CN108478852A - A kind of pharmaceutical products, the carrier for loading pharmaceutical products, hydrogel and its method - Google Patents

Abstract

A pharmaceutical product, a carrier for loading the pharmaceutical product, a hydrogel and a method thereof, belonging to the gel field. Hydrogel is a transparent biomaterial that can be injected by cross-linking proteins under the action of enzymes. It has good elasticity and biocompatibility, and has broad application prospects.

Description

A pharmaceutical product, carrier for loading pharmaceutical products, hydrogel and method thereof

technical field

The invention relates to the field of gel materials, in particular to a hydrogel and a method thereof.

Background technique

Silk fibroin is rich in sources and low in cost. Its main components are amino acids, and it also has non-irritating, good dissolved oxygen permeability, and controllable biodegradability. Silk fibroin can support various cell adhesion, growth and differentiation in vitro; in vivo, silk fibroin also has good tissue compatibility. Therefore, silk fibroin has a wide range of applications in the fields of tissue engineering, biomedicine, drug sustained release and optical imaging. Recently, silk fibroin has been approved by the FDA for clinical use. However, the untreated regenerated silk fibroin material is easily soluble in water due to its large random coil structure, and it needs to be modified by physical or chemical methods before it can be practically applied. After physical modification (ethanol immersion, pH, high temperature and metal ions, etc.), the silk fibroin film is more brittle, has poor flexibility, and cannot be bent and folded. The chemical treatment method can effectively improve the stability and mechanical properties of the film by forming a covalently bonded network structure within and between the silk fibroin molecules. However, common chemical cross-linking reagents such as glutaraldehyde and diglycidyl ether can cause certain cytotoxicity, which limits their application in the field of biomaterials.

With the deepening of biotechnology research on the functional modification of polymer materials, the research reports on improving the properties of silk fibroin materials by enzymatic technology are not uncommon. Chinese invention patent "A method for preparing silk fibroin/elastin composite membrane material by enzymatic method" (publication number CN 105218842A) uses β-tyrosinase to convert amino acids in silk fibroin, increasing tyrosine residues in silk fibroin The amount of silk fibroin/elastin film was prepared by tyrosinase-catalyzed oxidation of silk fibroin and elastin cross-linking. However, phenol is used in the amino acid conversion process, and its residue will affect the biocompatibility of the material.

Hydrogel is a special material with a three-dimensional polymer network structure, which can absorb a large amount of water, swell and keep the original structure from being dissolved. At present, it is widely used in drug sustained release, tissue engineering materials, artificial skin, injectable materials, etc. At present, silk fibroin hydrogels can be prepared by physical methods (ultrasound, changing pH, surfactant induction, etc.) The glue takes a long time, the β-sheet structure increases, and finally forms a milky white hydrogel with poor elasticity. The hydrogel formed by chemical method has better elasticity, but the chemical small molecules brought into the system need to be removed twice, and the biocompatibility is poor, so it is difficult to be used as a biomaterial that can be injected into the body.

The information disclosed in this background section is only intended to enhance the understanding of the general background of the invention, and should not be considered as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art.

Contents of the invention

Based on the deficiencies of the prior art, the present invention provides a pharmaceutical product, a carrier for loading the pharmaceutical product, a hydrogel and a method thereof, so as to partially or completely improve or even solve the above problems.

The present invention is achieved like this:

In a first aspect, embodiments of the present invention provide a hydrogel.

Hydrogel is a biomaterial that can be used by injection. The hydrogel is formed by cross-linking the protein contained in the raw material by using a complex cross-linking agent. Among them, the protein contains tyrosine, and the complex cross-linking Agents include horseradish peroxidase (Peroxidase Horseradish, HRP) and hydrogen peroxide.

In the second aspect, the embodiments of the present invention provide a carrier for carrying medical products.

The carrier for loading medical products is made of hydrogel, wherein the medical products include one or more of antibacterial drugs, analgesics and growth factors.

In a third aspect, the embodiments of the present invention provide a pharmaceutical preparation.

Pharmaceutical products are made of hydrogel, and pharmaceutical products include dressings, injectable materials in vivo, fillers in vivo, tissue repair materials, and isolation materials.

In a fourth aspect, embodiments of the present invention provide a method for preparing a hydrogel.

The preparation method comprises: adding horseradish peroxidase to the dispersion liquid in which the protein obtained from raw materials is dispersed to dissolve the protein, and then adding hydrogen peroxide to carry out cross-linking reaction.

Beneficial effect:

The hydrogel provided in the embodiment of the present invention is an enzyme-crosslinked hydrogel, which has certain transparency, injectability, good elasticity and good cell compatibility. The hydrogel can be directly used on the surface of various wounds (such as burns, ulcers, bedsores, etc.) to promote wound healing and isolation protection. In addition, hydrogel can also be used as a drug carrier, loaded with various antibacterial drugs, analgesics and growth factors, to release drugs slowly, and to prevent infection, relieve pain and promote healing of wounds. Among the materials used to make the hydrogel, both the matrix (protein-containing raw material) and the enzymatic cross-linking components can be and are preferably natural biological sources, so that the hydrogel can be obtained safely, reliably and conveniently sterilized. Moreover, it can be used as a medical dressing, an injectable material in the body, a filler in the body, and used in tissue repair and other fields, and has a good application prospect.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Fig. 1 is the physical figure of the silk fibroin hydrogel of embodiment 1～5 of the present invention;

Fig. 2 is the gelation time figure of the hydrogel material in the embodiment of the present invention 1～3;

Fig. 3 is a light transmittance diagram of hydrogels in different states in Example 2 of the present invention.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The pharmaceutical products, the carrier for loading the pharmaceutical products, the hydrogel and the method thereof are described in detail below:

The hydrogel provided by the embodiment of the present invention is a biomaterial that can be used in an injection. The hydrogel is formed by cross-linking the protein contained in the raw material by using a composite cross-linking agent, wherein the protein contains tyrosine, and the composite cross-linking agent includes horseradish peroxidase and hydrogen peroxide.

Among the raw materials for making hydrogels, horseradish peroxidase, assisted by a small amount of hydrogen peroxide, can catalyze the tyrosine residues in silk fibroin to generate phenolic oxygen free radicals, triggering cross-linking between silk fibroin molecules. In a short time, the reaction system can form a gel.

The raw material therein can be various biological materials, for example, silk, which can be derived from different types of silkworms such as silkworm, tussah silkworm, and celestial silkworm. The raw material may not only be silk as a raw material, but also a product processed from silk, for example, one or more of silk fibroin, collagen, and gelatin.

As mentioned above, the hydrogel provided by the embodiments of the present invention has broad application prospects in the fields of medicine and pharmacy.

For example, in some examples, the hydrogel is used as a carrier, and the obtained hydrogel is directly shaped or made into a carrier with a certain shape by adding other pharmaceutically acceptable excipients. By loading the pharmaceutical products on the carrier, the medication efficiency and safety of the pharmaceutical products can be improved. The pharmaceutical products include one or more of antibacterial drugs, pain relievers and growth factors. The aforementioned loading of the pharmaceutical product on the carrier may be directly dispersing (coating, spraying, etc.) the pharmaceutical product (in the form of powder, tablet, spray, etc.) on the surface or inside of the hydrogel.

In addition, in addition to using the hydrogel as a carrier as described above, the hydrogel can also be directly used as a pharmaceutical product. That is, the pharmaceutical product is made of hydrogel. Among them, pharmaceutical products include dressings, injectable materials in the body, fillers in the body, tissue repair materials, and isolation materials.

In the embodiment of the present invention, a method for making the hydrogel is also provided. The preparation method comprises: adding horseradish peroxidase to the dispersion liquid in which the protein obtained from raw materials is dispersed to dissolve the protein, and then adding hydrogen peroxide to carry out cross-linking reaction.

In some examples, the aforementioned dispersion liquid is an aqueous solution of silk fibroin, the raw material is silk, and the protein is silk fibroin. In particular, the aqueous silk fibroin solution can be prepared by dissolving degummed silk in water.

When silk is used as the raw material for making the hydrogel, the method for dissolving degummed silk in water includes: dissolving silk fibroin fibers obtained after degumming silk in a solvent, wherein the solvent is lithium bromide aqueous solution or ternary aqueous solution.

In an alternative example, the ternary aqueous solution consists of calcium chloride, ethanol and water. Preferably, the ternary aqueous solution is prepared from calcium chloride, ethanol and water in a molar ratio of 1:2:8. The concentration of the lithium bromide aqueous solution can be selected to be 9.3 mol/L.

In addition, in order to promote the dissolution of silk fibers, in an optional example, silk fibers are dissolved in a solvent under heating conditions, wherein the heating temperature is 50-80°C, more preferably 50-70°C. °C, most preferably 70 to 75 °C.

The aforementioned crosslinking reaction is preferably carried out under heating conditions. Preferably, heating reaction is carried out after adding hydrogen peroxide, and the conditions of heating reaction are: temperature 10-50°C, silk fibroin concentration 1-8%, horseradish peroxidase 5-20U/mL, hydrogen peroxide 0.05-10mg/mL, The time is 10-120 minutes.

More preferably, after the hydrogen peroxide is added and before the catalyzed cross-linking reaction, the preparation method further includes vacuum defoaming the blended system of the dispersion liquid, horseradish peroxidase and hydrogen peroxide.

It should be noted that in the process of making the hydrogel, the transparency and elastic modulus of the hydrogel can be adjusted according to the concentration of silk fibroin. At the same time, the cross-linking reaction time in the production process can be controlled by the concentration of horseradish peroxidase, hydrogen peroxide concentration and action temperature.

As an example, silk is used to make hydrogels.

The method comprises the following steps: (a) degumming natural silk, dissolving the degummed silk with lithium bromide or a ternary solution, and then dialysis, filtering and concentrating to obtain silk fibroin aqueous solution (stock solution); (b) preparing ( Dilute stock solution) a certain concentration of silk fibroin aqueous solution, add horseradish peroxidase and hydrogen peroxide aqueous solution in turn, and mix evenly; (c) after vacuum degassing the blend system, put it under a certain temperature condition to obtain silk fibroin hydrogel glue.

Wherein, the preparation method of silk fibroin aqueous solution in the step (a) is specifically: place the silk fibroin fiber after degumming in CaCl2-ethanol-water ternary solution or 9.3mol/L lithium bromide solution, heating makes the temperature of solution be controlled at 50 ~80°C, stirred until dissolved, then dialyzed with deionized water for 36~72h, filtered, centrifuged to remove impurities, air-dried and concentrated to obtain silk fibroin aqueous solution, the CaCl ₂ -ethanol-water ternary solution contained CaCl ₂ , ethanol The ratio of the material quantity of water and water is 1:2:8.

According to the above scheme, the mass concentration of silk fibroin in step (b) is 1-8%, horseradish peroxidase is 5-20 U/ml, and hydrogen peroxide is 2-20 mg/ml.

According to the above scheme, in step (c), stir well at room temperature and then vacuum defoam, inject the mixed solution into the mold, and process at a temperature ranging from 10 to 50°C for 10 to 120 minutes to obtain a shaped silk fibroin hydrogel.

According to the test, the inventors believe that the hydrogel provided by the embodiments of the present invention has various advantages including but not limited to the following:

(1) The natural renewable polymer material silk fibroin is used as the raw material. The material has a wide range of sources and is a natural degradable polymer. The degradation product has good biocompatibility and low immunogenicity.

(2) Using enzymatic cross-linking, the phenolic oxygen free radicals generated by tyrosine residues form stable covalent cross-links between silk fibroin macromolecules, which promotes the rapid formation of silk fibroin hydrogels, It has good stability and expands its application range in biological materials.

(3) The silk fibroin hydrogel finally prepared in the present invention can obtain elastic transparent injectable hydrogel through controlled release preparation conditions.

(4) The preparation process of the injectable hydrogel provided by the present invention is simple and easy, the conditions are easy to control, and it can be sterilized by filtration, so it has a good application prospect.

The pharmaceutical product, the carrier for loading the pharmaceutical product, the hydrogel and the method thereof of the present invention will be further described in detail in conjunction with the examples below.

Example 1

A silk fibroin hydrogel material is prepared by the following preparation method.

Firstly, the natural silk is degummed, dissolved, dialyzed, filtered and concentrated to obtain silk fibroin with a mass fraction of 4%. Among them, the dissolution process is to mix the silk fiber obtained after degumming with a ternary solution composed of CaCl ₂ -ethanol-water, and keep it warm for 0.5h at a temperature of 75°C _; The amount ratio of substances is 1:2:8.

Secondly, take the silk fibroin solution with a mass fraction of 1%, add horseradish peroxidase and hydrogen peroxide successively therein under slow stirring, wherein the final concentration of horseradish peroxidase in the blended solution is 10U/ml, hydrogen peroxide The concentration is 0.6mg/ml.

Again, the blended solution was quickly defoamed in vacuum and placed in a 37°C water bath. After a period of time, silk fibroin hydrogel can be obtained.

Example 2

A silk fibroin hydrogel material is prepared by the following preparation method.

Firstly, the natural silk is degummed, dissolved, dialyzed, filtered and concentrated to obtain silk fibroin with a mass fraction of 4%. Among them, the dissolution process is to mix the silk fiber obtained after degumming with a ternary solution composed of CaCl ₂ -ethanol-water, and keep it warm for 0.5h at a temperature of 75°C _; The amount ratio of substances is 1:2:8.

Secondly, take the silk fibroin solution with a mass fraction of 2%, add horseradish peroxidase and hydrogen peroxide successively therein under slow stirring, wherein the final concentration of horseradish peroxidase in the blended solution is 10U/ml, hydrogen peroxide The concentration is 0.6mg/ml.

Again, the blended solution was quickly defoamed in vacuum and placed in a 37°C water bath. After a period of time, silk fibroin hydrogel can be obtained.

Example 3

A silk fibroin hydrogel material is prepared by the following preparation method.

Firstly, the natural silk is degummed, dissolved, dialyzed, filtered and concentrated to obtain silk fibroin with a mass fraction of 4%. Among them, the dissolution process is to mix the silk fiber obtained after degumming with a ternary solution composed of CaCl2-ethanol-water, and keep it warm for 0.5h at a temperature of 75°C; in the ternary solution, the content of CaCl2, ethanol and water The volume ratio is 1:2:8.

Secondly, take the silk fibroin solution with a mass fraction of 3%, add horseradish peroxidase and hydrogen peroxide successively therein under slow stirring, wherein the final concentration of horseradish peroxidase in the blended solution is 10U/ml, hydrogen peroxide The concentration is 0.6mg/ml.

Again, the blended solution was quickly defoamed in vacuum and placed in a 37°C water bath. After a period of time, silk fibroin hydrogel can be obtained.

Comparative example 1

A kind of hydrogel, its production process is the same as embodiment 3, and the difference is that hydrogen peroxide is not added.

Comparative example 2

A kind of hydrogel, its production process is the same as embodiment 3, and difference is not to add horseradish peroxidase and hydrogen peroxide.

Test example 1

The macroscopic morphology of the hydrogels obtained in Examples 1-3 and Comparative Examples 1-2 was tested by conventional methods, and the results are shown in FIG. 1 . As shown in the figure, the silk fibroin hydrogels obtained in Examples 1-3 have high transparency, while the silk fibroin hydrogels obtained in Comparative Examples 1 and 2 are milky white. Through artificial measurement, the thickness of its hydrogel is about 5mm.

The three figures in the upper part of Fig. 1 show the transparent effect of the hydrogel prepared in the preferred example of the present invention.

Among the five figures in the lower part of Fig. 1, 1-1% SF represents the silk fibroin solution that adopts 1% mass fraction to make hydrogel in embodiment 1; 2-2% SF represents that adopts 2% mass fraction silk fibroin in embodiment 2 Silk fibroin solution makes hydrogel; 3-1%SF means adopting the silk fibroin solution of 3% mass fraction in embodiment 3 to make hydrogel; 4-3%SF means adopting the silk fibroin of 3% mass fraction in comparative example 1 Solution to make hydrogel; 5-3% SF means using 3% mass fraction of silk fibroin solution to make hydrogel in Comparative Example 2. 3D means 3 days, SF means silk fibroin.

Test example 2

The gel times of Examples 1-3 were recorded by the inversion method, and the results are shown in Figure 2, while the gel times (37°C) of Comparative Examples 1 and 2 were 40h and 42h respectively (not listed in the figure). It can be seen from the figure that the enzymatically catalyzed cross-linking reaction significantly shortens the gelation time of silk fibroin and improves the injectability of silk fibroin materials.

Test example 3

A T9CS ultraviolet-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd.) was used to measure the full-band transmittance of visible light in different states of Example 2. Among them, deionized water was used as the control group. It can be seen from the spectrogram that in the range of visible light, the light transmittance of silk fibroin hydrogel is relatively high, ranging from 50% to 80%.

The research results show that the gel time of silk fibroin hydrogel prepared by enzymatic catalytic cross-linking method is obviously shortened, and the transparency of the formed hydrogel is high. This kind of transparent silk fibroin hydrogel material prepared by the present invention expands the application range of silk fibroin biomedical materials, and can be used as medical dressings, injectable materials in vivo, fillers in vivo, and tissue repair and other fields, and has good properties. Application prospect.

While particular embodiments of the invention have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. a kind of hydrogel, which is characterized in that the hydrogel is a kind of biomaterial that can be used in a manner of injection, The hydrogel is so that the protein cross being contained in raw material is formed by using multiple crosslinking agent, wherein the protein Containing tyrosine, the multiple crosslinking agent includes horseradish peroxidase and hydrogen peroxide.

2. hydrogel according to claim 1, which is characterized in that the raw material includes silk.

3. hydrogel according to claim 1, which is characterized in that the protein includes in fibroin, collagen and gelatin It is one or more.

4. a kind of for loading the carriers of pharmaceutical products, which is characterized in that the pharmaceutical products include antimicrobial, anodyne and It is one or more in growth factor, the carrier by as described in any one of claims 1 to 3 hydrogel make and At.

5. a kind of pharmaceutical products, which is characterized in that the pharmaceutical products are by the water as described in any one of claims 1 to 3 Gel is made, and the pharmaceutical products include dressing, internal injectable materials, internal filler, tissue renovation material, isolation Material.

6. a kind of production method of hydrogel as described in any one of claims 1 to 3, which is characterized in that the making Method includes：To be dispersed be added from the dispersion liquid of the protein obtained in the raw material horseradish peroxidase make it is described Proteolytic adds hydrogen peroxide and carries out cross-linking reaction.

7. the production method of hydrogel according to claim 6, which is characterized in that the dispersion liquid is silk fibroin aqueous solution, The raw material is silk, and the protein is fibroin, and the silk fibroin aqueous solution makes by the following method：Make described after degumming Silk is dissolved in water；

Preferably, the method that the silk after degumming is dissolved in water is made to include：It is fine by fibroin is obtained after the natural silk degumming Dimension is dissolved in solvent, and the solvent is lithium bromide water solution or ternary aqueous solution, and the ternary aqueous solution is by calcium chloride, ethyl alcohol And water composition, it is highly preferred that the fibroin fiber is to be dissolved in the solvent under heating conditions, wherein heating Temperature is 50~80 DEG C, further preferably 50~70 DEG C, most preferably 70~75 DEG C.

8. the production method of hydrogel according to claim 7, which is characterized in that the ternary aqueous solution is by chlorination Calcium, ethyl alcohol and water are according to 1:2:8 molar ratio forms, a concentration of 9.3mol/L of the lithium bromide water solution.

9. the production method of hydrogel according to claim 7, which is characterized in that carry out cross-linking reaction after hydrogen peroxide is added It is to carry out under heating conditions, and the condition for heating reaction is：

10~50 DEG C of temperature, fibroin be a concentration of 1~8%, 5~20U/mL of horseradish peroxidase, 0.05~10mg/mL of hydrogen peroxide, 10~120min of time.

10. the production method of hydrogel according to claim 9, which is characterized in that after hydrogen peroxide is added, urged Before changing cross-linking reaction, the production method further includes by the dispersion liquid, the horseradish peroxidase and the hydrogen peroxide Co-mixing system handled by vacuum defoamation.