Wenkai
Wang
a,
Dongdong
Lu
a,
Mingning
Zhu
a,
Jennifer M.
Saunders
a,
Amir H.
Milani
a,
Steven P.
Armes
b and
Brian R.
Saunders
*a
aSchool of Materials, University of Manchester, MSS Tower, Manchester, M13 9PL, UK. E-mail: Brian.saunders@manchester.ac.uk
bDepartment of Chemistry, The University of Sheffield, Dainton Building, Brook Hill, Sheffield, South Yorkshire S3 7HF, UK
First published on 13th April 2018
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 EA- and 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–MMA–GMA NPs was studied and both remarkable deformability and excellent recovery were observed. The gels were strongly pH-responsive and had tensile breaking strains of up to 420% with a 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.
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 pKa of the constituent copolymer chains.34 Macroscopic gels were prepared in the absence of any small molecule crosslinker by covalent interlinking of the pre-formed MGs to produce doubly-crosslinked microgels (DX MGs)33 (see Scheme 1(a)). As discussed in detail elsewhere35 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 but there no literature examples of highly deformable polyacid gels prepared from dispersed NPs without added small molecule monomers.
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 sacrificial3 or reversible37 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. The present gels differ substantially from vinyl-functionalised micellar-based gels that contained –COOH groups and required added small monomers for their formation19,20 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.
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−1. Engineering stress (σ) and strain values (ε) 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−1 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.
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Copolymer | Particle type | MAAa/mol% | MMAa/mol% | EAa/mol% | GMA/mol% | pKab | d TEM /nm [CV] | d z /nm [PDI] |
---|---|---|---|---|---|---|---|---|
a Calculated from potentiometric titration data shown in Fig. S1 (ESI). b Apparent pKa values were obtained from Fig. S1 (ESI). c Number-average diameters determined from TEM images. CV is the coefficient of variation. d Mean (z-average) diameter determined by DLS at pH 4.0. e These particles were internally crosslinked with BDDA. | ||||||||
PMAA–EA–GMA | NP | 57.0 | — | 29.8 | 13.2 | 5.9 | 81 [21] | 94 [0.08] |
PMAA–MMA–GMA | NP | 75.9 | 15.5 | — | 8.6 | 5.8 | 66 [25] | 74 [0.26] |
PEA–MAA–GMAe | MG | 32.0 | — | 61.0 | 7.0 | 6.2 | 62 [17] | 67 [0.04] |
TEM images confirmed that the as-made PMAA–EA–GMA (Fig. 1a) and PMAA–MMA–GMA (Fig. 1b) NPs were spherical and had number-average diameters, dTEM, of 81 and 66 nm, respectively. (Larger area TEM images are shown in Fig. S2(a) and (b), ESI†.) There was some minor debris present for PMAA–MMA–GMA (Fig. 1(b)). The control PEA–MAA–GMA MGs were spherical with a dTEM value of 62 nm. DLS data for PMAA–EA–GMA and PMAA–MMA–GMA NPs indicated dz values of 94 and 74 nm, respectively, at pH 4.0. The dz value for PEA–MAA–GMA MG was 67 nm at pH 4.0. The dz values were greater than the dTEM values for each of the three systems because DLS is biased towards larger particles for size distributions of finite width.
Increasing the aqueous dispersion pH had a dramatic effect on the NP morphology. TEM images for PMAA–EA–GMA and PMAA–MMA–GMA NPs deposited from aqueous solution at pH 7.4 (Fig. 1(d) and (e), respectively) are consistent with the expected NP disassembly (see also lower magnification TEM images in Fig. S2(d) and (e), ESI†). Nevertheless, some of the original NPs were still present (red arrows). This suggests that some (albeit relatively low) degree of self-crosslinking has occurred. Self-crosslinking has been reported for PNIPAM MGs by Lyon et al.39 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 Fig. S2(f), ESI†) where only fully intact spheres were present. These TEM observations demonstrate that omitting divinyl crosslinker leads to substantial loss of the original NP morphology on raising the solution pH above the NP pKa.
DLS was employed to study the pH-triggered disassembly of dispersed PMAA–EA–GMA and PMAA–MMA–GMA NPs in Fig. 2(a) and (b), respectively. As the solution pH is gradually increased, the NP size distributions shift to larger diameters and then become bimodal at pH 6.8 before shifting back to smaller diameters at pH 10. (Unfortunately, reliable DLS data could not be measured for the PMAA–EA–GMA NPs at pH 10.) These data indicate initial NP swelling at pH 6.8–7.4, followed by almost complete NP disassembly at pH 10. In striking contrast, the control MG DLS size distributions (Fig. 2(c)) remain monomodal and show only an increase in mean diameter at pH 6.8, which remained constant at higher pH.
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Fig. 2 DLS size distributions recorded at selected pH values for (a) PMAA–EA–GMA NPs, (b) PMAA–MMA–GMA NPs and (c) PEA–MAA–GMA MGs. |
The pH-dependence for the dz and polydispersity index (PDI) of the NPs is shown in Fig. S3(a) and (b) (ESI†) for completeness. These data show that the onset of NP swelling occurs at a critical solution pH that lies close to their pKa values (5.8–5.9). The NP dz values are less reliable at higher pH values because the PDI values were very high (i.e., greater than 0.30). The dz data obtained for the pH-responsive MGs indicate a significant increase in size near the particle pKa (of 6.2) followed by a plateau (Fig. S3(c), ESI†). To gain further insight into the NP swelling process, pH-dependent electrophoretic mobilities (μ) were measured for PMAA–MMA–GMA NPs via aqueous electrophoresis (Fig. S4, ESI†). The μ 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 dz (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.
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Fig. 3 (a) Uniaxial compressive stress–strain curves for Gel-MMA-7.4, Gel-EA-7.4 and a comparable DX MG–EA system reported earlier.45 The insets show and expanded view that includes the strain region used to calculate the modulus values (0–10%) as well as representative images of each gel. Scale bars: 5 mm. (b) Breaking strain and modulus values for the three gels from (a). |
The compression stress–strain data obtained for Gel-EA-7.4 and Gel-MMA-7.4 are compared to those previously reported45 for a DX MG gel prepared from PEA–MAA–GMA MGs. The MG used 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. The GMA content of the PEA–MAA–GMA MG precursor particles used to prepare the DX MG–EA gel was 3.5 mol%. If GMA-crosslinking controlled the mechanical properties, a more deformable gel would be expected compared to the present gels. In contrast, Gel-EA-7.4 and Gel-MMA-7.4 had higher breaking strains and hence were more 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.
Concentrated dispersions of conventional MGs prepared with a crosslinker (e.g., BDDA) form strong physical gels when the solution pH exceeds the particle pKa 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, the building blocks for gel formation are fundamentally different from those of the previously studied DX MGs.33
SEM images were obtained for freeze-dried Gel-MMA-X (X = 5.8, 7.4 and 10.0) samples and showed highly porous morphologies (see Fig. 4(a)–(c)). Digital image analysis indicated mean pore diameters of 4.0 ± 0.9, 7.2 ± 1.5 and 15.4 ± 4.5 μm, respectively. Such pores originate from ice formation.48 Control of pore size is relevant for membrane or tissue scaffold application.48,49 This 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. Such “hindered” ionisation is well-documented for polyacid hydrogels.53
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 pKa. 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 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 (νe). The modulus (E) is related to νe by
E = 3νekTϕ21/3 | (2) |
![]() | (3) |
Gel | Modulusa/kPa | Stress-at-break/kPa | Breaking-strain/% | ν e /× 10−23 m−3 | x c |
---|---|---|---|---|---|
a The polymer volume fraction used for these experiments was 9.0 vol%. b Number-density of elastically-effective chains (see text). c Average number of segments between crosslinks (see text). d Sample did not fail even at the maximum strain recorded by our instrument. | |||||
Gel-MMA-5.8 | 35.6 | 133 | 66.6 | 64.3 | 1![]() |
Gel-MMA-7.4 | 21.1 | 215 | 72.0 | 38.1 | 2![]() |
Gel-MMA-10 | 3.09 | >1170d | >93.0d | 5.6 | 13![]() |
We probed the mechanism responsible for the excellent deformability of Gel-MMA-5.8 and Gel-MMA-10 using cyclic compression measurements (Fig. S7, ESI†). The area calculated from the difference in the loading and unloading curves is the hysteresis. These area values represent only 13 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 (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.
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Fig. 6 Summary of hydrogel mechanical properties: (a) tensile stress–strain data, (b) tensile breaking strains and (c) Young's moduli for the three new gels reported herein. (d) Gel-MMA-10.0 was also prepared in the form of strings that could be tied in knots and were able to withstand substantial stretching without breaking. A video showing this manipulation is available in the ESI.† |
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:33,34 (i) the omission of a crosslinking comonomer and (ii) the higher MAA contents of the NPs. The absence of any crosslinker should enable faster diffusion of the GMA reagent throughout the precursor NPs during the coupling reaction between GMA and a minor fraction of the carboxylic acid groups on the monomer repeat units (Scheme S1, ESI†). In principle, this should result in copolymer chains containing a uniform distribution of vinyl groups. Furthermore, their relatively high MAA content means that the NPs should swell strongly and undergo almost complete disassembly when the solution pH is raised beyond the NP pKa, which has the effect of further distributing the crosslinking points and increasing xc. Combined with the substantial interpenetration of the copolymer chains and greater extent of NP disassembly in alkaline solution, a relatively uniform distribution of elastically-effective chains can be expected for these hydrogels (as depicted in Scheme 1(b)). The excellent deformability obtained for the optimised formulation (Gel-MMA-10) arises because more complete disassembly can be achieved at this higher pH. This interpretation is supported by a representative TEM image recorded for the parent PMAA–MMA–GMA dispersion deposited from a mildly alkaline (pH 10) solution (Fig. S8, ESI†) which confirmed that very few NPs remained intact under such conditions.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sm00325d |
This journal is © The Royal Society of Chemistry 2018 |