CN113929975B

CN113929975B - Chitosan-protein hydrogel composition, hydrogel, preparation method and medical application thereof

Info

Publication number: CN113929975B
Application number: CN202111416003.XA
Authority: CN
Inventors: 刘丽; 郭志刚; 姜楠; 付博
Original assignee: TIANJIN CHEST HOSPITAL; Nankai University
Current assignee: TIANJIN CHEST HOSPITAL; Nankai University
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-06-24
Anticipated expiration: 2041-11-25
Also published as: CN113929975A

Abstract

The invention provides a chitosan-protein hydrogel composition, which comprises a first solution and a second solution, wherein the first solution comprises a dithiopyridine modified chitosan derivative, and the second solution comprises reducing bovine serum albumin with naked sulfydryl modification. The invention also provides a hydrogel prepared from the composition, a medicinal preparation taking the hydrogel as a carrier and preparation methods thereof. One medical use of the hydrogel composition is as a scaffold material for the treatment of myocardial infarction, which has high biocompatibility and suitable degradation properties.

Description

Chitosan-protein hydrogel composition, hydrogel, preparation method and medical application thereof

Technical Field

The invention belongs to the field of biological materials, and particularly relates to an injectable hydrogel, a preparation method and application thereof.

Background

Myocardial Infarction (MI) is a common ischemic heart disease, which is characterized by necrosis of Myocardial cells due to ischemia and hypoxia after coronary sclerosis and/or blockage, replacement of the Myocardial cells with scar fibrous tissues only, thinning of the wall of the heart, enlargement of the heart chambers, progressive decline of the heart function, and finally heart failure. There is currently a clinical lack of effective means of treating MI. In recent years, a beneficial exploration is that angiogenesis promoting factors are used for inducing angiogenesis of tissues in an infarct area, and researchers at home and abroad find that Basic fibroblast growth factor (bFGF) has the effects of promoting angiogenesis of ischemic tissues, accelerating establishment of collateral circulation, reducing myocardial infarction area and improving cardiac function. However, bFGF injected into ischemic tissue sites has problems of rapid in vivo diffusion, short half-life, difficulty in maintaining biological activity, inability to achieve myocardial repair as desired, etc., thereby limiting its use. With the continuous development of tissue engineering research, the injectable scaffold material loaded with the angiogenesis promoting factors is directly injected to the myocardial injury part for myocardial repair, and the wound is small, so that the clinical application is facilitated, and the method becomes one of the research hotspots of myocardial tissue engineering. The hydrogel as a novel biomedical injectable scaffold plays an important role in myocardial repair. The three-dimensional network cross-linked structure of the hydrogel can replace a degraded Extracellular matrix (ECM), save the dying myocardium, prevent myocardial cells around infarction from necrosis caused by loss of the support of the ECM, and play a certain mechanical supporting role on the damaged myocardium, so that the ventricle is thickened, the pressure is reduced, and the ventricular remodeling is inhibited.

At present, the injectable chitosan hydrogel is used as a natural scaffold material most commonly used in myocardial tissue engineering, has the advantages of good biocompatibility, biodegradability, cell adhesion, sterilization, adhesion prevention and the like, and is already applied to the injectable myocardial tissue engineering. However, the existing chitosan hydrogel has many problems in mechanical properties, stability and the like, and although the chemical crosslinking method can solve the problems, the conventional method uses some toxic and difficultly removable small molecular crosslinking agents such as formaldehyde, glyoxal, carbodiimide and the like, so that the crosslinked injectable chitosan hydrogel with excellent mechanical properties, no toxic or side effect and good biocompatibility is obtained and still has the technical difficulty in the field.

Disclosure of Invention

The invention aims to provide a medical injectable hydrogel stent which does not introduce toxic substances, is completely compatible with a human body, is degradable in the human body and is a safe material for delivering drugs to the human body.

It is another object of the present invention to provide a pharmaceutical composition for treating myocardial infarction, which is a therapeutically active drug, loaded on a non-toxic degradable chitosan-protein hydrogel.

It is a further object of the present invention to provide a process for the preparation of the above-described stents and other therapeutic pharmaceutical compositions.

According to a first aspect of the present invention, there is provided a chitosan-protein hydrogel composition comprising a first solution and a second solution, wherein the first solution comprises a dithiopyridine-modified chitosan derivative, and the second solution comprises reduced bovine serum albumin having a naked thiol group.

According to a second aspect of the present invention there is provided a chitosan-protein hydrogel cross-linked from a dithiopyridine modified chitosan derivative and a reducing bovine serum albumin having a naked thiol group.

The third aspect of the present invention provides a method for preparing the above chitosan-protein hydrogel composition, comprising the steps of:

1) adding the aqueous solution of 2-iminothiolane hydrochloride into the aqueous solution of the chitosan derivative, and reacting to obtain a sulfhydryl modified chitosan aqueous solution;

2) adding N, N-Dimethylformamide (DMF) solution of dipyridyl disulfide into the solution, dialyzing for two days by PBS buffer solution after the reaction is finished, and freeze-drying to obtain the dipyridyl disulfide modified chitosan derivative;

3) preparing the dithiodipyridine-modified chitosan derivative obtained in the step 2) into a buffer solution to obtain a first solution;

4) dissolving bovine serum albumin in PBS buffer solution, adding tris (2-carbonylethyl) phosphate dissolved in the PBS buffer solution, and degassing and stirring in an ice water bath;

5) and 4) dialyzing the reaction product in cold PBS buffer solution for two days, freeze-drying to obtain the PBS buffer solution of the reducing bovine serum albumin, preparing into the second solution, and separately storing the first solution and the second solution.

The fourth aspect of the present invention provides a method for preparing a chitosan-protein hydrogel, comprising uniformly mixing the first solution and the second solution, and reacting at 32 to 40 ℃.

Preferably, the concentration of the dithiopyridine-modified carboxymethyl chitosan in the first solution is 3-5 w/v%, and the concentration of the reducing bovine serum albumin in the second solution is 0.75-3 w/v%.

Preferably, the first solution and the second solution are mixed in equal volumes.

In a fifth aspect of the present invention, there is provided a use of the above chitosan-protein hydrogel composition in a medical material for treating myocardial infarction, the medical material comprising chitosan-protein hydrogel and basic fibroblast growth factor prepared in situ from the first and second solutions.

In one embodiment, the reducing bovine serum albumin and dithiodimercapto modified carboxymethyl chitosan are used at a mass ratio of substantially 1: 1.

In one embodiment, the first solution and the second solution are both PBS buffer solutions, pH7.4, and concentration 100 mM.

Drawings

FIG. 1 is an infrared spectrum of each of the substances of interest in example 1;

FIG. 2 is a graph of the results of rheometer measurements on the product of example 3, showing the effect of the molar ratio of dithiodipyridine to thiol and the solid content of dithiodipyridine-modified chitosan hydrogel on the mechanical properties of the hydrogel.

FIG. 3 is an electron micrograph of the hydrogel obtained in example 3.

FIG. 4 shows the results of the evaluation of cytotoxicity of the hydrogel by the CKK-8 method.

FIG. 5 is a graphical representation of the results of the degradation test of the hydrogel obtained in example 3.

FIG. 6 shows the echocardiography test results of the rat experiment for myocardial infarction treatment effect.

FIG. 7 is a graph showing the results of histomorphometric measurements of four groups of rat heart tissue specimens.

FIG. 8 shows immunohistochemical staining and blood vessel count results.

Detailed Description

In the chitosan-protein hydrogel composition of the first aspect of the present invention, the first solution comprises a dithiopyridine-modified chitosan derivative, and the second solution comprises a reducing bovine serum albumin, which has a naked thiol group. The first solution and the second solution are stored separately prior to use of the composition.

In the present invention, the chitosan derivative is a derivative which modifies chitosan to have a desired water solubility, and the derivative includes carboxylated chitosan and chitosan salts, examples of carboxylated chitosan include but are not limited to carboxymethyl chitosan, and chitosan salts include but are not limited to chitosan hydrochloride, chitosan quaternary ammonium salt, chitosan lactate, chitosan glutamate, and carboxymethyl chitosan is most preferable in the present invention. In the case of carboxymethyl chitosan, the molecular weight of the chitosan derivative may be between 10 and 30 ten thousand. In a typical embodiment, carboxymethyl chitosan having a molecular weight of 15 to 25 ten thousand is used. When the molecular weight is less than 10 ten thousand, the mechanical properties of the resulting hydrogel deteriorate. In the molecular weight range of 10-30, the desired water solubility can be obtained by adjusting the degree of carboxymethylation. In the present invention, the degree of carboxylation is not more than 95%, preferably not more than 90%. Since too high a carboxylation will excessively consume reactive groups (e.g., hydroxymethyl and amino groups) in the chitosan derivative, making further modification difficult. In a preferred embodiment of the invention, carboxymethyl chitosan with a molecule of 18-22 ten thousand and a degree of carboxymethylation in the interval of 75-85% is used.

One feature of the present invention is that the chitosan derivative is modified to have a dithiopyridine group on the molecular chain, and the dithiopyridine group can chemically bond with the biomacromolecule cross-linking agent provided in the present invention to form a network structure to form hydrogel. The dithiopyridine group can be directly linked to the chitosan derivative by a suitable reaction, for example, to a free amino group or hydroxymethyl group, or indirectly linked to the aforementioned amino group or hydroxymethyl group via a physiologically acceptable group, in other words, a small molecule (motif) with a dithiopyridine group is linked to the amino group or hydroxymethyl group.

In a most preferred embodiment of the invention, the dithiopyridine group is formed by the attachment of 2-iminothiolane hydrochloride to the amino group of chitosan, see reaction scheme I.

In the scheme, a sulfhydryl-modified carboxymethyl chitosan (CMCS-SH) is obtained in the first step of reaction, and then the sulfhydryl-modified carboxymethyl chitosan (CMCS-S-S-Py) is obtained by utilizing the reactivity of the sulfhydryl to react with dithiodipyridine. The implementation method has good advantages, and can completely convert the sulfhydryl-modified carboxymethyl chitosan obtained in the first step into the dithiopyridine-modified carboxymethyl chitosan by utilizing efficient sulfhydryl-disulfide exchange reaction.

In a specific aspect, in the resulting dithiopyridine-modified carboxymethyl chitosan, each chitosan molecule is incorporated5 to 40Typical examples of the dithiopyridine functional group, preferably 7 to 30, for example 7 to 20, show that when each chitosan molecule carries an integer number of dithiopyridine groups in the range of 8 to 15, moldability and mechanical properties of the resulting hydrogel are most desirable.

In the present invention, Bovine Serum Albumin (BSA) is modified so that the resulting modified product has a naked thiol group (-SH), and is referred to as reducing Bovine Serum Albumin (BSA) in the present specification. In one embodiment, the reduced bovine serum albumin is obtained by reacting bovine serum albumin with Tris (2-carbonylethyl) phosphine hydrochloride (TCEP), see equation II.

This modification has the particular advantage of being non-toxic and providing thiol groups that are highly reactive under physiological conditions and readily cross-link by reaction with the dithiopyridine groups in the chitosan derivative. The crosslinked network formed has suitable mechanical properties and can be degraded in the desired time.

In a preferred embodiment of the invention, each reducing bovine serum albumin contains 5-20 naked thiol groups, preferably 8-12 naked thiol groups.

In the chitosan-protein hydrogel composition of the present invention, typically the first solution and the second solution are PBS buffer solutions, wherein the concentration of the first solution is preferably 3-5 w/v%, most preferably 3 w/v%. The concentration of the second solution is preferably 0.75-3 w/v%, most preferably 3 w/v%.

The chitosan-protein hydrogel composition can be used for preparing injectable hydrogel on site, forming a scaffold in a human body and being used for tissue repair. The preparation is administered by mixing the desired medicinal materials with the first solution and the second solution at a mass ratio of 1:1, and injecting into human body. In another variation, the reducing bovine serum albumin is prepared as a lyophilized powder, which is dissolved in the PBS buffer of the desired drug prior to injection, and then mixed with the first solution uniformly. For this purpose, a pharmaceutical composition may be provided, which further comprises one or more desired drugs (antibiotics, anticancer drugs, growth factors, etc.) in one of the first solution and the second solution, and is used by mixing the first solution and the second solution uniformly and injecting them into the body to form a drug-carrying scaffold material at the corresponding site.

A typical application of the composition is that the composition is used for preparing a hydrogel scaffold loaded with basic fibroblast growth factor (bFGF), and the hydrogel scaffold is injected into a myocardial damage part of a heart to repair the myocardium. In a preferred embodiment, the first solution is prepared as PBS buffer (pH 7.4) having a solid content of 3-5 w/v%, the second solution is prepared as PBS buffer (pH 7.4) having a solid content of 0.75-3 w/v%, the first two solutions are used and mixed homogeneously in equal volumes, and the desired amount of bFGF is introduced, and then the resulting mixture is injected into the body over the desired period of time.

In a second aspect of the present invention, there is also provided a chitosan-protein hydrogel formed by cross-linking a dithiopyridine-modified chitosan derivative and a reducing bovine serum albumin having a naked thiol group. The hydrogel is also loaded with one or more substances having beneficial physiological activities, so that the delivery of the physiologically active substance to a predetermined site of the human or animal body at a controlled rate can be achieved, for example, a hydrogel patch, which is applied to the outside of the human or animal body for slow delivery of one or more physiologically active substances to the subcutaneous tissue, such as a mask for beauty, a patch for treatment of skin diseases, a coagulant for hemostasis.

In the chitosan hydrogel, the chitosan derivative is preferably carboxymethyl chitosan, and the molecular weight can be between 10 and 30 ten thousand. In a typical embodiment, carboxymethyl chitosan with a molecular weight of 15-25 ten thousand is used. When the molecular weight is less than 10 ten thousand, the mechanical properties of the resulting hydrogel deteriorate. In the molecular weight range of 10-30, proper water solubility can be obtained by adjusting the degree of carboxymethylation. In the present invention, the carboxylation degree is not preferably more than 95%, preferably not more than 90%. Since too high a degree of carboxylation excessively consumes reactive groups (e.g., hydroxymethyl and amino groups) in the chitosan derivative, thereby increasing the difficulty of subsequent modification. In a preferred embodiment of the invention, carboxymethyl chitosan with a molecule of 18-22 million and a degree of carboxylation in the interval of 75-85% is used.

Specific ways of introducing the dithiopyridine group into the chitosan derivative are as described in detail above. In the dithiopyridine-modified chitosan derivative, each chitosan derivative molecule may contain 5 to 40 dithiopyridine functional groups, with preferred amounts as set forth above.

Preferably, the reducing bovine serum albumin is modified from bovine serum albumin so that each protein molecule contains 5-20 naked sulfhydryl groups, and the sulfhydryl groups can efficiently exchange and react with the dithiopyridine groups to be crosslinked into a porous network structure. In one embodiment, the reducing bovine serum albumin is obtained by reacting bovine serum albumin with tris (2-carbonylethyl) phosphate.

In one preferred embodiment, the mass ratio of the dithiopyridine-modified chitosan derivative to the reducing bovine serum albumin is such that the molar ratio of the dithiopyridine group to the thiol group is 1: 2.

The third aspect of the present invention relates to a method for preparing the aforementioned chitosan-protein hydrogel composition, comprising: 1) adding the aqueous solution of 2-iminothiolane hydrochloride into the aqueous solution of the chitosan derivative, and reacting to obtain a sulfhydryl modified chitosan aqueous solution; 2) adding dithiodipyridine into the solution, dialyzing with PBS buffer solution after the reaction is finished, and freeze-drying to obtain dithiodipyridine-modified chitosan derivative; 3) preparing the disulfide bipyridine modified chitosan derivative obtained in the step 2) into a buffer solution to obtain a first solution; 4) dissolving bovine serum albumin in PBS buffer solution, adding tris (2-carbonylethyl) phosphate dissolved in the PBS buffer solution, and degassing and stirring in an ice water bath; 5) and 4) dialyzing the reaction product in cold PBS buffer solution, freeze-drying to obtain the PBS buffer solution of the reducing bovine serum albumin, preparing into the second solution, and separately storing the first solution and the second solution.

In the present invention, the molecular weight of the chitosan derivative is usually between 15 and 30 ten thousand. In one embodiment, carboxymethyl chitosan is used, the molecular weight is 18-22 ten thousand, the carboxylation rate is 75-85%, and the mass ratio of the carboxymethyl chitosan to 2-iminosulfane hydrochloride and dithiodipyridine is 1: 0.015-0.030: 0.86-0.18, preferably 1: 0.025-0.30: 0.13-0.18.

In step 4), the mass ratio of bovine serum albumin to tris (2-carbonylethyl) phosphate hydrochloride is 1: 0.03-0.05, preferably 1: 0.04, wherein the dosage ratio of the chitosan derivative to the bovine serum albumin is substantially 1: 1.

In step 2) and step 5), dialysis is preferably performed with a PBS buffer solution having a pH of 6.0 and 10 mM.

In a fourth aspect of the present invention, there is provided a method for preparing a chitosan-protein hydrogel, which comprises mixing the first solution and the second solution uniformly, and reacting at 32-40 ℃ for a desired time. In a preferred embodiment, the concentration of the dithiopyridine-modified carboxymethyl chitosan in the first solution is 3 to 5 w/v%, and the concentration of the reducing bovine serum albumin in the second solution is 0.75 to 3 w/v%. When the concentration of the reducing bovine serum albumin is 3 w/v%, the first solution and the second solution are preferably mixed in equal volumes.

The following examples further illustrate embodiments of the invention, and the details of the operations therein are not to be construed as limitations of the embodiments of the invention.

EXAMPLE 1 preparation of dithiopyridine-modified carboxymethyl Chitosan

0.5g of carboxymethyl chitosan (CMCS, molecular weight 20 ten thousand, degree of carboxylation about 80%) was dissolved in 45mL of deionized water, and 1mL of 2-iminosulfane hydrochloride (13.4mg) dissolved in deionized water was added to the carboxymethyl chitosan solution under argon. And (3) reacting for 4 hours at 37 ℃ to obtain the sulfhydryl modified carboxymethyl chitosan (CMCS-SH), and measuring that each carboxymethyl chitosan molecule contains 9 sulfhydryl functional groups on average by using an Ellman reagent method. 5mL of a dithiodipyridine (77.1mg) solution dissolved in DMF was added to the above solution, and reacted for 12 hours. The reaction product was dialyzed against PBS buffer (pH 6.0,10mM) for two days (MWCO 12kDa), and lyophilized to give dithiopyridine-modified carboxymethyl chitosan (CMCS-S-Py).

The Ellman reagent method is utilized to determine the content of sulfydryl in the carboxymethyl chitosan modified by the dithiopyridine so as to indirectly calculate the content of the dithiopyridine in the carboxymethyl chitosan, and the calculation result is that each carboxymethyl chitosan molecule contains 9 dithiodipyridine functional groups on average. Meanwhile, Fourier transform infrared spectroscopy (FT-IR) is utilized to carry out on carboxymethyl chitosan and carboxyl modified by sulfydrylMethyl chitosan and dithiopyridine modified carboxymethyl chitosan were characterized, and it can be seen from FIG. 1 that the length of the dithiopyridine modified carboxymethyl chitosan is 940cm^-1The C-H bending vibration absorption peak of the benzene ring appears, and the dithiopyridine is proved to be successfully modified on the carboxymethyl chitosan molecule.

Example 2 preparation of reducing bovine serum albumin

0.5g of Bovine Serum Albumin (BSA) was dissolved in 50mL of PBS buffer (pH 8.0,10mM), tris (2-carbonylethyl) phosphate (TCEP, 19.3mg) dissolved in 1mL of PBS buffer was added, the mixture was degassed and stirred in an ice-water bath for 5 hours, and the reaction product was dialyzed in cold PBS buffer (pH 6.0,10mM) for two days, and freeze-dried to obtain reducing bovine serum albumin (rBSA). The Ellman reagent method was used to determine that on average there were 6 thiol functional groups per BSA molecule.

EXAMPLE 3 preparation of disulfide-bond Cross-Linked Chitosan hydrogel

Preparing a series of thiol-modified carboxymethyl chitosan 1 (3-5% w/v%) and reducing bovine serum albumin (3-5 w/v%) solutions with different concentrations by using PBS buffer solution, uniformly mixing the two solutions at room temperature in equal volume, and reacting at 37 ℃ for 10min to obtain the disulfide bond crosslinked chitosan hydrogel.

FIG. 2 is a graph showing the results of a rheometer test of the molar ratio of dithiodipyridine to thiol and the effect of the solid content of dithiodipyridine-modified carboxymethyl chitosan on the mechanical properties of hydrogel. As can be seen from FIG. 2, the mechanical properties of the hydrogel were best when the molar ratio of dithiopyridine to thiol was 1:2 and the solids content of dithiodipyridine-modified carboxymethyl chitosan was 3%.

The microstructure of the hydrogel obtained using the molar ratios of dithiopyridine to thiol was observed by transmission electron microscopy. FIG. 3 is a partial photograph in which A is 2:1, B is 1:1, and C is 1: 2. As can be seen from FIG. 3, when the molar ratio of dithiopyridine to thiol is 1:2, the hydrogel has more pore structures due to the increase of the crosslinking degree, and the pore structures are beneficial to the subsequent loading of growth factors. In subsequent experiments, the hydrogel is prepared under the conditions that the molar ratio of the dithiopyridine to the sulfydryl is 1:2 and the solid content of the dithiodipyridine modified carboxymethyl chitosan is 3%.

Cytotoxicity test

The cytotoxicity of the hydrogel was evaluated by the CKK-8 method, as shown in FIG. 4. NIH 3T3 fibroblasts were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in CO ₂5% in an incubator at 37 ℃. The hydrogel was placed in 48-well culture plates and washed repeatedly with sterile PBS. NIH 3T3 fibroblasts⁴One cell/well was inoculated onto the surface of sterile hydrogel and incubated at 37 ℃. After

days

1, 3 and 5, CCK-8 reagent was added to each well and incubated for 4 h. Proliferation of 3T3 fibroblasts was assessed by measuring the Optical Density (OD) value of the cell suspension at 450nm at the microplate reader, with cells seeded on anhydrous gel tissue culture plates as controls. As can be seen from fig. 4, the hydrogel was almost non-cytotoxic to NIH 3T3 fibroblasts.

In vitro degradation experiments

The in vitro degradation experiment simulates the physiological environment of glutathione (glutathione concentration: 0.5mM) in a living body, and the degradation performance of the hydrogel is reflected by the change of the gel quality. Freeze-drying the prepared hydrogel, weighing W₀Adding 1.5ml of water into the hydrogel in the centrifuge tube, placing the centrifuge tube into a constant temperature shaking table at 37 ℃, and swelling for 90 min. Pouring out water from the centrifuge tube, adding 1.5mL PBS (pH7.4, 10mM) containing 0.5mM glutathione, placing in a constant temperature shaking table at 37 deg.C, reacting for 2,6, 10, 24, 48, 72h, thoroughly washing off excessive enzyme and salt, freeze drying, and weighing W₁The weight loss ratio was calculated by the following formula, three sets of parallel samples were made at each time point, and the experimental results are shown in fig. 5. The experimental results showed that the hydrogel was completely degraded within 48 hours.

Weight loss ratio (W)₀-W₁)/W₀

Preparation of disulfide bond cross-linked chitosan hydrogel loaded with basic fibroblast growth factor

Dissolving 3mg of reducing bovine serum albumin in 0.1mL of PBS buffer containing 27 mu g of basic fibroblast growth factor bFGF, dissolving 3mg of sulfhydryl-modified carboxymethyl chitosan in 0.1mL of PBS buffer, uniformly mixing the two solutions at room temperature, and reacting for 5min at 37 ℃ to obtain the disulfide bond crosslinking injectable chitosan hydrogel loaded with the basic fibroblast growth factor.

Test of Effect on treating myocardial infarction

SD rats (250-300 g) were anesthetized with 2% isoflurane, ventilated with an endotracheal tube ventilator, prepared for skin, and subjected to thoracotomy. The pericardium was cut open and the pleuroperitoneal cavity was squeezed to expose the heart sufficiently as shown in figure 8. No. 6-0 non-invasive slide ligature is performed on the junction of the conical center intersection of the left auricle and the pulmonary artery and the connecting line of the left apex of the heart, the lower edge of the left auricle is 2 mm. The left ventricular wall becomes pale, the ventricular wall motion is weakened, and the ST segment of the II lead under the electrocardiographic monitoring is obviously raised, which indicates that the preparation of the myocardial infarction model is successful.

After the myocardial infarction model is built, the myocardial infarction model is randomly divided into 4 groups (n is 10): phosphate Buffered Saline (PBS), bFGF (basic fibroblast growth factor), hydrogel (hydrogel), and bFGF-loaded hydrogel (bFGF-hydrogel).

An implantation operation step: three injection sites (below the left atrium, in the middle of the left ventricle and at the apex of the heart) were selected at the border region of the myocardial infarction, and Phosphate Buffered Saline (PBS), bFGF, hydrogel (hydrogel) and bFGF-loaded hydrogel (bFGF-hydrogel) were injected into the myocardium of each group of rats, respectively. After injection was complete, the chest was closed and marked. The local infection and adhesion can be prevented by injecting penicillin into abdominal cavity for 1 week.

Cardiac function was examined in each group of animals 4 weeks after surgery using cardiac ultrasound. After anesthesia, each group of animals were fixed in the supine position, preserved, and subjected to echocardiography examination. Obtaining a left ventricular long axis section and a left ventricular short axis section beside the sternum, performing parallel M-mode ultrasound, measuring left ventricular end-diastolic internal diameter (LVIDd) and end-systolic internal diameter (LVIDs), and calculating left ventricular short axis shortening rate (LVFS) and ejection fraction (LVEF). The data measured in 3-6 cardiac cycles were averaged and the results are shown in fig. 6, where a is the typical rat cardiac ultrasound image in different experimental groups after 4 weeks and B to E are the graphical results of the cardiac ultrasound related parameters of four groups of rats after 4 weeks.

From FIG. 6(A), it can be seen that LVEF and LVFS values of PBS group are lowest and cardiac function is significantly decreased, and from FIG. 6(B), it can be seen that LVEF value (62.76 + -3.56%) of bFGF-hydrogel group rats is significantly higher than that of bFGF group (55.53 + -4.00%), hydrogel group (57.58 + -1.66%), PBS group (54.60 + -5.63%), P < 0.06; from FIG. 6(C), it can be seen that the LVFS value (29.94 + -2.22%) of the bFGF-hydrogel group rats is significantly higher than that of the bFGF, hydrogel and PBS groups (25.59 + -2.34, 26.51 + -1.11% and 24.97 + -3.35%, respectively, P < 0.06); from FIG. 6(D), it can be seen that the LVIDd value of the rats in the bFGF-hydrogel group is the least increased (7.74. + -. 0.42mm), and is lower than that of the bFGF group (8.45. + -. 0.34mm), the hydrogel group (8.32. + -. 0.61mm) and the PBS group (8.79. + -. 0.23mm), and P is less than 0.05; from FIG. 6(E), it can be seen that the increase in LVIDs values was minimal (5.24. + -. 0.40mm) in the bFGF-hydrogel group, and was lower than that in the other three groups (5.94. + -. 0.72mm, 5.82. + -. 0.66mm and 6.43. + -. 0.51mm, and P <0.05), respectively, and that the heart function was improved in the bFGF-hydrogel group rats compared with the other three groups.

Tissue morphology detection

After 4 weeks of operation, rat heart tissue samples were collected from each group of rats and myocardial infarction area measurements were performed. Each group of specimens is embedded with OTC, and after being frozen for 20s by liquid nitrogen, the specimens are continuously sliced (with the thickness of 5 μm) from the apex to the fundus according to a short axis plane, so that the apex, the fundus and the center of the peduncle area can be cut. And performing Masson three-staining on each group of specimens, and then detecting the deposition degree of the collagen fibers in the myocardial infarction area. The degree of collagen fiber deposition in the myocardial infarction area is expressed by the percentage of the fibrosis area in the left ventricular wall area, and is analyzed and calculated by RS Image Pro Image analysis software. In FIG. 7, A is a photograph showing each of the fibrotic tissue (blue) and normal tissue (red) by Masson staining, scale bar, 100 μm; b is a photograph of the results of TUNEL apoptosis assay showing DAPI (blue fluorescence, normal cells) and TUNEL (green fluorescence, apoptotic cells) at a scale bar of 20 μm. C is the percentage area of left ventricular fibrosis (fibrosis) analysis and D is the percentage apoptotic index (apoptosis).

As can be seen from FIG. 7, the degree of fibrosis in the myocardial infarction region of the rats of the bFGF-hydrogel group was significantly suppressed compared to the other three groups.

TUNEL apoptosis assay

The samples of each group were subjected to OCT embedding, frozen sections (thickness 5 μm) and apoptosis in infarct junction area was detected using TUNEL kit, and the apoptotic cells were observed under high power microscope (x 400 times). Selecting 3 slices from each specimen, randomly selecting 5 non-overlapping fields from each slice, counting the positive cells with brown particles in the cell nucleus, and calculating the apoptosis index (apoptosis index): the apoptosis index is the number of single-visual field positive nuclei/single-visual field total nuclei multiplied by 100%, the experimental results are shown in fig. 7(B, D), and it can be seen from the figure that the number of apoptotic cells in the myocardial infarction region of the bFGF-hydrogel group rats is obviously reduced compared with the other three groups.

Immunohistochemical staining and blood vessel counting

After OCT embedding, each group of samples is frozen and sliced (the thickness is 5 mu m), endogenous peroxidase is blocked and goat serum is sealed, primary anti-vWAg (1:200) and corresponding secondary antibodies are dripped, DAB color development, hematoxylin counterstaining, and microvasculature (the diameter is 20-100 mu m) at the infarct part is observed and counted under a high-power microscope. 5 slices are taken from each group of specimen, 5 high-power visual fields are taken from each slice, the measurement results are averaged, the experimental result is shown in figure 8, and the figure shows that the number of the microvascular in the myocardial infarction area of the rat in the bFGF-hydrogel group is obviously increased compared with other three groups,

the sulfhydryl-modified bovine serum albumin macromolecular cross-linking agent is used for replacing the traditional micromolecular cross-linking agent which has toxic and side effects and is difficult to remove, such as formaldehyde, glyoxal and the like, and the sulfhydryl-disulfide-crosslinked injectable chitosan hydrogel is obtained by carrying out mild and efficient sulfhydryl-disulfide exchange reaction with the disulfide-modified carboxymethyl chitosan. The material is easy to prepare and low in cost. The hydrogel can be degraded under the condition of glutathione in vivo, and the controlled release of growth factors in the gel is realized.

Claims

1. A chitosan hydrogel composition comprises a first solution and a second solution, wherein the first solution comprises a dithiopyridine modified water-soluble chitosan derivative, the second solution comprises reducing bovine serum albumin, the reducing bovine serum albumin has a sulfydryl group, the first solution is PBS buffer solution of the dithiopyridine modified chitosan derivative, the concentration of the PBS buffer solution is 3-5 w/v%, the second solution is PBS buffer solution of the reducing bovine serum albumin, the concentration of the PBS buffer solution is 0.75-3 w/v%, and the mass ratio of the dithiopyridine modified chitosan derivative to the reducing bovine serum albumin is such that the molar ratio of the dithiopyridine group to the sulfydryl group in the composition is 1: 2.

2. The chitosan hydrogel composition of claim 1, wherein the chitosan derivative is carboxymethyl chitosan having a molecular weight of 10 to 30 ten thousand.

3. The chitosan hydrogel composition of claim 1, wherein the dithiopyridine modified chitosan derivative is a small molecule with a dithiopyridine group attached to the amino group of the chitosan derivative.

4. The chitosan hydrogel composition of claim 1, wherein the chitosan derivative has 8 to 15 dithiopyridine functional groups per molecule, obtained by reacting the chitosan derivative with 2-iminothiolane hydrochloride and dithiopyridine in sequence, and the reduced bovine serum albumin is preferably obtained by reacting bovine serum albumin with tris (2-carbonylethyl) phosphonium hydrochloride.

5. A chitosan hydrogel formed by crosslinking the dithiopyridine-modified chitosan derivative of any one of claims 1 to 4 with reducing bovine serum albumin.

6. A method for preparing the chitosan hydrogel composition of any of claims 1 to 4, comprising the steps of:

2) adding dithiodipyridine into the solution, dialyzing with PBS buffer solution after the reaction is finished, and freeze-drying to obtain the dithiodipyridine modified chitosan derivative;

3) preparing the dithiopyridine modified chitosan derivative obtained in the step 2) into a buffer solution to obtain a first solution;

7. The method of claim 6, wherein the chitosan derivative has a molecular weight of 15 to 30 million and a mass ratio of 2-iminothiolane hydrochloride to dithiodipyridine of 1: 0.015 to 0.030: 0.86 to 0.18, and the chitosan derivative and bovine serum albumin are used in a mass ratio of 1: 0.03 to 0.05 in step 4).

8. Use of the chitosan hydrogel composition of any one of claims 1 to 4 in the preparation of a medical material for the treatment of myocardial infarction, wherein said medical material comprises chitosan hydrogel and basic fibroblast growth factor prepared in situ from said first and second solutions.

9. A pharmaceutical formulation comprising the chitosan hydrogel of claim 1 and one or more therapeutically active substances which are blood clotting agents.