CN109503863B

CN109503863B - Injectable hydrogel and preparation method and application thereof

Info

Publication number: CN109503863B
Application number: CN201811395263.1A
Authority: CN
Inventors: 柯晓; 彭长农; 王小庆
Original assignee: Shenzhen Sun Yixian Cardiovascular Hospital (shenzhen Institute Of Cardiovascular Diseases)
Current assignee: Shenzhen Sun Yixian Cardiovascular Hospital (shenzhen Institute Of Cardiovascular Diseases)
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2021-07-27
Anticipated expiration: 2038-11-21
Also published as: CN109503863A

Abstract

The invention discloses an injectable hydrogel, which is prepared from raw materials including chitosan, beta-sodium glycerophosphate and glucan. The invention also discloses a preparation method of the injectable hydrogel, which comprises the following steps: (1) adding chitosan into HCL solution for fully dissolving to obtain chitosan solution; (2) adding glucan into the chitosan solution prepared in the step (1), and fully dissolving to prepare a composite solution of glucan and chitosan; (3) providing a beta-sodium glycerophosphate aqueous solution, dropwise adding the beta-sodium glycerophosphate aqueous solution into the composite solution obtained in the step (2), and fully dissolving to obtain a composite sol of chitosan, beta-sodium glycerophosphate and glucan; (4) and (4) placing the composite sol obtained in the step (3) in a constant-temperature water bath for carrying out gel reaction to prepare the chitosan, beta-sodium glycerophosphate and glucan composite hydrogel, thus obtaining the chitosan-beta-sodium glycerophosphate-glucan composite hydrogel. The hydrogel has excellent biocompatibility, can be gelled into a solid state in situ when the temperature is raised to 37 ℃, and has good mechanical properties.

Description

Injectable hydrogel and preparation method and application thereof

Technical Field

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

Background

MI (myocardial infarction) is an acute and severe cardiac condition resulting from sudden interruption of blood circulation in a portion of the myocardium, which is damaged by the inability to obtain sufficient oxygen. Currently, poly (N-isopropylacrylamide) (PNIPAM) and injectable hydrogels formed by modifying the same are widely used for treating myocardial infarction. The hydrogel is a gel with a reticular structure and using water as a dispersion medium, has wide application in the biomedical field, and has been proved by a large number of reports in the literature to be very suitable for repairing myocardial infarction. The current studies show that the injectable hydrogels used in myocardial infarction include poly (N-isopropylacrylamide), hyaluronic acid, polyesters, etc. The hydrogel can provide an environment and a structure similar to extracellular matrix, has good biocompatibility, is often used as a carrier of cells, provides support and nutrition for cell growth, and is widely applied in tissue engineering.

Stem cells have the functions of self-replication and differentiation, and thus are often combined with biomaterials to repair damaged parts, so that the cells are regenerated. Stem cells have been developed for decades, and have been studied and applied to repair of injuries of various tissues of the human body, and in myocardial repair, BMSCs (bone marrow mesenchymal stem cells), BADSCs (brown adipose derived stem cells), hCSCs (human cardiac stem cells), and the like are available. Stem cells are an important component in tissue engineering and regenerative medicine. "Shangqingqing" tea^【1】And the like, provides a technical scheme that the hyaluronic acid hydrogel wraps the mesenchymal stem cells to improve the cardiac function of the rat after myocardial infarction, wherein the hyaluronic acid is formed into injectable solution through chemical crosslinkingThe water-jetting gel is retained in the damaged part of the heart by means of the viscosity of the water-jetting gel, and the mesenchymal stem cells of the bone marrow are proliferated and differentiated to generate new cardiac muscle cells, so that the cardiac function after the myocardial infarction is improved.

However, injectable hydrogels have various gelling mechanisms, gel is formed by chemical crosslinking, a crosslinking agent is needed, and the crosslinking agent has certain toxicity, so that the biocompatibility of the gel is affected, and the biocompatibility of the hydrogel is often not excellent enough; physical gel depends on hydrogen bonds, hydrophilicity and hydrophobicity, electrostatic force and the like during formation, acting force is weak, the gel formed through a physical mechanism is generally poor in mechanical property and mismatched with a damaged tissue, or the problem of poor stability exists, and the gel can spontaneously form after being placed for a long time; the stem cell technology has the defects that cells are difficult to obtain and culture in large quantities, the stem cells need to keep dry in the culture process, the requirement on the growth environment is high, and the like.

Reference documents:

1. shangqing, Zhou Jian, Likai and the like, the hyaluronic acid hydrogel wraps the mesenchymal stem cells to improve the cardiac function of the rat after myocardial infarction [ J ] Chinese tissue engineering research, 2018, 22(5) 675-679.

Disclosure of Invention

Based on the problems, the invention aims to overcome the defects of the prior art and provide an injectable hydrogel which has excellent biocompatibility, wherein hydrophobic bonds, hydrogen bonds and electrostatic force enable the hydrogel to have temperature sensitivity, the hydrogel is liquid at room temperature and can be mixed with UCMSCs, the hydrogel can be gelled into a solid state in situ when the temperature is raised to 37 ℃, the gelling time is short, the mechanical property is good, the preparation is simple, and the hydrogel is a drug carrier with great potential.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides an injectable hydrogel prepared from raw materials including chitosan, sodium beta-glycerophosphate and dextran. The injectable temperature-sensitive hydrogel is liquid hydrogel at room temperature, can be directly injected into a human body through a needle, and forms solid gel in situ at 37 ℃, so that the injectable temperature-sensitive hydrogel is an ideal tissue engineering material, has good mechanical property, is simple and convenient to operate, and reduces the injury of a patient caused by an operation.

Preferably, the mass ratio of the chitosan to the sodium beta-glycerophosphate to the glucan is 4: 10: 1 to 4.

Preferably, the feedstock also comprises hydrochloric acid.

Preferably, the mass ratio of the hydrochloric acid to the chitosan is 1: 6.

in a second aspect, the present invention provides a method for preparing the above hydrogel, comprising the steps of:

(1) adding chitosan into HCL solution for fully dissolving to obtain chitosan solution;

(2) adding glucan into the chitosan solution prepared in the step (1), and fully dissolving to prepare a composite solution of glucan and chitosan;

(3) providing a beta-sodium glycerophosphate aqueous solution, dropwise adding the beta-sodium glycerophosphate aqueous solution into the composite solution obtained in the step (2), and fully dissolving to obtain a composite sol of chitosan, beta-sodium glycerophosphate and glucan;

(4) and (4) placing the composite sol obtained in the step (3) in a constant-temperature water bath for carrying out gel reaction to prepare the chitosan, beta-sodium glycerophosphate and glucan composite hydrogel, thus obtaining the chitosan-beta-sodium glycerophosphate-glucan composite hydrogel. The CS/DEX/beta-GP (chitosan/glucan/beta-sodium glycerophosphate) injectable temperature-sensitive hydrogel prepared by the method has more excellent mechanical property and biological property, and is suitable for repairing the injury after myocardial infarction.

Preferably, the mass ratio of chitosan, sodium beta-glycerophosphate and dextran in the composite sol of the step (3) is 4: 10: 1 to 4. The inventor of the application finds that the survival rate of the cells co-cultured with the composite hydrogel exceeds 100% through a plurality of tests when the mass concentration of the chitosan, the beta-sodium glycerophosphate and the glucan in the composite hydrogel is 0.1-0.5 mg/ml.

Preferably, the mass ratio of the hydrochloric acid to the chitosan in the step (1) is 1: 6.

preferably, the temperature of the thermostatic water bath in the step (4) is 37 ℃.

In a third aspect, the present invention provides the use of the above hydrogel for the preparation of a medicament for the treatment of myocardial infarction or for the repair of myocardium.

In a fourth aspect, the present invention provides a medicament for treating myocardial infarction, which comprises the hydrogel according to the first aspect of the present invention and Umbilical Cord Mesenchymal Stem Cells (UCMSCs) loaded with the hydrogel. It should be noted that the stem cells adopted in the present invention are UCMSCs, and in the prior art, BMSCs (mesenchymal stem cells in bone marrow) are generally used, but the UCMSCs are derived from umbilical cord tissue, so that the risk of contamination by a source of disease is reduced, and the cell viability is guaranteed; secondly, the stem cells are very rich, the proliferation capacity is strong and is superior to BMSCs, the immunogenicity is lower than that of the BMSCs, the acquisition is easy, no damage is caused to the puerpera and the infant, and no ethical dispute exists; the UCMSCs have strong proliferation and differentiation capacities, have wide clinical application prospect in the aspect of tissue engineering, and can be applied to the heart injury repair of myocardial infarction.

In conclusion, the beneficial effects of the invention are as follows:

the hydrogel material has injectability, the chitosan and the glucan enable the hydrogel material to have excellent biocompatibility, hydrophobic bonds, hydrogen bonds and electrostatic force enable the gel to have temperature sensitivity, the hydrogel material is liquid at room temperature and can be mixed with UCMSCs, when the temperature is raised to 37 ℃, the hydrogel material can be subjected to in-situ gelling to be solid, the gelling time is short (only 3min), the preparation is simple, and the hydrogel material is a drug carrier with great potential.

Drawings

FIG. 1 is a flow chart of a hydrogel test of the present invention;

FIG. 2 is a schematic diagram of the liquid-solid transition of a hydrogel;

FIG. 3 is a Fourier infrared spectrum of the product of each step;

FIG. 4 is a CS/2.0Dex/β -GP rheometer (temperature-modulus);

FIG. 5 is a scanning electron micrograph of a UCMSCs-loaded hydrogel scaffold;

FIG. 6 is a graph showing the statistical results of cell activities after co-culturing composite hydrogels with different concentrations and cells.

Detailed Description

The invention relates to the field of tissue engineering and regenerative medicine, and the inventor of the application develops a novel injectable hydrogel by utilizing chitosan, glucan and beta-sodium glycerophosphate, wherein the novel injectable hydrogel is an injectable temperature-sensitive natural biological material and is used for carrying UCMSCs (umbilical cord mesenchymal stem cells) to repair heart injury after myocardial infarction. The hydrogel coated stem cells are injected into a body, and damaged myocardial tissues can be repaired and regenerated due to the characteristics of the stem cells. The hydrogel is prepared by adopting chitosan and adding glucan for modification and carrying out physical crosslinking, has more excellent biocompatibility and simple preparation process compared with PNIPAM, and is suitable for future industrial development.

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the concentrations of the reagents in the present invention are mass concentrations.

Example 1

One embodiment of the preparation method of the injectable temperature-sensitive hydrogel CS/beta-GP/DEX (chitosan/beta-sodium glycerophosphate/dextran) comprises the following steps:

1) diluting concentrated laboratory hydrochloric acid, and preparing enough 0.1mol/L HCL solution;

2) accurately weighing 400mg of chitosan, dissolving the chitosan in 18ml of 0.1mol/L hydrochloric acid, stirring and dissolving the chitosan overnight to prepare a chitosan solution;

3) accurately weighing 500mg of beta-GP, dissolving the beta-GP in a certain amount of deionized water, stirring, shaking, fully dissolving, adding a proper amount of deionized water, fixing the volume to 1ml, preparing a beta-GP aqueous solution, and standing for 1h at 4 ℃;

4) accurately weighing 10mg, 20mg, 30mg and 40mg of glucan, respectively dissolving the glucan in 1.8ml of CS solution, stirring the solution to be fully dissolved, and then placing the solution at 4 ℃ for 1 h;

5) sucking 0.2ml of GP solution in the step 3), slowly adding the GP solution into the CS/DEX composite solution, stirring while dripping, and stirring for 10 minutes to respectively prepare CS/DEX/beta-GP composite sol with the concentration of 0.5W/V%, 1.0W/V%, 1.5W/V% and 2.0W/V%;

6) placing the CS/DEX/beta-GP composite sol in a constant temperature water bath at 37 ℃ for gel reaction to prepare the CS/DEX/beta-GP composite hydrogel.

Example 2 use of the hydrogel obtained in example 1

(1) Extraction and culture of UCMSCs (umbilical cord mesenchymal stem cells)

1) Taking umbilical cord blood of healthy full-moon newborn: after the umbilical cord is collected, the umbilical cord is treated within 6h, the part with clamping marks and extravasated blood on both sides is cut off, and the periphery of the umbilical cord and the inner cavity of umbilical vein are fully washed by PBS buffer solution containing double antibodies;

2) culturing UCMSCs by a planting method: cutting blood vessels along the lumen of the umbilical vein longitudinally, peeling off the intima of the umbilical vein, and cutting the remaining tissue into 3.0-5.0 mm³The small pieces were attached to the bottom wall of a T25 flask previously moistened with 1mL of complete medium (DF 12 medium containing 2% by volume of fetal bovine serum, 40% MCDB201, 10. mu.g/L of platelet-derived growth factor, 10. mu.g/L of basic fibroblast growth factor, 10. mu.g/L of epidermal growth factor), the bottom wall was faced upward, and the flask was placed at 37 ℃ in a 5% by volume CO atmosphere₂Turning over after staying overnight in a saturated humidity incubator;

3) 1mL of the above medium was added every 24 hours, and the whole volume was changed for 72 hours, and then 2 times per week. Observing the climbing condition of adherent cells around the sticking block, and removing the sticking block after 2 weeks;

4) colonies were counted on day 14 of culture and over 50 cells counted as colonies. When the cell is fused to 70-80%, digesting with digestive juice containing 1.25g/L trypsin and 1g/L EDTA, and then (2.5-5.0) × 10³/cm²The density is inoculated and subcultured, and counted as the 1 st generation (P1) cells;

5) the cells after passaging were designated as P2, P3 and P4 … by continuing the culture in the complete medium

(2) Construction of animal model and animal experiment

1) Pre-sterilized hydrogel (hydrogel prepared in example 1) was mixed with UCMSCs for use as a treatment group;

2) 70 male Wistar rats with the age of 4 weeks are provided, and the mass is 180-220 g. Animals were randomly divided into 5 groups, sham (n-10), PBS (n-15), PBS + UCMSCs (n-15), hydrogel + UCMSCs (n-15);

3) after anesthetizing a rat by intraperitoneal injection of 2% pentobarbital sodium (20mg/kg), preparing skin, performing tracheal intubation, opening the chest in the middle, after exposing the heart, threading and ligating the PBS group, the PBS + UCMSCs group, the hydrogel group and the hydrogel + UCMSCs group at the near end of the left anterior descending branch by using a 5-0 non-invasive silk thread, and not ligating the group in a sham operation group;

4) after successful molding, 75 μ L of liquid was injected into the infarcted myocardium border in 3 portions as follows:

the PBS group was injected with 75 μ L PBS;

the PBS + UCMSCs group was injected with 75 μ L of PBS containing UCMSCs;

the hydrogel group was injected with 75 μ L of hydrogel;

the hydrogel + UCMSCs group was injected with 75 μ L of UCMSCs and hydrogel cocktail.

And then the chest wall is sutured layer by layer, the thoracic cavity is closed by negative pressure air suction, and after the animal recovers spontaneous respiration, the cannula is pulled out, and the respirator is stopped. Intramuscular injection 50X 10⁴U/d penicillin 3d to prevent infection;

5) the myocardial recovery condition is represented by parameters such as an echocardiogram, an electrocardiogram, LVEF (left ventricular ejection fraction) and LVFS (left ventricular shortening rate);

6) immunohistochemistry, tissue section and other experiments show that the UCMSCs are differentiated, the area of the myocardial infarction area is equal.

EXAMPLE 3 hydrogel Performance testing prepared in example 1

The test content is shown in the flow chart of fig. 1.

(1) Fourier Infrared Spectroscopy testing

FTIR Fourier infrared spectrum is a spectrum showing molecular vibration, can identify functional groups in a substance to be detected, and can prove whether a target product is successfully synthesized or not by performing infrared test on products and raw materials obtained in each step.

A proper amount of a substance to be tested (namely the hydrogel prepared in example 1, 2.0W/V% of CS/DEX/beta-GP) and potassium bromide are taken, ground and tabletted, and analyzed by a spectrometer, and the obtained result is shown in figure 3.The CS curve: 3000cm^-1-3500cm^-1Is O-H telescopic vibration absorption band with amino group at 3400cm^-1-3500cm^-1Vibration of 2872cm in extension and contraction^-1Respectively are the stretching vibration absorption peak of aliphatic C-H; 1674cm^-1C is an O stretching vibration absorption peak; 1604cm^-1Is the N-H deformation vibration absorption peak; 1383cm^-1Is the symmetric deformation vibration absorption peak of C-CH 3; 1322cm^-1Is a C-N stretching vibration absorption peak; 1251cm^-1Is C-O stretching vibration absorption peak on six-membered ring: 1086cm^-1Is the C-O stretching vibration absorption peak. beta-GP of 3365cm^-1Is an O-H stretching vibration peak of 1080cm^-1The position is an asymmetric telescopic vibration absorption peak of a phosphate radical; 968cm^-1The symmetric stretching vibration of phosphate radical is adopted. DEX O-H stretching vibration corresponds to 3340cm^-1Absorption peak at position, stretching vibration of C-H bond to 2922cm^-1Generates a characteristic absorption peak at 1641cm^-1The absorption peaks at (b) correspond to the C ═ O bonds of — CHO in the sample, and these three characteristic peaks indicate that DEX has characteristic absorption peaks of carbohydrates.

CS/DEX/β -GP: after DEX was added, the O-H and N-H absorption peaks red shifted, indicating hydrogen bonding with dextran. Bending vibration of C-H bond 1446cm^-1Reduced to 1427cm^-1Indicating that chitosan forms hydrogen bonds with the dextran molecular chain. In addition, CS/DEX/beta-GP is at 1000-1500cm^-1The absorption peak was broader, probably due to interaction between DEX and CS and β -GP.

(2) The rotational rheometer can characterize the product modulus, G' the elastic modulus of the sample, and G "the viscous modulus of the sample. When G '> G' is present, a gel is considered to form, as can be seen in FIG. 4, CS/2.0Dex/β -GP forms a gel at 37 ℃; as can be seen from FIG. 2, the CS/2.0Dex/β -GP [ wherein CS is 2.0 (W/V)%, GP is 5.0 (W/V)%, and dextran Dex is 2.0 (W/V)% ] hydrogel changed from a liquid state to a solid state due to the change of temperature.

(3) Stem Cells (UCMSCs) were grown on the scaffolds (i.e., the hydrogel prepared in example 1) by adhesion, and the morphology of the cells and the scaffolds (i.e., the hydrogel) was determined by field emission scanning electron microscopy (fig. 5).

(4) Cell proliferation and toxicity detection by the hydrogel of the present invention

The detection method comprises the following steps: cells and material (hydrogel prepared by the method of example 1) were co-cultured, and Cell Counting Kit-8 (CCK-8 for short) reagent was used to analyze Cell proliferation and toxicity. The method comprises the following specific steps:

1) taking umbilical cord blood of healthy full-moon newborn: the umbilical cord is treated within 6h after being collected, the part with clamping marks and extravasated blood on both sides is cut off, and the periphery of the umbilical cord and the inner cavity of umbilical vein are fully washed by PBS buffer solution containing double antibodies.

2) Culturing UCMSCs by using a planting method: cutting blood vessels along the lumen of the umbilical vein longitudinally, peeling off the intima of the umbilical vein, and cutting the remaining tissue into 3.0-5.0 mm³The small pieces were attached to the bottom wall of a T25 flask previously moistened with 1mL of complete medium (DF 12 medium containing 2% by volume of fetal bovine serum, 40% MCDB201, 10. mu.g/L of platelet-derived growth factor, 10. mu.g/L of basic fibroblast growth factor, 10. mu.g/L of epidermal growth factor), the bottom wall was faced upward, and the flask was placed at 37 ℃ in a 5% by volume CO atmosphere₂In a saturated humidity incubator, turned over overnight. 1mL of the above medium was added every 24 hours, and the whole volume was changed for 72 hours, and then 2 times per week.

3) And (5) observing the climbing condition of adherent cells around the patch, and removing the patch after 2 weeks. Colonies were counted on day 14 of culture and over 50 cells counted as colonies. When the cell is fused to 70-80%, digesting with digestive juice containing 1.25g/L trypsin and 1g/L EDTA, and then (2.5-5.0) × 10³/cm²The density was inoculated and subcultured, and counted as the 1 st (P1) cell. After further culturing in the complete medium, the cells after passaging were designated as P2, P3 and P4.

The experimental results are shown in fig. 6, and it can be seen from the figure that the survival rate of the cells (UCMSCs) is above 90%, which indicates that the hydrogel material of the present invention has good biocompatibility and no toxicity to the cells (UCMSCs).

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An injectable hydrogel, characterized in that the hydrogel is prepared by a method comprising the steps of:

(1) adding chitosan into a hydrochloric acid solution for fully dissolving to prepare a chitosan solution;

(4) placing the composite sol obtained in the step (3) in a thermostatic water bath at 37 ℃ for gel reaction to prepare chitosan, beta-sodium glycerophosphate and glucan composite hydrogel;

the mass ratio of the chitosan, the beta-sodium glycerophosphate and the glucan in the composite sol obtained in the step (3) is 4: 10: 1-4;

the mass ratio of the hydrochloric acid to the chitosan in the step (1) is 1: 6.

2. use of the hydrogel of claim 1 for the preparation of a medicament for treating myocardial infarction or repairing myocardium.

3. A drug for treating myocardial infarction, which comprises the hydrogel according to claim 1 and umbilical cord mesenchymal stem cells loaded with the hydrogel.