Polysaccharide-based double network injectable hydrogels, methods of making and uses thereof

Abstract

This invention discloses a polysaccharide-based dual-network injectable hydrogel, its preparation method, and its applications. The method involves first sulfated microcrystalline cellulose with sulfamic acid, then oxidizing the sulfated microcrystalline cellulose with sodium periodate, followed by crosslinking the aldehyde-modified sulfated cellulose with borax, and finally mixing it with carboxymethyl chitosan to undergo a Schiff base reaction to obtain the dual-network hydrogel. The polysaccharide-based dual-network injectable hydrogel of this invention possesses excellent biocompatibility, mechanical properties, and a good porous structure. Its three-dimensional network structure has the ability to support three-dimensional cell growth. The dual network composed of two reversible bonds enables the hydrogel to be injectable and self-healing, allowing for cell delivery via minimally invasive surgery. It has broad application prospects in in vitro cell culture, disease modeling and research, drug screening, and tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a polysaccharide-based dual-network injectable hydrogel, its preparation method, and its application. Background Technology

[0002] The liver is a crucial hub for many physiological processes and plays a vital role in various bodily functions. In recent years, the incidence of liver diseases has been rising, becoming a public health challenge and imposing a significant burden on the socioeconomic system. Cell therapy, breaking through the limitations of traditional surgery and drug treatments, is an emerging and promising regenerative approach that treats liver diseases or damage through cell replacement.

[0003] Hydrogel materials possess high hydrophilicity and can mimic the 3D network structure of the natural extracellular matrix (ECM), providing mechanical support for cells. However, rigid hydrogels have limited flexibility, resulting in large surgical wounds and increased risks. To address these drawbacks, injectable hydrogels can not only directly penetrate deep tissues, creating a favorable microenvironment through minimally invasive surgery to protect the viability and function of delivered cells, but also fill irregular wounds. Their self-healing hydrogels, with their reversible dynamic connectivity network, can autonomously restore their network structure, protecting them from shear damage during injection and maintaining the retention and integrity of implants in vivo. This increases application time and enhances the potential for biomedical applications, leading to their widespread use in drug delivery and tissue engineering within the biopharmaceutical field.

[0004] Natural hydrogels possess excellent biocompatibility, making the prepared hydrogels promising for broad applications in the biomedical field. Natural polysaccharides, rich in carboxyl groups, are more easily modified to introduce new functional groups, thus altering their bioactivity. However, hydrogels formed from traditional natural polysaccharides exhibit weak mechanical properties, limiting their use. Reference 1 (Maharjan B. Regenerated cellulose nanofiber reinforced chitosan hydrogelscaffolds for bone tissue engineering. Carbohydrate Polymers, 2021.) overcomes the limitation of insufficient mechanical strength exhibited by natural hydrogels by incorporating cellulose nanofibers into chitosan (CS) hydrogels. However, the intermolecular forces in this hydrogel are insufficient to form a stable network structure, preventing gelation through self-crosslinking and requiring freeze-drying followed by rehydration. Reference 2 (… M A. Crosslinked oxidized-nanocellulose / chitosan hydrogels as a scaffold matrix for mesenchymal stem cell growth. Cellulose, 2024.) A hydrogel was prepared by oxidizing cellulose in a one-pot process and then crosslinking it with chitosan. Studies showed that this hydrogel promoted cell adhesion, growth, and proliferation. However, the hydrogel material prepared by this method exhibited inhomogeneity and lacked injectability and self-healing properties, significantly limiting its applications. Summary of the Invention

[0005] The purpose of this invention is to provide a polysaccharide-based dual-network injectable hydrogel, its preparation method, and its applications. This method uses microcrystalline cellulose as a matrix, improves cellulose solubility and biocompatibility through sulfation and aldehyde modification, and utilizes borate ester bonds and Schiff base dual-network self-crosslinking to obtain a self-healing hydrogel with good mechanical properties, injectability, and biocompatibility.

[0006] The technical solution for achieving the objective of this invention is as follows:

[0007] A method for preparing a polysaccharide-based dual-network injectable hydrogel includes the following steps:

[0008] (1) Dissolve aminosulfonic acid in an organic solvent by stirring, add microcrystalline cellulose (MCC) powder, heat and stir until the cellulose fibers are fully esterified, add ethanol to terminate the reaction, centrifuge and wash, add NaOH solution to adjust the pH of the suspension to neutral, and then add water for dialyzing to obtain sulfated microcrystalline cellulose (SMCC) solution.

[0009] (2) Add sodium periodate aqueous solution to SMCC solution, adjust pH to 2-6, heat and stir until cellulose fibers are fully aldehyde-treated, add ethylene glycol to terminate the reaction, add NaOH solution to adjust the pH of suspension to neutral, and then add water for dialyzing to obtain aldehyde-treated sulfated cellulose (OSMCC) solution.

[0010] (3) Add borax to OSMCC solution, adjust pH=7.6, stir and crosslink. After the reaction is complete, add water for dialyzing, freeze dry to obtain B-OSMCC powder;

[0011] (4) Dissolve B-OMCC powder and carboxymethyl chitosan (CMC) powder in PBS solution to form a uniform precursor solution, wherein the concentration of B-OMCC is 5% to 10% and the concentration of CMC is 3%. Mix the two precursor solutions in a 1:1 volume ratio and crosslink them to obtain a polysaccharide-based dual-network injectable hydrogel.

[0012] Preferably, in step (1), the molar ratio of MCC to aminosulfonic acid is 1:1 to 1:10, more preferably 1:4; the organic solvent is N,N-dimethylformamide (DMF).

[0013] Preferably, in step (1), the heating temperature is 30-60°C, more preferably 50°C; and the stirring time is 4-12 hours, more preferably 5 hours.

[0014] Preferably, in step (2), the concentration of the sodium periodate aqueous solution is 0.1 mol / L to 0.5 mol / L, more preferably 0.3 mol / L.

[0015] Preferably, in step (2), pH = 5; heating temperature is 30-60℃, more preferably 60℃; stirring time is 1-5 hours, more preferably 3 hours.

[0016] Preferably, in step (3), the concentration of borax is 0.1 to 0.3 mol / L, more preferably 0.1 mol / L.

[0017] Preferably, in step (4), the B-OMCC concentration is 7.5%.

[0018] The present invention also provides a polysaccharide-based dual-network injectable hydrogel prepared by the above preparation method.

[0019] Furthermore, the present invention provides the application of the above-mentioned polysaccharide-based dual-network injectable hydrogel as a biomimetic scaffold for three-dimensional culture of liver cells.

[0020] Preferably, the liver cells are selected from mouse hepatocyte line AML12, mouse primary liver cells, etc., and more preferably mouse primary liver cells.

[0021] This invention modifies cellulose by sulfation, mimicking the chemical structure of extracellular matrix polysaccharide glycosaminoglycans (GAGs), and simultaneously introduces aldehyde groups and borate ester bonds to construct a dual-network self-crosslinking structure, resulting in a polysaccharide-based dual-network injectable hydrogel with self-crosslinking, injectability, and self-healing properties. The introduction of sulfate groups enhances the binding capacity of cellulose to growth factors, strengthening its regulation of liver cells, and also increases the material's anticoagulant properties, leading to a material beneficial for cell growth. Furthermore, the dual-network crosslinking mimics the softness of a real liver while maintaining injectability, showing potential application in exogenous cell therapy.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention uses microcrystalline cellulose and carboxymethyl chitosan as raw materials. Both are biomass macromolecules, which are good biomaterials, have no biotoxicity, have good biocompatibility, and the preparation process is simple and easy to carry out.

[0024] (2) This invention modifies cellulose by sulfation by mimicking the chemical structure of glycosaminoglycans. On the one hand, it improves the ability of cellulose to bind growth factors and enhances the regulation of liver cells. On the other hand, it increases the anticoagulant properties of the material, thus obtaining a scaffold material that is conducive to cell growth.

[0025] (3) In this invention, MCC is modified by sulfation and aldehyde, and a biomimetic polysaccharide-based three-dimensional culture medium with injectable and self-healing properties is prepared by physical cross-linking of CMC with Schiff base without adding chemical cross-linking agent, laying the foundation for exogenous cell therapy.

[0026] (4) The polysaccharide-based dual-network injectable hydrogel prepared in this invention has soft mechanical properties comparable to those of a real liver, providing a favorable environment for the in vitro culture of liver cells. Its injectable nature allows for cell delivery via minimally invasive surgery, greatly reducing the surgical risks in cell therapy. Attached Figure Description

[0027] Figure 1 The images show actual photos of the polysaccharide-based dual-network injectable hydrogels prepared in Examples 1, 2, and 3.

[0028] Figure 2 Infrared spectra of the precursor liquids prepared in Examples 1, 2, and 3;

[0029] Figure 3 The precursor solutions B-OSMCC and borax prepared in Examples 1, 2, and 3 1 H NMR spectrum;

[0030] Figure 4 The viscosity of the polysaccharide-based dual-network injectable hydrogel prepared in Example 2 is shown as a function of shear rate.

[0031] Figure 5 The shear strain scanning curve of the polysaccharide-based dual-network injectable hydrogel prepared in Example 2;

[0032] Figure 6 Compression curves of the polysaccharide-based dual-network injectable hydrogels prepared in Examples 1, 2, 3 and Comparative Example 2;

[0033] Figure 7 The images show the cytotoxicity of CCK8 cells after 1, 3, and 5 days following injection of the polysaccharide-based dual-network injectable hydrogel prepared in Example 4; where High, Middle, and Low correspond to Examples 3, 2, and 1, respectively.

[0034] Figure 8 This is a cell viability assay diagram showing the cells encapsulated in the polysaccharide-based dual-network injectable hydrogel prepared in Example 5 3 days after injection.

[0035] Figure 9 The images show SEM images of the polysaccharide-based dual-network injectable hydrogel prepared in Example 5 and SEM images of primary hepatocytes cultured on the polysaccharide-based dual-network injectable hydrogel for 3 days. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0037] Example 1

[0038] (1) Weigh 9.7g of sulfamic acid, the esterification reagent, and add 200mL of DMF. Stir until dissolved. Weigh 6g of MCC powder and add it to the sulfamic acid solution. Heat in a 50℃ water bath for 5 hours. After the reaction is complete, add a certain amount of ethanol to stop the reaction. After centrifuging and washing three times, add NaOH solution to adjust the pH of the suspension to neutral. Dialyze in deionized water for three days to obtain SMCC solution.

[0039] (2) Weigh 12.83 g of sodium periodate and dissolve it in 100 mL of deionized water. Place the solution in a 60°C water bath and stir until dissolved. Then add 100 mL of SMCC solution and adjust the pH to 5. Oxidize for 3 h. After the reaction is complete, add ethylene glycol to terminate the reaction. Add NaOH solution to adjust the pH of the suspension to neutral. Dialyze the solution in deionized water for three days to obtain OSMCC solution. The sodium periodate concentration is 0.3 M, the reaction pH is 5, and the reaction time is 3 h.

[0040] (3) Borax was added to the OSMCC solution to adjust the pH to 7.6. The mixture was stirred for 30 min, and after the reaction was completed, it was dialyzed in deionized water for three days. The solution was then dried in a freeze dryer to obtain a white cotton-like substance of B-OSMCC. The concentration of borax was 0.1 M.

[0041] (4) Dissolve B-OSMCC in PBS solution to prepare a 5 w / v B-OSMCC solution and adjust the pH to 7. Dissolve CMC in PBS solution to prepare a 3 w / v CMC solution and adjust the pH to 7.

[0042] (5) Take 200 μL of B-OSMCC solution and CMC solution into a 1.5 mL PE tube, mix them quickly and evenly to prepare an injectable polysaccharide-based dual-network injectable hydrogel.

[0043] Example 2

[0044] This embodiment is largely the same as Embodiment 1, except that in step (4), the concentration of B-OSMCC is 7.5%.

[0045] Example 3

[0046] This embodiment is largely the same as Embodiment 1, except that the B-OSMCC concentration in step (4) is 10%.

[0047] Figure 1 The images show physical specimens of the polysaccharide-based dual-network injectable hydrogels prepared in Examples 1, 2, and 3. The hydrogels exhibit a color change from light yellow to dark yellow as the proportion of SMCC increases, due to the formation of C@N chromophores. This is common in hydrogels formed via Schiff base reactions. With increasing O-SMCC precursor solution concentration, the degree of cross-linking increases, resulting in more chromophores in the hydrogel and a deeper color.

[0048] Figure 2 The infrared spectra of the precursor solutions prepared in Examples 1, 2, and 3 are shown. After oxidation with sodium periodate, SMCC showed a peak density of 1725 cm⁻¹. -1 The aldehyde absorption peak. The stretching vibration peak of OH from 3300 cm⁻¹. -1 Moved to 2978cm -1 This is due to the formation of boronic acid ester bonds;

[0049] Figure 3 B-OSMCC and borax in the precursor solutions prepared in Examples 1, 2, and 3 1 The 1H NMR spectrum showed that B-OSMCC exhibited a characteristic peak of boric acid at 19.49 ppm.

[0050] Figure 4 The figure shows the viscosity of the polysaccharide-based dual-network injectable hydrogel prepared in Example 2 as a function of shear rate. As the shear rate increases, the composite viscosity of the hydrogel decreases rapidly, indicating that the prepared material is injectable because it contains a large number of non-covalent and dynamic covalent bonds. These bonds are broken by shear force, making the hydrogel properties closer to those of a fluid, as shear force causes the hydrogel to thin, facilitating extrusion.

[0051] Figure 5The storage modulus (G') and loss modulus (G") of the polysaccharide-based dual-network injectable hydrogel prepared in Example 2 were verified by rheological recovery tests at 37°C, based on the changes in G' and G" at higher and lower strains, to evaluate the self-healing ability of the hydrogel. Under high dynamic strain (100%), the hydrogel's G' decreased and fell below the loss modulus (G") due to the collapse of the hydrogel network. Under low strain (0.1%), the hydrogel's G' gradually recovered to its initial value, indicating that the network structure of the hydrogel gradually recovered. The fact that G' essentially recovered to its initial value during this repeated process indicates that the mechanical properties of the hydrogel remained essentially unchanged before and after self-healing. This is due to the presence of a large number of non-covalent and dynamic covalent bonds in the hydrogel, which exhibits rapid and complete self-repair ability.

[0052] Comparative Example 1

[0053] This comparative example is largely the same as Example 2, except that step (2) aldehyde formation of sulfated cellulose and step (3) crosslinking of borate ester bonds are not performed. Further mixing of 7.5% SMCC precursor solution and 3% CMC precursor solution failed to produce a hydrogel material. Therefore, aldehyde formation of sulfated cellulose and crosslinking of borate ester bonds are key steps in forming a stable hydrogel network.

[0054] Comparative Example 2

[0055] This comparative example is roughly the same as Example 2, except that the crosslinking of borate ester bonds in step (3) is not performed. The hydrogel material is prepared from 7.5% OSMCC precursor liquid and 3% CMC precursor liquid.

[0056] Figure 6 The compression curves of the dual-network hydrogels prepared in Examples 1, 2, 3 and Comparative Example 1 are shown. As a connection between cells and tissues, the scaffold material needs to have certain compression properties to withstand external pressure and maintain the original shape and integrity of the tissue, and also needs a certain mechanical flexibility to simulate the biophysical properties of the extracellular matrix in vivo in order to perform normal biological functions.

[0057] As the concentration of B-OSMCC precursor fluid in the hydrogel increases, the pressure required for the same deformation gradually increases, indicating that the strength of the composite material gradually increases and it has better support capacity. The hydrogel prepared by OSMCC precursor fluid in Comparative Example 2 has excessively strong mechanical properties and is unsuitable for cell growth. The hydrogel prepared by B-OSMCC precursor fluid, through a double network cross-linking of Schiff base and borate ester bonds, makes the rigid hydrogel more flexible and closer to the strength of a real liver, thus better mimicking the in vivo environment and promoting cell growth.

[0058] Example 4

[0059] (1) The two hydrogel precursor solutions of Example 2 were filtered through a 0.22 μm filter head for sterilization;

[0060] (2) Mix 500 μL of B-OSMCC precursor solution with 500 μL of CMC precursor solution to prepare a hydrogel;

[0061] (3) Prepare the extract at a concentration of 0.1 g / mL according to the national standard GB / T 16886.12;

[0062] (4) The absorbance intensity, i.e. the cell proliferation, was determined using the CCK-8 (CCK-8, Dojindo Laboratories, Rockville, MD, USA) kit.

[0063] Figure 7 The graphs show the CCK-8 cytotoxicity assays of AML12 cells cultured in the extract of the material prepared in Example 4 for 1, 3, and 5 days, respectively. High, Middle, and Low correspond to Examples 2, 4, and 1, respectively. The graphs show good cell growth viability, and no significant difference was observed compared to the control. This indicates that the hydrogel has good biocompatibility.

[0064] Example 5

[0065] (1) The two hydrogel precursor solutions of Examples 1, 2 and 3 were filtered through a 0.22 μm filter head for sterilization;

[0066] (2) Take 100 μL of B-OSMCC precursor solution and mix it with an appropriate number of primary mouse hepatocytes;

[0067] (3) A hydrogel was prepared by mixing B-OSMCC precursor solution containing cells with 100 μL of CMC precursor solution.

[0068] (4) Inject the hydrogel into a cell culture plate at a volume of 50 μL and culture it in complete culture medium for 3 days.

[0069] (5) Cytotoxicity was assessed using a live / dead cell double staining kit (Calcein-AM / PI), and the samples were imaged and observed using a laser confocal microscope.

[0070] Figure 8 The image shows the cell viability experiment 3 days after injection of mouse primary hepatocytes encapsulated in hydrogel in Example 5. The results indicate that the hydrogel prepared in Example 2 is more suitable for the growth of primary hepatocytes, the material has no toxic effect on cells, and has good cell compatibility.

[0071] Figure 9The images shown are SEM images of the hydrogel material in Example 5 and SEM images of primary hepatocytes cultured on the material for 3 days. The images show that the cells have normal morphology, further demonstrating that the hydrogel provides a good cellular microenvironment for hepatocytes.

Claims

1. A method for preparing a polysaccharide-based dual-network injectable hydrogel, characterized in that, Includes the following steps: (1) Dissolve aminosulfonic acid in an organic solvent by stirring, add microcrystalline cellulose powder, heat and stir until the cellulose fibers are fully esterified, add ethanol to terminate the reaction, centrifuge and wash, add NaOH solution to adjust the pH of the suspension to neutral, and then add water for dialyzing to obtain sulfated microcrystalline cellulose solution; (2) Add sodium periodate aqueous solution to sulfated microcrystalline cellulose solution, adjust pH=2~6 and heat and stir until cellulose fibers are fully aldehyde-treated. Add ethylene glycol to terminate the reaction, add NaOH solution to adjust the pH of the suspension to neutral, and then add water for dialyzing to obtain aldehyde-treated sulfated cellulose solution. (3) Add borax to the aldehyde-modified sulfated cellulose solution, adjust the pH to 7.6, stir and crosslink, add water for dialyzing after the reaction is complete, freeze dry to obtain B-OSMCC powder; (4) Dissolve B-OSMCC powder and carboxymethyl chitosan powder in PBS solution to form a uniform precursor solution, wherein the concentration of B-OSMCC is 5%~10% and the concentration of CMC is 3%. Mix the two precursor solutions in a 1:1 volume ratio and crosslink them to obtain a polysaccharide-based dual-network injectable hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of microcrystalline cellulose to aminosulfonic acid is 1:1 to 1:10; the organic solvent is N,N-dimethylformamide; the heating temperature is 30 to 60 °C; and the stirring time is 4 to 12 hours.

3. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of microcrystalline cellulose to aminosulfonic acid is 1:4; the heating temperature is 50 ℃; and the stirring time is 5 hours.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of sodium periodate aqueous solution is 0.1 mol / L to 0.5 mol / L, the heating temperature is 30 to 60 ℃, and the stirring time is 1 to 5 hours.

5. The preparation method according to claim 1, characterized in that, In step (2), the concentration of sodium periodate aqueous solution is 0.3 mol / L, pH=5; the heating temperature is 60 ℃; and the stirring time is 3 hours.

6. The preparation method according to claim 1, characterized in that, In step (3), the concentration of borax is 0.1~0.3 mol / L.

7. The preparation method according to claim 1, characterized in that, In step (4), the B-OMCC concentration is 7.5%.

8. The polysaccharide-based dual-network injectable hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the polysaccharide-based dual-network injectable hydrogel according to claim 8 as a biomimetic scaffold for three-dimensional culture of liver cells.

10. The application according to claim 9, characterized in that, The liver cells were selected from the mouse hepatocyte line AML12 and mouse primary liver cells.

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