Composite colloidal gel based on host-guest interaction and preparation method thereof

Abstract

The preparation method comprises the following specific preparation steps: respectively grafting a subject (beta-cyclodextrin) and an object (adamantane) on the surfaces of gelatin nanospheres through an amidation reaction to prepare beta-cyclodextrin gelatin nanospheres and adamantane gelatin nanospheres, then uniformly blending the beta-cyclodextrin gelatin nanospheres and the adamantane gelatin nanospheres, and preparing the composite colloidal gel based on the interaction of the subject and the object. The beta-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is prepared by adding an adamantane gelatin nanosphere into an object, centrifuging the obtained mixture, enabling the object and the object to be in full contact and react, and removing supernate, and the method is simple in preparation process and high in production efficiency, and can be used for preparing the beta-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel. The obtained composite colloidal gel has high mechanical strength and excellent self-repairing capability, injectability, degradability and biocompatibility; the prepared hydrogel can tolerate the physiological salt environment, is beneficial to application in the field of biomedicine, and has good self-repairing capability and high mechanical strength in the physiological salt environment, the surfactant environment and the serum environment.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials, and in particular relates to a composite colloidal gel based on host-guest interaction and a preparation method thereof. Background Art

[0002] Colloidal gel is a gel material system with a microscopic network structure, which is constructed by physical or chemical interactions between particles and takes micron or nano colloidal particles as the basic structural unit. It is widely used in biomedical engineering, 3D printing materials, cosmetics and other fields. Unlike traditional polymer gels, colloidal gels are formed by non-covalent forces between colloidal particles. When this interaction is reversible, colloidal gels have good self-healing ability, that is, colloidal gels exhibit liquid-like fluidity under destructive shear forces, and when the external force is removed, the material automatically restores its mechanical strength. This special rheological behavior makes this type of self-healing colloidal gel very important in the fields of tissue engineering and regenerative medicine.

[0003] Although the unique rheological behavior of colloidal gels makes them of great application value, this type of material system still has the following two problems that need to be solved: (1) Colloidal gels constructed by reversible weak forces generally have poor mechanical strength; (2) Colloidal gels constructed by forces such as electrostatic effects or the coordination effects of metal ion ligands are difficult to tolerate physiological salt environments, which seriously limits their application in biomedical environments. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned deficiencies of colloidal gels in the prior art and to provide a composite colloidal gel based on host-guest interaction and a preparation method thereof which has high mechanical strength, excellent self-repairing ability, tolerance to physiological salt environment, good degradability and biocompatibility.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention is to provide a method for preparing a composite colloidal gel based on host-guest interaction, comprising the following steps:

[0007] S1: preparing a gelatin nanosphere suspension with carboxyl groups on the surface, then adding an amino-containing β-cyclodextrin derivative to the gelatin nanosphere suspension, mixing and stirring evenly, then adding a cross-linking agent and stirring and reacting at room temperature, and obtaining a β-cyclodextrin gelatin nanosphere powder after the reaction product is purified by dialysis and freeze-dried;

[0008] S2: preparing a gelatin nanosphere suspension with carboxyl groups on the surface, then adding an amino-containing adamantane derivative to the gelatin nanosphere suspension, mixing and stirring evenly, adding a cross-linking agent and stirring at room temperature to react, and dialysis purification and freeze-drying the reaction product to obtain adamantane gelatin nanosphere powder;

[0009] S3: weighing appropriate amounts of β-cyclodextrin gelatin nanosphere powder and adamantane gelatin nanosphere powder, and dissolving them in a solvent to form a β-cyclodextrin gelatin nanosphere suspension and adamantane gelatin nanosphere suspension, respectively;

[0010] S4: mixing the β-cyclodextrin gelatin nanosphere suspension and the adamantane gelatin nanosphere suspension in an appropriate proportion to obtain a uniformly dispersed colloidal suspension; centrifuging the colloidal suspension and removing the supernatant to obtain a composite colloidal gel.

[0011] According to the present invention, in steps S1 and S2, the concentration of the gelatin nanosphere suspension is 5 mg / mL to 20 mg / mL, and the pH is 4 to 7; the content of carboxyl groups in the gelatin nanospheres is 0.06 mmol / g to 0.10 mmol / g.

[0012] According to the present invention, in steps S1 and S2, the amino-containing β-cyclodextrin derivative is ethylenediamine β-cyclodextrin, peramino β-cyclodextrin or mono-6-O-amino-β-cyclodextrin, preferably ethylenediamine β-cyclodextrin; the amino-containing adamantane derivative is adamantane hydrochloride, adamantane methylamine hydrochloride, adamantane ethylamine hydrochloride or adamantane amidine hydrochloride, preferably adamantane hydrochloride.

[0013] According to the present invention, in step S1, the molar ratio of the amino-containing β-cyclodextrin derivative to the carboxyl groups contained in the gelatin nanospheres is 0.5:1 to 10:1; the molar ratio of the cross-linking agent to the carboxyl groups contained in the gelatin nanospheres is 1:1 to 10:1.

[0014] According to the present invention, in step S2, the molar ratio of the amino-containing adamantane derivative to the carboxyl groups contained in the gelatin nanospheres is 0.5:1 to 10:1; and the molar ratio of the cross-linking agent to the carboxyl groups contained in the gelatin nanospheres is 1:1 to 10:1.

[0015] According to the present invention, in steps S1 and S2, the cross-linking agent is selected from one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, carbodiimide and N-hydroxysuccinimide.

[0016] According to the present invention, in step S3, the solvent is deionized water, an aqueous solution containing 150 mM sodium chloride, a Tween 20 aqueous solution with a volume concentration of 1%, or a culture medium containing 10% fetal bovine serum.

[0017] According to the present invention, in step S4, the mass concentration of the colloidal suspension is 5% to 30%; the mass ratio of the adamantane gelatin nanospheres to the β-cyclodextrin gelatin nanospheres in the colloidal suspension is 0.2 to 5, preferably 1.

[0018] According to the present invention, in step S4, the relative centrifugal force during centrifugation of the colloidal suspension is 5000 to 15000 g. -1 , preferably 9000~13000g -1 The centrifugation time is 5 to 30 min, preferably 10 to 20 min.

[0019] The second aspect of the present invention is to provide a composite colloidal gel prepared by the above preparation method.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The composite colloidal gel of the present invention is constructed by two kinds of gelatin nanoparticles with a host (β-cyclodextrin) grafted on the surface and a guest (adamantane) grafted on the surface respectively through host-guest interaction, and has high mechanical strength and excellent self-repairing ability, injectability, degradability and biocompatibility; the elastic modulus of the obtained composite colloidal gel is higher than 10kPa, the self-repairing efficiency is ≥100%, and after the injection is completed, it can still maintain a certain mechanical strength.

[0022] 2. Compared with the colloidal gel constructed by electrostatic interaction and the coordination effect of metal ligands, the composite colloidal gel of the present invention can withstand the physiological salt environment, which is beneficial to its application in the biomedical field; it has good self-healing ability and high mechanical strength in the physiological salt environment, surfactant environment and serum environment.

[0023] 3. The preparation method of the present invention is simple and has high production efficiency. The preparation principle is applicable to similar composite colloidal gel systems, providing a design solution for the development of this large category of materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the experimental principle of forming a composite colloidal gel in the present invention (the inset is a photograph of the sample after gelation).

[0025] Figures 2A to 2C They are transmission electron microscope photos of gelatin nanospheres, adamantane gelatin nanospheres, and β-cyclodextrin gelatin nanospheres in Example 1, respectively. Scale bar = 1 μm.

[0026] Figure 3 The elastic modulus (G') and viscous modulus (G") of the colloidal suspension of β-cyclodextrin gelatin nanospheres and adamantane gelatin nanospheres change with time during the gelation process at room temperature.

[0027] Figure 4 This is the self-healing behavior of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel prepared in Example 1.

[0028] Figure 5 The self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared under different relative centrifugal forces.

[0029] Figure 6 The self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared under different relative centrifugation times.

[0030] Figure 7 The self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared by centrifugation at different colloidal suspension concentrations.

[0031] Figure 8 The self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared by centrifugation at different two-phase particle mass ratios.

[0032] Fig. 9 It is the self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel in physiological salt environment.

[0033] Fig.10 It is the self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel in a surfactant environment.

[0034] Fig.11 It is the self-healing behavior of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel in serum environment.

[0035] Fig.12 The injectability of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel.

[0036] Fig.13 The cytotoxicity of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel.

[0037] Fig.14 The biocompatibility of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel. DETAILED DESCRIPTION

[0038] The technical scheme of the present invention is clearly and completely described below by specific embodiments in conjunction with the accompanying drawings. It should be understood that the following embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.

[0039] Unless otherwise specified, the reagents used in the following examples are all conventional commercially available products. In the following examples, conventional desolvation methods were used to prepare gelatin nanospheres containing carboxyl groups on the surface, preferably gelatin nanospheres with a carboxyl content of 0.06 to 0.1 mmol / g.

[0040] like Figure 1 As shown, the present invention grafts the host (β-cyclodextrin) and the guest (adamantane) onto the surface of gelatin nanospheres respectively through amide reaction to prepare β-cyclodextrin gelatin nanospheres and adamantane gelatin nanospheres, and then uniformly blends the two, and then centrifuges the resulting mixture to allow the host and the guest to fully contact, thereby forming a uniform composite colloidal gel network.

[0041] Example 1

[0042] (1) Preparation of gelatin nanospheres:

[0043] First, gelatin nanospheres were prepared by desolvation method. The particle size of the obtained gelatin nanospheres was about 380 nm. The surface Zeta potential was measured to be about -7 mV in 5 mM HEPES aqueous solution (pH 7.4). The carboxyl content in the gelatin nanospheres was 0.08 mmol / g. The morphology of the gelatin nanospheres was as follows: Figure 2A shown.

[0044] (2) Preparation of diamond gelatin nanospheres:

[0045] Take 1g of gelatin nanospheres and disperse it in 100mL of deionized water to obtain a 10mg / mL gelatin nanosphere suspension, and adjust the pH value of the suspension to 6-7. Weigh 0.45g of adamantane hydrochloride, add it to the suspension, and stir thoroughly to obtain a mixed solution. Weigh 0.33g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (cross-linker), add it to the mixed solution, and stir the reaction for 3 days. After the product was dialyzed and purified in ultrapure water using a dialysis bag with a molecular weight cutoff of 8k to 14k, the particle size of the gelatin nanospheres grafted with adamantane was measured to be about 400nm, and the surface Zeta potential was measured to be about +4.5mV in a 5mM HEPES aqueous solution at pH7.4. Its surface morphology is as follows: Figure 2B shown.

[0046] (3) Preparation of β-cyclodextrin gelatin nanospheres:

[0047] Take 1g of gelatin nanospheres and disperse them in 100mL of deionized water to obtain a 10mg / mL gelatin nanosphere suspension, and adjust the pH value of the suspension to 6-7. Weigh 2.82g of ethylenediamine β-cyclodextrin, add it to the suspension, and stir thoroughly to obtain a mixed solution. Weigh 0.33g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (cross-linker), add it to the mixed solution, and stir the reaction for 3 days. After the product was dialyzed and purified in ultrapure water using a dialysis bag with a molecular weight cutoff of 8k to 14k, the particle size of the gelatin nanospheres grafted with β-cyclodextrin was measured to be approximately 390nm, and the surface Zeta potential was measured to be approximately +8mV in a 5mM HEPES aqueous solution at pH7.4. Its surface morphology is as follows: Figure 2C shown.

[0048] (4) Preparation of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel

[0049] A certain amount of the adamantane gelatin nanospheres and β-cyclodextrin gelatin nanospheres prepared in the above steps (2) and (3) are dispersed in deionized water to prepare a colloidal particle suspension with a colloidal particle mass concentration of 20%. The two are directly blended and quickly stirred to obtain a colloidal suspension in which the two-phase colloidal particles are evenly mixed. The mass concentration of the colloidal suspension is still 20%, wherein the mass ratio of the two-phase colloidal particles, that is, the mass ratio of the β-cyclodextrin gelatin nanospheres to the adamantane gelatin nanospheres is 1. The colloidal suspension is heated to 11000 g. -1 The mixture was centrifuged for 20 min and the supernatant was taken out with a pipette to obtain the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel.

[0050] The gelation process was detected by rheometer. Figure 3 The elastic modulus (G') and viscous modulus (G") of the colloidal suspension during gelation at room temperature are shown as a function of time.

[0051] Depend on Figure 3 The results show that at room temperature, the two-phase colloidal particles will quickly self-assemble and form a gel network. The elastic modulus (G') intersects with the viscous modulus (G") in a very short time and is always higher than the viscous modulus (G") thereafter. The intersection of the two is the turning point of the colloidal dispersion from a liquid fluid to a solid gel, thus confirming the rapid formation of the gel.

[0052] Example 2

[0053] This example is basically the same as Example 1, except that the crosslinking agents in steps (2) and (3) are 0.15 g of carbodiimide and 0.09 g of N-hydroxysuccinimide; and finally a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0054] Among them, the particle size of the adamantane gelatin nanospheres prepared in step (2) was measured to be about 380nm, the surface Zeta potential was measured to be about +3.2mV in a 5mM HEPES aqueous solution at pH7.4, and the grafting rate was 0.039mmol / g; the particle size of the β-cyclodextrin gelatin nanospheres prepared in step (3) was measured to be about 375nm, the surface Zeta potential was measured to be about +6.1mV in a 5mM HEPES aqueous solution at pH7.4, and the grafting rate was 0.045mmol / g.

[0055] Embodiments 3 to 8

[0056] Examples 3 to 8 are basically the same as Example 1, except that the concentration of the gelatin nanosphere suspension, the molar ratio of ethylenediamine β-cyclodextrin / gelatin nanosphere carboxyl content, the molar ratio of adamantane hydrochloride / gelatin nanosphere carboxyl content and the molar ratio of cross-linking agent / gelatin nanosphere carboxyl content in steps (2) and (3) are set as shown in Tables 1 and 2 below; and finally a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0057] Among them, it can be seen from the results of Table 1 and Table 2 that in steps (2) and (3), by changing the concentration of the gelatin nanosphere suspension, the molar ratio of ethylenediamine β-cyclodextrin / gelatin nanosphere carboxyl content, the molar ratio of adamantane hydrochloride / gelatin nanosphere carboxyl content or the molar ratio of cross-linking agent / gelatin nanosphere carboxyl content, adamantane gelatin nanospheres and β-cyclodextrin gelatin nanospheres with different particle sizes and different grafting rates can be obtained.

[0058] Table 1. Particle size and grafting rate of adamantane gelatin nanospheres obtained in Examples 3 to 8

[0059]

[0060] Table 2 Particle size and grafting rate of β-cyclodextrin gelatin nanospheres prepared in Examples 3 to 8

[0061]

[0062] Example 9

[0063] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in embodiment 1, except that in step (4), the colloidal suspension is respectively heated to 5000 g and heated to 400 g. -1 The mixture was centrifuged for 20 min; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0064] Example 10

[0065] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in Example 1, except that in step (4), the colloidal suspension is respectively heated to 9000 g -1 The mixture was centrifuged for 20 min; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0066] Embodiment 11

[0067] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in embodiment 1, except that in step (4), the colloidal suspension is heated to 13000 g. -1 The mixture was centrifuged for 20 min; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0068] Example 12

[0069] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in Example 1, except that in step (4), the colloidal suspension is heated to 11000 g. -1 The mixture was centrifuged for 5 min; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0070] Embodiment 13

[0071] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in Example 1, except that in step (4), the colloidal suspension is heated to 11000 g. -1 The mixture was centrifuged for 10 min at 4 °C; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0072] Embodiment 14

[0073] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in Example 1, except that in step (4), the colloidal suspension is heated to 11000 g. -1 The mixture was centrifuged for 15 min at 4 °C; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0074] Embodiment 15

[0075] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in Example 1, except that in step (4), the colloidal suspension is heated to 11000 g. -1The mixture was centrifuged for 25 min at 4 °C; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0076] Example 16

[0077] The preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel in this embodiment is basically the same as that in Example 1, except that in step (4), the colloidal suspension is heated to 11000 g. -1 The mixture was centrifuged for 30 min at room temperature; finally, a composite colloidal gel of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres was obtained.

[0078] Embodiment 17

[0079] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that the mass concentration of the colloidal particles in the colloidal suspension in step (4) is 15%; and finally the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0080] Embodiment 18

[0081] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that the mass concentration of the colloidal particles in the colloidal suspension in step (4) is 25%; and finally, the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0082] Embodiment 19

[0083] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that the mass concentration of the colloidal particles in the colloidal suspension in step (4) is 30%; and finally the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0084] Embodiment 20

[0085] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that the mixing ratio of the two-phase particles in step (4) is the mass ratio of the two-phase particles: β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere = 0.2; and finally, a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0086] Embodiment 21

[0087] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that the mixing ratio of the two-phase particles in step (4) is the mass ratio of the two-phase particles: β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere = 5; and finally, a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0088] Embodiment 22

[0089] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that in step (4), the adamantane gelatin nanospheres and β-cyclodextrin gelatin nanospheres prepared in Example 1 are respectively dispersed in a 150 mM NaCl salt solution to prepare a colloidal particle suspension having a colloidal particle mass concentration of 20%; and finally, a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0090] Embodiment 23

[0091] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that in step (4), the adamantane gelatin nanospheres and β-cyclodextrin gelatin nanospheres prepared in Example 1 are respectively dispersed in a Tween20 aqueous solution with a volume concentration of 1% to prepare a colloidal particle suspension with a colloidal particle mass concentration of 20%; and finally, a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0092] Embodiment 24

[0093] In this embodiment, the preparation method of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is basically the same as that in Example 1, except that in step (4), the adamantane gelatin nanospheres and β-cyclodextrin gelatin nanospheres prepared in Example 1 are respectively dispersed in a culture medium containing 10% fetal bovine serum to prepare a colloidal particle suspension with a colloidal particle mass concentration of 20%; finally, a β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel is obtained.

[0094] Example 25: Self-healing behavior of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel prepared in Example 1

[0095] The self-healing behavior of the composite colloidal gel obtained in the above Example 1 was tested by a rheometer. The specific test method is as follows: first, an oscillation time scan is performed, and an external force with a frequency of 1 Hz and a strain of 0.5% is applied to the sample, and the elastic modulus (G') and viscous modulus (G") of the sample are tested. The G' value at this stage is the initial elastic modulus of the sample. Then, the applied strain is gradually increased from 0.1% to 1000%. In this process, the external force applied destroys the sample, and the elastic modulus (G') gradually decreases, and finally falls below the viscous modulus (G"), that is, the colloidal system transforms from a rigid solid to a viscous fluid, and the gel structure is destroyed. Subsequently, the external force is removed, and the recovery of the elastic modulus (G') of the sample is examined, and the percentage of the elastic modulus of the sample after recovery to its initial elastic modulus is defined as the self-healing efficiency of the gel. The test results are as follows: Figure 4 shown.

[0096] Depend on Figure 4 The results show that the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel has excellent self-healing ability. The elastic modulus of the gel recovers quickly after shear damage, and the elastic modulus after repair is higher than the initial elastic modulus, proving that the self-healing efficiency of the gel is even higher than 100%. Moreover, such self-healing behavior can occur repeatedly, and its elastic modulus can be quickly restored after multiple shear cycles are applied to the sample.

[0097] Under low shear force conditions, the elastic modulus (G') of the composite colloidal gel is higher than the viscous modulus (G") and remains stable, that is, the gel exhibits the rigid behavior of a solid. Then, as the shear force is gradually increased (strain increases from 0.1% to 1000%), the elastic modulus (G') gradually decreases and eventually falls below the viscous modulus (G"), the network structure of the surface gel is destroyed, and it transforms from a solid to a viscous fluid. After the destructive shear force on the gel is removed, the elastic modulus of the gel quickly recovers, even slightly higher than the initial elastic modulus, and this self-healing behavior is repeatable.

[0098] Embodiment 26

[0099] The self-healing behavior of the β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel obtained in Examples 9 to 24 was tested by a rheometer using the test method of Example 25. The specific test results are as follows:

[0100] (1) Self-healing behavior of composite colloidal gels prepared under different relative centrifugal forces

[0101] The self-healing behaviors of the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gels prepared in Examples 9 to 11 and Example 1 under different relative centrifugal forces are shown in FIG. Figure 5 shown.

[0102] Depend on Figure 5The results show that the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gels have excellent self-healing capabilities under different relative centrifugal forces, and the self-healing efficiency can reach or even exceed 100%. Under lower relative centrifugal forces, the subject (β-cyclodextrin) and the guest (adamantane) cannot fully contact and react, and cannot form a uniformly dispersed network structure, resulting in a lower elastic modulus of the gel.

[0103] (2) Self-healing behavior of composite colloidal gels prepared at different centrifugation times

[0104] The self-healing behaviors of the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gels prepared in Examples 12 to 16 and Example 1 at different relative centrifugation times are shown in FIG. Figure 6 shown.

[0105] Depend on Figure 6 The results show that with the increase of centrifugation time, the elastic modulus and viscous modulus of the composite colloidal gel increase until 20 minutes later, when the elastic modulus no longer increases with the increase of centrifugation time. Under a shorter centrifugation time, the host (β-cyclodextrin) and the guest (adamantane) cannot fully contact and react, and cannot form a uniformly dispersed network structure, resulting in a lower elastic modulus of the gel. After a certain time, the host (β-cyclodextrin) and the guest (adamantane) fully contact and react, and further increasing the centrifugation time thereafter cannot increase the elastic modulus of the composite colloidal gel.

[0106] (3) Self-healing behavior of composite colloidal gels prepared at different colloidal suspension concentrations

[0107] The self-healing behaviors of the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gels prepared in Examples 17 to 19 and Example 1 at different colloidal suspension concentrations are shown in FIG. Figure 7 shown.

[0108] Depend on Figure 7 The results show that the elastic modulus and viscous modulus of the composite colloidal gel obtained after centrifugation are basically the same when the mass concentration of the colloidal suspension is different. Under sufficient relative centrifugal force and centrifugation time, the host (β-cyclodextrin) and the guest (adamantane) fully contact and react to form a uniform network structure, and different mass concentrations of the colloidal suspension have no effect on the final composite colloidal gel.

[0109] (4) Self-healing behavior of composite colloidal gels prepared at different two-phase particle mass ratios

[0110] The self-healing behaviors of the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gels prepared in Examples 20-21 and Example 1 at different two-phase particle mass ratios are shown in FIG. Figure 8 shown.

[0111] Depend on Figure 8 The results show that when the mass ratio of adamantane gelatin nanospheres to β-cyclodextrin gelatin nanospheres is greater than 1, the elastic modulus (G') of the composite colloidal gel is low and the self-healing efficiency is also low. When the mass ratio is less than or equal to 1, the elastic modulus (G') and self-healing efficiency are both high. When the mass ratio of adamantane gelatin nanospheres to β-cyclodextrin gelatin nanospheres is relatively large, the number of the host (β-cyclodextrin) is less than the guest (adamantane), and sufficient connection cannot be made, resulting in an imperfect colloidal network structure. When the mass of adamantane gelatin nanospheres to β-cyclodextrin gelatin nanospheres reaches 1, the number of the host (β-cyclodextrin) is similar to that of the guest (adamantane) or there is a surplus of guests, and sufficient connection can be made to form a uniformly dispersed colloidal gel network.

[0112] (5) Self-healing behavior of composite colloidal gels prepared under different environments

[0113] The self-healing ability of the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared under different conditions in Examples 22 to 24 is as follows: Figures 9-11 As shown in Figure 2, the self-repair efficiency of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel in the self-repair cycle after multiple rounds of shear damage under different environments is shown in Figure 2. Figures 9-11 shown.

[0114] Depend on Figures 9-11 The results show that the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel has excellent self-healing ability in physiological salt environment, surfactant environment and serum environment, and the elastic modulus of the composite colloidal gel recovers quickly after shear damage.

[0115] As shown in Table 3, the self-healing efficiency of the composite gel in a physiological salt environment can still reach 90%, the self-healing efficiency in a surfactant environment can reach or even exceed 100%, and the self-healing efficiency in a serum environment can reach or even exceed 95%, and such self-healing behavior can occur repeatedly, and its elastic modulus can be quickly restored after applying multiple shear cycles to the sample. This proves that the composite colloidal gel of the present invention has good self-healing ability and high mechanical strength in a physiological salt environment, a surfactant environment, and a serum environment.

[0116] Table 3. Self-repair efficiency of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel in self-repair cycles after multiple rounds of shear damage under different environments

[0117]

[0118] Example 27: Injectability of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel

[0119] The β-cyclodextrin gelatin nanosphere / adamantane gelatin nanosphere composite colloidal gel prepared in Example 24 was injected using a common medical needle. The injection effect was as follows: Fig.12 shown.

[0120] Depend on Fig.12 The results show that the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared by the preparation method of the present invention proves that the material has good injectability and can be formed by extrusion.

[0121] Example 28: Biocompatibility of β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel.

[0122] The gelatin nanospheres, adamantane gelatin nanospheres, β-cyclodextrin gelatin nanospheres and β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel prepared in Example 1 were dispersed in deionized water to obtain uniformly dispersed suspensions.

[0123] The biocompatibility of the composite colloidal gel was investigated by in vitro cell experiments, and the specific implementation steps are as follows:

[0124] (I) Cell culture

[0125] Taking HUVEC cell culture as an example, in a proliferation medium (DMEM, containing 10% fetal bovine serum (FBS, Gibco)), at 37°C, 95% relative humidity and 5% CO2. The cell culture medium was replaced every three days. Before use, the cells were rinsed with phosphate buffered saline (PBS), detached using trypsin / EDTA solution (0.25% trypsin / 0.02% EDTA) for 1 minute, and suspended in the culture medium for use.

[0126] (Ⅱ) CCK8 method to determine the cytotoxicity of materials

[0127] The cell suspension prepared in step (I) was inoculated into a 96-well plate at a cell concentration of 5000 cells / well. After static culture for 24 hours, gelatin nanospheres, adamantane gelatin nanospheres, β-cyclodextrin gelatin nanospheres and β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres suspensions were added into the 96-well plate at different concentrations. A blank control group was set up at the same time. After 24 hours, the culture medium was replaced and CCK8 solution was added for co-culture. After 4 hours, the absorbance at a wavelength of 450 nm was measured by an enzyme reader to obtain the cytotoxicity of different materials. The test results are shown in FIG. Fig.13 shown.

[0128] Depend on Fig.13The results show that gelatin nanospheres have slight cytotoxicity at high concentrations, while adamantane gelatin nanospheres have reduced cytotoxicity, β-cyclodextrin gelatin nanospheres are completely non-cytotoxic, and the composite colloidal gel of the two is also completely non-cytotoxic.

[0129] (III) Live / dead fluorescence staining (LIVE / DEAD assay) to determine the biocompatibility of materials

[0130] The cell suspension prepared in step Ⅰ was inoculated into a 6-well plate at a cell concentration of 4*10^5 cells / well. After static culture for 24 hours, gelatin nanospheres, adamantane gelatin nanospheres, β-cyclodextrin gelatin nanospheres, and β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel suspension were added at a concentration of 35 mg / mL and co-cultured with the cells for 24 hours. Wash three times with sterile PBS before staining, add 2mM calcein (green fluorescence to mark live cells) and 3mM ethidium homodimer (red fluorescence to mark dead cells) at room temperature, and observe with a fluorescence microscope. The detection results are as follows: Fig.14 shown.

[0131] Depend on Fig.14 The results show that the β-cyclodextrin gelatin nanospheres / adamantane gelatin nanospheres composite colloidal gel has good biocompatibility and no cytotoxicity.

[0132] The technical solution of the present invention is described in detail above through specific embodiments. However, it is worth noting that based on the inventive concept of the present application, various equivalent changes, modifications or substitutions are obvious to those skilled in the art and should be included in the protection scope of the present application.

Claims

1. A method for preparing a composite colloidal gel based on host-guest interaction, characterized in that: The following steps are involved: S1: preparing a gelatin nanosphere suspension with carboxyl groups on the surface, then adding an amino-containing β-cyclodextrin derivative to the gelatin nanosphere suspension, mixing and stirring evenly, then adding a cross-linking agent and stirring and reacting at room temperature, and obtaining a β-cyclodextrin gelatin nanosphere powder after the reaction product is purified by dialysis and freeze-dried; S2: preparing a gelatin nanosphere suspension with carboxyl groups on the surface, then adding an amino-containing adamantane derivative to the gelatin nanosphere suspension, mixing and stirring evenly, adding a cross-linking agent and stirring at room temperature to react, and dialysis purification and freeze-drying the reaction product to obtain adamantane gelatin nanosphere powder; S3: weighing appropriate amounts of β-cyclodextrin gelatin nanosphere powder and adamantane gelatin nanosphere powder, and dissolving them in a solvent to form a β-cyclodextrin gelatin nanosphere suspension and adamantane gelatin nanosphere suspension, respectively; S4: mixing the β-cyclodextrin gelatin nanosphere suspension and the adamantane gelatin nanosphere suspension in an appropriate proportion to obtain a uniformly dispersed colloidal suspension; centrifuging the colloidal suspension and removing the supernatant to obtain a composite colloidal gel.

2. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In steps S1 and S2, the concentration of the gelatin nanosphere suspension is 5 mg / mL to 20 mg / mL, and the pH is 4 to 7; the content of carboxyl groups in the gelatin nanospheres is 0.06 mmol / g to 0.10 mmol / g.

3. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In steps S1 and S2, the amino-containing β-cyclodextrin derivative is ethylenediamine β-cyclodextrin, peramino β-cyclodextrin or mono-6-O-amino-β-cyclodextrin; the amino-containing adamantane derivative is adamantane hydrochloride, adamantane methylamine hydrochloride, adamantane ethylamine hydrochloride or adamantane amidine hydrochloride.

4. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In step S1, the molar ratio of the amino-containing β-cyclodextrin derivative to the carboxyl groups contained in the gelatin nanospheres is 0.5:1 to 10:1; and the molar ratio of the cross-linking agent to the carboxyl groups contained in the gelatin nanospheres is 1:1 to 10:

1.

5. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In step S2, the molar ratio of the amino-containing adamantane derivative to the carboxyl groups contained in the gelatin nanospheres is 0.5:1 to 10:1; and the molar ratio of the cross-linking agent to the carboxyl groups contained in the gelatin nanospheres is 1:1 to 10:

1.

6. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In steps S1 and S2, the cross-linking agent is selected from one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, carbodiimide and N-hydroxysuccinimide.

7. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In step S3, the solvent is deionized water, an aqueous solution containing 150 mM sodium chloride, a Tween 20 aqueous solution with a volume concentration of 1%, or a culture medium containing 10% fetal bovine serum.

8. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In step S4, the mass concentration of the colloidal suspension is 5% to 30%; the mass ratio of the adamantane gelatin nanospheres to the β-cyclodextrin gelatin nanospheres in the colloidal suspension is 0.2 to 5.

9. The method for preparing a composite colloidal gel based on host-guest interaction according to claim 1, characterized in that: In step S4, the relative centrifugal force of the colloidal suspension during centrifugation is 5000-15000g. -1 , the centrifugation time is 5 to 30 minutes.

10. A composite colloidal gel based on host-guest interaction, characterized in that: The composite colloidal gel is prepared by the preparation method according to any one of claims 1 to 9.

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