A cationic silk fibroin / sodium oxidized hyaluronic acid injectable hydrogel, its preparation method and application

Abstract

This invention discloses a cationic silk fibroin / oxidized sodium hyaluronate injectable hydrogel, its preparation method, and its applications. Using silk fibroin (SF) and sodium hyaluronate (SH) as main raw materials, SF is cationically modified to obtain CSF, and SH is oxidized to obtain OSH. Utilizing the Schiff base reaction between the amino groups in CSF and the aldehyde groups in OSH, a CSF / OSH injectable hydrogel with excellent adhesion, mechanical properties, self-healing ability, biocompatibility, and antibacterial properties can be formed without the addition of any cross-linking agent. The CSF / OSH injectable hydrogel provided by this invention can play an important role in the treatment of wound healing, bone defects, cardiovascular diseases, and brain diseases, and has broad application prospects in the fields of biomedicine and tissue engineering.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an injectable hydrogel of cationic silk fibroin / oxidized sodium hyaluronate, its preparation method, and its application. Background Technology

[0002] Hydrogels are three-dimensional network materials composed of one or more hydrophilic polymers and are used in many fields. Among them, hydrogels made of biodegradable polymers have very high hydrophilicity and biocompatibility due to their structure similar to the natural extracellular matrix. They also have physical properties and mechanical properties similar to living tissues and have important application value in the fields of biomedicine and tissue engineering.

[0003] Compared to chemically covalently cross-linked hydrogels, injectable hydrogels are a novel type of hydrogel cross-linked through dynamic covalent or non-covalent bonds. They can be injected into a designated site and gel in situ. The polymer molecular solution maintaining a sol state undergoes a sol-gel transition after reaching the lesion site via a syringe. It can yield and flow under shear force and self-repair damaged structures after stress removal. Therefore, it plays an important role in the treatment of wound healing, bone defects, cardiovascular diseases, and brain diseases. In addition to possessing the general characteristics of biomaterials—non-toxic, no adverse reactions, abundant supply, stable properties, easy storage and sterilization, good biocompatibility and tissue compatibility—injectable hydrogel materials also require good adhesion, mechanical properties, antibacterial properties, and injectability. For example, Yang Jintao et al. developed a biodegradable injectable hydrogel based on natural polysaccharides, combining the rapid Schiff base reaction between -NH2 and -CHO with the slow reaction between -NH2 and epoxy groups, which can rapidly gel to fill cavities and slowly self-reinforce and support tissue repair (Biomacromolecules. 2023, 24(7), 3345). Wu Yidong et al. rapidly prepared a hydrogel for wound healing by using the amino groups of methacryloylated chitosan and the aldehyde and carboxyl groups on aldehyde-modified hyaluronic acid to form reversible dynamic Schiff base bonds and electrostatic interactions, respectively (Chinese Journal of Biomedical Engineering, 2021, 40(5): 590), but it has disadvantages such as poor adhesion, low mechanical properties and no antibacterial properties. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide an injectable hydrogel of cationic silk fibroin / sodium oxyhyaluronate, its preparation method, and its applications. Low molecular weight PEI is used to modify SF, improving its mechanical properties and antibacterial activity. A cationic silk fibroin / sodium oxyhyaluronate (CSF / OSH) hydrogel is successfully prepared through dynamic covalent cross-linking between cationic silk fibroin (CSF) and sodium oxyhyaluronate (OSH). This hydrogel is injectable, adaptable to irregular wound healing, and spontaneously heals upon mechanical damage. The mechanical properties of this injectable hydrogel can be adjusted according to the mass ratio of cationic silk fibroin to sodium hyaluronate, and it possesses adjustable antibacterial properties and biocompatibility, demonstrating significant application potential in the biomedical field.

[0005] The above-mentioned objective of this invention is achieved through the following technical solutions:

[0006] The first aspect of this invention provides a method for preparing a cationic silk fibroin / oxidized sodium hyaluronate injectable hydrogel, comprising the following steps:

[0007] (1) At 0-4℃, silk fibroin (SF) was modified with polyethyleneimine (PEI) to obtain cationic silk fibroin (CSF) containing amino group (-NH2); sodium hyaluronate (SH) was oxidized to obtain oxidized sodium hyaluronate (OSH) containing aldehyde group (-CHO).

[0008] (2) The cationic silk fibroin solution and the sodium oxidized hyaluronic acid solution are mixed to obtain the cationic silk fibroin / sodium oxidized hyaluronic acid (CSF / OSH) injectable hydrogel.

[0009] This invention uses SF and SH as the main raw materials. Both SF and SH are natural polymers that can be degraded and absorbed by biological organisms without producing toxic or harmful substances, exhibiting good biocompatibility. This invention modifies SF with cations and oxidizes sodium hyaluronate. Starting from the molecular structure, it utilizes the Schiff base reaction between the amino groups in CSF and the aldehyde groups in OSH, which have strong oxidizing properties, to prepare an injectable, self-healing CSF / OSH hydrogel. Existing technologies typically use cross-linking agents to form hydrogels, but these agents are usually toxic. This invention obtains a safe and non-toxic CSF / OSH injectable hydrogel without adding toxic cross-linking agents.

[0010] Further, in step (1), the molecular weight of the silk fibroin is 100-150 kDa.

[0011] Further, in step (1), the molecular weight of the polyethyleneimine is 300-2000 Da.

[0012] Further, in step (1), the mass ratio of silk fibroin to polyethyleneimine is 100:(2-10).

[0013] In a specific implementation, the method of modifying SF with PEI is as follows: PEI solution and carboxyl activating reagent are added to the SF solution.

[0014] Furthermore, the concentration of SF in the SF solution is 5–10 mg / mL, and the concentration of PEI in the PEI solution is 0.05–2 mol / L.

[0015] Furthermore, the carboxyl activating agent is N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0016] Furthermore, the mass ratio of SF, NHS and EDC is 10:(1~3):(1~3).

[0017] Furthermore, in step (1), the reaction time is 5 to 7 hours.

[0018] Furthermore, step (1) includes a dialysis step after the reaction is completed.

[0019] Furthermore, the dialysis time is 48–72 hours.

[0020] Furthermore, in step (1), the sodium hyaluronate is an anionic polysaccharide with a weight-average molecular weight of 100-120 kDa.

[0021] Further, in step (1), the oxidation treatment method is as follows: sodium periodate (NaIO4) is added to the sodium hyaluronate solution, and an oxidation reaction is carried out under light-protected conditions to obtain the oxidized sodium hyaluronate containing aldehyde groups.

[0022] Sodium periodate was used to oxidize sodium hyaluronate, which reduced the reaction concentration of the sodium hyaluronate solution and increased the degree of oxidation of sodium hyaluronate.

[0023] Furthermore, the concentration of sodium hyaluronate in the sodium hyaluronate solution is 2.5–10 mg / mL.

[0024] Furthermore, the molar ratio of sodium periodate to sodium hyaluronate in the sodium hyaluronate solution is (0.5-1.5):1, preferably (1-1.25):1.

[0025] Furthermore, a sodium periodate solution can also be added to the sodium hyaluronate solution, wherein the concentration of sodium periodate in the sodium periodate solution is 0.07–0.1 g / mL.

[0026] Furthermore, the oxidation treatment is performed at room temperature.

[0027] Furthermore, the oxidation reaction takes 1 to 3 hours.

[0028] Furthermore, it also includes the step of adding ethylene glycol to terminate the oxidation reaction.

[0029] Furthermore, the mass ratio of ethylene glycol to sodium periodate is (1-3):1.

[0030] Furthermore, after the oxidation reaction is complete, the process also includes steps of dialysis purification and freeze-drying.

[0031] Furthermore, the dialysis time is 48–72 hours.

[0032] Further, in step (2), the concentration of cationic silk fibroin in the cationic silk fibroin solution is 4-6 wt%, and the concentration of sodium oxidized hyaluronic acid in the sodium oxidized hyaluronic acid solution is 4-6 wt%.

[0033] Further, in step (2), the mass ratio of the cationic silk fibroin to the oxidized sodium hyaluronate is 1:(0.5-3), preferably 1:(1-2).

[0034] Furthermore, in step (2), the mixing temperature is ambient temperature and does not exceed 40°C, and can be 20 to 40°C.

[0035] In a specific embodiment, the solution of cationic silk fibroin and the solution of sodium oxidized hyaluronic acid are respectively placed into the two tubes of a dual sampler, and the cationic silk fibroin / sodium oxidized hyaluronic acid injectable hydrogel is obtained by injection.

[0036] The second aspect of the present invention provides an injectable hydrogel of cationic silk fibroin / sodium oxidized hyaluronic acid obtained by the preparation method described in the first aspect.

[0037] The CSF / OSH injectable hydrogel provided by this invention has good mechanical properties, and the mechanical properties of the hydrogel can be adjusted by changing the mass ratio of CSF and OSH.

[0038] The CSF / OSH injectable hydrogel provided by this invention can largely mimic the three-dimensional network structure of the extracellular matrix, promote cell adhesion and proliferation, and endow the hydrogel scaffold with certain antibacterial properties.

[0039] The third aspect of this invention provides the application of the cationic silk fibroin / oxidized sodium hyaluronate injectable hydrogel described in the second aspect in tissue engineering.

[0040] The CSF / OSH injectable hydrogel provided by this invention can be injected into irregularly shaped wounds to form gels in situ and spontaneously heal when damaged by external forces. At the same time, the hydrogel has good adhesion, mechanical properties, antibacterial properties and injectability, and plays an important role in the treatment of wound healing, bone defects, cardiovascular diseases and brain diseases.

[0041] The beneficial effects of this invention are:

[0042] This invention modifies SF with low molecular weight PEI to prepare amino-containing CSF, changing the surface charge of SF to a positive charge. This not only enhances the mechanical properties of the three-dimensional network structure of the hydrogel, slows down the degradation rate, and promotes cell proliferation, but also endows the hydrogel scaffold with certain antibacterial properties. The CSF / OSH injectable hydrogel provided by this invention does not require the addition of any cross-linking agent. It cross-links with the aldehyde groups of OSH through a Schiff base reaction between the amino groups on CSF and OSH, forming a hydrogel with injectable, self-healing, and antibacterial functions, showing broad application prospects in the fields of biomedicine and tissue engineering. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the dual sampler and its injection method in Example 1.

[0044] Figure 2 The infrared absorption spectra of SF and CSF in Example 1 are shown.

[0045] Figure 3 The above are the 1H NMR spectra of PEI, SF and CSF in Example 1.

[0046] Figure 4 The infrared absorption spectra of SH and OSH in Example 1 are shown.

[0047] Figure 5 The above are the hydrogen nuclear magnetic resonance spectra of SH and OSH in Example 1.

[0048] Figure 6 The images show cross-sectional SEM images of the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8. Specifically, a1, a2, and a3 are from Example 6, with scale bars of 100 μm, 50 μm, and 20 μm, respectively; b1, b2, and b3 are from Example 7, with scale bars of 100 μm, 50 μm, and 20 μm, respectively; c1, c2, and c3 are from Example 1, with scale bars of 100 μm, 50 μm, and 20 μm, respectively; and d1, d2, and d3 are from Example 8, with scale bars of 100 μm, 50 μm, and 20 μm, respectively.

[0049] Figure 7The figures show the test results of scanning the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 using a rheometer. Among them, (a) is the data graph of G' (storage modulus) and G" (loss modulus) obtained by time scanning of CSF / OSH injectable hydrogels with different mass ratios; (b) is the data graph of G' and G" obtained by frequency scanning of CSF / OSH injectable hydrogels with different mass ratios; (c) is the data graph of G' and G" obtained by oscillatory strain scanning of CSF / OSH injectable hydrogel of Example 6; (d) is the data graph of G' and G" obtained by oscillatory strain scanning of CSF / OSH injectable hydrogel of Example 7; (e) is the data graph of G' and G" obtained by oscillatory strain scanning of CSF / OSH injectable hydrogel of Example 1; and (f) is the data graph of G' and G" obtained by oscillatory strain scanning of CSF / OSH injectable hydrogel of Example 8.

[0050] Figure 8 The figures show the test results of alternating strain scanning of the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 using a rheometer, where (a) is Example 6, (b) is Example 7, (c) is Example 1, and (d) is Example 8.

[0051] Figure 9 This is a diagram of the self-healing test of the CSF / OSH injectable hydrogel prepared in Example 1.

[0052] Figure 10 The image shows the shear frequency scan data of the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8.

[0053] Figure 11 The diagram shows the antibacterial effects of the SF / SH hydrogel prepared as a blank control group, the CSF / OSH injectable hydrogel prepared as a comparative example 1, and the Escherichia coli prepared as an example 1.

[0054] Figure 12 The CCK-8 assay results of L929 cells after co-culturing the blank control group, the SF / SH hydrogel extract prepared in Comparative Example 1, and the CSF / OSH injectable hydrogel extract prepared in Example 1 with L929 cells for 1, 3, and 5 days are shown in the figure. Detailed Implementation

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0056] This invention provides a method for preparing a cationic silk fibroin / oxidized sodium hyaluronate injectable hydrogel, comprising the following steps:

[0057] (1) At 0-4℃, silk fibroin (SF) was modified with polyethyleneimine (PEI) to obtain cationic silk fibroin (CSF) containing amino group (-NH2); sodium hyaluronate (SH) was oxidized to obtain oxidized sodium hyaluronate (OSH) containing aldehyde group (-CHO).

[0058] (2) The cationic silk fibroin solution and the sodium oxidized hyaluronic acid solution are mixed to obtain the cationic silk fibroin / sodium oxidized hyaluronic acid (CSF / OSH) injectable hydrogel.

[0059] In a specific implementation, in step (1), the method of modifying SF with PEI is as follows: PEI solution and carboxyl activating reagent are added to the SF solution.

[0060] In a specific embodiment, the carboxyl activating agent is N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0061] In step (1), the reaction equation for PEI modification of SF is as follows:

[0062]

[0063] In the presence of EDC, the carboxyl groups of aspartic acid and glutamic acid in the side chain of PEI and SF are activated and undergo a nucleophilic reaction with the -N=C- double bond in EDC to generate an unstable intermediate, an O-isocyanate derivative. Subsequently, the free hydroxyl group (-OH) on NHS undergoes an alcoholysis reaction with the unstable intermediate to generate a stable intermediate, and the amino group of PEI undergoes an ammonolysis reaction with this product. Thus, the -COOH group of the SF side chain is linked to the -HN2 group of PEI via an amide bond, modifying the SF side chain to obtain CSF containing -NH2.

[0064] In a specific embodiment, in step (1), the oxidation treatment method is as follows: sodium periodate (NaIO4) is added to the SH solution to carry out an oxidation reaction to obtain the OSH containing the aldehyde group.

[0065] In step (1), the reaction equation for the preparation of OSH from SH is as follows:

[0066]

[0067] The C-C bonds in the vicinal diol of the SH structural unit can be oxidized by NaIO4, followed by ring-opening cleavage to generate an aldehyde group. NaIO4 is unstable and decomposes easily in light; therefore, this oxidation reaction must be carried out under light-protected conditions to proceed smoothly.

[0068] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0070] Example 1

[0071] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel includes the following steps:

[0072] (1) Take 400 mL of SF (molecular weight 100 kDa) solution with a concentration of 5 mg / mL and equilibrate in an ice bath for 30 min. Then add 12 mL of PEI (molecular weight 1800 Da) solution with a concentration of 10 mg / mL and then quickly add 20 mL of NHS and EDC with a concentration of 10 mg / mL. After reacting in an ice bath for 6 h, let the solution stand overnight and then dialyze it in deionized water for 3 days to obtain CSF solution. Store at 4-8℃ for later use.

[0073] (2) Dissolve 0.5 g of SH powder in 100 mL of deionized water. After the SH is completely dissolved, weigh NaIO4 powder (the molar ratio of NaIO4 to SH is 1.25:1) and add it to the SH solution. Mix and stir for 2 h under light-protected conditions. Then add 1.0 mL of ethylene glycol to the solution and stir for 1 h to terminate the reaction. After the reaction is complete, put it into a dialysis bag and dialyze it into deionized water for three days to purify it. After dialysis, freeze-dry for two days to obtain a white spongy solid, which is OSH. Store at 4-8℃ for later use.

[0074] (3) Dissolve OSH in deionized water to obtain an OSH solution. Place the CSF solution (5wt%) and OSH solution (5wt%) into tubes A and B of the dual-syringe, respectively. The mass ratio of OSH in the OSH solution to CSF ​​in the CSF solution is 2:1. Injectable CSF / OSH hydrogel is obtained by injection. A schematic diagram of the dual-syringe and its injection method is shown below. Figure 1 As shown.

[0075] Figure 2 The images show the infrared absorption spectra of SF and CSF in Example 1. Figure 3The nuclear magnetic resonance hydrogen of PEI, SF and CSF in Example 1 1 1H-NMR spectrum. Figure 2 As can be seen from the data, after PEI modification, SF is at 1642 cm⁻¹ -1 1235cm -1 and 944cm -1 The absorption peaks at 1634 cm⁻¹ are respectively directed towards 1634 cm⁻¹ -1 1228cm -1 and 951cm -1 The shift indicates that the carboxyl group on the SF side chain reacts with the -NH2 group of PEI to form a new amide bond. In CSF... 1 In the H-NMR spectrum, a new proton peak appeared at point k in the range of δ to 3.14 ppm. This peak corresponds to the chemical shift of the methylene proton adjacent to the amide bond in the -CONHCH2CH2NH group, indicating that the -COOH on the SF side chain reacted with the -NH2 of PEI to form an amide bond, thus demonstrating the successful preparation of CSF.

[0076] Figure 4 The following are the Fourier Transmission-Infrared (FT-IR) spectra of SH and OSH in Example 1. Figure 5 The images show the 1H NMR spectra of SH and OSH in Example 1. The SH structure contains many hydroxyl groups, which readily form hydrogen bonds with electron-donating groups such as -OH and -NH2. The more hydrogen bonds there are, the broader the stretching vibration peak of -OH becomes. Figure 4 3288cm -1 The absorption peak at 1740 cm⁻¹ is the stretching vibration peak of OSH and SH; in addition, the FT-IR spectrum of OSH at 1740 cm⁻¹ is... -1 There is a relatively weak absorption peak, which belongs to the C=O bending vibration absorption peak in the ketone or aldehyde structure. This indicates that the vicinal diol structure of SH was successfully oxidized to generate two aldehyde groups. From Figure 5 As can be seen, OSH exhibits a relatively small new peak at δ ~ 4.85-5.00 ppm (points a and b) compared to SH. This corresponds to the formation of a hemiacetal proton H by the aldehyde group and the adjacent hydroxyl group, while no corresponding new peak was observed in the spectrum of SH. This indicates that sodium periodate oxidizes the vicinal diol structure of SH to generate an aldehyde group, demonstrating the successful preparation of OSH.

[0077] Example 2

[0078] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (2), the molar ratio of NaIO4 to SH is 0.5:1.

[0079] Example 3

[0080] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (2), the molar ratio of NaIO4 to SH is 0.75:1.

[0081] Example 4

[0082] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (2), the molar ratio of NaIO4 to SH is 1:1.

[0083] Example 5

[0084] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (2), the molar ratio of NaIO4 to SH is 1.5:1.

[0085] Example 6

[0086] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (3), the mass ratio of OSH in the OSH solution to CSF ​​in the CSF solution is 0.5:1.

[0087] Example 7

[0088] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (3), the mass ratio of OSH in the OSH solution to CSF ​​in the CSF solution is 1:1.

[0089] Example 8

[0090] A method for preparing a cationic silk fibroin / oxidized sodium hyaluronate (CSF / OSH) injectable hydrogel is basically the same as that in Example 1, except that in step (3), the mass ratio of OSH in the OSH solution to CSF ​​in the CSF solution is 3:1.

[0091] Comparative Example 1

[0092] A method for preparing a silk fibroin / sodium hyaluronate (SF / SH) hydrogel includes the following steps: mixing an SF solution (concentration of 5 wt%) and an SH solution (concentration of 5 wt%), wherein the mass ratio of SH in the SH solution to SF in the SF solution is 2:1, to obtain an SF / SH hydrogel.

[0093] The aldehyde concentration and oxidation degree of OSH in Examples 1-5 were tested. The oxidation degree was determined by potentiometric titration with hydroxylamine hydrochloride. The amino group on hydroxylamine hydrochloride readily reacts with the aldehyde group on OSH to form an oxime and release HCl. The aldehyde concentration was calculated by titrating the released HCl with NaOH. The test results are shown in Table 1.

[0094] Table 1. Data on the determination of aldehyde concentration and oxidation degree on OSH by hydroxylamine hydrochloride titration method.

[0095]

[0096] As shown in Table 1, when the molar ratio of NaIO4 to SH gradually increases from 0.5:1 to 1.5:1, the concentration of aldehyde groups and the degree of oxidation on SH gradually increase. When the molar ratio increases from 1.25:1 to 1.5:1, the degree of oxidation no longer increases and remains at around 47%. This may be because when the degree of oxidation reaches 47%, the concentration of aldehyde groups on OSH is very high, making it easy to form a hemiacetal structure with adjacent hydroxyl groups, thus preventing further reaction between the hydroxyl groups on SH and NaIO4, and therefore the degree of oxidation cannot be further increased. When the molar ratio of NaIO4 to SH is 1.25:1, the prepared OSH contains more aldehyde groups, which is beneficial for preparing injectable hydrogels.

[0097] The cross-sections of the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 were characterized using scanning electron microscopy (SEM). The characterization results are as follows: Figure 6 As shown, the internal structure of the CSF / OSH injectable hydrogels is porous. The internal pore sizes of the CSF / OSH injectable hydrogels with OSH to CSF ​​mass ratios of 0.5:1, 1:1, 2:1, and 3:1 are 155.8±9.8 μm, 185.1±22.3 μm, 208.0±13.8 μm, and 224.8±15.9 μm, respectively, and the internal porosities are 46.6±2.5%, 47.6±11%, 55.4±8.6%, and 56.6±11.4%, respectively. With the increase of OSH content, both the pore size and porosity of the CSF / OSH injectable hydrogels increase. This may be because changing the OSH content actually alters the crosslinking density of CSF and aldehyde-modified sodium hyaluronate, leading to changes in the internal pore size of the gel. Injectable hydrogels with an OSH to CSF ​​mass ratio of 2:1 often exhibit elliptical or irregular polygonal pore shapes, with uniform pore size and relatively smooth pore walls.

[0098] The CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 were subjected to time, frequency, and oscillatory strain scans using a rheometer. Figure 7The figures show the test results of scanning the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 using a rheometer. (a) shows the G' (storage modulus) and G" (loss modulus) data obtained from time scanning of CSF / OSH injectable hydrogels with different mass ratios; (b) shows the G' and G" data obtained from frequency scanning of CSF / OSH injectable hydrogels with different mass ratios; (c) shows the G' and G" data obtained from oscillatory strain scanning of the CSF / OSH injectable hydrogel of Example 6; (d) shows the G' and G" data obtained from oscillatory strain scanning of the CSF / OSH injectable hydrogel of Example 7; (e) shows the G' and G" data obtained from oscillatory strain scanning of the CSF / OSH injectable hydrogel of Example 1; and (f) shows the G' and G" data obtained from oscillatory strain scanning of the CSF / OSH injectable hydrogel of Example 8. Figure 7 As shown in Figure (a), all four hydrogels exhibited significant solid properties (G' > G"). The G' values ​​of the CSF / OSH injectable hydrogels with OSH to CSF ​​mass ratios of 0.5:1, 1:1, 2:1, and 3:1 were 72 Pa, 194 Pa, 350 Pa, and 301 Pa, respectively, under 1% oscillating strain. This indicates that the CSF / OSH injectable hydrogel with an OSH to CSF ​​mass ratio of 2:1 possesses the optimal cross-linking network. With increasing OSH content, both G' and G" of the gel also showed an increasing trend. The G' value of the hydrogel reached its maximum (350 Pa) when the OSH to CSF ​​mass ratio was 2:1. This indicates that at this point, the aldehyde and amino groups inside the hydrogel were comparable, resulting in the most imine bonds and the most stable three-dimensional network structure.

[0099] Four groups of hydrogels were tested at different frequencies with a fixed oscillatory strain (1%). The G' of the four hydrogels remained almost stable in the frequency range of 0.1–100 rad / s, and in all cases, G' was significantly higher than G"0. Figure 7 As shown in (b). Furthermore, when the mass ratio of OSH to CSF ​​is 2:1, the gel has the highest G', indicating that this group of gels has the best cross-linking network, which is consistent with the gel time results.

[0100] from Figure 7As shown in (c) to (f), the G' and G" of the OSH / CSF injectable hydrogel remain almost stable within the oscillating strain range of 0.1% to 50%. As the oscillating strain gradually increases, the G' of the hydrogel decreases, and the curves of G' and G" intersect, indicating the collapse of the internal network structure of the hydrogel. This suggests that the hydrogel transforms from a solid with gel properties to a liquid-like structure with fluid properties. The critical strain of the gel is highest (345%) when the mass ratio of OSH to CSF ​​is 2:1, and lowest (65%) when the mass ratio is 3:1. This indicates that the hydrogel has the most imine bonds and the tightest cross-linking when the mass ratio of OSH to CSF ​​is 2:1, resulting in the highest critical strain. A higher critical strain at a mass ratio of OSH to CSF ​​of 2:1 indicates that it can withstand greater deformation.

[0101] Alternating strain scanning was performed on the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 using a rheometer. Figure 8 The figures show the results of alternating strain scanning tests performed on the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8 using a rheometer, where (a) is Example 6, (b) is Example 7, (c) is Example 1, and (d) is Example 8. Figure 8 As can be seen, when the strain increases from 1% to 1000%, G" > G', and the internal structure of the gel gradually disintegrates; when the strain recovers from 1000% to 1%, G' > G" and both G' and G" return to their original values, indicating that the OSH / CSF hydrogel reconstructs its own structure and achieves self-healing.

[0102] Figure 9 This image shows a self-healing experiment of the CSF / OSH injectable hydrogel prepared in Example 1. The OSH / CSF injectable hydrogel was cut in half, with one half stained with methyl orange. Small pieces of the different colored hemigels were made and assembled together, then observed at room temperature without any further intervention. Figure 9 As can be seen, the two hydrogels were cut into small pieces and merged into a single unit. When placed tightly together, their surface became smooth. The two dye molecules clearly interpenetrated at the weld joint, indicating that polymer molecular chains moved between adjacent hydrogel segments. At this point, applying a pulling force to the hydrogel with tweezers revealed that the weld joint remained tightly connected without breakage. This is likely because when cracks occur, the imine bonds break, exposing free aldehyde and amide groups at the interface. Due to their dynamic and reversible nature, the exposed amino and aldehyde groups can reconstruct new imine bonds, allowing the hydrogel to recover from the crack. This further demonstrates the excellent self-healing ability of the CSF / OSH injectable hydrogel.

[0103] Figure 10 The image shows the shear frequency scan data of the CSF / OSH injectable hydrogels prepared in Examples 1 and 6-8. The composite viscosity of the OSH / CSF hydrogel decreases with increasing shear frequency, indicating that it has pseudoplasticity, is easy to inject into the application, and has excellent injectability.

[0104] Figure 11 The figures show the antibacterial effects of the blank control group (CON), the SF / SH hydrogel prepared in Comparative Example 1, and the CSF / OSH injectable hydrogel prepared in Example 1 against Escherichia coli. The SF / SH hydrogel has a certain antibacterial effect, and the antibacterial rate of the CSF / OSH injectable hydrogel increases with the addition of cationic silk fibroin, which proves that the CSF / OSH injectable hydrogel has excellent antibacterial properties against Escherichia coli.

[0105] Figure 12 The images show the CCK-8 assay results of L929 cells (mouse fibroblasts) after co-culturing the blank control group (Control), the SF / SH hydrogel extract prepared in Comparative Example 1, and the CSF / OSH injectable hydrogel extract prepared in Example 1 with L929 cells (mouse fibroblasts) for 1, 3, and 5 days. The extracts were prepared according to the national standard GB / T16886.5-2017 / ISO10993-12. After 1 day of culture, the OD value of the CSF / OSH injectable hydrogel was significantly higher than that of the blank control group (without hydrogel) and the SF / SH hydrogel. After 3 days of culture, the OD values ​​of all groups increased significantly, with the OD value of the CSF / OSH injectable hydrogel showing a highly significant difference from the blank control group and the SF / SH hydrogel. After 5 days of culture, the OD values ​​of all groups increased further. The OD values ​​of the blank control group and the SF / SH hydrogel showed a significant difference, and the OD value of the CSF / OSH injectable hydrogel showed a highly significant difference from the SF / SH hydrogel. This demonstrates that the CSF / OSH injectable hydrogel has no obvious toxic side effects on L929 cells and can promote the proliferation of L929 cells.

[0106] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a cationic silk fibroin / sodium oxidized hyaluronic acid injectable hydrogel, characterized in that, Includes the following steps: (1) The silk fibroin was modified with polyethyleneimine at 0~4 ℃ to obtain cationic silk fibroin containing amino groups; sodium hyaluronate was oxidized to obtain sodium oxyhyaluronate containing aldehyde groups; the method of modifying silk fibroin with polyethyleneimine was as follows: polyethyleneimine solution and carboxyl activating reagent were added to the silk fibroin solution; the carboxyl activating reagent was N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; (2) The cationic silk fibroin solution and the sodium oxidized hyaluronic acid solution are mixed to obtain the cationic silk fibroin / sodium oxidized hyaluronic acid injectable hydrogel; The concentration of cationic silk fibroin in the solution is 4-6 wt%, and the concentration of sodium oxidized hyaluronic acid in the solution is 4-6 wt%; the mass ratio of cationic silk fibroin to sodium oxidized hyaluronic acid is 1:(0.5-3).

2. The preparation method according to claim 1, characterized in that, In step (1), the molecular weight of the silk fibroin is 100~150 kDa.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of silk fibroin to polyethyleneimine is 100:(2~10).

4. The preparation method according to claim 1, characterized in that, In step (1), the oxidation treatment method is as follows: sodium periodate is added to the sodium hyaluronate solution, and an oxidation reaction is carried out under light-protected conditions to obtain the oxidized sodium hyaluronate containing aldehyde groups.

5. The preparation method according to claim 4, characterized in that, The concentration of sodium hyaluronate in the sodium hyaluronate solution is 2~3 mg / mL.

6. The preparation method according to claim 4, characterized in that, The molar ratio of sodium periodate to sodium hyaluronate in the solution is (0.5~1.5):

1.

7. The cationic silk fibroin / oxidized sodium hyaluronate injectable hydrogel obtained by the preparation method according to any one of claims 1 to 6.

Images