Modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel, preparation method and application thereof

Abstract

The application discloses a modified cellulose nanofiber reinforced double-crosslinked immunomodulatory hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biomaterials and biomedical engineering. The main body of the composite hydrogel is prepared by grafting gelatin on oxidized sodium alginate and dopamine, the aldehyde group on the oxidized sodium alginate and the amino group on the gelatin can form a Schiff base bond, borax and the oxidized sodium alginate and the vicinal diol structure on the modified cellulose nanofiber can form a borate ester bond, and the double-crosslinked structure enhances the mechanical properties of the hydrogel. The borate ester bond and the Schiff base bond are both dynamic chemical bonds and can respond to pH changes, endowing the hydrogel with self-healing properties and the ability of pH-responsive drug release, so that the hydrogel can quickly release liposomes with anti-inflammatory functions in the early inflammatory environment of the cartilage injury which is acidic, and restore the original state after the fracture caused by joint movement, and the double-crosslinked structure guarantees the stability of the whole hydrogel.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and biomedical engineering technology, and particularly relates to a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, its preparation method and application. Background Technology

[0002] Cartilage defects caused by various pathological conditions such as trauma, genetic factors, and bone diseases result in progressive joint pain, disability, and even death for millions of people worldwide, imposing a huge clinical and socioeconomic burden. Due to the complex structure of articular cartilage and its lack of blood vessels and lymphatic tissue, it cannot receive effective nutritional supply, thus limiting its self-healing ability. Current clinical treatments still face risks such as poor quality regenerated cartilage, immunogenicity, and infection during secondary surgery. Therefore, the clinical treatment of cartilage defects remains a significant challenge. To address this, tissue-engineered scaffolds combining cells or growth regulators have made significant progress in treating cartilage injuries. Among these, hydrogels, as a primary scaffold type, offer advantages such as good biocompatibility, excellent degradation performance, and the ability to deliver drugs in a sustained-release manner.

[0003] It is noteworthy that current methods for cartilage repair using hydrogels focus primarily on the cartilage regeneration phase, neglecting the inflammatory phase preceding regeneration. In the early stages of cartilage injury, the recruitment of neutrophils and activation of macrophages lead to the release of large amounts of inflammatory mediators such as proteases and reactive oxygen species, playing a crucial role in the formation of the inflammatory microenvironment. Without intervention, the continuous recruitment and activation of immune cells results in excessive inflammation associated with chronic tissue damage, ultimately leading to osteoarthritis. Hydrogels lacking immunomodulatory functions cannot precisely regulate the early inflammatory microenvironment of cartilage, thus limiting its regenerative function.

[0004] Hydrogels, with their unique three-dimensional structure, highly tunable physicochemical properties, and excellent biocompatibility, are the optimal choice for loading active substances and drugs. By altering the material composition and cross-linking mechanism of hydrogels, unique physical properties can be imparted, enabling them to respond to abnormal biochemical conditions at the lesion site. This, in turn, influences the hydrogel's network structure, achieving controlled drug release and reducing drug toxicity. Single-crosslinked hydrogels exhibit poor mechanical properties, and while double-crosslinking can improve compressive strength, it still falls short of meeting the mechanical requirements for articular cartilage repair. Furthermore, the short residence time of hydrogels within the joint cavity and the fragmentation caused by joint movement remain technical challenges for hydrogels used in cartilage repair. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, its preparation method, and its applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, the raw materials of which include sodium oxidized alginate (OSA), dopamine-grafted gelatin (Gel-DA), modified cellulose nanofibers, borax, drug-loaded targeted liposomes and water in a ratio of (16-64) g: (32-128) g: (1-2) g: (8-32) mol: (50-200) μmol: 1 L;

[0008] The modified cellulose nanofibers are cellulose nanofibers modified with mono-(6-amino-6-deoxy)-β-cyclodextrin (NH2-β-cyclodextrin);

[0009] The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel has a double-crosslinked structure and pH responsiveness.

[0010] Technical Principle: This invention uses sodium alginate oxidized and dopamine-grafted gelatin to prepare the main body of a composite hydrogel. The aldehyde groups on sodium alginate oxidized and the amino groups on gelatin can form Schiff base bonds, while the vicinal diol structures on borax, sodium alginate oxidized, and modified cellulose nanofibers can form borate ester bonds. This double cross-linked structure enhances the mechanical properties of the hydrogel. Both borate ester bonds and Schiff base bonds are dynamic chemical bonds and can respond to pH changes, endowing the hydrogel with self-healing properties and the ability to release drugs in a pH-responsive manner. This allows the hydrogel to rapidly release anti-inflammatory liposomes in the acidic inflammatory environment of early cartilage injury and to recover its original state after fracture caused by joint movement. The double cross-linked structure ensures the overall stability of the hydrogel. In addition, the modification of the aldehyde groups and the effect of hydrogen bonding endow the hydrogel with certain adhesive properties, allowing the hydrogel to remain at the joint cartilage defect site for a long time.

[0011] Furthermore, the preparation method of the modified cellulose nanofibers includes the following steps:

[0012] Dissolve mono-(6-amino-6-deoxy)-β-cyclodextrin in water, then add a hydrophobic drug, stir in the dark to obtain solution A;

[0013] Carboxylated cellulose nanofibers (CCNF) were dispersed in water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to carry out a carboxyl activation reaction to obtain solution B;

[0014] Solution B is added to solution A to carry out an amidation reaction, thereby obtaining the modified cellulose nanofibers.

[0015] Further, the mass ratio of the mono-(6-amino-6-deoxy)-β-cyclodextrin to the hydrophobic drug is (5-10):1; and / or,

[0016] The stirring temperature in the dark is 25-37℃, and the stirring time in the dark is 24-48h.

[0017] Further, the mass ratio of the carboxylated cellulose nanofibers, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(1-3):(0.8-3); and / or,

[0018] The carboxyl activation reaction is carried out at a temperature of 25-37°C for 0.5-2 hours; and / or,

[0019] The mass ratio of the carboxylated cellulose nanofibers to mono-(6-amino-6-deoxy)-β-cyclodextrin is 100:(1-2); and / or,

[0020] The amidation reaction is carried out at a temperature of 25-37°C for 24-48 hours.

[0021] Furthermore, the method for preparing the drug-loaded targeted liposomes includes the following steps:

[0022] S1. Egg yolk lecithin, cholesterol, anti-inflammatory drugs, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2000-folic acid (DSPE-PEG2000-FA) are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is then subjected to rotary evaporation to obtain a liposome film.

[0023] S2. The liposome membrane is subjected to hydration and ultrasonic treatment in sequence, and then filtered to obtain the drug-loaded targeted liposome.

[0024] Further, in step S1, the mass ratio of the egg yolk lecithin, cholesterol, anti-inflammatory drug, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2000-folic acid is (60-100):(12-30):(3-5):(1-2); and / or,

[0025] The anti-inflammatory drug is selected from one or more of dimethyl fumarate, itaconic acid, and celecoxib.

[0026] Further, in step S1, the temperature of the rotary evaporation is 40-50℃, the rotation speed is 100-200 rpm, and the evaporation time is 0.5-1h.

[0027] Further, in step S2, the hydration treatment temperature is 40-50℃, and the hydration treatment time is 0.5-1h; and / or,

[0028] The ultrasonic treatment power is 100-150W, and the ultrasonic treatment time is 1-3 minutes; and / or,

[0029] The filter membrane has a pore size of 0.1-0.45 μm.

[0030] This invention provides a method for preparing a modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel as described above, comprising the following steps:

[0031] Sodium oxidized alginate, dopamine-grafted gelatin, borax and modified cellulose nanofibers were mixed in water to obtain a hydrogel precursor solution.

[0032] The drug-loaded targeted liposomes were added to the hydrogel precursor solution and mixed to obtain the modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel.

[0033] This invention also provides the application of the modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel as described above in the preparation of cartilage repair materials.

[0034] Compared with the prior art, the present invention has the following advantages and technical effects:

[0035] This invention uses sodium alginate oxide, dopamine-grafted gelatin, borax, and cellulose nanofibers modified with mono-(6-amino-6-deoxy)-β-cyclodextrin as raw materials to prepare hydrogels, and uses egg yolk lecithin, cholesterol, anti-inflammatory drugs, and DSPE-PEG2000-FA as raw materials to prepare drug-loaded targeted liposomes. By combining the hydrogels and drug-loaded targeted liposomes, on-demand release of liposomes is achieved.

[0036] In this invention, the double cross-linked structure and modified cellulose nanofibers can enhance the mechanical properties of the hydrogel, the introduction of aldehyde groups can enhance the adhesion of the hydrogel, and the pH responsiveness of borate ester bonds and Schiff base bonds endows the hydrogel with the properties of controlled-release liposomes.

[0037] The modified cellulose nanofibers in this invention can encapsulate different hydrophobic cartilage regeneration molecules, and the liposomes can load different anti-inflammatory drugs. The combination of the two achieves the regulation of early cartilage inflammation and the regeneration of cartilage tissue in the later stage.

[0038] The raw materials used in this invention, sodium alginate, gelatin, and cellulose nanofibers, all possess excellent biocompatibility. The dual dynamic cross-linking of Schiff base bonds and borate ester bonds endows the hydrogel with self-healing properties and pH responsiveness. Aldehyde groups, hydroxyl groups, and carboxyl groups contribute to the hydrogel's adhesiveness, enabling its long-term retention within the joint cavity. DSPE-PEG2000-FA-modified liposomes exhibit suitable particle size, potential, and good stability, not only improving the solubility of anti-inflammatory drugs but also enabling precise drug delivery to macrophages, thereby regulating macrophage polarization and the release of reactive oxygen species. NH2-β-cyclodextrin-modified cellulose nanofibers enhance the solubility and long-term release of cartilage regeneration drugs and significantly improve the mechanical properties of the hydrogel. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0040] Figure 1 The images show the microstructure morphology of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0041] Figure 2 The stress-strain curves of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2 are shown.

[0042] Figure 3 The compressive modulus of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2;

[0043] Figure 4 The image shows the self-healing performance of the hydrogel prepared in Example 1.

[0044] Figure 5 The image shows the adhesion performance of the hydrogel prepared in Example 1; where AC represents the adhesion effect of suspending different weights, D represents the adhesion effect of the hydrogel on pig skin, EG represents the adhesion effect under different bending directions, and H represents the retention effect of the hydrogel on pig articular cartilage defects.

[0045] Figure 6 The image shows the pH response of the hydrogel prepared in Example 1; where A represents the release rate of liposomes at pH=7.4 and B represents the release rate of liposomes at pH=5.5.

[0046] Figure 7 The images shown are particle size analysis, potential analysis, and transmission electron microscopy (TEM) images of the drug-loaded targeted liposomes prepared in step 1) of Example 1; where A represents particle size analysis, B represents potential analysis, and C represents TEM image. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] This invention provides a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, characterized in that the raw materials include sodium alginate oxidized, dopamine-grafted gelatin, modified cellulose nanofibers, borax, drug-loaded targeted liposomes, and water in a ratio of (16-64) g: (32-128) g: (1-2) g: (8-32) mol: (50-200) μmol: 1 L.

[0050] The modified cellulose nanofibers are mono-(6-amino-6-deoxy)-β-cyclodextrin modified cellulose nanofibers;

[0051] The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel has a double-crosslinked structure and pH responsiveness.

[0052] In a preferred embodiment, the method for preparing the modified cellulose nanofibers includes the following steps:

[0053] Dissolve mono-(6-amino-6-deoxy)-β-cyclodextrin in water, then add a hydrophobic drug, stir in the dark to obtain solution A;

[0054] Carboxylated cellulose nanofibers were dispersed in water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to carry out a carboxyl activation reaction to obtain solution B;

[0055] Solution B is added to solution A for amidation to obtain the modified cellulose nanofibers. This invention utilizes an amidation reaction to graft NH₂-β-cyclodextrin onto the side chains of cellulose nanofibers. The hydrophobic cavity of β-cyclodextrin allows for the encapsulation of different cartilage regeneration-promoting drugs through host-guest interactions, enhancing the solubility of hydrophobic drugs and achieving long-term release that matches the drug's degradation rate with the hydrogel. Furthermore, the modified cellulose nanofibers possess extremely high specific surface area and excellent mechanical properties, enabling them to form a tight bond with the hydrogel network through physical interactions (such as hydrogen bonding) or chemical crosslinking (such as borate ester bonds). The introduction of modified cellulose nanofibers significantly improves the mechanical properties of the hydrogel. On one hand, the high strength and high modulus of the nanofibers effectively transfer and disperse external stress, preventing the hydrogel from breaking or deteriorating under stress; on the other hand, the tight bond between the modified cellulose nanofibers and the hydrogel network increases the hydrogel's toughness and ductility, allowing it to withstand deformation over a wider range without damage.

[0056] In a preferred embodiment, the mass ratio of the mono-(6-amino-6-deoxy)-β-cyclodextrin to the hydrophobic drug is (5-10):1.

[0057] In a preferred embodiment, the hydrophobic drug is a hydrophobic drug that promotes cartilage regeneration; the hydrophobic drug that promotes cartilage regeneration is selected from one or more of Kartogenin (KGN), TD-198946 and BNTA.

[0058] In a preferred embodiment, the temperature for stirring in the dark is 25-37°C, and the stirring time in the dark is 24-48 hours.

[0059] In a preferred embodiment, the mass ratio of the carboxylated cellulose nanofibers, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:(1-3):(0.8-3).

[0060] In a preferred embodiment, the carboxyl content of the carboxylated cellulose nanofibers is 1.2-3 mmol / g.

[0061] In a preferred embodiment, the temperature of the carboxyl activation reaction is 25-37°C, and the time of the carboxyl activation reaction is 0.5-2 hours.

[0062] In a preferred embodiment, the mass ratio of the carboxylated cellulose nanofibers to mono-(6-amino-6-deoxy)-β-cyclodextrin is 100:(1-2).

[0063] In a preferred embodiment, the amidation reaction is carried out at a temperature of 25-37°C for 24-48 hours.

[0064] In a preferred embodiment, the method for preparing the drug-loaded targeted liposomes includes the following steps:

[0065] S1. Egg yolk lecithin, cholesterol, anti-inflammatory drugs, and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000-folic acid are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is then subjected to rotary evaporation to obtain a liposome film;

[0066] S2. The liposome membrane is sequentially subjected to hydration and ultrasonic treatment, followed by filtration to obtain the drug-loaded targeted liposomes. The liposomes prepared in this invention can load different anti-inflammatory drugs, improve the solubility of hydrophobic drugs, and the modification with DSPE-PEG2000-FA enables the liposomes to target the highly expressed folic acid receptors on the surface of M1 macrophages, achieving precise delivery of anti-inflammatory drugs, improving drug utilization efficiency, and reducing drug side effects.

[0067] In a preferred embodiment, in step S1, the mass ratio of the egg yolk lecithin, cholesterol, anti-inflammatory drug and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2000-folic acid is (60-100):(12-30):(3-5):(1-2).

[0068] In a preferred embodiment, in step S1, based on the amount of egg yolk lecithin used being 60-100 mg, the amount of the organic solvent used is 20-30 mL.

[0069] In a preferred embodiment, the anti-inflammatory drug is selected from one or more of dimethyl fumarate, itaconic acid, and celecoxib.

[0070] In a preferred embodiment, the organic solvent is selected from one or more of anhydrous ethanol, chloroform, and methanol.

[0071] In a preferred embodiment, in step S1, the temperature of the rotary evaporation is 40-50°C, the rotation speed is 100-200 rpm, and the evaporation time is 0.5-1 h.

[0072] In a preferred embodiment, in step S2, the temperature of the hydration treatment is 40-50°C, and the hydration treatment time is 0.5-1h.

[0073] In a preferred embodiment, in step S2, the power of the ultrasonic treatment is 100-150W, and the ultrasonic treatment time is 1-3 minutes.

[0074] In a preferred embodiment, in step S2, the pore size of the filter membrane is 0.1-0.45 μm.

[0075] In a preferred embodiment, the raw materials for preparing the oxidized sodium alginate include sodium alginate and sodium periodate in a mass ratio of 5:(1-4).

[0076] In a further preferred embodiment, the method for preparing oxidized sodium alginate includes the following steps: dispersing sodium alginate in anhydrous ethanol to obtain a sodium alginate dispersion; dissolving sodium periodate in distilled water to obtain a sodium periodate solution; adding the sodium periodate solution dropwise to the sodium alginate dispersion, stirring and reacting at room temperature in the dark for 1-8 hours, then adding ethylene glycol to terminate the oxidation reaction, pouring the reaction product into anhydrous ethanol and filtering to obtain oxidized sodium alginate; dissolving the oxidized sodium alginate in distilled water, dialyzing to remove residual sodium periodate and ethylene glycol from the oxidized sodium alginate, and finally freeze-drying to obtain purified OSA.

[0077] In a preferred embodiment, the amount of sodium alginate used is 5g, and the amount of ethylene glycol used is 2.5mL; the filtration device is a Buchner funnel; the dialysis bag for dialysis is 3500Da, and the dialysis time is 5 days; the freeze-drying temperature is -65℃, and the time is 3 days.

[0078] In a preferred embodiment, the preparation method of the dopamine-grafted gelatin includes the following steps: using gelatin and dopamine hydrochloride as raw materials, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide as catalysts, the dopamine-grafted gelatin is prepared by amidation reaction.

[0079] In a preferred embodiment, the mass ratio of gelatin to dopamine hydrochloride is 2:(1-2); the mass ratio of gelatin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 2:(0.25-0.75):(0.2-0.4); the amidation reaction is carried out at a temperature of 37-40°C for 24-48 hours.

[0080] In a further preferred embodiment, the preparation method of the dopamine-grafted gelatin includes the following steps: dissolving gelatin in distilled water, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, adjusting the pH of the solution to acidic with hydrochloric acid to obtain a gelatin solution; dissolving dopamine hydrochloride in distilled water and adding it dropwise to the gelatin solution to carry out an amidation reaction, followed by dialysis and freeze-drying to obtain the dopamine-grafted gelatin.

[0081] In a preferred embodiment, the pH of the solution is adjusted to 5.5 with hydrochloric acid; the dialysis bag used for dialysis has a capacity of 3500 Da, and the dialysis time is 5 days; the freeze-drying temperature is -65°C, and the time is 3 days.

[0082] Embodiments of the present invention provide a method for preparing a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel as described above, comprising the following steps:

[0083] Sodium oxidized alginate, dopamine-grafted gelatin, borax and modified cellulose nanofibers were mixed in water to obtain a hydrogel precursor solution.

[0084] The drug-loaded targeted liposomes were added to the hydrogel precursor solution and mixed to obtain the modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel.

[0085] Embodiments of the present invention also provide the application of the modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel as described above in the preparation of cartilage repair materials.

[0086] In this embodiment of the invention, room temperature refers to "25±2℃".

[0087] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0088] The abbreviations used in the following embodiments specifically refer to:

[0089] OSA: Oxygenated sodium alginate;

[0090] Gel-DA: Dopamine hydrochloride-grafted gelatin;

[0091] CCNF: Carboxylated cellulose nanofibers;

[0092] CK: NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating KGN;

[0093] CT: NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating TD198946.

[0094] Example 1

[0095] A method for preparing a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, comprising the following steps:

[0096] 1) Preparation of targeted liposomes loaded with dimethyl fumarate

[0097] 60 mg of lecithin, 30 mg of cholesterol, 5 mg of dimethyl fumarate, and 2 mg of DSPE-PEG2000-FA were weighed and dissolved in a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform. After thorough mixing, the mixture was transferred to a round-bottom flask and distilled under reduced pressure at 40 °C and 200 rpm for 1 h to form a liposome film. 5 mL of distilled water was added to the liposome film, and the film was dispersed at 40 °C for 45 min. The film was then sonicated at 150 W for 2 min, and subsequently filtered through 0.45 μm, 0.22 μm, and 0.1 μm filters to obtain targeted liposomes loaded with dimethyl fumarate.

[0098] 2) Preparation of OSA

[0099] 5g of sodium alginate was weighed and dispersed in anhydrous ethanol to obtain a sodium alginate dispersion. 3.25g of sodium periodate was dissolved in distilled water and added dropwise to the sodium alginate dispersion. The mixture was stirred at room temperature in the dark for 6 hours. Then, 2.5mL of ethylene glycol was added to the system to terminate the oxidation reaction. The reaction product was poured into anhydrous ethanol and filtered using a Buchner funnel. The solid obtained after filtration was oxidized sodium alginate. The oxidized sodium alginate was dissolved in distilled water and dialyzed against a 3500Da dialysis bag for 5 days. The purified OSA was obtained by freeze-drying at -65℃ for 3 days.

[0100] 3) Preparation of Gel-DA

[0101] 2g of gelatin was completely dissolved in 100mL of distilled water at 60℃. Then, 0.5g of EDC and 0.3g of NHS were added. The pH of the solution was adjusted to 5.5 with 1M hydrochloric acid, and stirring was continued for 30min to obtain a gelatin solution. 1g of dopamine hydrochloride was dissolved in 4mL of distilled water and added dropwise to the above gelatin solution. The mixture was stirred at 37℃ for 48h, dialyzed for 3 days using a 3500Da dialysis bag, and then freeze-dried at -65℃ for 3 days to obtain Gel-DA.

[0102] 4) Preparation of NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating KGN

[0103] 10 mg of NH2-β-cyclodextrin was dissolved in 10 mL of distilled water, and 1 mg of KGN was added. The mixture was stirred at room temperature in the dark for 24 h to obtain a KGN-encapsulated NH2-β-cyclodextrin solution. 1 g of CCNF (carboxyl content 1.2 mmol / g) was dispersed in 50 mL of distilled water, and 1.24 g of EDC and 0.92 g of NHS were added. The pH of the solution was adjusted to 5.5 with 1 M hydrochloric acid, and stirring was continued for 30 min to obtain a CCNF dispersion. The KGN-encapsulated NH2-β-cyclodextrin solution was added dropwise to the CCNF dispersion, and stirring was continued at room temperature for 24 h. The solution was dialyzed for 3 days using a 3500 Da dialysis bag, and then freeze-dried at -65 °C for 3 days to obtain KGN-encapsulated NH2-β-cyclodextrin-modified cellulose nanofibers.

[0104] 5) Preparation of OSA / Gel-DA / CK@Lip hydrogel

[0105] Borax was added to the purified OSA obtained in step 2) to obtain a mixture of OSA and borax; Gel-DA obtained in step 3) was dissolved in distilled water to prepare a Gel-DA solution; the NH2-β-cyclodextrin-modified cellulose nanofibers encapsulated with KGN obtained in step 4) were dispersed in water to obtain a modified cellulose nanofiber dispersion. The mixture of OSA and borax, the Gel-DA solution and the modified cellulose nanofiber dispersion were mixed and then added to the targeted liposomes loaded with dimethyl fumarate prepared in step 1). After thorough mixing, a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel was obtained, denoted as OSA / Gel-DA / CK@Lip. The ratio of OSA, borax, Gel-DA, NH2-β-cyclodextrin-modified cellulose nanofibers encapsulated with KGN, the targeted liposomes loaded with dimethyl fumarate and water was 0.032 g : 0.016 mol : 0.096 g : 0.002 g : 0.1 μmol : 1 mL.

[0106] Example 2

[0107] A method for preparing a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, comprising the following steps:

[0108] 1) Preparation of targeted liposomes loaded with itaconic acid

[0109] Weigh 60 mg of lecithin, 30 mg of cholesterol, 5 mg of itaconic acid, and 2 mg of DSPE-PEG2000-FA and dissolve them in a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform. After thorough mixing, transfer the mixture to a round-bottom flask and distill under reduced pressure at 40 °C and 200 rpm for 1 h to form a liposome film. Add 5 mL of distilled water to the liposome film and disperse it at 40 °C for 45 min. Then sonicate it at 150 W for 2 min and filter it sequentially through 0.45 μm, 0.22 μm, and 0.1 μm filter membranes to obtain itaconic acid-loaded targeted liposomes.

[0110] Steps 2)-5) are the same as in Example 1.

[0111] Example 3

[0112] A method for preparing a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel, comprising the following steps:

[0113] Steps 1)-3) are the same as in Example 1;

[0114] 4) Preparation of NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating TD-198946

[0115] 10 mg of NH2-β-cyclodextrin was dissolved in 10 mL of distilled water, and 1 mg of TD-198946 was added. The mixture was stirred at room temperature in the dark for 24 h to obtain an NH2-β-cyclodextrin solution encapsulated with TD-198946. 1 g of CCNF (carboxyl content 1.2 mmol / g) was dispersed in 50 mL of distilled water, and 1.24 g of EDC and 0.92 g of NHS were added. The pH of the solution was adjusted to 5.5 with 1 M hydrochloric acid, and stirring was continued for 30 min to obtain a CCNF dispersion. The NH2-β-cyclodextrin solution encapsulated with TD-198946 was added dropwise to the CCNF dispersion, and stirring was continued for 24 h. The solution was dialyzed for 3 days using a 3500 Da dialysis bag, and then freeze-dried at -65 °C for 3 days to obtain NH2-β-cyclodextrin-modified cellulose nanofibers encapsulated with TD-198946.

[0116] Step 5) is the same as in Example 1.

[0117] Comparative Example 1

[0118] A method for preparing a modified cellulose nanofiber-reinforced double crosslinked hydrogel, comprising the following steps:

[0119] Borax was added to purified OSA to obtain a mixture of OSA and borax; Gel-DA was dissolved in distilled water to prepare a Gel-DA solution; NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating KGN were dispersed in water to obtain a modified cellulose nanofiber dispersion. The above mixture of OSA and borax, Gel-DA solution and modified cellulose nanofiber dispersion were mixed and thoroughly stirred to obtain a modified cellulose nanofiber-reinforced bi-crosslinked hydrogel; wherein, the ratio of OSA, borax, Gel-DA, NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating KGN and water was 0.032g∶0.016mol∶0.096g∶0.002g∶1mL; the preparation method of OSA, Gel-DA and NH2-β-cyclodextrin-modified cellulose nanofibers encapsulating KGN was the same as in Example 1, and the obtained modified cellulose nanofiber-reinforced bi-crosslinked hydrogel was denoted as OSA / Gel-DA / CK.

[0120] Comparative Example 2

[0121] A method for preparing a double crosslinked hydrogel, comprising the following steps:

[0122] Borax was added to purified OSA to obtain a mixture of OSA and borax; Gel-DA was dissolved in distilled water to prepare a Gel-DA solution; the above mixture of OSA and borax was mixed with the Gel-DA solution and stirred thoroughly to obtain a bi-crosslinked hydrogel; wherein, the ratio of OSA, borax, Gel-DA and water was 0.032g∶0.016mol∶0.096g∶1mL; the preparation methods of OSA and Gel-DA were the same as in Example 1, and the obtained bi-crosslinked hydrogel was denoted as OSA / Gel-DA.

[0123] The hydrogels prepared in Examples 1-3 have similar properties. The following description and comparison will only use the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2 as examples.

[0124] 1. Microstructure of hydrogels

[0125] The microstructure of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2 was observed using scanning electron microscopy. The results are shown in [Figure number missing]. Figure 1 .

[0126] Figure 1 The images show the microstructure morphology of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2. Figure 1 As can be seen, the hydrogels prepared in Example 1, Comparative Example 1 and Comparative Example 2 all have a network porous structure with through-pores, indicating that the addition of modified cellulose nanofibers and drug-loaded targeted liposomes did not affect the three-dimensional structure of the hydrogels.

[0127] 2. Mechanical property testing of hydrogels

[0128] The hydrogels from Example 1, Comparative Example 1, and Comparative Example 2 were fabricated into cylinders with a diameter of 14 mm and a height of 7 mm. The hydrogels were compressed using a universal mechanical testing instrument at a compression rate of 2 mm / min, and the mechanical data were recorded. The data processing results are shown below. Figure 2-3 .

[0129] Figure 2 Stress-strain curves of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2 are shown. Figure 2 It can be seen that during 80% of the compressive strain process, the compressive stress of Example 1 and Comparative Example 1 is higher than that of Comparative Example 2.

[0130] Figure 3 The compressive modulus of the hydrogels prepared in Example 1, Comparative Example 1, and Comparative Example 2. Figure 3 It is evident that the compression modulus of Example 1 and Comparative Example 1 is significantly higher than that of Comparative Example 2. Figure 2 and Figure 3 This indicates that the addition of modified cellulose nanofibers enhances the mechanical properties of the hydrogel, while the addition of drug-targeting liposomes has no significant effect on the mechanical properties of the hydrogel.

[0131] 3. Self-healing properties of hydrogels

[0132] The self-healing properties of the hydrogel prepared in Example 1 are as follows: Figure 4 As shown, a piece of hydrogel was placed in a petri dish and cut along the middle to obtain two pieces of hydrogel. The two pieces of hydrogel were then overlapped and kept still. After 30 minutes, the hydrogels could be picked up with tweezers, and the overlapped hydrogels were bonded together without any obvious gaps, proving that the hydrogel prepared by this invention has self-healing ability.

[0133] 4. Hydrogel Adhesion Properties

[0134] The adhesive properties of the hydrogel prepared in Example 1 are as follows: Figure 5 As shown, a 1 mL piece of hydrogel was adhered between two pieces of pigskin. After the hydrogel solidified, weights of different weights were hung under one side of the pigskin. When lifted, the two pieces of pigskin did not separate. Another piece of hydrogel was adhered to the pigskin, and the bending direction of the pigskin was changed. The hydrogel still showed good adhesion, proving that the hydrogel prepared in this invention has excellent adhesive properties.

[0135] A circular defect with a diameter of 6 mm and a depth of 1.5 mm was drilled in the trochlear groove of a pig knee joint using a drill. The hydrogel prepared in Example 1 was injected into the defect and allowed to settle. Pig articular cartilage was then placed laterally or longitudinally, and the gelation and adhesion of the hydrogel at the defect were tested. The results are as follows: Figure 5As shown in section H, the experimental results demonstrate that the hydrogel can fill circular defects and gel, while also exhibiting good adhesion; the hydrogel does not fall off when placed longitudinally on the knee joint. Even after 7 days, the hydrogel remains firmly adhered to the defect, proving that it can remain in the joint defect for a long period.

[0136] 5. pH responsiveness of hydrogels

[0137] The hydrogel prepared in Example 1 was used to conduct a liposome sustained-release experiment to detect the sustained-release effect of the hydrogel on liposomes. The pH responsiveness of the hydrogel was evaluated by the release of liposomes at different pH values. The specific experimental procedure was as follows: 0.5 mL of hydrogel was immersed in 5 mL of PBS solution and gently shaken continuously at 37°C. 1 mL of PBS solution was collected on days 1, 2, 3, 4, 5, 6, and 7, and 1 mL of PBS solution was added to make up the difference. The concentration of released liposomes in the solution was detected using a UV spectrophotometer, and the release rate was calculated. The results are shown below. Figure 6 As shown.

[0138] Figure 6 The image shows the pH-responsiveness of the hydrogel prepared in Example 1; where A represents the release rate of liposomes at pH 7.4, and B represents the release rate of liposomes at pH 5.5. Figure 6 As can be seen, the hydrogel slowly releases liposomes at pH 7.4, with a release rate of 76.27% by day 7, while at pH 5.5, the release rate of liposomes exceeds 70% by day 3. These results indicate that liposome release is relatively slow at pH 7.4, while rapid release occurs within 3 days at pH 5.5, demonstrating that the hydrogel in Example 1 possesses both sustained-release drug capability and pH responsiveness.

[0139] 6. Physical properties of liposomes

[0140] The drug-loaded targeted liposomes prepared in step 1) of Example 1 were characterized by dynamic light scattering (DLS) and transmission electron microscopy. The results are as follows: Figure 7 As shown.

[0141] Figure 7 The images show particle size analysis, potential analysis, and transmission electron microscopy (TEM) images of the drug-loaded targeted liposomes prepared in step 1) of Example 1; where A represents particle size analysis, B represents potential analysis, and C represents TEM image. Figure 7 As can be seen, the drug-loaded targeted liposomes prepared in Example 1 exhibit a spherical sac-like structure with an average particle size of 158.3±2.5nm and an average zeta potential of -30.04±0.52mV.

[0142] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel, characterized in that, The raw materials include sodium oxidized alginate, dopamine-grafted gelatin, modified cellulose nanofibers, borax, drug-loaded targeted liposomes, and water in a ratio of (16-64) g: (32-128) g: (1-2) g: (8-32) mol: (50-200) μmol: 1 L. The modified cellulose nanofibers are mono-(6-amino-6-deoxy)-β-cyclodextrin modified cellulose nanofibers; The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel has a double-crosslinked structure and pH responsiveness.

2. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 1, characterized in that, The method for preparing the modified cellulose nanofibers includes the following steps: Dissolve mono-(6-amino-6-deoxy)-β-cyclodextrin in water, then add a hydrophobic drug, stir in the dark to obtain solution A; Carboxylated cellulose nanofibers were dispersed in water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to carry out a carboxyl activation reaction to obtain solution B; Solution B is added to solution A to carry out an amidation reaction, thereby obtaining the modified cellulose nanofibers.

3. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 2, characterized in that, The mass ratio of the mono-(6-amino-6-deoxy)-β-cyclodextrin to the hydrophobic drug is (5-10):1; and / or, The stirring temperature in the dark is 25-37℃, and the stirring time in the dark is 24-48h.

4. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 2, characterized in that, The mass ratio of the carboxylated cellulose nanofibers, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(1-3):(0.8-3); and / or, The carboxyl activation reaction is carried out at a temperature of 25-37°C for 0.5-2 hours; and / or, The mass ratio of the carboxylated cellulose nanofibers to mono-(6-amino-6-deoxy)-β-cyclodextrin is 100:(1-2); and / or, The amidation reaction is carried out at a temperature of 25-37°C for 24-48 hours.

5. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 1, characterized in that, The method for preparing the drug-loaded targeted liposomes includes the following steps: S1. Egg yolk lecithin, cholesterol, anti-inflammatory drugs, and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000-folic acid are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is then subjected to rotary evaporation to obtain a liposome film; S2. The liposome membrane is subjected to hydration and ultrasonic treatment in sequence, and then filtered to obtain the drug-loaded targeted liposome.

6. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 5, characterized in that, In step S1, the mass ratio of egg yolk lecithin, cholesterol, anti-inflammatory drug, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2000-folic acid is (60-100):(12-30):(3-5):(1-2); and / or, The anti-inflammatory drug is selected from one or more of dimethyl fumarate, itaconic acid, and celecoxib.

7. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 5, characterized in that, In step S1, the temperature of the rotary evaporation is 40-50℃, the rotation speed of the rotary evaporation is 100-200 rpm, and the time of the rotary evaporation is 0.5-1h.

8. The modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel according to claim 5, characterized in that, In step S2, the hydration treatment temperature is 40-50℃, and the hydration treatment time is 0.5-1 hour; and / or, The ultrasonic treatment power is 100-150W, and the ultrasonic treatment time is 1-3 minutes; and / or, The filter membrane has a pore size of 0.1-0.45 μm.

9. A method for preparing a modified cellulose nanofiber-reinforced double-crosslinked immunomodulatory hydrogel as described in any one of claims 1-8, characterized in that, Includes the following steps: Sodium oxidized alginate, dopamine-grafted gelatin, borax and modified cellulose nanofibers were mixed in water to obtain a hydrogel precursor solution. The drug-loaded targeted liposomes were added to the hydrogel precursor solution and mixed to obtain the modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel.

10. The application of a modified cellulose nanofiber-reinforced double crosslinked immunomodulatory hydrogel as described in any one of claims 1-8 in the preparation of cartilage repair materials.

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