Preparation method of self-assembled polypeptide bionic microcarrier

Abstract

The invention belongs to the technical field of biological materials, and relates to a high-biocompatibility polypeptide microcarrier as well as a preparation method and cell culture application thereof. In order to solve the problems of insufficient biocompatibility and poor cell adaptability of the traditional microcarrier, collagen and polypeptide are adopted to construct a composite matrix, a water phase (containing polypeptide / collagen solution) and an oil phase (containing Span 80 vegetable oil) are emulsified to form a microsphere precursor, and after heating for preliminary stabilization, the microsphere precursor is sequentially crosslinked by a crosslinking agent to form a composite microsphere. And forming the microcarrier with a three-dimensional network structure. Biocompatible materials are used in the whole process, the surface characteristic of the obtained microcarrier is beneficial to cell attachment, and degradation products do not have toxic or side effects. Experiments show that the microcarrier can significantly promote proliferation and spreading of mouse fibroblasts, and is suitable for a three-dimensional cell culture system in tissue engineering and regenerative medicine.

Description

Technical fields:

[0001] This invention relates to the field of microcarrier material preparation, specifically to a microcarrier for three-dimensional cell culture and its preparation method, particularly a method for preparing a biomimetic polypeptide microcarrier with high biocompatibility. Background technology:

[0002] Microcarriers are biofunctional microsphere scaffolds used in cell culture, amplification, and delivery, and are widely used in 3D cell culture. An ideal microcarrier should possess good biocompatibility, controllable degradability, suitable surface properties to promote cell adhesion and expansion, and sufficient mechanical strength. Currently, commercially available microcarriers are mostly prepared from materials such as dextran, gelatin, and polystyrene. However, these materials have some inherent drawbacks: synthetic polymers (such as polystyrene) have poor biocompatibility and degradability, and their degradation products may induce inflammatory responses; furthermore, many existing microcarriers lack cell-specific recognition sites on their surfaces, resulting in low cell adhesion efficiency and difficulty in simulating the real extracellular matrix (ECM) environment.

[0003] Peptide materials have become a research hotspot due to their excellent biocompatibility, biodegradability, and designable bioactivity. Through molecular design, specific cell adhesion sequences can be introduced into peptide chains, thereby preparing biomimetic materials that specifically promote cell adhesion and proliferation. However, how to fabricate these water-soluble peptide materials into uniformly sized, structurally stable, and bioactive microspheres using a mild and controllable process remains a challenging problem. Traditional emulsion crosslinking methods often use toxic chemical crosslinking agents (such as glutaraldehyde), and residual crosslinking agents can be toxic to cells. Therefore, developing a method that uses fully biocompatible materials, employs a green crosslinking process, and can prepare biomimetic peptide microcarriers with high cell affinity has significant scientific and application value. Summary of the Invention:

[0004] The primary objective of this invention is to provide a simple, mild method for preparing biomimetic polypeptide microcarriers using biocompatible materials throughout the process. By designing the polypeptide sequence and introducing other biocompatible materials, biomimetic microcarriers meeting specific requirements can be effectively prepared. The microcarriers are then cross-linked and solidified, resulting in microcarriers with high biocompatibility and good stability. The entire preparation process is simple, controllable, environmentally friendly, and pollution-free. The resulting biomimetic polypeptide microcarriers have a uniform particle size distribution, require no additional surface modification, and can be used in various fields such as cell culture, drug delivery, and tissue engineering scaffolds.

[0005] This invention provides a method for preparing biomimetic polypeptide microcarriers, comprising the following steps:

[0006] S1. Polypeptide microspheres were prepared using the W / O emulsification-evaporation method;

[0007] S2. Crosslink and cure the microcarrier obtained in step S1.

[0008] Preferably, in step S1, the W / O emulsification method involves mixing a polypeptide solution with an oil phase containing an emulsifier to obtain a W / O type emulsion, emulsifying at high temperature, dehydrating at high temperature, and then washing with an organic solvent to obtain polypeptide microspheres immersed in an organic solvent.

[0009] Preferably, the concentration of the polypeptide solution is 1-5%, for example 1%, 2%, 3%, 4%, 5%; the volume ratio of the polypeptide solution to the oil phase is 1:10 to 1:15, for example 1:10, 1:11, 1:12, 1:13, 1:14, 1:15.

[0010] Preferably, the concentration of the emulsifier is 0.5-5%, for example, 0.5%, 1%, 2%, 3%, 4%, or 5%; the high-temperature emulsification temperature is 50-70°C, for example, 50°C, 55°C, 60°C, 65°C, or 70°C; the emulsification time is 0.5-2 hours, for example, 0.5 hours, 1 hour, 1.5 hours, or 2 hours; the stirring speed during emulsification is 300-800 rpm, for example, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, or 800 rpm; the oil phase is one or a combination of vegetable oil, mineral oil, and paraffin wax; the emulsifier is one or a combination of Tween 80, Span 80, and stearic acid; and the organic solvent is one or a combination of petroleum ether, isopropanol, acetone, and anhydrous ethanol.

[0011] Preferably, the cross-linking curing in step S2 specifically involves immersing the polypeptide microcarrier in an ethanol-water solution containing genipin and cross-linking curing for 12–96 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, or 90 hours.

[0012] Preferably, the concentration of genipin is 0.1-5%, the concentration of ethanol in the aqueous ethanol solution is 80-100%, and the crosslinking temperature is 4-50°C, for example, 10°C, 20°C, 30°C, or 40°C.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. This invention proposes a method for preparing polypeptide microcarriers. This invention uses biocompatible components throughout the entire process from raw materials to technology. The aqueous core consists of biomimetic polypeptides and collagen, which simulate the composition of the natural extracellular matrix.

[0015] 2. This invention proposes a method for preparing polypeptide microcarriers, wherein the cross-linking agent genipin is a natural plant-derived cross-linking agent with cytotoxicity far lower than that of the traditional chemical cross-linking agent glutaraldehyde.

[0016] 3. The present invention proposes that the microcarrier prepared by combining the W / O emulsification evaporation method with the above crosslinking process has a wrinkled structure on the surface, which greatly increases the specific surface area and is conducive to cell adhesion.

[0017] 4. The cell adhesion sequence (such as RGD) designed on the biomimetic polypeptide raw material for preparing polypeptide microcarriers in this invention provides specific binding sites for cells, and works synergistically with collagen to efficiently promote cell adhesion, spreading and proliferation.

[0018] 5. The microcarriers prepared by this invention can not only be used for the expansion of adherent cells (such as stem cells, fibroblasts, etc.), but their degradability also makes them an excellent choice for controlled release carriers of drugs or growth factors, and they have great potential in the construction of tissue engineering scaffolds. Attached image description:

[0019] Figure 1 Image (a) of the polypeptide microcarrier prepared in Example 1 under an optical microscope and surface structure diagram (b) under a scanning electron microscope (SEM);

[0020] Figure 2 The particle size distribution diagram of the polypeptide microcarriers prepared in Example 1;

[0021] Figure 3 Infrared spectra of the polypeptide microcarriers prepared in Example 1 before and after crosslinking;

[0022] Figure 4 The change in water solubility of the polypeptide microcarrier prepared in Example 1 before and after crosslinking;

[0023] Figure 5 Example 3 shows the changes in cell proliferation on polypeptide microcarriers (a) and the evaluation of the cytotoxicity of polypeptide microspheres by the CCK-8 assay (b).

[0024] Figure 6 This is a laser confocal image of cells attached and growing on polypeptide microcarriers, as shown in Example 4. Detailed implementation method:

[0025] The present invention will be further described in detail below through embodiments. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0026] Example 1. Preparation of biomimetic polypeptide microspheres

[0027] This embodiment relates to the preparation of biomimetic polypeptide microspheres, specifically including the following steps:

[0028] 1) Preparation of aqueous phase: Accurately weigh 30 mg of biomimetic self-assembled peptide, 12 mg of collagen and 3 mg of polylysine, dissolve them in 1.5 mL of deionized water, vortex and then sonicate in an ultrasonic instrument until they are completely dissolved to obtain an aqueous phase solution with a concentration of 30 mg / mL.

[0029] 2) Preparation of the oil phase: Dissolve 100 μL of Span-80 in 10 mL of Arowana oil, vortex until homogeneous, to obtain an approximately 1% Span-80 oil solution. Separately, take 10 mL of Arowana oil, add 100 mg of stearic acid, and stir in a 60°C water bath until completely dissolved. Then add the above Span-80 oil solution to the mixture and stir vigorously at 1000 r / min for 20 minutes to obtain a homogeneous mixed oil phase.

[0030] 3) Emulsification: Place the mixed oil phase under a mechanical stirrer and slowly add the aqueous phase solution dropwise to the oil phase at a rate of 1 mL / min using a syringe pump at a speed of 500 rpm. After the addition is complete, continue shear emulsification for 10 minutes to form a stable W / O type emulsion.

[0031] 4) Curing and precipitation: Transfer the emulsion to a 60°C water bath and react for 4 hours under mechanical stirring at 500 r / min until the aqueous phase is completely evaporated and the microspheres are precipitated. Then wash with petroleum ether and anhydrous ethanol alternately.

[0032] 5) Crosslinking and curing: Immerse the microspheres in 3 mL of 90% ethanol solution containing 0.5% genipin and crosslink in a water bath at 37°C for 48 hours. After crosslinking, wash three times with anhydrous ethanol to remove residual genipin.

[0033] Microscopic images of the polypeptide microcarriers obtained in Example 1 are shown below. Figure 1 As shown in (a), the particles are plump and uniform in size, and their surface structure is as follows: Figure 1 As shown in (b), the wrinkled structure on its surface can be observed, with a large surface area for cells to attach and spread.

[0034] Example 2. The prepared polypeptide microspheres were characterized.

[0035] The cross-linked polypeptide microcarriers were washed with anhydrous ethanol, photographed under an optical microscope, and their particle size was statistically analyzed using ImageJ. The results are as follows: Figure 2 As shown, the particle size of the microcarriers is mainly distributed between 150 and 300 μm.

[0036] Example 3. Counting and viability determination of cells grown on the surface of the prepared polypeptide microspheres.

[0037] Pretreatment of biomimetic peptide microcarriers: The peptide microcarriers were immersed in 75% ethanol until completely precipitated, then washed three times with PBS, and finally incubated overnight in culture medium. Seeding of mouse embryonic fibroblasts: Mouse embryonic fibroblasts were seeded in T25 culture flasks and cultured in a humid environment of 37°C and 5% CO2. When cell confluence reached 80%, cells were harvested by trypsin digestion with EDTA. The peptide microcarriers were seeded at a concentration of 10–15% per well at the bottom of a 48-well plate, with 1–2 × 10⁶ cells per well. 4 Inoculate cells.

[0038] Cell counting: Microcarriers were collected on days 1, 3, 5, and 7, and cells were counted after digestion with trypsin containing EDTA. The results are as follows: Figure 5 As shown in (a), the cells proliferated better on the microcarriers compared to group 2D.

[0039] Cell viability: On days 1, 3, 5, and 7, 10 μl of CCK-8 was added to each well, and the cells were resuspended and incubated in a cell culture incubator for 40 min. Then, 70 μL of the supernatant was aspirated, and the OD value was measured at 450 nm using a microplate reader. A curve was plotted as shown below. Figure 5 As shown in (b), cell viability was significantly enhanced compared to group 2D.

[0040] Example 4. Staining of cell viability and mortality on the surface of the prepared polypeptide microspheres.

[0041] Pretreatment of peptide microcarriers: The peptide microcarriers were immersed in 75% ethanol until completely precipitated, then washed three times with PBS, and finally incubated overnight in culture medium. Seeding of mouse embryonic fibroblasts: Mouse embryonic fibroblasts were seeded in T25 culture flasks and cultured in a humid environment of 37°C and 5% CO2. When cell confluence reached 80%, cells were harvested by trypsin digestion with EDTA. Peptide microcarriers were seeded at a concentration of 10–15% per well at the bottom of a 96-well plate, and then cells were seeded at a concentration of 5 × 10³–2 × 10⁴ per well. After 2 days of culture, cells were stained with Calcein-AM / PI cell liveness / death staining kit, and cell attachment on the microcarriers was observed under a confocal microscope. Results are as follows: Figure 6 As shown, the absence of red fluorescence in the image indicates good biocompatibility of the microcarrier. The presence of green fluorescence on the surface of the microcarrier indicates that cells have successfully attached to the microcarrier and maintained good activity, and that the microcarrier provides a good 3D environment for cell growth.

[0042] Based on the above-described ideal examples of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing a self-assembled polypeptide biomimetic microcarrier, characterized in that, Includes the following steps: S1. Polypeptide microspheres were prepared using a W / O emulsification-evaporation method. S2. Crosslink and cure the microcarrier obtained in step S1.

2. The method for preparing a self-assembled peptide biomimetic microcarrier according to claim 1, wherein the W / O emulsification evaporation method in step S1 involves mixing a peptide solution with an oil phase containing an emulsifier to obtain a W / O type emulsion, emulsifying at high temperature, dehydrating at high temperature, and then washing with an organic solvent to obtain peptide microspheres immersed in an organic solvent.

3. The method for preparing a self-assembled peptide biomimetic microcarrier according to claim 1, wherein the peptide solution concentration is 1-5% and the volume ratio of the peptide solution to the oil phase is 1:10-1:

15.

4. A method for preparing a self-assembled polypeptide biomimetic microcarrier according to claims 1-2, wherein the emulsifier concentration is 0.5-5%, the high-temperature emulsification temperature is 50-70℃, the emulsification time is 0.5-2h, the stirring speed during emulsification is 300-800rpm, and the dehydration time is 2-4h; 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; and the organic solvent is one or a combination of petroleum ether, isopropanol, acetone, and anhydrous ethanol.

5. A method for preparing a self-assembled peptide biomimetic microcarrier according to claim 1, wherein the cross-linking curing in step S2 specifically comprises: immersing the peptide microcarrier in a solution containing a cross-linking agent for cross-linking curing.

6. A method for preparing a self-assembled polypeptide biomimetic microcarrier according to claim 5, wherein the crosslinking agent is selected from at least one of genipin, anhydrous calcium chloride, and 1,4-butanediol diglycidyl ether.

7. The method for preparing a self-assembled peptide biomimetic microcarrier according to claims 1-6, wherein the obtained biomimetic peptide microcarrier has a particle size of 100-300 μm.

8. The method for preparing a self-assembled polypeptide biomimetic microcarrier according to claims 1-7, wherein the prepared biomimetic polypeptide gel microcarrier can be used for cell culture.

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