Quercetin nanofiber anti-inflammatory hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a quercetin nanofiber anti-inflammatory hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: physically mixing nanofibers formed by self-assembly of a natural flavonoid compound quercetin with polyvinyl alcohol, and then carrying out oriented freezing, salting-out, pre-stretching and swelling strategies to obtain the hydrogel. Wherein due to the introduction of the quercetin nanofibers, the mechanical strength and the more directional network structure of the hydrogel are improved, and the inflammatory reaction caused after the hydrogel is implanted in vivo is effectively relieved based on the excellent anti-oxidation and anti-inflammatory effects of the quercetin nanofibers. Then, salting-out, pre-stretching and swelling are carried out, so that the hydrogel achieves the mechanical property required by the tendon and good biocompatibility, and therefore, the hydrogel can be more effectively applied to regeneration of a 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, a preparation method thereof, and an application thereof. Background Art

[0002] Tendons are a type of dense connective tissue that connects muscles to bones and play functions of force transmission, joint stabilization, and shock absorption in body movement. Among them, tendon tissue has relatively few blood vessels and relatively low cell activity. When it is 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 replacement of damaged tendons with prosthetic devices. However, these methods generally have problems such as donor site morbidity, inflammatory reactions, limited sources of potential disease transmission, and strong immune rejection. Therefore, there is an urgent need for more effective treatment technologies and biomaterials for tendon repair.

[0003] Hydrogels are a type of soft and wet material with a three-dimensional network structure. Through component structure regulation, while endowing them with the required mechanical properties, they also have good flexibility, biocompatibility, etc., making them one of the most popular and representative biomaterials in the field of tissue engineering. However, at present, 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 a dimethyl sulfoxide solvent, and after complete dissolution, uniformly add it to deionized water, stir under uniform conditions, and then 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, and the obtained mixed solution is subjected to directional freezing, salting out, pre-stretching, and swelling to obtain a quercetin nanofiber anti-inflammatory hydrogel.

[0010] In the present invention, a hydrogel is obtained by physically mixing nanofibers formed by self-assembly of the natural flavonoid quercetin with polyvinyl alcohol and then performing 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 about 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, so as to be 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 cut-off molecular weight of the 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 a 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] The present invention also provides a quercetin nanofiber anti-inflammatory hydrogel prepared by the above preparation method. The hydrogel prepared by the present invention has a network structure parallel to tendons, has a strong tensile strength, and has good antioxidant and anti-inflammatory effects and biocompatibility, can promote the anti-inflammatory effect at the tendon defect site, and helps to promote tendon repair.

[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 hydrogen-bonded with the abundant hydroxyl groups on 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-inflammatory. 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 Optical picture and Tyndall effect picture of quercetin self-assembled nanofiber solution.

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

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

[0025] Figure 4 UV picture of quercetin self-assembled nanofiber.

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

[0027] Figure 6 、 7 Stress-strain curve graphs of the hydrogels prepared in the examples and comparative examples.

[0028] Figure 8 Optical images of the hydrogel prepared in Example 1 under torsion, bending, folding and capable of pulling a dumbbell weighing up to 5 kg.

[0029] Figure 9 Results 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 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 mode

[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 complete dissolution at room temperature, add it to deionized water uniformly, and then stir at a constant speed of 600 rpm for 5 h. Place it in a dialysis bag (the cut-off molecular weight 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 red-shifted to 264 nm and 383 nm. The reason for the red-shift of the ultraviolet-visible absorption spectrum of the nanofibers may be due to the π-π stacking in the self-assembly of quercetin.

[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 anti-inflammatory hydrogel of quercetin nanofibers

[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 to form a solid and then taken out, placed in a 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 jig and kept for 10 min and then taken off, and swollen in deionized water for 24 h to remove the ions remaining on the hydrogel to obtain the anti-inflammatory hydrogel of quercetin nanofibers (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 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 to form a solid and then taken out, placed in a 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 monomers were taken and added to deionized water to prepare a quercetin solution. The quercetin solution was added to a 15 wt% 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 1.5 mol / L sodium citrate solution for 24 h for salting out, and then swollen in deionized water for 24 h 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 added to deionized water at a constant speed, and then stirred at a constant speed of 500 rpm for 6 h, and dialyzed in a dialysis bag for 72 h to obtain a quercetin self-assembled nanofiber solution with a concentration of 0.2 wt%.

[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 300 rpm for 1.5 h to obtain a 10 wt% polyvinyl alcohol solution.

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

[0057] 20 mL of the 0.2 wt% quercetin self-assembled nanofiber solution was mixed with 40 g of the 10 wt% polyvinyl alcohol solution. The resulting mixed solution was placed in a silica gel mold, orientationally frozen into a solid, taken out, placed in a 1.0 mol / L sodium citrate solution for 24 hours for salting out, and then stretched on a fixture at a speed of 50 mm / min to 120% and held for 10 min and then taken down, and swollen in deionized water for 24 h 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 added to deionized water at a constant speed, and then stirred at a constant speed of 700 rpm for 4 h, and dialyzed in a dialysis bag for 72 h to obtain a quercetin self-assembled nanofiber solution with a concentration of 0.5 wt%.

[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 resulting mixed solution was placed in a silica gel mold and orientation-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 held for 10 min and then removed, and swollen in deionized water 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 set to 50%, 100%, and 150% respectively, 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 was tested at a tensile speed of 100 mm / min.

[0069] Figure 6 、 7 is the stress-strain curve of the prepared hydrogel. From Figure 6 it can be seen that 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 more than 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] The prepared hydrogel was clamped at both ends with tweezers to twist, bend, and fold the hydrogel, and optical images were taken. The weighted photography of the hydrogel was done by tying a cotton rope and a rubber band to a barbell, and then using a vise to clamp the hydrogel on the rubber band and lift it up, and then taking an optical image after the barbell was off the ground.

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

[0074] (3) Cytotoxicity test

[0075] The cell compatibility test was performed using a CCK-8 kit. The test method included incubating L929 cells at 1×10 4 The cells were inoculated at a density of 100 μL / well in a 96-well plate. After incubation for 24 and 48 hours, 0.1 g of PVA and PQNF200 hydrogels soaked in serum-free medium for 24 hours were added respectively. The simple medium was the negative control group, and the medium containing cells was the positive control group. The culture was continued for 24 hours. Then, the materials and medium were removed, and 100 μL of 10% CCK-8 solution was added to each well. After incubation for 1 hour, the 96-well plate was placed in an ELISA instrument to detect the OD value at 450 nm. The calculation results showed that the relative cell proliferation rates of the PQNF200 hydrogel prepared in Example 1 were 97.24% and 100.48%, respectively (see Figure 9 ), proving that the hydrogel has good cell compatibility.

[0076] (4) Hemolysis rate test

[0077] Fresh blood was collected from the abdominal aorta of SD rats and placed in a sodium heparin anticoagulation blood collection tube. 3 mL of blood was added to 30 mL of normal saline and centrifuged at 1500 rpm for 15 minutes. The blood was washed three times until the supernatant had no obvious red color. The lower layer of red blood cells was resuspended with normal saline and diluted to a volume fraction of 2%. 5 mL of 2% red blood cell suspension and hydrogel sample (0.1 g) were added to a 10 mL centrifuge tube. In addition, a negative control group was set up with a 2% red blood cell suspension diluted with normal saline solution, and a positive control group was set up with a 2% red blood cell suspension diluted with sterile water. All samples were incubated at 37°C for 2 hours, then centrifuged at 1500 rpm for 15 minutes, the supernatant was aspirated and placed in a 1.5 mL centrifuge tube for photographing and recording, and a UV spectrophotometer was used to measure the absorbance of the supernatant at 545 nm and calculate the hemolysis rate. 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 indicate that the PQNF200 hydrogel has good blood compatibility.

[0078] (5) Anti-inflammatory effect

[0079] To simulate a highly inflammatory environment, macrophages were induced with 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 the normal medium was set as the blank control group. After co-incubation for 24 hours, the supernatant was discarded, and the cells were washed twice with PBS. Then 500 μL of DCFH-DA solution with a concentration of 100 μM was added to each well, and the cells were incubated at 37 °C in the dark for 20 minutes. Subsequently, the cells were 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 means 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 indicates that the addition of quercetin self-assembled nanofibers greatly reduces 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, forming 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 a dose of 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 medical iodophor was applied for disinfection. 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 of approximately 4.0 mm in length was created at 0.5 cm near the calcaneus with 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, the 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) dissolving quercetin in dimethyl sulfoxide solvent, adding deionized water at a uniform speed after being fully dissolved, stirring at a uniform speed and dialyzing to obtain a quercetin self-assembled nanofiber solution; (2) adding polyvinyl alcohol into 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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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