Preparation method and application of ovalbumin transplant based on hexafluoroisopropanol / trifluoroacetic acid system

Abstract

The present invention discloses a method for preparing an ovalbumin transplant based on a hexafluoroisopropanol / trifluoroacetic acid solvent system, and the specific process includes: (1) preparing an ovalbumin solution; (2) preparing an ovalbumin bionic fiber scaffold; (3) preparing an ovalbumin membrane; (4) preparing an ovalbumin sponge scaffold; (5) placing the ovalbumin solution under high temperature to form a gel. The present invention uses a mixture of hexafluoroisopropanol and trifluoroacetic acid as an ovalbumin solvent, and a transplant with good biocompatibility can be prepared by different methods. The transplant preparation method is simple, easy to implement, short in cycle, safe and non-toxic, and has high transplant molding stability, which is convenient for large-scale production, and the transplant can be applied to different tissue regeneration according to its characteristics.

Description

Technical Field

[0001] The invention belongs to the field of medical biomaterials implantable in human body in tissue engineering and regenerative medicine, and specifically relates to a preparation method and application of an ovalbumin transplant based on a hexafluoroisopropanol / trifluoroacetic acid system. Background Art

[0002] As tissue and organ damage seriously threatens human health. Therefore, effective tissue and organ damage repair and regeneration is a problem that needs to be solved urgently in clinical practice. At present, people mainly rely on various tissue engineering grafts to achieve the repair of damaged tissues and organs. Among them, the grafts used are mainly made of natural biomaterials or synthetic polymer materials with good biocompatibility and biodegradability. However, judging from the current results, the repair effect of these artificial grafts is still difficult to achieve the repair effect of ideal autologous transplants, and cannot fully meet clinical needs. Therefore, it is urgent to develop new biomaterials for use in tissue engineering.

[0003] Among the many polymers that can be used to prepare transplants, natural polymers with high bioactivity and availability, simple operation and low production cost should be the first choice. Protein-based natural polymers are used in tissue engineering because of their similarity to natural extracellular matrix elements. However, although protein-based biomaterials are bioactive and support cell responses such as attachment, proliferation and migration, their sources are often limited or require complex extraction and purification procedures, which makes them expensive or difficult to obtain. Egg white is a low-cost, easily available direct protein-based material, and this material has the characteristics of combining proteins and growth factors. Egg white protein can mediate cell adhesion and growth, and enhance its ability to establish interactions with biomaterials. It can also mimic the effects of nerve growth factor (NGF) by inducing axon growth in mammalian cells such as PC12 cells, which indicates that there are bioactive components similar to NGF in egg white.

[0004] Ovalbumin (OVA) is a glycoprotein extracted from egg white, accounting for 54% of the total amount of egg white protein. It is the most abundant protein in egg white and has genetic similarity with human serum albumin. Ovalbumin contains a variety of bioactive components. Due to its bioactive components, the protein has a variety of biological activities, such as wound healing, cell growth promotion and antibacterial properties. Due to the characteristics of OVA being non-immunogenic, tolerable, antibacterial, non-toxic and biodegradable, it has been used as an adjuvant for ointments for centuries to treat various diseases. In addition, OVA also has excellent biocompatibility, better promotes cell attachment and proliferation, and can be degraded into safe byproducts in the body. At present, little is known about the application of ovalbumin grafts in tissue engineering, and the prepared ovalbumin grafts also have various process problems. For example, some researchers have tried to prepare OVA fibers by electrospinning, but the prepared fibers have problems such as short fibers and small quantities. During the experiment, it was found that the conventional hexafluoroisopropanol as the solvent of ovalbumin had poor solubility and was difficult to prepare into an OVA solution with a certain viscosity. The use of trifluoroacetic acid as the electrospinning solution of ovalbumin was too volatile and corrosive, making the OVA solution unstable and difficult to shape.

[0005] In order to solve the above problems and improve the molding stability of ovalbumin transplants, the present invention uses hexafluoroisopropanol and trifluoroacetic acid mixed in a certain proportion as a solution. Trifluoroacetic acid can dissolve ovalbumin powder well, and hexafluoroisopropanol can increase the stability and formability of the solution. Ovalbumin porous sponge scaffolds, biomimetic fiber mesh scaffolds, films and gels with good biocompatibility and biodegradability can be prepared through different process methods, thereby achieving the effect of promoting regeneration of ovalbumin transplants after damage to different tissues or organs. Summary of the invention

[0006] Purpose of the invention: Technical problems to be solved by the present invention: According to existing research, ovalbumin contains a variety of bioactive ingredients, so it has the properties of promoting wound healing, cell growth and antibacterial properties. Due to its excellent biocompatibility, ovalbumin has been used in the repair of bone, skin and soft tissue injuries, but the prepared ovalbumin transplants, such as fiber scaffolds, mostly need to be mixed with other polymers to prepare composite nanofibers, and pure ovalbumin electrospun fibers have the problem of small amount of fibers, short fibers or no filaments. Further development is needed for the application in the repair of injuries to different tissues or organs. In view of the above problems, the present invention is committed to providing a method for preparing an ovalbumin transplant based on a hexafluoroisopropanol / trifluoroacetic acid system.

[0007] Technical solution: A method for preparing an ovalbumin transplant based on a hexafluoroisopropanol / trifluoroacetic acid system, comprising:

[0008] Step 1, weighing a certain amount of egg white protein powder and dissolving it in an organic solvent, stirring it on a magnetic stirrer at room temperature for 1-2 days to fully dissolve it, to prepare a transparent egg white protein solution;

[0009] Step 2: Use a 5mL electrospinning syringe to absorb the ovalbumin solution, place the syringe on the syringe pump, and use the receiving distance, voltage, electrospinning solution solubility, electrospinning time, and electrospinning jet speed as parameters to electrospin onto the metal foil using electrospinning technology. The resulting film is a randomly arranged continuous nanofiber structure with a fiber width of 500-1500nm. Place the prepared ovalbumin fiber scaffold in a culture dish for cross-linking treatment;

[0010] Step three, use an acidic solution to adjust the pH of the ovalbumin solution, pour the solution into a 24-well plate, and degas the ovalbumin solution under vacuum to eliminate bubbles. Freeze at a gradient low temperature, transfer to a freeze dryer, and use freeze-drying technology to shape the biomaterial solution to prepare an ovalbumin sponge scaffold. Place the prepared ovalbumin sponge scaffold in a culture dish and adjust the pH of the scaffold to neutral. Place the prepared ovalbumin sponge scaffold in a culture dish and perform cross-linking treatment;

[0011] Step 4: Use a 1mL syringe to drop the ovalbumin solution onto the smooth surface and place it in a 37°C air drying oven for 4-48 hours to solidify the ovalbumin solution into a film. Place the prepared ovalbumin film in a culture dish for cross-linking treatment;

[0012] Step 5, heating the ovalbumin solution in a high-temperature water bath for 30 minutes, and quickly transferring it to an ice water bath to obtain an ovalbumin gel;

[0013] Step 6: Place the prepared ovalbumin gel in a culture dish and perform cross-linking treatment to further prepare an ovalbumin graft with good mechanical and biodegradability.

[0014] The electrospinning solvent in step 1 is a mixed solvent of hexafluoroisopropanol and trifluoroacetic acid. Trifluoroacetic acid can dissolve egg white protein powder well, while hexafluoroisopropanol can increase the stability and spinnability of the solution. The volume ratio between the two is 1:1-3:1, and the concentration of the prepared egg white protein solution is 10-20%.

[0015] The metal foil in step 2 can be titanium foil, aluminum foil, tin foil or stainless steel foil.

[0016] The spinnability of the electrospinning solution in step 2 is determined by trying a series of conditions (voltage, temperature, distance to the collector, solution jet flow rate, needle model, solution concentration) in the spinning device. The receiving end of the electrospinning is a planar receiving device, the voltage parameter range is 20-30kV, the electrospinning solution concentration is 8-15%, the receiving distance is 10-30cm, the electrospinning time is 5-120min, the electrospinning propulsion speed is 10-30μL / min, the prepared fiber diameter is 100-1500nm, and the length is 1-500mm. Due to different indoor temperature, humidity, etc., adjustments can be made to the best parameters for the actual fiber.

[0017] The acidic solution in step 3 is an acetic acid solution, the acetic acid concentration is 1-3M, and the pH is adjusted to 3-5.

[0018] The sponge scaffold prepared in step three has a porous mesh structure and has certain shape memory properties.

[0019] In step 3, the ovalbumin sponge scaffold was washed in 0.1 M sodium dihydrogen phosphate (Na2HPO4) for 2 hours, and the pH of the scaffold was adjusted to 7.0. It was then rinsed in double distilled water (ddH2O) four times, each for 30 minutes, and finally rinsed with phosphate buffer for 24 hours, and then gradient freeze-dried;

[0020] The natural polymer film in step 4, especially the protein film, has some unfavorable properties, such as brittleness, high water solubility and poor water vapor permeability. The egg white protein film prepared in step 4 solves the problem of film flexibility and does not need to add plasticizers to the structure.

[0021] The film samples obtained after the ovalbumin film prepared in step 4 is cross-linked with EDC and ethanol solutions of different concentrations have different transparencies.

[0022] In step five, the temperature is 70-90° C., and the hardness, elasticity, water retention and thermal stability of the ovalbumin gel can be changed by adding succinic anhydride of different concentrations.

[0023] The ovalbumin graft prepared by the present invention is placed in a culture dish and cross-linked with a cross-linking agent solution to change its water solubility and enhance its mechanical properties. The cross-linking agent used is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and ethanol solution, the concentration of EDC is 5-10mM, and the concentration of ethanol is 70%.

[0024] Beneficial effects: The present invention uses hexafluoroisopropanol and trifluoroacetic acid mixed in a certain proportion as a solvent. Trifluoroacetic acid can dissolve egg white protein powder well, while hexafluoroisopropanol can increase the stability and formability of the solution. By changing the parameters, different forms of egg white protein transplants with good biocompatibility and biodegradability can be prepared through different process methods.

[0025] The ovalbumin graft used in tissue engineering prepared by the preparation method of the present invention can be prepared into different forms of grafts according to the needs after different tissues or organs are damaged. For example, an ovalbumin film with antibacterial and good air permeability can be prepared for skin trauma; a macroporous sponge scaffold with good compression resistance can be prepared for cartilage and bone tissue damage; an injectable ovalbumin gel can be prepared for spinal cord injury; and an ovalbumin nanofiber scaffold that can mimic the natural extracellular matrix can be prepared for nerve damage. It can better promote the repair and regeneration of tissues after damage.

[0026] The graft preparation method used in the present invention can be electrospinning, freeze drying, solvent volatilization and gel technology, and the specific preparation process can be adjusted according to the selected preparation method. The ovalbumin tissue engineering graft prepared by the preparation method can be in different forms such as scaffolds, membranes or gels.

[0027] The ovalbumin transplant based on the hexafluoroisopropanol / trifluoroacetic acid system prepared by the present invention can also be loaded with growth factors or bioactive molecules.

[0028] The ovalbumin transplant prepared by the invention based on the hexafluoroisopropanol / trifluoroacetic acid system can improve the shaping stability of the transplant.

[0029] The egg white protein powder used in the present invention is easy to obtain, simple to operate and has low production cost.

[0030] The preparation method adopted by the present invention is relatively simple, and the preparation of the ovalbumin transplant based on the hexafluoroisopropanol / trifluoroacetic acid system has good repeatability.

[0031] The ovalbumin transplant based on the hexafluoroisopropanol / trifluoroacetic acid system prepared by the present invention is beneficial to supporting cell responses such as attachment, proliferation and migration.

[0032] The ovalbumin transplant prepared by the invention based on the hexafluoroisopropanol / trifluoroacetic acid system has relatively stable chemical properties and good biocompatibility.

[0033] The ovalbumin transplant based on the hexafluoroisopropanol / trifluoroacetic acid system prepared by the present invention can have a porous structure, which is beneficial to the nutrient transport of new tissue cells and the material exchange with the surrounding environment.

[0034] The tissue engineering graft prepared by the invention has a unique structure and has broad application prospects in tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the preparation of an ovalbumin solution based on a hexafluoroisopropanol / trifluoroacetic acid system according to an embodiment of the present invention.

[0036] Figure 2 This is a schematic diagram of preparing an ovalbumin nanofiber scaffold using the electrospinning technology described in an embodiment of the present invention.

[0037] Figure 3 This is a schematic diagram of preparing an ovalbumin sponge scaffold using the freeze-drying technology described in an embodiment of the present invention.

[0038] Figure 4 Schematic diagram of preparing ovalbumin membrane by the casting method described in an embodiment of the present invention.

[0039] Figure 5 This is a schematic diagram of the preparation of ovalbumin gel according to an embodiment of the present invention.

[0040] Figure 6 , Figure 7 As described in the embodiment of the present invention, ovalbumin nanofibers were prepared by electrospinning at a flow rate of 15 μL / min, 20 μL / min under the conditions of HFIP:TFA=2:1, OVA solution concentration of 10%, electrospinning time of 5 min, needle distance of 15 cm, needle model of 20, and voltage of 20 kV.

[0041] Figure 8 This is a scanning electron microscope image of the ovalbumin membrane described in the embodiment of the present invention. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention will be described clearly and completely below so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] Preparation of an ovalbumin biomimetic nanofiber scaffold loaded with nerve growth factor

[0045] After blending the ovalbumin powder and the nerve growth factor at a mass ratio of 2:1, they were added to a hexafluoroisopropanol / trifluoroacetic acid mixed solvent at a concentration of 10%, and the ratio of hexafluoroisopropanol to trifluoroacetic acid was 2:1. Stirring on a magnetic stirrer for 1d to fully dissolve it, the ovalbumin blended electrospinning solution was prepared, and the ovalbumin blended electrospinning solution was transferred to a 5mL electrospinning special syringe and placed on a syringe pump. The electrospinning parameters were voltage: 20kV, distance from the needle to the receiving end: 15cm, flow rate: 15μL / min, and electrospinning time: 60min. After the electrospinning was completed, a randomly arranged continuous ovalbumin nanofiber scaffold loaded with nerve growth factor was obtained on the tin foil. Then, the tin foil was placed in a 37°C vacuum drying oven for drying to completely volatilize the hexafluoroisopropanol and trifluoroacetic acid. The prepared ovalbumin nanofiber scaffold was placed in a culture dish, and a 5mM EDC crosslinker solution was prepared with 70% ethanol solvent. The ovalbumin fiber scaffold was soaked at 4°C for 24 hours to prepare a water-insoluble ovalbumin fiber scaffold sample, which was then washed with double distilled water for 3 days to remove excess EDC and urea byproducts.

[0046] Preparation of a three-dimensional elastic ovalbumin biomimetic nanofiber scaffold

[0047] Ovalbumin powder was added to a hexafluoroisopropanol / trifluoroacetic acid mixed solvent at a concentration of 10%, with a ratio of hexafluoroisopropanol to trifluoroacetic acid of 2:1. Stir on a magnetic stirrer for 1d to fully dissolve it to obtain an ovalbumin electrospinning solution, which was transferred to a 5mL electrospinning syringe and placed on a syringe pump. The electrospinning parameters were voltage: 25kV, distance from the needle to the receiving end: 15cm, flow rate: 20μL / min, and electrospinning time: 60min. After electrospinning, a three-dimensional elastic, randomly arranged continuous ovalbumin nanofiber scaffold was obtained on the tin foil. Then, the tin foil was placed in a 37°C vacuum drying oven for drying to completely volatilize the hexafluoroisopropanol and trifluoroacetic acid. The prepared ovalbumin nanofiber scaffold was placed in a culture dish, and a 5mM EDC crosslinker solution was prepared with 70% ethanol solvent. The ovalbumin fiber scaffold was soaked at 4°C for 24 hours to prepare a water-insoluble ovalbumin fiber scaffold sample, which was then washed with double distilled water for 3 days to remove excess EDC and urea byproducts.

[0048] Preparation of an antibacterial and healing-promoting ovalbumin patch

[0049] Ovalbumin powder was added to a hexafluoroisopropanol / trifluoroacetic acid mixed solvent at a concentration of 10%, with a ratio of hexafluoroisopropanol to trifluoroacetic acid of 2:1. Stir on a magnetic stirrer for 1 day to fully dissolve it to obtain an ovalbumin solution. Use a syringe to drop 50 μL of the blended solution onto a small round glass slide and place it in an air drying oven at 37°C overnight. The prepared ovalbumin film was placed in a culture dish, and a 5mM EDC crosslinker solution was prepared with 70% ethanol solvent. The ovalbumin film was soaked for 24 hours at 4°C to prepare an ovalbumin film sample with high transparency, and washed with double distilled water for 3 days to remove excess EDC and urea byproducts. The ovalbumin film has good antibacterial properties, and antimicrobial peptides are loaded on the ovalbumin film. Antimicrobial peptides are pure natural peptides that have a killing effect on some viruses, fungi, and cancer cells, so they can improve immunity and accelerate the wound healing process.

[0050] Preparation of a 3D drug-loaded elastic ovalbumin sponge scaffold

[0051] Ovalbumin powder was added to a hexafluoroisopropanol / trifluoroacetic acid mixed solvent at a concentration of 10%, with a ratio of hexafluoroisopropanol to trifluoroacetic acid of 2:1. The ovalbumin solution was prepared by stirring on a magnetic stirrer for 1 day to fully dissolve it. The pH of the ovalbumin solution was adjusted to pH=3 using a 2M acetic acid solution. The solution was degassed under vacuum to eliminate bubbles. The prepared solution was poured into a 24-well plate and placed at 4°C for 4 hours. The sample was then frozen overnight at -20°C, transferred to a -80°C refrigerator for 2 hours, and transferred to a freeze dryer to prepare an ovalbumin porous scaffold. The ovalbumin scaffold was cross-linked with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in 70% ethanol at 4°C to increase the molar ratio of EDC to the free COOH group of ovalbumin (1:4). The molar ratio of free carboxylic acid groups to EDC was obtained from the amino acid sequence of ovalbumin. The scaffolds were then washed in 0.1 M sodium dihydrogen phosphate (Na2HPO4) for 2 h, followed by four 30-min rinse steps in double distilled water (ddH2O), and finally rinsed with phosphate buffered saline for 24 h. The ovalbumin sponge scaffolds were immersed in 5 mg / mL metformin solution for 4 h to prepare a 3D drug-loaded ovalbumin sponge scaffold.

[0052] Preparation of an ovalbumin sponge scaffold with shape memory properties

[0053] Ovalbumin powder was added to a hexafluoroisopropanol / trifluoroacetic acid mixed solvent at a concentration of 10%, with a ratio of hexafluoroisopropanol to trifluoroacetic acid of 2:1. The ovalbumin solution was prepared by stirring on a magnetic stirrer for 1 day to fully dissolve it. The pH of the ovalbumin solution was adjusted to pH = 3 using a 2M acetic acid solution. The solution was degassed under vacuum to eliminate bubbles. The prepared solution was poured into a 24-well plate and placed at 4°C for 4 hours. The sample was then frozen overnight at -20°C, transferred to a -80°C refrigerator for 2 hours, and transferred to a freeze dryer to prepare an ovalbumin macroporous scaffold. The ovalbumin scaffold was cross-linked with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in 70% ethanol at 4°C, and the concentration of EDC was 5mM. The scaffolds were then washed in 0.1 M sodium phosphate (Na2HPO4) for 2 h, followed by four 30-min rinses in double distilled water (ddH2O), and finally rinsed in phosphate buffered saline for 24 h and freeze-dried. An ovalbumin sponge scaffold with shape memory properties was prepared.

Claims

1. A method for preparing an ovalbumin transplant based on a hexafluoroisopropanol / trifluoroacetic acid system, characterized in that: The steps include: Step (1) dissolving a certain amount of egg white protein powder in an organic solvent, stirring on a magnetic stirrer at room temperature for 1-2 days to fully dissolve it, to prepare an egg white protein solution; Step (2) transferring the ovalbumin solution into a syringe, placing the syringe on a syringe pump, and electrospinning the solution onto a metal foil using electrospinning technology to prepare an ovalbumin nanofiber scaffold; Step (3) degassing the ovalbumin solution under vacuum to eliminate bubbles, adjusting the pH of the ovalbumin solution, pouring the solution into a container, freezing it at a gradient low temperature, transferring it to a freeze dryer, and freeze-drying the ovalbumin into a shape using a freeze-drying technique to obtain an ovalbumin sponge scaffold; placing the prepared ovalbumin sponge scaffold into a culture dish, and adjusting the pH of the scaffold to neutral; Step (4) using a syringe to drop a certain volume of ovalbumin solution onto the smooth surface, and placing it in an air drying oven at 37° C. for 4-48 hours to allow the ovalbumin solution to solidify into a film; Step (5) heating the ovalbumin solution in a high-temperature water bath for 30 minutes, and quickly transferring it to an ice water bath to obtain an ovalbumin gel; Step (6) placing the prepared ovalbumin graft into a culture dish, and adding a crosslinker solution dropwise with a pipette to ensure that the material is immersed but not overflowed, thereby further preparing an ovalbumin graft with good mechanical and biodegradability; The organic solvent in step (1) is a mixture of hexafluoroisopropanol and trifluoroacetic acid; trifluoroacetic acid can dissolve egg white protein powder well, while hexafluoroisopropanol can increase the stability and electrospinnability of the solution, and the volume ratio of hexafluoroisopropanol to trifluoroacetic acid is 3:1-1:1; the concentration of the prepared egg white protein solution is 10-20%; The spinnability of the electrospinning solution in step (2) is determined by trying the following series of conditions in a spinning device: voltage, temperature, distance to the collector, solution injection flow rate, needle model, and solution concentration; the voltage parameter range is 20-30 kV, the receiving distance is 10-30 cm, the electrospinning time is 5-120 min, the electrospinning propulsion speed is 10-30 μL / min, the prepared fiber diameter is 100-1500 nm, the length is 1-500 mm, and the nanofibers are uniformly continuous and randomly distributed; The metal foil used in step (2) can be aluminum foil, tin foil, titanium foil or stainless steel foil, the thickness of the foil is 10-300 μm, the surface of the foil is smooth, and the roughness is 1-10 nm; In the step (3), acetic acid is used to adjust the pH of the solution to a pH of 3-5; the prepared sponge scaffold has a porous mesh structure and a certain shape memory property; In the step (3), the ovalbumin sponge scaffold is washed in 0.1M disodium hydrogen phosphate (Na2HPO4) for 2 hours, the pH of the scaffold is adjusted to 7.0, and then rinsed four times with double distilled water (ddH2O), each time for 30 minutes, and finally rinsed with phosphate buffer for 24 hours, and then gradient freeze-dried; In the step (4), 10-200 μL of the ovalbumin solution is added dropwise each time, and the ovalbumin film prepared has good flexibility; In the step (5), the high temperature is 70-90° C., and the hardness, elasticity, water retention and thermal stability of the ovalbumin gel can be changed by adding succinic anhydride of different concentrations; In the step (6), the ovalbumin graft is cross-linked with different molar amounts of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in 70% ethanol at 4°C, and the molar ratio of EDC to the free COOH group of ovalbumin is 0-10; the purpose of cross-linking with a cross-linking agent solution is to change its biodegradability and enhance its mechanical properties.

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