Fibronectin preparation and preparation method thereof

Abstract

The present invention relates to the technical field of fibronectin preparations, and particularly discloses a fibronectin preparation, which comprises 1-5 mg / ml of hydrolyzed collagen, 0.5-5 mg / ml of pullulan, and 0.4 mg / ml of a fibronectin stock solution. The pullulan and the hydrolyzed collagen are added into the fibronectin stock solution, the pullulan and the hydrolyzed collagen play a synergistic role, and a protective net is constructed through a barrier and a hydration layer, so that denaturation of the fibronectin can be reduced, and turbidity or precipitation formed by protein agglomeration can be avoided; the stability, the biocompatibility and the effect of the fibronectin preparation are obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of macromolecular protein application, and particularly relates to a fibronectin preparation and a preparation method thereof. Background Art

[0002] Fibronectin is an important extracellular matrix glycoprotein involved in processes such as cell adhesion, migration, differentiation, and tissue repair. Conventional fibronectin is extracted from plasma or obtained through recombinant DNA technology and is widely used in biopharmaceuticals, regenerative medicine, and cosmetics. Maintaining fibronectin's stability is crucial to its function.

[0003] However, fibronectin is thermodynamically unstable and easily affected by changes in ambient temperature. It is prone to aggregation and degradation in a short period of time, especially at room temperature. The protein in the fibronectin aqueous solution easily aggregates to form flocculent precipitates. To prevent fibronectin degradation, it is usually necessary to store and handle it at low temperatures (-20°C), and the conditions of the purification step need to be controlled and must be carried out at temperatures below 4°C. The process of extracting fibronectin from plasma is complex and time-consuming, involving multiple purification steps, which also places high demands on the storage, transportation, and use of fibronectin. Moreover, because fibronectin is very sensitive to environmental conditions such as pH and salt concentration, it is easy for fibronectin to be inactivated or denatured in cosmetics or pharmaceutical preparations due to environmental changes. At the same time, because it is difficult to disperse evenly, it is easy to aggregate, resulting in turbidity or precipitation, affecting the normal use of the product.

[0004] In order to solve the above problems, reagents and systems have been developed to improve the stability of fibronectin aqueous solutions, such as by introducing buffer solution systems, surfactants, antioxidants, etc. However, the introduction of the above reagents is mainly to inhibit the denaturation of fibronectin. Although they also have an improvement effect on improving the stability of fibronectin, the long-term stability effect in actual application is not good. Therefore, it is necessary to develop a new stabilization system to enable fibronectin to maintain stability and good performance in aqueous solution dispersion systems and freeze-dried systems for a long time. Summary of the Invention

[0005] In view of the defects and problems of current fibronectin preparations such as poor stability and variability, the present invention provides a fibronectin preparation and a preparation method thereof.

[0006] The invention provides a fibronectin stabilizer comprising hydrolyzed collagen and pullulan.

[0007] The above-mentioned fibronectin stabilizer, the pullulan has a molecular weight of 200,000 Da.

[0008] The above-mentioned fibronectin stabilizer has a molecular weight of 500-2000 Da of the hydrolyzed collagen.

[0009] The present invention also provides a fibronectin preparation comprising 1-5 mg / ml of hydrolyzed collagen, 0.5-5 mg / ml of pullulan, and 0.4 mg / ml of fibronectin stock solution.

[0010] The concentration of hydrolyzed collagen in the above-mentioned fibronectin preparation is 1-5 mg / ml.

[0011] The concentration of pullulan in the above-mentioned fibronectin preparation is 1-5 mg / ml.

[0012] The concentration of pullulan in the above-mentioned fibronectin preparation is 1 mg / ml.

[0013] The above-mentioned fibronectin preparation, the fibronectin stock solution includes 0.4 mg / ml fibronectin, 5% glycerol and 1% hyaluronic acid.

[0014] The preparation method of the above-mentioned fibronectin preparation is to fully stir and dissolve pullulan and hydrolyzed collagen in deionized water, mix them to obtain a mixed solution of pullulan and hydrolyzed collagen, and then add fibronectin stock solution to prepare a fibronectin preparation, so that the hydrolyzed collagen in the fibronectin preparation is 1-5 mg / ml, pullulan is 0.5-5 mg / ml, and the fibronectin stock solution is 0.4 mg / ml.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention adds pullulan and hydrolyzed collagen to the fibronectin stock solution, and through the synergistic effect of pullulan and hydrolyzed collagen, significantly improves the stability, biocompatibility and efficacy of the fibronectin preparation. Pullulan can wrap the fibronectin molecules in the solution to form a physical barrier, bind to the polar groups of fibronectin through hydrogen bonds, reduce the hydrophobic interaction of the protein, promote solubilization, improve protein solubility, reduce aggregation between proteins, and at the same time reduce the damage of the external environment to the protein structure and maintain its biological activity; and hydrolyzed collagen is rich in a variety of hydrophilic amino acids, which can form a stable hydration layer around the fibronectin. The two work together to build a protective network through the barrier and hydration layer, which can not only reduce the denaturation of fibronectin, but also avoid protein aggregation to form turbidity or precipitation, thereby maintaining the stability of the fibronectin preparation.

[0017] 2. The present invention can maintain stable properties of the fibronectin preparation for a long time by regulating and optimizing the addition amounts of pullulan and hydrolyzed collagen, without turbidity and flocculent precipitation, thus providing a basis for the application of ready-to-use fibronectin.

[0018] 3. The fibronectin preparation prepared by the present invention can be directly freeze-dried as the stock solution of freeze-dried powder without the need to add other protective agents and excipients, is not affected by the compounding temperature and system, and is more widely and conveniently used.

[0019] 4. The pullulan used in the preparation of the fibronectin preparation of the present invention is a natural water-soluble polysaccharide produced by fungi. It has natural ingredients and excellent biocompatibility. It does not need to be separated or removed when used in skin care products and biomedical products.

[0020] 5. The fibronectin preparation of the present invention has stable properties and does not require complicated dissolution and processing steps. It can be directly added to the product in proportion when used without adjusting the product preparation process, saving costs and making it more convenient to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1 is a comparison diagram of the fibronectin preparation of the present invention and a blank control; in the figure, A is the fibronectin preparation of the present invention, and B is the control. DETAILED DESCRIPTION

[0022] In order to solve the problems of poor stability and variability of current fibronectin preparations, the present invention provides a fibronectin preparation and a preparation method thereof. The present invention will be further described below with reference to specific examples.

[0023] Example 1: In this example, fibronectin solutions with different ratios were prepared. The fibronectin stock solution used in the solution included 0.4 mg / ml fibronectin, 5% glycerol, and 1% hyaluronic acid. The components and proportions of the fibronectin solutions in each test group are shown in Table 1 below.

[0024] Table 1 Components and ratios of fibronectin solutions in different experimental groups

[0025]

[0026] After the preparation is completed, the concentration of the fibronectin aqueous solution prepared above is adjusted to 0.4 mg / ml and stored at -20°C for future use.

[0027] The fibronectin solution stored at -20°C was taken and thawed as the experimental sample for the high-temperature accelerated coagulation experiment. The experimental group and the control group were set up. After mixing evenly, the fibronectin solutions of the control group and the experimental group were sterilized respectively; then, the absorbance value of the fibronectin aqueous solution of the experimental group and the control group at a wavelength of 278 nm was detected by UV-visible spectrophotometer. The absorbance value was the starting absorbance value of each group, recorded as Abs0.

[0028] The fibronectin aqueous solutions of the experimental group and the control group were sealed in glass sample bottles, respectively, and then placed in a constant temperature cell culture incubator at 37°C. The fibronectin aqueous solutions of the experimental group and the control group were placed in a constant temperature cell culture incubator, respectively.

[0029] The absorbance value at 280 nm after different time t (1d, 2d, 3d, 5d, 8d, 11d and 14d) (the glass sample bottle needs to be slightly shaken before measurement) is recorded as Abs t .

[0030] According to the formula ΔAbs=Abs t -Abs0 (ΔAbs represents the absorbance change rate, ΔAbs is a positive value) calculates the absorbance change rate of the experimental group samples and the control group samples at different placement times.

[0031] During the long-term high-temperature accelerated coagulation experiment, the sedimentation caused by fibronectin aggregation will affect the light scattering behavior, and the sample absorbance may first increase and then decrease. Therefore, the maximum value of the absorbance change rate within 14 days (ΔAbs max ) as a basis for assessing the stability of fibronectin aqueous solutions. Furthermore, because fibronectin aqueous solutions are prone to aggregation and precipitation after prolonged storage, the fibronectin in the solution aggregates to form large amounts of flocculent or flaky precipitates that settle at the bottom of the sample bottle. Even with gentle shaking before absorbance measurement, the solution's absorbance may still decrease. Therefore, if the ΔAbs value calculated according to the above formula is negative, the result is disregarded.

[0032] △Abs of fibronectin solution in different experimental groups max The results are shown in Table 2 below.

[0033] Table 2 △Abs of fibronectin solution in different experimental groups after 14 days max

[0034]

[0035]

[0036] From the observation and Table 1, it can be seen that when the amount of pullulan added is large, the fibronectin solution will have precipitation or a small amount of flocculent matter, and the △Abs of the fibronectin solution is max When the amount of pullulan added was low, the prepared fibronectin solution had a clear appearance without obvious precipitation and flocculent formation, indicating that the amount of pullulan added had a great influence on the stability of the fibronectin solution.

[0037] Example 2: In this example, a fibronectin solution prepared at a ratio of 1 mg / ml hydrolyzed collagen, 5 mg / ml pullulan, and 0.4 mg / ml fibronectin stock solution was used as the test group and prepared into a lyophilized powder. Separate control groups were prepared by adding trehalose, mannitol, and dextran to the fibronectin stock solution to prepare lyophilized powders. The components and ratios of the lyophilized preparations for each control group are shown in Table 5 below.

[0038] Table 5 Components and ratios of freeze-dried preparations in different control groups

[0039] Group Fibronectin stock solution Trehalose Mannitol Glucan Control group 1 15ml 3% w / v -- -- Control group 2 15ml -- 2% w / v -- Control group 3 15ml -- -- 1％ Control group 4 15ml 3% w / v 2% w / v 1％

[0040] Note: “--” means not added.

[0041] Each preparation was filtered through a 0.22 μm filter membrane and dispensed into vials (1.5 ml per vial). The vials were quickly pre-frozen in a liquid nitrogen tank, and then freeze-dried and immediately filled with nitrogen and sealed. The vials were then stored at -20°C in the dark.

[0042] Electron microscopic observation showed that the freeze-dried powder prepared in each group was loose and porous powder without agglomeration.

[0043] After the freeze-dried preparations prepared in each group were added with equal amounts of buffer, they were completely dissolved within 2 minutes, and the solutions were clear without precipitation.

[0044] Based on the above test results, it is shown that the fibronectin preparation prepared by the present invention can be directly used as a lyophilized stock solution to prepare a lyophilized powder, and the same protective effect can be achieved without the need to add additional protective agents and excipients.

[0045] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fibronectin stabilizer, characterized in that: Includes hydrolyzed collagen and pullulan.

2. The fibronectin stabilizer according to claim 1, characterized in that: The molecular weight of the pullulan is 200,000 Da.

3. The fibronectin stabilizer according to claim 1, characterized in that: The molecular weight of the hydrolyzed collagen is 500-2000Da.

4. A fibronectin preparation, characterized in that: It includes hydrolyzed collagen 1-5 mg / ml, pullulan 0.5-5 mg / ml, and fibronectin stock solution 0.4 mg / ml.

5. The fibronectin preparation according to claim 4, characterized in that: The concentration of hydrolyzed collagen in the fibronectin preparation is 1-5 mg / ml.

6. The fibronectin preparation according to claim 4, characterized in that: The concentration of pullulan in the fibronectin preparation is 1-5 mg / ml.

7. The fibronectin preparation according to claim 6, characterized in that: The concentration of pullulan in the fibronectin preparation is 1 mg / ml.

8. The fibronectin preparation according to claim 4, characterized in that: The fibronectin stock solution includes 0.4 mg / ml fibronectin, 5% glycerol and 1% hyaluronic acid.

9. The method for preparing the fibronectin preparation according to any one of claims 4 to 8, wherein: Pullulan and hydrolyzed collagen were separately dissolved in deionized water with sufficient stirring, and the mixture was mixed to obtain a mixed solution of pullulan and hydrolyzed collagen, which was then added to a fibronectin stock solution to prepare a fibronectin preparation, such that the hydrolyzed collagen content in the fibronectin preparation was 1-5 mg / ml, the pullulan content was 1-50 mg / ml, and the fibronectin stock solution was 0.4 mg / ml.

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