BMP2 protein protection solution, BMP2 protein preparation and preparation method thereof

Abstract

This invention first provides a protein protection solution for long-term transportation, storage, and use of bone morphogenetic protein BMP2 at room temperature, as well as a BMP2 protein preparation and its preparation method. The BMP2 protein protection solution and BMP2 protein preparation provided by this invention provide an acidic environment with a pH of 4.0-6.8, and the arginine contained therein protects and enhances the osteogenic induction activity of BMP2 at room temperature (25°C) and 37°C.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a bone morphogenetic protein BMP2 protein protection solution, a BMP2 protein preparation and a preparation method thereof. Background Art

[0002] Bone morphogenetic protein (BMP2) is a member of the bone morphogenetic protein family and has multiple biological functions. It can not only induce bone formation in the body, but also is an essential cell regulatory factor in the embryonic development stage.

[0003] BMP is mainly distributed in the bone tissue of animals. BMP has been isolated from the decalcified bone matrix of cattle, rabbits, pigs, sheep, dogs, monkeys and humans, and has also been extracted from the teeth and kidney tissues of humans and rabbits. In nature. There are more than 20 BMP subtypes that have been discovered. Except for BMP1, they all belong to the TGFβ superfamily. They are very similar to each other in terms of genes, protein sequences or their higher-level structures, as well as biological functions. There is a high degree of conservation between the BMP genes of various animals, suggesting that this is a very conservative and important family of regulatory molecules in the evolutionary process. Among the BMP subtypes that have been cloned, there are seven very conservative cysteine ​​residues in the primary structure, just like TGFβ. These residues form three pairs of intrachain disulfide bonds and one pair of interchain disulfide bonds. These disulfide bonds play a decisive role in maintaining the natural active conformation of the molecule. After the disulfide bonds are opened with a reducing agent, their bone induction activity will be completely lost. Natural BMP2 is a glycoprotein, but the glycosylation modification of the BMP molecule has nothing to do with its osteoinductive activity, and deglycosylation does not affect the activity of BMP2.

[0004] The full length of human BMP2 cDNA is 1191bp; it encodes 396 amino acids and consists of three parts: signal peptide sequence, propeptide region and mature peptide. Among them, the mature peptide monomer has 114 amino acids and the isoelectric point is about: 8.6~8.8. BMP2 not only plays a vital role in the development and repair of hard tissues, but also widely participates in the occurrence and maturation of almost all organs and tissues. Due to its strong bone induction activity, BMP2 is often compounded with some carrier materials such as bioceramics (hydroxyapatite, tricalcium phosphate, bioactive glass), natural tissue materials (collagen, decalcified bone matrix), and organic polymer materials (polylactic acid, hydroxypropionic acid polymer, etc.) for large bone defect repair, spinal fusion surgery, treatment of refractory fractures such as mid-tibial fractures or old fractures. Both in animal experiments and clinical experiments, the results have been significantly better than those of the control group. With the further deepening of basic experiments and clinical research, it can be expected that BMP2 will have broad application prospects.

[0005] BMP2 has the disadvantages of short half-life in the body, easy diffusion to surrounding tissues or loss through blood diffusion, which makes it unable to act on the body stably for a long time. In order to enhance the persistence and stability of BMP2 at the site of action, the commonly used method is to prepare BMP2 using genetic recombination technology so that it can specifically bind to the surface of extracellular matrix materials, natural polymer materials, synthetic polymer materials and metal materials.

[0006] BMP2 is difficult to dissolve under physiological conditions. Acidic solutions can improve the solubility of BMP2, but the pH of existing BMP2 protective agents is extremely acidic. This is not conducive to the osteogenic effect of BMP2 on cells or tissues. It has been reported that oral L-arginine (L-arginine) promotes the growth of height in short children. 【1】 Arginine can provide nitric oxide (NO), which is a highly diffusible free radical. As a second messenger and neurotransmitter, it is an important regulator of cell-to-cell information transmission. NO can regulate the proliferation and functional activity of osteoblasts, thus playing an important role in maintaining bone morphology and bone reconstruction. 【2】 . And L-arginine can promote osteoporotic fracture healing and improve osteoporosis 【3】 There are also reports on the application of arginine combined with chitosan and DNA nanoparticle controlled release PELA microspheres in osteogenesis 【4】 Arginine is also widely used as a protein protectant. For example, arginine can improve the stability of hemoglobin pigment. 【5】 The addition of arginine can also improve the oxidative stability and gel properties of myofibrillar protein, as well as the oxidative stability of meat protein, and improve the tenderness and water retention of meat products. 【6】 , Kim NA et al. 【7】 Introduced that arginine can be used as a protective agent in protein liquid preparations 【7】 However, arginine solution is alkaline, which greatly reduces the solubility of BMP2 compared with acetic acid solution, which is not conducive to the application of BMP2. Therefore, it is necessary to develop a composition that contains arginine, does not affect the solubility of BMP2, and provides a mild pH for long-term transportation, storage, and use of bone morphogenetic protein BMP2 solution or lyophilized powder.

[0007] Reference Index:

[0008] 【1】Jiang Mingyu. Study on the effect and mechanism of oral arginine in promoting linear growth of long bones[D]. Fudan University.

[0009] 【2】Hao Yingjie, Wang Zhen, Li Xiuqun, et al. Effects of L-arginine on rat osteoblasts[J]. Chinese Journal of Experimental Surgery, 2007, 24(9): 1.

[0010] 【3】Zhang Shixu, Lu Xuesheng, Shi Changhao, et al. Effects of L-arginine on bone healing and blood biochemistry in osteoporotic fractures[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(28): 6.

[0011] 【4】Xu Xiaolong. Preparation of arginine-chitosan / DNA nanoparticle controlled release PELA microspheres and experimental study on their osteoinductive efficacy[D]. Southern Medical University, 2016.

[0012] 【5】Song Xuan. Research on the stability and application of arginine-hemoglobin pigment[D]. Northwest Agriculture and Forestry University.

[0013] 【6】Ma Wenhui, Li Baoling, Cao Yungang, Huang Junrong. Effects of different concentrations of arginine on the oxidative stability and gel properties of myofibrillar protein[C] / / The 17th Annual Meeting of the Chinese Society of Food Science and Technology.

[0014] 【7】Kim NA.[Int J Pharm,2016,513(1-2):26] Summary of the invention

[0015] The invention provides a solution or freeze-dried powder for ensuring the osteoinductive activity of bone morphogenetic protein BMP2 protein during long-term transportation and storage at room temperature.

[0016] The present invention first provides a method for preparing a composition for protecting the stability of BMP2 protein, which comprises:

[0017] 1) Dissolve an appropriate amount of arginine in water to make the arginine concentration 0.001-0.5M;

[0018] 2) Add appropriate amount of acid and adjust the pH value to 4.0-6.8 to obtain BMP2 protein protection solution.

[0019] Preferably, the arginine concentration is 0.001-0.5M; more preferably, the arginine concentration is 0.01M, or 0.05M.

[0020] Further preferably, in step 2), the acid is acetic acid.

[0021] The present invention also provides a BMP2 protein protection solution prepared according to the above preparation method.

[0022] Another aspect of the present invention provides a method for preparing a BMP2 protein preparation, which comprises: dissolving a BMP2 protein composition in the above-mentioned BMP2 protein protection solution to obtain a BMP2 protein solution.

[0023] Preferably, in the BMP2 protein composition, the BMP2 protein content is 0 mg / ml-20 mg / ml.

[0024] More preferably, the ratio of BMP2 protein to BMP2 protein protection solution is 0 mg / ml-15 mg / ml in mg:mL.

[0025] In another embodiment according to the present invention, it also includes:

[0026] The BMP2 protein solution is placed in a freeze dryer for freeze drying to obtain BMP2 protein freeze-dried powder. When reconstituted, protein protection solution is added to the BMP2 freeze-dried powder. The ratio of BMP2 protein freeze-dried powder to protein protection solution is 0 mg / ml-15 mg / ml in mg:mL.

[0027] The present invention further provides a BMP2 protein preparation prepared according to the above preparation method.

[0028] The beneficial effects of the above technical solution of the present invention are as follows:

[0029] In the above scheme, the BMP2 protein protection solution provided by the present invention provides an acidic environment with a pH value of 4.0-6.8. The arginine contained in the solution protects the protein activity of BMP2 stored at room temperature 25°C and 37°C, thereby providing a good osteogenic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 After the BMP2 protein was stored in the protein protection solution prepared according to Examples 1 and 2 of the present invention at 25°C for 1 month, 2 months, 4 months and 8 months, the alkaline phosphatase ALP activity detection kit was used to detect its osteogenic induction activity on iMEF cells, and the absorbance value at a wavelength of 405 nm was detected. The larger the absorbance value, the higher the alkaline phosphatase ALP activity, which reflects the higher the BMP2 activity, and the smaller the absorbance value, the lower the alkaline phosphatase ALP activity, which reflects the lower the BMP2 activity. After the protein protection solution 1 and 2 stored BMP2 at 25°C for 1 / 2 / 4 / 8 months, its osteogenic activity was significantly higher than that of conventional acetic acid and hydrochloric acid.

[0031] Figure 2 After the BMP2 protein was stored in the protein protection solution prepared according to Examples 1 and 2 of the present invention at 37°C for 1 month, 2 months, 4 months and 8 months, the alkaline phosphatase ALP activity detection kit was used to detect its osteogenic induction activity on iMEF cells, and the absorbance value at a wavelength of 405 nm was detected. The larger the absorbance value, the higher the alkaline phosphatase ALP activity, which reflects the higher the BMP2 activity, and the smaller the absorbance value, the lower the alkaline phosphatase ALP activity, which reflects the lower the BMP2 activity. After the protein protection solution 1 and 2 stored BMP2 at 37°C for 1 / 2 / 4 / 8 months, its osteogenic activity was significantly higher than that of conventional acetic acid and hydrochloric acid.

[0032] Figure 3iMEF cells cultured under different conditions were stained with Alizarin Red to show mineralized nodules. The darker the color of Alizarin Red staining, the stronger the osteogenic induction activity of BMP2, and the lighter the color of Alizarin Red staining, the weaker the osteogenic induction activity of BMP2. As shown in the figure, protein protection solutions 1 and 2 make BMP2 have stronger activity in promoting the osteogenic differentiation of iMEF cells.

[0033] Figure 4 This is an experiment on ectopic induction of osteogenesis in rat muscle pouch. X-ray examination at 4 weeks showed obvious development. Protein protection solutions 1 and 2 made the silk fibroin osteogenic material containing BMP2 have stronger ectopic osteogenesis performance. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0035] Reagents and materials:

[0036] BMP2 dry powder was purchased from GenScript Biotech Co., Ltd.

[0037] ALP (alkaline phosphatase) activity detection kit was purchased from Biotech Co., Ltd.

[0038] Alizarin red staining kit was purchased from Solarbio

[0039] BCA protein detection kit was purchased from Bio-Tech Biotech Co., Ltd.

[0040] SD rats were purchased from the Animal Experiment Center of Chongqing Medical University.

[0041] Example 1 Preparation method of BMP2 protein protective solution

[0042] Prepare BMP2 protein protection solution 1:

[0043] 1) Preparation step: dissolving or diluting arginine and acetic acid so that the BMP2 protein protection solution composition finally contains 0.01 M / L arginine and acetic acid, and adjusting the pH value of the protein protection solution to 4.5 by using acetic acid.

[0044] 2) Example:

[0045] Weigh 0.1742 g of arginine, dissolve it in 10 mL of double distilled water, and adjust the pH to 4.5 with acetic acid.

[0046] Example 2 Preparation method of BMP2 protein protective solution

[0047] 1) Prepare BMP2 protein protection solution 2:

[0048] Arginine and acetic acid are dissolved or diluted so that the BMP2 protein protection solution composition finally contains 0.05 M / L arginine and acetic acid, and the pH value of the protein protection solution is adjusted to 6.0 with acetic acid.

[0049] 2) Example: Weigh 0.871 g of arginine and dissolve it in 10 mL of double distilled water. Adjust the pH to 6.0 with acetic acid.

[0050] Example 3 Protein protection solution performance test

[0051] Dissolve 0.05mg BMP2 dry powder in 0.1ml BMP2 protein protection solution 1, 0.1ml BMP2 protein protection solution 2, 0.1ml 50mM acetic acid and 0.1ml 1mM hydrochloric acid. After fully dissolved, divide 0.1ml of solution into 4 1.5ml EP tubes, 25ul each, and then put them into a -80° refrigerator for pre-freezing overnight and then put them into a vacuum freeze dryer for freeze drying. After freeze-drying, place them in a 25°C constant temperature incubator and store them for 1 month, 2 months, 4 months and 8 months respectively. Then take out one tube each and treat iMEF cells for 3 days. The ALP (alkaline phosphatase) activity detection kit was used to detect the expression of alkaline phosphatase in the cells to show the activity of BMP2 after being stored in different protein protection solutions for different periods of time. The results are as shown in the figure. Figure 1 As shown, protein protection solutions 1 and 2 can make the osteogenic activity of BMP2 significantly higher than that of conventional acetic acid and hydrochloric acid after storage at 25°C for 1 / 2 / 4 / 8 months.

[0052] Example 4 Protein protection solution performance test

[0053] Dissolve 0.05mg BMP2 dry powder in 0.1ml BMP2 protein protection solution 1, 0.1ml BMP2 protein protection solution 2, 0.1ml 50mM acetic acid and 0.1ml 1mM hydrochloric acid. After fully dissolved, divide 0.1ml of solution into 4 1.5ml EP tubes, 25ul each, and then put them into a -80° refrigerator for pre-freezing overnight and then put them into a vacuum freeze dryer for freeze drying. After freeze-drying, place them in a 37°C constant temperature incubator and store them for 1 month, 2 months, 4 months and 8 months respectively. Then take out one tube each and treat iMEF cells for 3 days. The ALP (alkaline phosphatase) activity detection kit was used to detect the expression of alkaline phosphatase in the cells to show the activity of BMP2 after being stored in different protein protection solutions for different periods of time. The results are as shown in the following figure. Figure 2 As shown, protein protection solutions 1 and 2 can make the osteogenic activity of BMP2 significantly higher than that of conventional acetic acid and hydrochloric acid after storage at 37°C for 1 / 2 / 4 / 8 months.

[0054] Example 5 Alizarin red staining experiment to detect the performance of protein protection solution

[0055] iMEF cells cultured under different conditions were stained with Alizarin Red to show mineralized nodules. The cell culture conditions in the Alizarin Red staining study were similar to those in the iMEF cell ALP activity detection experiment. About 24 hours after inoculation, the culture medium was converted to osteogenic induction medium to induce bone formation, and protein protection solution 1, protein protection solution 2, 50mM acetic acid at pH 3.5, and 1mM hydrochloric acid at pH 3.5 were prepared according to Examples 1, 2, and 4, and BMP2 stored at 37°C for 8 months was dissolved respectively, and then added to the 6-well plates with iMEF cells. After 13 days of cell culture, the cells were stained with an Alizarin Red staining kit (Solarbio, China). When Alizarin Red dye reacts with calcium ions, an orange-red complex can be formed. The results are as shown in Figure 3 The protein protection solutions 1 and 2 shown above make BMP2 have stronger activity in promoting osteogenic differentiation of iMEF cells.

[0056] Example 6: Experimental test on ectopic induction of osteogenesis in rat muscle bag to detect the performance of protein protective solution

[0057] Female SD rats weighing about 300 g (purchased from the Animal Experiment Center of Chongqing Medical University) were selected, and 100 ul of protein protection solution 1, 100 ul of protein protection solution 2 and 100 ul of 50 mM acetic acid at pH 3.5 were prepared according to Examples 1, 2, and 4, and BMP2 stored for 8 months at 37°C was dissolved respectively. 100 ul of double distilled water without BMP2 was used as a blank control, and the results were added dropwise to 200 ul of silk fibroin material, sterilized with ethylene oxide, and implanted into the quadriceps femoris muscle bag of rats. After 4 weeks, X-ray photographs were taken to detect osteogenesis.

[0058] The specific experimental steps are as follows: 1. Perform intraperitoneal injection anesthesia on SD rats, prepare the skin of the surgical area and disinfect it; 2. Select the middle and upper sides of the back of the left and right thighs of the rats, make an incision about 1 cm long, separate the fascia to expose the quadriceps femoris of the rats, bluntly separate the quadriceps femoris muscles, and make a muscle bag with a length and depth of 0.5 cm along the direction of the muscle fibers (do not penetrate the muscle); 3. Implant the silk protein material containing BMP-2, and at the same time implant the silk protein material containing the same volume of double distilled water as a negative control, suture the muscle bag and skin, and mark it. X-ray examination at 4 weeks showed that osteogenic materials would promote ectopic bone formation, and X-ray examination showed obvious development, but protein protection solutions 1 and 2 made the silk protein osteogenic material containing BMP2 have stronger ectopic bone formation performance, see Figure 4 .

[0059] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a BMP2 protein preparation, characterized in that: include: Dissolving the BMP2 protein composition in a BMP2 protein protection solution to obtain a BMP2 protein solution; The BMP2 protein protection solution is obtained by the following method: 1) Dissolve an appropriate amount of arginine in water to make the arginine concentration 0.01M or 0.05M; 2) Add appropriate amount of acetic acid and adjust the pH value to 4.0-6.8 to obtain BMP2 protein protection solution; In the BMP2 protein composition, the BMP2 protein content is 0.5 mg / ml-20 mg / ml.

2. The method for preparing the BMP2 protein preparation according to claim 1, characterized in that: In terms of mg:mL, the ratio of BMP2 protein to BMP2 protein protection solution is 0.5 mg / ml-20 mg / ml.

3. The method for preparing the BMP2 protein preparation according to claim 1, characterized in that: Also includes: The BMP2 protein solution is freeze-dried to obtain BMP2 protein freeze-dried powder.

4. The BMP2 protein preparation obtained by the preparation method according to any one of claims 1 to 3.

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