 2.4. UV–Vis spectroscopy A scanning electron microscope (SEM), JEOL (JSM-5300) was used to observe specimen morphologies. The POEGDMA samples were prepared by freeze-drying, using an Edwards Freeze Dryer System (England) consisting of a freeze-drying unit and an Edwards E2M8 high vacuum pump. POEGDMA xerogel samples were swollen in pH 7.4 buffer solution to equilibrium. Afterwards, they were pre-frozen in a freeze dryer at −80 °C and freeze-dried for 24 h in the vacuum. The freeze-dried samples were gold sputter coated under vacuum prior to the SEM measurements.

UV–Vis measurements were performed for different OEGDMA monomer concentrations (1, 3, 5, 10, 25 and 50 wt%) in demi-water and in pH 2.2 and 7.4 buffer solutions. Absorption spectra and the transmittance at 650 nm were recorded in steps of 2 °C during a heating run from 4 °C to 80 °C, with a heating rate of 0.2 °C min−1, using a Shimadzu 1800 UV–Vis spectrophotometer equipped with a temperature controller. The LCST values were determined at the temperatures showing an optical transmittance of 50 wt% (Soppimath et al., 2005); strictly speaking, this is the cloud point (CP) of the solution (Tang et al., 2015).

3. Results and discussion

2.5. FTIR spectroscopy

3.1. UV–Vis spectroscopy

OEGDMA based structures were studied by Fourier Transform Infrared spectroscopy in the attenuated total reflectance mode (ATRFTIR). The spectra were recorded on a Nicolet FTIR spectrometer (IS 50) at room temperature in the wavenumber range of 4000–400 cm−1 with a resolution of 4 cm−1. The spectra are average values of three identically prepared samples randomly selected.

Fig. 1a and 1b represent digital optical pictures of 50 wt% OEGDMA550 monomer in water and water/ethanol (1/1, by volume) before and after shaking the solutions, respectively; while OEGDMAwater/ethanol mixture remains fully transparent, OEGDMA-water mixture which initially shows a phase separation (designated by an arrow in Fig. 1a) after shaking turns to completely turbid. The transition from clear aqueous to a completely turbid solution with temperature in UV–Vis measurements is observed as a large decrease in transmittance (Fig. 1c). On the other hand, the transmittance of OEGDMAwater/ethanol solution which is close to 100% remained unchanged with temperature clearly showing the influence of ethanol addition to the mixture. As it was expected, the addition of short-chain alcohols, such as ethanol, increases the solubility of the monomer (Padmanabhan and Kritchevsky, 1971). This is an important step that should precede the radiation synthesis of the hydrogels since increased monomer solubility generates efficient and rapid polymerisation and crosslinking kinetics during irradiation (Lutz, 2008; Suljovrujic and Micic, 2015). In such a way the results showed the high conversion of the monomers into polymer networks. The thermoresponsive behaviour of hydrogels investigated in this paper lies in the nature of OEGDMA monomer. Namely, due to the presence of EG unit, structures such as OEGDMA exhibit a thermoreversible transition from a soluble (solved coil) to an insoluble phase (collapsed globule) upon heating, the effect is known as LCST. To determine an LCST, the cloud point (CP) method was used (Bebis et al., 2011); it is based on the fact that solutions turn from clear to turbid at certain temperatures, which is reflected in the quantitative measurement as a large decrease in transmittance. The temperature at which transmittance is 50% is defined as an LCST. The influence of the concentration of the monomer solution on the LCST value under physiological conditions is significant and well investigated thus far (Bebis

2.6. Swelling study Swelling measurements were performed over a broad temperature range from 5 to 80 °C and in a wide range of pH buffer solutions (2.2–9.0) important for biomedical studies. The buffer solutions were prepared at room temperature using chemicals given in Section 2.1 following the procedure described in more details previously (Micic, 2015). Due to the fact that the swelling of PEG polymers is highly sensitive to the presence of dissolved ions (Jiang et al., 2018; Zhang et al., 2014), the ionic strengths of all buffers were adjusted to 0.1 mol dm−3 by addition of KCl. As the temperature rises, molecular vibrations increase resulting in the ability of water to ionize and form more hydrogen ions; as a result the pH will decrease. For that reason when pH drops for more than 0.2 with temperature, buffers are adjusted to the initial pH (Micic, 2015). The swelling ratios and kinetics of the hydrogels were measured gravimetrically. The equilibrium degree of swelling (qe) was calculated as qe = (We − Wo)/ Wo , where We is a weight of the swollen hydrogel at equilibrium and Wo is a weight of the xerogel. The average value from five different specimens of the same hydrogel was taken. In order to obtain more reliable data from temperature measurements, the xerogel samples were swollen to equilibrium at 5 °C and the weight was measured. Furthermore, the temperature was raised in increments of 2.5 °C every 24 h which is the period sufficient to reach swelling equilibrium. 39

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35

(a)

oC

35

(b)

oC

H2O HO Eth. 2

H2O HO Eth. 2

100

Transmittance (%)

OEGDMA/solvent ratio 50/50 wt%

50 H O-Eth. HO

(c)

0 10

20

30

T (ºC)

40

(d)

Transmittance (%)

100

(Constantin et al., 2011); therefore homopolymeric hydrogels with less than 10 wt% of OEGDMA were not considered in this study. Exposure of OEGDMA-water/ethanol mixture to high-energy radiation leads to polymerisation and crosslinking, i.e., to the creation of homopolymeric POEGDMA hydrogels. Addition of ethyl alcohol to the mixture increases the solubility of the monomer and suppresses phase separation and therefore generates uniform, efficient and rapid polymerisation and crosslinking kinetics during irradiation (Rogic Miladinovic et al., 2018). Furthermore, difunctional structures, i.e., structures with polymerisable end-groups at both ends, such as OEGDMA, allow fast and intensive crosslinking; due to the presence of two vinyl groups, both polymerisation and crosslinking of OEGDMA occur simultaneously. The gel fraction is considered to be a quantitative indicator of the efficiency of network formation. The high conversion of the OEGDMA monomer into a crosslinked polymer was obtained even at lower doses (8 kGy), while the saturation in the gel fraction was observed for 25 kGy. For both doses, gravimetric analysis indicated a high sol-gel conversion and the gel fraction higher than 90% (Table 1). Specifically, for a dose of 25 kGy, the gel fraction of all POEGDMA homopolymeric hydrogels was higher than 99%, the matrices were strong enough to be easily handled and also possessed characteristics suitable for different applications; in addition, this is the standard sterilization dose. Therefore, it can be concluded that solution (water/ethanol) in the prepolymerisation mixture was not able to significantly disrupt the network connectivity and in all cases, the gel formation occurred. Complete sol-gel conversion for 25 kGy was confirmed by FTIR spectroscopy as well; the ATR-FTIR spectra of pure OEGDMA monomer and POEGDMA-100 gels obtained by gamma radiation after 8 and 25 kGy are illustrated in Fig. 2a. Since nonappearance of broad OH stretching absorption peak in POEGDMA-100 around 3400 cm−1 indicated the absence of water, peak at 1640 cm−1 which is present in the pure OEGDMA monomer is completely due to vibration of the resident C˭C band and there is no contribution of OH group from water which also has an absorption peak around 1640 cm−1. For irradiation dose of 8 kGy, a trace of the C˭C band is still present, while after 25 kGy complete absence of C˭C absorption peak around 1640 cm−1 is clearly noticeable. This indicated that radiation-induced polymerisation and crosslinking of OEGDMA monomers, which involved the breaking of terminal carbon-carbon double bonds, were complete for 25 kGy. Since the gel content may considerably differ depending on type and conditions of synthesis this can also have significant influence on toxicological response and biocompatibility; namely, toxicology analysis has shown that hydrogels in most circumstances require large gel content or even washing steps to ensure the cytocompatibility, mainly due to the presence of unreacted monomer after the synthesis (Geever et al., 2008). The ATR-FTIR spectra has also indicated a change in the chemical environment in the vicinity of the C˭O bond; a shift of the peak at ~1716 cm−1 (monomer) to ~1728 cm−1 (hydrogel) is evident from Fig. 2a. The OEGDMA monomer contains C˭O bonds conjugated with the adjacent C˭C bonds. In the POEGDMA hydrogels, the C˭C bond was converted to a C–C bond and the C˭O bonds are no longer conjugated giving rise to the revealed shift in agreement with previous observations (Bäckström et al., 2012). After synthesis C˭O peak was clearly shifted to 1728 cm–1 without any residual peak remaining at 1716 cm–1 indicating that practically all OEGDMA monomers were used up in the radiation-induced synthesis and effectively no C˭O bonds conjugated with C˭C bonds remained. According to the previous discussion, in further investigations, we used POEGDMA homopolymeric hydrogels with very large gel content (> 99%) obtained for absorbed dose of 25 kGy. Fig. 2b shows the FTIR spectra of different POEGDMA homopolymeric hydrogels obtained by swelling of xerogels in demi-water for 1 min, which were used for identification of relevant functional group peaks. Investigated hydrogels showed beside the broad OH stretching absorption peak around 3400 cm−1 characteristic absorption bands

OEGDMA 10 wt%

OEGDMA

50

1 wt% 3 wt% 5 wt% 10 wt% 25 wt%

0 0

10

pH 7.4 20

30

40

50

60

70

Temperature (ºC)

(e)

35 oC

25 wt% 10 wt% 5 wt%

3 wt% 1 wt%

pH 7.4 Fig. 1. Digital optical picture of 50 wt% OEGDMA monomer in water and water/ethanol (a) before and (b) after shaking the solutions, respectively. (c) Transmission profile of 10 wt% OEGDMA-water and OEGDMA-water/ethanol solutions measured by UV–Vis spectroscopy. (d) LCST profiles of OEGDMA for different monomer concentrations in pH 7.4 buffer solution, measured by UV–Vis spectroscopy. Transmittance value of 50% (cloud point) is defined as the LCST. (e) Digital optical picture of different OEGDMA monomer concentrations in pH 7.4 buffer solution.

et al., 2011; Constantin et al., 2011). Nevertheless, it needs to be determined for each individual thermoresponsive component; the temperature dependence of the transmittance for different OEGDMA monomer concentrations in pH 7.4 buffer solution is shown in Fig. 1d. It is evident that the LCST value determined by the CP method increases with decreasing OEGDMA concentration; obtained values are about: 19 °C (25 wt%), 27 °C (10 wt%), 36 °C (5 wt%) and 48 °C (3 wt%). In the more concentrated solutions, the opalescence was more accentuated than in diluted ones. Digital optical picture of different OEGDMA monomer concentrations confirmed this, as well (Fig. 1e). Noticed LCST behaviour was consistent with other studies concerning thermoresponsive polymers and small organic molecules (Constantin et al., 2011; Loh, 2013). 3.2. Radiation synthesis, sol-gel conversion and FTIR spectroscopy The synthesis was performed from OEGDMA550-solvent mixtures with different monomer feed ratios, from 10 to 100 wt% of OEGDMA, by gamma radiation and five different POEGDMA homopolymers were fabricated and labelled in Table 1. In general, physically and chemically crosslinked hydrogels used in biomedical applications were prepared starting from more concentrated polymer solutions (at least 5–10 wt%) 40

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(a)

(b)

Transmittance (a.u.)

POEGDMA-25 kGy

POEGDMA homopolymeric hydrogels to water significantly increased. Conformation for this can be more clearly observed in Fig. 2c. Namely, absorption peaks of OH groups for POEGDMA-100 xerogel left in water for 10 min are lower than for POEGDMA-10 xerogel immersed in water for only 1 min. Nevertheless, swelling data obtained for POEGDMA homopolymeric hydrogels and given in Table 1 can give the most valuable information about hydrogel response to water. Equilibrium swelling degree (qe) of the investigated POEGDMA hydrogels sharply decays with an increase in the OEGDMA monomer content in monomer/solvent mixture prior to radiation-induced synthesis. Thus, with the increase in OEGDMA monomer content in the prepolymerisation mixture from 10 to 100 wt% a decrease in the swelling capacity of the obtained hydrogels is more than an order of magnitude. Swelling data presented at two different temperatures (20 and 50 °C) for the POEGDMA hydrogels show that observed swelling behaviour is preserved despite a significant decrease in the swelling capacity with temperature.

POEGDMA-100 POEGDMA-75

POEGDMA-8 kGy

POEGDMA-50 POEGDMA-25

OEGDMA

POEGDMA-10 WATER O-H

WATER O-H C=O C=C

4000

3000 1800

1700

O-H

3000

1600

Wavenumber (cm-1) Transmittance (a.u.)

(c)

C=O O-H C=C

C-H

C-H C-O C-C

2000

1000

Wavenumber (cm-1)

O-H

O-H

O-H

xerogel

POEGDMA-100

swelling 1 min swelling 10 min xerogel

POEGDMA-10

3.3. Thermal and swelling study of the POEGDMA-10 hydrogel

swelling 1 min WATER

4000

3500

3000

2500

2000

1500

1000

The absorption of water is one of the most important factors determining applications of hydrogels. For thermoresponsive hydrogels, large alteration in the hydrophilic/hydrophobic balance and the transition of polymer pendant chains from spread coil state into collapsed globule upon heating are reflected in the emergence of hydrogel turbidity and squeezing of water from the hydrogel's network (Micic et al., 2016). The thermoresponsive swelling behaviour of the POEGDMA-10 hydrogel is presented in Fig. 2d and the obtained VPTT value is 62.5 °C at physiological pH which is far above temperatures significant for biomedical applications. In general, LCST/VPTT of OEG based polymer/hydrogel is governed by the EG chain length of the monomer, i.e., by a number of EG units in pedant chains of polymer/hydrogel (Lutz and Hoth, 2006; Sun and Wu, 2013). With a suitable choice of monomer, it is possible to tune the VPTT of hydrogels to lower (e.g. body) temperatures. Another possible design methodology (at which we work at the present) is based on copolymerisation of OEG-based functional monomers with more hydrophobic thermoresponsive monomers in order to prepare well-defined copolymeric hydrogels with composition-dependent thermoresponsivity (Paris and QuijadaGarrido, 2009; Roy et al., 2013). For VPTT determination besides the swelling method, thermal analysis was used as well. DSC heating scan of the hydrogel swollen to equilibrium in pH 7.4 buffer solution is presented as an insert 1 (Fig. 2d). For the POEGDMA-10 hydrogel the onset point of the endotherm indicates VPTT around 64 °C. Therefore, it is evident that DSC results are in a good agreement with those derived from the swelling method. Since the change from the spread coil state into the collapsed globule upon heating is reflected in hydrogel turbidity additional information about VPTT can be obtained visually. Digital optical picture of the POEGDMA-10 swollen disc at 50 °C and 70 °C is presented as an insert 2 (Fig. 2d). By increasing the temperature from 50 °C it is noticed that heating leads to the occurrence of turbidity in a disc that becomes completely turbid at 70 °C. Comparing this optical transition with temperature swelling and thermal behaviour, a clear distinction is noticed in the dynamics of the investigated hydrogel. In order to investigate the influence of the hydrogel composition and external conditions (pH and temperature) on the swelling properties the comprehensive swelling study was performed. The influence of pH on the swelling behaviour and VPTT is presented in Fig. 3. The equilibrium swelling degree (qe) of the POEGDMA-10 hydrogel, as a function of pH, at different temperatures ranging from 5 to 80 °C is presented in Fig. 3a. It can be observed that POEGDMA-10 hydrogel is practically pH insensitive at temperatures < 50 °C, while pH sensitivity was noticed with further temperature increase. The effect of pH sensitivity is the most noticeable at temperatures close to 60 °C. Digital optical picture taken for POEGDMA-10 swollen discs in two different buffer solutions (pH 2.2 and 7.4) at 60 °C is presented as an insert 1

500

Wavenumber (cm-1)

6

POEGDMA-10

(d) 50 oC

qe

70 oC

4

Temperature (ºC)

2

0

0

20

40

endo

Heat Flow

0

60

80

63.8 ºC

62.5 oC pH 7.4

20

40

60

80

Temperature (ºC) Fig. 2. (a) FTIR spectra of the pure OEGDMA monomer and POEGDMA-100 hydrogel obtained by gamma irradiation dose of 8 and 25 kGy. (b) FTIR spectra for all POEGDMA hydrogels obtained by swelling of xerogels in demi-water for 1 min (c) FTIR spectra of the POEGDMA-100 and POEGDMA-10 xerogels and hydrogels. For a better understanding of the differences in the spectra among hydrogels containing water, an FTIR spectrum of demi-water is given, as well. The scans were shifted vertically for clarity. (d) Equilibrium swelling degree (qe) of the POEGDMA-10 hydrogel in pH 7.4 buffer solution as a function of temperature. The insert 1 is a digital optical picture of the POEGDMA-10 hydrogel in pH 7.4 buffer solution at 50 °C and 70 °C. The insert 2 is a DSC heating scan obtained for POEGDMA-10 hydrogel with the heating rate of 1 °C min−1.

between 3000 and 2800 cm−1 which correspond to the stretching of aliphatic CH, CH2, and CH3 groups. In 2000–500 cm−1 region beside the C˭O stretching vibration at 1730 cm−1, and absorption band at 1640 cm−1 due to bending of HOH groups as well as vibration of the residual C˭C groups, numerous absorption bands between 1500 and 700 cm−1, which mainly correspond to the stretching and bending of CH, CH2, CH3, C-C, C-O, O˭C-O and OH groups were clearly observed from the spectra, as well. Despite the great similarity between the presented spectra, some significant differences are also evident and can be mostly attributed to the presence of water in POEGDMA homopolymeric hydrogels. Difference in broad OH stretching absorption peaks above 3200 cm−1 and bellow 750 cm−1 as well as in HOH bending absorption peak around 1640 cm−1 can be attributed to different affinity of the obtained hydrogels to water; with decrease in the initial monomer/solvent feed ratio, i.e., with decrease in the OEGDMA monomer content in mixture prior to synthesis, affinity of final

41

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POEGDMA-10

(a) 6

influence, VPTT was obtained from swelling measurements and presented as a function of buffer solution pH. From Fig. 3c it is evident that VPTT values obtained for acidic and basic buffer solutions were about few degrees above those observed around neutral pH. It should be highlighted that neither in non-ionic OEGDMA monomer nor in nonionic polymer networks, such as POEGDMA, pH-sensibility is expected and this holds for temperatures much lower than transition temperatures. On the other hand, when approaching to LCST/VPTT pH starts to play a significant role which cannot be neglected. According to Becer et al. (2008) this is most likely due to slightly stronger interactions of the EG pendant chains with hydroxide ions or hydronium protons than with water. However, since in thermoresponsive non-ionic polymers and networks a number of interactions (such as the polymer-polymer and water-polymer hydrophobic interactions, as well as the waterpolymer and water-water H-bonds) play a key role this scarce explanation is partly questionable. Namely, it is also probable that ions do not interact with EG chains directly, but rather influence water structure leading to redistribution of hydrogen bonds in the system and to weakening of polymer-water interactions (Olejniczak et al., 2016; Pastorczak et al., 2014). Therefore, the additional studies are required to fully elucidate the mechanism of the observed effect.

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3.4. Scanning electron microscopy (SEM) and VPTT of the POEGDMA hydrogels Swelling capacity of the investigated POEGDMA hydrogels showed a progressive decay with the increase in the OEGDMA monomer content in the monomer/solvent mixture prior to radiation-induced synthesis (Table 1). This decay is significant and for hydrogels with the utmost distinction in the monomer content, POEGDMA-10 (qe ≅ 5) and POEGDMA-100 (qe ≅ 0.3), the difference in the equilibrium swelling degree is about 20 times at room temperature. Nevertheless, such difference in the swelling capacity also results in different microstructure and porosity. Fig. 4 shows the SEM microstructures of lyophilized samples; surface microstructures are presented on the left and crosssections on the right-hand side. The morphological analysis was focused on different POEGDMA samples swelled to equilibrium at room temperature in a physiological buffer solution, allowing a better investigation of the correlation between microstructure and the swelling properties. The SEM image for POEGDMA-10 hydrogel demonstrates a homogeneous porous structure with large pores as expected, since qe ≅ 5 at room temperature (20 °C); the pores formed on the surfaces are also uniformly distributed throughout the hydrogel. ImageJ software was used to measure the morphological parameters such as pore size and accordingly the mean pore size for the observed surface of the hydrogel was calculated. It can be seen that the hydrogel exhibited relatively regular porous structures with diameters of pores in the 10–15 µm range. The large decrease in the average size of the micropores with relatively uniform distribution is clearly visible in Fig. 4, from top to bottom. Thus, for the POEGDMA-100 sample, in comparison with POEGDMA-10 microstructure, the absence of porosity for small magnification (×750) is evident. Therefore, the porosity of the obtained hydrogels can be accurately controlled by altering the OEGDMA monomer content in the initial mixture. Furthermore, the large temperature-induced change in the swelling behaviour would also result in a significant change in the microstructure of the sample (Fig. 5a). The morphological analysis was focused on samples swollen to the equilibrium swelling degree below and above the VPTT because they showed significant differences in the swelling capacity, thus allowing a better insight in the correlation between microstructure and swelling properties. Therefore, SEM images of lyophilized POEGDMA-10 microstructures swelled to equilibrium at 20 °C and 70 °C are presented as inserts in Fig. 5a; the transition from relatively regular porous structures with large diameters of pores in the 10–15 µm range (insert 1) to rather obscure morphology with uniform but less defined and small defects up to 2 µm (insert 2) is shown for

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Fig. 3. (a) Effect of pH on the equilibrium swelling degree of the POEGDMA-10 hydrogel at different temperatures in the range from 5 to 80 °C. (b) Equilibrium swelling behaviour of the POEGDMA-10 hydrogel in different buffer solutions (pH 2.2 and 7.4) as a function of temperature. The insert 1 is a digital optical picture of the POEGDMA-10 hydrogel at 60 °C in buffer solutions pH 2.2 and 7.4. The insert 2 is a CP profile of OEGDMA (3 wt% monomer concentration) in buffer solutions with different pH (2.2 and 7.4). (c) VPTT of the POEGDMA-10 hydrogel as a function of buffer solution pH.

(Fig. 3b). Observed difference in turbidity along with previously revealed variance in pH sensitivity can be connected with VPTT. To clarify this, the equilibrium swelling degree (qe) of the POEGDMA-10 hydrogel as a function of temperature for two buffer solutions (pH 2.2 and 7.4) is presented in Fig. 3b. Despite the fact that swelling dynamics are very much alike, it can be observed that decay in qe was shifted to higher temperatures for acidic buffer solution and this shift affects the VPTT. Since OEGDMA does not possess pH sensitive groups the observed pH sensitivity undoubtedly originates from the monomer as LCST shows similar behaviour with pH (Insert 2 in Fig. 3b); for the OEGDMA concentration of 3 wt%, LCST measured at pH 2.2 (53 °C) is a few degrees higher than in the case of pH 7.4 (48 °C). Alike situation was also observed in the case of some OAG-based polymers; thus, for OEGMA and oligo(ethylene glycol) ethyl ether methacrylate (OEGEMA) with different molecular weights, as well as for oligo(propylene glycol) methacrylate (OPGMA) the LCST values are lower in neutral than in acidic or basic solutions (Becer, 2009; Becer et al., 2008; Suljovrujic and Micic, 2015). In order to provide a better overview of the pH 42

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Fig. 4. SEM micrographs at ×750 magnification of surface and cross-section microstructures of different POEGDMA homopolymeric hydrogels swollen to equilibrium at room temperature and in pH 7.4 buffer solution before lyophilisation are presented on the left and right-hand side, respectively.

magnification (×750). These pores may enlarge the ratio surface area of networks, which is a favour to increase the contact points of hydrophilic chains and solvents, thus enabling small molecules, e.g. water molecules to penetrate through the network easily and to retain in such pores (Huang et al., 2013). By increase in the temperature the sample suffers a large change in hydrophilic/hydrophobic balance and therefore releases higher amounts of the water. Thus, by comparing the sample swollen at 70 °C (qe ≅ 0.5) with the microstructure observed at 20 °C (qe ≅ 5) the absence of porosity was confirmed. The microstructural investigation supports the fact that the porosity of POEGDMA thermosensitive hydrogels can be controlled not only by the initial composition but also by the external conditions such as temperature. Comparison of the temperature dependent swelling behaviour of different POEGDMA hydrogels shows a clear distinction in the swelling dynamics of investigated hydrogels. From Figs. 5a and 5b two important conclusions can be derived. Firstly, the initial difference in the swelling capacity of the hydrogels can be accurately controlled by altering the OEGDMA monomer content in the initial mixture. Secondly, all hydrogels possess thermoresponsive nature and VPTT with no respect to the preparation conditions. The equilibrium swelling degree (qe) of POEGDMA as a function of OEGDMA monomer content in the prepolymerisation mixture is presented in Fig. 5c; the exponential increase in qe with a decrease in the OEGDMA monomer content in the initial mixture is obtained with no respect to temperature. The reason for that is in the nature of difunctional structures which allow fast and intensive crosslinking dynamics. Dilution of difunctional monomers in the prepolymerisation mixture decreases a probability for crosslinking during synthesis thus resulting in decreased crosslinking density and at some point in increased polymerisation induced phase separation (PIPS) in the obtained hydrogels. PIPS is a kinetic phenomenon, reflecting the competition between the rate of polymerisation, which is

Fig. 5. (a) Equilibrium swelling degree (qe) of different POEGDMA hydrogels in pH 7.4 buffer solution as a function of temperature. The inserts on the righthand side represent SEM micrographs of lyophilized POEGDMA-10 hydrogel swollen to equilibrium in pH 7.4 buffer solution at 20 °C (at the top) and 70 °C (at the bottom). (b) Equilibrium swelling degree (qe) as a function of temperature for four different OEGDMA based hydrogels (POEGDMA-25, POEGDMA-50, POEGDMA-75 and POEGDMA-100). The point at which a significant change in the slope of the curve occurs is the VPTT. (c) Equilibrium swelling degree (qe) of OEGDMA, at different temperatures and in pH 7.4 buffer solution as a function of initial OEGDMA wt%. The results were fitted with the exponential growth functions with R2 ≥ 0.95. (d) VPTT of POEGDMA in pH 7.4 buffer solution as a function of OEGDMA content (wt%).

forming the network, and the rate of phase separation (Okay, 2000; Williams et al., 1997). When the polymerisable species in an initially homogeneous solution undergo polymerisation to the point where the growing chains become sufficiently long such that their water solubility decreases below the amount of water in the polymerising mixture, phase separation occurs. The final structure of hydrogel results from a balance between these two competing rate processes (polymerisation and phase separation). According to Wu et al. (2010) and Poplawska et al. (2014), the PEGDA700 and PEGDMA550 crosslinked structures exhibited PIPS when the water content of the prepolymerisation mixture was greater than 60 and 50 wt%, respectively. Despite the fact that the temperature dependent swelling dynamics are qualitatively quite similar since all POEGDMA hydrogels have inverse thermoresponse and volume phase transition temperature (VPTT), it can be clearly observed from Fig. 5b that VPTT was shifted to the lower temperature with an increase in OEGDMA monomer content in the initial mixture. Therefore, it is evident that OEGDMA monomer

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content in prepolymerisation mixture will also affect the VPTT. Fig. 5d demonstrates a clear structure-property relationship; it is obvious that POEGDMA hydrogels follow a simple rule in their thermoresponsive behaviour showing a linear increase in VPTT with a decrease in the weight percentage of OEGDMA monomer content in the initial mixture prior to radiation-induced synthesis. However, it should be noted that the change in VPTT is less than 7 °C for all investigated hydrogels indicating that this phenomenon should be carefully treated and interpreted.

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4. Conclusions In this paper, gamma radiation was used to successfully produce POEGDMA hydrogels by varying the monomer/solvent feed ratio prior to synthesis; a complete screening in the initial composition was elaborated from 10 to 100 wt% of OEGDMA and five different homopolymeric hydrogels were fabricated. FTIR analyses indicated that radiation-induced polymerisation and crosslinking of OEGDMA monomers which involved the breaking of terminal carbon-carbon double bonds was completed for a dose of 25 kGy, while sol-gel measurements revealed the gel fraction higher than 99% for the investigated networks at this dose. The thermoresponsive swelling behaviour of POEGDMA-10 hydrogel was investigated in detail and the obtained VPTT value was found far above temperatures significant for biomedical applications (62.5 °C) and is in a good agreement with DSC data. The pH influence on the swelling behaviour of POEGDMA-10 was also investigated. It should be underlined that in non-ionic polymer networks such as POEGDMA pH-sensitivity is not expected and this holds for temperatures much lower than VPTT. As approaching to the VPTT the influence of pH cannot be neglected; obtained VPTT values for acidic and basic buffer solutions were found about few degrees above those observed around the neutral one. UV–Vis data indicate that this effect originates from OEGDMA monomer and in the hydrogel networks can be connected with OEG pendant chains. Nevertheless, the additional studies are required to fully elucidate the observed pH behaviour. In general, the swelling data obtained for all investigated POEGDMA homopolymeric compositions showed thermoresponsive behaviour, inverse thermoresponse and VPTT. This behaviour is significantly influenced by initial preparation conditions prior to synthesis. Lower crosslinking density (i.e., lesser OEGDMA monomer content and higher water content in the prepolymerisation mixture) leads to the higher water sorption of the hydrogel and consequently the difference in the equilibrium swelling degree of the obtained hydrogels can be more than an order of magnitude. A linear VPTT decrease with monomer content was observed, as well. The microstructure and porosity of the obtained networks can be controlled by the composition of prepolymerisation mixture as well as by the external conditions such as temperature thus underlying their potential use as membrane separation systems. Mechanical properties, network parameters and biocompatibility of the obtained thermoresponsive POEGDMA homopolymeric hydrogels will be further examined. Acknowledgement This work has been supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grants no. 172026 and No. III45005). The authors are also gratefully acknowledged to Mr. Ivan Radovic for his relevant contribution. References Bäckström, S., Benavente, J., Berg, R., Stibius, K., Larsen, M., Bohr, H., Helix-Nielsen, C., 2012. Tailoring properties of biocompatible PEG-DMA hydrogels with UV light. Mater. Sci. Appl. 03, 425–431. https://doi.org/10.4236/msa.2012.36060. Badi, N., 2017. Non-linear PEG-based thermoresponsive polymer systems. Prog. Polym.

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