Highly deformable hydrogels constructed by pH-triggered polyacid nanoparticle disassembly in aqueous dispersions

Most hydrogels are prepared using small-molecule monomers but unfortunately this approach may not be feasible for certain biomaterial applications. Consequently, alternative gel construction strategies have been established, which include using covalent inter-linking of preformed gel particles, or microgels (MGs). For example, covalently interlinking pH-responsive MGs can produce hydrogels comprising doubly crosslinked microgels (DX MGs). We hypothesised that the deformability of such DX MGs was limited by the presence of intra-MG crosslinking. Thus, in this study we designed new nanoparticle (NP)-based gels based on pH-swellable NPs that are not internally crosslinked. Two polyacid NPs were synthesised containing methacrylic acid (MAA) and either ethyl acrylate (EA) or methyl methacrylate (MMA). The PMAA-EA and PMAA-MMA NPs were subsequently vinyl-functionalised using glycidyl methacrylate (GMA) prior to gel formation via free-radical crosslinking. The NPs mostly disassembled on raising the solution pH but some self-crosslinking was nevertheless evident. The gels constructed from the EAand MMA-based NPs had greater breaking strains than a control DX MG. The effect of varying the solution pH during curing on the morphology and mechanical properties of gels prepared using PMAA-MMAGMA NPs was studied and both remarkable deformability and excellent recovery was observed. The gels were strongly pH-responsive and had tensile breaking strains of up to 420% with a 2 compressive strain-at-break of more than 93%. An optimised formulation produced the most deformable and stretchable gel yet constructed using NPs or MGs as the only building block.


INTRODUCTION
Hydrogels are water-rich polymer networks that continue to attract wide interest in the literature 1-3 .
This activity is largely driven by their potential applications which include biomaterials 4,5 , water purification 6 , photonics 7 and electronic skin 8 . Hydrogels are usually prepared from the bottom up via statistical copolymerisation of (small-molecule) monomers and crosslinkers 9 . Alternatively, preformed soluble linear chains can be covalently linked together 10 . Combined approaches have also been explored, whereby multiple networks are formed 11 or hydrogels are prepared using nanoparticles (NPs) as crosslinkers 12 . Recently, hydrogels have also been prepared without any covalent crosslinking 13 . Of particular interest are pH-responsive polyacid hydrogels containing high levels of carboxylic acid groups. Such gels can exhibit strong pH-responsive or electrolyteresponsive swelling [14][15][16][17][18][19][20] . They also offer potential biomaterial applications, because the pHtriggered swelling transition can be tuned to correspond to physiological conditions, especially for gels based on methacrylic acid (MAA) [21][22][23][24] . Whilst excellent mechanical properties have been reported for such gels 25 , one common limitation in the context of biomaterials applications is that gel construction methods rely on the use of small molecule monomers 14,[17][18][19][20][25][26][27][28][29][30][31][32] . In this study we introduce a new generic approach that enables the rational design of highly deformable pHresponsive hydrogels using aqueous dispersions of pre-formed polyacid NPs as the only gel building blocks.
A new approach to polyacid hydrogel construction utilising pH-responsive copolymer microgels (MGs) as building blocks was introduced in 2011 at Manchester 33 . Such MGs are typically lightly crosslinked colloidal particles that are usually prepared with a crosslinking comonomer and significant swelling occurs when the solution pH approaches the pK a of the constituent copolymer chains 34 . Macroscopic gels were prepared in the absence of any small molecule crosslinker by 3 covalent interlinking of the pre-formed MGs to produce doubly-crosslinked microgels (DX MGs) 33 (see Scheme 1(a)). As discussed in detail elsewhere 35 this DX MG concept differs from gels constructed from small molecule monomers and MG particle macro-crosslinkers 19,20,25 . Gel assembly using only pre-formed MGs is a potential advantage for biomaterials applications because using small molecules as building blocks may present cytotoxicity challenges in vivo. DX MGs exhibit suitable mechanical properties for potential application in the field of intervertebral disc repair 24  (b) pH-triggered disassembly of vinyl-functionalised nanoparticles (NPs) and subsequent hydrogel formation. This study focuses on gels formed via Scheme 1(b) but the results are compared to DX MGs prepared via Scheme 1(a). Note that no free monomer is present in either (a) or (b). A major driver for development of new gels is enhanced mechanical properties.
Unfortunately, heterogeneity in the elastically-effective chain length is common for hydrogels. This results in gel embrittlement because stress is concentrated at the shortest chains 11 . In principle, highly stretchable tough gels can be obtained by making all the elastically-effective chains of similar length 36 . Alternatively, tough gels can be prepared by introducing sacrificial 3 or reversible 37 crosslinks to dissipate energy. Our previous DX MGs consisted of interlinked MGs that comprise two populations of elastically-effective chains; intra-MG chains as well as inter-MG chains (Scheme 1(a)). Here, we sought to substantially reduce the former population by preparing pH-swellable nanoparticles (NPs) without added crosslinker (Scheme 1(b)). We hypothesised that such gels should contain only one major population of elastically-effective chains which was expected to improve gel deformability.
MGs are usually prepared using a divinyl crosslinker 38 . However, seminal studies by the Lyon group have shown that N-isopropylacrylamide (NIPAM) MGs can be prepared in the absence of such comonomers. 39,40 Moreover, such self-crosslinked MGs can be ultrasoft and highly deformable 41 . Although the precise mechanism responsible for MG self-crosslinking has not been established, it seems likely that chain transfer to polymer is responsible. 39,40 . In the present study, we use two different formulations comprising either ethyl acrylate (EA) or methyl methacrylate (MMA) to prepare the first examples of self-crosslinked polyacid NPs (Scheme 1(b)).
Our approach to gel construction is summarised in Scheme 1(b). We seek to enhance pH-triggered particle swelling by preparing MAA-rich NPs with no intra-particle crosslinker. Thus, these NPs simply contain mainly MAA plus a minor proportion of either MMA or EA comonomer. They were subsequently vinyl-functionalised by reaction of a minor fraction of the MAA units with glycidyl methacrylate (GMA; see Scheme S1, ESI †). On raising the solution pH the vinyl-functionalised NPs initially swell and eventually undergo disassembly. The interpenetrating copolymer chains were then crosslinked to afford deformable pH-responsive gels. This new approach employs preformed NPs to deliver vinyl-functionalised polyacid chains on demand using pH as the trigger. 5 The present gels differ substantially from vinyl-functionalised micellar-based gels that contained -COOH groups and required added small monomers for their formation 19,20 and also from gels constructed using a combination of vinyl-functionalised nanogels as crosslinkers and small molecule monomers such as NIPAM 25 .
Herein, we first characterise the two NP systems and study their pH-triggered disassembly. The effect of varying the solution pH on the dilute dispersion properties is compared to that for a control pH-responsive MG. We then compare the mechanical properties of gels constructed from such NPs at pH 7.4 to data previously reported for a related DX MG. In order to understand and optimise the influence of NP disassembly on gel mechanical properties the effect of the preparation pH is then investigated. These gels are shown to be the most deformable (and toughest) that have been constructed entirely from polyacid NP building blocks to date. This study is an important step in the development of injectable NP-based injectable gels for high toughness applications such as cartilage repair.

Synthesis of vinyl-functionalised polyacid nanoparticles
The synthesis of the precursor NPs was conducted using a modified "seed-feed" emulsion 6 polymerisation method to minimise self-crosslinking. For preparation of PMAA-MMA NPs a comonomer mixture (35.0 g) comprising MMA (14.0 g, 0.14 mol) and MAA (21.0 g, 0.25 mol) was used. Seed formation was achieved using 5.0 g of this comonomer stock solution, which was added to water (480 g) containing SDS (2.4 g, 8.0 mmol) and then TEMED solution (1.0 ml of 11.0 wt.% solution) and APS (5.0 g of a 11.0 wt.% solution) were added. The seed was prepared at 50 ˚C with continuous mechanical stirring for 15 min. A total of 20.0 g of the comonomer solution was then added at a uniform rate of 0.50 ml/min over 40 min and the temperature was raised to 60 o C. APS (2.75 g of a 11.0 wt% solution) was added and the remaining comonomer solution was fed into the mixture at the same addition rate. Particle growth continued for a further 1 h at 60 o C. The crude product was purified by extensive dialysis using water. GMA (1.25 g, 9.0 mmol.) was added to the purified precursor NP dispersion (100 g, 3.0 wt.%) and the ring-opening reaction (Scheme S1, ESI †) was allowed to proceed for 8.0 h at pH ~ 5.0. Unreacted GMA was removed by extensive dialysis using water. An identical protocol was used for the preparation of PMAA-EA-GMA NPs.

Nanoparticle disassembly studies
NaOH solution was added to NP dispersions to achieve the pH required. Dynamic light scattering (DLS) measurements were obtained after 24 h equilibration at 25 o C.

Hydrogel preparation
Aqueous NaOH solution (4.0 M) was added to the NPs

Physical Measurements
DLS measurements were conducted using a 50 mW He-Ne laser operating at 633 nm with a standard avalanche photodiode (APD) and 901 detection optics connected to a Malvern Zetasizer 7 Nano ZS90 autocorrelator. This instrument set-up provided the z-average diameter (d z ) as well as the electrophoretic mobility. Potentiometric titration was conducted using a Mettler Toledo DL15 titrator. Titrations were performed in the presence of aqueous NaCl (0.010 M) as background electrolyte and the titration solution was NaOH (0.10 M). TEM images were obtained using an FEI Tecnai Bio Twin instrument (100 kV) and samples were prepared using 300 mesh copper TEM grids. The dispersion concentration used was 0.0010 wt,% and uranyl acetate staining (0.25 wt% solution) was used. SEM measurements were obtained using a Philips XL30 FEG SEM instrument.
Grids were coated with a thin layer of either platinum or carbon to prevent sample charging.
Uniaxial compression measurements were performed using an Instron series 5569 load frame equipped with a 100 N compression testing head. The modulus was calculated using the gradient of the stress-strain data measured over the first 10% strain. Cyclic compression experiments were conducted at 10 mm/s. Engineering stress () and strain values () are reported. Uniaxial tensile stress-strain measurements were conducted using an Instron series 5569 load frame equipped with a 10 N compression testing head at a rate of 10 mm/s using dog-bone shaped molds with a central rectangular region of length 20 mm. The width and thickness were both 4.0 mm, while paddle regions had a width of 17.5 mm. Gel samples for the qualitative tensile tests involving knot formation were prepared using a cylindrical mold.
Gel swelling measurements were conducted by immersing gel samples in buffer for 24 h. The volume swelling ratios (Q) were calculated using gravimetry according to the following equation. (1) where Q m is the mass swelling ratio. The ρ s and ρ p values are the densities of the solvent (water) and polymer, respectively. The values used for water and the DX MGs were 1.00 and 1.20 g cm -3 , respectively. 8

Nanoparticle characterisation and pH-triggered disassembly
Two types of MAA-rich, crosslinker-free NPs were prepared to demonstrate the versatility of our new approach for the construction of deformable gels using NP building blocks: PMAA-EA-GMA and PMAA-MMA-GMA. Potentiometric titration data (Fig. S1, ESI †, and Table 1) Table 1). a Calculated from potentiometric titration data shown in Figure S1 (ESI, †). b Apparent pK a values were obtained from Figure S1.  were still present (red arrows). This suggests that some (albeit relatively low) degree of selfcrosslinking has occurred. Self-crosslinking has been reported for PNIPAM MGs by Lyon et al. 39 10 However, it has not been previously observed for polyacid MGs. Self-crosslinking most likely involves chain transfer to polymer. Following the approach adopted by Lyon et al., we used TEMED at low temperature to minimise this side reaction during particle formation and growth.
However, it was not possible to completely eliminate self-crosslinking. TEM images obtained for these NPs differ markedly from those obtained for the control MG particles at pH 7.4 ( Fig. 1(f) and 12 pH values because the PDI values were very high (i.e., greater than 0.30). The d z data obtained for the pH-responsive MGs indicate a significant increase in size near the particle pK a (of 6.2) followed by a plateau (Fig. S3(c), ESI †). To gain further insight into the NP swelling process, pH-dependent electrophoretic mobilities (were measured for PMAA-MMA-GMA NPs via aqueous electrophoresis (Fig. S4, ESI †). The  magnitude increased strongly when the pH exceeded 6.5, which is attributed to the formation of anionic carboxylate groups. This critical pH coincided with a dramatic increase in d z (Fig. S3(b), ESI †), which suggests that NP disassembly occurs as a result of a pH-triggered increase in electrostatic repulsion between anionic PMAA-MMA-GMA chains.

Effect of comonomer type on hydrogel mechanical properties
Hydrogels were constructed from concentrated EA-or MMA-based NP dispersions at pH 7.4 using free-radical crosslinking (Scheme 1(b)). respectively.] The as-made gels were transparent and easily supported their own weight (see inset of Fig. 3(a)). Compressive stress-strain data for these gels are shown in Fig. 3(a) and Table 2. Gel-EA-7.4 and Gel-MMA-7.4 had breaking strain values of 45 % and 72 %, respectively ( Fig. 3(b)). The latter gel was softer and more deformable than the former. This implies that the elastically-effective chains are relatively long for Gel-MMA-7.4, which is reasonable given the compositions of the parent NPs used to construct these gels. PMAA-MMA-GMA NPs had a lower GMA content (8.6 mol%) compared to the PMMA-EA-GMA NPs (13.2 mol%).  to prepare that gel (denoted as DX MG-EA) had an almost identical composition and particle size as those shown in Fig. 1(c) and Table 1 Gel-EA-7.4 14 deformable gels (Fig. 3). Gel-EA-7.4 and Gel-MMA-7.4 exhibited lower moduli compared to DX MG-EA ( Fig. 3(b)). Accordingly, Gel-EA-7.4 and Gel-MMA-7.4 had lower number densities of elastically-effective chains compared to DX MG-EA, which accounts for the enhanced gel properties. We attribute the superior deformability of Gel-MMA-7.4 and Gel-EA-7.4 to the absence of any divinyl crosslinker within the NP building blocks.

Effect of preparation pH on nanoparticle-based hydrogel properties
A key aim of this study was to examine our hypothesis that the absence of intra-particle crosslinking would produce stronger, more resilient hydrogels compared to the previously studied DX MGs. 45, 46 . Gel-MMA-7.4 was selected for further study because it had a much higher breaking strain than Gel-EA-7.4 (Table 2) According to the DLS studies ( Fig. 2(b)), these three pH values of 5.8, 7.4 and 10 correspond to NPs that are slightly swollen, partially dissociated and mostly dissociated, respectively.
Concentrated dispersions of conventional MGs prepared with a crosslinker (e.g., BDDA) form strong physical gels when the solution pH exceeds the particle pK a because of MG particle swelling 33 . In contrast, the present PMAA-MMA-GMA NPs exhibited only a modest increase in viscosity at pH 5.8, 7.4 and 10 and did not form strong physical gels (see Fig. S5, ESI †). These results demonstrate that the NPs did not restrict each other's motion significantly during macrogel network formation. The NPs were not swollen at pH 5.8, whereas they became swollen, and underwent disassembly at higher pH, with interpenetration of the dissolved copolymer chains ( Fig.   2(b)). Transparent gels were obtained after curing at each pH (see insets in Fig. 4(a) -(c)). Thus, 15 the building blocks for gel formation are fundamentally different from those of the previously studied DX MGs 33 . parameter also provides useful qualitative information concerning the mechanical properties of the gels. In our previous work on DX MGs, it was shown that the pore size increased as the gel modulus is reduced 50 . Here, larger pores were obtained at higher pH, suggesting that the gels became less stiff (lower moduli). These images suggest that increasing the preparation pH leads to more deformable gels because gel stiffness is often inversely related to deformability.
FTIR spectroscopy was used to probe the extent of ionisation of the carboxylic acid groups and spectra were recorded for freeze-dried gels prepared at pH 5.8, 7.4 and 10 ( Fig. 4(d) -(f)). Bands at 1710 and 1544 cm -1 are assigned to carboxylic acid and anionic carboxylate groups, respectively 51,52 . As the gel preparation pH increased the relative -COO -/-COOH absorbance ratio for these bands increased from 0.3 (pH 5.8) to 1.5 (pH 7.4) and to 4.0 (at pH 10.0). Hence, the MAA residues gradually became neutralised, as expected. Notably, neutralisation was not complete at pH 7.4.
The three Gel-MMA-X (X = 5.8, 7.4 and 10) samples were pH-responsive (Fig. S6, ESI †) and exhibited their lowest volume swelling ratios (Q) at pH 5.0, which is well below the particle pK a .
These gels swelled strongly and attained Q values of 40 -47 when equilibrated in buffer with a pH of 7.4 or pH 10. The swelling is driven by the formation of anionic carboxylate groups, which leads to inter-chain electrostatic repulsion as discussed above in relation to Fig. S4 (ESI †). Increasing the solution pH further to pH 10 did not significantly change the Q value for Gel-MMA-10 which is presumably because the -COOH groups were fully neutralised. However, the Q values for the Gel-MMA-5.8 and Gel-MMA-7.4 gels were significantly lower. The corresponding FTIR spectra ( Fig.   4(d) and (e)) provided evidence for incomplete neutralisation of the latter gels. We propose that a higher intra-gel ionic strength (and hence electrostatic screening) occurred for those gels as a result of the higher proportions of carboxylic acid groups that required neutralisation when subsequently swollen in pH 10 buffer.
The preparation pH had a major effect on the mechanical properties of the gels. Fig. 5(a) shows 17 compression stress-strain data for Gel-MMA-5.8, Gel-MMA-7.4 and Gel-MMA-10, which exhibited breaking strains (and gel moduli) of 66% (35.6 kPa), 72% (21.1 kPa) and > 93% (3.09 kPa), respectively (Table 3). Hence, these three gels exhibited significantly greater deformability as they became less stiff. These data enabled calculation of the number-density of elastically-effective chains ( e ). The modulus (E) is related to  e by (2) where k, T and  2 are Boltzmann's constant, absolute temperature and the polymer volume fraction within the gel, respectively 54 . Note that this analysis assumes that there is only one significant source of crosslinking (which is from GMA) and ignores the minor proportion of self-crosslinking.
where M o is the average repeat unit molecular weight (93 g/mol). The data are shown in Table 3.
Increasing the solution pH during gel synthesis dramatically increased x c . The network breaking strain (and hence deformability) scales with x c 55 . This relationship explains why Gel-MMA-10 did not fail at the maximum strain measurable by our instrument (93%). The breaking strain of this new hydrogel is much greater than any value reported for DX MGs 24,33,34,50,56 . Furthermore, Gel-MMA-10 showed excellent recovery as shown by a qualitative deformation test (Fig. 5(c)). Note that recovery is the ability of a sample to show a reduction in residual strain after loadingunloading 57 .   Gel-MMA-10 19 and 17% of the respective areas under the loading curves for Gel-MMA-5.8 and Gel-MMA-10.
These data reveal modest hysteresis and hence relatively low energy dissipation. There was also no residual strain, which confirms excellent recovery for these hydrogels. If either reversible or dynamic crosslinks made a major contribution, then much higher hysteresis and significant residual strain would be expected. 58 Consequently, covalent crosslinking is primarily responsible for the observed mechanical properties. Covalent crosslinks are mainly formed via free-radical coupling of the vinyl groups on the chains (plus a minor proportion of self-crosslinks remaining within the NPs, as discussed above).
Not only did the three Gel-MMA-X samples possess good to excellent mechanical properties when subjected to compression, they also proved to be remarkably stretchable. This property enabled tensile stress-strain data to be determined. We emphasise that this is the first time that such data has been reported for MAA-rich hydrogels constructed from NP (or MG) building blocks without using added small molecule monofunctional or difunctional comonomers. Tensile stress-strain data ( Fig.   6(a)) showed approximately linear behaviour with only moderate strain-hardening. (The tensile data are summarised in Table 4.) Interestingly, such behaviour is qualitatively similar to that reported for cartilage 59 . The tensile breaking strain and Young's modulus values are shown in Fig. 6(b) and 6(c), respectively. The stretchability (as determined by the breaking strain) proved to be very sensitive to the gel preparation pH. Thus, Gel-MMA-5.8, Gel-MMA-7.4 and Gel-MMA-10 could be stretched to 200, 340 and 520% of their original lengths, respectively. The gel moduli also showed the same trend indicated by the compression data ( Table 3). The high breaking strain obtained for the hydrogel prepared at pH 7.4 (Gel-MMA-7.4) together with its remarkable toughness (49.4 kJ m -3 , Table 4) are very encouraging for future biomaterials applications. The breaking strain obtained for Gel-MMA-10 compares favourably to data recently reported for alternative next-generation hydrogels 60,61 . Indeed, Gel-MMA-10 strings could be readily tied in knots and stretched to more than 250% of its original length without damage (See Fig. 6(d) and video, ESI †), which is unprecedented for this class of gel.  What is the origin of the high deformability of these gels? There are two key differences between the NPs used in the present study and the MGs reported previously: 33

CONCLUSIONS
In summary, a versatile new NP-based approach that does not require small molecule comonomers is reported for the preparation of highly deformable hydrogels with excellent recovery. These MMA-or EA-based NPs contain very high MAA contents and can be vinyl-functionalised using GMA. Triggered NP disassembly occurs on increasing the pH and subsequent crosslinking leads to the formation of highly deformable and strongly pH-responsive polyacid hydrogels. The MMAbased gels proved to be more deformable than the EA-based gels. The mechanical properties of the MMA-based gels could be tuned by adjusting the preparation pH. The most deformable gel (Gel-MMA-10) had a strain-at-break of 420%. Importantly, the deliberate omission of any intra-particle crosslinker enabled a substantial improvement in both gel deformability and toughness compared to previously reported DX MGs. These new NP-based hydrogels are comparable to next-generation 22 gels constructed using small molecule monomers in terms of their mechanical performance. Future work will investigate the use of lower synthesis temperatures (e.g., 37 o C) in order to target injectable highly deformable gels for load-bearing tissue and possibly cartilage repair applications.