Preparation method for coating protein on surface of needle-shaped silicon dioxide

Abstract

The invention relates to the field of biological materials, in particular to a preparation method of needle-shaped silicon dioxide coated with protein on the surface, which comprises the following steps: S1, treating artificially synthesized needle-shaped silicon dioxide by using a sodium hydroxide aqueous solution; then modifying the amount of the needle-shaped silicon dioxide by using a 3-aminopropyl methyl diethoxy silane modifier; s2, preparing a protein solution; s3, coating the surface of the needle-shaped silicon dioxide with protein by using EDC and NHS; and S4, centrifuging, washing and drying. The method has the beneficial effects that the operation is simple and convenient, the reaction condition is mild (protein denaturation is avoided), the biocompatibility is excellent (non-toxic crosslinking agent residues are left), and the method is suitable for industrial production of biomedical carriers, cosmetic active ingredient loading and tissue engineering materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of material preparation, in particular to a preparation method for artificially preparing a needle-shaped silicon dioxide material surface coated with a protein sample. The product can be widely used in biomedical, cosmetics, tissue engineering and other fields with high requirements for the uniformity of carrier morphology and biological activity. BACKGROUND

[0002] As an inorganic material with unique morphology, needle-shaped silicon dioxide has become an ideal carrier for bioactive substances due to its high specific surface area, good chemical stability, low toxicity and biocompatibility. Hydrolyzed collagen and collagen protein are natural high molecular substances with excellent biological activity, and have good moisturizing properties, biocompatibility and cell affinity. For example, collagen is the main component of the skin, which can promote cell proliferation and tissue repair; hydrolyzed collagen has a smaller molecular weight and is more easily absorbed, and can play a moisturizing and anti-wrinkle role in cosmetics. Coating such proteins on the surface of needle-shaped silicon dioxide can combine the advantages of both, not only realizing stable loading of proteins by utilizing the carrier characteristics of needle-shaped silicon dioxide, but also expanding its application scenarios by taking advantage of the biological activity of proteins. In the prior art, there has been some progress in the research on coating proteins on the surface of silicon dioxide, but there are still many deficiencies in the coating of needle-shaped silicon dioxide and hydrolyzed collagen, collagen protein and other proteins. The reasons are as follows: (1) In the prior art, the combination of proteins and silicon dioxide relies on physical adsorption, resulting in weak binding force and easy desorption in complex systems; (2) Some chemical coupling methods are prone to denaturation of proteins due to harsh reaction conditions (such as high temperature and strong acid and alkali); at the same time, the large molecular weight and complex structure of proteins make it difficult for existing methods to achieve uniform coating on the surface of needle-shaped silicon dioxide, affecting the performance stability of the product; (3) The prior art generally uses naturally occurring needle-shaped silicon dioxide. Such naturally occurring silicon dioxide is of various types, and the surface active sites are active, which further exacerbates the defects of uneven coating and poor binding force, and cannot meet the requirements of consistency of raw materials for industrial mass production.

[0003] In addition, it should be noted that some of the existing methods for coating proteins on needle-shaped silicon dioxide use toxic cross-linking agents, which limits the application of the product in the fields of biomedicine and cosmetics. At present, there is a need for needle-shaped silicon dioxide surface coated with proteins in many fields, but the existing technology has many defects. Therefore, there is a need for non-toxic "needle-shaped silicon dioxide coated with proteins", and the coating effect of a single silicon dioxide surface is required to be good, and the coating effect of multiple silicon dioxide in a row is required to be consistent, and the coating process is required to be simple, and most importantly, it can be mass-produced.

[0004] Therefore, developing a method that uses highly consistent artificially prepared needle-shaped silica as a carrier to achieve uniform and firm protein coating, preserve bioactivity, is simple to operate, has excellent biocompatibility, and can be mass-produced industrially is of great research significance and application value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a method for preparing protein coating on needle-shaped silica. This method solves the problems of easy detachment, uneven coating, poor biocompatibility, and poor performance stability of existing needle-shaped silica when coating proteins, which prevents large-scale industrial production.

[0006] The objective of this invention is achieved through the following technical solution: a method for preparing protein-coated needle-shaped silica, comprising the following steps: S1. Pretreatment of artificially synthesized needle-shaped silica; Artificial needle-shaped silica with uniform morphology and an impurity content ≤0.5% was selected. This needle-shaped silica was placed in a 0.15 g / mL sodium hydroxide aqueous solution and stirred or sonicated at room temperature for a period of time. It was then washed with deionized water until neutral and dried at 80℃-100℃. The dried needle-like silica was dispersed in a mixture of anhydrous ethanol and water, and a 3-aminopropylmethyldiethoxysilane modifier was added. The mixture was refluxed at 50℃-70℃ for a period of time. After centrifugation, the silica was washed with anhydrous ethanol and dried at 80℃-100℃ to obtain aminated needle-like silica. The volume ratio of the 3-aminopropylmethyldiethoxysilane modifier to the weight of the artificial needle-like silica was (10:1~10). S2, Preparation of protein solution; Hydrolyzed collagen was dissolved in deionized water and stirred until homogeneous to prepare a solution with a concentration of 2 mg / mL to 8 mg / mL. The pH of the solution was then adjusted using MES buffer solution with a pH of 4.0 to 6.0. S3, coating reaction; Aminated needle-shaped silica was mixed with a protein solution, and EDC and NHS were added. The molar ratio of EDC to protein was (1.5-4):1, and the molar ratio of NHS to EDC was (1-1.5):1. The mixture was stirred and reacted in the dark at 20℃-30℃ for a period of time. S4, Post-processing; After the reaction was completed, the product was centrifuged at 3000-5000 r / min to collect the solid product. It was then washed repeatedly with deionized water in MES buffer (pH=4.0-6.0) and vacuum dried at 40℃-50℃ for a period of time to obtain a product with protein coated on needle-shaped silica.

[0007] Further, in S1: the mixture is stirred or ultrasonically treated at room temperature for 1-3 hours, washed with deionized water until neutral, and dried at 80-100℃ for 6-12 hours.

[0008] Further, in S1: the dried artificial needle-like silica is dispersed in anhydrous ethanol and water, 3-aminopropylmethyldiethoxysilane modifier is added, and the mixture is refluxed at 50℃-70℃ for 4h-8h. After centrifugation, the mixture is washed 3-5 times with anhydrous ethanol and dried at 80℃-100℃ for 6h-12h to obtain aminated needle-like silica.

[0009] Furthermore, in S1: the alkaline washing time is 2 h; the volume ratio of 3-aminopropylmethyldiethoxysilane modifier to the weight of artificial needle-like silica is (10:2), and the modification time is 6 h.

[0010] Furthermore, in S2: the concentration of the hydrolyzed collagen solution is 5 mg / mL, and the pH of the MES buffer is 5.0.

[0011] Further, in S3: aminated needle-shaped silica is mixed with a protein solution, and EDC and NHS are added, wherein the molar ratio of EDC to protein is (1.5-4):1, and the molar ratio of NHS to EDC is (1-1.5):1, and the reaction is carried out at 20℃-30℃ in the dark with stirring for 3h-8h.

[0012] Furthermore, in S3: the solid-liquid ratio of the aminated needle-like silica to the protein solution is 1:40 (g / mL), the molar ratio of EDC to protein is 3:1, the molar ratio of NHS to EDC is 1.2:1, the reaction temperature is 25℃, and the reaction time is 5 h.

[0013] Further, in S4: after the reaction is completed, the product is centrifuged at 3000-5000 r / min for 5-10 min, the solid product is collected, and it is washed 4-6 times alternately with MES buffer (pH=4.0-6.0) and deionized water. The product is then vacuum dried at 40 ℃-50 ℃ for 8 h-16 h to obtain a product with protein coated on needle-shaped silica surface.

[0014] Furthermore, in S4: the centrifugation speed is 4000 r / min, the centrifugation time is 8 min; the drying temperature is 40℃, and the drying time is 8 h.

[0015] It should be noted that in this scheme, EDC refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and NHS refers to N-hydroxysuccinimide.

[0016] It should be noted that the artificially synthesized needle-like silica referred to in this scheme refers to silica synthesized in a serial manner: Step 1, Raw material pretreatment: Cut the purple bud flower raw material into blocks with a diameter of 0.1-1.0 cm, sterilize and set aside; Step 2, Microbial fermentation: Chemoheterotrophic bacteria are inoculated into liquid culture medium for activation and amplification to obtain amplified bacterial solution; the amplified bacterial solution is mixed with the sterilized purple flower block after step 1, and then placed in a constant temperature shaker for fermentation and culture at a certain temperature and speed for a period of time. When placed in a constant temperature shaker, the material is continuously fermented and cultured for 5-10 days at a rotation speed of 50-200 rpm and a temperature of 16-37 ℃. Furthermore, the blocky material with a diameter gradually increasing from 0.1 cm to 1.0 cm in step 1 corresponds to the rotation speed gradually decreasing from 200 rpm to 50 rpm, that is, the rotation speed is negatively correlated with the aspect ratio of the resulting sponge spicules - the higher the rotation speed, the lower the aspect ratio. The chemoheterotrophic bacteria used were selected from one of the following genera: Ochrobactrum spp., Bacillus subtilis, Clostridium spp., and Aenibacillus mucilaginosus. Among them, the chemoheterotrophic bacteria used were Ochrobactrum spp.; The liquid culture medium was selected from Luria-Bertani liquid medium, nutrient broth (NA) liquid medium, or Nutrient Broth (NB) liquid medium. Step 3, Product separation and purification: After fermentation, the fermentation mixture is first screened through a certain mesh size to remove floating impurities, then allowed to settle and the supernatant is discarded. Then, it is washed and screened through a series of mesh sizes with gradually smaller meshes, and the mesh size of the washing screen is smaller than that of the primary screen. The residue on the final mesh screen is collected. After settling and discarding the supernatant, the mixture was sequentially washed and screened using 100-mesh, 200-mesh, and 400-mesh sieves with progressively increasing mesh sizes. The residue retained on the 400-mesh sieve was collected. Step 4, product drying: The retentate obtained in step 3 is dried to obtain silica sponge spicules; the silica sponge spicules are artificially synthesized needle-shaped silica, with a length of 70-150 μm and an aspect ratio of 10.9±1.3 to 13.7±1.1.

[0017] The present invention has the following advantages: (1) Good coating effect; Specifically, the single artificial needle-shaped silica is evenly coated in various parts, and the overall surface after coating is relatively smooth and uniform (good effect), with only a very small number of protrusions on the surface (good effect). In a batch of production, the uniformity of the surface of each artificial needle-shaped silica tends to be consistent (good results). Moreover, the coating effect was still good even after high-speed centrifugation during the observation, indicating high coating stability (good effect). (2) It is simple to prepare and can be mass-produced; The preparation method of this scheme is not complicated, and the effect of each needle-shaped silica coating protein in a batch production is excellent. It is suitable for mass industrial production and meets the current market needs (compared to the large number of defective products in existing preparation methods, the pass rate of this scheme is very high). (3) Compared with traditional cross-linking agents that use coupling adsorption, this solution is non-toxic and has good biocompatibility and bioactivity. Attached Figure Description

[0018] Figure 1 This is a transmission electron microscope image of a synthetic needle-shaped silica sample in this invention that is not coated with protein. Figure 2 Transmission electron microscopy (TEM) image of the protein-coated needle-shaped silica sample prepared for Example 1 (general morphology); Figure 3 Transmission electron microscopy (TEM) image (single-root morphology) of the protein-coated needle-shaped silica sample prepared for Example 1. Figure 4 The effect of different concentrations of hydrolyzed collagen on the loading of protein samples coated on needle-shaped silica surfaces; Figure 5 The effect of different modifier concentrations on the loading of protein samples coated on needle-shaped silica surfaces. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0020] This solution provides a method for preparing protein coated on needle-shaped silica surfaces, comprising the following steps: S1. Pretreatment of artificially synthesized needle-shaped silica: Artificial needle-shaped silica was placed in a sodium hydroxide aqueous solution with a concentration of 0.15 g / ml, stirred or sonicated at room temperature for 1-3 h, washed with deionized water until neutral, and dried at 80-100 ℃ for 6-12 h. The artificially prepared needle-shaped silica had a uniform morphology and an impurity content of ≤0.5%, and had more stable surface active sites compared with naturally sourced needle-shaped silica. The dried artificial needle-shaped silica was dispersed in anhydrous ethanol and water, and 3-aminopropylmethyldiethoxysilane modifier was added (the volume ratio of 3-aminopropylmethyldiethoxysilane modifier to the weight of artificial needle-shaped silica was (10:1~10)). The mixture was refluxed at 50℃-70℃ for 4h-8h, then centrifuged, washed 3-5 times with anhydrous ethanol, and dried at 80℃-100℃ for 6h-12h to obtain aminated needle-shaped silica.

[0021] S2, Preparation of protein solution; Hydrolyzed collagen was dissolved in deionized water and stirred until homogeneous to prepare a solution with a concentration of 2 mg / mL to 8 mg / mL. The pH of the system was adjusted with MES buffer (pH=4.0-6.0).

[0022] Preferably, the concentration of the hydrolyzed collagen solution is 5 mg / mL. The MES buffer solution has a pH of 5.0.

[0023] S3, coating reaction; Aminated needle-like silica was mixed with a protein solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added. The molar ratio of EDC to protein was (1.5-4):1, and the molar ratio of NHS to EDC was (1-1.5):1. The mixture was stirred in the dark at 20℃-30℃ for 3-8 hours.

[0024] Preferably, the solid-liquid ratio is 1:40 (g / mL), the molar ratio of EDC to protein is 3:1, the molar ratio of NHS to EDC is 1.2:1, the reaction temperature is 25 °C, and the reaction time is 5 h.

[0025] S4, Post-processing; After the reaction was completed, the product was centrifuged at 3000-5000 rpm for 5-10 min, and the solid product was collected. It was washed 4-6 times alternately with MES buffer (pH=4.0-6.0) and deionized water, and then vacuum dried at 40-50 ℃ for 8-16 h to obtain a product with protein coated on needle-shaped silica.

[0026] Preferably, the centrifugation speed is 4000 r / min, the centrifugation time is 8 min, the drying temperature is 40 ℃, and the drying time is 8 h.

[0027] The following specific embodiments further illustrate this solution (if any parameters are not mentioned in the following embodiments, the preferred parameters in the above solution or the parameter range set in the above solution may be used; if any detailed steps are not mentioned, the detailed steps in the above solution shall be used).

[0028] (Example 1) This embodiment discloses a method for preparing peptides coated on needle-shaped silica surfaces, comprising the following preparation steps: S1. Pretreatment of needle-shaped silica.

[0029] Weigh 0.1 g of artificially prepared needle-shaped silica sample (artificial needle-shaped silica with uniform morphology and impurity content ≤0.5%) and place it in a beaker. Then weigh sodium hydroxide and add it to a beaker containing 10 mL of deionized water to prepare a sodium hydroxide aqueous solution with a concentration of 0.15 g / mL. Stir the solution magnetically at room temperature to make it homogeneous. Then add 2 mL of sodium hydroxide aqueous solution to the beaker containing the needle-shaped silica sample. Then stir or sonicate at room temperature for 2 h (1 to 3 h is acceptable) to remove surface impurities. Then wash repeatedly with deionized water until neutral, and dry at 80℃ (80~100℃ is acceptable) for 6 h (6~12 h is acceptable); The dried needle-like silica was dispersed in 2 mL of anhydrous ethanol and 2 mL of water, and 0.3 mL of 3-aminopropylmethyldiethoxysilane was added. The mixture was stirred at 50 °C (50-70 °C is acceptable) for 4 h (4-8 h is acceptable). After the reaction was completed, the mixture was centrifuged, washed 3 times (3-5 times is acceptable) with anhydrous ethanol, and dried at 80 °C (80-100 °C is acceptable) for 6 h (6-12 h is acceptable) to obtain aminated needle-like silica.

[0030] S2, Preparation of protein solution.

[0031] Weigh 0.06 g of hydrolyzed collagen and add 10 mL of MES buffer (pH 5-6 is acceptable) as a solvent. Stir well to form an MES solution of hydrolyzed collagen (the concentration can be in the range of 2 mg / mL-8 mg / mL).

[0032] S3, coating reaction.

[0033] Weigh 19.17 mg of EDC and 11.51 mg of NHS (within the range of (1-1.5):1) and add them to the MES solution of hydrolyzed collagen (the molar ratio of EDC to protein is within the range of (1.5-4):1), and stir until homogeneous. Add the aminated needle-like silica sample obtained in S1 to the solution stirred in step S3, and then place them together on a magnetic stirrer and stir at 30 °C for 4 hours.

[0034] S4, Post-processing.

[0035] After stirring in S3, allow the solution to stand. After standing, centrifuge at 4000 r / min for 8 min (centrifugation at 3000-5000 r / min for 5-10 min is also acceptable). Then wash alternately with MES buffer (pH=4.0, pH=6.0) and deionized water 4 times (4-6 times is also acceptable). The washed white product is then dried at 40℃ (40-50℃ is also acceptable) for 8-16 h to obtain a protein sample coated with needle-like silica. The morphology of the prepared protein-coated needle-like silica sample was analyzed. A protein-coated needle-like silica sample with a loading of 93% was obtained.

[0036] (Example 2) By replacing the amount of 3-aminopropylmethyldiethoxysilane modifier in Example 1 with 0.1 mL, while keeping the rest of the reaction process and conditions unchanged, a protein sample coated with needle-like silica with a loading of 19% can be obtained.

[0037] (Example 3) By replacing the amount of 3-aminopropylmethyldiethoxysilane modifier in Example 1 with 1 mL, while keeping the rest of the reaction process and conditions unchanged, a protein sample with a 99% loading of needle-like silica surface can be obtained.

[0038] (Example) In Example 1, hydrolyzed collagen was replaced with collagen protein, while the rest of the reaction process and conditions remained unchanged. This yielded a protein sample coated on a needle-like silica surface.

[0039] (Example 5) By replacing the amount of hydrolyzed collagen in Example 1 with 0.02 g, while keeping the rest of the reaction process and conditions unchanged, a protein sample coated on a needle-like silica surface with a loading of 22.4% can be obtained.

[0040] (Example 6) By replacing the amount of hydrolyzed collagen in Example 1 with 0.08 g, while keeping the rest of the reaction process and conditions unchanged, a protein sample coated on a needle-shaped silica surface with a loading of 99.1% can be obtained.

[0041] (Test example) Detection Example 1: The artificially synthesized needle-shaped silica selected in Example 1 was observed using a transmission electron microscope before it was coated with peptides. Figure 1 As shown: the lengths and diameters of the needle-like silica needles are relatively uniform.

[0042] The sample from Example 1, after being coated with a protein solution on the surface of needle-shaped silica, was observed using a transmission electron microscope, such as... Figure 2 As shown: it can be seen that each needle-shaped silica is coated with protein, and the thickness of the protein coating on the surface of each needle-shaped silica is consistent; the coating of a single needle-shaped silica is relatively uniform at different locations.

[0043] When the surface of the single needle-shaped silica coated with protein in Example 1 is magnified and observed, it can be seen that the surface of the single silica is generally smooth (with few additional protrusions), indicating that the coating effect is exceptionally good.

[0044] Example 2: The loading of proteins onto the surface of needle-shaped silica was determined by adjusting the concentration of hydrolyzed collagen. The results are as follows: Figure 4 As shown.

[0045] Example 3: The loading of proteins onto the surface of needle-shaped silica was determined by using different concentrations of modifiers. The results are as follows: Figure 5 As shown.

[0046] Based on the results of test cases 2 and 3, relevant personnel should select appropriate peptide concentrations and appropriate modifier concentrations as needed.

[0047] The above embodiments only illustrate preferred implementation methods, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this invention, and these all fall within the protection scope of this invention.

Claims

1. A method for preparing protein-coated needle-shaped silica, characterized in that: Includes the following steps: S1. Pretreatment of artificially synthesized needle-shaped silica; Artificial needle-shaped silica with uniform morphology and an impurity content ≤0.5% was selected. This needle-shaped silica was placed in a 0.15 g / mL sodium hydroxide aqueous solution and stirred or sonicated at room temperature for a period of time. It was then washed with deionized water until neutral and dried at 80℃-100℃. The dried needle-like silica was dispersed in a mixture of anhydrous ethanol and water, and a 3-aminopropylmethyldiethoxysilane modifier was added. The mixture was refluxed at 50℃-70℃ for a period of time. After centrifugation, the silica was washed with anhydrous ethanol and dried at 80℃-100℃ to obtain aminated needle-like silica. The volume ratio of the 3-aminopropylmethyldiethoxysilane modifier to the weight of the artificial needle-like silica was (10:1~10). S2, Preparation of protein solution; Hydrolyzed collagen was dissolved in deionized water and stirred until homogeneous to prepare a solution with a concentration of 2 mg / mL to 8 mg / mL. The pH of the solution was then adjusted using MES buffer solution with a pH of 4.0 to 6.

0. S3, coating reaction; Aminated needle-shaped silica was mixed with a protein solution, and EDC and NHS were added. The molar ratio of EDC to protein was (1.5-4):1, and the molar ratio of NHS to EDC was (1-1.5):

1. The mixture was stirred and reacted in the dark at 20℃-30℃ for a period of time. S4, Post-processing; After the reaction was completed, the product was centrifuged at 3000-5000 r / min to collect the solid product. It was then washed multiple times with deionized water in MES buffer (pH=4.0-6.0) and vacuum dried at 40℃-50℃ for a period of time to obtain a product with protein coated on needle-shaped silica.

2. The method for preparing protein-coated needle-shaped silica according to claim 1, characterized in that: In S1: the mixture is stirred or ultrasonically treated at room temperature for 1-3 hours, washed with deionized water until neutral, and dried at 80-100℃ for 6-12 hours.

3. The method for preparing protein-coated needle-shaped silica according to claim 2, characterized in that: In S1: The dried artificial needle-shaped silica was dispersed in anhydrous ethanol and water, and 3-aminopropylmethyldiethoxysilane was added as a modifier. The mixture was refluxed at 50℃-70℃ for 4-8 hours, centrifuged, washed 3-5 times with anhydrous ethanol, and dried at 80℃-100℃ for 6-12 hours to obtain aminated needle-shaped silica.

4. The method for preparing protein-coated needle-shaped silica according to claim 3, characterized in that: In S1: the alkaline washing time is 2 h; the volume ratio of 3-aminopropylmethyldiethoxysilane modifier to the weight of artificial needle-like silica is (10:2), and the modification time is 6 h.

5. The method for preparing protein-coated needle-shaped silica according to claim 3, characterized in that: In S2: the concentration of the hydrolyzed collagen solution is 5 mg / mL, and the pH of the MES buffer is 5.

0.

6. The method for preparing protein-coated needle-shaped silica according to claim 3, characterized in that: In S3: Aminated needle-shaped silica was mixed with a protein solution, and EDC and NHS were added. The molar ratio of EDC to protein was (1.5-4):1, and the molar ratio of NHS to EDC was (1-1.5):

1. The mixture was stirred in the dark at 20℃-30℃ for 3-8 hours.

7. The method for preparing protein-coated needle-shaped silica according to claim 6, characterized in that: In S3: The solid-liquid ratio of the aminated needle-shaped silica to the protein solution was 1:40 (g / mL), the molar ratio of EDC to protein was 3:1, the molar ratio of NHS to EDC was 1.2:1, the reaction temperature was 25℃, and the reaction time was 5 h.

8. The method for preparing protein-coated needle-shaped silica according to claim 6, characterized in that: In S4 as described above: After the reaction was completed, the product was centrifuged at 3000-5000 r / min for 5-10 min, and the solid product was collected. It was washed 4-6 times alternately with MES buffer (pH=4.0-6.0) and deionized water, and then dried under vacuum at 40 ℃-50 ℃ for 8 h-16 h to obtain a product with protein coated on needle-shaped silica.

9. The method for preparing protein-coated needle-shaped silica according to claim 8, characterized in that: In S4, the centrifugation speed is 4000 r / min, the centrifugation time is 8 min, the drying temperature is 40 ℃, and the drying time is 8 h.

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