Thermoresponsive Hydrogels Based on Telechelic Polyelectrolytes

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Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Thermoresponsive Hydrogels Based on Telechelic Polyelectrolytes: From Dynamic to “Frozen” Networks Constantinos Tsitsilianis,*,† George Serras,† Chia-Hsin Ko,‡ Florian Jung,‡ Christine M. Papadakis,‡ Maria Rikkou-Kalourkoti,§ Costas S. Patrickios,§ Ralf Schweins,∥ and Christophe Chassenieux⊥ †

Department of Chemical Engineering, University of Patras, 26504 Patras, Greece Physik-Department, Physik weicher Materie, Technische Universität München, James-Franck Str. 1, 85748, Garching, Germany § Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus ∥ Large Scale Structures Group, Institut Laue-Langevin, DS/LSS, 71 Avenue des Martyrs, CS 20 156, 38042 Grenoble, France ⊥ Le Mans Université, IMMM UMR CNRS6283, Département Polymères, Colloı̈des, Interfaces, av. O. Messiaen, Cedex 9 72085 Le Mans, France ‡

S Supporting Information *

ABSTRACT: A novel thermoresponsive gelator of (B-co-C)-b-A-b(B-co-C) topology, comprising a poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) weak polyelectrolyte as central block, end-capped by thermosensitive poly(triethylene glycol methyl ether methacrylate/n-butyl methacrylate) [P(TEGMA-co-nBuMA)] random copolymers, was designed and explored in aqueous media. The main target of this design was to control the dynamics of the stickers by temperature as to create an injectable hydrogel that behaves as a weak gel at low temperature and as a strong gel at physiological temperature. Indeed, at low temperatures, the system behaves like a viscoelastic complex fluid (dynamic network), while at higher temperatures, an elastic hydrogel is formed (“frozen” network). The viscosity increases exponentially upon heating, about 5 orders of magnitude from 5 to 45 °C, which is attributed to the exponential increase of the lifetime of the self-assembled stickers. The integration of thermo- and shear responsive properties in the gelator endows the gel with injectability. Moreover, the gel can be rapidly recovered upon cessation of the applied stress at 37 °C, simulating conditions similar to those of injection through a 28-gauge syringe needle. All these hydrogel properties render it a good candidate for potential applications in cell transplantation through injection strategies.

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INTRODUCTION Hydrogels constitute a very attractive class of soft matter, receiving a lot of attention due to a plethora of applications in various fields of daily life, for instance food and cosmetics as well as in advanced technologies, e.g., biomedicine.1 Particularly for the latter, the applications concern three major directions, namely tissue engineering, stem cell transplantation, and controlled drug delivery. For these applications, a special category of hydrogels is required, namely injectable hydrogels,2 in order to avoid surgical treatment.3−5 Hydrogel injectability can be achieved by designing suitable macromolecular topologies allowing the assembly of the chains to occur “in situ”, forming a three-dimensional (3D) network.6 For such an approach, the key point is the mode of chain cross-linking after injecting a solution, containing the chosen suitable constituents. One of the strategies developed so far is to design block copolymers bearing associative, potentially hydrophobic blocks (stickers) that are actuated upon applying a stimulus like temperature, pH, light, etc.7 These associative block copolymers are water-soluble and molecularly dissolved in aqueous media while they self-assemble under specific conditions forming a spanning 3D network, constituted of interconnected © XXXX American Chemical Society

physically cross-linked nanodomains. Thus, the potential stickers are in fact stimuli-responsive polymers that undergo a sharp phase transition upon applying a trigger.8,9 Macromolecular engineering offers a variety of synthetic approaches, relying on the so-called “living” controlled polymerization methods that lead to well-defined associative copolymers, enabling to form 3D reversible networks in aqueous environments.10 Among a vast variety of macromolecular architectures, ABA triblock copolymer comprising a long hydrophilic central block, end-capped by shorter A blocks, that undergo association, is the simplest, well-defined, and widely used topology as injectable gelators.11 By using weak electrolytes as monomer repeating units for the B block, pHsensitive associative triblock copolymers, also named telechelic polyelectrolytes, have been designed and used as efficient gelators.12−18 Thanks to the extended conformation of the weak polyelectrolytes in their fully ionized form, these gelators can form hydrogels at low polymer concentrations, exhibiting Received: January 25, 2018 Revised: February 21, 2018

A

DOI: 10.1021/acs.macromol.8b00193 Macromolecules XXXX, XXX, XXX−XXX

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enabling to bridge the hydrophobic nanodomains of the endblocks easier, lowering in that way the percolation concentration as has also been shown by simulation. Therefore, they are more efficient gelators than their uncharged counterparts.13,22,50 Additionally, PTEGMA is a thermosensitive polymer, displaying a LCST at about 52 °C. 51 The copolymerization of this monomer with nBuMA (about 50%) was expected to endow the end-stickers with thermosensitivity manifested below the physiological temperature. The main target of this design is to control the dynamics of the stickers by temperature as to create an injectable hydrogel that behaves as a weak gel at room temperature and as a strong gel at physiological temperature. These kinds of hydrogels are good candidates for cell transplantation applications, through injection strategies, which requires a weak gel to protect the cells during injection and a stronger gel after injection to immobilize the created scaffold in the targeting position of the host tissue.52

unique rheological properties. Besides their pH-controlled properties that can be used to provide injectability, an additional interesting property for this purpose is the strong shear-thinning effect that they exhibit at very low shear rates/ strains.18,19 Thus, reversible shear-thinning hydrogels represent an alternative strategy to afford injectability. Recently, the incorporation of weak electrolytes (acidic or basic) into the hydrophobic end-blocks of a telechelic polyelectrolyte, through statistical copolymerization [(A-coB)-b-A-b-(A-co-B) topology], resulted in a highly pH-controlled tunability of the exchange dynamics of the hydrophobic stickers.20−23 This concept could be used to design efficient gelators, targeting pH-triggered injectable hydrogels. The strategy of using as building blocks a random copolymer, instead of a pure one, has been recognized as an advanced capability of designing precisely demanding polymer-based selfassembly properties,24−30 comprising micelles and gels. Thermoresponsive block copolymers constitute another class of gelators endowed with injectability, induced by temperature variation. In most of the cases, these copolymers incorporate polymeric blocks exhibiting lower critical solution temperature (LCST).31−38 Suitable macromolecular topologies for designing associative gelators dictates the LCST stickers (A) as outer (e.g., A-b-B-b-A)39−42 or grafted blocks (B-g-A).43 Poly(Nisopropylacrylamide) (PNIPAM) is a representative kind of thermoresponsive polymers that turn from hydrophilic to hydrophobic above ca. 32 °C (lower than the physiological temperature of 37 °C) which is suitable for biomedical applications. For this reason, it has been widely used as a model LCST building block. However, the sol−gel critical temperature is established at temperatures well above the LCST which, in some cases, exceeds the physiological temperature, rendering it out of potential applications.44−46 The reason is that LCST is defined at infinite molar mass and in most of the cases the method used (turbidimetry) gives Tc which depends on Mn. Usually the stickers are of low Mn and higher temperature is needed to induce enough hydrophobicity to form the network junctions with reasonable lifetime (higher than experimental time). Moreover, hydrophobicity depends on the degree of dehydration. For instance, in PNIPAM at 32 °C the hydrated monomeric units are still about 90% and becomes 40% at 55 °C.47 To overcome this problem, many other LCST polymers have been studied as stickers.48 Alternatively, statistical copolymers of NIPAM (or other monomers) with hydrophobic monomers exhibit lower LCST values and, in turn, lower sol−gel transition temperatures.49 The latter strategy displays some advantages with respect to the homopolymer stickers, namely precise control of the sol−gel transition through suitable selection of the comonomer composition and, more importantly, control of the stickers’ hydrophobicity that affect their exchange dynamics and network functionality, which in turn control the rheological properties of the hydrogel. In this work, a novel telechelic polyelectrolyte comprising a poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA), end-capped by poly(triethylene glycol methyl ether methacrylate (TEGMA), incorporating the hydrophobic n-butyl methacrylate (nBuMA) monomer, P(TEGMA-co-nBuMA), was designed and explored in aqueous media. The central PDMAEMA block is a pH-sensitive cationic polyelectrolyte, and it was chosen to take advantage of the efficient gel properties of telechelic polyelectrolytes. In this case the central blocks exhibit stretched conformation (higher effective length)

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EXPERIMENTAL SECTION

Materials. The monomers 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%), n-butyl methacrylate (nBuMA, 99%), and tri(ethylene glycol) methyl ether methacrylate (TEGMA, 98%), calcium hydride (CaH2, 90−95%), and 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH, 95%) were purchased from Aldrich, Germany. 2,2′Azobis(isobutyronitrile) (AIBN, 95%), N,N-dimethylformamide (DMF, 99%), and deuterated chloroform (CDCl3) were purchased from Merck, Germany. Tetrahydrofuran (THF, 99.8%, both HPLC and reagent grade) was purchased from Scharlau, Spain. DMAEMA, nBuMA, and TEGMA were passed through basic alumina columns to remove the polymerization inhibitor and any other acidic impurities, and they were stirred overnight over CaH2 to remove the last traces of moisture. This was done in the presence of added DPPH, a free radical inhibitor, to prevent undesired thermal polymerization. The monomers and solvent were freshly distilled under reduced pressure prior to the polymerizations. 1,4-Bis[2-(thiobenzoylthio)prop-2-yl]benzene (TBTPB) was synthesized according to the literature.53 Polymer Synthesis by RAFT Polymerization. Synthesis of the PDMAEMA Homopolymer. 10.00 mL of DMAEMA (9.32 g, 0.06 mol), 1,4-bis[2-(thiobenzoylthio)prop-2-yl] benzene (TBTPB) chain transfer agent (275.9 mg, 5.92 × 10−4 mol), and AIBN radical initiator (60.6 mg, 3.70 × 10−4 mol) in 9.75 mL of DMF were transferred to a 50 mL round-bottomed flask fitted with a glass valve and containing a magnetic stirring bar. The system was degassed by three freeze− pump−thaw cycles and was subsequently placed in an oil bath thermostated at 70 °C for 20 h. Synthesis of the Terpolymer. The polymerization procedure for the synthesis of the terpolymer P(nBuMA-co-TEGMA)-b-PDMAEMA-bP(nBuMA-co-TEGMA) is described below. 5.0 g of the PDMAEMA linear homopolymer (Mn = 25 400 g mol−1, Đ = 1.63, 3.94 × 10−4 mol of dithioester groups) and AIBN (40.4 mg, 2.46 × 10−4 mol) were dissolved in toluene (1.1 mL) and were transferred to a 50 mL roundbottomed flask, fitted with a glass valve, containing nBuMA (1.26 mL, 1.12 g, 7.88 mmol), TEGMA (1.78 mL, 1.84 g, 7.88 mmol), and a magnetic stirring bar. The solution was degassed by three freeze− pump−thaw cycles and was subsequently placed in an oil bath at 65 °C for 24 h. The resulting copolymer (Mn = 31 000 g mol−1, Đ = 2.05) was purified by precipitation in methanol and dried in vacuo for 48 h. Polymer Characterization. Gel Permeation Chromatography (GPC). Samples of the linear homopolymer and the linear terpolymer were characterized by GPC to obtain their molecular weight distributions (MWD) and calculate from those the average MWs and Đ. GPC was performed on a Polymer Laboratories chromatography equipped with an ERC-7515A refractive index (RI) detector and a PL Mixed “D” column. The mobile phase was THF delivered at a flow rate of 1 mL min−1 using a Waters 515 isocratic pump. The MW calibration curve was based on eight narrow MWD linear PMMA B

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Macromolecules standards of MWs of 850, 2810, 4900, 11 550, 30 530, 60 150, 138 500, and 342 900 g mol−1 also from Polymer Laboratories. Nuclear Magnetic Resonance (NMR) Spectroscopy. The composition of the linear terpolymer was determined by 1H NMR spectroscopy using a 300 MHz Avance Bruker NMR spectrometer equipped with an Ultrashield magnet. 1H NMR spectroscopy was also used to determine the monomers conversions. In all cases, the solvent was CDCl3 containing traces of tetramethylsilane (TMS) which was used as an internal reference. Rheology. Rheological measurements were carried out using a stress-controlled rheometer AR-2000ex (TA Instruments) equipped with a cone and plate geometry (diameter = 20 mm with an angle of 4°, truncation 111 μm). The samples were loaded at low temperature (5−10 °C) where they exhibit viscoelastic behavior. The linear viscoelastic regime was established first by oscillatory strain sweeps using a frequency of 1 Hz. Oscillatory frequency sweeps and creep measurements were carried out in the linear regime. The rheometer was thermally regulated (±0.1 °C) by the Peltier controlling system. To prevent changes in concentrations from water evaporation during the experiments, a solvent trap was used. Small-Angle Neutron Scattering (SANS). SANS experiments were performed at the instrument D11 at the Institut Laue-Langevin (ILL) in Grenoble, France. The incident neutrons had a wavelength λ = 6.0 Å with a spread of 9%. The 3He gas detector had an area of 96 cm × 96 cm and a pixel size of 7.5 mm × 7.5 mm. A q-range from 0.002 to 0.52 Å−1 was covered. q is the momentum transfer, q = 4π sin(θ/2)/λ, with θ being the scattering angle. Samples were mounted in quartz glass cells (Hellma Analytics) with a neutron path of 0.5 mm. At the end of each run, the sample transmission was measured. Boron carbide was used for measurement of the dark current and H2O for the detector sensitivity and calibration of the intensity. The scattered intensity curves were azimuthally averaged and corrected for background scattering from the solvent-filled cell and parasitic scattering with the software package LAMP.54 The sample was measured while heating from 10 to 50 °C in steps of 5 or 10 °C. Measurements were performed using a copper sample holder and an inner flow circuit, which was connected to a thermostat. The holder is a massive copper holder for accurate measurements over a wide T range, with precision of T measurement below 0.5 °C. After each temperature change, a thermal equilibration time of 15 min was applied. The measuring times were 45 min, 5 min, and 3 min at the sample−detector distances (SDDs) of 28.00 m, 8.00 m, and 1.50 m, respectively. Sample Preparation. Samples were prepared by mixing a weighted amount of the copolymer with triply distilled water (3D water) adjusted at constant pH 6.5. Attention was given to keep the polymer concentration fixed at 4 wt %. For SANS, a sample of 4 wt % was prepared by weighing appropriate amounts of the copolymer in a vial and adding deuterium oxide (D2O). The samples were homogenized by centrifuging several times. Modeling of the Scattering Curves. To investigate the internal structure of the associated polymer system, in dependence on temperature at fixed pH, the SANS curves were fitted with the following model function:

I(q) = I0Pmic(q)SHS(q) + SOZ(q) + PPorod(q) + Ibkg

Fcs(q) =

1 ⟨Vmic⟩

∫0

∞

f (rcore)Fcs 2(q) drcore

qrcore

+

3Vmic(ρshell − ρsolv )j1 (qrmic) qrmic

(3) where Vcore and Vmic are the volumes of the core and the entire micelle. ρcore, ρshell, and ρsolv are the scattering length densities of the core, the shell, and the solvent; their choice is described below. j1(x) = (sin x − x cos x)/x2, and rmic = rcore + t is the micellar radius. In eq 2, the form factor is normalized by the average micelle volume ⟨Vmic⟩ = 4π⟨rmic3⟩/ 3. f(rcore) is the distribution function of the core radius, modeled by a Schulz distribution:56 f (r ) = (z + 1)z + 1u z

exp[− (z + 1)u] rcore ̅ Γ(z + 1)

(4) −2

where u = rcore/rc̅ ore (rc̅ ore is the average core radius). z = p − 1 with p = σ/rc̅ ore and σ2 the variance of the distribution. The core polydispersity p was not kept constant during fitting. It changed from 0.32 at 10 °C to 0.19 at 50 °C. The Percus−Yevick hard-sphere structure factor is used to describe the intermicellar interactions:57

SHS(q) =

1 1 + 24ηHSG(2RHSq)/(2RHSq)

(5)

The hard-sphere radius, RHS, is half the average center-to-center distance between two particles, and ηHS is the volume fraction of the correlated particles. The function G(x) is given by

2x sin x + (2 − x 2) cos x − 2 sin x − x cos x +δ 2 x x3 4 2 3 − x cos x + 4[(3x − 6) cos x + (x − 6x) sin x + 6] +ε x5 (6) with the help functions G(x) = γ

γ=

(1 + 2ηHS)2 4

(1 − ηHS)

,

δ=

− 6ηHS(1 + ηHS /2)2 (1 − ηHS)4

,

ε=γ

ηHS 2 (7)

The Ornstein−Zernike structure factor reads58 SOZ(q) =

IOZ 1 + (ξq)2

(8)

where ξ is the correlation length of concentration fluctuations and IOZ the scaling factor. These fluctuations give rise to scattering at high q values. Scattering from large, but finite, aggregates of micelles appears at low q values. We adopted Porod’s law to fit this contribution:59 PPorod(q) =

KP qα

(9)

KP is the scaling factor and α the Porod exponent. The latter was found at 2.7 at all temperatures. For ρsolv, the scattering length density (SLD) of D2O, ρD2O = 6.38 × −6 −2 10 Å , was used.60 The value of ρcore was fixed in each fit, and the other fitting parameters were varied. The set of parameters giving the smallest χ2 value was used as starting values with a finer variation of ρcore. In the last run, the step size of ρcore was 0.03 × 10−6 Å−2. The q-resolution was taken into account during fitting.61 The SANS curves were modeled using the SasView software.62

(1)

I0 is a scaling factor and Pmic(q) is the form factor of spherical micelles formed by hydrophobic association of the end blocks of the copolymer. The hard-sphere structure factor, SHS(q), describes their correlation. SOZ(q) is the Ornstein−Zernike structure factor describing the concentration fluctuations in the micellar shell. PPorod(q) describes the scattering of large aggregates formed by the micelles. Ibkg denotes the background. The form factor of a core−shell particle, Pmic(q), having a core with a polydisperse radius, rcore, and a uniform shell thickness, t,55 reads

Pmic(q) =

3Vcore(ρcore − ρshell )j1 (qrcore)

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RESULTS AND DISCUSSION Preparation of (B-co-C)-b-A-b-(B-co-C) Thermoresponsive Telechelic Polyelectrolytes. The strategy implemented in this work involved the preparation of thermoresponsive telechelic polyelectrolytes with tunable hydrophobicity of the (B-co-C)-b-A-b-(B-co-C) topology. 2-(Dimethylamino)ethyl

(2)

with C

DOI: 10.1021/acs.macromol.8b00193 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Synthetic Procedure Followed for the Preparation of the P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMA-coTEGMA) Triblock Terpolymer

methacrylate (DMAEMA) was chosen as the C monomer, nbutyl methacrylate (nBuMA) was chosen as the A monomer, and tri(ethylene glycol) methyl ether methacrylate (TEGMA) was chosen as the B monomer. For the preparation of the methacrylate terpolymer, a two-step reversible addition− fragmentation chain transfer polymerization (RAFT) was employed (Scheme 1). In the first step, TBTPB was used as bifunctional chain transfer agent and 2,2′-azobis(isobutylnitrile) (AIBN) was used as the initiator to produce the poly[(2dimethylamino)ethyl methacrylate] (PDMAEMA) central block. In the second step, extension of the PDMAEMA homopolymer was performed using the produced PDMAEMA as macro-chain-transfer agent for the copolymerization of nBuMA and TEGMA at a 3 M monomer concentration in toluene, at 65 °C and at a constant molar ratio of initiator to CTA of 0.625. The resulting terpolymer was characterized in terms of its molecular weight and composition via GPC and 1H NMR spectroscopy, respectively, and the characterization results are presented in Table 1. As shown in Figure S1 and Table 1, after the second addition of monomers, the MWD was shifted to higher molecular weights, indicating successful extension of the central PDMAEMA block. Characterization using GPC (calibrated with PMMA standards) indicated that the Mn values were higher than the theoretical MWs due to partial deactivation of the active sites. For this reason the triblock copolymer is contaminated by about 15 wt % of the dead PDMAEMA polymer precursor as estimated from the GPC traces (Figure S1). The composition of the resulting terpolymer, as determined from 1H NMR spectroscopy, is in good agreement with the theoretical one. We note here that each of the P(nBuMA18-co-TEGMA15) end-blocks is a random copolymer bearing about 18 −(CH2)3−CH3 (hydrophobic) and 15 −[(CH2)2−O]3−CH3 (hydrophilic) side groups.

Table 1. Molecular Weight and Composition of the Linear Terpolymer and Its Precursor, Synthesized by RAFT Polymerization theor mol %

expt mol %a

GPC

polymer structure

nBu

TEG

nBu

TEG

theor Mw

PDMAEMA P(nBuMA-coTEGMA)-bPDMAEMA-bP(nBuMA-coTEGMA)

0 14

0 14

0 19

0 16

15700 23200

Mn

Mw/Mn

25400 31000

1.63 2.05

a

The composition of monomers refers to to the whole polymer. The composition of monomers in the end-blocks is 54 mol % nBuMA.

Rheological Investigation. Aqueous solutions at different polymer concentrations at room temperature showed strong thickening ability of the P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMA-co-TEGMA) triblock terpolymer, confirming our design expectation to prepare a polymer that is able to create a 3D network in aqueous media. Provided that the focus of this study was to explore the thermoresponsiveness of this thickener and the influence of temperature on its rheological behavior, the concentration was fixed at 4 wt %, which is well above the percolation threshold, ensuring the formation of a 3D spanning polymeric network. Moreover, since the central block is a weak cationic polyelectrolyte and thus the potential network elastic chains are pH-sensitive, the measurements were accomplished at fixed pH 6.5 in the vicinity of the pKa = 6.9 of the PDMAEMA, with a degree of ionization of about 0.55.63 A rapid evaluation of the effect of temperature on the mechanical response of the system was attempted by creep experiments, carried out at constant stress (10 Pa) within the linear viscoelastic regime. In Figure 1, the creep compliance, J(t), is plotted as a function of time for various temperatures D

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terminal relaxation zone is visible, and the terminal relaxation time, τ, can be determined from the inverse radial frequency (1/ω = 1/2πf) at the crossover point of the moduli. This behavior characterizes a viscoelastic fluid. As the temperature increases, the crossover frequency is shifted continuously toward lower frequency values, and at elevated temperatures above 20 °C, the terminal zone is no longer visible within the frequency range investigated, implying very long relaxation times. Thus, the viscoelastic behavior is transformed into a nearly elastic one, revealing the formation of a hydrogel at elevated temperatures. Frequency−temperature superposition was attempted for both moduli (and tan δ, Figure S2) which is demonstrated in Figure 3a as G′ and G″ versus the reduced frequency ωαT, with

Figure 1. Creep compliance, J(t), as a function of time of a 4 wt % P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMA-co-TEGMA) aqueous solution at pH 6.5 for various temperatures, given in the graph.

between 10 and 45 °C. As seen at first glance, the behavior of the system changes remarkably with temperature, passing from a viscoelastic response (high slope at long times) at low temperatures to an elastic response at high temperatures, approaching the behavior of a soft solid (the slope tends to zero). Subsequently, oscillatory shear experiments were carried out in the linear regime. Plots of the frequency dependence of the storage (G′) and loss (G″) modulus, obtained at different temperatures, are given in Figure 2, where two main types of behavior can be distinguished. At low temperatures, the

Figure 3. (a) Time−temperature superposition master curve of G′ (square) and G″(circle) obtained in the temperature range from 5 to 45 °C with a reference temperature of 5 °C. (b) Temperature dependence of the shift factors, aT. The dashed lines are guides for the eye.

a reference temperature of 5 °C. The data can be superposed reasonably well which means that the same mechanism is at play for the relaxation of the macroscopic strain, regardless of the temperature. The crossover of the moduli occurs at a reduced frequency of 0.16 rad/s, which allows us to estimate the shortest relaxation time τ at 6.3 s at the lowest temperature investigated, 5 °C. We note, however, that the distribution of the relaxation times is broad, providing the absence of liquid state at the lowest temperature (i.e., G′ ∼ ω2 and G″ ∼ ω, at low frequencies) and the fact that G′ displays an increase with frequency rather than a plateau value at high frequencies, which may be ascribed to the molecular polydispersity of the stickers.20,22 The shift factor αT is plotted versus temperature in Figure 3b. As seen, the data can be fitted linearly below and above 20 °C, showing two activation processes. Using Arrhenius analysis (ln αT vs 1/T, see Figure S3), the activation energy was estimated to 78 and 246 kJ/mol below and above 20 °C, respectively. This remarkable increase of the activation energy may be attributed to the significant increase of the hydrophobicity of the stickers (end-blocks), imposing slow exchange dynamics.

Figure 2. Storage (G′) and loss (G″) modulus as a function of frequency of a 4 wt % P(nBuMA-co-TEGMA)-b-PDMAEMA-bP(nBuMA-co-TEGMA) triblock terpolymer at different temperatures and pH 6.5. E

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Macromolecules These findings could support a dynamic-to-frozen hydrogel transition at 20 °C. The elastic modulus increased with the reduced frequency approaching a plateau at elevated temperatures. According to rubber elasticity theory for percolated networks, the storage modulus is proportional to the number density of elastically active chains (bridges), n, i.e., G = nkBT (where kB is Boltzmann’s constant).64 Thus, for the experimental value of the high frequency elastic modulus, we could estimate the percentage of the elastic (bridges), namely about 11.8% for a 4 wt % polymer sample. Although this is still a low value, it is sufficient to build a percolated flowerlike micellar network as indicated by the rheological data. From the above dynamic mechanical data, the terminal relaxation times were calculated by using either crossover radial frequencies, τ = 1/ωc (T < 20 °C), or shift factors, τ = τrefaT (T > 20 °C), and are plotted against temperature in Figure 4. As clearly shown, the relaxation time increases by more than 4 orders of magnitude in the temperature range investigated.

Figure 5. Temperature dependence of the zero shear viscosity, η0, extracted from creep (triangles) by the formula η0 = Gelτ (circles), by the formula η0 = η0(T10)aT(T10) (squares) of a 4 wt % P(nBuMA-coTEGMA)-b-PDMAEMA-b-P(nBuMA-co-TEGMA) aqueous solution at pH 6.5. Insert: linear plot of η0 vs T.

temperature cycles for a given sample loaded to the rheometer. The time needed for the equilibration of the temperature after the temperature jump was lower than 1 min. In Figure 6, the frequency-dependent modulus shows that the system exhibited indeed reversible responsiveness upon sudden temperature variation and transformed rapidly and reversibly from a viscoelastic liquid at 12 °C to a nearly elastic soft solid at the physiological temperature. In addition to the above experiment, a temperature ramp was also performed with a heating/cooling rate of 1 °C/min. Figure 7 presents the evolution of the moduli upon varying temperature for a heating/cooling cycle. As observed, the system responds reversibly with negligible hysteresis. The elastic modulus increases slightly, about 2 times, from 5 to 45 °C, implying some rearrangement of the polymer associated structure. Moreover, the strength of the network increases noticeably since tan δ decreases by a factor of 3 as seen in Figure 7b. Obviously, the incorporation of the thermosensitive TEGMA (LCST-type) monomer into the hydrophobic nBuMA stickers of the telechelic polyelectrolyte modified the hydrophobic interactions of the end-blocks, endowing the formed hydrogel with remarkable thermosensitivity. As it has been shown previously, the relaxation time is correlated with the exchange dynamics of the sticky end-blocks of the gelator which influence remarkably the rheological properties of the self-assembling hydrogel.11 The presence of the hydrophilic LCST-type monomers in the end-blocks lowers the lifetime of the physical cross-links, imposing relatively fast exchange of the stickers and therefore rendering the structure dynamic. Thanks to the thermosensitivity of TEGMA, the hydrophobicity of the stickers and, in turn, their exchange time increase upon heating, approaching eventually a “frozen network” (very slow dynamics) at the highest temperatures investigated (see Scheme 2). Structural Investigation by SANS. It is well-known that amphiphilic triblock copolymers bearing hydrophobic associative end-blocks tend to form a transient micellar network constituted of hydrophobic domains (micellar cores) interconnected by bridging chains. The building block of the network is thus a flowerlike micelle, and scattering techniques may give the characteristic sizes of the network, i.e., the radius of the micellar cores and the average distance between them.16 In order to investigate if the network structure of the hydrogel is influenced by temperature, SANS experiments were

Figure 4. Temperature dependence of the characteristic relaxation time of a 4 wt % P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMAco-TEGMA) aqueous solution at pH 6.5. The dashed line is a guide for the eye.

The temperature dependence of the zero-shear viscosity was evaluated using oscillatory data via the equation η0 = Gelτ, where Gel was taken as the storage modulus at 1 Hz (well above the crossover frequency) and from the creep data at sufficiently long times (much higher than the relaxation times) where steady state could be achieved. Provided that η0 values from oscillatory and creep measurements coincide at 10 °C, we also evaluated η0 using shift factors with 10 °C as a reference temperature, that is, η0 = η0(T10)aT(T10). All these data are plotted in Figure 5. The shear viscosity increases remarkably, about 5 orders of magnitude in the temperature range from 10 to 45 °C, indicating a strong thermothickening effect. Displaying the data in a linear scale (inset of Figure 5), we observe a steep viscosity enhancement above 20 °C in consistence with the dynamic-to-frozen hydrogel transition discussed below. Given that the elastic modulus increases only slightly in this temperature range, the main mechanism of this remarkable thermothickening effect is accounted for by the slowdown of the exchange dynamics of the stickers upon increasing T due to their enhanced hydrophobicity above their LCST. Reversibility of the Thermoresponsiveness. In order to evaluate the reversibility of the thermoresponsiveness of the hydrogel, consecutive oscillatory shear measurements were performed by alternating low (12 °C) and high (37 °C) F

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Figure 6. Oscillatory shear (G′ and G″ vs frequency) upon temperature switch between 12 and 37 °C of 4 wt % P(nBuMA-co-TEGMA)-bPDMAEMA-b-P(nBuMA-co-TEGMA) aqueous solution at pH 6.5.

Figure 8. SANS data of a 4 wt % polymer in aqueous media of pH 6.6 at the temperatures given. Symbols: experimental data; lines: model fits. The curves are shifted vertically by factors of 1.5. In the experimental data, only every second point is shown.

of the micelles. The smooth decay at q > 0.08 Å−1 is mainly due to the Ornstein−Zernike structure factor, which describes concentration fluctuations in semidilute or concentrated polymer solutions. The forward scattering at q < 0.01 Å−1 is due to large-scale inhomogeneities of the network. We chose to fit a model comprising expressions for spherical core−shell micelles correlated by a hard-sphere structure factor, the Ornstein−Zernike term describing concentration fluctuations of the polymer solution in the shell and between the micellar cores, and a Porod term describing forward scattering due to fractal aggregates of the flowerlike micelles. Because of the thermoresponsive nature of the end blocks forming the micellar cores, these may contain D2O. Thus, one cannot assume that the scattering length density of these cores is the same as in the bulk and that it is independent of temperature. Since the parameters are strongly correlated and artifacts may arise when fitting them all at once, we proceeded as follows: The SLD of the core, ρcore, was varied step by step, and all other parameters were fitted while ρcore was fixed at each step. The set of parameters giving the minimum value of χ2 was used as a starting point for the next round of fitting in which ρcore was varied in smaller steps around such identified value. A few rounds were carried out in this way, until the step size was as small as 0.03 × 10−6 Å−2. Fits of good quality are obtained over the whole q range. The contributions to the model are shown at an example in Figure S4 of the Supporting Information. The dependence on temperature of the most important fitting parameters is shown in Figure 9. The core radius increases from 2.4 nm at 10 °C to 3.9 nm at 50 °C (Figure 9a). The micellar radius, rmic, increases from 6.2 nm at 10 °C to 9.4 nm at 50 °C, which goes along with an increase of the shell thickness from 3.8 nm at 10 °C to 5.5 nm at 50 °C. The hard-sphere radius, indicative of the center-to-

Figure 7. Storage and loss modulus (a) and tan δ (b) versus temperature in a heating/cooling cycle with a rate of 1 °C/min of a 4 wt % P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMA-coTEGMA) aqueous solution at pH 6.5.

Scheme 2. Schematic Illustration of the Physically CrossLinked P(TEGMA-co-nBuMA) End-Blocks of the Gelatora

a

The hydrophilic side chains of TEGMA are dehydrated reversibly upon heating and turn to hydrophobic, increasing hence the lifetime of the junctions and slightly the network functionality, which leads eventually to a “frozen” structure at elevated temperatures.

performed with a 4 wt % polymer solution in D2O at pH 6.6. D2O was chosen to maximize the scattering contrast between the polymer and the solvent. A heating run was carried out from 10 to 50 °C in steps of 5 or 10 °C. As Figure 8 demonstrates, the scattering curves feature a maximum at ca. 0.027 Å−1 at 10 °C which moves to ∼0.023 Å−1 at 50 °C. At higher q values, shoulders appear which reflect the form factor G

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TEGMA) segments are incorporated into the core of the micelles due to the increasing hydrophobicity of the P(nBuMAco-TEGMA) end-blocks (Scheme 2). The increase of RHS and thus the distance between micelles with temperature seem to be consistent with an increase of the aggregation number with temperature. Consequently, more stretched conformations should be expected resulting from the steric hindrance effect that is induced by a higher number of PDMAEMA chains in the shell part of the micelles. The average distance between the centers of the micellar cores is about 20−23 nm. Comparing with the contour length of the copolymer, it is deduced that the degree of extension of the bridging chains is about 50%. The volume fraction of correlated micelles η is relatively low, meaning that the interactions between the micelles are weak for the 4 wt % polymer concentration. This could explain the low percentage of the bridging chains of the formed transient network. Coevaluating the rheological and SANS data, we conclude that the basic structure of a flowerlike micellar network changes only slightly with temperature, and the strong effect on the viscosity is mainly due to the lifetime of the network junctions which increases by several orders of magnitude upon heating. This is the consequence of the increasing hydrophobicity of the end-blocks of the copolymer, as expected from their macromolecular design. Shear Responsiveness and Injectability. In order to evaluate the shear responsiveness of the hydrogel and its ability to self-heal rapidly after injection, the hydrogel was subjected to alternating shear rates, simulating conditions similar to those of injection through a 28-gauge syringe needle. The simulated shear rate for such injection was estimated to 17.25 s−1, assuming a flow rate of 1000 μL/min through a needle of internal radius of 0.0925 mm.66 Figure 10 shows the time

Figure 9. Results from SANS on a 4 wt % polymer solution in D2O. (a) Ornstein−Zernike correlation length ξ (closed black squares), core radius rcore (open red circles), micellar radius rmic (close green triangles up), and hard-sphere radius RHS (open blue triangles down). (b) Volume fraction from the hard-sphere structure factor. (c) Scattering length densities of the core (closed red circles) and of the shell (open red circles).

center distance between the micelles, increases from 10.1 nm at 10 °C to 11.5 nm at 50 °C. The Ornstein−Zernike correlation length increases from 0.27 nm at 10 °C to 0.87 nm at 50 °C. The spatial correlation between the micelles becomes stronger, as indicated by the increasing value of the volume fraction from the hard-sphere structure factor (Figure 9b). However, the values of 0.16−0.20 are relatively low. Strong forward scattering indicates that the network is not homogeneous, but the connected micelles form large clusters. The SLD of the core (related to the content of D2O in the core) increases from 4.0 × 10−6 to 4.4 × 10−6 Å−2 and then decreases smoothly with temperature. This may be attributed to the integration of the TEGMA moieties within the hydrophobic cores upon heating as approaching LCST which remain partially hydrated close to LCST.65 Upon further heating above LCST, progressive partial dehydration seems to occur due to the increasing hydrophobicity of the end-blocks that imposes partial expulsion of the water molecules from the P(nBuMA-co-TEGMA) nanodomains. Additionally, the Porod exponent was determined at 2.7, remarkably lower than 4, which is found for compact particles with a smooth surface. We suggest that the micelles form loosely connected clusters. The increase of rcore with temperature may have two reasons. First, a progressive dehydration of the TEGMA moieties might result in a better integration of the end-blocks into the hydrophobic domains. Second, the aggregation number may increase upon heating, meaning that more P(nBuMA-co-

Figure 10. Shear viscosities versus time at alternating shear rates 0.005 s−1 (at 18 °C), 17.25 s−1 (at 18 °C), and 0.005 s−1 (at 37 °C) of a 4 wt % P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMA-co-TEGMA) aqueous solution at pH 6.5.

sweep of the apparent viscosity at alternating, constant low (0.005 s−1) and high (17.25 s−1) shear rates. Obviously, the hydrogel exhibited a remarkable shear-thinning effect at the temperature of injection (18 °C), as the viscosity drops instantaneously 2 orders of magnitude, responding to the sudden shear rate jump. Interestingly, high viscosity was instantaneously recovered upon lowering the shear rate to its initial value at 37 °C. Moreover, a thixotropic behavior can be observed probably due to long relaxation times at the temperature of injection and physiological temperature. As observed, the viscosity increases continuously at the physiological temperature, and upon cessation of the applied stress, H

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6.5. Creep, oscillatory, and flow measurements in the temperature range of 5−45 °C revealed a strong thermothickening behavior. At low temperatures the system behaves like a viscoelastic complex fluid (dynamic network) while at higher temperatures an elastic hydrogel was formed (“frozen” network). More importantly, the viscosity increased exponentially upon heating, about 5 orders of magnitude from 5 to 45 °C, while the elastic modules increased slightly in the same temperature range, implying that the basic network structure does not change remarkably. Thus, the main reason for this strong viscosity augmentation is the exchange dynamics of the stickers that change significantly upon heating. SANS experimental data, well fitted with a spherical core− shell particle form factor and correlated by a hard-sphere structure factor, showed some evolution of the various characteristic sizes of the flowerlike micellar network, namely micellar core (network junctions), shell thickness, and intercore distance (hard-sphere radius), with increasing temperature. These results were understood as an increase of the network functionality (number of stickers per hydrophobic core) upon increasing the hydrophobicity of the end-blocks of the triblock gelator), in qualitative agreement with the increasing exchange dynamics of the stickers observed by rheology. The hydrogel exhibits a drop of the shear induced viscosity at about 10 Pa upon applying a shear rate of 17.25 s−1 at 18 °C, and it is recovered rapidly upon cessation of the applied stress at 37 °C, simulating conditions similar to those of injection through a 28-gauge syringe needle. Moreover, the strength of the network increased with time when resting at 37 °C, facilitating immobilization of the gel in the position of injection. The combination of thermo- (weak gel at room temperature and strong gel at physiological temperature) and shear-induced gel injectability renders this hydrogel suitable for potential applications in tissue engineering. For instance, these kinds of hydrogels are good candidates for cell transplantation through injection strategies, which requires a weak gel to protect the cells during injection and a stronger gel after injection to immobilize the created scaffold in the targeting position of the host tissue. It should be mentioned that although we do not provide cell viability tests, all the monomers incorporated in this terpolymer exhibit biocompatibility. As is shown in this work, the requirements of this kind of hydrogels can be met in simple amphiphilic triblock copolymer topologies, combining thermo- and shear-responsive properties. The gel properties can be tailored by the easy design of the molecular characteristics of the polymeric gelator through controlled polymerization methods. The key strategy was the appropriate design of the sticky (hydrophobic) end-blocks. Instead of using LCST polymeric sequences, we replaced them with a random copolymer incorporating associative hydrophobic moieties. Thus, the gelling system did not exhibit a temperature-induced sharp sol−gel transition (case of pure LCST-type stickers) but a weak-to-strong gel transition which is required for carrying sensitive cells to the host tissue by injection.

the viscosity should approach the zero-shear viscosity value which is much higher (about 1.8 × 106 Pa·s) as extracted from the data of Figure 5. Finally, the behavior of the elastic modulus upon disrupting− recovery of the hydrogel was explored by designing two consecutive experiments. The hydrogel was subjected to a strain sweep at 18 °C (temperature of injection) far beyond the linear regime, followed by a time sweep at 37 °C (body temperature) with a strain amplitude of 10%, within the linear regime. The time needed to switch the temperature from 18 to 37 °C was 45 s. As can be seen in Figure 11, G′ becomes lower

Figure 11. Strain sweep at 18 °C and 1 Hz and subsequent time sweep at 37 °C with applying strain within the linear viscoelastic regime (10%) of a 4 wt % P(nBuMA-co-TEGMA)-b-PDMAEMA-b-P(nBuMA-co-TEGMA) aqueous solution at pH 6.5. Inset: time evolution of elastic modulus in linear plot.

than G″ at high strain in the first part of the experiment, implying disruption of the network structure, hence allowing the liquid to flow. Upon a sudden decrease of the strain amplitude to 10%, the hydrogel rapidly recovered at 37 °C, since the elastic modulus is much higher than the storage modulus, and their values are slightly higher than those at 18 °C in the linear regime. Using a linear scale (Figure 11, inset), it is seen that the elastic modulus increases slightly with time, implying that rearrangements of the structure continuously take place after the nearly instantaneous recovery of the network which likely is due to the high relaxation times. The observed evolution of the modulus and, in turn, the network strengthening after injection is beneficial for the purpose of cell transplantation.

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CONCLUSIONS A novel thermoresponsive gelator of (B-co-C)-b-A-b-(B-co-C) topology, comprising a PDMAEMA weak polyelectrolyte as central block, end-capped by thermosensitive P(TEGMA-conBuMA) random copolymers, was designed and explored in aqueous media. The triblock terpolymer was synthesized by reversible addition−fragmentation chain transfer (RAFT) controlled polymerization method. The end-blocks are sticky due to the presence of the hydrophobic nBuMA moieties (54%) which drives the triblock to self-assemble in water, forming a percolated three-dimensional network of flowerlike micelles at elevated concentrations. The second TEGMA monomer within the end-blocks is of LCST type, since the corresponding PTEGMA homopolymer exhibits a LCST at about 52 °C and was chosen to endow the stickers with thermosensitivity. The temperature-dependent gelling properties of the P(TEGMA-co-nBuMA)-b-PDMAEMA-b-P(TEGMA-conBuMA) triblock terpolymer were investigated at polymer concentration of 4 wt % (above percolation) and at fixed pH

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b00193. GPC characterization, tan δ versus ω at different temperatures, and time−temperature superposition I

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master curve (tan δ vs ωaT), Arrhenius plot of the shift factor aT, and example of the best fit to the SANS scattering curves (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.T.). ORCID

Constantinos Tsitsilianis: 0000-0002-7265-9037 Christine M. Papadakis: 0000-0002-7098-3458 Costas S. Patrickios: 0000-0001-8855-0370 Christophe Chassenieux: 0000-0002-3859-8277 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Dr. Margarita A. Dyakonova for her assistance in the SANS experiments. C.T. expresses his gratitude for the invitation and the financial support of his stay at the ̈ Département Polymères, Colloides et Interfaces, of Le Mans Université.

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