Stress relaxation time in-situ adjustable hydrogel and preparation method thereof

Abstract

The invention discloses stress relaxation time in-situ adjustable hydrogel and a preparation method thereof.The preparation method comprises the following steps that Azo-HA, poly-beta-CD-RGD and polyvinyl alcohol are taken and dissolved in a buffer solution, a mixed solution is prepared, and the concentration of Azo-HA in the mixed solution is 6-8%, the concentration of poly-beta-CD-RGD in the mixed solution is 3-5%, and the concentration of polyvinyl alcohol in the mixed solution is 3-5%; standing and solidifying the mixed solution under a dark condition to obtain hydrogel; azo-HA is (E)-4-((2-boron phenyl) diazenyl) benzoic acid modified hyaluronic acid; the poly (beta-CD-GD) is a copolymer of Ac-beta-CD and acryloylated RGD peptide, and the Ac-beta-CD is acrylated beta-cyclodextrin. The invention provides a novel stress relaxation time in-situ adjustable hydrogel and a preparation method thereof, and provides a novel strategy to decouple stress relaxation and rigidity of the hydrogel.

Description

Technical Field

[0001] The invention relates to the technical field of biocompatible dynamic hydrogels, and in particular to a hydrogel with in-situ adjustable stress relaxation time and a preparation method thereof. Background Art

[0002] Extracellular matrix (ECM) and cells make up our tissues. ECM not only provides mechanical support for cells, but also drives biochemical and / or biophysical signals. At the same time, cells sense the mechanical signals of ECM and reshape it. This interactive process regulates cell behavior and phenotype. Therefore, a comprehensive and in-depth understanding of the interaction between cells and ECM helps to study cell behavior and fate.

[0003] Hydrogels have been widely used as a substitute for natural ECM to reveal the mechanisms of various cell behaviors. It has been shown that the stiffness of hydrogels affects cell activity and function, such as cell morphology, proliferation, migration, differentiation, stemness, etc. However, natural ECM is viscoelastic, has dynamic mechanical properties, and exhibits time-dependent responses to external mechanical loads, which are not found in elastic hydrogels. Therefore, hydrogels with dynamic networks can better mimic the viscoelastic properties of natural ECM. In addition to stiffness, stress relaxation has been shown to guide cell behavior in hydrogels. A variety of hydrogels with decoupled stiffness and stress relaxation have been developed to further explore how stress relaxation affects cell fate. For example, Mooney et al. used a double cross-linking strategy to prepare alginate hydrogels with adjustable stress relaxation time and almost constant stiffness to regulate the spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) by using polymers of different molecular weights and different concentrations of calcium. Heilshorn et al. designed hydrogels based on the different kinetics of reactive groups, with faster dissociation / association kinetics of aldehydes and slower dissociation / association kinetics of benzaldehyde, and adjusted the ratio of aldehydes to benzaldehyde to decouple stress relaxation time and stiffness of the hydrogels. However, these decoupling methods are not in situ and cannot simulate complex changes in viscoelastic tissues, such as the enhanced ECM viscoelasticity caused by the accumulation of advanced glycation end products (AGEs) in type 2 diabetes (faster stress relaxation but no change in stiffness). Therefore, in vitro cell scaffolds with in situ decoupled stiffness and stress relaxation can provide insights into how the ECM regulates cell behavior.

[0004] The viscoelastic properties of hydrogels are determined by the kinetics and thermodynamics of cross-linking. Hydrogel systems with decoupled stiffness and stress relaxation require changes in the kinetics that determine the dissociation / association rates while maintaining thermodynamic stability that controls the equilibrium. Azobenzene (Azo) is a reversibly photoresponsive molecule that can transform from the trans to the cis form under UV irradiation and from the cis to the trans form under visible light. Azobenzene can enter the hydrophobic cavity of β-cyclodextrin (β-CD), form supramolecular interactions in the trans configuration, and escape from the cavity in the cis configuration. Kawashima et al. found that the Lewis acidity of catechol borane could be affected by the E / Z isomerization of the azo group, and Kalow et al. successfully achieved the sol-gel transition of hydrogels cross-linked with azobenzene boronic acid and diol-terminated polyethylene glycol polymers due to the different binding affinities between trans / cis and diol. Based on these results, we speculate that trans-azophenylboronic acid can form a supramolecular complex with β-CD. After UV irradiation, cis-azophenylboronic acid comes out of β-CD and combines with diols to form new cross-links. In this way, the dissociation / association rate of the hydrogel can be changed, while the cross-linking density can be kept unchanged by regulating the hydrogel components, thereby achieving decoupling of stress relaxation and stiffness. Summary of the invention

[0005] The purpose of the present invention is to provide a hydrogel with in-situ adjustable stress relaxation time and a preparation method thereof in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a method for preparing a hydrogel with in-situ adjustable stress relaxation time, comprising the following steps: dissolving Azo-HA, poly-β-CD-RGD and polyvinyl alcohol in a buffer solution to prepare a mixed solution, wherein the concentrations of Azo-HA, poly-β-CD-RGD and polyvinyl alcohol in the mixed solution are 6-8%, 3-5% and 3-5% respectively according to mass volume percentage; allowing the mixed solution to stand and solidify in the dark to obtain the hydrogel;

[0008] The Azo-HA is (E)-4-((2-borylphenyl)diazenyl)benzoic acid modified hyaluronic acid;

[0009] The poly β-CD-GD is a copolymer of Ac-β-CD and acrylated RGD peptide, and the Ac-β-CD is acrylated β-cyclodextrin.

[0010] In the above preparation method, the mixed solution is allowed to stand overnight at 35-38° C. in the dark to solidify, thereby obtaining the hydrogel.

[0011] In the above preparation method, the buffer is PBS buffer.

[0012] In the above-mentioned preparation method, the (E)-4-((2-borylphenyl)diazenyl)benzoic acid is a compound having the following chemical structural formula:

[0013]

[0014] The above-mentioned preparation method, the preparation method of Azo-HA comprises the following steps:

[0015] Dowex resin is added to tetrabutylammonium hydroxide, stirred and filtered to obtain Dowex TBA resin; sodium hyaluronate is dissolved in deionized water, Dowex TBA resin is added, stirred and mixed, and Dowex resin is removed by filtering to obtain HA-TBA solution;

[0016] The HA-TBA solution is frozen and lyophilized to obtain a polymer HA-TBA; (E)-4-((2-borylphenyl)diazenyl)benzoic acid, HA-TBA and 4-dimethylaminopyridine are dissolved in anhydrous DMSO, and after complete dissolution, di-tert-butyl dicarbonate is added to the above solution; the mixture is heated to 40-50° C. and maintained under nitrogen protection for 20-28 hours; after cooling to room temperature, the solution is dialyzed with DMSO, NaCl and deionized water to remove all unreacted small organic compounds; the solution is then frozen and lyophilized to obtain an orange solid polymer, namely Azo-HA.

[0017] The preparation method of the poly β-CD-GD comprises the following steps: preparing an aqueous solution containing Ac-β-CD, acrylamide, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate and acryloyl RGD peptide, and irradiating the solution with 405 nm light for 8 to 14 minutes; filtering the mixture to obtain a clear solution; freezing and freeze-drying the solution to obtain a solid polymer, namely the poly β-CD-RGD.

[0018] In the above-mentioned preparation method, the concentration of each substance in the aqueous solution is, by mass volume percentage, 9-11% Ac-β-CD, 1.5-2.5% acrylamide, 1.0-2.0% lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate and 0.8-1.2% acryloyl RGD.

[0019] The preparation method of Ac-β-CD comprises the following steps: dissolving β-cyclodextrin in DMF and adding TEA to the solution; stirring, cooling to 0°C, and adding acrylic acid; after sufficient reaction, filtering the mixture to obtain a clear solution; then, concentrating the solution in vacuo, and dropping into acetone to precipitate modified β-cyclodextrin; washing the precipitate with acetone, and drying to obtain a white solid, namely Ac-β-CD.

[0020] A second aspect of the present invention provides a hydrogel with in-situ adjustable stress relaxation time, which is prepared by any of the preparation methods described above.

[0021] The beneficial effects of the present invention are:

[0022] The present invention prepares a dynamic hydrogel with in situ decoupled kinetics and thermodynamics. Rheological tests show that the mechanical strength of the hydrogel before and after illumination is 900Pa, but the stress relaxation time is 10 times different. We then study how the kinetic changes of the dynamic hydrogel affect the spreading and tube-forming behavior of human umbilical vein endothelial cells (HUVEC). Our experimental results show that compared with hydrogels with slower dissociation / association rates, dynamic hydrogels with faster dissociation / association rates are more conducive to the spreading and tube-forming behavior of HUVEC, and the kinetics of expansion and tube formation can be accelerated by in situ conversion of slow kinetic hydrogels into fast kinetic hydrogels. The present invention's research proves that compared with hydrogels with slower kinetics, hydrogels with faster kinetics are more conducive to integrin aggregation, leading to the recruitment of vinculin and the formation of large focal adhesions (FA), thereby supporting rapid cell spreading and tube formation. The present invention provides a new strategy to decouple hydrogel stress relaxation and stiffness to design dynamic hydrogels to simulate the viscoelastic changes of tissues in vitro, emphasizes the importance of hydrogel dynamic changes to cell behavior, and provides a new hydrogel with in situ adjustable stress relaxation time and a preparation method thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Characterization of poly β-CD-RGD: a) GPC chromatogram of poly β-CD-RGD; b) FT-IR spectrum of poly β-CD-RGD; at 3447 and 1655 cm -1 Strong absorption peaks were observed at 932 and 575 cm-1, indicating that the polymer contained acrylamide. -1 Place.

[0024] Figure 2 a) Azo-HA in D2O 1 H-NMR spectrum; b) Absorption spectrum changes of Azo-HA under 365nm and 475nm light irradiation; c) Absorption spectrum changes of Azo-HA under 10 cycles of 365nm and 475nm light irradiation; d) Host-guest interaction diagram of Azo-HA(E) and β-CD; e) 2DNOESY of β-CD / Azo-HA(E) complex in D2O 1 HNMR spectrum; f) First-order kinetic analysis and Arrhenius plot of thermal isomerization of Azo-HA(Z) in H2O.

[0025] Figure 3The stoichiometry of the 1(E) / β-CD complex: a) UV-visible absorption spectrum of the mixed solution of 1(E) and β-CD (1(E)+β-CD=2×10 -5 M); b) Job's plot of 1(E) and β-CD.

[0026] Figure 4 Rheological properties of hydrogels: a, b and c) frequency scan (1% strain), time scan (1% stress) and stress relaxation curve (0.5% strain) of S-hydrogel (7% Azo-HA, 3.5% poly-β-CD-RGD and 3.5% PVA); d, e and f) frequency scan (1% strain), time scan (1% stress) and stress relaxation curve (0.5% strain) of F-hydrogel (7% Azo-HA, 3.5% p-β-CD and 3.5% PVA); g, h and i) frequency scan (1% strain), time scan (1% stress) and stress relaxation curve (0.5% strain) of E-hydrogel (2.5% HAMA).

[0027] Figure 5 Characterization of hydrogels with adjustable in situ stress relaxation time: a) FT-IR spectra of Azo-HA, PVA and S-hydrogel; b) changes in the absorption spectrum of hydrogels under 365nm and 475nm light; c) changes in the absorption spectrum of hydrogels after 6 cycles of irradiation at 365nm and 475nm light; d) SEM images of F-hydrogel and S-hydrogel; e) frequency scans of F-hydrogel and S-hydrogel (1% strain); f) stress relaxation curves of F-hydrogel, S-hydrogel and E-hydrogel (0.5% strain); g) G' and G" of F-hydrogel and S-hydrogel; h) G' and τ of hydrogels after 6 cycles of irradiation at 365nm and 475nm light. 1 / 2 change.

[0028] Figure 6 Shown are the cell viability of HUVECs exposed to the hydrogel extracts for 7 days.

[0029] Figure 7 Figure 4 shows that stress relaxation in situ tunable hydrogels regulate the spreading of HUVECs: a) Immunofluorescence staining images of F-actin (green) show the phenotypic changes of HUVECs during spreading culture in 5 independent hydrogels, scale bar, 20 μm; b, d, f, h and j) Quantitative analysis of the circularity of HUVECs during 30 hours of culture in 5 independent hydrogels, data were analyzed by ordinary one-way ANOVA: ns, not significant, *p<0.05, **p<0.01, ***p<0.001,

[0030] ****p<0.0001; n=50 cells; c, e, g, i and k) Quantification of spreading area of ​​HUVECs during 30 h culture in 5 independent hydrogels, data were analyzed by ordinary one-way ANOVA: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n=50 cells.

[0031] Figure 8 Shows that rapid stress relaxation hydrogels promote the formation of FAs: a) Schematic diagram of light-responsive hydrogels with repeated stress relaxation gradients regulating HUVEC cell spreading; b) Immunofluorescence staining images showing the phenotypic changes of HUVECs on hydrogels with repeated stress relaxation gradients, scale bar, 100 μm; c) Quantitative analysis of the circularity of hydrogels with repeated stress relaxation gradients, data analysis was performed using ordinary one-way ANOVA: ****p<0.0001; n=17 cells; d) Immunofluorescence images of YAP in HUVECs on F-hydrogels, S-hydrogels, and E-hydrogels. Scale bar = 30 μm; e) Quantitative analysis of YAP nuclear localization (intensity ratio between nucleus and cytoplasm) in HUVECs on F-hydrogel, S-hydrogel and E-hydrogel, error bars, mean ± standard error, data were analyzed by ordinary one-way ANOVA: *p < 0.05, ****p < 0.0001; n = 15 cells; f) Immunofluorescence images of p-FAK (green) and F-actin (red) in HUVECs on F-hydrogel, S-hydrogel and E-hydrogel, scale bar = 30 μm; g) Average fluorescence intensity (M) of p-FAK in HUVECs on F-hydrogel, S-hydrogel and E-hydrogel Quantitative analysis of FI), error bars, mean ± standard error; data were analyzed by ordinary one-way ANOVA: *p<0.05, **p<0.01, ****p<0.0001; n=16, 18 and 19 cells; enlarged images of p-FAK immunofluorescence images in h)-g), scale bar = 5 μm; i) Quantitative analysis of the mean area (MA) of FA in HUVECs on F-hydrogel, S-hydrogel and E-hydrogel, error bars, mean ± standard error, data were analyzed by ordinary one-way ANOVA: **p<0.001, ****p<0.0001; n=16, 18 and 19 cells.

[0032] Fig. 9Figure 4 shows that the stress relaxation time in situ adjustable hydrogel regulates the tube-forming behavior of HUVEC: a) Optical microscopy images show the tube morphology of HUVEC incubated in 5 independent hydrogels, scale bar, 100 μm; Immunofluorescence staining images of F-actin (green) show the tube morphology of HUVEC incubated in b) F-hydrogel, c) S-hydrogel and d) E-hydrogel, after 45 hours of culture in 4 independent hydrogels, e) total tube area and f) total tube length were quantitatively analyzed, and the data were analyzed by ordinary one-way ANOVA: *p<0.05, **p<0.01; data are from 3 independent experiments; after 45 hours of culture in F-hydrogel, S-hydrogel and E-hydrogel, g) total tube area and h) total tube length were quantitatively analyzed, and the data were analyzed by ordinary one-way ANOVA: **p<0.01, ***p<0.001,

[0033] ****p<0.0001; data are from 3 independent experiments; i) Immunofluorescence images of VE-cad (green) in HUVECs on F-hydrogel, S-hydrogel, and E-hydrogel, scale bar = 30 μm; j) Representative immunofluorescence staining of VE-cad (green) and cell nuclei (blue) in HUVECs on F-hydrogel, S-hydrogel, and E-hydrogel, error bars, mean ± standard error; data were analyzed by ordinary one-way ANOVA: *p<0.05, ****p<0.0001; n = 15 cells. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Example 1

[0037] 1. Materials and Instruments

[0038] All chemicals were commercially available and used without further purification. NMR spectra were obtained using a Bruker AM-400 spectrometer. UV-Vis spectra were recorded on a Varian Cary 500 (1 cm quartz cell). Lyophilization was performed on a DGJ-10C freeze dryer. Scanning electron microscopy (SEM) measurements were performed using a Zeiss Ultra 55 microscope at an accelerating voltage of 30 kV. The rheological properties of the hydrogels were measured using a Physica MCR101 rheometer. Oscillating time scans and frequency scans were performed for a fixed strain of 1% within the linear viscoelastic region (LVE). Bright field and fluorescence field images were taken using a laser scanning confocal microscope (STELLARIS 8) under the specified conditions of X40 or X10.

[0039] 2. Methods

[0040] 2. Synthesis of methyl 14-nitrosobenzoate (S1)

[0041] 3 g (20 mmol) of methyl 4-aminobenzoate was dissolved in 66 mL of DCM. 20 g (40 mmol) of Oxone was added to 250 mL of deionized water and then DCM was added after complete dissolution. The reaction was aged for 1 hour under high stirring. During the reaction, the two-phase solution turned an intense neon green color. The product was extracted with water (1x), 1 M HCl (2x) and 1 M NaOH (3x). The organic layer was dried over magnesium sulfate and concentrated in vacuo to give methyl 4-nitrosobenzoate (yield 65%). 1 H NMR (400 MHz, chloroform-d) δ 8.30 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 3H), 3.98 (s, 3H).

[0042] 2.2 Synthesis of methyl (E)-4-((2-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)phenyl)diazinyl)benzoate (S2)

[0043] 4.4 g (24 mmol) of S1 and 6.1 g (28 mmol) of 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline were added to 100 mL of DCM, to which 20 mL of acetic acid was added. The reactants were stirred at room temperature for 24 hours. After several hours of reaction, the solution turned black. The organic layer was washed with 1 M HCl (3x), collected and dried over magnesium sulfate, and concentrated in vacuo to remove acetic acid. The collected solid was diluted in hexane and the deposited by-products were removed. The filtrate was vacuum concentrated to about 100 mL, filtered again, and the collected filtrate was vacuum concentrated to obtain an orange solid, which was recrystallized twice from hexane. Finally, a red crystalline solid (61% yield) was obtained. 1 HNMR (400MHz, CDCl3) δ8.19 (d, J=8.7Hz, 2H), 7.94 (d, J=8.6Hz, 2H, 7.81 (d, J.=8.0, 1.1Hz, 1 H), 7.75(d, J / 1.5Hz, 1H, 7.56–7.52(m, 1H), 7.50–7.46(m, 1Hs), 3.96(s, 3H), 1.36(s, 12H).

[0044] 2.3 Synthesis of (E)-(2-((4-(methoxycarbonyl)phenyl)diazinyl)phenyl)boronic acid (S3)

[0045] 5g (10mmol) S2 is dissolved in 250mL THF, 9g (40mmol) NaIO4 is added in 62mL deionized water and transferred in THF. After stirring for 30 minutes, 8mL 1M HCl is added in the above solution. Reactant is stirred at room temperature overnight. After reaction is completed, filter solution and remove THF in vacuum. Gained solution is diluted with deionized water and filtered to obtain orange solid. Wash solid with 100mL acetonitrile, filter and dry solid to obtain S3 (91% yield). 1 HNMR (400MHz, DMSO-d6) δ8.19 (d, J=8.4Hz, 2H), 7.96 (d, J=8.4Hz, 2Hs), 7.92 (d, J.6.3Hz, 1H), 7.83 (s, 2H), 7.59–7.53 (m, 3H), 3.91 (s, 3H).

[0046] 2. Synthesis of 4-((2-borylphenyl)diazenyl)benzoic acid (Compound 1(E))

[0047] 3g (10mmol) of 1 was dissolved in 282mL of methanol, and 1g (40mmol) of LiOH was dissolved in deionized water and transferred to methanol. The reactant was stirred at room temperature overnight. Methanol was removed in vacuo, and the remaining solution was neutralized with 1M HCl to obtain a precipitated product. After filtering the solution, the solid was dried to obtain the product (97% yield), obtaining compound 1 (E). 1 HNMR (400MHz, DMSO-d6) δ13.25 (s, 1H), 8.17 (m, 2H), 7.98-7.90 (m, 3H), 7.82 (s, 2H) and 7.62-7.52 (m, 1H). The synthetic route of compound 1 is as follows:

[0048]

[0049] 2.5 Synthesis of (E)-4-((2-borylphenyl)diazenyl)benzoic acid modified hyaluronic acid (Azo-HA)

[0050] 12.5 g of Dowex resin was added to 24.5 mL of 1.5 M tetrabutylammonium hydroxide (TBAOH). After stirring for 30 minutes, Dowex TBA resin was obtained by filtration. Sodium hyaluronate (HA-Na, 1.0 g) was dissolved in 100 mL of deionized water, and 10 g of Dowex TBA resin was added to the above solution. After stirring and mixing for 3 hours, the Dowex resin was removed by filtration to obtain a HA-TBA solution. The solution was frozen and lyophilized to obtain a polymer HA-TBA. 0.674 g of compound 1 (E), 0.52 mg (1 equivalent) of HA-TBA and 76 mg (0.75 equivalent) of 4-dimethylaminopyridine (DMAP) were dissolved in 52 mL of anhydrous DMSO at room temperature. After complete dissolution, 0.615 mL (3.2 equivalents) of BOC2O (di-tert-butyl dicarbonate, CAS: 24424-99-5) was added to the above solution. The mixture was heated to 45°C and kept under nitrogen for 24 hours. After cooling to room temperature, the solution was dialyzed directly with DMSO, NaCl (aq) and deionized water to remove all unreacted small organic compounds. The solution was then frozen and lyophilized to obtain an orange solid polymer, which was (E)-4-((2-borylphenyl)diazenyl)benzoic acid modified hyaluronic acid, denoted as Azo-HA. 1 H NMR (600 MHz, D2O) quantified the degree of modification from the integration ratio between the azobenzene group (δ = 7.4-8.60, 10H) and the HA backbone (δ = 3.20-4.20, 10H).

[0051] 2.6 Synthesis of Acrylated β-cyclodextrin (Ac-β-CD)

[0052] Dissolve 10g of β-cyclodextrin (β-cyclodextrin, abbreviated as β-CD) in 150mL of DMF (N,N-dimethylformamide), and add 7mL of triethanolamine (TEA) to the solution. After stirring the above solution and cooling it to 0°C, add 5mL of acrylic acid. After reacting for 12 hours, filter the mixture to obtain a clear solution. Then, concentrate the solution in vacuo to about 20mL and drop it into acetone to precipitate the modified cyclodextrin. Wash the precipitate several times with acetone and dry it for 3 days to obtain a white solid, that is, acrylated β-cyclodextrin (Ac-β-CD). 1 H NMR (400MHz, DMSO-d6) δ 6.26 (d, J=49.2Hz, 1H), 5.81 (d, J=34.6Hz, 4H), 4.85 (s, 1H).

[0053] Synthesis of 2.7Ac-β-CD and acryloyl RGD peptide copolymer (poly β-CD-GD)

[0054] An aqueous solution containing 10% (w / v) Ac-β-CD, 2% (w / v) acrylamide, 1.5% (w / v) lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) and 1% (w / v) acryloyl RGD peptide was prepared and irradiated with 405 nm light for 10 minutes. The mixture was filtered to obtain a clear solution. Then, the solution was frozen and lyophilized to obtain a white solid polymer (i.e., poly β-CD-RGD, whose characterization diagram is shown in FIG. Figure 1 The M measured by GPC n =242425,M w =369312,M p =343168, polydispersity = 1.523. Fourier transform infrared spectrum (cm -1 )3447, 1655, 932, 575.

[0055] Acrylated RGD peptide (Pep-RGDfKAC) is an RGD peptide modified with an acrylic acid group (Arg-Gly-Asp) and was purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0056] 2.8 Determination of the stoichiometric ratio of 1(E) / β-CD complexes

[0057] Job's plot was determined based on UV-Vis data obtained in PBS (containing a small amount of methanol). A series of mixed solutions were prepared with the same total molar concentration (1(E)+β-CD=2×10 -5 M), but the molar ratio of compound 1 (E) and β-CD was different.

[0058] 2.9 Preparation of viscoelastic hydrogels

[0059] Azo-HA, poly-β-CD-GD and PVA (polyvinyl alcohol) were dissolved in PBS buffer (pH=7.4) respectively, and then uniformly mixed (final concentration: 7% (w / v) Azo-HA, 3.5% (w / v) poly-β-CD-GD, 3.5% (w / v) PVA). The hydrogel was placed in a dark environment at 37° C. overnight to obtain a viscoelastic hydrogel, which is the in-situ adjustable stress relaxation time hydrogel of the present invention.

[0060] 2.10 Preparation of elastic hydrogel

[0061] Methacryloyl hyaluronic acid (HAMA) was dissolved in PBS (pH = 7.4) and then mixed with LAP (final concentration: 2.5% (w / v) HAMA, 0.25% (w / v) LAP). The solution was filled into a mold and irradiated at 405 nm for 2 minutes. An elastic hydrogel was obtained, which was subsequently used as a control group without relaxation properties in cell experiments.

[0062] 2.11 Swelling ratio of viscoelastic hydrogels

[0063] The viscoelastic hydrogels were transformed from F-hydrogels to S-hydrogels by irradiation with 365 nm light for 15 min. The swelling ratios of F-hydrogels and S-hydrogels were measured by immersing the hydrogels in DMEM in a dark environment at 37 °C for 48 h. The swelling ratio was calculated by the following formula:

[0064]

[0065] Where W t and W0 represent the weights of the swollen hydrogel and the initial hydrogel, respectively.

[0066] 2.12 Mechanical properties

[0067] The sample was placed between the bottom rheometer plate and a 20 mm plate (2 mm thick). The experiments were performed at 37°C. Oscillation time sweeps and frequency sweeps were performed at 1% strain. Stress relaxation sweeps were performed at 0.5% strain.

[0068] 1.13 Cell culture

[0069] HUVEC cells were purchased from Shanghai Jinyuan Biotechnology Co., Ltd. and cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, 10099141C) and 1% penicillin / streptomycin. The cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0070] 1.14 Cytotoxicity and Cytophototoxicity

[0071] Prepared HAG was immersed in DMEM at 37°C for 72 hours, and the extract was collected for cytotoxicity measurement. HUVEC was seeded in 98-well plates at a concentration of 40,000 cells per well and cultured in DMEM (10% FBS) at 37°C, 5% humidified CO2 for 24 hours. The culture medium was replaced with extract or control DMEM. At 1, 3, 5 and 7 days, cells were incubated for 2 hours at 37°C with 10 μL cell counting kit-8 (CCK-8) in 5% humidified CO2. The absorbance at 450nm was measured by a microplate reader (Molecular Devices, Sunnyvale, CA).

[0072]

[0073] Among them A i is the absorbance of cells treated with hydrogel extraction solution, and A0 is the absorbance of blank control.

[0074] 1.15 HUVEC tube formation experiment

[0075] HUVEC cells were seeded on the hydrogel at a density of 10,000 cells / mL and cultured with complete culture medium supplemented with 30 ng / mL vascular endothelial growth factor (VEGF) and 30 ng / mL basic fibroblast growth factor (bFGF). The morphological changes of HUVEC cells were observed using a bright field microscope every 15 h.

[0076] 2.16 Immunofluorescence staining

[0077] The samples were fixed with pre-cooled 4% paraformaldehyde for 10 minutes, then permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 5% skim milk for 30 minutes. The samples were incubated with primary antibodies (diluted in 5% skim milk) overnight at 4°C. The secondary antibodies were diluted in PBS, and the samples were incubated with antibodies for 2 hours at room temperature. Nuclei were counterstained with DAPI and filamentous actin was counterstained with phalloidin (TraKine TM F-actin staining kit, KTC4009).

[0078] 2.17 Image Analysis

[0079] Images were analyzed using ImageJ (64-bit) (National Institutes of Health). To analyze YAP nuclear localization, the mean grayscale value in the nucleus was divided by the corresponding mean in the cytoplasm. To analyze pFAK and vinculin, background removal and thresholding were used to exclude cytoplasmic signals, and FMI was recorded. The tube length and tube area of ​​HUVEC cells were analyzed using Angio Tool.

[0080] 3 Results

[0081] 3.1 Construction of hydrogels with in situ adjustable stress relaxation time

[0082] Based on Kalow's work, we first synthesized an azobenzene derivative with the boric acid located at the ortho position of the azo group, and then obtained hyaluronic acid (Azo-HA) grafted with an azobenzene derivative by esterification reaction, with a modification degree of 32% ( Figure 2 a), and measured the photoswitching ability of Azo-HA. Figure 2As shown in (b), after 5 min of 365 nm light irradiation, Azo-HA isomerized from E to Z, with a decrease in absorbance at 340 nm and an increase in absorbance at 422 nm, which are attributed to π→π* and n→π* electronic transitions, respectively. The isomerization from Z to E occurs under visible light (475 nm, 2 min) or heating. Under alternating irradiation of 365 nm UV light and 475 nm blue light, the isomerization between E and Z can be repeated at least 10 times with negligible absorption changes, showing considerable photoisomerization reversibility and fatigue resistance ( Figure 2 c). The thermal half-life of Azo-HA was estimated using UV-visible spectroscopy at 37 °C in the dark based on first-order kinetics ( Figure 2 f), the thermal half-life of Azo-HA is 26.7 hours.

[0083] Azobenzene derivatives have a strong binding affinity with β-cyclodextrin (β-CD) and will form inclusion complexes. In order to reveal the stoichiometric ratio between 1(E) and β-CD, corresponding UV-Vis tests were performed, such as Figure 3 As shown, the absorption maximum of the 1(E) / β-CD system corresponds to 0.5 on the horizontal axis, indicating a 1:1 complexation. 1 H NOESY spectroscopy was used to further understand the interaction between Azo-HA and β-CD. The strong cross peaks (marked by the dashed box) between the azobenzene groups of Azo(E)-HA (8.0-8.5 ppm) and the internal protons of β-CD (3.5-4.0 ppm) confirmed the host-guest interaction ( Figure 2 d and e).

[0084] The preparation of the hydrogel was achieved by uniformly mixing Azo-HA, poly-β-CD-RGD and polyvinyl alcohol (PVA) in phosphate buffered saline (PBS, pH 7.4) under physiological conditions (37°C). We named the hydrogel based on host-guest interaction as fast stress relaxation hydrogel (F-hydrogel) and the hydrogel based on dynamic covalent bonds as slow stress relaxation gel (S-hydrogel). The hydrogel was finally composed of 7% (w / v) Azo-HA, 3.5% (w / v) poly-β-CD-RGD and 3.5% (w / v) PVA. Fourier transform infrared spectroscopy (FT-IR) analysis confirmed the formation of S-hydrogel, with characteristic peaks located at 1430-1355cm 1 , indicating the presence of BO bonds. 1108cm -1 is the peak of CO stretching vibration in borate esters, which is one of the characteristic absorption peaks of borate esters ( Figure 5 a). We then used UV-visible absorption spectroscopy to ensure that the azobenzene derivative groups in the hydrogel could be photoisomerized smoothly. Figure 5As shown in Figure b, after irradiation with 365nm light for 15 minutes, the absorbance at 340nm decreased, indicating that the azobenzene derivative group in the hydrogel successfully isomerized from E to Z. After the hydrogel was treated with 475nm light for 30 minutes, the absorbance at 340nm returned to the initial state, and the absorption change was negligible, indicating that the azobenzene derivative group isomerized from Z to E. Under alternating irradiation with 365nm ultraviolet light and 475nm blue light, the isomerization between E and Z can be repeated at least 6 times, showing excellent photoisomerization reversibility and fatigue resistance ( Figure 5 c).

[0085] Rheological tests showed that the two hydrogels in different states had similar mechanical strength (G'), about 900 Pa, but after irradiation with 365 nm light, the stress relaxation time of S-hydrogel (240 s) was ten times that of F-hydrogel (24 s), indicating that we successfully decoupled the G' and τ of the hydrogels. 1 / 2 , achieving in situ regulation of hydrogel stress relaxation time ( Figure 5 e and g, rheological data see Figure 4 ). Scanning electron microscopy images showed that F-hydrogel, S-hydrogel and E-hydrogel all exhibited typical macroporous structures ( Figure 5 d), no obvious pore size difference was found among the different hydrogels, which is due to the similarity of G' among the three hydrogels. G' and τ of F-hydrogel and S-hydrogel 1 / 2 The hydrogel remained stable under 365 and 475 nm light irradiation for 6 cycles, which proved that the hydrogel had good fatigue resistance ( Figure 5 h). To evaluate the swelling ratio of F-hydrogel and S-hydrogel, we immersed the hydrogel in DMEM medium at 37°C and recorded the mass change of the hydrogel within 48 hours. Since the initial G' of F-hydrogel and S-hydrogel were similar, F-hydrogel and S-hydrogel had similar swelling ratios after reaching swelling equilibrium. In addition, we also prepared elastic hydrogels (E-hydrogels) using hyaluronic acid methacryloyl and covalently linked acryloyl RGD for subsequent cell experiments. The obtained E-hydrogel, F-hydrogel, and S-hydrogel had similar G' (rheological data see Figure 4 ), but the E-hydrogel did not show a complete stress relaxation trend within 300 seconds ( Figure 5 f).

[0086] 3.2 In situ tunable stress relaxation time hydrogel regulates the spreading behavior of HUVEC

[0087] Cell spreading is one of the earliest interactions between cells and the extracellular matrix (ECM). The morphological changes of cells during spreading can provide important information about the interaction between cells and ECM and the mechanical response of cells to ECM, which is affected by the mechanical properties of ECM. As reported by Bian et al., dynamic hydrogels with short stress relaxation times lead to rapid spreading and mechanical response of cultured human mesenchymal stem cells. To further understand the interaction between cells and matrix, we observed the dynamic hydrogels seeded with different τ 1 / 2 The spreading behavior of human umbilical vein endothelial cells (HUVEC) on the viscoelastic hydrogel surface was investigated. In vitro cell experiments were performed to preliminarily evaluate the cytotoxicity of the hydrogel. Figure 6 It can be seen that the cell survival rate remained above 90% within 7 days, indicating that the hydrogel had no obvious inhibitory effect on cell growth and proliferation.

[0088] from Figure 7 As can be seen in a, spreading cells were observed in F-hydrogel after 10 hours, but cells incubated on S-hydrogel still did not spread before 20 hours. Cell roundness analysis showed that cells in F-hydrogel had smaller roundness and larger spreading area compared with cells in S-hydrogel ( Figure 7 bc, Figure 7 hi). Compared with cells on viscoelastic hydrogels, cells in E-hydrogels exhibited a non-spreading morphology within 30 h ( Figure 7 a) Larger cell roundness and smaller spreading area ( Figure 7 j and k), which is due to the longer stress relaxation time of E-hydrogel. These findings suggest that short stress relaxation time is more conducive to cell spreading than long stress relaxation time.

[0089] Next, the S-hydrogels were irradiated with visible light at 20 h and 10 h, respectively. Figure 7 a), which transformed it into F-hydrogel. After visible light irradiation, the cells began to spread, the roundness decreased, and the spreading area increased, indicating that the in situ stress relaxation changes of the viscoelastic hydrogel successfully affected the spreading behavior of HUVEC ( Figure 7 d, e, f and g).

[0090] To further demonstrate the in situ regulation of HUVEC spreading behavior, we cultured HUVECs on S-hydrogels and modulated the stress relaxation properties of different regions using photomasks ( Figure 8 a). Surprisingly, a spreading morphology of HUVECs can be observed in the illuminated area, but the cells are still round in the masked area ( Figure 8 b). Roundness analysis further confirms this result ( Figure 8 c).

[0091] 3.3 Fast stress relaxation hydrogel promotes the formation of focal adhesions

[0092] Focal adhesions (FAs) are plasma membrane-associated macromolecular complexes that contact the surrounding extracellular matrix (ECM) through integrin receptors. Integrins bind to the ECM through specific motifs in the RGD sequence, leading to FA formation by inducing nuclear localization of mechanosensory transcription factors such as Yes-associated protein (YAP) and integrin clustering, as well as recruitment of structural proteins such as vinculin and the signaling protein FA kinase (FAK). To explore the role of stress relaxation time on cell spreading and tube formation, we studied FA formation.

[0093] Higher nuclear localization of YAP is associated with increased FA formation, therefore, we stained HUVECs for endogenous YAP. Immunostaining and quantitative analysis of YAP nuclear localization showed ( Figure 8 d and e) F-hydrogel induced higher YAP nuclear intensity than S-hydrogel and E-hydrogel, and HUVECs on S-hydrogel showed a slight increase in YAP nuclear density compared with E-hydrogel.

[0094] Then, the formation of integrin clusters in different hydrogel networks was investigated. After 24 h of culture, increased integrin aggregation was found in F-hydrogels, indicating that the rapid stress relaxation hydrogel promoted the interaction of integrins with the hydrogel. The aggregation of integrins led to the recruitment of vinculin and ultimately the formation of mature FAs. The formation of stable FAs is essential for cytoskeletal remodeling and cell morphological changes. Immunostaining showed that the lack of stable FAs led to weak interactions with the hydrogels and non-spreading morphology. In addition, the cells cultured on F-hydrogels formed larger FAs than S-hydrogels and E-hydrogels after 24 h.

[0095] The process of integrin aggregation and activation begins with the binding between integrins and RGD sites and induces the recruitment of paxillin and FAK to FAs via mechanical transduction. Then, phosphorylation and subsequent activation of FAK promotes the aggregation and external translocation of integrins at the cell edge, accompanied by actin polymerization and myosin activation. This process ultimately promotes the aggregation of vinculin and the formation of mature FAs and increases cell motility. Therefore, phosphorylated FAK (pFAK) was stained to study its activation effect with different hydrogels. Larger FAs ( Figure 8 fi), representing a stronger interaction between HUVEC and F-hydrogel. This result suggests that hydrogels with fast stress relaxation time can promote the formation of larger FAs and the phosphorylation of FAK.

[0096] 3.4 In situ adjustable stress relaxation time hydrogel regulates HUVEC tube-forming behavior

[0097] To form HUVEC tubes, HUVECs seeded on different hydrogels were cultured with DMEM containing 30 ng / mL vascular endothelial growth factor (VEGF) and 30 ng / mL basic fibroblast growth factor (bFGF), and the kinetics of tube formation were observed. After 45 h of incubation, mature tubes were observed in F-hydrogels with a total tube area of ​​2.4 x 105 μm 2 , the total tube length was 3.0x104μm, but in S-hydrogel (total tube area was 1.2x105μm 2 , total tube length 1.8x104μm) and E-hydrogel (total tube area 6.7x104μm 2 , tube length 1.1x104μg) was observed to have a smaller tube area and shorter tube length ( Fig. 9 a, g, and h). Next, we added visible light in situ at 15 and 30 h to alter the kinetics of tube formation. Because the hydrogels transitioned earlier from S-hydrogels to F-hydrogels, 15 h of irradiation resulted in longer tubes and larger tube areas than 30 h of irradiation ( Fig. 9 a, e, and f). These findings suggest that hydrogels with fast stress relaxation times are beneficial for HUVEC tube formation.

[0098] Cadherins are a large family of transmembrane proteins with more than 30 members. VE-cadherin (VE-cad) is an endothelial-specific cell-cell adhesion molecule that stabilizes endothelial junctions through homologous binding to the extracellular region of VE-cad and has been shown to be a major component of endothelial adhesion junctions. Therefore, we investigated the expression of VE-cad by HUVECs in F-hydrogels, S-hydrogels, and E-hydrogels. Immunostaining and quantitative analysis of VE-cad showed that cells in F-hydrogels expressed more VE-cad than those in S-hydrogels and E-hydrogels. Fig. 9 i and j), this result indicates that the hydrogel with faster dynamic properties is beneficial for the VE-cad expression of HUVECs, thereby forming a mature tubular morphology.

[0099] 4 Analysis

[0100] In this work, we successfully prepared hydrogels with in situ tunable stress relaxation time based on host-guest interactions and different kinetic constants of boronate esters, which varied 10-fold under visible light and demonstrated that the dynamics of hydrogels are an important factor controlling cell behavior. Our experimental results show that F-hydrogels with faster dissociation / association rates facilitate cell spreading and tube formation compared with S-hydrogels with slower dissociation / association rates, while elastic hydrogels showed lower cell spreading and tube formation than viscoelastic hydrogels in the same time frame. In addition, visible light can change the dynamics of cell spreading and tube formation because the dynamic properties of hydrogels are altered in situ by visible light. The precise regulation of the cell spreading process can be explained by mechanotransduction based on integrin aggregation and FA formation. Our hydrogels with in situ tunable stress relaxation time provide a new strategy to decouple hydrogel stress relaxation and stiffness, as well as a convenient and controllable platform for studying the effects of hydrogel dynamics on cell behavior.

Claims

1. A method for preparing a hydrogel with in-situ adjustable stress relaxation time, characterized in that: The steps include: Azo-HA, poly-β-CD-RGD and polyvinyl alcohol are dissolved in a buffer solution to prepare a mixed solution, wherein the concentrations of Azo-HA, poly-β-CD-RGD and polyvinyl alcohol in the mixed solution are 6-8%, 3-5% and 3-5% respectively according to mass volume percentage; the mixed solution is allowed to stand and solidify in the dark to obtain the hydrogel; The Azo-HA is (E)-4-((2-borylphenyl)diazenyl)benzoic acid modified hyaluronic acid; The poly β-CD-GD is a copolymer of Ac-β-CD and acrylated RGD peptide, and the Ac-β-CD is acrylated β-cyclodextrin.

2. The preparation method according to claim 1, characterized in that: The mixed solution is allowed to stand overnight at 35-38° C. in the dark to solidify, thereby obtaining the hydrogel.

3. The preparation method according to claim 1, characterized in that: The buffer is PBS buffer.

4. The preparation method according to claim 1, characterized in that: The (E)-4-((2-borylphenyl)diazenyl)benzoic acid is a compound having the following chemical formula:

5. The preparation method according to claim 1, characterized in that: The preparation method of Azo-HA comprises the following steps: Dowex resin is added to tetrabutylammonium hydroxide, stirred and filtered to obtain Dowex TBA resin; sodium hyaluronate is dissolved in deionized water, Dowex TBA resin is added, stirred and mixed, and Dowex resin is removed by filtering to obtain HA-TBA solution; The HA-TBA solution is frozen and lyophilized to obtain a polymer HA-TBA; (E)-4-((2-borylphenyl)diazenyl)benzoic acid, HA-TBA and 4-dimethylaminopyridine are dissolved in anhydrous DMSO, and after complete dissolution, di-tert-butyl dicarbonate is added to the above solution; the mixture is heated to 40-50° C. and maintained under nitrogen protection for 20-28 hours; after cooling to room temperature, the solution is dialyzed with DMSO, NaCl and deionized water to remove all unreacted small organic compounds; the solution is then frozen and lyophilized to obtain an orange solid polymer, namely Azo-HA.

6. The preparation method according to claim 1, characterized in that: The preparation method of the poly-β-CD-GD comprises the following steps: An aqueous solution containing Ac-β-CD, acrylamide, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate and acryloyl RGD peptide is prepared and irradiated with 405 nm light for 8 to 14 minutes; the mixture is filtered to obtain a clear solution; the solution is frozen and lyophilized to obtain a solid polymer, namely poly β-CD-RGD.

7. The preparation method according to claim 1, characterized in that: In the aqueous solution, the concentration of each substance is 9-11% Ac-β-CD, 1.5-2.5% acrylamide, 1.0-2.0% lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate and 0.8-1.2% acryloyl RGD according to mass volume percentage concentration.

8. The preparation method according to claim 7, characterized in that: The preparation method of Ac-β-CD comprises the following steps: β-cyclodextrin is dissolved in DMF, and TEA is added to the solution; after stirring and cooling to 0°C, acrylic acid is added; after sufficient reaction, the mixture is filtered to obtain a clear solution; then, the solution is vacuum concentrated and dropped into acetone to precipitate modified β-cyclodextrin; the precipitate is washed with acetone and dried to obtain a white solid, namely Ac-β-CD.

9. A hydrogel with in-situ adjustable stress relaxation time, characterized in that: The preparation method is described in any one of claims 1 to 8.

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