A method for preparing recombinant collagen antioxidant microspheres without cross-linking agent

Abstract

The application discloses a method for preparing recombinant collagen antioxidant microspheres without cross-linking agent, comprising the following steps: 1, dissolving the recombinant collagen and chitosan in deionized water containing glacial acetic acid as an aqueous phase; dissolving petroleum ether containing a surfactant as an oil phase; 2, injecting the oil phase into the aqueous phase, emulsifying and stirring through a homogenizer, reducing the temperature of the system, and obtaining an emulsion; 3, blending a fixing agent, water and anhydrous ethanol, and precooling to obtain a fixing solution; 4, adding the fixing solution into the emulsion for solidification, and washing the solidified microspheres; 5, immersing the microspheres into a tannic acid aqueous solution, 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 dispersibility, uniform and controllable size, and the toxicity problem of the cross-linking agent is avoided, so that the recombinant collagen microspheres can be applied to medical devices, tissue engineering, drug delivery and other fields as a filler, and have a wide commercial application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials, and particularly relates to a method for preparing recombinant collagen antioxidant microspheres without a crosslinking agent. BACKGROUND

[0002] Collagen is the most abundant structural protein in the body, widely present in tissues such as skin, bone, cartilage, and tendon, playing a supporting and connecting role. It is composed of three spiral chains and has high strength and elasticity. In tissue repair and regenerative medicine, collagen is of great concern due to its biocompatibility and degradability. Bioengineering technology can produce recombinant collagen, which is applied in wound healing, tissue engineering, and drug delivery, becoming an important material to replace natural collagen.

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

[0004] Chitosan is a natural polysaccharide obtained by deacetylation of chitin, widely present 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, etc. In addition, chitosan can promote cell adhesion and tissue regeneration, and has antibacterial, hemostatic, and anti-inflammatory biological activities, becoming a material of great concern 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 of injection, and may also cause local tissue compression or irritation, causing adverse reactions. While too small microspheres may be phagocytosed by macrophages, affecting their stability and action time in the body.

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

[0007] Therefore, in view of the above status, it is urgent to develop a method for preparing recombinant collagen antioxidant microspheres without a crosslinking agent to overcome the deficiencies in current practical applications. SUMMARY

[0008] The technical problem solved by the present application is to provide a method for preparing recombinant collagen antioxidant microspheres without cross-linking agent to solve the problems of the prior art.

[0009] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a method for preparing recombinant collagen antioxidant microspheres without cross-linking agent, characterized in that it comprises the following steps:

[0010] Step one: dissolve recombinant collagen and chitosan in deionized water containing glacial acetic acid as an aqueous phase; and dissolve petroleum ether containing a surfactant as an oil phase;

[0011] Step two: inject the oil phase into the aqueous phase, emulsify and stir by a homogenizer, and then reduce the system temperature to obtain an emulsion;

[0012] Step three: blend a fixing agent, water and anhydrous ethanol, and pre-cool to obtain a fixing solution;

[0013] Step four: add the pre-cooled fixing solution in step three to the emulsion in step two for solidification, and then wash the solidified microspheres with deionized water;

[0014] Step five: transfer the washed microspheres in step four into a tannic acid aqueous solution for soaking, then wash with deionized water, freeze-dry, and obtain recombinant collagen microspheres with uniform size.

[0015] The method for preparing recombinant collagen antioxidant microspheres without cross-linking agent, 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%; and the mass percentage of the surfactant in the oil phase is 0.1% to 20%.

[0016] The method for preparing recombinant collagen antioxidant microspheres without cross-linking agent, 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 method for preparing recombinant collagen antioxidant microspheres without cross-linking agent, characterized in that the molecular weight of the chitosan in step one is 30000 to 500000 Da.

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

[0019] The method for preparing the recombinant collagen antioxidant microspheres without cross-linking agent has the characteristics that the petroleum ether in step one is a medical grade, and the boiling range is 30-60 DEG C, 60-90 DEG C or 90-120 DEG C.

[0020] The method for preparing the recombinant collagen antioxidant microspheres without cross-linking agent has the characteristics that the volume ratio of the water phase and the oil phase in step two is 1: (4-8), the rotating speed of the homogenizer is 1000-15000 rpm, and the system temperature is reduced to-20 DEG C to-80 DEG C after the stirring is finished.

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

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

[0023] The method for preparing the recombinant collagen antioxidant microspheres without cross-linking agent has the characteristics that the average particle size of the recombinant collagen microspheres in step five is 40-600 mu m.

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

[0025] 1. Combination of the properties of raw materials: the unique properties of chitosan (insoluble in water but soluble in acid) are combined with the recombinant collagen to prepare the microspheres by emulsification, the pH value is adjusted to neutral or alkaline by the fixing agent for solidification, and the stability and uniformity of the microspheres are ensured.

[0026] 2. Enhanced elasticity and antioxidant capacity: after the microspheres are soaked in the tannic acid solution, the tannic acid is adsorbed and hydrogen bonds are formed with the amino groups of chitosan, this process not only enhances the elastic modulus of the microspheres, but also endows the microspheres with excellent antioxidant capacity, and the biological function of the microspheres is improved.

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

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

[0029] 5. No cross-linking agent, avoiding toxicity issues: This invention avoids the use of cross-linking agents, overcoming the toxicity problems that traditional cross-linking agents may cause, ensuring the biocompatibility of the microspheres, and making them more suitable for application in the medical device industry.

[0030] 6. Wide range of applications: Due to its excellent performance, the recombinant collagen antioxidant microspheres prepared by this invention can be used as fillers in medical devices, tissue engineering, drug delivery and other fields, and have a wide range of commercial application prospects.

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[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 The elastic modulus is the recombinant collagen antioxidant microspheres prepared in Example 1 of this invention.

[0034] Figure 3 The DPPH scavenging rate is the value of the recombinant collagen antioxidant microspheres prepared in Example 1 of this invention.

[0035] Figure 4 The results of the MTT assay are for the recombinant collagen microspheres prepared in Example 1 of this invention.

[0036] Figure 5 The results show the experimental findings of L929 cell migration promoted by the recombinant collagen microspheres prepared in Example 1 of this invention.

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

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

[0039] Example 1

[0040] The present embodiment provides a method for preparing recombinant collagen antioxidant microspheres without crosslinking agent, specifically comprising the following steps:

[0041] Step one, take 2 mL of glacial acetic acid, 1 g of chitosan (100000 Da) and 0.5 g of recombinant collagen into 100 mL of deionized water to obtain a mixed solution as an aqueous phase; 2.5 g of Tween-80 is added into 400 mL of petroleum ether (boiling range 60-90℃) as an oil phase; the recombinant collagen is a macromolecular type I recombinant collagen, which is composed of a short amino acid sequence (GAPGAPGSQGAPGL) from natural human type I collagen as a repeating unit and repeated 52 times, the collagen has good water solubility, the molecular weight is 78862 Da, and is purchased from Xi'an Juzi Biological Gene Technology Co., Ltd.;

[0042] Step two, inject the oil phase into the aqueous phase, emulsify and stir by a homogenizer, and then add liquid nitrogen to reduce the system temperature to-40℃ after stirring; the rotation speed of the homogenizer is 10000 rpm, and the stirring time is 1 h;

[0043] Step three, blend 2 g of NaOH (or KOH), 5 mL of water and 50 mL of anhydrous ethanol and pre-cool to-40℃ to obtain a fixing solution;

[0044] Step four, add the pre-cooled fixing solution to the pre-cooled emulsion for solidification, and then wash the solidified microspheres with deionized water;

[0045] Step five, place the washed microspheres into a 10 wt% tannic acid solution, soak for 12 h, wash with deionized water after soaking, and freeze-dry to obtain recombinant collagen antioxidant microspheres with uniform size.

[0046] Example 2

[0047] The present embodiment provides a method for preparing recombinant collagen antioxidant microspheres without crosslinking agent, specifically comprising the following steps:

[0048] Step one, take 0.025 mL glacial acetic acid, 0.5 g chitosan (500000 Da) and 2.5 g recombinant collagen into 10 mL deionized water to obtain a mixed solution as an aqueous phase; 16 g Span 60 (or Span 80) is added into 80 mL petroleum ether (boiling range 90℃-120℃) as an oil phase; the recombinant collagen is recombinant collagen type II, which is composed of human collagen type II full-length alpha 1 chain sequence, has good water solubility, and is purchased from Xi'an Juzi Biological Gene Technology Co., Ltd.;

[0049] Step two, the oil phase is injected into the water phase and emulsified by a homogenizer, then liquid nitrogen is added to reduce the system temperature to-80℃ after stirring; the rotation speed of the homogenizer is 1000 rpm, and the stirring time is 2 h;

[0050] Step three, 0.03 g potassium bicarbonate (or sodium bicarbonate), 5 mL water and 25 mL anhydrous ethanol are blended and pre-cooled to-80℃ to obtain a fixing liquid;

[0051] Step four, the pre-cooled fixing liquid is added to the pre-cooled emulsion to solidify, and then the solidified microspheres are washed with deionized water;

[0052] Step five, the washed microspheres are placed in a 20 wt% tannic acid solution for 0.1 h, then washed with deionized water, and freeze-dried to obtain recombinant collagen antioxidant microspheres with uniform size.

[0053] Example 3

[0054] The embodiment provides a method for preparing recombinant collagen antioxidant microspheres without crosslinking agent, which specifically comprises the following steps:

[0055] Step one, take 0.3 mL glacial acetic acid, 0.001 g chitosan (30000 Da) and 0.5 g recombinant collagen into 10 mL deionized water to obtain a mixed solution as an aqueous phase; 0.06 g Tween-60 is added into 60 mL petroleum ether (boiling range 30℃-60℃) as an oil phase; the recombinant collagen is recombinant collagen type III, which is composed of a short amino acid sequence from natural human collagen type III as a repeating unit for multiple repetitions, wherein the short amino acid sequence (GERGAPGFRGPAGPNGIPGEKGPAGER) is repeated for 30 times, and the collagen has good water solubility, which is purchased from Xi'an Juzi Biological Gene Technology Co., Ltd.;

[0056] Step two, the oil phase is injected into the water phase and emulsified by a homogenizer, then liquid nitrogen is added to reduce the system temperature to-20℃ after stirring; the rotation speed of the homogenizer is 15000 rpm, and the stirring time is 1.5 h;

[0057] Step three, blend 0.24 g of sodium carbonate (or potassium carbonate), 4 mL of water and 20 mL of anhydrous ethanol and pre-cool to -20℃ to obtain a fixing solution;

[0058] Step four, add the pre-cooled fixing solution to the pre-cooled emulsion for solidification, and then wash the solidified microspheres with deionized water;

[0059] Step five, place the washed microspheres in a 0.01 wt% tannic acid solution for 72 h, then wash with deionized water, freeze-dry, and obtain uniform-sized recombinant collagen antioxidant microspheres.

[0060] The properties of the recombinant collagen antioxidant microspheres prepared by the method of the present application were determined using the recombinant collagen antioxidant microspheres prepared in Example 1 as an example.

[0061] 1. Morphology determination

[0062] The SEM photograph of the recombinant collagen microspheres of Example 1 was taken using a field emission scanning electron microscope (GeminiSEM 300, Carl Zeiss, Germany), as shown in Figure 1 The results show that the microspheres have a diameter of about 50 μm and a dense pore size on the surface.

[0063] 2. Elastic modulus

[0064] The elastic modulus of the microspheres before and after soaking was investigated by atomic force microscope uniaxial compression experiment. The elastic modulus of the microspheres after soaking increased to 20 Kpa, and the results show that tannic acid soaking improves the elastic modulus of the microspheres. Figure 2 ).

[0065] 3. DPPH scavenging ability

[0066] The antioxidant capacity of the microspheres before and after soaking with tannic acid was determined by DPPH, and the results show that the antioxidant capacity of the collagen microspheres is significantly improved by the soaking method. Figure 3 ).

[0067] 4. Biocompatibility

[0068] MTT and cell scratch methods were used to prepare the leaching solution according to the recommended method of GB / T 16886.5-2017 for in vitro cell compatibility test, Figure 4 and Figure 5 The results show that the leaching solution of the recombinant collagen antioxidant microspheres prepared in Example 1 can effectively promote the proliferation and migration of mouse fibroblasts (L929), and the cell proliferation rate 24 h after soaking is 120.2%, and the cell proliferation rate 24 h after soaking is 130.5, indicating that it has good biocompatibility.

[0069] The liver, spleen and kidney of the rats were examined by H&E staining 8 weeks after the recombinant collagen type I antioxidant microspheres were implanted into the skin, and the results showed that the liver, spleen and kidney of the rats had no obvious lesions before and after the recombinant collagen type I antioxidant microspheres were soaked in tannic acid, indicating that the recombinant collagen type I antioxidant microspheres had good biocompatibility.

[0070] The method for preparing the recombinant collagen type I antioxidant microspheres without a crosslinking agent provided by the application significantly simplifies the preparation process, is easy to operate, and is easy to popularize and apply. The recombinant collagen type I microspheres prepared by the method not only have good dispersibility and uniform size, ensuring the consistency and stability of product quality, but also have excellent antioxidant performance. These advantages make the microspheres have wide application potential in the medical device industry, especially in occasions requiring high-performance fillers, and can significantly improve the performance and reliability of products, providing strong support for the innovation and development of medical devices.

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

Claims

1. A method for preparing recombinant collagen antioxidant microspheres without cross-linking agents, characterized in that, Includes the following steps: Step 1: Dissolve recombinant collagen and chitosan in deionized water containing glacial acetic acid as the aqueous phase; use petroleum ether containing surfactants as the oil phase; Step 2: Inject the oil phase described in Step 1 into the aqueous phase, and emulsify and stir using a homogenizer. After stirring, lower the system temperature to obtain an emulsion. Step 3: Mix the fixative, water, and anhydrous ethanol and pre-cool to obtain a fixative solution; the fixative 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 pre-cooling temperature is -20℃ to -80℃; Step 4: Add the pre-cooled fixative from Step 3 to the emulsion from Step 2 for curing, and then wash the cured microspheres with deionized water. Step 5: Transfer the washed microspheres from Step 4 to a tannic acid aqueous solution for soaking, then wash with deionized water, freeze-dry, and obtain recombinant collagen microspheres with uniform size.

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

3. A method for preparing recombinant collagen antioxidant microspheres without cross-linking agents according to claim 1 or 2, characterized in that, The recombinant collagen mentioned 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.

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

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

6. The method for preparing recombinant collagen antioxidant microspheres without cross-linking agents according to claim 1, characterized in that, The petroleum ether mentioned in step one is pharmaceutical grade, with a boiling range of 30℃~60℃, 60℃~90℃, or 90℃~120℃.

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

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

9. The method for preparing recombinant collagen antioxidant microspheres without cross-linking agents 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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