A composite material of hydroxyapatite loaded silk fibroin and a preparation method thereof

Abstract

The present application relates to the technical field of medical dressings, and discloses a composite material of hydroxyapatite loaded silk fibroin and a preparation method thereof, which comprises the following steps: 1) dissolving soluble silk fibroin with a dissolving agent; 2) performing mechanical wave treatment on the silk fibroin solution; 3) polarizing hydroxyapatite powder in a direct current field air; and 4) uniformly dispersing the polarized hydroxyapatite in the silk fibroin hydrogel by vacuum embedding to obtain a stable composite material of hydroxyapatite loaded silk fibroin. The present application adopts ultrasonic mediation to make silk fibroin into gel, and the gelation speed is fast and the gel is uniform. The present application significantly increases the sunscreen performance and repair performance of the material, significantly improves the film-forming property and stability of the hydrogel, and simultaneously provides multiple functions such as sunscreen, anti-oxidation and moisturizing repair by synergistically combining the ultraviolet absorption property of hydroxyapatite with the film-forming property and antioxidant property of silk fibroin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical dressings, in particular to a composite material loaded with silk fibroin by hydroxyapatite and a preparation method. BACKGROUND

[0002] Gels can simulate the aqueous environment of the extracellular matrix, providing a microenvironment for cell adhesion, proliferation and differentiation, and are an ideal material for tissue injury regeneration, repair and reconstruction. However, the performance of current hydrogel products for skin dressings is single, so more research is being done on new needs of hydrogel users. Silk fibroin can form a dense nanofiber membrane covering the skin surface, reflect and scatter ultraviolet rays, form physical sun protection, and form a more uniform film, with stronger ultraviolet blocking ability. In addition, it contains tyrosine, tryptophan, phenylalanine and other amino acids, which can directly absorb ultraviolet rays, cover the full spectrum, inhibit tyrosinase activity, and reduce melanin production. The unique crystal structure of hydroxyapatite can enhance the scattering and reflection ability of ultraviolet rays, and its porous surface can load functional components to synergistically reduce free radical damage and improve skin feel. Chinese invention CN119770371A mixes a hydroxyapatite silk fibroin metal ion mixed solution with sericin to obtain a composition that can improve the sunscreen performance of sunscreen agents, which can avoid direct contact between sunscreen agents and the skin and reduce the sensitization and skin irritation of chemical sunscreen agents. Chinese invention CN117208871A discloses a dual-functional hydroxyapatite powder with ultraviolet absorption and antibacterial properties. Ce 3+ , Ag + , Zn 2+ are substituted for part of the calcium in hydroxyapatite, giving it high-efficiency ultraviolet absorption and antibacterial functions. Therefore, the present application combines the physical shielding performance of hydroxyapatite with the loading / film-forming properties of silk fibroin, breaking through the performance limitations of single materials, and the composite system has sunscreen, antibacterial, antioxidant and repair functions, and prolongs the sunscreen duration through a slow-release mechanism. SUMMARY

[0003] The purpose of the present application is to provide a composite material loaded with silk fibroin by hydroxyapatite and a preparation method.

[0004] The scheme of the present application is:

[0005] A preparation method of a composite material loaded with silk fibroin by hydroxyapatite, comprising the following steps:

[0006] 1) Dissolve the soluble silk fibroin with a dissolving agent to obtain a silk fibroin solution with a physiological concentration of 0.1-30% and a pH range of 4-7.4;

[0007] 2) the silk fibroin solution is subjected to mechanical wave treatment, the hydrophobic interaction is changed to promote the formation of beta sheet structure, thereby accelerating the physical crosslinking of gel stabilization; the gelation time is regulated by controlling the pH value of the solution, the low pH value accelerates the gelation kinetics, the silk fibroin solution is rapidly gelled, and the silk fibroin hydrogel is obtained;

[0008] 3) the hydroxyapatite powder is polarized in a direct current field air, and polarized hydroxyapatite is obtained;

[0009] 4) the polarized hydroxyapatite is uniformly dispersed in the silk fibroin hydrogel through vacuum embedding, and a stable hydroxyapatite-loaded silk fibroin composite material is obtained.

[0010] The soluble silk fibroin is one of a silk fibroin freeze-dried sponge or a silk fibroin freeze-dried powder;

[0011] As a preferred technical solution, the dissolving agent is one of deionized water or a salt solution.

[0012] As a preferred technical solution, the mechanical wave treatment in the step 2) is ultrasonic treatment, the ultrasonic power ratio is 50% to 100%, the ultrasonic time is 10 min to 30 min, and the temperature is 0 to 60 DEG C.

[0013] As a preferred technical solution, the hydroxyapatite is a hydroxyapatite microsphere, the hydroxyapatite microsphere is formed by a spraying mechanism, and the particle size range is 10 to 60 mu m.

[0014] As a preferred technical solution, in the step 3), the polarization conditions of the hydroxyapatite powder are 1 to 4 kVcm -1 , the polarization temperature is 400 to 800 DEG C, and the polarization time is 1 to 4 h.

[0015] As a preferred technical solution, in the step 4), the embedding speed of the vacuum embedding is 10 to 100 rpm, and the embedding time is 2 to 10 min.

[0016] As a preferred technical solution, in the step 4), the mass of the hydroxyapatite accounts for 0.1 to 30% of the mass of the silk fibroin.

[0017] The application further discloses a hydroxyapatite-loaded silk fibroin composite material prepared by the hydroxyapatite-loaded silk fibroin composite material and a preparation method and application thereof.

[0018] The application further discloses an application of the hydroxyapatite-loaded silk fibroin composite material in medical dressings.

[0019] Due to the adoption of the above technical scheme, the composite material of hydroxyapatite loaded silk fibroin and the preparation method, 1) soluble silk fibroin is dissolved by a dissolving agent to obtain a silk fibroin solution with a physiological concentration of 0.1-30% and a pH range of 4-7.4; 2) the silk fibroin solution is subjected to mechanical wave treatment to change the hydrophobic effect and promote the formation of a beta-fold structure, thereby accelerating the physical cross-linking of gel stabilization; the gelation time is controlled by adjusting the pH value of the solution to make the silk fibroin solution gel quickly to obtain a silk fibroin hydrogel; 3) hydroxyapatite powder is polarized in a direct current field air to obtain polarized hydroxyapatite; 4) the polarized hydroxyapatite is uniformly dispersed in the silk fibroin hydrogel by vacuum embedding to obtain a stable composite material of hydroxyapatite loaded silk fibroin.

[0020] Advantages of the present application:

[0021] (1) The present application adopts ultrasonic mediation for silk fibroin gelation, and by adjusting the pH value and reaction temperature of the solution, direct ultrasonic gelation can be realized without further incubation for gelation, and the gelation speed is fast and the gel is uniform;

[0022] (2) Compared with the pure silk fibroin hydrogel, the composite gel material added with the polarized hydroxyapatite maintains its biocompatibility and permeability, and significantly increases the sunscreen performance and repair performance of the material;

[0023] (3) The present application adopts the method of vacuum embedding to disperse the hydroxyapatite microspheres in the silk fibroin gel to obtain a paste compound, which has a three-dimensional structure and a pore structure connected to each other, thereby significantly improving the film-forming property and stability of the hydrogel;

[0024] (4) The present application synergizes the ultraviolet absorption property of hydroxyapatite with the film-forming property and antioxidant property of silk fibroin, and simultaneously provides multiple functions such as sunscreen, antioxidant and moisturizing repair. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is the ultraviolet absorption spectrum of the composite material of hydroxyapatite loaded silk fibroin in the experiment of the present application; wherein, HAP is hydroxyapatite, and SF-HAP is the composite material of hydroxyapatite loaded silk fibroin;

[0026] Figure 2 It is the 0 min observation record photo of the film-forming test in the experiment of the present application;

[0027] Figure 3 It is the 10 min observation record photo of the film-forming test in the experiment of the present application;

[0028] Figure 4 It is the 20 min observation record photo of the film-forming test in the experiment of the present application;

[0029] Figure 5 Figure 1 is a photograph of the 0 min observation record for the moisturizing test in the experiment of the present application;

[0030] Figure 6 Figure 2 is a photograph of the 10 min observation record for the moisturizing test in the experiment of the present application;

[0031] Figure 7 Figure 3 is a photograph of the 10 min observation record for the TSB solid medium scratch test without product coating after placement for 10 min in the moisturizing test in the experiment of the present application;

[0032] Figure 8 Figure 4 is a photograph of the 10 min observation record for the TSB solid medium scratch test with product coating after placement for 10 min in the moisturizing test in the experiment of the present application. DETAILED DESCRIPTION

[0033] In order to make up for the above deficiencies, the present application provides a composite material loaded with silk fibroin by hydroxyapatite and a preparation method to solve the problems in the above background art.

[0034] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application is further described below in combination with specific embodiments.

[0035] Embodiment 1

[0036] A composite material loaded with silk fibroin by hydroxyapatite and a preparation method, comprising the following steps:

[0037] 1) Dissolving soluble silk fibroin with a dissolving agent to obtain a silk fibroin solution with a physiological concentration of 0.1% and a pH range of 4;

[0038] 2) Mechanically treating the silk fibroin solution to change the hydrophobic effect and promote the formation of β-sheet structure, thereby accelerating the physical cross-linking of gel stabilization; controlling the pH value of the solution to regulate the gelation time, so as to quickly gel the silk fibroin solution and obtain a silk fibroin hydrogel;

[0039] 3) Polarizing hydroxyapatite powder in a direct current field in air to obtain polarized hydroxyapatite;

[0040] 4) Uniformly dispersing the polarized hydroxyapatite in the silk fibroin hydrogel by vacuum embedding to obtain a stable composite material loaded with silk fibroin by hydroxyapatite.

[0041] The soluble silk fibroin is a silk fibroin lyophilized sponge;

[0042] The dissolving agent is deionized water.

[0043] The mechanical wave treatment in the 2) step is ultrasonic treatment, the ultrasonic power ratio is 50%, the ultrasonic time is 10 min, and the temperature is 0°C.

[0044] The hydroxyapatite is hydroxyapatite microspheres, the hydroxyapatite microspheres are made by a spraying mechanism, and the particle size range is 10 μm.

[0045] In the 3) step, the polarization conditions of the hydroxyapatite powder are 1 kVcm -1 , the polarization temperature is 400°C, and the polarization time is 1 h.

[0046] In the 4) step, the embedding speed of the vacuum embedding is 10 rpm, and the embedding time is 2 min.

[0047] In the 4) step, the mass of the hydroxyapatite accounts for 0.1% of the mass of the silk fibroin.

[0048] Embodiment 2

[0049] A composite material loaded with hydroxyapatite and silk fibroin and a preparation method thereof, comprising the following steps:

[0050] 1) Dissolving soluble silk fibroin with a dissolving agent to obtain a silk fibroin solution with a physiological concentration of 30%, and the pH range is 7.4;

[0051] 2) Treating the silk fibroin solution with mechanical wave treatment to change the hydrophobic effect and promote the formation of a β-fold structure, thereby accelerating the physical cross-linking of gel stabilization; controlling the pH value of the solution to regulate the gelation time, so that the silk fibroin solution is quickly gelled to obtain a silk fibroin hydrogel;

[0052] 3) Polarizing hydroxyapatite powder in a direct current field in air to obtain polarized hydroxyapatite;

[0053] 4) Uniformly dispersing the polarized hydroxyapatite in the silk fibroin hydrogel through vacuum embedding to obtain a stable composite material loaded with hydroxyapatite and silk fibroin.

[0054] The soluble silk fibroin is a silk fibroin freeze-dried powder;

[0055] The dissolving agent is a salt solution.

[0056] The mechanical wave treatment in the 2) step is ultrasonic treatment, the ultrasonic power ratio is 100%, the ultrasonic time is 30 min, and the temperature is 60°C.

[0057] The hydroxyapatite is hydroxyapatite microspheres, the hydroxyapatite microspheres are made by a spraying mechanism, and the particle size range is 60 μm.

[0058] In the 3) step, the polarization conditions of the hydroxyapatite powder are 4 kVcm-1 polarization temperature 800℃, polarization time 4h.

[0059] In the step 4), the embedding speed of the vacuum embedding is 100 rpm, and the embedding time is 10 min.

[0060] In the step 4), the mass of the hydroxyapatite accounts for 30% of the mass of the silk fibroin.

[0061] Embodiment 3

[0062] A composite material of hydroxyapatite loaded silk fibroin and a preparation method thereof, comprising the following steps:

[0063] 1) dissolving soluble silk fibroin with a dissolving agent to obtain a silk fibroin solution with a physiological concentration of 15%, and the pH range is 6.5;

[0064] 2) performing mechanical wave treatment on the silk fibroin solution to change the hydrophobicity and promote the formation of β-sheet structure, thereby accelerating the physical cross-linking of gel stabilization; controlling the pH value of the solution to regulate the gelation time, so that the silk fibroin solution is quickly gelled to obtain a silk fibroin hydrogel;

[0065] 3) polarizing hydroxyapatite powder in a direct current field air to obtain polarized hydroxyapatite;

[0066] 4) uniformly dispersing the polarized hydroxyapatite in the silk fibroin hydrogel by vacuum embedding to obtain a stable composite material of hydroxyapatite loaded silk fibroin.

[0067] The soluble silk fibroin is a silk fibroin freeze-dried powder;

[0068] The dissolving agent is deionized water.

[0069] In the step 2), the mechanical wave treatment is ultrasonic treatment, the ultrasonic power ratio is 70%, the ultrasonic time is 20 min, and the temperature is 20℃.

[0070] The hydroxyapatite is a hydroxyapatite microsphere, and the hydroxyapatite microsphere is made by a spraying mechanism, and the particle size range is 30μm.

[0071] In the step 3), the polarization conditions of the hydroxyapatite powder are 2kVcm -1 polarization temperature 600℃, polarization time 2h.

[0072] In the step 4), the embedding speed of the vacuum embedding is 80 rpm, and the embedding time is 5 min.

[0073] In the step 4), the mass of the hydroxyapatite accounts for 15% of the mass of the silk fibroin.

[0074] Experiment

[0075] Test 1

[0076] The silk fibroin solution gelation test is carried out by the following method:

[0077] 1) Dissolve the soluble silk fibroin with a dissolving agent to obtain a silk fibroin solution with a concentration of 1%, and control the pH at 5; quantitatively divide the silk fibroin solution into beakers of the same size, insert the ultrasonic head 2 cm below the liquid surface, adjust the power intensity and time, and ultrasonically treat the silk fibroin solution.

[0078] 2) Mechanically treat the silk fibroin solution to change the hydrophobic effect and promote the formation of β-sheet structure, thereby accelerating the physical crosslinking of gel stabilization; control the solution pH value to regulate the gelation time, so that the silk fibroin solution quickly gels to obtain a silk fibroin hydrogel. The soluble silk fibroin is a silk fibroin lyophilized powder.

[0079] The dissolving agent is deionized water.

[0080] The temperature in the step 2) is 30°C.

[0081] In this experiment, the effects of ultrasonic power and temperature on the gelation time of silk fibroin solution are verified, the gelation time of silk fibroin after ultrasonic treatment under different conditions is observed by adjusting the ultrasonic power and ultrasonic time, the ultrasonic power is 500W, the power intensity is 60%~100%, and the ultrasonic time is 10~20min; the results are as follows in Table 1.

[0082] Table 1:

[0083]

[0084] Test 2

[0085] In the test, the effect of solution pH value on the gelation time of silk fibroin solution is verified, the pH value of silk fibroin solution is regulated by adjusting the concentration of salt solution, and the gelation time of silk fibroin solution after ultrasonic treatment under different concentrations is observed.

[0086] The test method is as follows:

[0087] (1) Dissolve the soluble silk fibroin lyophilized sponge in deionized water at room temperature to prepare silk fibroin solutions with concentrations of 0.5%, 1%, 2%, 4% and 8%, and stir uniformly to obtain clear and transparent silk fibroin solutions.

[0088] (2) Measuring 20 ml of the different concentrations of the silk fibroin solution prepared in step (1), and treating the silk fibroin solution with ultrasonic waves at an ultrasonic power of 1000 watts and an ultrasonic power ratio of 80% for 10 min, with the solution temperature controlled below 60°C during the ultrasonic treatment, and then incubating the solution at 50°C to form a gel, thereby obtaining a silk fibroin hydrogel.

[0089] (3) Measuring the hydroxyapatite microspheres with a particle size range of 10-60 μm, and adding the hydroxyapatite microspheres into the silk fibroin hydrogel, and placing the mixture into a vacuum embedding tank at an embedding speed of 100 rpm for 4 min, thereby obtaining a hydroxyapatite-loaded silk fibroin composite material with uniform dispersion. Figure 1

[0090] Test 3

[0091] Film formation test

[0092] 0.1 mL of the product was spread on a glass slide, and the glass slide was placed at 37°C. The glass slide was observed every 10 min to determine whether the product formed a film, and the observation time and phenomena were recorded.

[0093]

[0094] Test 4

[0095] Moisture retention test

[0096] 0.1 mL of the product was spread on a 2 cm diameter circular TSB solid culture medium, and the culture medium was placed in a drying oven for accelerated drying at a temperature of 100°C and an ambient humidity. The drying degree of the culture medium was observed after 10 min. If the drying degree of the culture medium covered by the product was significantly lower than that of the control group, it was determined that the product had moisture retention.

[0097]

[0098] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.​

Claims

1. A method for preparing a composite material of silk fibroin supported on hydroxyapatite, characterized in that, Includes the following steps: 1) Dissolve soluble silk fibroin in a solvent to obtain a silk fibroin solution with a physiological concentration of 0.1-30% and a pH range of 4-7.4; 2) The silk fibroin solution is subjected to mechanical wave treatment to change the hydrophobic interaction and promote the formation of β-sheet structure, thereby accelerating the physical cross-linking of gel stability; the gelation time is regulated by controlling the solution pH value, and the gelation kinetics are accelerated at low pH value, so that the silk fibroin solution is rapidly gelled to obtain silk fibroin hydrogel. 3) Hydroxyapatite powder is polarized in a DC field air to obtain polarized hydroxyapatite; 4) The polarized hydroxyapatite is uniformly dispersed in the silk fibroin hydrogel by vacuum embedding to obtain a hydroxyapatite-loaded silk fibroin composite material.

2. The method for preparing a composite material of silk fibroin supported by hydroxyapatite as described in claim 1, characterized in that: The solvent is either deionized water or a salt solution.

3. The method for preparing a composite material of silk fibroin supported by hydroxyapatite as described in claim 1, characterized in that: In step 2), the mechanical wave treatment is ultrasonic treatment, with an ultrasonic power ratio of 50% to 100%, an ultrasonic time of 10 to 30 minutes, and a temperature of 0 to 60°C.

4. The method for preparing a composite material of silk fibroin supported by hydroxyapatite as described in claim 1, characterized in that: The hydroxyapatite is hydroxyapatite microspheres, which are produced by spraying and have a particle size range of 10–60 μm.

5. The method for preparing a composite material of silk fibroin supported by hydroxyapatite as described in claim 1, characterized in that: In step 3), the polarization conditions of the hydroxyapatite powder are 1–4 kV / cm. -1 The polarization temperature is 400–800℃, and the polarization time is 1–4h.

6. The method for preparing a composite material of silk fibroin supported by hydroxyapatite as described in claim 1, characterized in that: In step 4), the vacuum embedding speed is 10-100 rpm and the embedding time is 2-10 min.

7. The method for preparing a composite material of silk fibroin supported by hydroxyapatite as described in claim 1, characterized in that: In step 4), the hydroxyapatite accounts for 0.1-30% of the silk fibroin protein mass.

8. A hydroxyapatite-loaded silk fibroin composite material prepared by the method for preparing a hydroxyapatite-loaded silk fibroin composite material as described in any one of claims 1-7.

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