A quercetin nanofiber anti-inflammatory hydrogel and its preparation method and application

Abstract

The present invention discloses a quercetin nanofiber anti-inflammatory hydrogel, its preparation method and application, including the steps: a hydrogel is obtained by physically mixing nanofibers formed by self-assembly of the natural flavonoid quercetin with polyvinyl alcohol and then performing orientation freezing, salting out, pre-stretching and swelling strategies. Among them, the introduction of quercetin nanofibers improves the mechanical strength of the hydrogel and a more oriented network structure, and also effectively alleviates the inflammatory reaction brought about after implantation in the body based on its excellent antioxidant and anti-inflammatory effects. Then, salting out, pre-stretching and swelling enable the hydrogel to achieve the mechanical properties required for tendons and good biocompatibility, so as to be more effectively applied to the regeneration of tendon tissue structure.

Description

Technical Field

[0001] The present invention belongs to the field of hydrogel biomaterials, and particularly relates to a quercetin nanofiber anti-inflammatory hydrogel, its preparation method and application. Background Art

[0002] Tendons are dense connective tissues that connect muscles to bones and play functions of force transmission, joint stabilization and shock absorption in body movement. Among them, tendon tissues have relatively few blood vessel distributions and relatively low cell activities. When they are damaged, these cells are difficult to respond quickly and initiate an effective repair process. Therefore, once a tendon is damaged, its self-healing process is often difficult. Existing treatment methods for tendon injuries include surgical suture, autologous tendon transplantation, allogeneic tendon transplantation, xenogeneic tendon transplantation, and prosthetic device replacement of damaged tendons. However, these methods generally have problems such as donor site morbidity, inflammatory reactions, limited sources of potential disease transmission, and strong immune rejection. Therefore, more effective treatment technologies and biomaterials are urgently needed for tendon repair.

[0003] Hydrogels are a class of soft, wet materials with a three-dimensional network structure. Through component structure regulation, while endowing them with the required mechanical properties, they also have good flexibility, biocompatibility and other properties, making them one of the most popular and representative biomaterials in the field of tissue engineering. However, currently, the use of hydrogels for tendon repair only solves the mechanical problem. The problems of the compatibility between the material and tendon tissue after artificial tendon replacement and the chronic inflammation during the repair process of tendon injury are one of the main challenges in the current research and development of biomaterials. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned existing technologies, the purpose of the present invention is to provide a quercetin nanofiber anti-inflammatory hydrogel. The introduction of quercetin self-assembled nanofibers in this hydrogel not only improves the mechanical strength of the hydrogel and a certain directional network structure arrangement, but also endows the hydrogel with pharmacological effects such as anti-inflammation. The hydrogel prepared by the present invention can be used as an artificial tendon replacement at the tendon defect site and can relieve the inflammatory problems brought about after artificial tendon replacement.

[0005] The present invention is achieved through the following technical solutions:

[0006] A preparation method of a quercetin nanofiber anti-inflammatory hydrogel, comprising the following steps:

[0007] (1) Dissolve quercetin in dimethyl sulfoxide solvent, and after complete dissolution, add it to deionized water at a constant speed. After stirring under constant speed conditions, dialyze to obtain a quercetin self-assembled nanofiber solution;

[0008] (2) Add polyvinyl alcohol to deionized water and stir to obtain a polyvinyl alcohol solution;

[0009] (3) Mix the quercetin self - assembled nanofiber solution and the polyvinyl alcohol solution. The obtained mixed solution is subjected to directional freezing, salting - out, pre - stretching, and swelling to obtain the quercetin nanofiber anti - inflammatory hydrogel.

[0010] In this invention, a hydrogel is obtained by physically mixing nanofibers formed by self - assembling the natural flavonoid quercetin with polyvinyl alcohol followed by directional freezing, salting - out, pre - stretching, and swelling strategies. Among them, the introduction of quercetin nanofibers not only improves the mechanical strength and more oriented network structure of the hydrogel, but also effectively alleviates the inflammatory reaction brought after implantation in the body based on its excellent antioxidant and anti - inflammatory effects. Then, through salting - out, pre - stretching, and swelling, the hydrogel achieves the mechanical properties and good biocompatibility required for tendons, thus being more effectively applied to the regeneration of tendon tissue structures.

[0011] Preferably, in step (1), the concentration of the quercetin self - assembled nanofiber solution is 0.02 - 1.0 wt%.

[0012] Preferably, in step (1), the molecular weight cut - off for dialysis is 8000 - 14000 Da.

[0013] Preferably, in step (2), the mass fraction of the polyvinyl alcohol solution is 10 - 15 wt%, and the stirring speed is 200 - 500 rpm.

[0014] Preferably, in step (3), the mass fraction of quercetin in the quercetin nanofiber anti - inflammatory hydrogel is 0.04 - 0.5 wt%, and the mass fraction of polyvinyl alcohol is 10 - 12 wt%.

[0015] Preferably, in step (3), the reagent used for salting - out is the sodium citrate solution, and the concentration of the sodium citrate solution is 1.0 - 2.0 mol / L.

[0016] Preferably, in step (3), the pre - stretching length is 50 - 500%.

[0017] This invention also provides a quercetin nanofiber anti - inflammatory hydrogel prepared by the above - mentioned preparation method. The hydrogel prepared by this invention has a network structure arranged in parallel like tendons, has a strong tensile strength, and has good antioxidant and anti - inflammatory effects and biocompatibility, which can promote the anti - inflammatory effect at the tendon defect site and contribute to the repair of tendons.

[0018] The present invention also provides the application of the above-mentioned quercetin nanofiber anti-inflammatory hydrogel in artificial tendons. The hydrogel prepared by the present invention can be used as an artificial tendon replacement at the tendon defect site. Its flexible structure will not compress the surrounding tissues after implantation, and can relieve the inflammatory problems brought about after artificial tendon replacement, which is helpful for the repair of tendon injury sites.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) In the method of the present invention, the natural flavonoid quercetin is first self-assembled into nanofibers, and then the nanofibers are subjected to the abundant hydrogen bonding of polyvinyl alcohol. Finally, the hydrogel obtained by the strategies of directional freezing, salting out and pre-stretching. By introducing the nanofibers formed by self-assembly of natural substances into the hydrogel matrix, the hydrogel not only improves the mechanical strength of the hydrogel, but also endows the hydrogel with pharmacological effects such as anti-inflammation. The hydrogel prepared by the method of the present invention can be used as an artificial tendon replacement at the tendon defect site, and can relieve the inflammatory problems brought about after artificial tendon replacement.

[0021] (2) The preparation method of the present invention is simple, the preparation conditions are mild and controllable, the raw materials are cheap and easy to obtain, and the flexible structure of the obtained hydrogel will not compress the surrounding tissues after implantation, which is helpful for the repair of tendon injury sites. <{ Description of the Drawings

[0022] Figure 1 It is the optical picture and Tyndall effect picture of the quercetin self-assembled nanofiber solution.

[0023] Figure 2 It is the TEM picture of the quercetin self-assembled nanofiber.

[0024] Figure 3 It is the SEM picture of the quercetin self-assembled nanofiber.

[0025] Figure 4 It is the ultraviolet picture of the quercetin self-assembled nanofiber.

[0026] Figure 5 It is the SEM picture of the hydrogel prepared in Example 1.

[0027] Figure 6 、 7 It is the stress-strain curve graph of the hydrogels prepared in the examples and comparative examples.

[0028] Figure 8 It is the optical images of the torsion, bending, folding of the hydrogel prepared in Example 1 and the ability to pull a dumbbell weighing up to 5 kg.

[0029] Figure 9 It is the result graph of the cytotoxicity test of the hydrogel prepared in Example 1.

[0030] Figure 10 Graph of the hemolysis rate test results for the hydrogel prepared in Example 1.

[0031] Figure 11 Graph of the results of measuring the ROS content by DCFH-DA staining for the polarization of lipopolysaccharide-induced macrophages by the hydrogel prepared in Example 1.

[0032] Figure 12 Schematic diagram of the rat tendon replacement with the hydrogel prepared in Example 1.

[0033] Figure 13 Macroscopic pictures of the SD rat tendon injury model constructed with the hydrogel prepared in Example 1 and the implanted hydrogel replacement. Detailed implementation manners

[0034] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1:

[0036] (1) Preparation of quercetin self-assembled nanofiber (QNF) solution

[0037] Dissolve a certain amount of quercetin in dimethyl sulfoxide solvent. After fully dissolving at room temperature, add it to deionized water at a constant speed, and then stir at a constant speed of 600 rpm for 5 h. Place it in a dialysis bag (the molecular weight cut-off is 8000 - 14000 Da) and dialyze for 72 h to obtain a 0.3 wt% quercetin self-assembled nanofiber solution;

[0038] Figure 1 Optical pictures and Tyndall effect pictures of the quercetin self-assembled nanofiber solution. From Figure 1 It can be seen that through the self-assembly of quercetin, a quercetin micelle solution is prepared. When the micelles are irradiated with a red laser beam, an obvious light beam path can be observed, and the Tyndall effect is produced.

[0039] From Figure 2 , 3 TEM and SEM images, it can be seen that quercetin forms a nanofiber structure through self-assembly.

[0040] Figure 4 UV diagram of the quercetin self-assembled nanofiber. According to Figure 4It can be seen that quercetin (QUE) has two characteristic absorption peaks at 256 nm and 373 nm respectively. In the ultraviolet-visible absorption spectrum of quercetin self-assembled nanofibers (QNF), these two peaks are redshifted to 264 nm and 383 nm. The reason for the redshift of the ultraviolet-visible absorption spectrum of the nanofibers may be due to the π-π stacking in the quercetin self-assembly.

[0041] (2) Preparation of polyvinyl alcohol (PVA) solution

[0042] A certain amount of polyvinyl alcohol was added to deionized water, and then stirred at 90 °C and 400 rpm for 2 h to obtain a 15 wt% polyvinyl alcohol solution.

[0043] (3) Preparation of quercetin nanofiber anti-inflammatory hydrogel

[0044] After mixing 20 mL of 0.3 wt% quercetin self-assembled nanofiber solution with 40 g of 15 wt% polyvinyl alcohol solution, the obtained mixed solution was placed in a silica gel mold for orientation freezing into a solid and then taken out, placed in 1.5 mol / L sodium citrate solution for salting out for 24 hours, and then stretched to 200% at a speed of 50 mm / min on a fixture and kept for 10 min and then taken down, and placed in deionized water for swelling for 24 h to remove the ions remaining on the hydrogel to obtain quercetin nanofiber anti-inflammatory hydrogel (PQNF200).

[0045] Figure 5 SEM image of the prepared hydrogel. According to Figure 5 It can be seen that from the longitudinal cross-section SEM image, a more tightly arranged continuous fiber bundle can be observed more clearly, proving the existence of an oriented structure in the gel structure.

[0046] Comparative Example 1:

[0047] Steps (1) and (2) are the same as those in Example 1.

[0048] (3) After mixing 20 mL of 0.3 wt% quercetin self-assembled nanofiber solution with 40 g of 15 wt% polyvinyl alcohol solution, the obtained mixed solution was placed in a silica gel mold for orientation freezing into a solid and then taken out, placed in 1.5 mol / L sodium citrate solution for salting out for 24 hours, and placed in deionized water for swelling for 24 h to remove the ions remaining on the hydrogel to obtain PQNF hydrogel.

[0049] Comparative Example 2:

[0050] Quercetin unassembled monomers were taken and added to deionized water to prepare a quercetin solution. The quercetin solution was added to a 15wt% polyvinyl alcohol solution and mixed. The resulting mixed solution was placed in a silica gel mold, orientationally frozen into a solid, taken out, placed in a 1.5mol / L sodium citrate solution for salting out for 24h, and then placed in deionized water for swelling for 24h to remove the ions remaining on the hydrogel to obtain a PQ hydrogel.

[0051] Example 2:

[0052] (1) Preparation of quercetin self-assembled nanofiber (QNF) solution

[0053] A certain amount of quercetin was dissolved in dimethyl sulfoxide solvent. After being fully dissolved at room temperature, it was uniformly added to deionized water, and then stirred at a constant speed of 500rpm for 6h, and placed in a dialysis bag for dialysis for 72h to obtain a quercetin self-assembled nanofiber solution with a concentration of 0.2wt%.

[0054] (2) Preparation of polyvinyl alcohol (PVA) solution

[0055] A certain amount of polyvinyl alcohol was added to deionized water, and then stirred at 90°C and 300rpm for 1.5h to obtain a 10wt% polyvinyl alcohol solution.

[0056] (3) Preparation of quercetin nanofiber anti-inflammatory hydrogel

[0057] 20 mL of the 0.2wt% quercetin self-assembled nanofiber solution was mixed with 40 g of the 10wt% polyvinyl alcohol solution. The resulting mixed solution was placed in a silica gel mold, orientationally frozen into a solid, taken out, placed in a 1.0mol / L sodium citrate solution for salting out for 24 hours, and then stretched to 120% at a speed of 50mm / min on a fixture and held for 10min and then taken down, and placed in deionized water for swelling for 24h to remove the ions remaining on the hydrogel to obtain a quercetin nanofiber anti-inflammatory hydrogel.

[0058] Example 3:

[0059] (1) Preparation of quercetin self-assembled nanofiber (QNF) solution

[0060] A certain amount of quercetin was dissolved in dimethyl sulfoxide solvent. After being fully dissolved at room temperature, it was uniformly added to deionized water, and then stirred at a constant speed of 700rpm for 4h, and placed in a dialysis bag for dialysis for 72h to obtain a quercetin self-assembled nanofiber solution with a concentration of 0.5wt%.

[0061] (2) Preparation of polyvinyl alcohol (PVA) solution

[0062] A certain amount of polyvinyl alcohol was added to deionized water, and then stirred at 90 °C and 500 rpm for 1 h to obtain a 12 wt% polyvinyl alcohol solution;

[0063] (3) Preparation of quercetin nanofiber anti-inflammatory hydrogel

[0064] After mixing 20 mL of a 0.5 wt% quercetin self-assembled nanofiber solution with 40 g of a 12 wt% polyvinyl alcohol solution, the obtained mixed solution was placed in a silica gel mold and orientationally frozen into a solid, then taken out, placed in a 1.2 mol / L sodium citrate solution for salting out for 24 hours, and then placed on a fixture and stretched to 180% at a speed of 50 mm / min and kept for 10 min and then taken off, and placed in deionized water for swelling for 24 h to remove the ions remaining on the hydrogel to obtain the quercetin nanofiber anti-inflammatory hydrogel.

[0065] Examples 4 - 6::

[0066] The stretching lengths were respectively set to 50%, 100%, and 150%, and the rest was the same as in Example 1, and quercetin nanofiber anti-inflammatory hydrogels PQNF50, PQNF100, and PQNF150 were obtained respectively. Test example: Performance test

[0067] (1) Tensile strain performance:

[0068] The tensile strain performance was tested by a computerized peel force testing machine. The testing method included: First, the taken-out hydrogel was cut into rectangular strips with a length of 30 mm, a width of 5 mm, and a thickness of 2 mm, and then placed in the testing machine fixture of a 50 N force sensor, and the tensile performance test was carried out at a tensile speed of 100 mm / min.

[0069] Figure 6 、 7 is the stress-strain curve of the prepared hydrogel. As Figure 6 can be seen, with the increase of the pre-stretching length, the mechanical strength of the hydrogel also increased synchronously. When the pre-stretching length reached 200%, the tensile strength of the hydrogel reached above 10 MPa, meeting the mechanical requirements of the tendon.

[0070] According to Figure 7 it can be seen that the introduction of quercetin self-assembled nanofibers in Comparative Example 1 compared with the introduction of quercetin monomers in Comparative Example 2 increased the tensile strength from 1.25 MPa to 3.61 MPa. In Example 1, by pre-stretching to 200%, the mechanical properties of the hydrogel were improved by 3 to 10 times compared with Comparative Example 1 and Comparative Example 2 respectively, the stress reached 10.4 MPa, and the strain reached 520%. It shows that the introduction of quercetin self-assembled nanofibers into the hydrogel matrix greatly improves the mechanical strength of the hydrogel, and through the optimization of the pre-stretching length, the mechanical properties are further enhanced.

[0071] (2) Mechanical properties

[0072] Use tweezers to hold both ends of the prepared hydrogel and perform twisting, bending, and folding operations on the hydrogel, and take optical pictures of it. For the weight-bearing shooting of the hydrogel, tie a cotton rope and a rubber band to a barbell, then use a vise to clamp the hydrogel sleeved on the rubber band and lift it up. When the barbell is lifted off the ground, take optical pictures of it.

[0073] Figure 8 The optical images of the prepared hydrogel for twisting, bending, folding, and being able to pull a dumbbell weighing 5.0 kg show that the hydrogel can be twisted, bent, and folded well, indicating that it has good elasticity. The hydrogel can bear a barbell weighing 5 kg, indicating that it has good toughness.

[0074] (3) Cytotoxicity test

[0075] The cell compatibility experiment was tested using a CCK-8 kit. The test method includes inoculating L929 cells into a 96-well plate at a cell density of 1×10 4 cells / well. After incubating for 24 and 48 h respectively, add 0.1 g of PVA and PQNF200 hydrogels soaked in serum-free medium 24 h in advance. The pure medium is the negative control group, and the medium containing cells is the positive control group. Continue to culture for 24 h. Then remove the materials and medium, add 100 μL of 10% CCK-8 solution to each well. After incubating for 1 h, place the 96-well plate in an enzyme-labeling instrument to detect the OD value at 450 nm. The calculation results show that the relative cell proliferation rates of the PQNF200 hydrogel prepared in Example 1 are 97.24% and 100.48% respectively (see Figure 9 ), which proves that the hydrogel has good cell compatibility.

[0076] (4) Hemolysis rate test

[0077] Take fresh blood from the abdominal aorta of SD rats into a heparin sodium anticoagulant blood collection tube. Take 3 mL of blood and add it to 30 mL of physiological saline. Centrifuge at a speed of 1500 rpm for 15 minutes and repeat the washing three times until the supernatant is not significantly red. Resuspend the lower-layer red blood cells with physiological saline and dilute to a volume fraction of 2%. Add 5 mL of 2% red blood cell suspension and hydrogel sample (0.1 g) to a 10 mL centrifuge tube. In addition, set up a negative control group as a 2% red blood cell suspension diluted with physiological saline solution, and a positive control group as a 2% red blood cell suspension diluted with sterile water. All samples are incubated at 37 °C for 2 hours, then centrifuged at a speed of 1500 rpm for 15 minutes. Absorb the supernatant and place it in a 1.5 mL centrifuge tube, take pictures and record, and use an ultraviolet spectrophotometer to measure the absorbance of the supernatant at 545 nm and calculate the hemolysis rate. As Figure 10As shown, the hydrogel exhibited an extremely low hemolysis rate, all lower than 5%. It was observed that the red blood cell suspension treated with pure water was red, indicating that the red blood cells were ruptured, while the supernatant of the red blood cell suspension treated with the hydrogel was colorless and transparent, similar to the normal saline group. These results indicated that the PQNF200 hydrogel had good blood compatibility.

[0078] (5) Anti-inflammatory effect

[0079] To simulate a highly inflammatory environment, macrophages were induced using LPS. RAW 264.7 cells were seeded in 24-well plates at a cell density of 2×105 cells / well. After incubation for 24 hours, the original medium was replaced with 500 μL of fresh medium containing 500 ng / mL LPS to induce the cells. At the same time, two groups of cells were additionally intervened with PVA and PQNF200 hydrogel respectively. And a normal medium was set as the blank control group. After co-incubation for 24 hours, the supernatant was discarded, washed twice with PBS, and 500 μL of DCFH-DA solution with a concentration of 100 μM was added to each well, and incubated at 37 °C in the dark for 20 minutes. Subsequently, it was washed twice with PBS, and then photographed and observed under a fluorescence microscope. As Figure 11 shown, the macrophages in the LPS group emitted strong green fluorescence, which meant that a large amount of ROS was produced by RAW264.7 cells under the stimulation of LPS. In contrast, almost no green fluorescence was observed in the PQNF200 group. This indicated that the addition of quercetin self-assembled nanofibers greatly reduced the oxidative stress caused by the respiratory burst of cells.

[0080] (6) Rat tendon replacement experiment

[0081] SD rats were anesthetized by intraperitoneal injection, and the hair on the right hind heel was shaved before surgery. Initially, an incision was made along the midline at the rear of the right heel to fully expose the tendon, creating a 4.0-mm-long gap. Then, the PQNF200 hydrogel prepared in Example 1 was used for rat tendon replacement (see Figure 12 ). The hydrogel sample was sutured to the end of the notch using the modified Kessler method, and then the skin was sutured.

[0082] Before surgery, the body weight of the rats was weighed, and 20% urethane was intraperitoneally injected into the rats at approximately 0.5 mL / 100 g. After the rats were anesthetized, their limbs were fixed on the rat board, the hair on the right hind limb was shaved and disinfected with medical iodine. Under sterile conditions, an approximately 2-cm skin incision was made, the subcutaneous fascia was separated, and the Achilles tendon was exposed. A full defect approximately 4.0 mm long was created at 0.5 cm near the calcaneus using a scalpel. The appropriately sized hydrogel was sutured to the Achilles tendon using the modified Kessler suture method with 6-0 sutures, and then the dermis and epidermal tissues were sutured in sequence with 4-0 sutures. According to Figure 13It can be seen that due to the structure similar to tendon orientation and the corresponding pharmacological activity of quercetin, this hydrogel can more effectively promote tendon injury healing.

Claims

1. A method for preparing a quercetin nanofiber anti-inflammatory hydrogel, characterized in that: The following steps are involved: (1) Dissolve quercetin in dimethyl sulfoxide solvent, add deionized water at a uniform speed after it is fully dissolved, stir at a uniform speed and dialyze to obtain a quercetin self-assembled nanofiber solution; (2) adding polyvinyl alcohol to deionized water and stirring to obtain a polyvinyl alcohol solution; (3) The quercetin self-assembled nanofiber solution was mixed with the polyvinyl alcohol solution, and the resulting mixed solution was subjected to oriented freezing, salting out, pre-stretching and swelling to obtain the quercetin nanofiber anti-inflammatory hydrogel.

2. The method for preparing the quercetin nanofiber anti-inflammatory hydrogel according to claim 1, wherein: In step (1), the concentration of the quercetin self-assembled nanofiber solution is 0.02-1.0 wt%.

3. The method for preparing the quercetin nanofiber anti-inflammatory hydrogel according to claim 1, wherein: In step (1), the molecular weight cut-off of the dialysis is 8000-14000 Da.

4. The method for preparing the quercetin nanofiber anti-inflammatory hydrogel according to claim 1, wherein: In step (2), the mass fraction of the polyvinyl alcohol solution is 10% to 15 wt%, and the stirring speed is 200 to 500 rpm.

5. The method for preparing the quercetin nanofiber anti-inflammatory hydrogel according to claim 1, wherein: In step (3), the mass fraction of quercetin in the quercetin nanofiber anti-inflammatory hydrogel is 0.04-0.5 wt%, and the mass fraction of polyvinyl alcohol is 10-12 wt%.

6. The method for preparing the quercetin nanofiber anti-inflammatory hydrogel according to claim 1, wherein: In step (3), the reagent used for salting out is sodium citrate solution, and the concentration of the sodium citrate solution is 1.0-2.0 mol / L.

7. The method for preparing the quercetin nanofiber anti-inflammatory hydrogel according to claim 1, wherein: In step (3), the pre-stretching length is 50-500%.

8. A quercetin nanofiber anti-inflammatory hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the quercetin nanofiber anti-inflammatory hydrogel according to claim 8 in artificial tendon.

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