Preparation method of porous gelatin microcarrier
By sublimating ice crystals during the preparation of microcarriers, and cross-linking and curing, the difficulties in controlling pore structures and pollution problems in the prior art are solved, and porous gelatin microcarriers with high porosity and mechanical stability are achieved, which are suitable for a variety of biological applications.
Patent Information
- Application Number
- CN202411732954.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-02-28
AI Technical Summary
During the freeze-drying process of existing microcarriers after low temperature treatment, it is difficult to effectively control the size and mechanical stability of the pore structure, and chemical pore making agents are often used to introduce contaminants.
Gelatin microspheres were prepared by W/O emulsification method, and the production and sublimation of ice crystals were used to directly form a porous structure after low temperature treatment, and then cross-linking and curing were performed to control the freezing temperature to adjust the pore size.
The high porosity and mechanical stability of porous gelatin microcarriers are achieved, and the contamination of chemical pore-making agents is avoided. The process is environmentally friendly and pollution-free. It is suitable for cell culture and drug delivery and other fields.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of microcarrier material preparation, and in particular to a method for preparing a porous gelatin microcarrier. Background Art
[0002] Microcarriers are microsphere scaffolds with biological functions in cell culture, expansion and delivery, and are widely used in the field of 3D cell culture. Among them, porous microcarriers with internal and external through-hole structures can allow cells to expand on the surface and inside of the microcarriers, protect cells from shear forces, provide a larger specific surface area and growth space for cell growth, and facilitate the entry of nutrients and the discharge of metabolites.
[0003] Gelatin is a natural polymer material obtained by hydrolysis of animal collagen. It has good biocompatibility, biodegradability, adhesion, and no toxic side effects. In recent years, gelatin microspheres have been increasingly used in the field of tissue engineering for cell storage, transportation, and proliferation due to their easy availability, ease of preparation, and superior biocompatibility.
[0004] The microcarriers are cryogenically treated and then freeze-dried. The pore size can be adjusted by controlling the freezing temperature. The ice crystals formed during the cryogenic freezing process are used as template pore-forming agents, and then the ice crystals are sublimated by freeze drying to form a porous structure. Compared with the pore-forming method of adding chemical pore-forming agents, this method does not introduce additional chemicals. The porous structure can be obtained by sublimating ice crystals. No additional cleaning steps are required, and the process is environmentally friendly and pollution-free. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing a porous gelatin microcarrier, wherein the microcarrier is subjected to low temperature treatment and then freeze-dried, and a porous structure is directly formed by the generation and sublimation of ice crystals inside the microspheres. By changing the freezing temperature, the size of the pore structure can be effectively controlled. After the porous structure is formed, the microcarrier is cross-linked and solidified, and the microcarrier prepared in this way has a high porosity and strong mechanical stability. The entire preparation process is simple, controllable, green, environmentally friendly and pollution-free. The porous gelatin microcarrier finally obtained has a uniform pore size distribution, does not require additional surface modification, and can be used in various fields such as cell culture and drug delivery.
[0006] The present invention provides a method for preparing a porous gelatin microcarrier, comprising the following steps:
[0007] S1, gelatin microspheres were prepared by W / O emulsification method;
[0008] S2, treating the gelatin microspheres at low temperature and drying them to obtain porous gelatin microcarriers with a pore size of 2 to 60 μm;
[0009] S3, cross-linking and curing the microcarriers obtained in step S2.
[0010] Preferably, the pore size of the gelatin microcarrier is 2 to 60 μm, for example, 2 μm, 5 μm, 8 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 55 μm, or 60 μm.
[0011] Preferably, the W / O emulsification method in step S1 is to mix the gelatin solution and the oil phase containing the emulsifier to obtain a W / O type emulsion, emulsify at low temperature, add anhydrous ethanol for dehydration, then wash with an organic solvent, and dry to obtain dry gelatin microspheres.
[0012] Preferably, the concentration of the gelatin solution is 5-30%, for example 5%, 10%, 15%, 20%, 25%, 30%; the volume ratio of the gelatin solution to the oil phase is 1:3-1:8, for example 1:3, 1:4, 1:5, 1:6, 1:7, 1:8.
[0013] Preferably, the concentration of the emulsifier is 0.5-5%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%; the temperature of low-temperature emulsification is 4--20°C, for example, 4°C, 0°C, -5°C, -10°C, -15°C, -20°C; the emulsification time is 1-4h, for example, 1h, 2h, 3h, 4h; the stirring speed during emulsification is 300-800rpm, for example, 300rpm, 400rmp, 500rpm, 600rmp, 700rpm, 800rmp; the oil phase is one or a combination of vegetable oil, mineral oil, and paraffin; the emulsifier is one or a combination of Tween 80, Span 80, and stearic acid; the organic solvent is one or a combination of petroleum ether, isopropanol, acetone, and anhydrous ethanol.
[0014] Preferably, the low temperature treatment in step S2 is to freeze the hydrated gelatin microspheres, and the freezing method is specifically any one of the following two methods: (1) spreading the hydrated gelatin microsphere solution to a thickness of 3-5 mm and freezing it at -20 to -80°C for 2 to 24 hours; (2) freezing the hydrated gelatin microspheres at -100 to -200°C for 1 to 10 minutes, preferably under liquid nitrogen conditions.
[0015] Preferably, the hydrated gelatin microspheres are prepared by immersing dried gelatin microspheres in deionized water to allow them to absorb water and swell, the deionized water temperature is 0 to 20°C, for example, 0°C, 5°C, 10°C, 15°C, 20°C; the immersion time is 1 to 30 min, for example, 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min.
[0016] Preferably, the drying method in step S2 is freeze drying, and the temperature during freeze drying is -10 to -80°C, for example, -20°C, -30°C, -40°C, -50°C, -60°C, -70°C; and the time is 12 to 48 hours.
[0017] Preferably, the cross-linking and curing in step S3 is specifically: immersing the gelatin microcarriers forming a porous structure in an ethanol aqueous solution containing genipin, and cross-linking and curing for 12 to 96 hours, for example, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 55 hours, 60 hours, 75 hours, 80 hours, 85 hours, and 90 hours.
[0018] Preferably, the concentration of genipin is 0.1-5%, the concentration of ethanol in the ethanol aqueous solution is 50-90%, and the cross-linking temperature is 4-50°C, such as 10°C, 20°C, 30°C, 40°C.
[0019] The present invention has the following beneficial effects:
[0020] The present invention proposes a method for preparing porous gelatin microcarriers, and gelatin microspheres are subjected to low-temperature treatment and freeze-drying, and a gelatin microcarrier with an internal and external through-structure is successfully developed. This unique structural design provides cells with a larger attachment space, increases the density of cell culture, and realizes large-scale cell expansion. By controlling the freezing temperature, the present invention can achieve regulation of the pore size of the microcarrier to meet the culture requirements of different cell types, avoids the use of organic solvents or other harmful chemicals, and realizes a green and environmentally friendly pore-making process. In addition, the microcarrier is cross-linked after forming a porous structure. This step not only maintains the high porosity of the microcarrier, but also improves its mechanical strength. This innovative preparation method improves the performance of the microcarrier and expands its application in fields such as drug delivery and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The morphologies of the porous gelatin microcarriers prepared in Examples 1-4 under a scanning electron microscope (SEM), Figure (A) is Example 3, Figure (B) is Example 2, Figure (C) is Example 1, and Figure (D) is Example 4.
[0022] Figure 2 This is the particle size distribution diagram of the porous gelatin microcarrier prepared in Example 1.
[0023] Figure 3 This is the pore size distribution diagram of the porous gelatin microcarrier prepared in Example 1.
[0024] Figure 4 This is the infrared spectra of the porous gelatin microcarrier prepared in Example 1 before and after cross-linking.
[0025] Figure 5The water solubility changes of the porous gelatin microcarriers prepared in Example 1 before and after cross-linking.
[0026] Figure 6 This is the cell proliferation on the porous gelatin microcarrier of Example 5.
[0027] Figure 7 This is a laser confocal image of the cells in Example 5 attached and growing on the porous gelatin microcarrier. DETAILED DESCRIPTION
[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0029] Example 1
[0030] Emulsification into spheres: Add 4 g of gelatin particles into deionized water at 60°C and stir to completely dissolve them to obtain a 20% gelatin aqueous solution, which is then mixed with liquid paraffin containing 1 ml of Span 80 at a volume ratio of 1:4 to obtain a W / O type emulsion. Emulsify at 4°C for 2 h with a stirring speed of 500 rpm. After emulsification, add anhydrous ethanol for dehydration, then wash alternately with petroleum ether and anhydrous ethanol, and dry to obtain dry gelatin microspheres.
[0031] Pore making: Soak the dried gelatin microspheres in deionized water at 4°C for 10 min to allow them to absorb water and swell to obtain hydrated gelatin microspheres. Spread the hydrated gelatin microsphere solution to a thickness of 3 mm, freeze it at -80°C for 2 h, and then freeze-dry it at -10°C for 24 h to form a porous structure.
[0032] Cross-linking and curing: The gelatin microcarriers with a porous structure are immersed in a 90% ethanol aqueous solution containing genipin, the genipin concentration is 0.5%, and cross-linking and curing is performed for 36 hours. After the cross-linking is completed, the microcarriers are washed with anhydrous ethanol and deionized water, and freeze-dried to obtain the porous gelatin microcarriers.
[0033] Example 2
[0034] Emulsification into spheres: Add 4 g of gelatin particles into deionized water at 60°C and stir to completely dissolve them to obtain a 20% gelatin aqueous solution, which is then mixed with liquid paraffin containing 1 ml of Span 80 at a volume ratio of 1:4 to obtain a W / O type emulsion. Emulsify at 4°C for 2 h with a stirring speed of 500 rpm. After emulsification, add anhydrous ethanol for dehydration, then wash alternately with petroleum ether and anhydrous ethanol, and dry to obtain dry gelatin microspheres.
[0035] Pore making: The dried gelatin microspheres were soaked in deionized water at 4°C for 10 min to allow them to absorb water and swell to obtain hydrated gelatin microspheres. The hydrated gelatin microsphere solution was spread to a thickness of 3 mm, frozen at -60°C for 2 h, and then freeze-dried at -10°C for 24 h to form a porous structure.
[0036] Cross-linking and curing: The gelatin microcarriers with a porous structure are immersed in a 90% ethanol aqueous solution containing genipin, the genipin concentration is 0.5%, and cross-linking and curing is performed for 36 hours. After the cross-linking is completed, the microcarriers are washed with anhydrous ethanol and deionized water, and freeze-dried to obtain the porous gelatin microcarriers.
[0037] Example 3
[0038] Emulsification into spheres: Add 4 g of gelatin particles into deionized water at 60°C and stir to completely dissolve them to obtain a 20% gelatin aqueous solution, which is then mixed with liquid paraffin containing 1 ml of Span 80 at a volume ratio of 1:4 to obtain a W / O type emulsion. Emulsify at 4°C for 2 h with a stirring speed of 500 rpm. After emulsification, add anhydrous ethanol for dehydration, then wash alternately with petroleum ether and anhydrous ethanol, and dry to obtain dry gelatin microspheres.
[0039] Pore making: Soak the dried gelatin microspheres in deionized water at 4°C for 10 min to allow them to absorb water and swell to obtain hydrated gelatin microspheres. Spread the hydrated gelatin microsphere solution to a thickness of 3 mm, freeze it at -20°C for 2 h, and then freeze-dry it at -10°C for 24 h to form a porous structure.
[0040] Cross-linking and curing: The gelatin microcarriers with a porous structure are immersed in a 90% ethanol aqueous solution containing genipin, the genipin concentration is 0.5%, and cross-linking and curing is performed for 36 hours. After the cross-linking is completed, the microcarriers are washed with anhydrous ethanol and deionized water, and freeze-dried to obtain the porous gelatin microcarriers.
[0041] Example 4
[0042] Emulsification into spheres: Add 4 g of gelatin particles into deionized water at 60°C and stir to completely dissolve them to obtain a 20% gelatin aqueous solution, which is then mixed with liquid paraffin containing 1 ml of Span 80 at a volume ratio of 1:4 to obtain a W / O type emulsion. Emulsify at 4°C for 2 h with a stirring speed of 500 rpm. After emulsification, add anhydrous ethanol for dehydration, then wash alternately with petroleum ether and anhydrous ethanol, and dry to obtain dry gelatin microspheres.
[0043] Pore making: The dried gelatin microspheres were soaked in deionized water at 4°C for 10 min to allow them to absorb water and swell to obtain hydrated gelatin microspheres. The hydrated gelatin microspheres were cold-extracted in liquid nitrogen for 5 min and then freeze-dried at -10°C for 24 h to form a porous structure.
[0044] Cross-linking and curing: The gelatin microcarriers with a porous structure are immersed in a 90% ethanol aqueous solution containing genipin, the genipin concentration is 0.5%, and cross-linking and curing is performed for 36 hours. After the cross-linking is completed, the microcarriers are washed with anhydrous ethanol and deionized water, and freeze-dried to obtain the porous gelatin microcarriers.
[0045] The porous gelatin microcarriers prepared in Examples 1-4 were observed by scanning electron microscopy. Figure 1 As shown, the change of freezing temperature affects the pore size. As the freezing temperature decreases, the growth rate of ice crystals slows down, the nucleation rate accelerates, the pore size decreases, and the distribution becomes more uniform.
[0046] Example 5
[0047] The porous gelatin microspheres prepared in Example 1 were characterized and tested.
[0048] 5.1 Particle size statistics
[0049] The cross-linked porous gelatin microcarriers were washed alternately with ethanol and deionized water, placed under an optical microscope, photographed, and the particle size was counted using Image J. The results are as follows: Figure 2 As shown, the particle size of microcarriers is mainly distributed between 150 and 300 μm.
[0050] 5.2 Aperture Statistics
[0051] The aperture of the SEM images was counted using Image J. The results are as follows: Figure 3 As shown, the pore size distribution of the microcarriers is uniform, concentrated around 20 μm.
[0052] 5.3 Fourier infrared spectroscopy test
[0053] The porous gelatin microcarriers before and after cross-linking were freeze-dried and the samples were prepared by potassium bromide tableting. The infrared data of each group were normalized using Origin. Figure 4 As shown in the infrared spectrum of gelatin microcarriers without genipin cross-linking, 3434 cm -1 The broad peak is attributed to the stretching vibration of the OH or NH group of gelatin amide, i.e., the characteristic peak of amide A band. -1 The peak is attributed to the stretching vibration of C=O in amide I band. After cross-linking of genipin, the amide A band shifts to a lower wave number, indicating that NH participates in hydrogen bonding. After polymerization of genipin, its ring-opening dihydropyran ring forms a new aldehyde group, which reacts with gelatin amino groups via Schiff base to form C=N. After cross-linking, the amide I band shifts from 1634 cm -1 Move to 1628cm -1 .
[0054] 5.4 Water solubility test
[0055] Take an appropriate amount of uncrosslinked porous gelatin microcarriers and add them to the ampoule. Add deionized water to the ampoule, shake for 1 to 2 minutes, and let it stand. Take another appropriate amount of crosslinked porous gelatin microcarriers and add them to the ampoule and repeat the above steps. Observe the microcarriers after standing for 30 minutes and take photos to record the results. Figure 5 As shown in the figure, the microcarriers are soluble in water before cross-linking and agglomerate; after cross-linking, the microcarriers are insoluble in water and have a complete shape. This shows that after cross-linking, the water solubility of the porous microcarriers can be changed, making them stable in aqueous solution.
[0056] 5.5 Cell counting and viability determination
[0057] Pretreatment of porous gelatin microcarriers: The porous gelatin microcarriers were immersed in 75% ethanol until they were completely precipitated, then washed three times with PBS, and finally immersed in culture medium and incubated overnight.
[0058] Inoculation of mouse embryonic fibroblasts: Inoculate mouse embryonic fibroblasts in T25 culture flasks and culture them in a humidified environment at 37°C and 5% CO2. When the cell confluence reaches 80%, digest the cells with trypsin containing EDTA and harvest the cells. Spread the porous gelatin microcarriers at a concentration of 10-15% per well on the bottom of a 48-well plate, with 1-2×10 4 Seeding cells.
[0059] Cell counting: The microcarriers were collected on days 1, 3, 5, and 7, and the cells were digested with trypsin containing EDTA and counted. Figure 6 As shown, the proliferation effect of cells on microcarriers was better than that on 2D group.
[0060] 5.6 Live and dead cell staining
[0061] Pretreatment of porous gelatin microcarriers: The porous gelatin microcarriers were immersed in 75% L-alcohol until they were completely precipitated, then washed three times with PBS, and finally immersed in culture medium and incubated overnight.
[0062] Inoculation of mouse embryonic fibroblasts: Inoculate mouse embryonic fibroblasts in T25 culture flasks and culture them in a humidified environment at 37°C and 5% CO2. When the cell confluence reaches 80%, digest the cells with trypsin containing EDTA and harvest the cells. Spread the porous gelatin microcarriers at a concentration of 10-15% per well on the bottom of a 96-well plate, and then add 5×10 3 ~2×10 4 Inoculate cells. After culturing for 2 days, stain with Calcein-AM / PI cell live-dead staining kit and observe the attachment of cells to the microcarriers under a confocal microscope. Figure 7As shown, there is no red fluorescence in the picture, indicating that the microcarrier has good biocompatibility, and there is green fluorescence on the surface of the microcarrier, indicating that the cells are successfully attached to the microcarrier and maintain good activity, and the microcarrier provides a good 3D environment for cell growth.
[0063] Based on the above ideal examples of the present invention, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the description.
Claims
1. A method for preparing a porous gelatin microcarrier, characterized in that: The following steps are involved: S1, gelatin microspheres were prepared by W / O emulsification method; S2, treating and drying the gelatin microspheres at low temperature to obtain porous gelatin microcarriers with a pore size of 2 to 60 μm; S3, cross-linking and curing the microcarriers obtained in step S2.
2. According to the method of claim 1, the W / O emulsification method in step S1 is to mix the gelatin solution and the oil phase containing the emulsifier to obtain a W / O type emulsion, emulsify at low temperature, add anhydrous ethanol for dehydration, then wash with an organic solvent, and dry to obtain dry gelatin microspheres.
3. According to the method of claim 2, the concentration of the gelatin solution is 5-30%, and the volume ratio of the gelatin solution to the oil phase is 1:3-1:
8.
4. According to the method of claim 2, the concentration of the emulsifier is 0.5-5%, the temperature of low-temperature emulsification is 4--20°C, the emulsification time is 1-4h, and the stirring speed during emulsification is 300-800rpm; the oil phase is one or a combination of vegetable oil, mineral oil, and paraffin; the emulsifier is one or a combination of Tween 80, Span 80, and stearic acid; the organic solvent is one or a combination of petroleum ether, isopropanol, acetone, and anhydrous ethanol.
5. According to the method of claim 1, the low temperature treatment in step S2 is to freeze the hydrated gelatin microspheres, and the freezing method is specifically any one of the following two methods: (1) the hydrated gelatin microsphere solution is spread to a thickness of 3-5 mm and frozen at -20 to -80°C for 2 to 24 hours; (2) the hydrated gelatin microspheres are frozen at -100 to -200°C for 1 to 10 minutes, preferably under liquid nitrogen conditions; the hydrated gelatin microspheres are prepared by soaking the dried gelatin microspheres in deionized water to make them absorb water and expand, the deionized water temperature is 0 to 20°C, and the soaking time is 1 to 30 minutes.
6. The method according to claim 1, wherein the drying method in step S2 is freeze drying, the temperature during freeze drying is -10 to -80°C, and the time is 12 to 48 hours.
7. According to the method of claim 1, the cross-linking and curing in step S3 specifically comprises: immersing the gelatin microcarriers having a porous structure in an ethanol aqueous solution containing genipin, and cross-linking and curing for 12 to 96 hours.
8. The method according to claim 7, wherein the concentration of genipin is 0.1-5%, the concentration of ethanol in the ethanol aqueous solution is 50-90%, and the cross-linking temperature is 4-50°C.
9. A porous gelatin microcarrier with a pore size of 2 to 60 μm obtained by the preparation method according to any one of claims 1 to 8.
10. Use of the porous gelatin microcarrier prepared by the method according to any one of claims 1 to 8 in cell culture.