CN121574434A

CN121574434A - A novel phenylboronic acid-modified chitosan microcarrier, its preparation method and application

Info

Publication number: CN121574434A
Application number: CN202511674589.8A
Authority: CN
Inventors: 轩昆; 郭皓; 杨晓雪
Original assignee: Air Force Medical University
Current assignee: Air Force Medical University
Priority date: 2025-11-14
Filing date: 2025-11-14
Publication date: 2026-02-27

Abstract

This invention relates to the field of biotechnology, specifically to a novel phenylboronic acid-modified chitosan microcarrier, its preparation method, and its applications. The phenylboronic acid-modified chitosan microcarrier is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and then cross-linking them. The microcarrier has a particle size of approximately 100 μm. Scanning electron microscopy analysis shows that the microcarrier exhibits a distinct spherical porous morphology with uniform pore shape and size, providing a three-dimensional environment for cell growth. Its moderate hardness, elasticity, and pore wall thickness are more conducive to cell growth and nutrient transport, resulting in higher stability and better suitability for cell culture. It also demonstrates excellent biosafety and biocompatibility, making it more suitable for 3D cell culture systems.

Description

Novel phenylboronic acid modified chitosan microcarrier, preparation method and application

Technical Field

The invention relates to the technical field of biology, in particular to a novel phenylboronic acid modified chitosan microcarrier, a preparation method and application.

Background

The generation of human stem cell derived spheroids and organoids is an important step in addressing numerous medical, pharmacological and biological challenges. The conventional culture method is performed on a two-dimensional planar culture dish, cells are adhered to an artificial plastic or glass substrate, and only at the periphery thereof are in contact with other cells. The environment is physiologically non-uniform due to the absence of oxygen, nutrient or waste gradients. The cells are not allowed to stack together but are forced to form a monolayer morphology, which is not the natural morphology of all cell types. Establishing a co-culture in a plate may increase natural contact and communication between cells, but the two-dimensional surface still inhibits the ability of the cells to form a multi-dimensional structure. Therefore, 2D cultured planar layer cells cannot accurately mimic the growth environment of cells in vivo. With the development of technology, 3D suspension cell culture has become a key step in the construction of organoids. The 3D suspension cell culture is based on biological scaffold materials, and forms a three-dimensional organoid structure through autonomous self-assembly of cells or micro-environment regulation and control, so that morphology and physiology of cells are better simulated.

The microcarrier is used as a biological scaffold for culturing suspension cells, the porous structure and the characteristics of the material provide stable matrix microenvironment for the cells, and plays an important role in the processes of cell proliferation, drug delivery and organoid construction. Compared with a two-dimensional cell culture mode, the three-dimensional cell culture mode based on the microcarrier can simulate the in-vivo microenvironment, realize the rapid proliferation of cells, enhance the stem property and directional differentiation capability of stem cells, and realize the time-series regeneration of the cells, so that the time-series functional change of the stem cells is guided. At present, the existing cell microcarrier in the market can realize large-scale expansion of cells to a certain extent, but various enzymes are required to be added to degrade the microcarrier when the cells are finally obtained, so that single cells are obtained. Meanwhile, in the in-vivo application and delivery scene of cells, the degradation of microcarriers in vivo and the slow release of cells encounter a large bottleneck. Therefore, the development of a novel microcarrier capable of responding to tissue and organ and organism environment and thus degrading spontaneously has profound social and medical significance in stem cell expansion, stem cell performance improvement and stem cell delivery.

Aiming at the problems, the invention develops a phenylboronic acid modified chitosan (CS-FPBA) microcarrier synthesized based on an emulsion method, which not only can realize large-scale expansion of cells, but also has the characteristics of glucose and PH sensitivity, and can realize self-degradation and delivery and slow release of stem cells in organisms and individuals with specific diseases (diabetes mellitus).

Disclosure of Invention

The primary aim of the invention is to provide a novel phenylboronic acid modified chitosan microcarrier, which is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking.

Preferably, the phenylboronic acid modified chitosan microcarrier has a particle size of >100 μm.

The second object of the invention is to provide a preparation method of the phenylboronic acid modified chitosan microcarrier, which comprises the following steps:

(1) Dispersing medium-viscosity chitosan in acetic acid solution, dissolving completely, and activating;

(2) Slowly adding 4-formylphenyl boric acid into the solution obtained in the step (1), and stirring at room temperature;

(3) Dialyzing, regulating the pH to 5.0, removing redundant 4-formylphenyl boric acid, and freeze-drying to obtain a product CS-FPBA;

(4) Fully dissolving the CS-FPBA product obtained in the step (3), and stirring and adding gelatin and a cross-linking agent to obtain a reaction system;

(5) Liquid paraffin and Span 80 are stirred until being mixed uniformly, and the liquid paraffin and Span 80 are a solvent system;

(6) Slowly adding the reaction system in the step (4) into the solvent system in the step (5) under stirring, reacting, standing and collecting the precipitate;

(7) And (3) cleaning the precipitate obtained in the step (6), swelling with ultrapure water, freezing, and drying to obtain the final product CS-FPBA microcarrier.

Preferably, the concentration of 4-formylbenzene boric acid in step (2) is 0.02 mmol.

Preferably, the cross-linking agent in step (4) is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

Preferably, the gelatin concentration in step (4) is 5%.

Preferably, the volume-mass ratio of the liquid paraffin to Span 80 in the step (5) is 250:5.

Preferably, the reaction temperature in the step (6) is 30 ℃ and the reaction time is 48 hours.

Preferably, the washing in step (7) uses isopropyl alcohol and absolute ethyl alcohol.

The third object of the invention is to provide the application of the phenylboronic acid modified chitosan microcarrier or the phenylboronic acid modified chitosan microcarrier prepared by the preparation method in three-dimensional cell culture.

The invention has the beneficial effects that (1) the invention provides a novel phenylboronic acid modified chitosan microcarrier, which is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking. The particle size of the microcarrier is about 100 mu m, and the analysis of a scanning electron microscope shows that the microcarrier has definite spherical porous morphology, uniform pore shape and uniform pore size, and can provide a three-dimensional environment for cell growth. The hardness, elasticity and pore wall of the composite material are moderate, and the composite material is more beneficial to the growth of more cells and the transmission of nutrient substances.

(2) At pH 5.5, the degradation rate of the CS and CS-FPBA is higher, at pH 6.5, the degradation rate of CS is lower than that of CS-FPBA, at pH 7.4, the degradation rate of CS-FPBA is lower than that of CS, and the stability of CS-FPBA is higher. The optimal pH range of animal cell culture is 7-7.5, and CS-FPBA is more suitable for cell culture application. Under the same culture mode, cell proliferation in the CS-FPBA microcarrier environment was significantly better than CS.

(3) Optical microscopy confirmed that the cells adhered well to the porous structure of the microcarriers. Cells on microcarriers showed significant proliferation and form tight junctions within 1-9D, with significantly increased expression rates in 3D cultured SHED (SHED-CD) compared to two-dimensional cultured SHED. The SHED is cultured on CS-FPBA microcarrier, and after co-culture in a stirring bioreactor, the assembled SHED microcarrier is obtained, and the SHED maintains good bioactivity and differentiation potential. Therefore, CS-FPBA microcarriers show excellent biosafety and biocompatibility, and are more suitable for 3D cell culture systems.

Drawings

FIG. 1 is a schematic flow chart of synthesizing phenylboronic acid modified chitosan CS-FPBA microcarrier

FIG. 2 CS-FPBA general and microstructure

FIG. 3 CS in vitro degradation rate of CS-FPBA

FIG. 4 CS stability with CS-FPBA at different pH values

FIG. 5 CS and CS-FPBA in different solutions

FIG. 6 CS and CS-FPBA cell proliferation assay

FIG. 7 CS-FPBA microcarrier culture cell application

FIG. 8 CS-FPBA cell culture scanning electron microscope

FIG. 9 CS-FPBA cell proliferation assay

FIG. 10 CS-FPBA microcarrier cultured SHED cells still maintain the stem character of mesenchymal stem cells

FIG. 11 CS-FPBA microcarrier cultured SHED cells have excellent osteogenic potential

Detailed Description

The following describes the scope of the present invention in detail with reference to specific examples, and it should be noted that the scope of the present invention is not limited by the following examples.

It should be noted that in the examples below, unless otherwise specified, the methods are conventional and the reagents are commercially available.

Example one, a method for preparing a chitosan Carrier

The method comprises the following steps:

(1) Dispersing medium-viscosity Chitosan (CS) in 1% acetic acid solution, and fully dissolving to obtain stable chitosan alternate suspension;

(2) Freeze drying to obtain a product CS;

(3) Drying CS in an oven to 40min, soaking in 70% ethanol for 24: 24 h, and repeatedly cleaning with deionized water for 3 times;

(4) Adding 0.5% gelatin into the 1% synthetic product CS obtained in the step (3) after fully dissolving, and adding a cross-linking agent (EDC+NHS) into the mixture to form a reaction system;

(5) Stirring liquid paraffin 250 mL+5 g Span 80,330 at a constant speed until the liquid paraffin is uniformly mixed to form a solvent system;

(6) Slowly adding the reaction system in the step (4) into the solvent system in the step (5) under the condition of stirring;

(7) Standing at 30 ℃ after the reaction is carried out at 48 h, and collecting precipitate;

(8) And (3) sequentially cleaning the isopropanol and the absolute ethyl alcohol, swelling the dried product by ultrapure water, freezing, drying to obtain a final product CS microcarrier, and drying and storing the final product CS microcarrier.

Example two, preparation method of phenylboronic acid-chitosan carrier

The method comprises the following steps:

(1) Dispersing medium-viscosity Chitosan (CS) in acetic acid solution, dissolving completely, and activating for 4 hr;

(2) Slowly adding 0.02 mmol of 4-formylphenyl boric acid (FPBA) into the mixed solution obtained in the step (1), and stirring at room temperature for 12 hours;

(3) Dialyzing with distilled water in dialysis bag for 3 days, adjusting pH to 5.0, removing excessive FPBA, and lyophilizing to obtain CS-FPBA product;

(4) Adding 0.5% gelatin into the 1% synthetic product CS-FPBA obtained in the step (4) after fully dissolving, and adding a cross-linking agent (EDC+NHS) into the mixture to form a reaction system;

(8) And (3) sequentially cleaning the isopropanol and the absolute ethyl alcohol, swelling the ethanol by ultrapure water, freezing and drying to obtain a final product CS-FPBA microcarrier, and screening to obtain a microcarrier with the particle size of more than 100 um, and drying and storing the microcarrier.

The chitosan is a medical polymer material with rapid development, has the characteristics of low immunogenicity, degradation controllability, porosity and the like, and is added with 4-formylphenylboronic acid to prepare a microcarrier material which can be used for cell culture and enhancing cell differentiation for preparing the multifunctional chitosan-based scaffold for supporting stem cell proliferation and differentiation.

The preparation process is shown in figure 1, after dialysis, CS-FPBA is obtained as a dry powder by lyophilization (figure 2A) and stored in a desiccator under ambient conditions for later use. As can be seen, CS-FPBA microcarriers with particle size of about 100 μm were prepared (FIG. 2B). The scanning electron microscope analysis shows that the microcarrier shows a definite spherical porous morphology, has uniform pore shape and uniform pore size, and can provide a three-dimensional environment for cell growth. The hardness, elasticity and pore wall of the composite material are moderate, and the composite material is more beneficial to the growth of more cells and the transmission of nutrient substances.

Example III determination of in vitro degradation Rate of microcarriers

Microcarriers were studied for degradation properties using PBS buffer at room temperature. The support material in the dry state was first weighed (mass at this time denoted m ₁) and then the sample material was immersed in PBS buffer and incubated 40 d at 37 ℃. Samples were removed and washed with deionized water at designated time intervals. Finally, the rinsed sample was re-weighed by lyophilization (the mass at this time was noted as m ₂).

The degradation rate of the scaffold was calculated from the following formula:

DR = (m₁ m₂)/m₁×100%

The in vitro degradation rates of CS and CS-FPBA scaffolds after 40 days of PBS are shown in FIG. 3. The in vitro degradation rate of CS-FPBA is lower than CS, which shows that the chitosan-phenylboronic acid microcarrier prepared by the experiment is slow to degrade, and shows that the chitosan-phenylboronic acid microcarrier can maintain the adhesion, spreading and growth metabolism of cells on the surface of the microcarrier.

The high-quality microcarrier is required to be suitable for various culture systems and has certain stability. As can be seen from FIG. 4, the degradation rate of both CS and CS-FPBA was higher at pH 5.5, the degradation rate of CS was lower than that of CS-FPBA at pH 6.5, and the degradation rate of CS-FPBA was lower than that of CS at pH 7.4, resulting in higher stability of CS-FPBA. The material was soaked in solutions of different pH and its integrity was observed separately (fig. 5). The optimal pH range for animal cell culture is between 7 and 7.5, and CS-FPBA is more suitable for cell culture application according to observation.

Example IV, cell proliferation assay

After two microcarrier materials are obtained, the microcarrier materials are applied to cell culture detection, and cell proliferation is observed. A three-dimensional culture mode was constructed using a bioreactor, inoculated at 100 mg microcarriers, 2.5X10 ⁶ cell mass, medium 50 mL was inoculated the first day, and after 24H in variable speed mode (40 revolutions), medium was added to 75 mL the next day, and the culture was rotated in constant mode. After staining the cells, they were observed with a fluorescence microscope, and the staining was performed with attention to light-shielding. As can be seen from FIG. 6, the proliferation of cells in the CS-FPBA microcarrier environment was significantly better than that of CS under the same culture pattern.

Fifth embodiment (V),

2D cell culture method:

Placing the tissue in a culture dish, repeatedly washing with PBS until no obvious blood is on the surface of the tissue, discarding PBS, adding 1.5 ml collagenase into the culture dish, shearing the tissue into tissue blocks, digesting in a 5% CO ₂ cell incubator, adding an equal volume of culture solution, centrifuging at 800 turns to 5 min, inoculating to a 25 cm ² culture flask, mixing uniformly, placing in the cell incubator for culturing, discarding the original culture medium, washing 3 times with sterile PBS, adding pancreatin for digestion 3 min, adding a proper amount of culture solution, blowing and transferring to a 15 ml centrifuge tube for centrifugation, discarding the supernatant, and inoculating into a 75 cm ² culture flask according to a density of 1X 10 ⁶/flask for later use.

3D cell culture method:

The resultant microcarrier was subjected to ultraviolet sterilization in a sterile biosafety cabinet 6 h. Transferring the sterilized microcarrier into a 125 mL ventilated built-in impeller culture flask, and supplementing the culture medium. The flask was then placed on a 3Dmini bioreactor and rotated at a constant speed to ensure complete swelling of the microcarriers (figure 7). Dental pulp stem cells (seed) were inoculated into a microcarrier-containing flask at a concentration of 2.5X10 ⁶ cells/mL, and medium was added to 50mL for a period of 24h. The next day, medium was added to 75 mL and the culture was rotated in constant mode. The seed-microcarrier assemblies were then obtained for subsequent experiments. The 3D cultured SHED was obtained by digestion with 0.25% trypsin at 37℃for 5 min.

Protein sample preparation detection method:

Protein samples of 2D-cultured seed and 3D-cultured seed were prepared by treatment with RIPA lysis buffer at 4 ℃ for 20 min. Samples were centrifuged at 15 min, the supernatant was collected and protein concentration was quantified using BCA protein assay kit. Equal amounts of protein samples were mixed with loading buffer and PBS and loaded into protein electrophoresis pre-gel for protein validation.

Optical microscopy confirmed that the cells adhered well to the porous structure of the microcarriers. And observing the morphological structure of the 3D cultured cell spheres at different time points by adopting a scanning electron microscope. Observations showed that cells on microcarriers showed significant proliferation and form tight junctions within 1-9 d (FIG. 8). By using cells cultured in two dimensions as a control group, a significant increase in cell count was observed throughout the culture period (fig. 9A). The staining results showed a significant increase in expression rate in 3D cultured reed (reed-CD) compared to two-dimensional cultured reed (fig. 9B). The SHED was cultured on CS-FPBA microcarriers and after co-culture in a stirred bioreactor, assembled SHED microcarriers were obtained, which maintained good bioactivity and differentiation potential (FIG. 10). Our research further proves that after a three-dimensional culture system is established by microcarrier and SHED suspension culture, the SHED highly expresses bone formation development related proteins such as RUNX2 (figure 11), so that the development potential of the protein can be better maintained, and the protein has excellent bone formation capability. These results thus indicate that CS-FPBA microcarriers exhibit excellent biosafety and biocompatibility, and are more suitable for use in 3D cell culture systems.

In summary, the invention provides a novel phenylboronic acid modified chitosan microcarrier, which is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking. The particle size of the microcarrier is about 100 mu m, and the analysis of a scanning electron microscope shows that the microcarrier has definite spherical porous morphology, uniform pore shape and uniform pore size, and can provide a three-dimensional environment for cell growth. The hardness, elasticity and pore wall of the composite material are moderate, and the composite material is more beneficial to the growth of more cells and the transmission of nutrient substances. At pH 5.5, the degradation rate of the CS and CS-FPBA is higher, at pH 6.5, the degradation rate of CS is lower than that of CS-FPBA, at pH 7.4, the degradation rate of CS-FPBA is lower than that of CS, and the stability of CS-FPBA is higher. The optimal pH range of animal cell culture is 7-7.5, and CS-FPBA is more suitable for cell culture application. Under the same culture mode, cell proliferation in the CS-FPBA microcarrier environment was significantly better than CS. Optical microscopy confirmed that the cells adhered well to the porous structure of the microcarriers. Cells on microcarriers showed significant proliferation and form tight junctions within 1-9D, with significantly increased expression rates in 3D cultured SHED (SHED-CD) compared to two-dimensional cultured SHED. The SHED is cultured on CS-FPBA microcarrier, and after co-culture in a stirring bioreactor, the assembled SHED microcarrier is obtained, and the SHED maintains good bioactivity and differentiation potential. Therefore, CS-FPBA microcarriers show excellent biosafety and biocompatibility, and are more suitable for 3D cell culture systems.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.

Claims

1. A novel chitosan microcarrier modified with phenylboronic acid, characterized in that the chitosan microcarrier modified with phenylboronic acid is obtained by mixing 4-formylphenylboronic acid and medium-viscosity chitosan with gelatin and crosslinking them.

2. The chitosan microcarrier modified with phenylboronic acid as described in claim 1, wherein the particle size of the chitosan microcarrier modified with phenylboronic acid is >100 μm.

3. The method for preparing chitosan microcarriers modified with phenylboronic acid as described in claim 1, characterized in that it comprises the following steps:

(1) Medium-viscosity chitosan is dispersed in acetic acid solution, fully dissolved, and activated;

(2) Slowly add 4-formylphenylboronic acid to the solution obtained in step (1) and stir at room temperature;

(3) Dialyze, adjust pH to 5.0 to remove excess 4-formylphenylboronic acid, freeze dry to obtain product CS-FPBA;

(4) The product CS-FPBA obtained in step (3) is fully dissolved, and gelatin and crosslinking agent are added by stirring to form a reaction system;

(5) Stir the liquid paraffin and Span 80 until they are evenly mixed to form a solvent system;

(6) The reaction system described in step (4) is slowly added to the solvent system described in step (5) under stirring, the reaction is carried out, the mixture is allowed to stand, and the precipitate is collected.

(7) Cleaning step (6) The precipitate collected is swollen with ultrapure water, then frozen and dried to obtain the final product CS-FPBA microcarrier.

4. The preparation method according to claim 3, wherein the concentration of 4-formylphenylboronic acid in step (2) is 0.02 mmol.

5. The preparation method according to claim 3, wherein the crosslinking agent in step (4) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

6. The preparation method according to claim 3, wherein the gelatin concentration in step (4) is 5%.

7. The preparation method according to claim 3, wherein the volume-to-mass ratio of liquid paraffin and Span 80 in step (5) is 250:5.

8. The preparation method according to claim 3, wherein the reaction temperature in step (6) is 30°C and the reaction time is 48h.

9. The preparation method according to claim 3, wherein the cleaning in step (7) uses isopropanol and anhydrous ethanol.

10. The application of the phenylboronic acid-modified chitosan microcarrier as described in claim 1 or the phenylboronic acid-modified chitosan microcarrier prepared by any one of the preparation methods described in claims 3-9 in three-dimensional cell culture.