In situ formation of multiple stimuli-responsive poly[(methyl vinyl ether)-alt-(maleic acid)]-based supramolecular hydrogels by inclusion complexation between cyclodextrin and azobenzene

Abstract

Biocompatible external stimuli-responsive hydrogels are of interest for drug delivery, cell and gene therapy, tissue engineering, protein patterning, and other biomedical applications. In this work, based on the biocompatible polymer poly(methyl vinyl ether-alt-maleic acid) (P(MVE-alt-MA)), host polymer β-cyclodextrin-grafted P(MVE-alt-MA) (P(MVE-alt-MA)-g-β-CD) and guest polymer azobenzene-grafted P(MVE-alt-MA) (P(MVE-alt-MA)-g-azo) were first prepared. Then through taking advantage of the traditional host–guest interaction of β-cyclodextrin and azobenzene, novel multiple stimuli-responsive physical P(MVE-alt-MA)-g-β-CD/P(MVE-alt-MA)-g-azo supramolecular hydrogels were obtained after simply mixing the aqueous solutions of the host polymer and guest polymer. The formation of the supramolecular hydrogels was also confirmed by 2D-NOESY. This kind of supramolecular hydrogels not only exhibits photo-, pH- and thermo-sensitivity, but also cytocompatibility. Ovarian cancer SKOV3 cells can survive within different hydrogel layers, which were observed by confocal microscopy. These results suggest that this multiple stimuli-responsive P(MVE-alt-MA)-based supramolecular hydrogel may be expected to have a powerful potential in biomedical applications as a three-dimensional (3D) cell culture matrix or as a vehicle for the delivery of drugs and therapeutic cells.

---

1 Introduction

Stimuli-responsive hydrogels, as one of the most common intelligent biomaterials capable of responding to electric¹ and magnetic fields,² light,³ temperature changes,⁴ pH changes,⁵ chemical signals,⁶ and enzymes,⁷ are currently widely investigated for their enormous potential biomedical applications in the controlled release of drugs,⁸ the encapsulation and culture of cells,⁹ research on tumors and histopathology,¹⁰ in vitro drug resistance,¹¹ stem cell separation¹² and so on. Those in situ-formed physical hydrogels with noncovalent cross-linking formed by host–guest interactions have many attractive advantages because they often exhibit a reversible sol–gel transition when exposed to external stimuli and can be used in optical sensing and imaging,^13,14 drug and gene delivery,^15–19 and self-healing materials.^20–23 It is well known that cyclodextrins (CDs) are an ideal species for the development of new self-assembling systems.²⁴ The cone-shaped cavities of CDs can act as hosts for a great variety of guests such as anthraquinone, ferrocene, adamantane, azobenzene, and some of their derivatives to form self-assembling systems with the inclusion driven by geometric compatibility and hydrophobic interactions.²⁵ Azobenzene and its derivatives could be isomerized from the more stable trans to the less stable cis configuration by light irradiation, due to a unique light-induced trans–cis isomerization property. The sol-to-gel or gel-to-sol phase transition of the light-responsive gel systems which were assembled with azobenzene units is controlled by light-induced trans–cis isomerization of the azobenzene units.^26–29 CD/azo inclusion complex interaction is an ideal tool with which to construct light-responsive supramolecular hydrogels. For example, Harada et al. have reported the sol–gel phase transition of photoswitchable hydrogels which were assembled between azo-modified poly(acrylic acid) and CD-modified curdlan.^30–33 Stoddart et al. also reported an azobenzene-based light-responsive hydrogel system formed from a deoxycholic acid-modified β-cyclodextrin derivative and an azobenzene-branched poly(acrylic acid) copolymer.³⁴ Although there are some examples of light-responsive supramolecular hydrogels formed by inclusion complexation of CD-azo moieties, cell adhesive and biocompatible light-responsive supramolecular hydrogels are seldom reported. The application of hydrogels in biomedical fields was largely limited, such as in cell culture, transmission, and treatment.

As a hydrophilic, biocompatible, and bioadhesive polycarboxylic acid containing polymer, poly(methyl vinyl ether-alt-maleic acid) (P(MVE-alt-MA)) and its derivatives have many important applications in biotechnology, pharmacology and health care, for example as a thickening and suspending agent, denture adhesive, and adjuvant for transdermal patches and mouthwash,³⁵ and for the capture of viruses,^36–38 isolation of nucleic acids,³⁹ and cell encapsulation and culture.^40,41 Meanwhile, P(MVE-alt-MA) also has available carboxylic acid functional groups, which can provide polyanionic characteristics in the polymer backbone as well as ease of chemical modification for amide linkage with amine and ester linkage with a hydroxyl group.^42,43

Although there are some reports on chemically crosslinked P(MVE-alt-MA) hydrogels, such as poly(ethylene glycol) (PEG) crosslinked P(MVE-alt-MA) hydrogel,^44,45 to our knowledge, a report on a stimuli-responsive physically crosslinked only P(MVE-alt-MA)-based supramolecular hydrogel has not been found so far.

Here, we used P(MVE-alt-MA) as the only main chain to construct a novel multiple stimuli-responsive biocompatible supramolecular hydrogel by inclusion complexation between azo and β-CD. The properties of the P(MVE-alt-MA)-based hydrogel were characterized using rheological measurements and scanning electron microscopy (SEM). The formation of the P(MVE-alt-MA)-based supramolecular hydrogel was further confirmed using 2D-NOESY spectroscopy. The cytotoxicity of the hydrogels was tested by CCK-8 assay. The cells’ survival state was observed using confocal microscopy. The experimental results suggest that the P(MVE-alt-MA)-based supramolecular hydrogel has the following notable characteristics: (1) it has expanded the application range of P(MVE-alt-MA) as a novel multiple stimuli-responsive supramolecular hydrogel of P(MVE-alt-MA)-g-β-CD/P(MVE-alt-MA)-g-azo; (2) light irradiation-, pH value-, or temperature-controlled mechanical strength; (3) cytocompatibility proved by the in vitro 3D ovarian cancer SKOV3 cell culture experiments. So, the biocompatible multiple stimuli-responsive P(MVE-alt-MA)-based supramolecular hydrogel would be a powerful candidate for biomedical applications.

2 Experimental method

2.1 Materials and characterizations

Poly[(methyl vinyl ether)-alt-(maleic anhydride)] (P(MVE-alt-MAH)) (M_w = 1 080 000, M_n = 311 000, MDW = 3.47) was purchased from Sigma-Aldrich (Shanghai, China); ethylenediamine (EDA) and p-aminoazobenzene were purchased from Aladdin; p-toluenesulfonyl chloride (p-TsCl), N,N-dimethylformamide (DMF), β-cyclodextrin (β-CD), and dicyclohexylcarbodiimide (DCC) were obtained from Sinopharm Chemical Reagent Shanghai Co. Ltd. β-Cyclodextrin (β-CD) was dried overnight under vacuum at 80 °C, N,N-dimethylformamide (DMF) was dried with calcium hydride, and water was of deionized quality.

The Fourier transform infrared (FTIR) spectra were obtained using a Nicolet 5700 spectrometer (Thermo, U.S.A.) with a KBr pellet. One dimensional NMR spectra were recorded on a Mercury VX-300 spectrometer at 300 MHz (Varian) using D₂O as a solvent and TMS as an internal standard. Two dimensional NOESY (2D-NOSEY) NMR spectra were recorded on a Varian-Inova-600 NMR spectrophotometer at 30 °C. Sample solutions were prepared using D₂O. Hydrogel samples were analysed using an Ultra Plus field emission scanning electron microscope. Rheology analyses were performed on a TA-AR 1000 rheometer with parallel plate geometry (25 mm in diameter) at 25 °C. The phase transition behavior of the hydrogel was examined under photoirradiation using a ZF-I UV analyzer. Confocal microscopy images were obtained using an FV1000 confocal microscope.

2.2 Synthesis of P(MVE-alt-MA)-based host polymer and guest polymer

2.2.1 Synthesis of host polymer P(MVE-alt-MA)-g-β-CD. A fixed amount of P(MVE-alt-MAH) was added to 20 mL of deionized water in a 100 mL round-bottom flask and heated at 90 °C to hydrolyze it for 2 h to obtain a clear solution of P(MVE-alt-MA) with vigorous stirring. Then water was rotary evaporated and the P(MVE-alt-MA) solid obtained was dried for 48 h under vacuum at 80 °C.

TsCD (mono-6-(p-tolylsulfonyl)-β-cyclodextrin) was synthesized as reported in ref. 46. EDA-CD (ethylenediamine-β-cyclodextrin) was synthesized as reported in ref. 47. P(MVE-alt-MA)-g-β-CD was prepared by an amidation reaction between EDA-CD and the anhydride groups as follows: a fixed amount of EDA-CD was dissolved in dried DMF (25 mL), different amounts of P(MVE-alt-MA) (molar ratios of P(MVE-alt-MA) to EDA–CD were 1 : 0.67, 1 : 1.00, and 1 : 1.30) and DCC (1/2 equal molar to P(MVE-alt-MA)) were dissolved in dried DMF and were added to the EDA–CD solution, respectively. The mixture was stirred for 12 h at 80 °C under nitrogen. The formed DCU (1,3-dicyclohexylurea) was removed by filtration. The DMF was rotary evaporated and the solid obtained was dissolved in deionized water. Unreacted raw material and low molecular weight products were removed by dialysis with a cellulose membrane (molecular weight cut off of 1 × 10⁴ g mol⁻¹). And then P(MVE-alt-MA)-g-β-CD was isolated by freeze-drying. In total, three P(MVE-alt-MA)-g-β-CD samples with CD grafting rates (r_CD) of 0.084, 0.097, and 0.129 were synthesized, which were coded as HP1, HP2, and HP3, respectively. The CD grafting rate in P(MVE-alt-MA)-g-β-CD was calculated according to quantitative analysis of ¹H NMR spectra.

2.2.2 Synthesis of guest polymer P(MVE-alt-MA)-g-azo. P(MVE-alt-MA)-g-azo was prepared through an amidation reaction between p-aminoazobenzene and the carboxyl groups of P(MVE-alt-MA) in the presence of DCC. The procedure is described below: DCC (1/2 equal molar to P(MVE-alt-MA)) was added to the solution of P(MVE-alt-MA) in DMF (30 mL). After the formation of DCU, different amounts of p-aminoazobenzene (molar ratios of p-aminoazobenzene to P(MVE-alt-MA) were 0.67 : 1, 1.00 : 1, and 1.33 : 1) were then added to the above mixture, respectively. After additional stirring for 12 h at 60 °C under nitrogen, the formed DCU was removed by filtration. The reaction mixture was concentrated and precipitated into chloroform, and then the solid obtained was dissolved in deionized water. The low molecular weight products were removed by dialysis with a cellulose membrane (molecular weight cut off of 1 × 10⁴ g mol⁻¹). And then P(MVE-alt-MA)-g-azo was isolated by freeze-drying.⁴⁸ In total, three P(MVE-alt-MA)-g-azo samples with azo grafting rates (r_azo) of 0.175, 0.260, and 0.337 were synthesized, which were coded as GP1, GP2, and GP3, respectively. The azo grafting rate in P(MVE-alt-MA)-g-azo was calculated according to quantitative analysis of ¹H NMR spectra.

2.3 In situ hydrogel formation via inclusion complexes

The host polymer P(MVE-alt-MA)-g-β-CD and guest polymer P(MVE-alt-MA)-g-azo were dissolved in distilled water to form a homogeneous solution under neutral conditions. Then, three supramolecular hydrogels, Gel-1, Gel-2, and Gel-3, were obtained by simply mixing an aqueous solution of P(MVE-alt-MA)-g-β-CD and an aqueous solution of P(MVE-alt-MA)-g-azo at room temperature, where the molar ratio of azo/CD was kept at a constant of 1 : 1 and the total polymer concentration (C) was 0.075 g mL⁻¹. Mixing HP1 and GP1, HP2 and GP2, HP3 and GP3 respectively led to the formation of Gel-1, Gel-2, and Gel-3.

In supramolecular hydrogels Gel-1, Gel-2 and Gel-3, the total grafting rate of CD and azo was 0.113, 0.138, and 0.187, respectively. The details of the calculation are shown in the ESI.†

2.4 Observation of interior morphology of the hydrogels

The hydrogel samples were in situ formed in distilled water at room temperature, quickly frozen in liquid nitrogen and further freeze-dried in a Freezone 6 freeze drier (Labconco Corporation, USA) under vacuum at −42 °C for at least 48 h until all the solvent had sublimed. For SEM measurements, the freeze-dried hydrogels were sectioned carefully and coated with gold; their interior morphology was studied with a scanning electron microscope (LEO, 1530VP, Germany).

2.5 Rheological experiments

All the dynamics rheological data were checked as a function of strain amplitude to ensure that the measurements were performed in the linear viscoelastic region. The cone used had a diameter of 25 mm and an angle of 1°. A solvent trap was used to prevent evaporation of the solvent. The viscoelastic properties of the associative networks were determined by measuring changes in the storage modulus G′ and the loss modulus G′′, at 25 °C. Frequency experiments were performed at a stress located in the range of linear viscoelasticity in the frequency range of 0.1–100 Hz. To examine the frequency-dependent gelation behaviors of the in situ formed hydrogels via inclusion complexes, equal volumes of the aqueous solutions of P(MVE-alt-MA)-g-β-CD and P(MVE-alt-MA)-g-azo with a molar ratio of azo/CD of 1 : 1 were mixed at room temperature for 2 min. After transferring the mixed solution to the rheometer, the rheological analyses were subsequently performed from 0.1 to 100 Hz with a constant strain (10%) at 25 °C to study the mechanical strength of the in situ formed hydrogels. To investigate the temperature-dependent gelation behaviors of the in situ formed hydrogels, temperature sweeps were performed from 10 to 70 °C with a heating rate of 0.5 °C min⁻¹ at a constant shear frequency (1 Hz) and strain (10%).

2.6 Phase transition behavior

Associative networks were obtained by simply mixing at room temperature as above and photoirradiating with UV light (λ = 365 nm) for 15 minutes using a ZF-I UV analyzer. The hydrogels were regulated at pH 2.0, 4.5 and 7.4 and detected using an MCR rheometer.

2.7 Cell culture

A total of 5.0 mL of ovarian cancer cell (SKOV3) dispersion with a concentration of about 1 × 10⁵ cells per mL was mixed with 1.0 mL of P(MVE-alt-MA)-g-β-CD solution at pH = 7.4, and then 1.0 mL P(MVE-alt-MA)-g-azo solution at pH = 7.4 was added and stirred. As a result, the mixture gelated immediately. The cells included in the hydrogel were cultured in media containing 90% Rosewell Park Memorial Institute 1640 (RPMI-1640), 10% heat-inactivated fetal calf serum, and 100 units/mL of penicillin/streptomycin. The cultures were maintained in an incubator at 37 °C with a humidified atmosphere of 5% CO₂.

2.7.1 Cytotoxicity evaluation of hydrogels. In order to fully assess the cytotoxicity of the hydrogels as culture matrices, Gel-1, Gel-2, and Gel-3 were first diluted to prepare three samples with concentrations of 50 μg mL⁻¹, 100 μg mL⁻¹, and 200 μg mL⁻¹, respectively, and then SKOV3 cells were cultured in these diluted samples of Gel-1, Gel-2 and Gel-3 for 48 h. After 48 h, the optical density (OD) value at 450 nm was detected using a 2030 multilabel reader (Victor X4, Perkin-Elmer). All experiments were performed in triplicate.

2.7.2 Staining and distribution of cells in hydrogels. The distribution of the cells which were stained with DIO and cultured in Gel-1, Gel-2, and Gel-3 after 4 days were observed using the laser scanning confocal microscope at room temperature.

3 Results and discussion

3.1 Synthesis and characterization of P(MVE-alt-MA)-based polymers

The P(MVE-alt-MA)-based polymers were synthesized according to Scheme 1. To investigate the possibility of in situ sensitive hydrogel formation via the reaction of inclusion complexes, CD and azo groups were introduced to the structures of P(MVE-alt-MA).

Scheme 1 Synthesis procedures of P(MVE-alt-MA)-g-β-CD (1) and P(MVE-alt-MA)-g-azo (2).

P(MVE-alt-MA)-g-β-CD was synthesized via an amidation reaction between carboxyl groups and EDA–CD. P(MVE-alt-MA) was grafted with CD, and the grafting rate was determined using ¹H NMR as in ref. 49. The grafting rates of P(MVE-alt-MA)-g-β-CD were 0.084, 0.097 and 0.129. In the IR spectra of P(MVE-alt-MA)-g-β-CD shown in Fig. S1(A),† two absorption bands at 1724.5 and 1652.1 cm⁻¹ can be observed. The band at 1724.5 cm⁻¹ is attributed to the C=O of the acid groups from hydrolyzed anhydride groups, and the band at 1652.1 cm⁻¹ should be attributed to the C=O of the amide groups. The ¹H NMR spectra and peak assignments for P(MVE-alt-MA)-g-β-CD are shown in Fig. S1(C).† ¹H NMR (300 MHz, D₂O): δ 4.94 (s, 1H), 3.83–3.74 (t, 3H), 3.52–3.49 (m, 10H), 3.27–2.86 (m, 3H), 2.58 (s, 1H), 1.85 (s, 2H).

P(MVE-alt-MA)-g-azo was prepared via an amidation reaction between p-aminoazobenzene and carboxyl groups. The azo grafting rate was determined using ¹H NMR. The azo grafting rates of P(MVE-alt-MA)-g-azo were 0.175, 0.260 and 0.337. In the IR spectra of azobenzene-containing polymers shown in Fig. S1(B),† the band at 1710.6 cm⁻¹ is attributed to the C=O of the acid groups from hydrolyzed anhydride, and the band at 1589.1 cm⁻¹ should be attributed to the C=O of the amide groups. The ¹H NMR spectra and peak assignments for P(MVE-alt-MA)-g-azo are shown in Fig. S1(D).† ¹H NMR (300 MHz, D₂O): δ 7.80–6.70 (m, 5H), 3.53–3.24 (m, 6H), 3.06–2.89 (m, 3H), 2.58 (s, 1H), 1.81 (s, 2H).

3.2 In situ hydrogel formation via inclusion complexes

The supramolecular hydrogels were prepared via inclusion complexes between trans-azobenzene groups from guest polymer azobenzene-grafted P(MVE-alt-MA) (P(MVE-alt-MA)-g-azo), and cyclodextrin cavities from host polymer β-cyclodextrin-grafted P(MVE-alt-MA) (P(MVE-alt-MA)-g-β-CD). When P(MVE-alt-MA)-g-β-CD and P(MVE-alt-MA)-g-azo solutions with an azo/CD molar ratio of 1 : 1 were mixed at room temperature, the expected gelation was observed immediately. The detailed gelation process and phenomena are shown in Scheme 2. The hydrogel formation was validated by the inversion method.

Scheme 2 Assembly of host polymer P(MVE-alt-MA)-g-β-CD and guest polymer P(MVE-alt-MA)-g-azo.

2D-NOESY was employed to further confirm the formation of hydrogels, namely the occurrence of inclusion complexation of CD and azo moieties. The Nuclear Overhauser Effect (NOE) technique can be used in supramolecular chemistry because it is very sensitive to the interproton distance. Therefore, NOE measurements can provide direct evidence of the inclusion interaction.⁵⁰ Here, the host–guest assembly of the CD and azo groups in the supramolecular gels was investigated using 2D-NOESY spectra as shown in Fig. 1. The rectangles enclose the cross-peaks arising from interactions of the trans-azo group protons with the native β-CD annular H_3,5 protons. The P(MVE-alt-MA)-g-β-CD/P(MVE-alt-MA)-g-azo system shows that the inner protons of the β-CD unit are correlated to the protons of the azobenzene group in P(MVE-alt-MA)-g-azo. The results clearly indicate the immediate proximity between the protons of azo and those in the cavities of β-CD, which confirms that the azo groups are trapped by the cavities of the β-CD and the formation of inclusion complexes. Hence, the results show that the formation of the hydrogel depends on inclusion complexation in addition to the winding of long chains of the polymer itself.

Fig. 1 2D-NOESY spectra of P(MVE-alt-MA)-g-β-CD/P(MVE-alt-MA)-g-azo (Gel-2) in D₂O.

3.3 Interior morphology of hydrogels

The interior morphologies of these in situ-formed hydrogels are displayed in Fig. 2(A). As all of the hydrogels have a similar porous network structure, these supramolecular hydrogels also present an interconnected porous structure with pore sizes ranging from several microns to several tens of microns. This interconnected porous microstructure is believed to be beneficial for cell culture because it allows cell migration and the transport of nutrients and waste products in and out of the scaffold.⁵¹ It was found that the pore sizes of the hydrogels are not the same and the pore densities of the hydrogels are also different, which are related to the grafting rate of CD and azo. Because of the higher content of β-CD and azo functional groups in the hydrogels, the cross-linking density is higher, resulting in the more compact hydrogel structure. In contrast, a lower grafting rate of β-CD and azo functional groups in the hydrogels led to the looser porous structure.

Fig. 2 (A) The SEM images of P(MVE-alt-MA)-based hydrogels (Gel-1(a)(a′), Gel-2(b)(b′), Gel-3(c)(c′)) with scale bars of 100 μm for (a), (b) and (c), and 20 μm for (a′), (b′) and (c′). The hydrogels were prepared from P(MVE-alt-MA)-g-β-CD/P(MVE-alt-MA)-g-azo with a molar ratio of azo/CD of 1 : 1 with different grafting rates and fully swelled at room temperature for 48 h. Storage (G′) and loss (G′′) moduli as a function of frequency for Gel-1 (B), Gel-2 (C), Gel-3 (D) at 25 °C, pH = 7.0; (E) storage (G′) and loss (G′′) moduli as a function of temperature for Gel-3.

3.4 Rheological properties

The mechanical properties of the hydrogels were examined by oscillatory rheological measurements, and the corresponding frequency sweeps are presented in Fig. 2(B)–(D). Both the storage modulus (G′) and the loss modulus (G′′) became continuously increased with sweep frequency and G′ was also higher than G′′ at room temperature. Usually, the storage modulus represents the elasticity of a material and the loss modulus represents its viscosity. A higher storage modulus indicates that a real 3D network is formed, and the network has a stronger mechanical strength. Therefore, the higher storage modulus than loss modulus shown in Fig. 2(B)–(D) demonstrates the formation of gel materials. On the other hand, both the storage modulus and the loss modulus increased gradually, and the storage modulus increased more rapidly than the loss modulus with the increasing sweep frequency. One possible reason for this is that the polymer chains wind more and more closely, causing the storage modulus to be higher than the loss modulus. Since physical gelation occurred by inclusion complexation only during the formation of these in situ-formed hydrogels, the hydrogels would be physically cross-linked. Therefore, the mechanical strength of these in situ-formed hydrogels should be weaker than that of chemical hydrogels or a combination of chemical and physical polymer hydrogels. The frequency sweep data also revealed this point.

3.5 Phase transition behavior

The phase-transition behavior of the hydrogels was further investigated by light irradiation. Generally, photo-irradiation with ultraviolet (UV) light (λ = 365 nm) causes isomerization of the trans-azo group, whereas visible light (Vis light) (λ = 430 nm) causes isomerization of the azo group from cis to trans. In order to clarify the interaction between the β-CD unit and the trans-azo group, the hydrogels were photo-irradiated with UV light (λ = 365 nm) for 15 min. And then a change to sol was observed, which is shown in Fig. 3(A). After being exposed to visible light (λ = 430 nm), the sol became hydrogels once again. The phase-transition process can be repeated under alternating UV and visible light irradiation.

Fig. 3 (A) Schematic representation of the interactions of the β-CD unit with azo moieties on irradiation with UV (λ = 365 nm) and visible light (λ = 430 nm). Storage (G′) and loss (G′′) moduli as a function of frequency for Gel-3 at 25 °C, pH = 2.0, pH = 4.5, and pH = 7.4 before (B, D and F) and after (C, E and G) UV irradiation. Insets are photographs to illustrate mixtures of host polymer and guest polymer before and after UV irradiation.

The influence of pH value on the novel hydrogels was examined using oscillatory rheological measurements as shown in Fig. 3 (B)–(F). The change of G′ and G′′ did not follow the pH order, namely, the values of G′ and G′′ at different pH values followed the order pH 2.0 > pH 7.4 > pH 4.5. In addition, the hydrogel even formed aggregates at pH = 2.0 and part of the aqueous solution was squeezed out of the hydrogel network (as shown in the illustration in Fig. 3(B)). The change should come from both the inclusion complex interactions between the azo functional groups and the CD cavities and the hydrogen bond interactions between the acidic functions and the hydroxyl groups present on the CDs, which further enhances the mechanical strength. At pH = 4.5, the strength of the network is mainly due to the inclusion complex interactions because this is close to the pK_a value of the carboxylic groups. At pH = 7.4, the strength of the associative networks mainly comes from both the inclusion complex interactions and the electrostatic repulsion among the charges of the P(MVE-alt-MA)-based polymer chains, because the pH value is larger than the pK_a.⁵²

In order to further explore the role of the inclusion complex interactions and hydrogen bond interactions in the formation of the P(MVE-alt-MA)-based hydrogels, the rheological properties (G′ and G′′) of the hydrogels at different pH values (pH = 2.0, 4.5, and 7.0) were determined after and before UV light irradiation (Fig. 3). At pH = 2.0, after UV light irradiation for 15 min, G′ is still larger than G′′, though lower than G′ and G′′ before UV light irradiation. This result indicates that the additional hydrogen bond interactions between the acidic functions and the hydroxyl groups on CDs should mainly occur at low pH. At pH = 4.5 and pH = 7.4, closer to or higher than the pK_a, inclusion complex formation is mainly responsible for the strength of the hydrogel. Before UV irradiation, G′ is larger than G′′, but smaller than G′′ after UV irradiation. This indicates that the occurrence of obvious phase-transition can be caused by UV irradiation. Therefore, strong hydrogen bond interactions existed in the hydrogels, which play a synergistic role with the inclusion complex interactions in the formation of the supramolecular hydrogels.

The hydrogels were also affected by the temperature. Taking Gel-3 as an example, the change in G′ and G′′ of Gel-3 with the temperature was examined as shown in Fig. 2(E). The temperature sweeps present the relevant hydrogel’s behavior as follows: both the storage modulus and the loss modulus decreased as the temperature increased from 10 °C to 70 °C. The cross point of the storage modulus and the loss modulus are observed at 43.7 °C because the former decreased more rapidly than the latter. G′ < G′′, indicated the translation of gel to sol. The possible reason for this is that the inclusion interaction between β-CD and azo was destroyed by the higher temperature.

3.6 Cytotoxicity evaluation of hydrogels

Biocompatibility is very important for the application of materials as a 3D cell scaffold. The results of the cytotoxicity evaluation of the P(MVE-alt-MA)-based hydrogels for the SKOV3 cells are shown in Fig. S2,† and there is no obvious decrease in cell viability within the culture time in all nine samples. In other words, as a cell culture scaffold, the cytocompatibility of P(MVE-alt-MA)-based supramolecular hydrogels is satisfactory on the whole.

3.7 Distribution of cells in hydrogels

As 3D cell scaffold materials, cells should be able to live well within the hydrogels. The confocal microscope was used for further observation of the survival state and distribution of SKOV3 cells in the hydrogels. After DIO staining and culturing for 4 days in hydrogels Gel-1, Gel-2 and Gel-3, pictures were taken and are shown in Fig. 4. The cells proliferated well within different hydrogel layers. The distribution and survival state of the cells in Gel-1, Gel-2, and Gel-3 were different. The higher the total grafting rate of the hydrogel, the wider the cell distribution range, and the weaker the activity of the cells. On the contrary, the lower the total grafting rate of the hydrogel, the narrower the cell distribution range, and the stronger the activity of the cells.

Fig. 4 3D (A, B, C and D control) and z-axis maximum projection (A′, B′, C′ and D′ control) views of confocal microscopy images, (A), in Gel-1; (B), in Gel-2; (C), in Gel-3, and (D) as the control experiment. Cell viability (viable cells: green via DIO) and spatial distribution of SKOV3 cells encapsulated in the supramolecular hydrogels and cultured for 4 days after gelation.

4 Conclusions

We have successfully developed novel intelligent supramolecular hydrogels of P(MVE-alt-MA)-g-β-CD/P(MVE-alt-MA)-g-azo based on the host–guest interaction of β-CD and azo, in which P(MVE-alt-MA) was used as the only main chain. These supramolecular hydrogels present an interconnected porous structure and easily adjusted rheological properties. They are not only photoresponsive, but also are acid responsive and thermoresponsive. These P(MVE-alt-MA)-based supramolecular hydrogels were also proved to be cytocompatible and can be used to construct a scaffold for 3D cell culture. SKOV3 cells were alive during the culture and distributed at different depth levels in the supramolecular hydrogels. All of the above results proved that this type of supramolecular hydrogel is a potential platform for 3D cell cultures, cell separation and drug release in the future.

Acknowledgements

This work is supported by the National Basic Research Program of China (2011CB933503), the Special Project on Development of National Key Scientific Instruments and Equipment of China (2011YQ03013403), the Fundamental Research Funds for the Central Universities (CXLX13_118), NSFC (61179035, 6112702, 31200757) and Suzhou medical apparatus and new medicine Fund (ZXY201440).

Notes and references

1. K. Adesanya, E. Vanderleyden, A. Embrechts, P. Glazer, E. Mendes and P. Dubruel, J. Appl. Polym. Sci., 2014, 131, 41195–41203.
2. W. Zhao, K. Odelius, U. Edlund, C. Zhao and A. C. Albertsson, Biomacromolecules, 2015, 16, 2522–2528.
3. L. Chen, S.-G. Li, Y.-P. Zhao, Y.-C. Wang and Q.-W. Wang, J. Appl. Polym. Sci., 2005, 96, 2163–2167.
4. K. Ishida, T. Uno, T. Itoh and M. Kubo, Macromolecules, 2012, 45, 6136–6142.
5. J. M. Swann, W. Bras, P. D. Topham, J. R. Howse and A. J. Ryan, Langmuir, 2010, 26, 10191–10197.
6. L. Liu, X. L. Song, X. J. Ju, R. Xie, Z. Liu and L. Y. Chu, J. Phys. Chem. B, 2012, 116, 974–979.
7. E. Secret, S. J. Kelly, K. E. Crannell and J. S. Andrew, ACS Appl. Mater. Interfaces, 2014, 6, 10313–10321.
8. S. Das and U. Subuddhi, Ind. Eng. Chem. Res., 2013, 52, 14192–14200.
9. B. Yeon, M. H. Park, H. J. Moon, S. J. Kim, Y. W. Cheon and B. Jeong, Biomacromolecules, 2013, 14, 3256–3266.
10. C. T. Tsao, F. M. Kievit, K. Wang, A. E. Erickson, R. G. Ellenbogen and M. Zhang, Mol. Pharm., 2014, 11, 2134–2142.
11. D. Loessner, K. S. Stok, M. P. Lutolf, D. W. Hutmacher, J. A. Clements and S. C. Rizzi, Biomaterials, 2010, 31, 8494–8506.
12. J. P. Jung, A. J. Sprangers, J. R. Byce, J. Su, J. M. Squirrell, P. B. Messersmith, K. W. Eliceiri and B. M. Ogle, Biomacromolecules, 2013, 14, 3102–3111.
13. J. Yin, C. Li, D. Wang and S. Liu, J. Phys. Chem. B, 2010, 114, 12213–12220.
14. D. Wang, T. Liu, J. Yin and S. Liu, Macromolecules, 2011, 44, 2282–2290.
15. Z. Yang and B. Xu, J. Mater. Chem., 2007, 17, 2385–2393.
16. A. Maciollek, M. Munteanu and H. Ritter, Macromol. Chem. Phys., 2010, 211, 245–249.
17. M. E. Davis, Mol. Pharm., 2009, 6, 659–668.
18. M. E. Davis, J. E. Zuckerman, C. H. Choi, D. Seligson, A. Tolcher, C. A. Alabi, Y. Yen, J. D. Heidel and A. Ribas, Nature, 2010, 464, 1067–1070.
19. J. Zhang, H. Sun and P. X. Ma, ACS Nano, 2010, 4, 1049–1059.
20. T. Kakuta, Y. Takashima, M. Nakahata, M. Otsubo, H. Yamaguchi and A. Harada, Adv. Mater., 2013, 25, 2849–2853.
21. M. Zhang, D. Xu, X. Yan, J. Chen, S. Dong, B. Zheng and F. Huang, Angew. Chem., Int. Ed., 2012, 51, 7011–7015.
22. M. Nakahata, Y. Takashima, H. Yamaguchi and A. Harada, Nat. Commun., 2011, 2, 511–516.
23. K. Miyamae, M. Nakahata, Y. Takashima and A. Harada, Angew. Chem., Int. Ed., 2015, 54, 8984–8987.
24. A. Harada, A. Hashidzume and Y. Takashima, Adv. Polym. Sci., 2006, 201, 1–43.
25. F. van de Manakker, T. Vermonden, C. F. van Nostrum and W. E. Hennink, Biomacromolecules, 2009, 10, 3157–3175.
26. Y. Zhou, M. Xu, T. Yi, S. Xiao, Z. Zhou, F. Li and C. Huang, Langmuir, 2007, 23, 202–208.
27. C. T. L. Jr, K. A. Smith and T. A. Hatton, Macromolecules, 2004, 37, 5397–5405.
28. N. Koumura, M. Kudo and N. Tamaoki, Langmuir, 2004, 20, 9897–9900.
29. S. Yagai, T. Karatsu and A. Kitamura, Langmuir, 2005, 21, 11048–11052.
30. H. Yamaguchi, Y. Kobayashi, R. Kobayashi, Y. Takashima, A. Hashidzume and A. Harada, Nat. Commun., 2012, 3, 603–608.
31. I. Tomatsu, A. Hashidzume and A. Harada, Macromolecules, 2005, 38, 5223–5227.
32. I. Tomatsu, A. Hashidzume and A. Harada, J. Am. Chem. Soc., 2006, 128, 2226–2227.
33. S. Tamesue, Y. Takashima, H. Yamaguchi, S. Shinkai and A. Harada, Angew. Chem., Int. Ed., 2010, 49, 7461–7464.
34. Y. L. Zhao and J. F. Stoddart, Langmuir, 2009, 25, 8442–8446.
35. N. C. Sharma, H. J. Galustians, J. Qaquish, A. Galustians, K. N. Rustogi, M. E. Petrone, P. Chalnis, L. Garcia, A. R. Volpe and H. M. Proskin, Journal of clinical pediatric dentistry, 1999, 10, 131–134.
36. A. Sakudo, K. Baba, M. Tsukamoto, A. Sugimoto, T. Okada, T. Kobayashi, N. Kawashita, T. Takagi and K. Ikuta, Bioorg. Med. Chem., 2009, 17, 752–757.
37. A. Sakudo, K. Baba, M. Tsukamoto and K. Ikuta, Bioorg. Med. Chem. Lett., 2009, 19, 4488–4491.
38. R. Veyret, A. Elaissari, P. Marianneau, A. A. Sall and T. Delair, Anal. Biochem., 2005, 346, 59–68.
39. R. Veyret, A. Elaissari and T. Delair, Colloids Surf., B, 2006, 53, 78–86.
40. C. M. Gardner, N. A. Burke, T. Chu, F. Shen, M. A. Potter and H. D. Stover, J. Biomater. Sci., Polym. Ed., 2011, 22, 2127–2145.
41. D. A. Brafman, C. W. Chang, A. Fernandez, K. Willert, S. Varghese and S. Chien, Biomaterials, 2010, 31, 9135–9144.
42. M. A. Shahbazi, P. V. Almeida, E. Makila, A. Correia, M. P. Ferreira, M. Kaasalainen, J. Salonen, J. Hirvonen and H. A. Santos, Macromol. Rapid Commun., 2014, 35, 624–629.
43. L. Goetz, M. Foston, A. P. Mathew, K. Oksman and A. J. Ragauskas, Biomacromolecules, 2010, 11, 2660–2666.
44. T. Z. Zhang, J. S. Chen, Q. Y. Zhang, J. Dou and N. Gu, Colloids Surf., A, 2013, 422, 81–89.
45. T. R. Raj Singh, P. A. McCarron, A. D. Woolfson and R. F. Donnelly, Eur. Polym. J., 2009, 45, 1239–1249.
46. R. C. Petter, J. S. Salek, C. T. Sikorski, G. Kumaravel and F. T. Lin, J. Am. Chem. Soc., 1990, 112, 3860–3868.
47. Y. Y. Liu, X.-D. Fan and L. Gao, Macromol. Biosci., 2003, 3, 715–719.
48. D. B. Bernert, I. Böhm, K. Isenbügel, L. Schönenberg and H. Ritter, Polym. Int., 2012, 61, 413–417.
49. E. Renard, G. l. Volet and C. Amiel, Polym. Int., 2005, 54, 594–599.
50. J. Wang and M. Jiang, J. Am. Chem. Soc., 2006, 128, 3703–3708.
51. S. Lü, B. Li, B. Ni, Z. Sun, M. Liu and Q. Wang, Soft Matter, 2011, 7, 10763–10772.
52. A. M. Layre, G. Volet, V. Wintgens and C. Amiel, Biomacromolecules, 2009, 10, 3283–3289.

---

Footnote

† Electronic supplementary information (ESI) available: Characterization of P(MVE-alt-MA)-based polymer and results including cytotoxicity evaluation, etc. See DOI: 10.1039/c5ra22541h

---