Method for preparing recombinant collagen antioxidant microspheres without cross-linking agent

Abstract

The invention discloses a method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent. The method comprises the following steps: 1, dissolving recombinant collagen and chitosan in deionized water containing glacial acetic acid to serve as a water phase; petroleum ether containing a surfactant is used as an oil phase; 2, injecting the oil phase into the water phase, emulsifying and stirring through a homogenizer, and reducing the temperature of the system to obtain an emulsion; 3, a fixing agent, water and absolute ethyl alcohol are blended and precooled, and fixing liquid is obtained; 4, adding the stationary liquid into the emulsified liquid for curing, and washing the cured microspheres; and 5, transferring into a tannic acid aqueous solution to soak, then washing with deionized water, and freeze-drying to obtain the recombinant collagen microspheres with uniform size. The recombinant collagen microspheres prepared by the method have good dispersity and uniform and controllable size, avoid the toxicity problem of a cross-linking agent, can be applied to the fields of medical instruments, tissue engineering, drug delivery and the like as a filling agent, and have wide commercial application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent. Background Art

[0002] Collagen is the most abundant structural protein in the body and is widely found in skin, bones, cartilage, tendons and other tissues, playing a supporting and connecting role. It is composed of triple helical chains and has high strength and elasticity. In tissue repair and regenerative medicine, collagen has attracted much attention due to its biocompatibility and degradability. Bioengineering technology can produce recombinant collagen, which is used in wound healing, tissue engineering and drug delivery, and has become an important material to replace natural collagen.

[0003] Currently, the microsphere products on the market are mainly based on synthetic degradable materials. Biodegradable materials have been approved by the FDA for use in humans due to their good biocompatibility, degradability and safety. However, conventional microsphere materials usually use a variety of crosslinking agents, but their residual toxicity limits the further application of microsphere products.

[0004] Chitosan is a natural polysaccharide obtained by deacetylation of chitin, which is widely found in the exoskeleton of crustaceans. It has good biocompatibility, biodegradability and low toxicity, and is widely used in drug delivery, tissue engineering and wound dressing. In addition, chitosan can promote cell adhesion and tissue regeneration, and has biological activities such as antibacterial, hemostatic and anti-inflammatory, making it a material that has attracted much attention in biomedical engineering.

[0005] The particle size, shape, smoothness and dispersibility of microspheres will directly affect their application effect. If the microspheres are too large, it may increase the difficulty and pain during injection, and may also cause compression or irritation of local tissues, causing adverse reactions. Microspheres that are too small may be phagocytosed by macrophages, thus affecting their stability and duration of action in the body.

[0006] In addition, damaged tissue sites are often accompanied by oxidative stress, which leads to increased cell damage and inflammatory response. However, conventional microsphere materials lack antioxidant capacity and cannot neutralize free radicals, reduce the damage of oxidative stress to tissues, and promote more effective repair processes, which limits their potential for application in tissue repair and regenerative medicine.

[0007] Therefore, in view of the above situation, it is urgent to develop a method for preparing recombinant collagen antioxidant microspheres without cross-linking agent to overcome the shortcomings in current practical applications. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for preparing recombinant collagen antioxidant microspheres without a crosslinking agent in view of the shortcomings of the above-mentioned prior art. The recombinant collagen microspheres prepared by the method have good dispersibility and uniform and controllable size. Since no crosslinking agent is used, the toxicity problem of the crosslinking agent is avoided. The microspheres can be used as fillers in medical devices, tissue engineering, drug delivery and other fields, and have broad commercial application prospects.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent, characterized in that it comprises the following steps:

[0010] Step 1: dissolving recombinant collagen and chitosan in deionized water containing glacial acetic acid as the aqueous phase; using petroleum ether containing a surfactant as the oil phase;

[0011] Step 2: injecting the oil phase in step 1 into the water phase, emulsifying and stirring by a homogenizer, and lowering the system temperature after stirring to obtain an emulsion;

[0012] Step 3, mixing the fixative, water and anhydrous ethanol and precooling to obtain a fixative solution;

[0013] Step 4, adding the precooled fixing solution in step 3 to the emulsion in step 2 for solidification, and then washing the solidified microspheres with deionized water;

[0014] Step 5: Transfer the washed microspheres in step 4 to a tannic acid aqueous solution for immersion, then wash with deionized water, and freeze-dry to obtain recombinant collagen microspheres of uniform size.

[0015] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the volume percentage of glacial acetic acid in the aqueous phase in step one is 0.25% to 3%, the mass percentage of recombinant collagen is 0.01% to 25%, and the mass percentage of chitosan is 0.1% to 5%; the mass percentage of the surfactant in the oil phase is 0.1% to 20%.

[0016] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the recombinant collagen in step one is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen.

[0017] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the molecular weight of the chitosan in step one is 30,000 to 500,000 Da.

[0018] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the surfactant in step one is Tween-60, Tween-80, Span 60 or Span 80.

[0019] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the petroleum ether in step 1 is of pharmaceutical grade and has a boiling range of 30°C to 60°C, 60°C to 90°C or 90°C to 120°C.

[0020] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the volume ratio of the water phase to the oil phase in step 2 is 1:(4-8), the speed of the homogenizer is 1000rpm-15000rpm, and the system temperature is reduced to -20°C to -80°C after the stirring is completed.

[0021] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the fixing agent in step three is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate, the volume of anhydrous ethanol is 5 to 50 times the volume of deionized water, the mass of the fixing agent is 0.8 to 1.2 times the mass of glacial acetic acid, the mass concentration of the fixing agent in the fixing solution is 0.1% to 3.6%, and the precooling temperature is -20°C to -80°C.

[0022] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the mass concentration of the tannic acid aqueous solution in step five is 0.01% to 20%, and the immersion time is 0.1h to 72h.

[0023] The above-mentioned method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent is characterized in that the average particle size of the recombinant collagen microspheres in step five is 40 μm to 600 μm.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. Combination of raw material characteristics: The unique properties of chitosan (insoluble in water but soluble in acid) are combined with recombinant collagen to prepare microspheres through emulsification into spheres. The pH value is adjusted to neutral or alkaline by a fixative for solidification, ensuring the stability and uniformity of the microspheres.

[0026] 2. Enhance elasticity and antioxidant capacity: After the microspheres are soaked in tannic acid solution, they absorb tannic acid and form hydrogen bonds with the amino groups of chitosan. This process not only enhances the elastic modulus of the microspheres, but also gives them excellent antioxidant capacity and improves the biological function of the microspheres.

[0027] 3. Simple and efficient operation: The method of the present invention is simple, fast and easy to implement. Its process has no complicated steps, is suitable for large-scale production, and has low cost.

[0028] 4. Excellent dispersibility and controllable size: The recombinant collagen microspheres prepared by the method of the present invention have good dispersibility, uniform and controllable size, meet the needs of practical applications, and can meet the requirements of the medical device industry.

[0029] 5. No cross-linking agent, avoiding toxicity problems: The present invention avoids the use of cross-linking agents, overcomes the toxicity problems that may be caused by traditional cross-linking agents, ensures the biocompatibility of the microspheres, and makes them more suitable for application in the medical device industry.

[0030] 6. Broad application prospects: Due to their excellent performance, the recombinant collagen antioxidant microspheres prepared by the present invention can be used as fillers in medical devices, tissue engineering, drug delivery and other fields, and have broad commercial application prospects.

[0031] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a SEM image of the recombinant collagen antioxidant microspheres prepared in Example 1 of the present invention.

[0033] Figure 2 It is the elastic modulus of the recombinant collagen antioxidant microspheres prepared in Example 1 of the present invention.

[0034] Figure 3 It is the DPPH clearance rate of the recombinant collagen antioxidant microspheres prepared in Example 1 of the present invention.

[0035] Figure 4 This is the MTT test result of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0036] Figure 5 The results are as follows: The recombinant collagen microspheres prepared in Example 1 of the present invention promote the migration of L929 cells.

[0037] Figure 6 HE pathological sections of the liver, spleen and kidney of rats after the recombinant collagen microspheres prepared in Example 1 of the present invention were implanted subcutaneously for eight weeks. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0039] Example 1

[0040] This embodiment provides a method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent, which specifically includes the following steps:

[0041] Step 1: Take 2 mL of glacial acetic acid, 1 g of chitosan (100,000 Da) and 0.5 g of recombinant collagen and add them to 100 mL of deionized water to obtain a mixed solution as the aqueous phase; add 2.5 g of Tween-80 to 400 mL of petroleum ether (boiling range 60-90 ° C) as the oil phase; the recombinant collagen is a large molecular type I recombinant collagen, which is composed of a short amino acid sequence (GAPG AP GSQGAPGL) from natural human type I collagen as a repeating unit and is repeated multiple times, the number of repetitions is 52 times, the collagen has good water solubility, a molecular weight of 78862 Da, and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0042] Step 2: inject the oil phase into the water phase, and emulsify and stir the mixture using a homogenizer. After stirring, add liquid nitrogen to lower the system temperature to -40°C. The speed of the homogenizer is 10,000 rpm, and the stirring time is 1 hour.

[0043] Step 3: Mix 2 g NaOH (or KOH), 5 mL water and 50 mL anhydrous ethanol and precool to -40°C to obtain a stationary solution;

[0044] Step 4: adding the precooled fixing solution into the precooled emulsion for solidification, and then washing the solidified microspheres with deionized water;

[0045] Step 5: Place the washed microspheres into a 10 wt % tannic acid solution and soak for 12 hours. After soaking, wash with deionized water and freeze-dry to obtain recombinant collagen antioxidant microspheres of uniform size.

[0046] Example 2

[0047] This embodiment provides a method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent, which specifically includes the following steps:

[0048] Step 1: Take 0.025 mL of glacial acetic acid, 0.5 g of chitosan (500,000 Da) and 2.5 g of recombinant collagen and add them into 10 mL of deionized water to obtain a mixed solution as the aqueous phase; add 16 g of Span 60 (or Span 80) into 80 mL of petroleum ether (boiling range 90° C. to 120° C.) as the oil phase; the recombinant collagen is recombinant type II collagen, which is composed of the full-length α1 chain sequence from human type II collagen, and the collagen has good water solubility and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0049] Step 2: inject the oil phase into the water phase and emulsify and stir it with a homogenizer. After stirring, add liquid nitrogen to lower the system temperature to -80°C. The speed of the homogenizer is 1000 rpm and the stirring time is 2 hours.

[0050] Step 3: Mix 0.03 g potassium bicarbonate (or sodium bicarbonate), 5 mL water and 25 mL anhydrous ethanol and precool to -80°C to obtain a stationary solution;

[0051] Step 4: adding the precooled fixing solution into the precooled emulsion for solidification, and then washing the solidified microspheres with deionized water;

[0052] Step 5: Place the washed microspheres into a 20 wt % tannic acid solution and soak for 0.1 h. After soaking, wash with deionized water and freeze-dry to obtain recombinant collagen antioxidant microspheres of uniform size.

[0053] Example 3

[0054] This embodiment provides a method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent, which specifically includes the following steps:

[0055] Step 1: Take 0.3 mL of glacial acetic acid, 0.001 g of chitosan (30,000 Da) and 0.5 g of recombinant collagen and add them to 10 mL of deionized water to obtain a mixed solution as the aqueous phase; add 0.06 g of Tween-60 to 60 mL of petroleum ether (boiling range 30° C. to 60° C.) as the oil phase; the recombinant collagen is recombinant type III collagen, which is composed of a short amino acid sequence from natural human type III collagen as a repeating unit and is repeated multiple times, wherein the short amino acid sequence (GERGAPGFRGPAGPNGIPGEKGPAGER) is repeated 30 times, and the collagen has good water solubility and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0056] Step 2: inject the oil phase into the water phase and emulsify and stir it with a homogenizer. After stirring, add liquid nitrogen to lower the system temperature to -20°C. The speed of the homogenizer is 15000 rpm and the stirring time is 1.5 hours.

[0057] Step 3: Mix 0.24 g of sodium carbonate (or potassium carbonate), 4 mL of water and 20 mL of anhydrous ethanol and precool to -20°C to obtain a stationary solution;

[0058] Step 4: adding the precooled fixing solution into the precooled emulsion for solidification, and then washing the solidified microspheres with deionized water;

[0059] Step 5: Place the washed microspheres in a 0.01 wt% tannic acid solution and soak for 72 hours. After soaking, wash with deionized water and freeze-dry to obtain recombinant collagen antioxidant microspheres of uniform size.

[0060] Taking the recombinant collagen antioxidant microspheres prepared in Example 1 as an example, various properties of the recombinant collagen antioxidant microspheres prepared by the method of the present invention were measured.

[0061] 1. Morphology measurement

[0062] The recombinant collagen microspheres of Example 1 were photographed using a field emission scanning electron microscope (GeminiSEM 300, Car1 Zeiss, Germany). Figure 1 As shown, the results show that the diameter of the microspheres is about 50 μm and there are dense pores on the surface.

[0063] 2. Elastic modulus

[0064] The elastic modulus of the microspheres before and after immersion was investigated by atomic force microscopy uniaxial compression test. After immersion, the elastic modulus of the microspheres increased to 20Kpa. The results showed that tannic acid immersion increased the elastic modulus of the microspheres ( Figure 2 ).

[0065] 3. DPPH scavenging ability

[0066] The antioxidant capacity of the microspheres before and after soaking in tannic acid was determined by DPPH. The results showed that the antioxidant capacity of the collagen microspheres was significantly improved by soaking ( Figure 3 ).

[0067] 4. Biocompatibility

[0068] The extract was prepared according to the method recommended by GB / T 16886.5-2017 for in vitro cell compatibility testing using MTT and cell scratch methods. Figure 4 and Figure 5 The results showed that the recombinant collagen antioxidant microsphere extract prepared in Example 1 can effectively promote the proliferation and migration of mouse fibroblasts (L929). The cell proliferation rate was 120.2% 24 hours before immersion and 130.5 24 hours after immersion, indicating good biocompatibility.

[0069] Eight weeks after the recombinant type I collagen antioxidant microspheres were implanted in the skin, the liver, spleen, and kidneys of the rats were examined by H&E staining. The results showed that there was no obvious lesion in the liver, spleen, and kidneys of the rats before and after the recombinant type I collagen antioxidant microspheres were soaked in tannic acid, indicating that they have good biocompatibility.

[0070] The method for preparing recombinant collagen antioxidant microspheres without a crosslinking agent provided by the present invention significantly simplifies the preparation process, is simple and quick to operate, and is easy to promote and apply. The recombinant collagen microspheres prepared by this method not only have good dispersibility and uniform size, ensuring the consistency and stability of product quality, but also have excellent antioxidant properties. These advantages make the microspheres have a wide range of application potentials in the medical device industry, especially in situations where high-performance fillers are required, which can significantly improve the performance and reliability of the product and provide strong support for the innovation and development of medical devices.

[0071] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent, characterized in that: The following steps are involved: Step 1: dissolving recombinant collagen and chitosan in deionized water containing glacial acetic acid as the water phase; using petroleum ether containing a surfactant as the oil phase; Step 2: inject the oil phase in step 1 into the water phase, and emulsify and stir by a homogenizer. After stirring, lower the system temperature to obtain an emulsion; Step 3, mixing the fixative, water and anhydrous ethanol and precooling to obtain a fixative solution; Step 4, adding the precooled fixing solution in step 3 to the emulsion in step 2 for solidification, and then washing the solidified microspheres with deionized water; Step 5: Transfer the washed microspheres in step 4 to a tannic acid aqueous solution for immersion, then wash with deionized water, and freeze-dry to obtain recombinant collagen microspheres of uniform size.

2. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: In step 1, the volume percentage of glacial acetic acid in the aqueous phase is 0.25% to 3%, the mass percentage of recombinant collagen is 0.01% to 25%, and the mass percentage of chitosan is 0.1% to 5%; the mass percentage of the surfactant in the oil phase is 0.1% to 20%.

3. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1 or 2, characterized in that: The recombinant collagen described in step 1 is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen.

4. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: The molecular weight of the chitosan in step 1 is 30,000 to 500,000 Da.

5. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: The surfactant in step 1 is Tween-60, Tween-80, Span 60 or Span 80.

6. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: The petroleum ether described in step 1 is of pharmaceutical grade, with a boiling range of 30°C to 60°C, 60°C to 90°C or 90°C to 120°C.

7. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: In step 2, the volume ratio of the water phase to the oil phase is 1:(4-8), the speed of the homogenizer is 1000rpm-15000rpm, and the system temperature is lowered to -20°C--80°C after the stirring is completed.

8. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: The fixative in step 3 is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate, the volume of anhydrous ethanol is 5 to 50 times the volume of deionized water, the mass of the fixative is 0.8 to 1.2 times the mass of glacial acetic acid, the mass concentration of the fixative in the fixative solution is 0.1% to 3.6%, and the precooling temperature is -20°C to -80°C.

9. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: The mass concentration of the tannic acid aqueous solution in step 5 is 0.01% to 20%, and the soaking time is 0.1h to 72h.

10. The method for preparing recombinant collagen antioxidant microspheres without a cross-linking agent according to claim 1, characterized in that: The average particle size of the recombinant collagen microspheres described in step five is 40 μm to 600 μm.

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