CN114605676B

CN114605676B - Degeneration nucleus pulposus repair injectable hydrogel and application thereof

Info

Publication number: CN114605676B
Application number: CN202210447092.2A
Authority: CN
Inventors: 王玉; 王云兵; 孔清泉
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-01-25
Filing date: 2022-04-26
Publication date: 2023-06-09
Anticipated expiration: 2042-04-26
Also published as: CN114605676A

Abstract

The hydrogel precursor containing 8-12 parts by weight of phenylboronic acid and cyclodextrin grafted gelatin, 1 part by weight of tannic acid and 80-120 parts by weight of water, and the hydrogel formed by solidifying the hydrogel precursor have self-healing and injectability, have the effects of inhibiting inflammation and promoting nucleus repair when being injected into the degenerated nucleus, further compound curcumin-carrying micelle and cholesterol modified miRNA-21 inhibitor, can release anti-inflammatory drug curcumin and miRNA-21 inhibitor for promoting ECM regeneration according to different inflammation degrees in the nucleus according to local conditions, and promote the regeneration of the degenerated nucleus under double-tube condition, and have good application prospect.

Description

Degeneration nucleus pulposus repair injectable hydrogel and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to an injectable hydrogel for repairing a degenerated nucleus pulposus and application thereof.

Background

With the increasing aging of society, the incidence of degenerative diseases of the spine (such as lumbar disc herniation, lumbar spinal stenosis and the like) is continuously increasing, and lumbocrural pain has become one of the pain diseases with the highest incidence. Global low back and leg pain prevalence is reported to be as high as 12%; a WHO survey showed that 37% of adolescents developed lumbago at least once a month. Degeneration of the spine is a degeneration (degeneration, dehydration) of the intervertebral disc, which consists of an inner nucleus pulposus and surrounding annulus fibrosus. With the progressive progression of degenerative diseases of the spine, conventional conservative treatment only partially relieves clinical symptoms and most patients have to finally receive surgical treatment. Although the current operation modes aiming at the spinal degeneration diseases are numerous and continuously approach to minimally invasive, the problems of operation injury, postoperative recurrence, adjacent segment degeneration acceleration, inaccurate long-term curative effect and the like cannot be overcome in any operation mode, and the problems of heavy economic and social burdens are definitely brought to individuals and society. For patients in the early stage of disc degeneration, especially those who only show disc-derived lumbago, if the progress of disc degeneration can be delayed or even reversed in some way, the occurrence of spinal degeneration diseases can be reduced from the source. With the rapid development of bioengineering technology, it has become possible to regenerate and repair the nucleus pulposus of an intervertebral disc through nucleus pulposus tissue engineering technology.

In recent years, it has become possible to regenerate degenerated nucleus pulposus tissues by constructing nucleus pulposus replacement bioengineering scaffold materials which can be used as transport carriers for cells and biomacromolecules (drugs, cytokines, miRNAs, etc.), in combination with biomedical engineering techniques typified by regenerative medicine. Nucleus pulposus growthThe objective of biomedical engineering strategies is to inhibit internal inflammatory reactions, promote regeneration of nucleus pulposus cells, restore ECM synthesis/catabolism balance, ultimately truly restoring disc physiological function and meeting biomechanical requirements. During this process, the selection of nucleus replacement scaffold materials is central. The ideal nucleus replacement material should have good biological safety, biocompatibility, biomechanical characteristics similar to those of the natural nucleus, degradation performance matched with the autologous biological speed, and finally realize automatic biology, more importantly, the nucleus replacement material can be implanted into the intervertebral disc in a minimally invasive mode: injectability. Researchers always want to develop ideal nucleus replacement scaffold materials through research in aspects of material screening, modification and the like, but a large number of research results show that due to complex tissue structure in intervertebral discs, poor blood supply and complex mechanical environment, the existing materials such as natural materials: acellular matrix, alginate, polypeptide hydrogel, and synthetic materials: hyaluronic acid, PLGA and the like can not well meet the construction requirements of in vitro functionalized nucleus pulposus. Internationally, a large number of scientists have passed through a number of hyaluronic acid sponges including fibrin gel (Gelfoam)

HYAFF) and polypeptide hydrogel->

And the like, can carry a nucleus pulposus replacing bracket material product for cell growth for preclinical research, but at present, no mature functional nucleus pulposus replacing product is applied to clinic.

In recent years, drug delivery systems such as microspheres, nano-micelles, exosomes, acellular matrixes and the like are continuously tried to be used as scaffold materials for replacing the nucleus pulposus, but all the requirements cannot be met all the time due to the limitations of lack of mechanical properties, complex preparation process, expensive raw materials and the like. Hydrogel materials continue to receive attention in recent years and are widely used in a variety of tissue repair fields. The injectable hydrogel has inherent permeability, water absorbability, degradability, good biocompatibility, huge drug-carrying controlled release capability and mechanical supporting potential, and huge application potential in the selection of nucleus pulposus replacing stent materials.

On the one hand, disc degeneration is a series of complex sequential processes including inflammation mediation, ECM synthesis/catabolism disorder, fibrosis and the like, and hydrogel is simply implanted into the degenerated disc as a cell or a drug carrier to be far from adapting to the complex microenvironment, so that an intelligent response type hydrogel drug delivery system capable of participating in the degenerated disc repair process in the whole course and changing the physicochemical properties according to different microenvironment changes needs to be constructed, namely, the 'inflammation response type hydrogel drug delivery system' with two corresponding pH and ROS can rapidly sense and respond to the change and stimulation of the inflammation microenvironment, and the drug is accurately and controllably released to a disease part.

MicroRNA (miRNA), on the other hand, is a class of small non-coding RNAs consisting of 18-24 nucleotide sequences, which are widely found in eukaryotes and are involved in gene regulation. Mature mirnas direct RISC complex (mirsc) targeting mrnas with partially complementary sequences in the 3' -UTR region in the cytoplasm, leading to transcriptional silencing or mRNA degradation, which in turn regulate a variety of physiological activities of the cell. Imbalance in miRNA regulation plays an important role in the occurrence of disc degeneration. Numerous studies have shown that abnormal expression of various miRNAs can lead to inflammation of cells in the nucleus pulposus, degradation of ECM, apoptosis of nucleus pulposus, etc., and accelerate degeneration of the nucleus pulposus of the intervertebral disc through different mechanisms of action. Therefore, the method searches and explores the action targets and rules of the miRNAs, regulates the functions of nucleus pulposus cells by a miRNAs gene therapy method, and has great application prospect in the degeneration of nucleus pulposus.

miRNAs gene therapy can be classified into miRNA replacement therapy in the absence of miRNAs and miRNA inhibition therapy in the presence of overexpression according to the expression of the miRNAs themselves. However, whichever approach, how to accurately and stably transport exogenous mirnas or miRNA inhibitors to the target region is critical for the success or failure of miRNA gene therapy to degenerate the nucleus pulposus. Based on the special structural characteristics of no blood vessels in the intervertebral disc and various limitations of the systemic delivery of miRNAs (such as abnormal aggregation outside target organs, local low bioactivity of the target organs, multiple drug administration and the like), the method for systemic delivery of the miRNAs is not suitable for the gene therapy of the miRNAs of the nucleus pulposus. However, due to the special closed structure of the nucleus pulposus of the intervertebral disc, simple local injection of miRNAs can cause a series of problems such as overhigh local concentration, insufficient transfection efficiency, too fast clearance rate and the like. Therefore, a transport carrier suitable for locally delivering miRNAs in the disc of the intervertebral disc is searched, and an intelligent miRNAs drug delivery system is constructed by combining with the inflammatory response type hydrogel, so that the method has great scientific significance in degeneration nucleus pulposus miRNAs gene therapy.

Disclosure of Invention

The invention aims to provide an injectable hydrogel precursor capable of preparing a degenerated nucleus pulposus repair hydrogel and the degenerated nucleus pulposus repair hydrogel obtained by curing the hydrogel precursor.

The invention provides a hydrogel precursor, which comprises the following components in parts by weight:

8-12 parts of phenylboronic acid and cyclodextrin grafted gelatin, 1 part of tannic acid and 80-120 parts of water.

Further, the composite material comprises the following components in parts by weight:

10 parts of phenylboronic acid and cyclodextrin grafted gelatin, 1 part of tannic acid and 100 parts of water.

Further, the composition also comprises the following components in parts by weight:

cholesterol modified miRNA inhibitor 0.003-0.004 weight portions and/or nano micelle coated with curcumin 0.006-0.008 weight portions.

Preferably, the cholesterol-modified miRNA inhibitor is 0.0033 parts and/or curcumin-entrapped nano-micelle is 0.0066 parts.

Further, in the gelatin grafted with phenylboronic acid and cyclodextrin, the grafting ratio of phenylboronic acid is 17.+ -. 2.4%, and the grafting ratio of cyclodextrin is 3.13.+ -. 0.87%.

Further, the phenylboronic acid and cyclodextrin grafted gelatin is prepared by the following method:

(1) Reacting gelatin with 3-carboxyphenylboronic acid under the action of an activating agent and a condensing agent to obtain phenylboronic acid grafted gelatin;

(2) Reacting phenylboronic acid grafted gelatin with aminated beta-cyclodextrin under the action of an activating agent and a condensing agent to obtain phenylboronic acid and cyclodextrin grafted gelatin.

Preferably, the weight ratio of the gelatin and the 3-carboxyphenylboronic acid in the step (1) is 1 (0.1-0.5), and preferably 1:0.4;

and/or the weight ratio of phenylboronic acid grafted gelatin to aminated beta-cyclodextrin in step (2) is 1 (0.1-0.5), preferably 1:0.4.

Further, the activator is NHS and the condensing agent is EDC.

Further, the cholesterol-modified miRNA inhibitor is a cholesterol-modified miRNA-21 inhibitor.

Further, in the nano micelle coated with curcumin, the content of the curcumin is 10-20%.

Furthermore, the nano micelle is formed by self-assembly of polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer.

Further, the polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer is prepared by reacting polyethylene glycol and polylactic acid-glycolic acid copolymer with ketal; the ketal has the structure that:

further, the molecular weight of the polyethylene glycol is 2000, and the molecular weight of the polylactic acid-glycolic acid copolymer is 1000.

Further, the nano micelle coated with curcumin is formed by self-assembly of the following raw materials in parts by weight:

8-12 parts of polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer and 1 part of curcumin;

preferably 10 parts of polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer and 1 part of curcumin.

The invention also provides a hydrogel which is formed by curing reaction of the hydrogel precursor.

Further, the curing reaction conditions are to be allowed to stand at 20 to 30 ℃.

The invention also provides application of the hydrogel in the degeneration nucleus pulposus repair material.

Further, the degenerated nucleus repair material may be a nucleus replacement material and/or a nucleus regeneration promoting material.

The invention has the beneficial effects that: firstly, grafting phenylboronic acid BA and cyclodextrin CD to gelatin Gel side chains, self-assembling into hydrogel through the cross-linking effect of BA and tannic acid molecules (TA), and simultaneously wrapping curcumin-coated micelle MIC@Cur and cholesterol-modified miRNA inhibitor. The hydrogel can be implanted into the interior of a degenerated intervertebral disc in an injection mode, and under the stimulation of internal pH and ROS, the hydrogel is cracked, and MIC@Cur, MIC@Cur is released, and curcumin Cur is released by continuous cracking in the presence of ROS, so that the anti-inflammatory effect is exerted. Meanwhile, miRNA inhibitor wrapped by CD molecule through hydrophilic and hydrophobic effects can be slowly released and transferred into nucleus pulposus cells, so that the effect of promoting the regeneration of the nucleus pulposus cells ECM is continuously exerted.

According to the invention, the injectable hydrogel is implanted into the degenerated nucleus pulposus, so that the effects of inhibiting inflammation and promoting nucleus pulposus repair are achieved, and the curcumin-21 inhibitor modified by curcumin and cholesterol is further compounded, so that the curcumin serving as an anti-inflammatory drug and the miRNA-21 inhibitor (Antagomir) for promoting ECM regeneration can be released according to different inflammation degrees in the nucleus pulposus according to local conditions, and the degeneration nucleus pulposus regeneration is promoted under double-tube condition. Provides a new idea for repairing the degenerated nucleus pulposus, and finally avoids the risks and huge economic cost brought by the operation.

The hydrogel precursor is a composition which takes raw materials for preparing the hydrogel as components, and the components can be packaged independently and respectively or mixed together. When the components are mixed together, the injectable performance is achieved particularly in the form of a liquid composition which has not yet cured to form a solid hydrogel.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows the standard curves for a) 3-carboxyphenylboronic acid and b) aminated β -cyclodextrin.

Fig. 2 is a standard curve of curcumin.

FIG. 3 shows physicochemical properties of Gel-BA-CD polymer and MIC@Cur; a is Gel-BA-CD and hydrogel preparation molecular diagram. b is the preparation process of amphiphilic molecule mPEG-TK-PLGA with ROS response. c, d is the NMR scan result. e is the FTIR scan result. f is the UV absorption peak result. g is the particle size and potential results of the MIC@Cur and MIC (unloaded MIC) prepared. h is the result of particle size analysis. i is the UV analysis result of MIC@Cur. J is the MIC@Cur Transmission Electron Microscope (TEM) scanning result.

FIG. 4 shows the results of physical and chemical properties and microstructure characterization of Gel-BA-CD/TA hydrogels. a is a schematic drawing of gel formation of the hydrogel. b is Gel-BA-CD solution before TA addition and hydrogel after crosslinking. c is the hydrogel internal structure scanned by SEM. d is the injectability of the hydrogels of the invention. e is the self-healing properties of the hydrogels of the present invention. F-h is the rheological result of the hydrogel.

FIG. 5 is a graph showing the results of in vitro degradation of two drug loaded hydrogels and characterization of pH/ROS response performance; a is the in vitro degradation result of the hydrogel. b is DPPH antioxidation experiment of different hydrogels. c is the cleavage of the hydrogel under different circumstances. d is a schematic diagram of MIC@Cur construction. e Cur release curves under different circumstances. f is rhodamine fluorescence quenching experimental result. Schematic of the sustained release of g Antagomir. h Cy3 fluorescence labelled Antagomir release profile. i, j is in vivo fluorescence imaging of the rat.

FIG. 6 is an evaluation of the ECM regeneration effect of hydrogel nucleus pulposus cells. a is the proliferation of nucleus pulposus cells after TBHP stimulation cultured by different hydrogel leaching solutions. B is the result of differential expression of the gene of nucleus pulposus Col II, aggrecan and MMP-13 after TBHP stimulation by culturing different hydrogel leaching solutions. c, d is the immunofluorescence result of the MMP-13 protein expression of nucleus pulposus cells Col II after TBHP stimulation by culturing different hydrogel leaching solutions.

FIG. 7 shows hydrogel-enhanced macrophage polarization assay results. a-c are the rear-view observations of different groups of treated macrophages. d is the RT-PCR detection result after the macrophages are stimulated by different groups. E is the expression of the M2-specific protein CD206 after the stimulation of macrophages in different groups. f is the cell flow result. g is the result of the intracellular ROS scavenging assay.

FIG. 8 shows the measurement results of the anti-inflammatory effect of hydrogels. a is the expression of macrophage RAW264.7, which is incubated with different sets of hydrogel extracts, RT-PCR anti-inflammatory factor CD206, IL-10 and TNF-alpha. B is the expression condition of IL-10 and TNF-alpha detected by Western-blot. c, d is immunofluorescence detection of IL-10 and TNF-alpha expression. e is a cell migration Transwell experiment. f is a schematic diagram of Cur promoting macrophage polarization.

FIG. 9 shows the in vivo identification of the therapeutic effect of hydrogels on rat disc degeneration. a is to build a rat intervertebral disc degeneration model, implant different hydrogels and observe the degeneration nucleus pulposus repairing effect in 4 and 8 weeks. b, d and e are X-ray scanning results after operation for 4,8 weeks. c, f is the post-operative 4,8 week MRI scan result.

FIG. 10 shows the results of histological examination of the effect of hydrogels on rat disc degeneration treatment. a, b, c are HE staining, safranin O-fast green staining, and Col II immunohistochemical staining results. d, e, f are quantitative observations of histological grading results.

Detailed Description

The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.

Example 1 preparation of hydrogels with pH/ROS double response according to the present invention

1. Synthesis of phenylboronic acid grafted gelatin (Gel-BA)

5g of gelatin was weighed and dissolved in 500ml MES buffer. EDC (4.8 g,25 mmol), NHS (1.15 g,10 mmol/L) and 3-carboxyphenylboronic acid (2 g,12.5 mmol/L) were weighed and dissolved in gelatin solution and stirred well for 48h at 37 ℃. After filtering the impurities, adding a dialysis bag for dialysis for 3d, and changing water for 3-6 times per day. Freeze-drying after dialysis is completed. FIG. 1A shows a schematic view of the above

2. Synthesis of phenylboronic acid and Cyclodextrin grafted gelatin (Gel-BA-CD)

Weighing 5g of Gel-BA powder, fully dissolving, weighing EDC, NHS and 2g of amination beta-cyclodextrin molecules which are equal to the reaction, and synthesizing Gel-BA-CD through amide condensation reaction again, wherein the reaction time and the steps are the same as those described above.

3. Hydrogel with pH/ROS dual response

Gel-BA-CD molecules were fully dissolved in deionized water (10% w/v) and the pH was adjusted to neutral. Dissolving Tannic Acid (TA) A solution of TA was prepared at a mass concentration of 10% w/v. The Gel-BA-CD solution and 1/10 volume of TA solution were thoroughly stirred to immediately prepare a blank Hydrogel Gel-BA-CD/TA (designated as Control Hydrogel or Hydrogel).

EXAMPLE 2 preparation of the inventive Carrier hydrogel with pH/ROS double response

1. Phenylboronic acid and cyclodextrin grafted gelatin were prepared according to the procedure of

steps

1, 2 of example 1.

2. Preparation of curcumin (Cur) -loaded micelles (MIC@Cur)

20mg of amphiphilic molecule polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer (mPEG-TK-PLGA) and 2mg of Cur are weighed and dissolved in 1mL of DMSO solution, heated to 37 ℃, added into 10mL of deionized water dropwise while being stirred by ultrasound, and dialyzed continuously for 48 hours, and the MIC@Cur is obtained after filtration through a filter tip with the diameter of 0.22 um. FIG. 5d is a schematic diagram of MIC@Cur construction.

The source of mPEG-TK-PLGA is prepared by self, and the mPEG-TK-PLGA is prepared by reacting PEG with molecular weight of 2000 with PLGA with molecular weight of 1000 and TK: TK molecule, dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) according to 1:6:0.6 in 15ml of Dimethylsulfoxide (DMSO). After dissolving 463mg of PLGA molecules (1 kDA) in 4ml of DMSO, stirring thoroughly under nitrogen for 24h, adding 618mg of mPEG (2 kDA) molecules, and continuing the reaction for 24h. And (5) dialyzing and freeze-drying for later use. The reaction formula is shown in FIG. 1b.

3. Preparation of a Carrier hydrogel with pH/ROS Dual response

Gel-BA-CD molecules were fully dissolved in deionized water (10% w/v), pH was adjusted to neutral, and 66. Mu.g MIC@Cur and 33. Mu.g Antagomir (available from GenePharma Corp. (Shanghai, china.) molecular weight: 7998.268. Base sequence: 5 '-UAGCUUUUUUUAUGACUGAUGUUGA-chol-3') were added. Dissolving Tannic Acid (TA) A solution of TA was prepared at a mass concentration of 10% w/v. Gel-BA-CD solution and 1/10 volume of TA solution are fully stirred to prepare the carrier Hydrogel (named as Hydrogel & MIC@Ant).

EXAMPLE 3 preparation of the inventive Carrier hydrogel with pH/ROS double response

Referring to the preparation method of example 2, the preparation process of step (3) does not add Antagomir, and the hydrogel of hydro gel@MIC only encapsulating curcumin is prepared.

EXAMPLE 4 preparation of the inventive Carrier hydrogel with pH/ROS double response

Referring to the preparation method of example 2, MIC@Cur is not added in the preparation process of the step (3), and the hydrogel of Hydrogel@Ant which only encapsulates Antagomir is prepared.

The following experiments prove the beneficial effects of the invention.

Experimental example 1, grafting ratio of carboxyphenylboronic acid and cyclodextrin of the invention and MIC@Cur drug-loading rate

1. The method for calculating the grafting rate of carboxyphenylboronic acid and cyclodextrin comprises the following steps:

ultraviolet spectrum detection of 275nm ultraviolet absorption peak height under different 3-carboxyphenylboronic acid concentration and drawing of standard curve (as shown in figure 1 a), experiment shows that Gel-BA solution prepared in example 1 (concentration 625 ug/ml) has ultraviolet absorption peak of 0.83 at 275nm, BA content is about 110.1ug/ml calculated by standard curve, and grafting ratio is about 110.1/625=17.6%.

Ultraviolet spectrum detection of 490nm ultraviolet absorbance peak height at different concentrations of aminated beta-cyclodextrin and drawing of standard curve (as in fig. 1 b), after cleavage of Gel-BA-CD solution (20 mg/ml) prepared in example 1 by phenol-sulfuric acid method, the ultraviolet absorbance peak at 490nm was measured to be 1.57, CD content was calculated by standard curve to be about 626.2ug/ml, and thus grafting ratio was about 626.2/20000=3.1%.

2. MIC@Cur drug-loading rate

Ultraviolet spectrum detection of different curcumin dissolved in DMSO/H ₂ The mic@cur solution prepared in example 2 (concentration 146 ug/ml) has an ultraviolet absorption peak at 430nm of 1.427 as determined by experiments by plotting the height of the ultraviolet absorption peak at 430nm at the solution concentration of O (1:1) and plotting a standard curve (see fig. 2), the Cur content is about 22.10ug/ml as calculated by the standard curve, and the grafting ratio is about 22.10/146=15.13%.

Experimental example 2, gel-BA-CD and physicochemical characterization of MIC@Cur

1. The experimental method comprises the following steps: the physicochemical properties of the Gel-BA-CD polymer prepared in example 1 and the MIC@Cur prepared in example 2 of the present invention were subjected to a series of characterization by means of NMR, FTIR, UV, DLS, TEM and the like.

2. Experimental results:

the results are shown in FIG. 3.

FIG. 3 shows physicochemical properties of Gel-BA-CD polymer and MIC@Cur; from the Gel-BA-CD and hydrogel preparation molecular diagram in FIG. 1a, it is reflected that hydrogel is formed by cross-linking Gel-BA-CD molecule and TA molecule, the formed boron ester bond is broken under the condition of pH and ROS existence, and Antagomir is wrapped in CD by self-assembly action with host and guest of CD, and is released slowly.

FIGS. 3c,1d are NMR scans showing that 3-carboxyphenylboronic acid and CD molecules have been successfully grafted to gelatin molecule side chains by an amide reaction; and have successfully prepared mPEG-TK-PLGA amphiphilic molecules.

FIG. 3e shows the result of FTIR scanning, which indicates that the synthesized Gel-BA-CD molecule has a remarkable tendency at the characteristic peaks of C= O C = C B-O C-H, indicating that the synthesis of the Gel-BA-CD molecule is successful.

FIG. 3f shows the UV absorbance peak, indicating that the synthesized Gel-BA has a significant absorbance peak at 275nm, indicating that the synthesis of the Gel-BA molecule was successful.

FIG. 3g shows the particle size and potential results of MIC@Cur and MIC (empty MIC) prepared, suggesting that the particle size and potential of MIC become larger and smaller after drug loading.

Fig. 3h is a graph showing the results of particle size analysis, suggesting that the particle diameter appears uniform Shan Feng (normal distribution) in a neutral environment and multimodal (i.e., size maldistribution) in ROS environment.

FIG. 3i is a UV analysis of MIC@Cur, which suggests that MIC@Cur after drug loading exhibits the same characteristic absorption peak as Cur, suggesting that drug loading was successful.

FIG. 3J shows the MIC@Cur Transmission Electron Microscope (TEM) scan, which indicates that MIC@Cur appears as uniform circular particles in PBS (pH 7.4), and the particles lyse under ROS environment, releasing the loaded drug.

Experimental example 3, physicochemical Properties and microstructure characterization of Gel-BA-CD/TA hydrogel

1. The experimental method comprises the following steps: the internal structure of the Gel-BA-CD/TA hydrogel prepared in example 1 was observed by SEM, and self-healing property and injectability were verified for the Gel-BA-CD/TA hydrogel prepared in example 1; in addition, rheological tests were performed on Gel-BA-CD/TA hydrogels prepared in example 1 (Control Hydrogel) and on hydrogels prepared in example 2 (Hydrogel & MIC@Ant), and the rheological behavior of the two were compared.

2. Experimental results:

the results are shown in FIG. 4.

FIG. 4a is a schematic diagram showing Gel formation of hydrogel, and FIG. 4b is a Gel-BA-CD solution before TA addition, and a crosslinked hydrogel, and it can be seen that the cured hydrogel was successfully prepared in example 1 of the present invention.

Fig. 4c shows the internal structure of the hydrogel scanned by SEM, which suggests that the hydrogel is internally of a porous structure with uniform size, which is beneficial for drug entrapment.

Figure 4d shows that the hydrogel has excellent injectability through a thick and thin needle.

Fig. 4e shows that the hydrogel of the present invention also has good self-healing properties, and can self-heal to form a complete gel structure at the defect site after injection through a needle.

Fig. 4f-h show the rheological results of hydrogels, and the results indicate that the rheological properties of hydrogels added with two drugs (example 2) are similar to those of blank hydrogels (example 1), and the hydrogels have good shear thinning and self-healing properties, and the elastic modulus can reach about 1000Pa, so that the injectable properties of the hydrogels prepared in example 1 of the invention are not affected after the hydrogels are further loaded with the drugs.

Experimental example 4, extra-aqueous gel degradation and characterization of pH/ROS response Properties

1. The experimental method comprises the following steps:

(1) The blank hydrogel prepared in example 1 and the aqueous carrier gel prepared in example 2 were placed in PBS solution and subjected to degradation experiments in a constant temperature shaker at 37 ℃ while the blank hydrogel prepared in example 1 was subjected to degradation experiments at ph= 5,H ₂ O ₂ (1 mM) an accelerated degradation experiment was performed.

(2) DPPH antioxidant test was performed on the hydrogels prepared in examples 1 to 4 and PVA hydrogels as a control.

(3) The hydrogel of example 1 was taken and added with PBS (pH 7.4), a solution (pH 5) of pH=5, and H, respectively ₂ O ₂ Solution (1 mM) (H ₂ O ₂ )、H ₂ O ₂ Solution and ph=5 solution (pH 5/H ₂ O ₂ ) In the course of which cleavage under different environmental conditions is observed.

(4) The aqueous carrier gel of example 2 was taken in PBS (pH 7.4), pH=5 solution (pH 5.0), H ₂ O ₂ Solution (1 mM) (H ₂ O ₂ )、H ₂ O ₂ Solution and ph=5 solution (pH 5+h ₂ O ₂ ) In the above, curcumin sustained release test was carried out at 37 ℃.

(5) To demonstrate the assembly of Gel-BA-CD with Antagomir, a rhodamine quench assay was performed:

to different concentrations of Gel-BA-CD solution (0, 2.5,5 mg/ml) was added rhodamine B (50 mg/ml) solution (2 ml total) and UV was observed for a change in the absorption peak at 550 nm. Then, 5mg/ml Gel-BA-CD mixed solution added with rhodamine B was selected, and Antagomir-21 (0, 2,5 uM) was added at different concentrations, and the change of the absorption peak at 550nm was observed again by UV.

(6) The hydrogel of example 4 (Antagomir was fluorescently labeled with Cy3 for ease of observation) was used for the slow release test at 37℃in a shaker, and only the gelatin modified with phenylboronic acid and tannic acid formed into a gel entrapping Antagomir (Cy 3 label) as a control was used for the slow release test under the same conditions.

Meanwhile, the two gels are injected into a rat body, and the slow release condition of Antagomir in the body is observed through fluorescence.

2. Experimental results:

the results are shown in FIG. 5.

(1) Fig. 5a shows in vitro degradation results of hydrogel, and the results indicate that the degradation rate of the hydrogel in PBS is slow, about 21d is needed for complete degradation, and the hydrogel can be rapidly degraded within 4d after the pH and ROS content are adjusted, so that the blank hydrogel and the carrier hydrogel prepared by the invention have good degradability, particularly can be accelerated to degrade under acidic ROS conditions, and have pH and ROS responsiveness.

(2) FIG. 5b shows the DPPH antioxidant test results of different hydrogels, which indicates that the added MIC hydrogel has the highest antioxidant capacity.

(3) FIG. 5c shows the cleavage of hydrogels under different conditions, showing almost complete cleavage in the presence of pH and ROS, in the liquid state, further demonstrating the pH, ROS responsiveness of hydrogels of the present invention.

(4) Fig. 5e shows the release profile of curcumin in different environments, and it can be seen that the curcumin-loaded hydrogel of the present invention can effectively realize the slow release of curcumin, has pH and ROS responsiveness, and increases the curcumin release under acidic and ROS conditions.

(5) FIG. 5f shows a rhodamine fluorescence quenching experiment, and the result shows that the rhodamine fluorescence is quenched continuously along with the increasing concentration of Gel-BA-CD, so that the rhodamine is wrapped by CD molecules in Gel-BA-CD to cause the fluorescence to disappear, and after the addition of Antagomir, the fluorescence is recovered, so that the Antagomir replaces the rhodamine molecules to enter the CD molecules, and the successful entrapment of the Antagomir is proved.

(6) FIG. 5g is a schematic of sustained release of Antagomir. FIG. 5h is a graph of Cy3 fluorescence labelled Antagomir release profile. FIG. 5i, j shows in vivo fluorescence imaging of rats, suggesting that slow release of up to 21d can be achieved after Cy 3-labeled Antagomir is encapsulated in Gel-BA-CD/TA hydrogel.

The experimental results show that the hydrogel prepared by the invention has good pH and ROS responsiveness, and is beneficial to slow release treatment of the entrapped medicine at the part to be repaired.

Experimental example 5 evaluation of the ECM regeneration Effect of hydrogel nucleus pulposus cells

1. The experimental method comprises the following steps:

nucleus pulposus cells stimulated with t-butanol hydroperoxide (TBHP) were cultured using the hydrogels of examples 1, 2 and 4, and the proliferation of the cells and the results of Col II, aggrecan and MMP-13 gene expression were observed.

After three hydrogels of control hydro, hydro@ant, hydro@mic & Ant (so should not be example 4 bar, or should not be one example 5, containing two drugs) were soaked in PBS for 24h in this part of the laboratory, the extracts were taken to treat TBHP-stimulated nucleus pulposus cells, and finally the cell proliferation and Col II, aggrecan, MMP-13 gene expression results were observed.

2. Experimental results:

as shown in fig. 6, fig. 6a shows the proliferation of nucleus pulposus cells after TBHP stimulation by culturing different hydrogel leaching solutions, and it can be seen that the blank hydrogel of example 1 has a certain effect of promoting proliferation of nucleus pulposus cells, and the effect of promoting proliferation of nucleus pulposus cells is remarkably improved after Antagomir is entrapped.

FIG. 6b shows the differential results of MMP-13 gene expression in nucleus pulposus ColII, aggrecan, after TBHP stimulation, in different hydrogel leaches. It can be seen that the blank gel prepared in example 1 has the effect of improving Col II, aggrecan and MMP-13 gene expression, the improvement effect of the gel loaded with Antagomir is obviously improved, and the improvement effect of curcumin on the gene expression can be further improved by compounding. FIGS. 6c, d are immunofluorescence results of MMP-13 protein expression in nucleus pulposus cells ColII after TBHP stimulation in various hydrogel leaches.

The results show that the hydrogel can effectively promote Col II and Aggrecan expression, inhibit MMP-13 protein expression and promote nucleus pulposus cell ECM regeneration.

Test example 6 hydrogel-facilitated macrophage polarization detection

1. The experimental method comprises the following steps:

the blank hydrogel prepared in example 1, the curcumin-loaded hydrogel prepared in example 3 and the curcumin nano micelle MIC@Cur used alone as a control are adopted to treat macrophages, and a Transwell experiment is carried out to verify the migration condition of cells.

Culturing macrophage RAW264.7, interfering with different groups for 24 hours, observing cell morphology under a microscope, detecting M2 polarization specific indexes CD206 and CD163 by using an RT-PCR technology, detecting M1 polarization specific indexes TNF-alpha and IL-1 beta, detecting CD206 protein expression by using a cell flow technology and an immunofluorescence result, and detecting intracellular ROS clearance by using DFCH-DA staining.

2. Experimental results:

the results are shown in FIG. 7.

Figures 7a-c are optical observations of different groups of treated macrophages, and measured aspect ratio values, and found that the blank gel and nanomicelles of example 1, when used alone, each had some intended effect of stimulating macrophages, whereas the MIC-coated hydrogel group stimulated macrophage polarization more significantly and effectively than the blank gel, and the curcumin nanomicelles mic@cur, when used alone.

FIG. 7d shows the results of RT-PCR assays after different groups of stimulated macrophages, suggesting that MIC loaded hydrogels effectively promote macrophage M2 polarization and fully exert anti-inflammatory effects compared to other groups.

FIG. 7e shows the expression of the M2-specific protein CD206 after the macrophage is stimulated by different groups, and the result shows that the blank gel and the nano-micelle of the example 1 have certain effect of promoting the polarization of the M2 of the macrophage when being singly used, and the hydrogel@MIC group can promote the polarization of the M2 of the macrophage more remarkably. The cell flow results of fig. 7f further confirm this conclusion. FIG. 8e shows the result of a cell migration Transwell experiment, and FIG. 8f shows that Cur promotes macrophage polarization. After the hydrogel disclosed by the invention is treated, M2 polarized macrophages can effectively promote migration of nucleus pulposus cells.

FIG. 7g is an intracellular ROS scavenging assay, demonstrating that the Hydrogel@MIC group can effectively inhibit ROS production following LPS stimulation of macrophages.

Test example 7 identification of the anti-inflammatory Effect of hydrogels

1. The experimental method comprises the following steps:

the blank hydrogel prepared in example 1, the double-carrier hydrogel prepared in example 2 and the curcumin-loaded hydrogel prepared in example 3 were used to characterize the expression of anti-inflammatory factors CD206, IL-10 and TNF-alpha of macrophage RAW264.7 after LPS treatment.

2. Experimental results:

FIG. 8a shows the expression of the RT-PCR anti-inflammatory factors CD206, IL-10 and TNF-alpha after LPS treatment of macrophage RAW264.7, and the expression of IL-10 and TNF-alpha by incubation with different sets of hydrogel extracts, FIG. 8b shows the detection of IL-10 and TNF-alpha by Western-blot, and FIGS. 8c and d show the detection of IL-10 and TNF-alpha by immunofluorescence. The results indicate that the blank hydrogel prepared in the embodiment 1 of the invention has obvious anti-inflammatory effect, and the hydrogel loaded with MIC@Cur and Antagomir-21 has more obvious improved anti-inflammatory effect.

Test example 8 in vivo identification of the therapeutic Effect of hydrogel rat disc degeneration

1. The experimental method comprises the following steps:

a rat intervertebral disc degeneration model is established, different hydrogels are implanted, and the degeneration nucleus pulposus restoration effect is observed through X-ray, MRI, histological staining (HE, safranin O-fast green, col II, MMP-13 immunohistochemical staining) and other results in 4 and 8 weeks, and a schematic diagram is shown in figure 9 a.

2. Experimental results:

fig. 9b, d and e show the results of the postoperative 4, 8-week X-ray scan, which illustrate that the blank Hydrogel prepared by the method can significantly recover the height of the intervertebral space, and further improves the recovery of the height of the intervertebral space after further drug loading, and the double drug loading has the best effect of recovering the hydro el & mic@ant.

Fig. 9c and f show MRI scanning results after 4 and 8 weeks of operation, which indicate that the blank Hydrogel prepared by the method can effectively increase the water content of the nucleus pulposus, recover the MRI signals of the nucleus pulposus, further improve the repairing effect after further encapsulating the medicine, and optimize the repairing effect of the double medicine-carrying Hydrogel & mic@ant group.

FIGS. 10a, b, c are HE staining, safranin O-fast green staining and Col II immunohistochemical staining results, and FIGS. 10d, e, f are quantitative observations of histological grading results, suggesting that the degenerative nucleus pulposus repair effect of the Hydrogel & MIC@Ant group is optimal.

In summary, the invention provides an injectable hydrogel formed by crosslinking phenylboronic acid and cyclodextrin grafted gelatin on a matrix under the action of tannic acid, which has the effects of inhibiting inflammation and promoting nucleus pulposus repair when being injected into the nucleus pulposus, further is compounded with curcumin micelle and cholesterol modified miRNA-21 inhibitor, can release anti-inflammatory drug curcumin and miRNA-21 inhibitor (Antagomir) for promoting ECM regeneration according to different inflammation degrees in the nucleus pulposus according to local conditions, and has good application prospect under double-tube condition.

Claims

1. The hydrogel precursor is characterized by comprising the following components in parts by weight:

8-12 parts of phenylboronic acid and cyclodextrin grafted gelatin, 1 part of tannic acid and 80-120 parts of water;

the phenylboronic acid and cyclodextrin grafted gelatin is prepared by the following method:

2. The hydrogel precursor of claim 1, comprising the following components in parts by weight:

3. The hydrogel precursor of claim 1 or 2, wherein the phenylboronic acid and cyclodextrin grafted gelatin has a phenylboronic acid grafting ratio of 17±2.4% and a cyclodextrin grafting ratio of 3.13±0.87%.

4. The hydrogel precursor of claim 1, wherein the weight ratio of gelatin to 3-carboxyphenylboronic acid in step (1) is 1 (0.1-0.5);

and/or the weight ratio of the phenylboronic acid grafted gelatin and the amino beta-cyclodextrin in the step (2) is 1 (0.1-0.5).

5. The hydrogel precursor of claim 4, wherein the weight ratio of gelatin to 3-carboxyphenylboronic acid of step (1) is 1:0.4;

and/or the weight ratio of phenylboronic acid grafted gelatin to aminated beta-cyclodextrin in step (2) is 1:0.4.

6. The hydrogel precursor of claim 1 wherein the activator is NHS and the condensing agent is EDC.

7. The hydrogel precursor of claim 1 or 2, further comprising the following components in parts by weight:

8. The hydrogel precursor of claim 7, wherein the cholesterol modified miRNA inhibitor is 0.0033 parts and/or the curcumin entrapped nano-micelle is 0.0066 parts.

9. The hydrogel precursor of claim 7 wherein the cholesterol modified miRNA inhibitor is a cholesterol modified miRNA-21 inhibitor.

10. The hydrogel precursor of claim 7, wherein the curcumin is present in the nano-micelle entrapped in an amount of 10% to 20% w/w.

11. The hydrogel precursor of claim 10 wherein the nanomicelles are self-assembled from polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer.

12. The hydrogel precursor of claim 11, wherein the polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer is prepared by reacting polyethylene glycol and polylactic acid-glycolic acid copolymer with ketal; the ketal has the structure that:

。

13. the hydrogel precursor of claim 12 wherein the polyethylene glycol has a molecular weight of 2000 and the polylactic acid-glycolic acid copolymer has a molecular weight of 1000.

14. The hydrogel precursor of claim 7, wherein the curcumin-entrapped nano-micelle is self-assembled from the following materials in parts by weight:

8-12 parts of polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer and 1 part of curcumin.

15. The hydrogel precursor of claim 14, wherein the curcumin-entrapped nano-micelle is self-assembled from the following materials in parts by weight:

10 parts of polyethylene glycol-ketal-polylactic acid-glycolic acid copolymer and 1 part of curcumin.

16. A hydrogel, characterized in that it is formed by curing the hydrogel precursor of any one of claims 1 to 15.

17. The hydrogel of claim 16, wherein the curing reaction conditions are resting at 20 to 30 ℃.

18. Use of the hydrogel of claim 16 or 17 in a degenerated nucleus repair material.

19. The use of claim 18, wherein the degenerated nucleus repair material is a nucleus replacement material and/or a nucleus regeneration promoting material.