Synthesis and characterisation of thermo-sensitive terpolymer ...

J Polym Res (2011) 18:2307–2324 DOI 10.1007/s10965-011-9644-0

ORIGINAL PAPER

Synthesis and characterisation of thermo-sensitive terpolymer hydrogels for drug delivery applications Jude I. Ngadaonye & Martin O. Cloonan & Luke M. Geever & Clement L. Higginbotham

Received: 1 February 2011 / Accepted: 7 June 2011 / Published online: 23 June 2011 # Springer Science+Business Media B.V. 2011

Abstract In this study, thermo-sensitive terpolymer hydrogels based on N-tert-butylacrylamide (NtBAAm), Nisopropylacrylamide (NIPAAm) and N-vinyl pyrrolidone (NVP) were successfully photopolymerised and characterised. 1-hydroxy-cyclohexylphenylketone (Irgacure 184) and 2-hydroxy-2-methyl-1-phenyl-propanone (Irgacure 2959) were used as light-sensitive initiators to initiate the reactions. Chemical structures of the hydrogels were confirmed using Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. The hydrogels were also characterised using modulated differential scanning calorimetry (MDSC) for their glass transition and phase transition temperatures. A single glass transition temperature (Tg) was observed, further confirming successful formation of a terpolymer. The hydrogels were thermo-responsive, exhibiting a decrease Electronic supplementary material The online version of this article (doi:10.1007/s10965-011-9644-0) contains supplementary material, which is available to authorized users. J. I. Ngadaonye : L. M. Geever : C. L. Higginbotham (*) Materials Research Group, Research Hub, Athlone Institute of Technology, Dublin Road, Athlone, Co., Westmeath, Ireland e-mail: [email protected] J. I. Ngadaonye e-mail: [email protected] L. M. Geever e-mail: [email protected] M. O. Cloonan Department of Chemistry, School of Science, Galway-Mayo Institute of Technology, Dublin Road, Galway, Ireland e-mail: [email protected]

in lower critical solution temperature (LCST) as the NtBAAm weight ratio was increased. Pulsatile swelling studies indicated that the hydrogels had thermo-reversible properties and the swelling properties were dependent on test temperature, monomer feed ratios and crosslinker content. The proposed hydrogel system could find applications in a broader field of gel/drug interaction, for the development of controlled release and targeted delivery devices. Keywords Hydrogel . Terpolymers . N-tertbutylacrylamide . Pulsatile swelling . Thermosensitive

Introduction Due to the significance of hydrogels in both theory and application, they have received increasing attention among researchers. They have also been employed in a vast amount of diverse applications. The high water content in a hydrogel allows it to be flexible and resemble biological tissues [1] and also allows selective diffusion of solutes through the hydrogel matrix. This characteristic is desired for controlled release drug delivery systems [2, 3]. Other desired characteristics of hydrogels for controlled release drug delivery applications include their response to change in the environment. In the past few decades, smart polymeric hydrogels that undergo reversible solubility or volume changes in response to external stimuli have attracted special attention. Depending on the nature of the pendant groups along the polymer chains, these hydrogels have the ability to respond to environmental stimuli such as temperature, pH, ionic strength and specific chemical compounds [4], while others have the ability to respond to applied electrical or magnetic fields.

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Many linear thermo-responsive polymers are soluble in water at low temperatures but become insoluble as the temperature rises above what is termed their lower critical solution temperature (LCST). The LCST of a temperature sensitive polymer corresponds to the disruption of the hydrogen bonds in a dilute aqueous solution, and collapse of single chains upon heating; a process usually referred to as the coil-to-globule transition. However, in a strict thermodynamic sense, it is a precipitation and dissolution temperature upon heating and cooling, respectively. It is well known that poly(N-isopropylacrylamide) (PNIPAAm) undergoes a rapid and reversible hydration-dehydration change in response to small temperature cycles around its LCST in aqueous media. The LCST value of PNIPAAm has been reported as 32–33 °C [5–8] which is an attractive range for biomedical applications, particularly drug delivery. In general, the water absorption capacity of PNIPAAm hydrogel is limited, which restricts its application in certain drug delivery applications. Highly absorbent hydrogels have excellent hydrophilic properties and high swelling ratios, and hence have been widely used in agriculture [9], as sorbents for the removal of heavy metals [10] and in biomedical applications [11]. Also due to their large pore size, they are also extremely useful as matrixes for the controlled release of macromolecular drugs and compounds such as therapeutic proteins, enzymes and DNA [2]. Copolymerisation with a hydrophilic monomer dramatically increases the water absorption capacity while usually reducing its sensitivity towards temperature change in an aqueous environment [12, 13]. Hydrogels based on N-isopropylacrylamide have been used for developing reversible temperature-controlled release systems. Generally, incorporation of a hydrophilic monomer leads to an increase in the LCST of a temperature-sensitive polymer and in the same vein, incorporation of a hydrophobic monomer leads to a decrease in LCST and/or the volume phase transition temperature (VPTT) [14, 15]. Liu & Zhu [16] investigated the effects of the chemical composition on the phase separations of the aqueous solutions of homo- and copolymers prepared from N-substituted acrylamides such as N-tert-butylacrylamide (NtBAAm), N-ethylacrylamide, and N,N-diethylacrylamide and reported that copolymers of NtBAAm possess LCST values depending on the hydrophilicity-hydrophobicity of the final polymer. Many hydrogels are prepared via different technologies with ultraviolet (UV) polymerisation or photopolymerisation been among the most commonly applied methods, due to its distinct advantages such as rapid cure, low curing temperature and low energy requirement. UV or visible light-initiated polymerisation reactions are a well-established technology for many industrial applications such as screen printing, wood coatings, pigmented coatings for textile applications and pigmented primary and secondary optical fibre coatings [17]. Photopolymerisation is also commonly used in a broad

C.L. Higginbotham et al.

range of biomedical applications such as dentistry, implants, scaffolds, bioadhesives and drug delivery systems [18–20]. One of the main reasons for this is that hydrogels can be synthesised at temperatures and pH conditions near physiological conditions and even in the presence of biologically active materials. The aim of this work was to prepare temperaturesensitive hydrogels that exhibit a high water uptake and sharp thermosensitivity in the vicinity of physiological temperature, via a UV-initiated polymerisation process. The hydrogels were prepared to imbibe a large amount of water so as to provide a main diffusional pathway for the permeation of solutes (drugs) through the polymer matrix.

Experimental Synthesis The monomers employed throughout this research were N-tertbutylacrylamide (NtBAAm, Sigma Aldrich), N-isopropylacrylamide (NIPAAm, TCI Europe), N-vinyl-2-pyrrolidone (NVP, Lancaster Synthesis) and N,N-dimethylacrylamide (DMAAm, Sigma Aldrich). All monomers were used as received. UV-curable formulations were prepared by dissolving the initiators in the neat (solvent and water-free) monomers. The two UV light-sensitive initiators used, 1hydroxy-cyclohexylphenylketone (Irgacure® 184) and 2hydroxy-2-methyl-1-phenyl-propanone (Irgacure® 2959), Ciba Specialty Chemicals, were added at 3 wt% with respect to the total weight of the monomers. The mixtures were continuously stirred until all the components were completely dissolved. The solutions were then pipetted into a silicone mould with disk impressions (for samples to be used in swelling studies). The mould was placed horizontally under two UVA 340 UV lamps (Q-panel products) and left for approximately 7 h in an enclosed environment under atmospheric conditions for the solution to cure. The gels were carefully turned over after approximately three and a half hours curing to ensure the entire surface area received the same radiation intensity during the photopolymerisation process. The samples were then dried in a vacuum oven at 30 °C for at least 24 h before use. The hydrogels described in the tables below were prepared using both photoinitiators, Irgacure® 184 and Irgacure® 2959. Table 1 shows the compositions of the copolymer hydrogels synthesised using NtBAAm, and either NVP or DMAAm, while Table 2 shows the compositions of the ternary polymer hydrogels that were fabricated using NtBAAm, NIPAAm and either NVP or DMAAm. Selected hydrogels investigated in this study were synthesised via chemical crosslinking using a more sophisticated

Synthesis and characterisation of thermo-sensitive terpolymers Table 1 Composition of physically-crosslinked copolymer hydrogels (Batch A) with NtBAAm and either NVP or DMAAm in their monomeric feed ratio (with 3 wt% photoinitiator content) NtBAAm (wt%)

NVP (wt%)

DMAAm (wt%)

10 20 30 40 50 10 20 30

90 80 70 60 50 – – –

– – – – – 90 80 70

UV curing system (Dr. Gröbel UV-Electronik GmbH). The aforementioned irradiation chamber is a controlled radiation source with 20 UV-tubes which provides a spectral range of between 315–400 nm. The ability to control the mode of operation of the irradiation chamber provides for three different intensity ranges; high intensity (10–13.5 mW/cm2), medium intensity (9 mW/cm2) and low intensity (0.6– 4.4 mW/cm2). These terpolymer hydrogels were again prepared using both Irgacure 184® and Irgacure 2959® photoinitiators, but at a concentration of 0.25 wt% with respect to the total weight of the monomers. The photoinitiator ratio was chosen following trials which showed satisfactory results at 0.25 wt%; the concentration of the photoinitiator was initially investigated at various concentrations (2 wt%, 1 wt%, 0.5 wt% and 0.25 wt%). The crosslinking agent used was polyethylene glycol 600 dimethacrylate (PEGDMA 600, Sigma Aldrich). Different quantities of crosslinker were employed with a view to investigating the effect of crosslinker content on hydrogel properties. Table 3 lists the hydrogel samples with differing monomeric feed ratios as were synthesised using the Dr. Gröbel UV-Elektronik GmbH system. The samples Table 2 Composition of physically-crosslinked terpolymer hydrogels (Batch B) with NtBAAm, NIPAAm, and either NVP or DMAAm in their monomeric feed ratio (with 3 wt% photoinitiator content) Hydrogel Name

NtBAAm (wt%)

NIPAAm (wt%)

NVP (wt%)

DMAAm (wt%)

H1

5

30

65

–

H2 H3 H4 H5 H6 H7 H8

5 10 15 5 5 10 15

35 30 20 30 35 30 20

60 60 65 – – – –

– – – 65 60 60 65

2309 Table 3 Composition of chemically crosslinked terpolymer hydrogels synthesised using the Dr. Gröbel UV-Elektronik GmbH system and the different ratios of crosslinker used (with 0.25 wt% photoinitiator content) Hydrogel Name

NtBAAm (wt%)

NIPAAm (wt%)

NVP (wt%)

(PEGDMA 600) (wt%)

H3 H3 H3 H4 H4 H4

10 10 10 15 15 15

30 30 30 20 20 20

60 60 60 65 65 65

0.1 0.25 0.4 0.1 0.25 0.4

were again dried in a vacuum oven at 30 °C for at least 24 h before use. Attenuated total reflectance Fourier transform infrared spectroscopy Attenuated total reflectance Fourier transform infrared spectroscopy was carried out on a Perkin Elmer Spectrum One fitted with a universal ATR sampling accessory. All data was recorded at room temperature, in the spectral range of 4000 to 650 cm−1, utilising a 16 scan per sample cycle and a fixed universal compression force. Nuclear magnetic resonance (NMR) spectroscopy NMR spectra were measured on a JEOL LAMBDA 400 MHz NMR machine with deuterated chloroform (CDCl3) as the solvent and tetramethysilane (TMS) as the reference. The samples were obtained by dissolving 60 mg of the polymer in 1 ml of CDCl3 over 2 days in a sealed vial. Assignments were supported by DEPT and COSY spectra. Sample H4 (15 wt% NtBAAm, 20 wt% NIPAAm, 65 wt% NVP; 3 wt% Irgacure 184) δH(CDCl3) 1.13 (d broad, Me, isopropyl), 1.34 (s, Me, t-butyl), 1.57–1.76 (m, polyvinyl methylene from NVP and polyvinyl methylene and methine from NtBAAm and NIPAAm), 2.00 (m, CH2, pyrrolidinone), 2.36 (m, CH2, pyrrolidinone, α to C=O), 3.10 (s, H2O), 3.32 (m, CH2 pyrrolidinone, α to N), 3.74 (m, CH, isopropyl), 3.94 (m, CH, polyvinyl methine from NVP), 6.47 (br, N-H, amide). δc 18.39 (CH2, pyrrolidinone), 21.52 (CH2), 22.59 (CH3, isopropyl), 28.76 (CH3, t-butyl), 31.81 (br, CH2, pyrrolidinone, α to C=O), 35.38 (br, CH2, polyvinyl methylene from NVP, NtBAAm, NIPAAm, overlap), 41.61 (CH, isopropyl), 41.96 (br, CH2, pyrrolidinone, α to N), 43.30 (CH), 44.89 (CH), 48.1 (br, CH, polyvinyl methine from NVP), 51.24 (C, t-butyl), 174–176 (C=O, amide). Sample H3 (10 wt% NtBAAm, 30 wt% NIPAAm, 60 wt% NVP; 3 wt% Irgacure 184) δH(CDCl3) 1.10 (d broad, Me,

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isopropyl), 1.28, 1.31 (Me, t-butyl), 1.56–1.76 (m, polyvinyl methylene from NVP and polyvinyl methylene and methine from NtBAAm and NIPAAm), 1.97 (m, CH2, pyrrolidinone), 2.33 (m, CH2, pyrrolidinone, α to C=O), 2.81 (s, H2O), 3.20 (m, CH2 pyrrolidinone, α to N), 3.74 (m, CH, isopropyl), 3.93 (m, CH, polyvinyl methine from NVP), 6.35 (br, N-H, amide). δc 18.39 (CH2, pyrrolidinone), 21.52 (CH2), 22.63 (CH3, isopropyl), 25.39 (CH2), 28.79 (CH3, t-butyl), 31.03, 31.73 (CH2, pyrrolidinone, α to C=O), 36.08 (br, CH2, polyvinyl methylene from NVP, NtBAAm, NIPAAm, overlap), 41.61–42.79 (CH, isopropyl, CH2, pyrrolidinone, α to N), 43.74 (CH), 44.93 (CH), 48.13 (br, CH, polyvinyl methine from NVP), 51.09, 51.28 (C, t-butyl), 174.10, 175.41, 175.69 (br, C=O, amide). The chemically crosslinked hydrogels were not soluble in chloroform, toluene, carbon tetrachloride, THF, acetonitrile or methanol and thus no NMR data was obtained. Glass transition determination Differential scanning calorimetric measurements were carried out using a TA Instruments 2920 DSC (AGB Scientific Ltd.) with MDSC capability and a refrigerated cooling system. Dried xerogel samples of between 8 and 10 mg were weighed out using a Sartorius scale, and tightly sealed into DSC pans. All measurements were conducted in crimped non-hermetic aluminum pans by heating the samples at a rate of 10 °C/min from 20 to 200 °C, with an empty crimped aluminum pan being used as the reference cell. The glass transition temperature was considered at the mid-point temperature of the endothermic drift in the heating curves. All DSC measurements were carried out nitrogen atmosphere to prevent oxidation. Calibration was previously performed on the machine using indium as standard. Preparation of aqueous copolymer solutions Homogeneous solutions of the hydrogels were prepared, by weighing appropriate amounts of the xerogels and distilled water, leaving these mixtures at room temperature for a period of hours/days, while applying gentle stirring with the use of magnetic stirrers. The aqueous polymer solutions were prepared for subsequent phase transition temperature measurements using cloud-point analysis and modulated differential scanning calorimetry (MDSC).

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tubes. The temperature was gradually increased at a rate of about 1 °C per minute until the solution began to turn cloudy. Cloud points were determined visually at the first sign of turbidity using a thermometer with an accuracy of ±0.2 °C. All tests were carried out in triplicate and pictures were taken of the samples to show the contrasting visual appearance of the solutions above and below phase transition temperature. Modulated differential scanning calorimetry The DSC method was also among the techniques used for the examination of the phase transition phenomena exhibited by these thermo-sensitive gels in aqueous solutions. The analyses were performed using the same MDSC machine apparatus stipulated in “Glass transition determination” section. Aqueous samples of the hydrogels of between 9–11 mg were weighed out and transferred by syringe into the DSC pans. Aluminium pans were crimped before testing, with an empty crimped aluminium pan being used as the reference cell. Calorimetric scans were carried out at temperatures ranging between 10 and 60 °C at a scanning rate of 1 °C/min under nitrogen atmosphere. Swelling studies Swelling and solubility testing The swelling and dissolution characteristics of the physical gels were investigated in duplicate at temperatures ranging between 20 and 60 °C. Samples of the polymer xerogels with a mass of 1.3±0.15 g were placed in a Petri dish; the Petri dish was filled with the appropriate solution (i.e. distilled water) and placed at 20 °C or in a fan-assisted oven at the required temperature. Petri dishes were covered with the lids and Petri seal (Diversified Biotech Ltd.) while in the oven to prevent evaporation. Periodically, excess polymer solution was removed after predetermined time intervals by pouring the solution through a Buchner funnel. The samples were blotted free of surface water with filter paper, and the wet weight of the gel sample was measured at room temperature. The samples were re-submerged in fresh distilled water, and returned to the bench or oven. The percentage that the hydrogels swelled was calculated using the formula below; Swellingð%Þ ¼ Wt =Wo  100

ð1Þ

Phase transition determination Cloud point measurement The cloud point temperatures were taken in a thermostable bath by immersing the solutions in 100 mm sealed glass test

where Wt is the weight of the swollen gel at a predetermined time and Wo is the dry mass of the gel. In order to provide a clear pictorial demonstration of the swelling behaviour of the gels and for comparative reasons, pictures of the swollen samples were taken after the removal of the

Synthesis and characterisation of thermo-sensitive terpolymers

polymer solution. This process was continued until the sample appeared to have dissolved or for up to 96 h. Swelling/deswelling kinetics The chemically crosslinked hydrogel samples were allowed to reach equilibrium swelling in distilled water at ambient temperature (following a similar procedure as in “Swelling and solubility testing” section). Upon attaining equilibrium swelling, hydrogels were then transferred into Petri dishes containing distilled water at a temperature of 60 °C. At predetermined time intervals, the samples were removed from the water, excess distilled water solution was removed by pouring the solution through a Buchner funnel and the surface of the hydrogel was wiped with wet filter paper and the hydrogel weighed. Pictures of the swollen samples were taken after the removal of the distilled water solution. After 48 h, the samples were again subjected to a room temperature swelling environment, and once more weighed at predetermined intervals after removing the distilled water, to determine the response of the hydrogels to environmental temperature change. This process was again repeated to investigate the cyclic swelling behaviour in response to alternating temperature change. The percentage swelling of the hydrogels was calculated as per Eq. 1. In order to examine the speed of response of the hydrogels, equilibrium swollen samples of the same hydrogels were subjected to temperature changes by quickly transferring them into distilled water kept at desired temperatures of 20 °C and 45 °C over one hour cycles. The weight changes of the hydrogels were recorded using the same method as described previously. The temperaturestimulating swelling-deswelling kinetics of the hydrogels was investigated at hourly intervals so as to ascertain how quickly the hydrogels respond to thermal stimuli over a relatively narrow temperature range and time interval.

Results and discussion Synthesis of samples All of the hydrogels in this study were synthesised using free-radical bulk polymerisation, which allowed the samples to be polymerised in the absence of any diluents under ultraviolet (UV) light. The commercially available CIBA photoinitiator, Irgacure® 2959 is a highly efficient radical photoinitiator for UV curing systems. Due to its tolerance over a wide range of cell types and chemical concentrations and its low cellular toxicity [18, 21], this photoinitiator has also been employed in the photopolymerisation of polymers and copolymers intended for biomedical and tissue engineering applications. Irgacure® 184 is also

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another efficient commonly used photoinitiator which is similar to Irgacure® 2959 as they are both α-hydroxyphenylketone photoinitiators. Homopolymers of N,N-dimethylacrylamide (DMAAm) and N-vinyl-2-pyrrolidone (NVP) were synthesised using both Irgacure® 184 and Irgacure® 2959 photoinitiators. This was carried out to ascertain whether the photoinitiators were suitable for polymerisation of these monomers in a nitrogen-free environment. Irgacure® 184 is well-known for having an excellent curing efficiency in air and is also claimed to be the most important commercial nonyellowing photoinitiator [22]. Eliminating the need for nitrogen during the synthesis would make the curing process much more cost-effective if it were to be applied on an industrial scale. Nitrogen is usually employed to maintain an inert environment during free-radical polymerisation processes because atmospheric oxygen strongly inhibits the chain reaction by scavenging both initiating and polymer radicals. Hence, the most effective way to overcome oxygen inhibition is to work in an inert atmosphere, by flushing with nitrogen [23, 24] or carbon dioxide [25]. Physically crosslinked hydrogels The more viable xerogels were transparent and glass-like in appearance after UV curing. NVP and N-tert-butylacrylamide (NtBAAm) were copolymerised at varied feed ratios using both initiators. The process allowed for successful polymerisation of the copolymers at low NtBAAm contents; NtBAAm/NVP (30:70; 20:80; 10:90). However, at higher ratios; NtBAAm/NVP (40:60; 50:50), the solid NtBAAm monomer became insoluble. Initial cloud point tests on aqueous samples of these copolymers in the ratios of 10:90, 20:80 and 30:70 did not yield satisfactory results. The samples did not turn completely opaque even at higher temperatures. Furthermore, MDSC tests did not detect any phase transition, hence these samples were not deemed satisfactory for further analysis (see also “Phase transition determination” section). Copolymerisation of DMAAm and NtBAAm was also attempted using both initiators, and this produced similar results; the NtBAAm was not soluble in monomer ratios as low as 20:80 (NtBAAm/DMAAm). Terpolymers with different monomer weight ratios were then synthesised in an effort to balance the hydrophobic and hydrophilic moieties to obtain hydrogels with a desired LCST. A batch of terpolymers; P(NtBAAm-NIPAAm-DMAAm) and P(NtBAAm-NIPAAm-NVP), were synthesised at varied feed ratios (see Table 2). The P(NtBAAm-NIPAAm-DMAAm) samples showed a considerable degree of yellowing, took longer time to cure and were also slightly tacky to the touch. The terpolymers of P(NtBAAm-NIPAAm-NVP) at different

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feed ratios displayed the desired characteristics. The physical appearance of the xerogels was good, and aqueous solution of the terpolymers exhibited LCSTs at temperatures ranging between 30 °C and 48 °C, as confirmed using cloud point and MDSC analyses (see Phase transition determination section). A previous study has shown that the lower the viscosity of a formulation, the higher the inhibitory effect of oxygen on the polymerisation due to faster oxygen diffusion [26]. As the NVP monomer used in this study is more viscous than the DMAAm monomer, this may account for the P(NtBAAm-NIPAAm-NVP) formulation curing with greater consistency than the P(NtBAAm-NIPAAMDMAAm) samples. The inclusion of NVP in photopolymer formulations has also been reported to dramatically reduce oxygen inhibition [27]. Polymerisation of all the physically crosslinked samples in this study took approximately 7 h and was carried out using the UV 340 curing system and at 3 wt% photoinitiator concentration as per supplier recommendation. Chemically crosslinked hydrogels Stimuli-responsive polymers have been utilised in various forms, one of which includes covalently crosslinked (permanent) hydrogels. Where linear polymers undergo inverse temperature solubility at the LCST, these types of hydrogels undergo thermoreversible swelling/deswelling at the transition temperature. This temperature which induces gel swelling/deswelling usually corresponds to the phase transition temperature of the physically crosslinked polymer. Importantly, chemical crosslinking affords the opportunity to increase the strength of a hydrogel and improve its mechanical properties for use in applications where dilution of the hydrogel matrix and diffusion of the polymer away from target sites are undesirable. Poly(NtBAAm-NIPAAm-NVP) terpolymers were prepared via UV polymerisation in the presence of polyethylene glycol 600 dimethacrylate (PEGDMA 600) as crosslinker. In this instance, the photoinitiator amounts were reduced to 0.25 wt% with respect to the total weight of the monomers as opposed to the 3 wt% employed in the formulation of the physical hydrogels. This initiator concentration, as earlier stated, was chosen after trials at various concentrations. The reduction of the photoinitiator content is desirable as the intended use of the hydrogels investigated in this study is drug-delivery related. The cellular toxicity of ultra violet lightsensitive photoinitiators has been widely investigated [18, 21, 28] and hence reducing the amount of photoinitiator employed is crucial from a toxicological point of view. The monomer compositions selected were based on the formulation which proved most effective for the physical hydrogels. Thus, chemically crosslinked hydrogels were prepared based on hydrogels H3 and H4 from Batch B (see Tables 2

C.L. Higginbotham et al.

and 3) as these samples exhibited LCSTs in distilled water that were relatively close to body temperature. The crosslinked samples were prepared using both Irgacure 184 and Irgacure 2959 photoinitiators in the presence of PEGDMA 600 in varied amounts. In this study, three different quantities of crosslinker were employed; 0.1 wt%, 0.25 wt% and 0.4 wt%, all with respect to the total weight of the monomers. All the chemically crosslinked samples used in this study were polymerised using the Dr. Grobel UV-Elektronik GmbH UV curing system. At the high intensity mode, the hydrogels cured relatively fast and exhibited the best aesthetic appearance of all the samples, requiring a curing time of 20 min. The use of more lamps (20 UV-tubes) and the high intensity range (10– 13.5 mW/cm2) accelerated the curing process when compared to the system utilising two UVA 340 lamps. Higher light intensities help increase efficiency when carrying out UV polymerisation in non-inert environments. Studer et al. [26] had reported an increase in reactivity ratio CO2/air from a value of 1.3 at a light intensity of 90 mW/cm2 up to 20 at 15 mW/cm2 in the UV curing of thick coatings. Fourier transform infrared spectroscopy (FTIR) The ATR-FTIR spectra of NtBAAm, NIPAAm, NVP and hydrogel H4 are illustrated in Fig. 1. The monomers displayed characteristic absorption bands at 1654/1626 cm−1 (NtBAAm) [29, 30], 1655/1619 cm−1 (NIPAAm) [31, 32] and 1694/1625 cm−1 (NVP) [33]. These bands are assigned to the carbonyl group C=O bond stretching vibration and the C=C bond stretching vibration. The disappearance of the C=C bond peaks in the H4 sample spectra indicates successful polymerisation of the terpolymers. The absorbance bands at 1555 cm−1 for NtBAAm monomer and 1547 cm−1 for NIPAAm are attributed to the secondary amide N-H bending [29, 31, 32]. The NtBAAm spectrum also showed absorption bands at approximately 1392/1359/1222 cm−1 which has been previously reported to be associated with the tertiary butyl groups (−C-(CH3)3) [30, 34, 35]. These bands appeared in the terpolymer spectrum, confirming the presence of NtBAAm in the structure. The characteristic double band for the isopropyl group at ∼1385 cm−1 and ∼1367 cm−1 [31, 36], which were discernable in the NIPAAm monomer spectrum, were also present in the terpolymer spectrum. Vinyl groups in the NIPAAm monomer could be identified at peaks between 986 and 916 cm−1 [37], while the NVP monomer exhibited strong peaks at 841 cm−1 and 980 cm−1 in agreement with reports that it shows strong peaks in the range of 800–1000 cm−1 corresponding to the mode of vinyl double bonds (CH2=CHR) [33, 38]. These characteristic monomeric peaks subsequently failed to appear in the

Synthesis and characterisation of thermo-sensitive terpolymers Fig. 1 FTIR spectra of the constituent monomers and the H4 hydrogel terpolymer

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Terpolymer

NIPAAm

%T NtBAAm

NVP

3400.0 3200

2800

2400

2000

1800

1600

1400

1200

1000

800

650.0

cm-1

H4 terpolymer spectra; further indicating polymerisation of the terpolymer. NMR spectroscopy, which is a much more powerful technique in the determination of the structure of unknown compounds and polymers, was also used to further elucidate the chemical structure of the terpolymers. Nuclear magnetic resonance (NMR) analysis The NMR spectra proved that all the monomers were incorporated into the polymeric structure. All peaks were assigned by the COSY spectrum (see Fig. 2). In the case of the sample H4 with 15 wt% NtBAAm, 20 wt% NIPAAm and 65 wt% NVP as monomers, the 1H NMR spectrum, Fig. 2, of the resultant polymer (washed sample) shows the methyl groups of the isopropyl function at 1.13 ppm and the methyl groups of the t-butyl function at 1.34 ppm. The integration shows a greater percentage of the isopropyl group in the terpolymer relative to the t-butyl group (1:1.36) in the hydrogel consistent with the higher percentage of the NIPAAm monomer used (1:1.50 moles). The methine proton of the isopropyl group appears at 3.74 ppm and is confirmed by the COSY spectrum (see Fig. 2). Evidence of the incorporation of the NVP monomer into the hydrogel is seen in the three peaks at 3.23 ppm, 2.36 ppm and 2.00 ppm corresponding to the three methylene protons of the pyrrolidinone ring. These chemical shifts are very similar to that reported for poly(N-vinyl-2pyrrolidone) in CDCl3 [39, 40]. The peak at 3.23 ppm corresponds to the protons α to the N of the lactam ring, the peak at 2.36 ppm is due to the protons α to the C=O function of the cyclic amide and the peak at 2.00 ppm is the

remaining methylene protons of the heterocycle. The polyvinyl methine proton arising from NVP is at 3.94 ppm, with an integration half that of the peaks due to the methylene protons of the pyrrolidinone ring. The polyvinyl methylene protons arising from NVP are at 1.57– 1.76 ppm. Based on the COSY spectrum this tails into the region close to the peak due to the methyl groups of the isopropyl function. The polyvinyl methylene and methine protons arising from NtBAAm and NIPAAm also overlap in the region 2.00–1.5 ppm. The amide N-H appears as a very broad and flat peak at 6.47 ppm. H2O appears in the spectrum at 3.10 ppm. The 13C spectrum of sample H4, Fig. 2, has three sets of peaks between 174–176 ppm consistent with the C=O of the amide group arising from all three monomers in the terpolymer. The methyl carbons of the t-butyl group are at 28.76 ppm while the methyl carbons of the isopropyl group are at 22.59 ppm. The methine carbon of the isopropyl group is at 41.61 ppm and the quaternary carbon of the tbutyl group is at 51.24 ppm. These assignments were further reinforced by comparing the 13C spectra of samples with different wt% of NtBAAm and NIPAAm, including samples in which one of the monomers was not used in the synthesis. Peaks between 18–43 ppm, combined with the DEPT 135 spectrum, are also consistent with the NVP monomer being incorporated into the hydrogel structure. The peaks centered at 41.96 ppm corresponds to the ring carbon α to the N of the lactam ring, the peaks centered at 31.81 ppm are due to the carbon α to the C=O function of the cyclic amide and the peaks centered at 18.39 ppm the remaining carbon of the heterocycle. The polyvinyl methylene carbon arising from NVP is centered at

2314 Fig. 2 1H, 13C, DEPT-135 and COSY spectra for hydrogel H4

C.L. Higginbotham et al.

Synthesis and characterisation of thermo-sensitive terpolymers

35.38 ppm and the polyvinyl methine carbon arising from NVP is centered at 48.1 ppm. The signals are again similar to the 13C spectrum reported for PVP in CDCl3 [41]. The peaks due to the polyvinyl methylene carbon and methine carbon arising from NtBAAm and NIPAAm are not distinct and in comparison to the reported 13C spectrum for polyacrylamide [42, 43], the former overlaps with the peaks that are centred at 35.38 ppm, and the latter with the peaks that are centred at 41.61 ppm. There are also signals at 43.30 ppm and 44.89 ppm, which based on the DEPT spectrum, could be due to the polyvinyl methine carbon arising from NtBAAm and NIPAAm. The 1H and the 13C NMR spectra for the polymer H3, Fig. 3, with 10 wt% NtBAAm, 30 wt% NIPAAm and 60 wt% NVP as monomers were similar to H4 consistent with the formation of a terpolymer. Based on the integration of the 1H NMR spectrum the mole ratio of the monomers incorporated into the polymer H4 is 1:1.36:2.43 (NtBAAm: NIPAAm: NVP) whereas the mole ratio of the monomers is 1:1.5:4.96 (66.5% mole fraction NVP). Thus 50.7% (mole fraction) of the hydrogel H4 is due to the NVP monomer. The ratio of the monomers incorporated into the hydrogel H3 is 1:3.31:3.61 (NtBAAm: NIPAAm: NVP) whereas the mole ratio of the monomers is 1:3.37:6.86 (61% mole fraction NVP). Thus 46.4% (mole fraction) of the hydrogel H3 is due to the NVP monomer. The higher mole fraction for sample H4, is consistent with the higher concentration of NVP used in the radical polymerisation. The literature reactivity ratios for these types of monomers [44, 45] are consistent with the formation of a random terpolymer as opposed to a blend of homopolymers. The reactivity ratios were reported to be 0.61 and 0.05 for acrylamide and N-vinylpyrrolidone, respectively [44] with N-alkylated acrylamides kinetically slower than acrylamide. The product of the reactivity ratios, indicate a tendency towards a random distribution of the monomers along the chains. The low incorporation of NVP into the terpolymer, relative to its high feed composition, is consistent with the lower reactivity ratio for NVP relative to the acrylamides [44]. This specificity in the radicalmonomer reaction has been related to dissimilarity in the polarisation properties of the vinyl monomer [46]. The radical formed as a result of addition to the NVP monomer is nucleophilic (σ-) and this carbon centred radical favours addition to the unsubstituted end (β-position) of the alkene function of NtBAAm and NIPAAm over addition to the unsubstituted end of the alkene function of NVP during propagation. This preference is due to the partial positive charge (σ+) on the β-vinyl carbon of NtBAAm and NIPAAm, due to resonance from the carbonyl group, and the partial negative charge (σ-) on the β-vinyl carbon of NVP, due to resonance from the lone pair of the nitrogen. This polar effect counteracts to some degree the higher

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concentration of NVP and is a well-documented factor in radical addition reactions [47]. The radical generated from the photoinitiators Irgacure 184 and 2959 are nucleophilic alkyl radicals (.CRR-OH) and they react faster with the more electron deficient vinyl group of NtBAAm and NIPAAm compared to the more electron rich vinyl group of NVP. This will also be a factor in the mole fraction of the hydrogel. Analysis by NMR shows that Irgacure 2959 is more effective for polymerisation than Irgacure 184 (3 wt%) for this chemical system and curing conditions, despite the lower number of moles used, as less unreacted monomer NVP was observed in the hydrogel when Irgacure 2959 was used. Irgacure 184 resulted in an 82% conversion of NVP into the hydrogel for sample H3, whereas Irgacure 2959 resulted in a 94% conversion. Sample H4, with a higher feed concentration of NVP, showed a 91% conversion of NVP with Irgacure 184. The mole fraction of NIPAAm in the hydrogel was found to decrease when Irgacure 2959 was used as the photoinitiator compared to Irgacure 184. The mole fraction of NtBAAm was found to increase and NVP decreased. When lower wt% of the photoinitiator was used the amount of the unreacted monomer NVP present in the unwashed polymer increased as expected. Sample H4 showed an 81% conversion of NVP with 1.0 wt% Irgacure 184 whereas a 0.25 wt% resulted in a 77% conversion. This compares to the above 91% conversion with 3 wt% Irgacure 184. NtBAAm and NIPAAm monomers were not detected in these samples due either to the lower wt% of the acrylamide monomers used or to their greater incorporation into the final polymeric structure due to the above kinetics or to both. The intensity of the lamp and irradiation time also reduced the amount of unreacted NVP monomer based on NMR analysis. High intensity on the Dr. Gröbel UVElectronik GmbH UV curing system gave a lower amount of unreacted monomer compared to the low intensity mode, including when the sample was irradiated for half the time at the high intensity. The UVA 340 UV lamps gave an intermediate result. The NMR spectra also show that the washing procedure (samples swelled in excess distilled water to equilibrium weight and the external solution changed daily) is very effective in removing the unreacted NVP monomer from the hydrogels. Samples H4 and H3 showed no monomers present after washing including when Irgacure 184 was used as the photoinitiator. Glass transition temperature The thermal behaviour of selected terpolymer samples was studied in duplicate using MDSC. Single endothermic transitions were observed for all the different hydrogel samples, which in conjunction with the NMR results

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Fig. 3

C.L. Higginbotham et al.

1

H and

13

C spectra for hydrogel H3

indicates that the hydrogels are terpolymers and not blends. Glass transition temperatures (Tg) ranging from about 77 °C to 102 °C were observed for all the samples tested. Glass

transition temperatures for PtBAAm, PNIPAAm and PVP have been reported at 108–128 °C, 111–135 °C and 54– 175 °C, respectively [36, 48–50]. Most blends would

Synthesis and characterisation of thermo-sensitive terpolymers

usually give two or more Tgs depending on the number of polymers in the blend. The average Tg of the linear samples were 79 °C and 97 °C, for H4 hydrogel synthesised using Irgacure 184 and Irgacure 2959, respectively. This is also consistent with the NMR result, which suggests increased polymerisation when Irgacure 2959 is employed, as a higher degree of polymerisation usually leads to an increase in Tg (Fig. 4). Phase transition determination The phase transition temperature can be determined by both DSC analysis, giving an endothermic transition peak, and by cloud point measurement, giving the cloud point value [51]. The phase transition of a temperature-sensitive polymer is an endothermic process corresponding to the disruption of the hydrogen bonds in the solution; hence the thermal analysis technique measures the heat resulting mainly from the breaking down of these hydrogen bonds between the water and the polymer [52]. The cloud point test visualises the clouding of the solution due to the precipitation of the polymer as the phase transition occurs. Copolymerisation of NIPAAm with the more hydrophobic monomer NtBAAm reduces the LCST of the resulting copolymer compared to that of PNIPAAm [53]. It has previously been reported that LCSTs for copolymers with monomer ratios NIPAAm/NtBAAm 85:15, NIPAAm/ NtBAAm 65:35, NIPAAm/NtBAAm 50:50, are 25 °C, 17 °C and 12 °C, respectively [54]. Liu & Zhu [16], by extrapolation of the experimental results of LCSTs of some

Fig. 4 MDSC curve for H4 hydrogels synthesised with Irgacure 2959 showing Tg

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copolymers, reported a hypothetical LCST value for PtBAAm homopolymer of −5 °C; hence making NtBAAm one of the most hydrophobic N-substituted acrylamide monomers. Cloud point determination The phase transition temperature of the physical hydrogel samples was measured first, using the cloud point technique by observing changes in the turbidity of the aqueous solution with increasing temperature (note LCST was taken at the onset of turbidity). All of the aqueous polymer samples were homogeneous and transparent at low temperatures, but once the temperature reached the phase transition temperature, the solutions became turbid. The copolymer samples listed in Table 1 did not exhibit the desired phase transition properties. The terpolymer samples synthesised with Irgacure® 184 precipitated in solutions at least 1 °C before those made using Irgacure® 2959. These samples synthesised with Irgacure 2959 photoinitiator also exhibited broader transitions by taking longer for their solutions to turn completely opaque. This is consistent with the above NMR analysis, which shows a lower degree of polymerisation with Irgacure 184. Since higher percentage of NVP monomer is involved in the polymerisation when Irgacure 2959 is used, the resultant terpolymer exhibits more hydrophilicity than the hydrogels synthesised with Irgacure 184. The P(NtBAAm-NIPAAm-DMAAm) samples did not appear as opaque as the P(NtBAAm-NIPAAm-NVP)

0.06

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Heat Flow (W/g)

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-0.06

97.38°C -0.08 50 Exo Up

70

90

110

130

Temperature (°C)

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Universal V3.0G TA Instruments

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samples as the temperature was increased above the cloud point temperature. In fact, there only remained a slight cloudiness, even at higher temperatures. This might be due to the high hydrophilicity of DMAAm which is one of the most hydrophilic N-substituted acrylamide monomers. A hypothetical LCST for the homopolymer of PDMAAm obtained by extrapolation was given as >200 °C [16]. The P(NtBAAm-NIPAAm-NVP) samples showed a much more distinct opacity at temperatures above the LCST. It was noted that none of the copolymer/terpolymer solutions in this study underwent thermo-reversible gelation above the cloud point temperature at the concentrations tested. Table 4 shows the cloud point temperatures as were observed using 5 wt% concentrations of aqueous polymer. Increase in the feed ratio of the NtBAAm had the most influence on the cloud point temperature of the P(NtBAAmNIPAAm-NVP) and P(NtBAAm-NIPAAm-DMAAm) samples. It was observed that the cloud point temperature of the terpolymers decreased as the ratio of the hydrophobic NtBAAm was increased, with respect to the other monomers. As the ratio of NtBAAm was increased from 5 wt% in both H1 and H2, to 10 wt% and 15 wt% in H3 and H4 respectively, the cloud point temperature subsequently decreased (H1 and H2: 48 °C; H3: 42 °C and H4: 37 °C). This shows the correlation between the LCST and the hydrophobicity of the polymer as would be expected. Modulated DSC analysis Conventional DSC provides information about the overall heat flow measured as a function of temperature or time while modulated DSC provides information about the reversing (heat capacity component of the total heat flow) and non-reversing (kinetic component of the total heat flow) characteristics of thermal events. Geever et al. [55] reported that the reversing heat flow yields a much more

Table 4 LCSTs of the terpolymer hydrogels as observed using the cloud point analysis Hydrogel Name/Composition

LCST (°C) LCST (°C) Irgacure Irgacure 184 2959

H1 (NtBAAm/NIPAAm/NVP 5/30/65)

48

49

H2 (NtBAAm/NIPAAm/NVP 5/35/60) H3 (NtBAAm/NIPAAm/NVP 10/30/60) H4 (NtBAAm/NIPAAm/NVP 15/20/65) H5 (NtBAAm/NIPAAm/DMAAm 5/30/65) H6 (NtBAAm/NIPAAm/DMAAm 5/35/60) H7 (NtBAAm/NIPAAm/DMAAm 10/30/60) H8 (NtBAAm/NIPAAm/DMAAm 15/20/65)

48 42 37 48 48 45 40

49 42 38 50 50 46 41

defined endotherm than the other signals and so provides much greater sensitivity in LCST determination, when compared with conventional DSC. Therefore, the LCST endotherm was examined using the reversing heat flow signal throughout this study. The tests were carried out at concentrations of 3 wt% and 5 wt% polymer solution. At 5 wt% polymer concentrations, the endothermic peaks appeared to be more distinct as would be expected. In this work, peak maximum values of the thermogram were recorded. Figure 5 shows a representative MDSC scan (at 5 wt% polymer concentration) of hydrogel H4. Results from the MDSC analysis (average from tests carried out in duplicate) are presented in Table 5. As well as shifting the LCST of the terpolymer hydrogels to higher temperatures, less NtBAAm content led to a broadening of the phase transition region on the MDSC curve. However MDSC tests did not detect a phase transition endotherm for the P(NtBAAm-NIPAAmDMAAm) hydrogel samples, again possibly due to the greater hydrophilicity of the DMAAm material as discussed in the previous section. In line with the intended drug delivery application of the hydrogels, the samples that had LCSTs close to physiological temperature (37 °C) were selected for further testing, namely hydrogels H3 and H4. The MDSC analyses detected phase transitions that corresponded well with the cloud point analysis results. Consistent with cloud point results, hydrogel samples synthesised using Irgacure 2959 showed phase transitions that were between 1–2.5 °C higher than those were Irgacure 184 was used. Swelling studies Physically crosslinked hydrogels The swelling and solubility properties of some of the physical terpolymer xerogels were investigated under differing environmental conditions, namely, room temperature (20 °C) and 60 °C. In contrast to permanent networks formed by chemical crosslinking, physically crosslinked hydrogels swell upon contact with a thermodynamically compatible solvent, only to dissolve with time. When the swollen polymer reaches a critical value, chain displacement sets in and the polymer begins to dissolve. This property was evident with the samples studied at the lowest test temperature; reaching a maximum swollen weight and eventually dissolving. The water solubility of physically crosslinked hydrogels is expected to increase with temperature, however for negative temperature sensitive polymers, their water solubility decreases with increase in temperature [56]. Each of the samples studied displayed similar characteristics with the highest percentage swelling in distilled water

Synthesis and characterisation of thermo-sensitive terpolymers Fig. 5 MDSC curve for hydrogel H4 at 5 wt% concentration showing its phase transition as represented by the reversing heat flow signal

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-0.040

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at 20 °C, which is well below the LCST of the samples (H3: 42 °C and H4: 37 °C). Also it was found that samples synthesised using Irgacure 2959 photoinitiator swelled to a greater degree than those made using Irgacure 184 at the lower temperature studied. For example, sample H3 fabricated using Irgacure 184, took 24 h to reach its maximum swollen weight (over 600%) and dissolved after about 48 h; but H3 synthesised using Irgacure 2959 took about 48 h to attain maximum swelling (over 800%) and a further 48 h to completely dissolve. A similar trend was observed with the H4 samples synthesised using both types of photoinitiator as can be observed in Figs. 6 and 7. This increase in maximum swelling for the hydrogel samples synthesised using Irgacure 2959 is consistent with the NMR results which show that more NVP is involved in the polymerisation when Irgacure 2959 is used, compared to Irgacure 184, hence the increase in hydrophilicity. The greater degree of polymerisation when Irgacure 2959 is employed as initiator may also have contributed to the time it took the hydrogels to reach equilibrium swelling and the

30

40

Temperature (°C)

50

60

Universal V3.0G TA Instruments

longer dissolution time. Rapid swelling in physically crosslinked hydrogels is generally associated with rapid dissolution. When immersed in aqueous solution at temperatures below their LCST, physically crosslinked hydrogels generally break down very quickly after reaching their maximum degree of swelling. On the other hand, samples of identical composition analysed at temperatures above their LCST can take a number of days to fully dissolve after attaining maximum swelling [55]. This may explain why hydrogel H4 at 60 °C (Fig. 7) appears to have broken down faster at the higher temperature, though if analysed over a longer time period this would prove not to be the case. At 20 °C, the sample is shown to have dissolved to nearly 200% of its maximum swollen size because that fraction of the sample still showed a thicker consistency than the swelling media, but practically it could be assumed

Table 5 Results of the MDSC analysis of the P(NtBAAm-NIPAAmNVP) hydrogels synthesised with Irgacure 184 Hydrogel Name

Cloud Point MDSC at 3 wt% MDSC at 5 wt% Test LCST (°C) in distilled H2O in distilled H2O LCST (°C) LCST (°C)

H1 H2 H3 H4

48 48 42 38

(5/30/65) (5/35/60) (10/30/60) (15/20/65)

47.72 47.60 41.11 36.54

47.37 47.38 41.32 36.26

Fig. 6 Swelling behaviour at different external temperatures of hydrogel H4 synthesised using Irgacure 184

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Fig. 7 Swelling behaviour at different external temperatures of hydrogel H4 synthesised using Irgacure 2959

that it had completely dissolved. When these samples were immersed in distilled water at 60 °C (well above their LCST), they did not swell to the same degree; became less soluble and had not dissolved even after 96 h. This is attributed to their inverse temperature dependent solubility, which is caused by the strong associative network formed due to the aggregation of the hydrophobic segments in the copolymer as a result of increasing temperature. These hydrogels have a short lifetime in aqueous media, therefore, would only be suitable for short-term drug release applications. Also, widespread application of physical hydrogels is often limited due to the weak mechanical strength and uncontrolled dissolution [57]. Subsequent studies in this research are therefore devoted to chemically crosslinked hydrogels only.

C.L. Higginbotham et al.

crosslinking degree. Previous work on the effect of crosslinking degree on the swelling behaviour of PNIPAAm hydrogels has shown that a lower degree of crosslinking leads to a higher swelling ratio [58]. However, high crosslink densities can significantly alter the physical properties of the hydrogel; particularly in terms of strength and brittleness. Thus, a compromise must be made in hydrogel design to achieve the optimum degree of swelling and mechanical properties for targeted applications. In this study, three different crosslinker ratios were employed in the fabrication of the crosslinked hydrogels; 0.1 wt%, 0.25 wt% and 0.4 wt%, with respect to the total weight of the monomers. The test temperatures used in this study, 20 °C and 60 °C, occur across the critical temperature values of the samples, as determined using cloud point and MDSC analyses. A typical illustration of the pulsatile swelling behaviour of these hydrogels in response to external temperature change can be seen in Fig. 8, using sample H4 that had been synthesised using Irgacure 2959. There was no apparent influence of photoinitiator type on the pulsatile swelling properties (see Fig. 9). The hydrogels were transparent in the swollen state at 20 °C. However, they changed from transparent to opaque and white upon immersion in distilled water at 60 °C, as can be observed in Fig. 10. Similar behaviour has been previously reported by Yoshida et al. [59] and was attributed to the formation of a heterogeneous structure having a denser surface skin than the interior of the gel matrix. Figure 8 displays the oscillatory swelling-shrinking kinetics of hydrogel H4 with varying crosslinker contents

Chemically crosslinked hydrogels Swelling Kinetics Temperature sensitive chemically crosslinked hydrogels are permanently crosslinked networks that undergo volume phase transition (swelling and shrinking) in aqueous media over a critical temperature range rather than dissolving. In an effort to determine the potential of these hydrogels for drug delivery applications where accomplishment of pulsatile release performances are desired, the on-off swelling behaviour of the hydrogels was investigated. This phenomenon involves the response of such environmentally sensitive hydrogels to pulses in temperature, and finds application in any situation where the release of a substance is triggered by a signal, such as a critical temperature. It is known that one of the main factors affecting the swelling ratio of chemically crosslinked polymers is the

Fig. 8 Pulsatile swelling behaviour of hydrogel H4 synthesised using Irgacure 2959 with varying crosslinker concentrations, in response to stepwise temperature changes between 20 °C and 60 °C

Synthesis and characterisation of thermo-sensitive terpolymers

Fig. 9 Pulsatile swelling behaviour of hydrogel H4, synthesised using Irgacure 184 and 2959 at 0.4 wt% crosslinker concentration, in response to stepwise temperature changes between 20 °C and 60 °C

over temperature cycles between 20 °C (below LCST) and 60 °C (above LCST) in distilled water. The effect of crosslink density is apparent, as the swelling degree decreased as the amount of crosslinker increased. Following immersion in distilled water at 20 °C for 168 h, H4 synthesised with Irgacure 2959 and 0.1 wt% crosslinker attained a swelling ratio of roughly 25, while the same hydrogel sample prepared using 0.25 wt% and 0.4 wt% crosslinker reached swelling ratios of approximately 18 and 14, respectively. The relatively lower swelling percentage attained during the second cycle is attributed to the shorter test period of 144 h, as opposed to the first cycle (168 h). A higher crosslink density intensifies a resistance to chain extension in the hydrogel structure, thereby reducing the equilibrium swelling degree as is evident. A lower crosslink density also creates a more porous structure (due to available space between macromolecular chains) which

Swelling (0hr)

Swelling (168hrs)

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enhances swelling and deswelling rates due to the ease with which water can diffuse in and out of the hydrogel matrix. Crosslinker concentration can also play a role in drug release with systems where the drug molecules are either very small or too large. Brazel & Peppas [60] reported that hydrogels with a higher degree of crosslinking and therefore a smaller mesh space would be better suited for releasing heparin (a low molecular weight drug) in a pulsatile manner. Wu et al. [61] also cited limited protein adsorption in hydrogels synthesised with higher crosslinker content due to increased network density in the gels and the large size of the protein molecules. A recurrent response is an important property for a temperature-sensitive hydrogel, as is a rapid response over a narrow temperature range. The oscillatory swelling and deswelling property of the hydrogels over one hour temperature cycles between 20 °C and 45 °C are displayed in Figs. 11 and 12. It was found that, although the hydrogels exhibited an oscillatory shrinking-swelling character, they did not shrink to the same degree as they did in the previous analysis, where temperature cycles of 20 °C and 60 °C were employed. This is due to the narrower temperature window and the shorter time allowed for response to the temperature change, which invariably has an effect on the pulsatile swelling behaviour. For instance, H3 (LCST ∼42 °C) synthesised using Irgacure 184 and 0.1 wt% crosslinker lost over 1,600% of its water content in just 2 h when immersed in distilled water at 60 °C but it lost only 230% in 1 h when immersed in pure water at 45 °C. The proximity of the higher test temperature (45 °C) to its phase transition temperature (42 °C) is likely the main reason for the reduced thermosensitivity compared to the H4 samples. Kaneko et al. [62] has previously reported that PNIPAAm-based gels shrunk much faster at higher shrinking temperatures, and stated that shrinking forces increase with increasing temperatures, as interior water is rapidly expelled by large internal accumulated pressures. It was also found that the swelling-deswelling cycles were accompanied by a slight decrease in the swelling ratio

Deswelling (48hrs)

Reswelling (144hrs)

Deswelling (24hrs)

Fig. 10 Pictures showing the swelling (at 20 °C) and deswelling (at 60 °C) of H4 hydrogel, synthesised using Irgacure 184 and 0.1 wt% crosslinker

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transition temperature ∼38 °C) showed more pronounced thermosensitivity (in response to the temperature cycles) absorbing and desorbing more water (see Fig. 12). The hydrophobic shrinking force of this hydrogel sample is assumed to have been enhanced by the higher ratio (15 wt%) of the NtBAAm monomer and lower LCST compared to H3. The oscillating behaviour of these hydrogels in response to temperature changes over a short time interval, while relatively maintaining their integrity, would be advantageous for practical applications in bioengineering and biotechnology.

Conclusion

Fig. 11 Oscillatory swelling-deswelling kinetics for H3 hydrogel, prepared with Irgacure 184 and different crosslinker dosages, in response to 1 h temperature cycles between 20 °C and 45 °C

(Fig. 12). This was due to the slower reswelling kinetics of the hydrogels when compared to the deswelling kinetics. Slow reswelling kinetics have also previously been observed in PNIPAAm hydrogels by Wu et al. [61]. Of the samples tested, the H4 hydrogel samples (phase

Fig. 12 Oscillatory swelling-deswelling kinetics for H3 and H4 hydrogel, prepared with Irgacure 184 and 0.1 wt% crosslinker dosages, in response to 1 h temperature cycles between 20 °C and 45 °C

In this work, temperature-sensitive terpolymer hydrogels based on N-tert-butylacrylamide, N-isopropylacrylamide and N-vinyl pyrrolidone monomers were fabricated via UV polymerisation, using two types of photoinitiator; Irgacure 184 and Irgacure 2959. The structural analysis of the hydrogels using NMR and FTIR spectroscopy confirmed the polymerisation of the feed components and the formation of a random terpolymer. Also from the NMR results, Irgacure 2959 proved to be more effective in polymerisation than Irgacure 184, as less unreacted monomer was observed. NMR results further showed that the hydrogel washing step completely extracts all unreacted monomers, which is very desirable from a toxicological point of view. Modulated differential scanning calorimetry was used in determining the phase transition temperature of the hydrogels which ranged from about 37 °C to 50 °C. Increase in the feed ratio of NtBAAm monomer had the most influence on the cloud point and transition temperatures of the terpolymer hydrogels. The hydrogels reached high equilibrium swelling ratios at room temperature and shrunk significantly at higher temperatures above their critical temperature. The swelling capacities and the thermosensitivity of the hydrogels were further examined through pulsatile swelling studies. The hydrogels were able to respond to temperature pulses across their critical temperatures, showing reversible swelling and deswelling behaviour. Furthermore, the swelling ratio of the hydrogels was adjusted by altering the amount of crosslinking agent employed during polymerisation. Considering these properties, it is expected that these thermosensitive P(NtBAAm-NIPAAm-NVP) hydrogels would be useful in drug delivery systems. Current work which focuses on toxicological tests on the hydrogels, drug incorporation and release studies are currently underway.

Synthesis and characterisation of thermo-sensitive terpolymers Acknowledgements This study was supported in parts by grants from both the Irish Department of Education (Core Research Strengths Enhancement-Technological Sector Research: Strand III) and the Athlone Institute of Technology research and development fund.

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