Freeze-drying treatment process suitable for bioactive coating

Abstract

The invention relates to a freeze-drying treatment process suitable for a bioactive coating. The freeze-drying treatment process comprises the following factors: coating freeze-drying pretreatment: preparing an excipient and an auxiliary material solution; in the pre-freezing part, the pre-freezing temperature, the shelf temperature before feeding and the pre-freezing time are determined; the freeze-drying time part comprises the fastest temperature point of one-time sublimation drying, the drying time of a high-speed section of one-time sublimation, the drying time of a low-speed section of one-time sublimation and the desorption drying time. And the vacuum degree part is one-time sublimation air doping amount. The method is simple, the technology is mature and feasible, the freeze-dried coating product is regular in appearance and extremely low in water content, the defects of an existing bioactive coating preparation technology are overcome, the actual application requirement is met, the drying technology in most bioactive coating preparation processes can be replaced, the biological activity of the coating is guaranteed, the service life of the coating is prolonged, and the application prospect is wide. The industrial application of the coating is facilitated, and powerful support is provided for related research.

Description

Technical Field

[0001] The invention relates to a freeze-drying process suitable for the preparation of a bioactive coating, and in particular to a freeze-drying method for a magnesium and titanium alloy composite coating modified with a protein or polypeptide, and belongs 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 bio-implantable materials. Due to the low corrosion resistance, high biodegradation rate and poor binding with human cells at the interface of magnesium and titanium alloys, it is extremely necessary to apply a layer of bioactive coating on their surface, and this coating must have good corrosion resistance and bioactivity, so that magnesium and titanium alloys can be better used to solve the problems existing in the field of bio-implantable materials. The commonly used bioactive components of this type of bioactive coating mainly include: proteins, peptides, cell growth factors, polysaccharides and organic small molecule drugs. Among them, protein is an important structural and functional unit of living organisms and has many special biological functions. Modifying bio-affinity protein substances on the surface of medical metals is of great significance to improving the bioactivity and biofunction of implants. At present, biological coatings based on protein and peptide modification are mostly in the experimental stage, and the actual application effect is not satisfactory. The reason lies in the inactivation of the coating protein modification and the long-term preservation of the coating bioactivity.

[0003] Vacuum freeze drying technology, referred to as freeze drying, is a process in which the material solution to be dried is first cooled and frozen to below the eutectic point until the material solution is completely frozen and does not contain any liquid phase, and then heat is provided to the frozen material. Under vacuum conditions, the water in the material solution is directly sublimated into gas, and then the water vapor is captured by a cold trap to finally obtain the dry material. Vacuum freeze drying is an important chemical process for material drying and separation that combines multiple technologies such as vacuum technology, drying technology, and refrigeration technology. The advantages of freeze drying technology over ordinary wet drying are: freeze drying is operated at extremely low temperatures, which can keep the physicochemical properties of the freeze-dried material basically unchanged, avoid protein denaturation caused by heating of biological materials, and ensure the biological activity of the material; the freeze-dried material is loose and porous, has a good physical shape, and a large specific surface area, and can quickly return to its original properties when used; freeze drying has a good drying effect, which can remove more than 95% to 99% of the water, and the sealed dried product can be stored for a long time without deterioration.

[0004] At present, the preparation process of bioactive coatings often ignores the important role of drying process, or adopts a more traditional drying process, which leads to premature inactivation of active protein modifiers in the coating, and the inactivated protein substances have a strong corrosive effect on medical metals, reducing the service life of medical metals. Therefore, the present invention develops a freeze-drying process suitable for bioactive coatings.

[0005] The Chinese patent "A super-hydrophobic coating based on protein particles and its preparation method" (publication number: CN112029146A) discloses a method for preparing a super-hydrophobic coating material based on protein particles, including the following specific steps: S1: pretreatment of porous substrate materials; S2: adsorption and fixation of protein particles; S3: modification of low surface energy materials. The porous substrate material with protein clusters needs to be dried after being soaked in activated sludge to remove most of the water. The conventional drying method is not conducive to maintaining the activity of protein particles, and has limitations in practical application.

[0006] The Chinese patent "A multifunctional composite coating for preparing magnesium alloy heart stent materials and its preparation method" (publication number: CN 114917414 A) discloses a multifunctional composite coating for preparing magnesium alloy heart stent materials and its preparation method, wherein the multifunctional composite coating has phosphorylated polybasic amino acids inside, and a coupling agent containing both thiol and carboxyl groups is introduced outside the phosphorylated polybasic amino acids, and a hydrophilic anionic polymer is introduced outside through the coupling action of the coupling agent containing both thiol and carboxyl groups, and finally coated on the surface of the magnesium alloy by the dipping-pulling method. The composite coating is washed with ethanol and then dried in N2 atmosphere, which ensures that the coating is oxidized, but the active components of the coating are easily inactivated in a high-temperature drying environment, reducing the biological function of the coating.

[0007] The Chinese patent "A method for preparing an antifouling coating on the surface of a composite polymer material" (publication number: CN116285682A) discloses that a protein coating is assembled on the surface of the material by oxidizing hydrogenated caffeic acid with sodium periodate to mediate bovine serum albumin. First, the oxidized hydrogenated caffeic acid rapidly aggregates with bovine serum albumin through hydrophobic interactions. At the same time, the hydrogen bonds and ionic bonds formed between the protein and the phenols enhance the cross-linking effect, and a stable hydrogenated caffeic acid-mediated bovine serum albumin coating is assembled. The hydrogenated caffeic acid-mediated bovine serum albumin coating exhibits good anti-cell and anti-platelet adhesion properties and has a certain antifouling effect. At the same time, the large number of carboxyl groups on the surface of the hydrogenated caffeic acid-mediated bovine serum albumin coating provide the possibility of secondary grafting of antifouling molecules. Polyethylene glycol is covalently grafted through an amidation reaction to obtain an antifouling coating on the surface of a composite polymer material. The reaction temperature of the preparation process is as high as 70°C, which is close to the protein inactivation temperature. It is difficult to ensure the activity of the protein by ordinary wet drying. 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 the bioactive coating, which can replace the drying process in most bioactive coating preparation processes, 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 suitable for a bioactive coating comprises the following steps:

[0011] 1) The pretreatment liquid is composed of mannitol and tert-butanol. Mannitol plays a role of skeleton support in the freeze-drying process to improve the freeze-drying efficiency. The role of tert-butanol is to combine with protein substances, improve the freeze-dried crystal form, form a unique needle-shaped crystal form, increase the freeze-dried surface area, and accelerate freeze-drying.

[0012] The mass volume of mannitol in the pretreatment solution is 8% W / V to 12% W / V, the mass volume of tert-butyl alcohol 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 inactivating.

[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, and 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 temperature of the freeze-drying chamber to -14.0±3°C and maintain it for 1 to 3 hours.

[0018] 5) Adjust the temperature of the freeze-drying chamber to -53.0±5.0℃ 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 firmly 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 temperature of the freeze drying chamber, slowly increase it from -53.0±5.0℃ to -28.0±5.0℃ in 4-6 hours; slowly increase it from -28.0±5.0℃ to -8.0±5.0℃ in 5-7 hours; maintain the temperature of the freeze drying chamber at -8.0±5.0℃ for 7-9 hours; slowly increase it from -8.0±5.0℃ to 0.0±5.0℃ in 3-5 hours, and maintain it under the vacuum conditions set in step 6) for 3-5 hours.

[0021] 8) Continue to adjust the temperature of the freeze drying chamber, slowly raise the temperature from 0.0±5.0℃ to 45.0±5℃ in 2-4 hours, and keep it under vacuum for 3-5 hours; the vacuum condition is a variable pressure state between 10 and 30Pa. Finally, the freeze drying chamber is evacuated to the ultimate vacuum at a constant temperature of 45.0±5℃; the main task of the desorption drying stage is to remove the bound water in the coating that is more difficult to remove, accounting for about 5% of the total water. At the same time, the maximum desorption drying temperature should not be higher than 50℃, which not only ensures the complete removal of the water in the protein coating, but also ensures that the protein components of the coating are not inactivated.

[0022] The set gas entrainment amount of the vacuum condition 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 indoor environment to normal pressure and take out the coating sample. After freeze drying, the coating sample has no tert-butyl alcohol residue in the gas phase measurement, eliminating the problem of removing tert-butyl alcohol.

[0024] After freeze-drying, no tert-butyl alcohol remained in the coating sample as determined by gas phase analysis.

[0025] The technical solution disclosed in the present invention realizes freeze drying of the coating product by pretreatment, freeze drying condition adjustment, etc. Thus, on the basis of ensuring the original activity of the coating, excess water is removed to ensure that the coating product does not lose 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, 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 through 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 solid 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 intervention 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 the existing bioactive coating preparation technology and meets the needs of practical applications. 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, and help the industrial application of the coating, providing strong support for related research. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 embodiments. The following embodiments are used to specifically illustrate the contents of the present invention. These embodiments are only general descriptions of the contents of the present invention and do not limit the contents of the present invention.

[0034] In view of the problems existing in the existing bioactive coating drying technology, the present invention innovates the coating pretreatment process and provides a freeze-drying process suitable for bioactive coatings. The present invention adjusts and optimizes the freeze-drying process parameters according to 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 section of sublimation, the drying time of the slow section of sublimation, and the desorption drying time.

[0038] Vacuum degree: The amount of gas mixed in the first 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, has a regular coating appearance, and has a low defective rate.

[0040] Embodiment 1:

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

[0045] 5) Adjust the temperature of the freeze drying chamber to -53.0°C and maintain it for 3 hours to quickly freeze the coated product. Continue to observe the freezing situation. Sufficient freezing time can make the crystals freeze more firmly and eliminate the hidden dangers of product melting and protein coating inactivation during the sublimation stage.

[0046] 6) Then, the equipment was evacuated at -53.0°C. The vacuum condition was gradually reduced from 40Pa to 20Pa, and the gas volume was set to 0.09mbar, which took 20 hours.

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

[0048] 8) Continue to adjust the temperature of the freeze drying chamber, slowly increase the temperature from 0.0°C to 45.0°C over 2 hours, and maintain it under vacuum for 3 hours. The vacuum degree is set to jump back and forth between 30Pa and 10Pa. Finally, the freeze drying chamber is vacuumed to a maximum at a constant temperature of 45.0°C. The main task of the desorption and drying stage is to remove the bound water in the coating that is more difficult to remove, accounting for about 5% of the total water. At the same time, the maximum desorption and drying temperature should not be higher than 50°C, which not only ensures the complete removal of water from the protein coating, but also ensures that the protein components of the coating are not inactivated.

[0049] 9) Turn off the freeze dryer, package the coated product under vacuum conditions, and take out the coated product after adjusting the freeze drying room environment to normal pressure. After freeze drying, the coating sample has no tert-butyl alcohol residue in the gas phase measurement, eliminating the problem of removing tert-butyl alcohol.

[0050] Embodiment 2:

[0051] 1) Preparation of pretreatment solution: The pretreatment solution contains 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 surface of the sample, and 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 temperature of the freeze drying chamber to -58.0°C and maintain it for 5 hours to quickly freeze the coated product. Continue to observe the freezing situation. Sufficient freezing time can make the crystals freeze more firmly and eliminate the hidden dangers of product melting and protein coating inactivation during the sublimation stage.

[0056] 6) Then, the equipment was evacuated at -58.0°C. The vacuum condition was gradually reduced from 40Pa to 20Pa, and the gas volume was set to 0.06mbar, which took 20 hours.

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

[0058] 8) Continue to adjust the temperature of the freeze drying chamber, slowly raise the temperature from -5.0°C to 40.0°C over 4 hours, and maintain it under vacuum for 5 hours. The vacuum degree is set to jump back and forth between 30Pa and 10Pa. Finally, the freeze drying chamber is vacuumed to a maximum at a constant temperature of 40.0°C. The main task of the desorption and drying stage is to remove the bound water in the coating that is more difficult to remove, accounting for about 5% of the total water. At the same time, the maximum desorption and drying temperature should not be higher than 50°C, which not only ensures the complete removal of water from the protein coating, but also ensures that the protein components of the coating are not inactivated.

[0059] 9) Turn off the freeze dryer, adjust the freeze drying room environment to normal pressure, and then take out the coated product. After freeze drying, the coating sample has no tert-butyl alcohol residue in the gas phase measurement, eliminating the problem of removing tert-butyl alcohol.

[0060] Embodiment 3:

[0061] 1) Preparation of pretreatment solution: The pretreatment solution is composed of mannitol and tert-butyl alcohol, wherein the mass volume of mannitol in the pretreatment solution is 8% W / V, the mass volume of tert-butyl alcohol 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 surface of the sample, and 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 for 1 hour.

[0065] 5) Adjust the temperature of the freeze drying chamber to -48.0℃ and maintain it for 3 hours to quickly freeze the coated product. Continue to observe the freezing situation. Sufficient freezing time can make the crystals freeze more firmly and eliminate the hidden dangers of product melting and protein coating inactivation during the sublimation stage.

[0066] 6) Then, the equipment was evacuated at -48.0°C. The vacuum condition was gradually reduced from 40Pa to 20Pa, and the gas volume was set to 0.12mbar, which took 20 hours.

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

[0068] 8) Continue to adjust the temperature of the freeze drying chamber, slowly increase the temperature from 5.0°C to 50.0°C over 3 hours, and maintain it under vacuum for 4 hours. The vacuum degree is set to jump back and forth between 30Pa and 10Pa. Finally, the freeze drying chamber is subjected to extreme vacuum at a constant temperature of 50.0°C. The main task of the desorption and drying stage is to remove the bound water in the coating that is more difficult to remove, accounting for about 5% of the total water. At the same time, the maximum desorption and drying temperature should not be higher than 50°C, which not only ensures the complete removal of water from the protein coating, but also ensures that the protein components of the coating are not inactivated.

[0069] 9) Turn off the freeze dryer, package the coated product under vacuum conditions, and take out the coated product after adjusting the freeze drying room environment to normal pressure. After freeze drying, the coating sample has no tert-butyl alcohol residue in the gas phase measurement, eliminating the problem of removing tert-butyl alcohol.

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-treating the coated product to be freeze-dried, placing the coated product to be dried in a drying container, spraying the room temperature pre-treatment liquid on the surface of the sample using a spray pot, and after the sample surface is evenly sprayed, quickly placing the sample on the freeze dryer shelf; 4) Adjust the temperature of the freeze-drying chamber to -14.0±3°C and maintain it for 1 to 3 hours; 5) Adjust the temperature of the freeze-drying chamber to -53.0±5.0°C and maintain it for 3 to 5 hours to quickly freeze the coating sample; 6) Then, the equipment is evacuated at -53.0±5.0℃; 7) Continue to adjust the temperature of the freeze drying chamber, slowly increase it from -53.0±5.0°C to -28.0±5.0°C in 4-6 hours; slowly increase it from -28.0±5.0°C to -8.0±5.0°C in 5-7 hours; maintain the temperature of the freeze drying chamber at -8.0±5.0°C for 7-9 hours; slowly increase it from -8.0±5.0°C to 0.0±5.0°C in 3-5 hours, and maintain it for 3-5 hours under the vacuum conditions set in step 6); 8) Continue to adjust the temperature of the freeze drying chamber, slowly raise the temperature from 0.0±5.0°C to 45.0±5°C over 2 to 4 hours, and maintain it under vacuum conditions for 3 to 5 hours; finally, draw an ultimate 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 suitable 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 solution is 8% W / V to 12% W / V, the mass volume of tert-butyl alcohol 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±2Pa to 20±2Pa, and the pressure drop does not exceed 1Pa 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. A 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 room environment to normal pressure.

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

Images