Diverse nanostructures and gel behaviours contained in a thermo- and dual-pH-sensitive ABC (PNIPAM–PAA–P4VP) terpolymer in an aqueous solution

Abstract

A thermo- and dual-pH-sensitive ABC triblock copolymer composed of poly(N-isopropylacrylamide)-block-poly(acrylic acid)-block-poly(4-vinyl pyridine) (PNIPAM-b-PAA-b-P4VP, NAV) was synthesized via sequential reversible addition–fragmentation chain transfer (RAFT) polymerization and subsequent hydrolysis. NAV, which had the property of multi-stimuli, containing thermo-sensitive PNIPAM block and opposite pH-sensitive PAA and P4VP blocks, formed diverse nanostructures in an aqueous solution by varying the temperature and pH. Nanostructures including large compound micelles (LCMs), vesicles, spherical micelles and 3D topologies were observed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). More significantly, the solation–gelation behaviour and morphology of the NAV system induced by temperature and pH were investigated by rheology and scanning electron microscopy (SEM), respectively. TEM and SEM revealed that the gelation behaviour of NAV resulted from the bridge effect of the ionized PAA chains between the hydrophobic P4VP cores (V-core) and PNIPAM cores (N-core) over the LCST of PNIPAM. The results obtained from the rheology measurements showed that the gelation temperature was independent of alkaline pH, but slightly tuned by the copolymer concentration or pH (only 6 to 7). Repeated sol–gel transition via temperature changes under alkaline conditions showed the good hydrogel recyclability of NAV.

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Introduction

Stimuli-responsive chemical or physical hydrogels with broadly tuneable characteristics are widely used in drug delivery, tissue engineering and other biomedical fields. The tuneable conditions include temperature, pH, light, ionic strength, enzymes and magnetic fields.^1–9 In general, physical hydrogels can be formed by a physical mixture, hydrophobic interactions, hydrogen bonding or ionic interactions, etc. Thus, its phase state can be reversibly transferred between sol and gel by changing the external conditions. When compared with chemical hydrogels formed by covalent cross-linking, physical hydrogels have more extensive applications, especially in the biomedical field such as site-specific drug delivery and medical injection.^2,10–13

Among all the physical stimuli-responsive hydrogels, the thermo- or pH-responsive hydrogel are especially useful in biomedical applications. A kind of well-known thermo-responsive polymer used widely in the smart hydrogel, poly(N-isopropylacrylamide) (PNIPAM) and its derivatives have been extensively studied because of their tuneable lower critical solution temperature (LCST) phase transition in aqueous solution between room temperature (25 °C) and human physiological temperature (37 °C).^14–20 For the pH-responsive polymer, due to the opposite hydrophilic and hydrophobic effects in the same pH environment, poly(acrylic acid) (PAA) and poly(2/4-vinyl pyridine) (P2/4VP) have also obvious applications in drug delivery and bio-hydrogel.^21–25 Therefore, a compound comprised of PNIPAM and PAA (or P2/4VP) will be of substantial interest and research value in biomedical fields. For instance, Gao and co-workers prepared a pH- and thermo-responsive poly(N-isopropylacrylamide-co-acrylic acid derivative) (P(NIPAM-co-AAD)) hydrogel, which can be self-regulated at above or below physiological temperature and pH. The bioavailability of hydrogel-encapsulated insulin via the oral administration reached 5.24 wt%, which is much higher than pure insulin solution given orally.²⁶ Wang et al. studied P4VP ultrathin hydrogels with remarkably high, fast and reversible swelling/shrinkage and sharp surface wettability transitions due to protonation/deprotonation in response to pH changes.²⁷

Over the past few years, a unique physical stimuli-responsive hydrogel model, ABA (or ABCBA) block copolymer containing hydrophilic mid-blocks and hydrophobic end-blocks has been extensively investigated.^28–33 However, these hydrogels are often single-responsive and have a higher minimum gelation concentration over 10 wt%. Zhou and co-workers explained the reason for this and designed a novel hydrogel formed from ABC block copolymers. This hydrogel could reduce the minimum gelation concentration to 5 wt%.³⁴ In the same year, they also reported another pH- and thermo-responsive hydrogel with linkage effect from poly(ethylene-alt-propylene)-block-poly(ethylene oxide)-block-poly(N-isopropyl acrylamide-co-acrylic acid) (PEP–PEO–P(NIPAM-co-AA)). However, this hydrogel could be prepared by temperature inducement with a narrow pH aqueous solution (pH 4–5).³⁵ In other words, in other pH conditions the gelation structure would be destroyed at any temperature. Thus, the application of this hydrogel would be probably limited due to the close contact between pH and temperature.

In this study, a novel micelle-based thermo- and dual-pH-sensitive hydrogel obtained from poly(N-isopropyl acrylamide)-block-poly(acrylic acid)-block-poly(4-vinylpyridine) (PNIPAM-b-PAA-b-P4VP, NAV) ABC triblock copolymer was synthesized. On a micro level, upon the variation of pH, the NAV triblock copolymer could also self-assemble into a series of particular structures, such as large compound micelles (LCMs), vesicles, aggregations and spherical micelles. On a macro level, the gelation of NAV could be independently triggered by regulating the temperature or alkaline pH. The NAV was comprised of PAA, P4VP and PNIPAM blocks, which formed hydrophilic bridges, pH-sensitive cores and thermo-sensitive cores, respectively (Fig. 1). At any temperature, the PAA bridges and P4VP cores could be formed or destroyed by a slight regulation of the weakly acidic pH. Similarly, the PNIPAM cores were controlled by temperature. In addition, the corresponding effects above could contribute to the generation of flower-like nanostructures and hydrogels at low and high concentrations of the copolymer, respectively. In a nutshell, this work may provide a general and efficient paradigm to fabricate a family of stimuli-responsive polymer in theoretical studies and practical applications.

Fig. 1 A schematic of the micro-nanostructures (drawn) and macro-hydrogel (filmed) of the NAV triblock copolymer with varying pH at 25 °C and 50 °C, respectively. The P4VP cores (marked V-core), ionized PAA chains (marked bridge) and PNIPAM cores (marked N-core) have a rapid response to pH and pH and temperature, respectively.

Experimental

Materials

N-Isopropylacrylamide (NIPAM, 97%, Aladdin China) was recrystallized from a mixture of toluene and hexane (1/7 by volume). 4-Vinyl pyridine (4VP, 99%, Aladdin China) and tert-butyl acrylate (tBA, 99%, Aladdin China) was passed through neutral alumina and distilled under reduced pressure to remove inhibitors prior to use. 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol. The chain transfer agent (CTA) 2-(2-cyanopropyl)dithiobenzoate (CPDB) was synthesized according to a literature procedure.³⁶ Trifluoroacetic acid (TFA, 99%, Aldrich China). Sodium hydroxide, diethyl ether, THF and 1,4-dioxane were purified by standard methods before use.

Synthesis of PNIPAM-b-PtBA-b-P4VP (NBV) via sequential RAFT polymerization

The triblock copolymer, PNIPAM-b-PtBA-b-P4VP (NBV), was synthesized via sequential RAFT polymerization, following the procedure shown in Scheme 1. The polymerization degree (D_p) of each product was calculated by ¹H NMR recorded in CDCl₃. In the first step, the homopolymer of PNIPAM with dithiobenzoate chain ends was prepared. A typical process was as follows: a 25 mL Schlenk flask was charged with NIPAM (6.0005 g, 53.01 mmol), CPDB (0.1360 g, 0.66 mmol), AIBN (0.0325 g, 0.20 mmol) and DMF (6 mL). After degassing via three freeze–pump–thaw cycles, the flask was immersed into a 70 °C oil bath and the mixture was stirred for 2 h. The reaction was terminated by immersing the flask into an ice bath. The product was precipitated twice with a 10-fold volume of ice–diethyl ether. The crude product was dialysed against distilled water using a dialysis tube with a cut-off molecular weight of 5000 Da for 48 h, then freeze-dried. ¹H NMR (CDCl₃, δ, ppm): 7.41 (para-C₆H₅, 1H), 7.57–7.59 (meta-C₆H₅, 1H), 7.96 (ortho-C₆H₅, 1H), from –CPDB (–CTA); 1.14 (–(CH₃)₂, 6H); 4.01 (–CH–, 1H), 1.65–2.16 (–CH–CH₂–, 3H). M_nNMR ∼ 7335 g mol⁻¹, D_p = 65. Other similar RAFT polymerization procedures and ¹H NMR signals were briefly described in the following sections.

Scheme 1 The synthetic routes used to prepare PNIPAM-b-PAA-b-P4VP via sequential RAFT polymerization and subsequent hydrolysis.

CTA-PtBA–PNIPAM diblock copolymer. CTA-PNIPAM (1.5005 g, 0.21 mmol), tBA (5.2458 g, 40.93 mmol), AIBN (0.0103 g, 0.06 mmol) and solvent (DMF, 6 mL). After three freeze–pump–thaw cycles in a 25 mL Schlenk flask, the mixture was stirred for 6 h in a 75 °C oil bath. Then, the product was purified and characterized. ¹H NMR (CDCl₃, δ, ppm): besides the H signals of PNIPAM, 1.44 (–(CH₃)₃, 9H), 1.65–2.16 (–CH–CH₂–, 3H) from PtBA. M_nNMR ∼ 30 405 g mol⁻¹, D_pPtBA = 180.

P4VP-b-PNIPAM-b-PtBA triblock copolymer. CTA-PtBA–PNIPAM (4.0020 g, 0.13 mmol), 4VP (1.1066 g, 10.62 mmol), AIBN (0.0065 g, 0.04 mmol) and solvent (DMF, 5 mL). The reaction was continued for 24 h in a 75 °C oil bath. After purification, the product was characterized by ¹H NMR in CDCl₃. ¹H NMR (CDCl₃, δ, ppm): besides the above H signals from PNIPAM and PtBA, 6.41 (ortho-C₅H₅N, 1H), 8.35 (meta-C₅H₅N, 1H), 1.65–2.16 (–CH–CH₂–, 3H) from P4VP. M_nNMR ∼ 38 531 g mol⁻¹, D_pP4VP = 85.

Acidic hydrolysis of the PNIPAM-b-PtBA-b-P4VP (NBV) triblock copolymer

Trifluoroacetic acid (CF₃COOH, 10-fold) was added dropwise into a dichloromethane (CH₂Cl₂) solution of PNIPAM-b-PtBA-b-P4VP triblock copolymer (C ∼ 150 mg mL⁻¹) at 0 °C under vigorous stirring. Then, the mixture was constantly stirred for 1 h at 0 °C and a further 24 h at room temperature.³⁷ After purification using a dialysis tube with a cut-off molecular weight of 20 000 Da for 48 h in distilled water. The triblock copolymer of PNIPAM-b-PAA-b-P4VP (NAV) was obtained after freeze-drying. ¹H NMR (DMSO-d₆, δ, ppm): besides the H signals from PNIPAM and P4VP, 12.24 (–COOH, 1H) from PAA. M_nNMR ∼ 29 015 g mol⁻¹, D_pPAA = 177. The target triblock copolymer was expressed as PNIPAM₆₃-b-PAA₁₇₇-b-P4VP₈₅.

Formation of the NAV aggregation solutions

The aggregation solutions of NAV triblock copolymer, including dilute and concentrated solutions, which were used in the morphology and rheology studies, respectively, were prepared by an ultrasonic-standing method in acid or alkaline aqueous media. For the morphological characterization, an initial dilute concentration (IC, 5 mg mL⁻¹) of NAV was obtained by dissolving the copolymer (50 mg) in 0.01 M HCl or NaOH aqueous (10 mL). Under ultrasonication, the other pH's NAV dilute solutions were prepared from IC by titration using 1–0.01 M HCl or 1–0.01 M NaOH aqueous media. In the rheology section, the designed concentrated NAV solutions were obtained from the initial concentration of 100 mg mL⁻¹ (∼10 wt%) of NAV using the same titration method with 0.1 M HCl. Finally, a series of NAV aggregation solutions were obtained after standing overnight.

Characterization

Fourier transform infrared (FTIR) spectra were obtained on an AVATAR 370 FT-IR spectrometer (Nicolet, USA). The D_p of the products was calculated using the ¹H NMR spectra recorded on a Bruker 500 MHz spectrometer using DMSO-d₆ or CDCl₃ as the NMR solvent. The turbidity behaviour of NAV was measured using a UV-visible spectrophotometer (UV-vis, Agilent, USA) at a wavelength of 500 nm. DLS measurements were performed on Zetasizer Nano ZSP (Malvern Instruments Ltd., Malvern, U.K.). Scattered light was collected at a fixed angle of 90° for a duration of ∼10 min. The rheological measurements were carried out using a LVDV-11 DV-II+Pro viscometer (Brookfield, USA) and the viscosity and temperature of the NAV concentrated solutions were obtained on viscometer under 100 RPM and 64# spindle.

The morphology of the NAV aggregations was observed using a JEOL JEM-1210 transmission electron microscope (TEM, Electron, Japan) operating at an accelerating voltage of 200 kV. The TEM samples were prepared by dripping a polymer solution onto 400 mesh copper grids pre-coated with carbon film. Importantly, during sample preparation, all the tools used and NAV solutions must be preheated at a predetermined temperature (25 °C and 50 °C) using an oven. Especially, the samples at 50 °C were carried out at 50 °C oven.

The solution and gelation morphology of NAV were investigated using a LEO 1530 VP Scanning Electron Microscope (SEM). The solation and gelation NAV samples were prepared by maintaining the polymer solution of 50 mg mL⁻¹ at 25 °C and 50 °C for 30 min, respectively. Immediately, the samples were frozen in liquid nitrogen and freeze-dried. Finally, the dried samples were sprayed gold on their surface and subsequently subjected to SEM.

Results and discussion

Synthesis of thermo- and pH-sensitive NAV triblock copolymers

RAFT polymerization is one of the attractive controlled/“living” radical polymerization methods used for the synthesis of block copolymers because of its efficient control over the molecular weight and distribution (MWD).³⁸ Plenty of papers have reported the use of RAFT polymerization to design diverse block copolymer using different CTAs. In this paper, according to the order: PNIPAM, PtBA and P4VP, the PNIPAM-b-PtBA-b-P4VP (NBV) triblock copolymer was first synthesized via sequential RAFT using 2-(2-cyanopropyl)dithiobenzoate (CPDB) as the CTA under an initiation temperature of 70–75 °C. Then, the PNIPAM-b-PAA-b-P4VP (NAV) triblock copolymer was obtained via an acidic hydrolysis reaction from NBV.³⁷ Each purified product was characterized by ¹H NMR and FT-IR spectroscopy.

Firstly, FT-IR spectra of NAV and the precursors are shown Fig. 2(a). All the spectra exhibit an absorption band at 2973 cm⁻¹ and 2930 cm⁻¹ from the –CH₃ and –CH₂– asymmetric stretching vibrations, respectively. The other main signals for the PNIPAM homopolymer (spectra A) include the peaks at 1650 cm⁻¹ and 1544 cm⁻¹ that are attributed to –C=O and –NH vibrational bands from –CONH, respectively.²⁹ The embed monomer tBA, the diblock copolymer of PNIPAM-b-PtBA show the absorption peak of –COOR at 1730 cm⁻¹ (spectra B). In the triblock copolymer of PNIPAM-b-PtBA-b-P4VP (spectra C), a series of new absorption peaks at 1601 cm⁻¹ and 1418 cm⁻¹ correspond to the –C=N and –C=C– stretching vibrations in the pyridine ring, respectively. In addition, the band at 993 cm⁻¹ belongs to the –CH bending vibration peaks within the pyridine ring.^39,40 In the NAV triblock copolymer (spectra D), a lower ratio of the –CH₃ (2973 cm⁻¹) and –CH₂– (2930 cm⁻¹) peak intensities and a red-shift of the –C=O bands from 1730 cm⁻¹ to 1717 cm⁻¹ suggest the successful hydrolysis of PtBA to form PAA. In addition, the loss of the –C=N bands arising from pyridine was probably due to the formation of hydrogen bonds between PAA and P4VP.⁴¹ Furthermore, the sodium PAA-copolymer (PNIPAM-b-PAAS-b-P4VP) spectra also show that the ester carbonyl peak disappeared and the –C=N (1601 cm⁻¹) arose again (Fig. S1†). Secondly, the chemical structure and polymerization degree (D_p) of NAV and its precursors were detected using ¹H NMR in DMSO-d₆ or CDCl₃ (Fig. 2 and S2†). The structure signals have already been mentioned in the Experimental section. Using the calculated ratio of the integral peak area between the methine protons of PNIPAM (marked b) and the para-C₆H₅ protons of CPDB (marked 1), the D_p of PNIPAM was estimated to be about 65. Similarly, the D_p values of the other stage polymers were also roughly calculated (see ESI†). In short, all the evidence proves the success of the synthesis of the NAV triblock copolymer.

Fig. 2 (a) The FT-IR spectra of (A) PNIPAM, (B) PNIPAM-b-PtBA, (C) PNIPAM-b-PtBA-b-P4VP and (D) PNIPAM-b-PAA-b-P4VP (NAV). (b) The ¹H NMR spectrum of NAV in DMSO-d₆.

The dual-responsive behaviours of the NAV triblock copolymer

In this section, the microscopic assembly behaviour of the NAV triblock copolymer was investigated in aqueous solution at different pH and temperature.

Isoelectric point (IEP). For the polyampholyte of NAV, the P4VP blocks are protonated in acidic solution, whereas the PAA segments are ionized in an alkaline solution. Thus, this will contribute to a charge change from positive to negative as the pH increases and the isoelectric point (IEP) will be located at the pH at which the polymer bears a zero net charge. At pH = 1.0, the steady aggregation of NAV displays a net positive charge with a zeta potential (ζ) of +40.7 eV, which is much higher than that found at pH 2.0 with a ζ of +25.9 eV because of the low number of protonated P4VP segments at pH 2.0 (Fig. 3). Destructively, a sharp change in the charge from positive to negative value occurred at pH ∼ 4.0, namely the IEP. Further, the absolute ζ value increased. The IEP at ∼4.0 was only a slight deviation from the theoretical value (4.56) calculated using the following analytical expression:⁴²
where R is the molar ratio of the acid to basic groups.

Fig. 3 Zeta potential of the NAV triblock copolymer in different pH solutions.

pH-Sensitivity of the NAV triblock copolymer. Holding the temperature constant, a series of pH values were firstly used to study the pH-responsive behaviour using a turbidity method and DLS (Fig. 4 and 5). In a pH 1.0 solution, the NAV was comprised of long hydrophobic PAA chains, short hydrophilic chains of PNIPAM and protonated P4VP at 25 °C. However, there was no thermo-responsive behaviour and phase separation, but a lower stable transmittance. DLS also detected a constant D_n at about 277 and 260 nm with narrow particle dispersion index (PDI) of 0.183 and 0.103 at 25 and 50 °C, respectively (Fig. 5). A possible explanation is that the NAV triblock copolymer formed different LCMs models (see the illustration in Fig. 1, marked by LCM₁-C and LCM₁-H at 25 °C and 50 °C, respectively). In addition, the theory of LCM₁-C was reported in 1999, by another name, “finite size clusters”.⁴³ By raising the temperature to 50 °C, the NAV triblock copolymer was predicted to display similar aggregation behaviour to P2VP–PAA–PnBMA with core–shell–corona structures.⁴⁴ However, the constant D_n and transmittance imply another LCMs structure-like PS₂₀₀–PAA₄ diblock copolymer in DMF/THF–water (Fig. S3(a)†).^45,46 At this moment, the hydrophobic PNIPAM and PAA chains play the same roles as the above PS segments and were randomly distributed in or out of the LCMs, resulting in a constant turbidity and aggregation size.

Fig. 4 The turbidity curves obtained for the NAV triblock copolymer solution at 2.0 mg mL⁻¹ as a function of pH.

Fig. 5 The intensity distribution of the hydrodynamic diameter (D_n) of NAV at 2.0 mg mL⁻¹ as a function of pH at 25 °C and 50 °C, respectively.

Near the IEP (pH 2, 4 and 6), the NAV shows abnormal turbidity behaviour. Firstly, at pH 2 and 4, the transmittance begins to increase after the first decrease with increasing temperature. The reason for the decrease between 26–30 °C is that the PNIPAM chains collapse after the LCST of NAV. However, as temperature the continually rises, the slight increase in transmittance shows a solubility improvement, probably due to the breaking of the H-bonds between the NIPAM and AA moieties. Secondly, when the pH increased to 6, NAV showed obvious thermo-sensitive behaviour between 30–40 °C. However, DLS detected a transition from small D_n with wide distribution at 25 °C to large D_n with narrow distribution at 50 °C. The TEM results explained that the opposite effect of size and distribution was due to the change in the NAV structure from tight spherical micelles to loose 3D networks (Fig. S3(b)†). Indeed, the tight structure was probably due to the H-bonding interactions that exist intra- or intermolecularly in NAV between the deionized PAA and PNIPAM segments.⁴⁷ When the temperature was increased, the H-bonds among the PAA and PNIPAM are first destroyed. Secondly, the H-bonding between H₂O and PNIPAM disappear, namely, the PNIPAM collapsed. The ionized PAA chains interpenetrate between the collapsed PNIPAM and P4VP chains, resulting in the formation of a loose 3D network.

In the alkaline region, the strong hydrophilicity of the completely ionized PAA offset the turbidity behaviour of PNIPAM. Thus, the transmittance remained constant with increasing temperature. However, the Z-ave of the micelles had a decreasing trend after the first increase with a more alkaline pH (Fig. S4†). The larger size was due to the stronger electrostatic repulsion effect caused by the gradually ionized PAA in the micelles.⁴⁴ However, a too alkaline pH contributed to excess counter-ions, resulting in a macromolecule electrostatic shielding effect.⁴³ This effect led to a decreased trend.

The thermo-sensitivity of the NAV triblock copolymer. PNIPAM, a thermo-sensitive polymer, will ensure NAV thermo-response characteristics. However, the turbidity curves show that the macro-behaviour of NAV occurred in only at pH 2, 4, and 6 (Fig. 4). This will induce us a depth discussion (Fig. 6). At pH = 2, DLS detected a synchronous increase in Z-ave and scattered light intensity (kcps) with increasing temperature, yet a significant decline in size after 40 °C. TEM revealed a transition of the NAV structure from vesicles, aggregations to LCMs (marked LCM₂ in Fig. 1) until a complete collapse of PNIPAM.^45,46,48,49 Before 40 °C, the size of the aggregations has a dominant impact on kcps.^50,51 After that, the increase in the density of the tight LCMs offsets the small-size effect, resulting in a constant kcps. In the IEP (pH ∼ 4) solution, higher temperature would strengthen the aggregation of unstable NAV polymer, which caused just a slight increase in the kcps. At pH = 6, the hydrophilic PNIPAM and ionized PAA promote the higher stability and solubility of NAV, resulting in a larger kcps. As stated in the previous paragraph, the high temperature causes a loose structure and low density, leading to a decline in the kcps (Fig. S5†).⁵² Under the other pH conditions, the Z-ave and kcps do not significantly change depending on the temperature (Fig. S5†). At pH = 1, the strong kcps proves the uniform and tight structure of the LCMs. Under alkaline conditions, due to the electrostatic repulsion, the looser micelles cause a lower kcps.

Fig. 6 The thermo-responsive behaviour of NAV triblock copolymer at pH = 2, 4 and 6: (a) the change in the average hydrodynamic diameter (Z-ave) with temperature, (b) the change of the derived count rate with temperature and (c) TEM images of NAV at pH = 2 at various temperatures. The test concentration of NAV triblock copolymer was 2.0 mg mL⁻¹.

Interestingly, when temperature was further than the LCST of PNIPAM, the highly ionized PAA segments will play bridging role between the hydrophobic PNIPAM and P4VP cores, which probably results in the following topology: (1) flower-like micelles; (2) dangling ending and (3) 3D network structure.^44,53,54 Especially, when the triblock copolymer concentration was increased to 5–15 wt%, the 3D network structure may lead to gelation behaviour.^28,55,56

The gel–sol behaviour of the NAV triblock copolymer. In order to investigate the gelation behaviour of NAV, the detection of topology was first tried by DLS in NAV aqueous solutions at pH = 8 (Fig. 7). At 25 °C, the intensity and number distribution of the NAV size only slightly increased upon increasing the copolymer concentration from 2 to 10 mg mL⁻¹. However, when the temperature was raised to 50 °C, a strong intensity distribution showed a larger size at the higher concentration of 10 mg mL⁻¹. Especially, the number distribution (∼400 nm) revealed a possible size rule for the gelation net points at 10 mg mL⁻¹. The net points contained the P4VP and PNIPAM cores and were bridged by the ionized PAA chains. Further, gelation behaviour was clearly observed by TEM and SEM at 5 mg mL⁻¹ and 50 mg mL⁻¹, respectively (Fig. 7(b) and (c)). The TEM images show a transition from uniform spherical micelles of 40–60 nm at 25 °C to an uneven distribution topology at 50 °C. Here, two factors may lead to a smaller size being detected by TEM than DLS, namely the dry state than wet: (1) evaporation of water from the wet to dry micelles on copper mesh⁵⁷ and (2) the disappearance of electrostatic repulsion formed by the ionized PAA in the dried micelles.^44,58,58 Ample evidence of gelation was also provided by SEM (Fig. 7(c)). At a concentration of 50 mg mL⁻¹, the blocky and silky structure corresponds to a flowing-state at 25 °C and free-standing hydrogel at 50 °C, respectively.

Fig. 7 (a) The intensity and number distribution of NAV used to detect the trace large-size particles dependant on concentration. (b) TEM images of the micelle solution at 5 mg mL⁻¹. (c) SEM images of the gelation behaviour at 50 mg mL⁻¹ (∼5 wt%). All tests are in NAV aqueous solutions of pH = 8 at 25 °C and 50 °C, respectively. In the images, ○ and | represent the “N/V cores” and “bridges”, respectively.

Concentration effect. We know that the concentration of polymer will have a significant impact on the gelation properties and practical applications. The gelation properties of NAV also depend closely on the copolymer concentration (Fig. S6†). As the concentration of NAV increased, the viscosity of the NAV system gradually increased. Using the inversion method, the NAV system underwent sol, weak gel, soft gel and free-standing gel transformations. The serious concentration effect was due to the more complex network the among interpenetrated multiplets. Besides, the significant increase in NAV viscosity with increasing temperature implies that all the listed concentrations are above the percolation concentration of NAV. Thus, the higher temperature resulted in the stronger gelation strength and lower mobility.⁵⁹

The pH-responsive gel–sol behaviour. At a concentration of 9 wt%, the pH-responsive behaviour of the NAV hydrogel was studied by rheology at a series of pH values (Fig. 8). Under alkaline conditions (pH > 7), the rheology curves show only a slight increase in the viscosity curve of the NAV system. This means that the electrostatic interactions of the ionized PAA have no significant effects on the gelation network structure of NAV. However, at a pH between 6 and 7, the viscosity curve displays a sharp migration in the temperature direction, namely pH-responsive behaviour. The pH-responsive behaviour mentioned already in the above paragraph was caused by the hydrogen bonds among the PAA and PNIPAM.^47,60 At pH = 5, the viscosity of the NAV system first increased, then dehydrated and finally stratified. This is highly consistent with the microscopic aggregation behaviour at the same pH. Of course, this behaviour may limit the application of the NAV hydrogel.

Fig. 8 The rheology of the NAV hydrogel: (a) the viscosity curves dependant on the pH at 9 wt%, (b) the repetitive sol–gel curves obtained for NAV at pH = 7.4 and 9 wt%. The viscosity measurements were carried out using 64# spindle with a constant rotation speed of 100 RPM and a constant heating/cooling rate of 1.0 °C min⁻¹; the viscosity and temperature of the NAV concentrated solutions were read on a viscometer.

The thermo-responsive gel–sol behaviour. The thermo-responsive behaviour of the NAV hydrogel was mainly due to the thermo-sensitive polymer PNIPAM. Higher temperatures cause stronger hydrophobic forces, contributing to the formation of a free-standing gel. As the temperature increases, the viscosity of the NAV hydrogel gradually increased. Namely, the sol–gel transition behaviour can be conveniently tuned by temperature. In addition, the behaviour has a close contact with the copolymer concentration and pH values (Fig. 8(a) and S6†). Further, at pH = 7.4, the cycling performance of the NAV hydrogel was studied by repeated heating–cooling experiments at 9 wt% (Fig. 8(b)). In the heating stage, the viscosity of NAV first increases slowly, then sharply rises and finally becomes constant. In addition, the opposite behaviour occurs in the cooling stage. When comparing the viscosity curves in heating and cooling steps, there was only ∼2 °C difference. The deviation may be caused by a lower rate of heat transfer in the gel than in sol. However, in any case, the constant gelation performance indicates good recyclability and application prospects.

Conclusions

The thermo- and dual-pH-sensitive ABC triblock copolymer of PNIPAM-b-PAA-b-P4VP (NAV) that forms a hydrogel upon changes in temperature or pH was successfully synthesized via sequential RAFT polymerization and subsequent hydrolysis. DLS and TEM showed that the NAV triblock copolymer in dilute solution at 25 °C goes through large compound micelles (LCMs), vesicles, aggregations and spherical micelles at pH 1, 2, 4, and over 6, respectively. When the temperature rises to 50 °C, the vesicles turn into another LCMs and the spherical micelles are converted into a 3D topology. Especially, under alkaline conditions, when the concentration of the NAV triblock copolymer was increased to 4–5 wt%, the macroscopic gelation behaviour of NAV was observed by the naked eye at 38–45 °C and the microcosmic gel net points and “bridges” between the PNIPAM and P4VP cores can be detected by SEM. Rheology measurements revealed that the gelation behaviour of the NAV hydrogel can be tuned slightly by the copolymer concentration and pH between 6 and 7. However, once the pH was over 7, the gelation behaviour would have nothing to do with pH values. Repeated heating–cooling experiments showed the good circulation performance of the NAV hydrogel. We hope that all our work on PNIPAM-b-PAA-b-P4VP can provide a general and efficient paradigm to fabricate a family of stimuli-responsive ABC copolymers with hydrophilic mid-block in theoretical studies and practical applications.

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Footnote

† Electronic supplementary information (ESI) available: Supplementary Fig. S1–S7: FT-IR, ¹H NMR spectra, turbidity curves triblock copolymer, DLS and TEM image of micelles. See DOI: 10.1039/c6ra14682a

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