A freeze-drying process suitable for bioactive coatings

Abstract

The present invention relates to a freeze-drying process suitable for bioactive coatings, including the following factors: coating pre-freeze treatment: excipient and auxiliary material solution; pre-freezing part: pre-freezing temperature, shelf temperature before feeding, pre-freezing time. Freeze-drying time part: the fastest temperature point of primary sublimation drying, primary sublimation high-speed section drying time, primary sublimation slow section drying time, desorption drying time. Vacuum degree part: primary sublimation air entrainment amount. The method of the present invention is simple, the process is mature and feasible, and the coating product after freeze-drying has a regular appearance and extremely low water content. It overcomes the shortcomings of the existing bioactive coating preparation technology, meets the needs of actual applications, can replace the drying process in the preparation process of most bioactive coatings, ensure the biological activity of the coating, extend the service life of the coating, help the industrial application of the coating, and provide strong support for related research.

Description

Technical Field

[0001] The present invention relates to a freeze-drying process suitable for the preparation of bioactive coatings, and in particular to a freeze-drying method for a magnesium and titanium alloy composite coating modified with proteins and polypeptides, belonging to the field of biomedical materials. Background Art

[0002] Medical metal materials such as magnesium and titanium alloys are widely used in the field of bioimplantable materials. Due to their low corrosion resistance, high biodegradation rate, and poor interfacial binding with human cells, applying a bioactive coating to their surface is extremely necessary. This coating must possess both good corrosion resistance and bioactivity. This will allow magnesium and titanium alloys to be better utilized to address current challenges in the field of bioimplantable materials. Commonly used bioactive ingredients in such coatings include proteins, peptides, cell growth factors, polysaccharides, and organic small molecule drugs. Proteins are important structural and functional units of living organisms and possess a variety of specialized biological functions. Modifying the surface of medical metals with biocompatible proteins is of great significance for enhancing the bioactivity and functionality of implantable materials. Currently, biocoatings based on protein and peptide modifications are mostly in the experimental stage, and their practical application results are less than satisfactory due to issues such as the inactivation of the coating protein modifications and the long-term preservation of the coating's bioactivity.

[0003] Vacuum freeze-drying, or freeze-drying for short, involves first cooling and freezing a solution of the material to be dried below the eutectic point until it is completely frozen and free of any liquid phase. Heat is then applied to the frozen material, causing the water in the solution to sublime directly into a gaseous state under vacuum conditions. This water vapor is then captured in a cold trap, resulting in the dried material. Vacuum freeze-drying is an important chemical process for drying and separating substances, combining multiple technologies, including vacuum, drying, and refrigeration. Freeze-drying offers several advantages over conventional wet drying methods: freeze-drying operates at extremely low temperatures, which essentially preserves the physicochemical properties of the freeze-dried material, preventing heat-induced protein denaturation in biological materials and ensuring their biological activity. The resulting freeze-dried material is porous, exhibits a well-defined physical shape, and has a large specific surface area, allowing it to quickly return to its original properties upon use. Freeze-drying offers excellent drying efficiency, removing over 95% to 99% of the water content. Once sealed, the dried product can be stored for extended periods without deterioration.

[0004] Current bioactive coating preparation processes often overlook the crucial role of drying, or employ more traditional drying processes. This leads to premature inactivation of the active protein modifiers in the coating. These inactivated proteins, in turn, can severely corrode the medical metal, reducing its lifespan. Therefore, the present invention has developed a freeze-drying process suitable for bioactive coatings.

[0005] The Chinese patent "A Super-Hydrophobic Coating Based on Protein Particles and Preparation Method" (Publication No. CN112029146A) discloses a method for preparing a super-hydrophobic coating material based on protein particles, comprising the following specific steps: S1: Pretreatment of the porous substrate material; S2: Adsorption and immobilization of the protein particles; S3: Modification of the low-surface-energy material. The porous substrate material containing protein clusters needs to be dried after being soaked in activated sludge to remove most of the moisture. Conventional drying methods are not conducive to maintaining the activity of the protein particles, and thus have practical limitations.

[0006] The Chinese patent "A Multifunctional Composite Coating for Magnesium Alloy Heart Stent Materials and Its Preparation Method" (Publication No.: CN 114917414 A) discloses a multifunctional composite coating for magnesium alloy heart stent materials and its preparation method. The multifunctional composite coating comprises a phosphorylated polybasic amino acid within the coating. A coupling agent containing both thiol and carboxyl groups is introduced onto the exterior of the phosphorylated polybasic amino acid. Through the coupling action of the coupling agent containing both thiol and carboxyl groups, a hydrophilic anionic polymer is introduced onto the exterior. Finally, the coating is coated onto the magnesium alloy surface via a dip-pull method. The composite coating is washed with ethanol and then dried in a nitrogen atmosphere to ensure oxidation. However, the active ingredients in the coating are easily inactivated in a high-temperature drying environment, which reduces the coating's biological function.

[0007] The Chinese patent "A Method for Preparing an Antifouling Coating on the Surface of a Composite Polymer Material" (Publication No. CN116285682A) discloses the use of sodium periodate to oxidize hydrogenated caffeic acid with bovine serum albumin (BSA) to assemble a protein coating on the surface of the material. The oxidized hydrogenated caffeic acid rapidly aggregates with the BSA through hydrophobic interactions. Simultaneously, hydrogen bonds and ionic bonds formed between the protein and the phenolic compound enhance crosslinking, resulting in a stable hydrogenated caffeic acid-mediated BSA coating. This hydrogenated caffeic acid-mediated BSA coating exhibits excellent anti-cell and anti-platelet adhesion properties, and possesses a moderate antifouling effect. Furthermore, the numerous carboxyl groups on the surface of the hydrogenated caffeic acid-mediated BSA coating provide potential for secondary grafting of antifouling molecules. Polyethylene glycol is then covalently grafted onto the surface of the composite polymer material via an amidation reaction, resulting in an antifouling coating. The preparation process utilizes a high reaction temperature of 70°C, near the protein inactivation temperature, making it difficult to maintain protein activity using conventional wet drying methods. Summary of the Invention

[0008] In order to overcome the shortcomings of existing bioactive coating preparation technology and meet practical application needs, the present invention designs a freeze-drying treatment process for bioactive coatings. This process can replace the drying process in the preparation process of most bioactive coatings, ensure the biological activity of the coating, extend the service life of the coating, and facilitate the industrial application of the coating.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A freeze-drying process for a bioactive coating comprises the following steps:

[0011] 1) The pretreatment liquid consists of mannitol and tert-butanol. Mannitol acts as a skeleton support during the freeze-drying process, improving the freeze-drying efficiency. Tert-butanol can combine with protein substances to improve the freeze-dried crystal form, forming a unique needle-shaped crystal form, increasing the freeze-dried surface area, and accelerating freeze-drying.

[0012] The mass volume of mannitol in the pretreatment liquid is 8% W / V to 12% W / V, the mass volume of tert-butanol is 3% W / V to 9% W / V, and the balance is water.

[0013] 2) Turn on the freeze dryer and set the shelf temperature before feeding to 0.0±5.0℃ to provide a high degree of supercooling for freeze drying. After drying, a hard gel layer can be formed on the surface of the protein coating to isolate the air and prevent the coating from inactivation.

[0014] 3) Pre-treat the coated product to be freeze-dried. Place the coated product to be dried in a drying container. Use a spray bottle to spray the room temperature pre-treatment liquid on the sample surface. After the sample surface is evenly sprayed, quickly place the sample on the freeze dryer shelf.

[0015] The product to be dried with the outer coating is a magnesium alloy or a titanium alloy.

[0016] The coating is protein-based.

[0017] 4) Adjust the freeze-drying chamber temperature to -14.0±3°C and maintain it for 1 to 3 hours.

[0018] 5) Adjust the freeze-drying chamber temperature to -53.0±5.0°C and maintain it for 3 to 5 hours to quickly freeze the coating sample; continue to observe the freezing condition. Sufficient freezing time can make the crystals freeze more solid and eliminate the hidden dangers of product melting and protein coating inactivation during the sublimation stage.

[0019] 6) Then, at -53.0±5.0℃, the equipment is evacuated; the vacuum condition is decreased from 40±2Pa to 20±2Pa, and the pressure drop does not exceed 1Pa per hour.

[0020] 7) Continue to adjust the freeze-drying chamber temperature, slowly increasing it from -53.0±5.0°C to -28.0±5.0°C over 4-6 hours; slowly increasing it from -28.0±5.0°C to -8.0±5.0°C over 5-7 hours; maintain the freeze-drying chamber temperature at -8.0±5.0°C for 7-9 hours; slowly increasing it from -8.0±5.0°C to 0.0±5.0°C over 3-5 hours, and maintain it under the vacuum conditions set in step 6) for 3-5 hours.

[0021] 8) Continue to adjust the freeze-drying chamber temperature, slowly raising it from 0.0±5.0°C to 45.0±5°C over 2-4 hours, and maintaining it under vacuum for 3-5 hours; the vacuum condition is a variable pressure state between 10 and 30 Pa. Finally, the freeze-drying chamber is subjected to ultimate vacuum at a constant temperature of 45.0±5°C. The desorption drying stage primarily removes the difficult-to-remove bound water in the coating, which accounts for approximately 5% of the total moisture. The maximum desorption drying temperature should not exceed 50°C to ensure complete removal of moisture from the protein coating while preserving the inactivation of the coating protein components.

[0022] The set air entrainment amount under the vacuum conditions in the above steps 6) to 7) is 0.09±0.03 mbar.

[0023] 9) Turn off the freeze dryer, package the outer coating sample under vacuum, adjust the freeze drying chamber environment to normal pressure, and remove the coating sample. After freeze drying, gas phase analysis shows that there is no tert-butyl alcohol remaining in the coating sample, eliminating the problem of tert-butyl alcohol removal.

[0024] After freeze-drying, gas phase determination showed that no tert-butyl alcohol remained in the coating sample.

[0025] The technical solution disclosed in the present invention realizes freeze-drying of the coating product through pretreatment and freeze-drying condition adjustment, thereby removing excess water while maintaining the original activity of the coating, ensuring that the coating product does not lose its activity for a long time.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention applies freeze-drying technology in the pharmaceutical field to the bioactive coating processing process, which greatly enhances the shelf life of the biological coating and ensures that the coating is not inactivated.

[0028] 2. The present invention selects tert-butanol as a freeze-drying auxiliary material suitable for protein coatings, which can form needle-shaped crystals and improve freeze-drying efficiency. At the same time, after freeze-drying, the tert-butanol is completely separated by gas phase detection, and has no effect on the properties of the product.

[0029] 3. The gel layer is a harmful phenomenon in the freeze-drying process. It is a hard layer formed on the surface of the freeze-dried product, which reduces the freeze-drying efficiency. However, in the present invention, a specific gel layer is intentionally formed to effectively block the entry of air and bacteria, further ensuring the activity and sterility of the coating.

[0030] 4. The method of the present invention is simple and the process is mature and feasible. The freeze-dried coating product has a regular appearance and extremely low water content, which overcomes the shortcomings of existing bioactive coating preparation technology and meets practical application needs. It can replace the drying process in the preparation process of most bioactive coatings, ensure the biological activity of the coating, extend the service life of the coating, facilitate the industrial application of the coating, and provide strong support for related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the structure of the protein coating after freeze-drying.

[0032] In the figure: 1-medical metal, 2-mannitol lyophilized layer, 3-protein coating + mannitol lyophilized layer. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the specific implementation methods of the present invention are further described below in conjunction with examples. The following examples are used to specifically illustrate the contents of the present invention. These examples are only general descriptions of the contents of the present invention and do not limit the contents of the present invention.

[0034] To address the challenges of existing bioactive coating drying technologies, the present invention innovates the coating pretreatment process and provides a freeze-drying process suitable for bioactive coatings. The present invention optimizes freeze-drying process parameters based on the performance characteristics of the bioactive coating, including the following factors:

[0035] Coating freeze-thaw treatment: excipient and auxiliary material solution; (excipient is mannitol, auxiliary material is tert-butyl alcohol)

[0036] Pre-freezing part: pre-freezing temperature, shelf temperature before feeding, pre-freezing time.

[0037] Freeze-drying time part: the fastest temperature point of sublimation drying, the drying time of the high-speed sublimation section, the drying time of the slow sublimation section, and the desorption drying time.

[0038] Vacuum degree: The amount of gas entrained during sublimation. It provides disturbance to the vacuum environment and promotes freeze-drying.

[0039] The optimized freeze-drying process takes a total of 45 hours, is energy-saving, produces a regular coating appearance, and has a low defective rate.

[0040] Example 1:

[0041] 1) Preparation of pretreatment solution: The pretreatment solution comprises 9% W / V of mannitol and 6% W / V of tert-butyl alcohol, with the balance being water.

[0042] 2) Turn on the freeze dryer and adjust the shelf temperature to 0.0°C before feeding.

[0043] 3) Pretreat the coated product to be freeze-dried. Place the magnesium alloy sample to be dried with the protein-based biological coating in a drying container. Use a spray bottle to spray the room temperature pretreatment liquid on the sample surface. After the sample surface is evenly sprayed, quickly place the sample on the freeze dryer shelf.

[0044] 4) Adjust the freeze-drying chamber temperature to -14.0°C and maintain it for 1 hour.

[0045] 5) Adjust the freeze-drying chamber temperature to -53.0°C and maintain it for 3 hours to rapidly freeze the coated product. Continuously monitor the freezing process; sufficient freezing time will ensure a more solid crystal freeze and eliminate the risk of product melting during the sublimation phase and inactivation of the protein coating.

[0046] 6) The equipment was then evacuated at -53.0° C. The vacuum condition was gradually reduced from 40 Pa to 20 Pa, with the air entrainment volume set at 0.09 mbar, for 20 hours.

[0047] 7) Continue to adjust the freeze-drying chamber temperature, slowly increasing it from -53.0°C to -28.0°C over 4 hours. Slowly increasing it from -28.0°C to -8.0°C over 5 hours. Maintain the freeze-drying chamber temperature at -8.0°C for 7 hours. Slowly increasing it from -8.0°C to 0.0°C over 3 hours, and maintaining it under vacuum for 3 hours.

[0048] 8) Continue to adjust the freeze-drying chamber temperature, slowly raising it from 0.0°C to 45.0°C over 2 hours and maintaining it under vacuum for 3 hours. The vacuum level is set to alternate between 30Pa and 10Pa. Finally, the freeze-drying chamber is subjected to ultimate vacuum at a constant temperature of 45.0°C. The main task of the desorption drying stage is to remove the difficult-to-remove bound water in the coating, which accounts for approximately 5% of the total water content. At the same time, the maximum desorption drying temperature should not exceed 50°C to ensure that the water in the protein coating is completely removed and the protein components in the coating are not inactivated.

[0049] 9) Turn off the freeze dryer, package the coated product under vacuum conditions, adjust the freeze drying chamber environment to normal pressure, and remove the coated product. After freeze drying, gas phase analysis shows that there is no tert-butyl alcohol remaining in the coating sample, eliminating the difficult problem of tert-butyl alcohol removal.

[0050] Example 2:

[0051] 1) Preparation of pretreatment solution: The pretreatment solution consists of mannitol and tert-butyl alcohol. The mass volume of mannitol in the pretreatment solution is 10% W / V, the mass volume of tert-butyl alcohol is 5% W / V, and the balance is water.

[0052] 2) Turn on the freeze dryer and adjust the shelf temperature to -5°C before feeding.

[0053] 3) Pretreat the coated product to be freeze-dried. Place the titanium alloy sample to be dried with the protein-based biological coating in a drying container. Use a spray bottle to spray the room temperature pretreatment liquid on the sample surface. After the sample surface is evenly sprayed, quickly place the sample on the freeze dryer shelf.

[0054] 4) Adjust the freeze-drying chamber temperature to -14.0°C and maintain it for 2 hours.

[0055] 5) Adjust the freeze-drying chamber temperature to -58.0°C and maintain it for 5 hours to rapidly freeze the coated product. Continuously monitor the freezing process; sufficient freezing time will ensure a more solid crystal freeze and eliminate the risk of product melting during the sublimation phase and inactivation of the protein coating.

[0056] 6) The equipment was then evacuated at -58.0° C. The vacuum condition was gradually reduced from 40 Pa to 20 Pa, with the air entrainment volume set at 0.06 mbar, for 20 hours.

[0057] 7) Continue to adjust the freeze-drying chamber temperature, slowly increasing it from -58.0°C to -33.0°C over 6 hours. Slowly increasing it from -33.0°C to -13.0°C over 7 hours. Maintain the freeze-drying chamber temperature at -13.0°C for 9 hours. Slowly increasing it from -13.0°C to -5.0°C over 5 hours, and maintaining it under vacuum for 5 hours.

[0058] 8) Continue to adjust the freeze-drying chamber temperature, slowly raising it from -5.0°C to 40.0°C over 4 hours and maintaining it under vacuum for 5 hours. The vacuum level is set to alternate between 30 Pa and 10 Pa. Finally, the freeze-drying chamber is subjected to a maximum vacuum at a constant temperature of 40.0°C. The main task of the desorption drying stage is to remove the difficult-to-remove bound water in the coating, which accounts for approximately 5% of the total water content. At the same time, the maximum desorption drying temperature should not exceed 50°C to ensure that the water in the protein coating is completely removed while also ensuring that the protein components in the coating are not inactivated.

[0059] 9) Turn off the freeze dryer, adjust the freeze drying chamber environment to normal pressure, and remove the coated product. After freeze drying, gas phase analysis shows that there is no tert-butyl alcohol remaining in the coating sample, eliminating the problem of tert-butyl alcohol removal.

[0060] Example 3:

[0061] 1) Preparation of pretreatment solution: The pretreatment solution is composed of mannitol and tert-butanol, wherein the mass volume of mannitol in the pretreatment solution is 8% W / V, the mass volume of tert-butanol is 5% W / V, and the balance is water.

[0062] 2) Turn on the freeze dryer and adjust the shelf temperature to 5.0°C before feeding.

[0063] 3) Pretreat the coated product to be freeze-dried. Place the titanium alloy sample to be dried with the protein-based biological coating in a drying container. Use a spray bottle to spray the room temperature pretreatment liquid on the sample surface. After the sample surface is evenly sprayed, quickly place the sample on the freeze dryer shelf.

[0064] 4) Adjust the freeze-drying chamber temperature to -14.0°C and maintain it for 1 hour.

[0065] 5) Adjust the freeze-drying chamber temperature to -48.0°C and maintain it for 3 hours to rapidly freeze the coated product. Continuously monitor the freezing process; sufficient freezing time will ensure a more solid crystal freeze and eliminate the risk of product melting during the sublimation phase and inactivation of the protein coating.

[0066] 6) The equipment was then evacuated at -48.0°C. The vacuum condition was gradually reduced from 40 Pa to 20 Pa, with the air volume set at 0.12 mbar, for 20 hours.

[0067] 7) Continue to adjust the freeze-drying chamber temperature, slowly increasing it from -48.0°C to -23.0°C over 5 hours. Slowly increase it from -23.0°C to -3.0°C over 6 hours. Maintain the freeze-drying chamber temperature at -3.0°C for 8 hours. Slowly increase it from -3.0°C to 5.0°C over 4 hours and maintain it under vacuum for 4 hours.

[0068] 8) Continue to adjust the freeze-drying chamber temperature, slowly raising it from 5.0°C to 50.0°C over 3 hours and maintaining it under vacuum for 4 hours. The vacuum level is set to alternate between 30 Pa and 10 Pa. Finally, the freeze-drying chamber is subjected to a maximum vacuum at a constant temperature of 50.0°C. The main task of the desorption drying stage is to remove the difficult-to-remove bound water in the coating, which accounts for approximately 5% of the total water content. At the same time, the maximum desorption drying temperature should not exceed 50°C to ensure that the water in the protein coating is completely removed and the protein components in the coating are not inactivated.

[0069] 9) Turn off the freeze dryer, package the coated product under vacuum conditions, adjust the freeze drying chamber environment to normal pressure, and remove the coated product. After freeze drying, gas phase analysis shows that there is no tert-butyl alcohol remaining in the coating sample, eliminating the difficult problem of tert-butyl alcohol removal.

Claims

1. A freeze-drying process suitable for bioactive coatings, characterized in that: The following steps are involved: 1) The pretreatment liquid comprises mannitol and tert-butyl alcohol; 2) Turn on the freeze dryer and set the shelf temperature before feeding to 0.0±5.0℃; 3) Pre-treat the coated product to be freeze-dried: Place the coated product to be dried in a drying container, spray the sample surface with the room temperature pre-treatment liquid using a spray bottle, and after the sample surface is evenly sprayed, quickly place the sample on the freeze dryer shelf; 4) Adjust the freeze-drying chamber temperature to -14.0 ± 3°C and maintain it for 1 to 3 hours; 5) Adjust the freeze-drying chamber temperature to -53.0±5.0°C and maintain it for 3-5 hours to quickly freeze the coating sample; 6) Then, evacuate the equipment at -53.0±5.0℃; 7) Continue to adjust the freeze-drying chamber temperature, slowly increasing it from -53.0±5.0°C to -28.0±5.0°C over 4-6 hours; slowly increasing it from -28.0±5.0°C to -8.0±5.0°C over 5-7 hours; maintain the freeze-drying chamber temperature at -8.0±5.0°C for 7-9 hours; slowly increasing it from -8.0±5.0°C to 0.0±5.0°C over 3-5 hours, and maintaining it under the vacuum conditions set in step 6) for 3-5 hours; 8) Continue to adjust the temperature of the freeze-drying chamber, slowly raising the temperature from 0.0±5.0°C to 45.0±5°C over 2-4 hours, and maintain it under vacuum for 3-5 hours; finally, draw a maximum vacuum when the freeze-drying chamber is kept at a constant temperature of 45.0±5°C.

2. A freeze-drying process for a bioactive coating according to claim 1, characterized in that: The product to be dried with the outer coating is a magnesium alloy or a titanium alloy.

3. A freeze-drying process for a bioactive coating according to claim 1 or 2, characterized in that: The coating is protein-based.

4. A freeze-drying process for a bioactive coating according to claim 1, characterized in that: The mass volume of mannitol in the pretreatment liquid is 8% W / V to 12% W / V, the mass volume of tert-butanol is 3% W / V to 9% W / V, and the balance is water.

5. A freeze-drying process for a bioactive coating according to claim 1, characterized in that: The vacuum condition in step 6) is to decrease gradually from 40±2 Pa to 20±2 Pa, with the pressure drop not exceeding 1 Pa per hour.

6. A freeze-drying process for a bioactive coating according to claim 1, characterized in that: The vacuum condition in step 8) is a variable pressure state between 10 and 30 Pa.

7. A freeze-drying process for a bioactive coating according to claim 5 or 6, characterized in that: The set air entrainment amount under the vacuum condition is 0.09±0.03 mbar.

8. The freeze-drying process for a bioactive coating according to claim 1, characterized in that: The outer coating samples were packaged under vacuum conditions, and the coating samples were taken out after adjusting the freeze-drying indoor environment to normal pressure.

9. A freeze-drying process for a bioactive coating according to claim 1, characterized in that: After freeze-drying, gas phase determination showed that no tert-butyl alcohol remained in the coating sample.

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