阅读说明：本技术 一种可长效药物缓释的丝素蛋白支架材料的制备方法 (Preparation method of silk fibroin scaffold material capable of realizing long-acting drug sustained release ) 是由 敖英芳 孙牧旸 胡晓青 于 2019-09-26 设计创作，主要内容包括：本发明涉及一种可长效药物缓释的丝素蛋白支架材料的制备方法,利用液氮速冻制得的丝素蛋白冷冻海绵作为缓释药物的载体,利用丝素蛋白低温自组装的特性实现药物包裹与缓释,并根据从丝素蛋白溶液制备到最终药物加载的技术路线,即差式热实验寻找玻璃化转变温度-药物添加-低温冷冻保存-解冻及使用,实现药物的长期可控缓释。同其他载药形式相比,本技术的实施方案无毒性添加成分,不需要化学交联剂和有机溶剂,载药率极高,并且本技术方案的制备工艺简单可控,适合大规模工业推广。(The invention relates to a preparation method of a silk fibroin scaffold material capable of realizing long-acting drug slow release, which utilizes a silk fibroin frozen sponge prepared by liquid nitrogen quick freezing as a carrier for slow release of a drug, utilizes the characteristic of low-temperature self-assembly of silk fibroin to realize drug wrapping and slow release, and realizes long-term controllable slow release of the drug according to the technical route from preparation of a silk fibroin solution to final drug loading, namely, differential thermal experiment searching for glass transition temperature, drug addition, low-temperature cryopreservation, thawing and use. Compared with other medicine carrying forms, the embodiment of the technology has no toxic additive components, does not need chemical cross-linking agents and organic solvents, has extremely high medicine carrying rate, and the preparation process of the technical scheme is simple and controllable, and is suitable for large-scale industrial popularization.)

1. A preparation method of a silk fibroin scaffold material capable of realizing long-acting drug slow release is characterized by comprising the following steps:

1) extraction of silk fibroin

Adding silkworm cocoon into boiled 0.01-0.03mol/L sodium carbonate solution, keeping boiling state, stirring and dissolving for 20-40min, taking out solid matter, washing with water, and drying at room temperature to obtain silk fibroin;

according to the mass ratio of silk fibroin to lithium bromide of 1: 4 weighing lithium bromide and preparing 9.3mol/L lithium bromide water solution;

adding a lithium bromide aqueous solution into a container with silk fibroin, completely covering the silk fibroin with the lithium bromide aqueous solution, and stirring at 50-70 ℃ for 3-5 h;

dialyzing the obtained brown viscous solution, centrifuging the collected liquid, and keeping the supernatant;

quickly freezing the supernatant with liquid nitrogen, and freeze-drying to obtain spongy silk fibroin;

2) phase transition temperature interval determination

According to the set concentration, the spongy silk fibroin is dissolved into a corresponding aqueous solution or salt solution and is sterilized for later use;

measuring the glass transition temperature of the spongy silk fibroin solution by a differential thermal experiment to obtain a phase transition temperature interval, wherein the temperature interval is usually different from 0 ℃ to-20 ℃;

3) preparation of silk fibroin scaffold material

Dissolving the silk fibroin sponge in the step 2) according to the corresponding concentration according to the requirement, blending the set medicine and the silk fibroin solution, and placing the obtained phase transition temperature interval obtained by a differential thermal experiment into the corresponding temperature for low-temperature freezing denaturation to obtain the silk fibroin scaffold material capable of slowly releasing the long-acting medicine.

2. The preparation method of the silk fibroin scaffold material capable of sustained release of long-acting drugs according to claim 1, wherein the preparation of the 9.3mol/L lithium bromide solution in step 1) comprises calculating the volume of the lithium bromide solution, weighing 60% of water for ice bath, slowly adding the lithium bromide into water while stirring, and supplementing the water to the calculated value after the lithium bromide is completely dissolved.

3. The preparation method of the silk fibroin scaffold material that can release drug slowly for a long time according to claim 1, wherein the dialysis method in step 1) is to change water every 12h, collect dialysate after three days, centrifuge twice at 4 ℃ and 9000rpm, and collect supernatant.

4. The method for preparing the silk fibroin scaffold material capable of releasing the drug slowly for the long term according to claim 1, wherein the sterilization in the step 2) is ultraviolet sterilization or cobalt 60 radiation sterilization.

5. The method for preparing the silk fibroin scaffold material capable of sustained release of long-acting drugs according to claim 1, wherein the set drug is one or a combination of more than one drug required to have a long-acting sustained release effect.

Technical Field

The invention belongs to the technical field of tissue injury repair, and particularly relates to a preparation method of a silk fibroin scaffold material capable of slowly releasing long-acting drugs.

Background

Drug therapy with various small molecule drugs and cytokines as the core is one of the key fields in clinical drug development at present. Besides the need of regulating the activity and efficacy of drugs, how to screen and construct proper drug controlled release carriers also has an important component in drug development. Drug carriers need to find a balance between burst and sustained release for different lesions and disorders. Except for the need of using drugs for disease treatment, the slow release and burst release of drugs have great significance in the field of tissue engineering.

When the tissue engineering scaffold is constructed, on one hand, the drugs are required to be utilized to rapidly recruit cells and inhibit local inflammation, and on the other hand, the drugs are required to be released for a long time to achieve the effect of stimulating cell differentiation and proliferation. The requirements of the traditional drug carrier are the same, and the drug-stent carrier also needs to adjust the degradation and drug release capabilities of the drug-stent carrier aiming at different tissue defects when a drug sustained-release system of tissue engineering is constructed.

In the field of tissue injury repair, sustained release of drugs is achieved mainly by physical encapsulation and chemical cross-linking. The physical package is mainly in the form of microspheres, and the preparation of the microspheres is realized by blending precursor solution of high polymer materials such as polylactic acid, polycaprolactone, polystyrene and the like with the medicament and mixing the medicament while the preparation of the microspheres is realized by emulsion polymerization and the like. Drug release is achieved in both diffusion and material degradation. Chemical crosslinking is to carry out covalent crosslinking on the drug and functional groups on the surface of the material, and realize the slow release of the drug by utilizing the degradation and decomposition of the material. Although these two methods have been extensively studied, each still has significant drawbacks. The drug-loaded microspheres are complex to prepare, require complex work to elute organic impurities, have drug-loading efficiency of less than 40 percent, and are not satisfactory in degradation. The chemical crosslinking has large drug loading limitation and limited crosslinking efficiency, and the problem of changing the drug effect exists when the drug is subjected to crosslinking modification. In addition, the realization of the slow release of the drug mainly depends on the synthesis of high polymer materials, and although the materials have simple preparation process and are beneficial to large-scale production, the materials have long degradation time, and the degradation products have side effects on organisms. How to realize the long-term slow release of the drugs through the research and development of drug-loaded materials still is one of the key directions of the research and development of the drugs at present.

Compared with synthetic polymer materials, the degradation product of the natural polymer material has small toxic and side effects and is easy to degrade and absorb by cells in vivo, so that the natural polymer material is more suitable for drug-loaded material selection. However, natural polymer materials are generally degraded quickly, and the preparation process of the materials is limited, so that the slow release of the drugs is difficult to realize. Silk fibroin, a natural protein material with long degradation time, can be subjected to enzymolysis by collagenase of an organism, and degradation products are the same as collagen. The protein is simple to extract and has extremely low immunogenicity, so the protein is an ideal drug sustained-release component material. The novel drug sustained-release system constructed around the silk fibroin has high clinical application value.

There are many drug sustained-release systems based on silk fibroin, and the drug-loaded forms which are researched more include hydrogel encapsulation and silk fibroin microsphere. In the text of Colloidal Stability of Silk fibrous nanoparticles Coated with functional Polymer for Effective Drug Delivery by Suhang Wang, acetone and Silk fibroin solution are blended to prepare Silk fibroin microspheres, and drugs are carried in a surface adsorption mode to realize slow release. In the text of Silk hydrogels for sustained delivery of anti-vascular growth factors (anti-VEGF) therapeutics, Bevacizumab is added to a Silk fibroin solution by Lovett, and the protein is induced by ultrasound to form hydrogel, thereby realizing long-term sustained release of the drug. In the invention patent of China with publication number CN109876147A of lie in preparation method and application of a silk fibroin microsphere drug sustained release carrier, after silk fibroin microspheres are obtained by using dialysis principle, drug loading is realized by using adsorption mode, and drug sustained release is carried out.

Although the technology can realize the drug slow release with the silk fibroin material as the main body, the two articles which utilize the protein microsphere adsorption principle to realize the drug slow release have poor slow release effect, nearly 50 percent of the drug is released in 12 hours, and the drug loading rate is also poor. Although Lovett works to realize long-term slow release of the drug and high drug loading rate, the preparation process is complex and has poor strength, which is not beneficial to the popularization of clinical work.

Disclosure of Invention

The invention aims to provide a preparation method of a silk fibroin scaffold material capable of realizing long-acting drug slow release, and aims to solve the problems that in the prior art, the drug slow release effect taking silk fibroin as a main body is poor, the preparation process is complex and the like.

The invention is realized by the following technical scheme:

a preparation method of a silk fibroin bracket material capable of realizing long-acting drug slow release comprises the following steps:

1) extraction of silk fibroin

Adding silkworm cocoon into boiled 0.01-0.03mol/L sodium carbonate solution, keeping boiling state, stirring and dissolving for 20-40min, taking out solid matter, washing with water, and drying at room temperature to obtain silk fibroin;

according to the mass ratio of silk fibroin to lithium bromide of 1: 4 weighing lithium bromide and preparing 9.3mol/L lithium bromide water solution;

adding a lithium bromide aqueous solution into a container with silk fibroin, completely covering the silk fibroin with the lithium bromide aqueous solution, and stirring at 50-70 ℃ for 3-5 h;

dialyzing the obtained brown viscous solution, centrifuging the collected liquid, and keeping the supernatant;

quickly freezing the supernatant with liquid nitrogen, and freeze-drying to obtain spongy silk fibroin;

2) phase transition temperature interval determination

According to the set concentration, the spongy silk fibroin is dissolved into a corresponding aqueous solution or salt solution and is sterilized for later use;

measuring the glass transition temperature of the spongy silk fibroin solution by a differential thermal experiment to obtain a phase transition temperature interval, wherein the temperature interval is usually different from 0 ℃ to-20 ℃;

3) preparation of silk fibroin scaffold material

Dissolving the silk fibroin sponge in the step 2) according to the corresponding concentration according to the requirement, blending the set medicine and the silk fibroin solution, and placing the obtained phase transition temperature interval obtained by a differential thermal experiment into the corresponding temperature for low-temperature freezing denaturation to obtain the silk fibroin scaffold material capable of slowly releasing the long-acting medicine.

In the step 1), the 9.3mol/L lithium bromide solution is prepared by firstly calculating the volume of the lithium bromide solution, weighing 60% of water for ice bath, slowly adding the lithium bromide into the water while stirring, and supplementing the water to the calculated value after the lithium bromide is completely dissolved.

The dialysis method in step 1) is that water is changed every 12h, the dialysate is collected after three days, the centrifugal speed is 9000rpm at 4 ℃, the dialysate is centrifuged twice, and the supernatant is collected.

The disinfection in the step 2) adopts ultraviolet disinfection or cobalt 60 radiation disinfection.

The set drug is one or the combination of more than one drug which needs to have long-acting slow-release effect.

The invention has the beneficial effects that:

the technology can realize the long-term controllable slow release of the drug, the release rate completely depends on the degradation rate of the stent, compared with other drug-carrying forms, the implementation scheme of the technology has no toxic additive components, does not need chemical cross-linking agents and organic solvents, and has extremely high drug-carrying rate. The preparation process of the technical scheme is simple and controllable, and is suitable for large-scale industrial popularization.

Drawings

FIG. 1 is a graph showing the results of a differential thermal experiment according to the present embodiment;

FIG. 2 is a graph showing the variation of the secondary structure of 5% silk fibroin in different processing environments;

FIG. 3 is a graph of the hydrolytic release profile of a 5% silk fibroin scaffold prepared by three protocols;

fig. 4 is a graph of the enzymatic sustained release of a 5% silk fibroin scaffold prepared by three schemes.

Detailed Description

The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention.

The application provides a preparation method of a silk fibroin scaffold material capable of realizing long-acting drug sustained release, which comprises the following steps:

1) extraction of silk fibroin

Shearing 10g Bombyx Bombycis into small pieces with scissors, boiling 0.02mol/L anhydrous sodium carbonate solution (2L), adding, stirring and dissolving for 30min while keeping boiling state, taking out the obtained white cotton-like solid, washing with water for several times, and oven drying at room temperature. After sufficient drying, weigh and mix silk fibroin: lithium bromide ═ 1: 4, weighing the corresponding lithium bromide according to the mass ratio. Preparing 9.3mol/L lithium bromide aqueous solution (calculating the volume of the liquid, then weighing about 60% of water, carrying out ice bath, slowly adding lithium bromide into the stirring water, and filling the volume to the calculated value after complete dissolution), and pouring the solution into a beaker filled with silk fibroin to ensure that the lithium bromide solution completely covers the protein. After which it was stirred at 60 ℃ for 4 h. The tan viscous solution was then dialyzed, water was exchanged every 12h, and after 3 days was collected and centrifuged. The centrifugation speed was 9000rpm, the temperature was kept at 4 ℃, the centrifugation was carried out twice, the impurities were discarded, and only the supernatant was retained. And (3) subpackaging the harvested supernatant, quickly freezing by using liquid nitrogen, and freeze-drying to obtain the silk fibroin freezing sponge which can be stored for a long time in a room-temperature drying environment.

2) Phase transition temperature interval determination

Dissolving silk fibroin sponge into corresponding aqueous solution or salt solution (such as PBS buffer solution) according to the set concentration, and sterilizing by ultraviolet rays or cobalt 60 radiation for later use; the specific set concentration is changed according to different degradation rates and drug-loaded release rates required by different tissue repairs.

Measuring the glass transition temperature of the fibroin solution by a differential thermal experiment to obtain a phase transition temperature interval, wherein the temperature interval is usually different from 0 ℃ to-20 ℃; in this example, we prepared a silk fibroin solution at 5% concentration and performed differential thermal experimental analysis. The experimental result shows that for the silk fibroin solution with the concentration, the temperature of-7.0 to-12.5 ℃ is a low-temperature phase transition region, and the silk fibroin solution is suitable for preparing frozen sponges.

3) Preparation of silk fibroin scaffold material

Blending the set drug and the silk fibroin solution prepared in the step 2), and placing the mixture into a corresponding temperature for low-temperature freezing denaturation according to a phase transition temperature interval obtained by a differential thermal experiment to obtain the silk fibroin scaffold material capable of slowly releasing the long-acting drug.

Detection method

In the technical scheme, the set drug refers to one or a combination of more than one drug which needs to have long-acting slow-release effect. In the following examples of the present application, rhodamine is used as an exemplary drug for experiments, but the technical scheme of the present application is not limited by space and the like, and other drugs are not described. And mixing rhodamine serving as a demonstration medicament of an experiment with the silk fibroin frozen sponge solution, putting the mixture into a corresponding temperature to perform low-temperature freezing denaturation according to a result obtained by a differential thermal experiment, taking the mixture out after 12-72 hours of time difference, and performing subsequent experimental characterization.

In order to verify the drug slow-release capacity of the scaffold material, silk fibroin hydrogel and alcohol-treated silk fibroin freeze-dried sponge are respectively prepared as a control group, and the silk fibroin hydrogel and the alcohol-treated silk fibroin freeze-dried sponge are used as the scaffold material for analysis. The silk fibroin hydrogel is prepared by blending 5% of silk fibroin, 1.68mM of hydrogen peroxide and 3U/mL of catalase. The preparation method of the silk fibroin freeze-dried sponge treated by alcohol comprises the following steps: the 5% silk fibroin solution is freeze-dried, soaked in ethanol for 4 hours, then washed with water for multiple times to remove the ethanol, and freeze-dried again.

The method for measuring the drug loading rate of the stent material comprises the steps of mixing rhodamine with the same concentration into the stent prepared from the three materials, preparing the corresponding stent by respective methods, carrying out full enzymolysis on the stent by pepsin K, and measuring the fluorescence intensity of the rhodamine by an enzyme-labeling instrument. And (3) obtaining a linear relation between the fluorescence intensity and the concentration of the rhodamine by drawing a standard curve, and carrying out the following calculation. The ratio of the rhodamine content of the scaffold to the initial content is the drug loading rate of the scaffold.

The slow release curve determination of the stent comprises two parts of hydrolysis and enzymolysis. The three brackets carry rhodamine with the same concentration, and after the preparation is finished, the brackets are respectively soaked by 3mL of PBS solution and 0.8U/mL of pepsin K, wherein the former bracket is used for carrying out hydrolysis experiments, and the latter bracket is used for carrying out enzymolysis experiments. Fluorescence intensity measurements were taken at 0.1mL per day, and tubes were supplemented with 0.1mL fresh PBS or pepsin K solution. After 10 days, high concentration pepsin K powder was put in to completely degrade the stent, and the final fluorescence intensity was measured. The hydrolysis and enzymolysis slow release curves of the stent are respectively displayed in the form of cumulative release ratio.

The result of the detection

Differential thermal test results

Differential thermal experiments were used to determine the low temperature phase transition region of the silk fibroin solution. In this example, we prepared a silk fibroin solution at 5% concentration and performed differential thermal experimental analysis. The experimental results show that for the silk fibroin solution with the concentration, the temperature of-7.0 to-12.5 ℃ is a low-temperature phase transition region, and the silk fibroin solution is suitable for preparing frozen sponges, as shown in figure 1.

Structural change of silk fibroin sponge in freezing process

As shown in FIG. 2, after freezing and freeze-drying silk fibroin in liquid nitrogen, it was frozen at 1650cm^-1There is a main peak on the left and right, which indicates that the main component is mainly Random protein segments (Random peptides). After treatment at-18 ℃ and lyophilization, the protein peak gradually reached 1658cm^-1The blue shift occurred from side to side, indicating that a secondary structure alpha helix (alpha-helix) was produced, at 1628cm^-1There was also a shoulder in the vicinity, indicating the formation of a secondary structure beta sheet (beta-sheet). In contrast, in the spectrogram of our sponge formed by freezing, it can be seen that the intensity of the fluorescence intensity is 1658cm^-1There is a distinct main peak. Compared with conventional method for preparing silk fibroin sponge, i.e. soaking in low-polarity solvent (methanol or ethanol) after lyophilization, the lyophilized sponge is 1628cm^-1The beta-sheet peak at (b) was significantly weaker than the alcohol treated group, indicating that the two sponges had distinct structures.

Drug loading rate determination of silk fibroin frozen sponge

By comparing the initial drug loading of the scaffold with the drug loading of the scaffold after enzymolysis, we find that the drug loading of the silk fibroin gel and the silk fibroin frozen sponge both reach 100%, while the drug loading of the silk fibroin alcohol-treated sponge is affected by ethanol and water elution and is only 83 +/-4.1%.

Drug sustained release curve of silk fibroin sponge

FIG. 3 shows a comparison of rhodamine sustained release curves for silk fibroin gel, silk fibroin frozen sponge, and alcohol-treated sponge. When the hydrolysis is carried out in PBS, the silk fibroin gel is completely released on the third day, the silk fibroin is denatured on the fourth day, the gel is changed from transparent to white, and the released rhodamine is re-adsorbed, so that the release rate of the accumulated medicament in the solution is continuously reduced. Both the silk fibroin frozen sponges and the alcohol treated sponges had excellent water stability, and no rhodamine was released after less than 10% of the release on the first day and the second day.

As shown in FIG. 4, when the enzyme is slowly released in pepsin K, the hydrogel is completely degraded and releases all rhodamine on the first day due to the degradation capability of the enzyme. The drug release and degradation of the silk fibroin frozen sponge are carried out synchronously, the release is over 35 percent on the ninth day, and the cumulative release rate reaches 38 +/-2.4 percent. The degradation stability of the silk fibroin alcohol-treated sponge is stronger, the degradation is slow in an enzyme solution, and only 15% of the silk fibroin alcohol-treated sponge is released in the ninth day. Compared with the sponge treated by alcohol, the silk fibroin freezing sponge developed by people has the same water stability, avoids the burst release of the drug, and is degraded more quickly in the environment of body fluid enzyme, thereby avoiding the problem of slow release of the drug.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.