Hyaluronic acid particle hydrogel and preparation method and application thereof

Abstract

The application belongs to the technical field of medical biomaterials, and particularly relates to a hyaluronic acid particle hydrogel as well as a preparation method and application thereof. The hyaluronic acid particle hydrogel is prepared by using hyaluronate and 1,4-butanediol diglycidyl ether as raw materials, and by reacting a mixed aqueous solution of the two in an oil phase containing a fluorine surfactant. The prepared hyaluronic acid particle hydrogel has excellent mechanical properties and elasticity, and the anti-enzymatic degradation capability is greatly improved. The hyaluronic acid particle hydrogel can also significantly reduce the generation of aging cells, reduce the expression levels of inflammatory factors IL-6 and IL-1beta in chondrocytes, and play an excellent anti-inflammatory role. Moreover, the preparation method is simple, the conditions are mild, and the method is suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical biomaterials. More particularly, it relates to a hyaluronic acid particle hydrogel, a preparation method and application thereof. BACKGROUND

[0002] Hyaluronic acid (HA) is a natural macromolecular chain polysaccharide and an important component of synovial fluid. In early osteoarthritis (OA) patients, the molecular weight of HA in the joint cavity decreases, and it loses its unique viscoelasticity, resulting in increased wear and tear between cartilages, and ultimately aggravating the development of OA. Studies have found that intra-articular injection of HA can reduce synovial inflammation and maintain synovial viscoelasticity, effectively relieving pain and protecting cartilage.

[0003] However, the current commercial HA supplement (such as ARTZ) has low molecular weight, poor mechanical strength and elasticity, and under the action of hyaluronidase, the in vivo residence time of these HA supplements is short and can only play a limited therapeutic role, thus requiring frequent injections, which may increase the chance of joint infection. To solve the above problems, researchers use crosslinking agents to crosslink HA to form a hydrogel. For example, a crosslinked hyaluronic acid gel is disclosed in a Chinese patent application, which uses hyaluronic acid and 1,4-butanediol diglycidyl ether (BDDE) to react at a low temperature below 0℃. The prepared crosslinked hyaluronic acid gel improves the mechanical strength to some extent, but the highest storage modulus is only 455.62 Pa, the highest loss modulus is only 106.64 Pa, the mechanical strength still needs to be improved, the difference between the storage modulus and the loss modulus is small, which proves that the elasticity is poor and the elasticity also needs to be further improved. In addition, it degrades by 41.72% in 4.5 hours, and the anti-enzymatic effect is poor. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the existing HA hydrogel, which has poor mechanical strength, elasticity and anti-enzymatic effect, and to provide a preparation method of a hyaluronic acid particle hydrogel. The prepared hyaluronic acid particle hydrogel can simultaneously improve its mechanical strength, elasticity and anti-enzymatic effect.

[0005] The purpose of the present application is to provide a hyaluronic acid particle hydrogel.

[0006] Another purpose of the present application is to provide the application of a hyaluronic acid particle hydrogel in the preparation of a drug for treating or preventing osteoarthritis.

[0007] The above purpose of the present application is achieved by the following technical solutions:

[0008] A preparation method of a hyaluronic acid particle hydrogel, comprising the following steps:

[0009] Dissolve hyaluronate in alkaline reagent, add 1,4-butanediol diglycidyl ether to dissolve, pour the obtained liquid as water phase into oil phase containing fluorine surfactant, stir at 35-45℃ until reaction is complete, sieve, and post-treat to obtain;

[0010] The ratio of the addition amount of hyaluronate to 1,4-butanediol diglycidyl ether is 1g:(0.8-1.2)ml.

[0011] The application creatively reacts the mixed aqueous solution of hyaluronate and 1,4-butanediol diglycidyl ether in oil phase containing fluorine surfactant, the fluorine surfactant has amphiphilic property, one end is hydrophilic group and the other end is hydrophobic group, the fluorine surfactant gathers at the boundary between water phase and oil phase, reduces the surface tension of water and the particle size of microspheres, and the microspheres are not easy to agglomerate due to charge problem, so that the obtained HA particle hydrogel has uniform porous structure, forms stable hydrogel, has excellent mechanical strength and elasticity, and has small particle size and uniform dispersion.

[0012] The use of specific addition amount ratio of hyaluronate and 1,4-butanediol diglycidyl ether makes the pores in the obtained HA particle hydrogel compact, thereby increasing the mechanical strength, improving the anti-enzymatic ability, and prolonging the residence time in vivo. If the addition amount of 1,4-butanediol diglycidyl ether is too small, the pores of the obtained HA particle hydrogel are loose, and the mechanical strength and anti-enzymatic ability are low; if the addition amount of 1,4-butanediol diglycidyl ether is too high, the pores of the obtained HA particle hydrogel are too compact, which is not conducive to injection.

[0013] Preferably, the oil phase is fluorine oil or vegetable oil.

[0014] Preferably, the volume ratio of the water phase to the oil phase is 1:10-15.

[0015] Preferably, the fluorine surfactant accounts for 2-6% of the mass of the oil phase.

[0016] Preferably, the fluorine surfactant is C5-18-perfluoroalkyl (FC-40).

[0017] Preferably, the hyaluronate is sodium hyaluronate.

[0018] Preferably, the alkaline reagent is 1-3% NaOH solution or NaHCO3 solution.

[0019] Preferably, the reaction time is 5-7h.

[0020] Preferably, the stirring speed is 300-500rpm.

[0021] The stirring speed is slow, and too low stirring speed cannot make the water phase uniformly dispersed in the oil phase, the microspheres will be oval and have the effect of adhesion, and the dispersion is uneven; too fast stirring speed will reduce the average particle size of the microspheres and reduce the yield of the microspheres in the range of 100-200 μm.

[0022] Preferably, the method of screening is grading screening using screens with different mesh sizes, so that the particle size of the final HA particle hydrogel is 100-200 μm.

[0023] Preferably, the method of post-treatment is washing and centrifugation.

[0024] More preferably, the method of washing is first washing the oil phase and fluorine surfactant on the surface of the hydrogel with perfluorohexyl ethyl alcohol (PFO), and then washing with pure water for 3-5 times.

[0025] The application further protects a hyaluronic acid particle hydrogel prepared by the above method.

[0026] Preferably, the particle size of the hyaluronic acid particle hydrogel is 100-200 μm.

[0027] The application further protects the use of the above hyaluronic acid particle hydrogel in the preparation of a drug for treating or preventing osteoarthritis.

[0028] Preferably, the drug is an injection.

[0029] The application has the following beneficial effects:

[0030] The application uses hyaluronate and 1,4-butanediol diglycidyl ether as raw materials, and reacts the mixed aqueous solution of the two in an oil phase containing a fluorine surfactant, to prepare a hyaluronic acid particle hydrogel with excellent mechanical properties and elasticity, greatly improved anti-enzymatic ability; can also significantly reduce the production of senescent cells, reduce the expression level of cell inflammatory factors IL-6 and IL-1β in chondrocytes, and play an excellent anti-inflammatory role; and the preparation method is simple, the conditions are mild, and is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a schematic diagram of the preparation process of the HA particle hydrogel in Example 1.

[0032] Figure 2 It is a nuclear magnetic resonance hydrogen spectrum diagram of the HA particle hydrogel obtained in the examples and comparative examples.

[0033] Figure 3 It is a test result diagram of the injectability of the HA particle hydrogel obtained in the examples and comparative examples.

[0034] Figure 4Particle size distribution and SEM images of HA particle hydrogels obtained in Examples and Comparative Examples, Figure 4 A is the particle size distribution chart of HA particle hydrogels in Example 1. Figure 4 B is the SEM images of HA particle hydrogels obtained in Examples 1-2 and Comparative Example 1.

[0035] Figure 5 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0036] Figure 6 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0037] Figure 7 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0038] Figure 8 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0039] Figure 9 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0040] Figure 10 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0041] Figure 11 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0042] Figure 12 A is the particle size distribution chart of HA particle hydrogels in Example 1.

[0043] Figure 13The figure shows the test results of the effect of HA particle hydrogel treatment on the number of SA-β-Gal positive cells in mouse cartilage tissue. Figure 13 A is an image of mouse cartilage tissue after SA-β-Gal staining following treatment with HA particle hydrogel; Figure 13 B is a statistical graph showing the number of SA-β-Gal positive cells in mouse cartilage tissue by HA particle hydrogel.

[0044] Figure 14 The figure shows the test results of the effect of HA particle hydrogel on the expression level of target genes in mice. Figure 14 A is for pl6 INK4a The influence of genes, Figure 14 B represents the effect on the P21 gene. Figure 14 C represents the effect on Nrf2, COX-2, and SOD. Figure 14 D represents the effect on IL-6 and IL-1β.

[0045] Figure 15 The figure shows the test results of the effect of HA particle hydrogel on the TLR-2 / NF-KB pathway. Figure 15 A and Figure 15 B represents p16 in chondrocytes under TNF-α stimulation or non-stimulation. INK4a TLR-2, NF-κBp65 and NF-κB p-p65 levels, Figure 15 C represents Western blot images of TLR-2 in chondrocytes induced by different concentrations of TNF-α. Figure 15 D is Figure 15 A and Figure 15 Quantitative analysis results of B.

[0046] Figure 16 The figure shows the test results of the effect of HA particle hydrogel on the expression of genes related to cartilage synthesis and degradation in mouse cartilage tissue cells. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] The fluorinated oil (PicoSurf-1) contains FC-40, which accounts for 5% of the fluorinated oil mass, and was purchased from Sphere Fluidics.

[0050] Preparation of hyaluronic acid particles hydrogel with 1 vol% BDDE content (1 n-HA)

[0051] Add 13.3 g of sodium hyaluronate powder into 100 ml of 1% NaOH solution, then add 1 ml of crosslinking agent BDDE, pour the fully dissolved liquid as the dispersed phase (aqueous phase) into 1 L of PicoSurf-1, react in a 40°C water bath, and stir at a constant speed of 400 rpm using a stirrer, keep stirring for 6 h; after the reaction is completed, use sieves of different mesh sizes to grade the obtained HA gel particles, and sieve out the gel particles between 100-200 pm; use pfo to clean the fluorine oil and fluorine surfactant on the surface of the gel particles, then wash the gel microspheres with pure water for 3-5 times, and centrifuge to obtain (the preparation process is shown in the schematic diagram Figure 1 ).

[0052] Preparation of hyaluronic acid particles hydrogel with 0.8 vol% BDDE content (0.8 n-HA)

[0053] Add 13.3 g of sodium hyaluronate powder into 100 ml of 1% NaOH solution, then add 0.8 ml of crosslinking agent BDDE, pour the fully dissolved liquid as the dispersed phase (aqueous phase) into 1 L of PicoSurf-1, react in a 40°C water bath, and stir at a constant speed of 400 rpm using a stirrer, keep stirring for 6 h; after the reaction is completed, use sieves of different mesh sizes to grade the obtained HA gel particles, and sieve out the gel particles between 100-200 pm; use pfo to clean the fluorine oil and fluorine surfactant on the surface of the gel particles, then wash the gel microspheres with pure water for 3-5 times, and centrifuge to obtain.

[0054] Preparation of hyaluronic acid particles hydrogel with 0.5 vol% BDDE content (0.5 n-HA)

[0055] Add 13.3 g of sodium hyaluronate powder into 100 ml of 1% NaOH solution, then add 0.5 ml of crosslinking agent BDDE, pour the fully dissolved liquid as the dispersed phase (aqueous phase) into 1 L of PicoSurf-1, react in a 40°C water bath, and stir at a constant speed of 400 rpm using a stirrer, keep stirring for 6 h; after the reaction is completed, use sieves of different mesh sizes to grade the obtained HA gel particles, and sieve out the gel particles between 100-200 pm; use pfo to clean the fluorine oil and fluorine surfactant on the surface of the gel particles, then wash the gel microspheres with pure water for 3-5 times, and centrifuge to obtain.

[0056] Preparation of hyaluronic acid particles hydrogel with 1.5 vol% BDDE content (1.5 n-HA)

[0057] The 13.3 g of sodium hyaluronate powder was added into 100 ml of 1% NaOH solution, and 0.5 ml of crosslinking agent BDDE was added. The well-dissolved liquid was poured into 1 L of PicoSurf-1 as the dispersed phase (aqueous phase), and reacted in a 40°C water bath while stirring with a stirrer at a constant speed of 400 rpm. The stirring reaction was maintained for 6 h. After the reaction was completed, the obtained HA gel particles were classified and screened using sieves of different mesh sizes, and the gel particles between 100-200 μm were screened out. The fluorine oil and fluorine surfactant on the surface of the gel particles were cleaned using pfo, and then the gel microspheres were washed 3-5 times with pure water, and centrifuged to obtain the product.

[0058] Structural characterization:

[0059] The hyaluronic acid particle hydrogels obtained in Examples 1-2 and Comparative Examples 1-2 were characterized by nuclear magnetic resonance hydrogen spectrum, as shown in Figure 2 : the hydrogen spectrum peaks corresponded to the structure one by one, proving the successful synthesis.

[0060] Example 3: Study on the injectability of hyaluronic acid particle hydrogel

[0061] The hyaluronic acid particle hydrogels obtained in Examples 1-2 and Comparative Examples 1-2 were filled into a syringe and extruded with a 260 μm needle, and the results are shown in Figure 3 .

[0062] As shown in Figure 3 : 1 n-HA of Example 1, 0.8 n-HA of Example 2, and 0.5 n-HA of Comparative Example 1 can be well extruded, while 1.5 n-HA of Comparative Example 2 cannot be extruded from a 260 μm needle, but can only be extruded through a 0.2 cm diameter syringe, and does not have injectability.

[0063] Since 1.5 n-HA of Comparative Example 2 does not have injectability, subsequent experiments were carried out using 1 n-HA of Example 1, 0.8 n-HA of Example 2, and 0.5 n-HA of Comparative Example 1.

[0064] Example 4: Study on the morphology and mechanical properties of hyaluronic acid particle hydrogel

[0065] Particle size test: The particle size distribution of 1 n-HA obtained in Example 1 was detected, and the results are shown in Figure 4 A, the particle size distribution of 1 n-HA was uniform, within the range of 90-150 nm.

[0066] Morphology test: The morphology of n-HA obtained in Examples 1-2 and Comparative Example 1 was observed by 10 kV electron scanning microscope (SEM).

[0067] After the sample reached swelling equilibrium, it was freeze-dried in liquid hydrogen for 72 h, the dried sample was taken out, a layer of ultra-thin gold layer was sprayed on the surface of the sample, and then the cross-sectional morphology of the freeze-dried gel was observed by SEM, and the results are shown in Figure 4 B.

[0068] As shown in Figure 4 B: all gels have a uniform porous structure, but the pores of 0.5 n-HA are loose and not tight, and the pore structure of 0.8 n-HA and 1.0 n-HA is relatively tight. Since the dense pore structure can increase the mechanical strength of the hydrogel, 0.8 n-HA and 1.0 n-HA have good mechanical strength, and 1 n-HA has the highest mechanical strength.

[0069] Measurement of rheological parameters modulus: The n-HA obtained in Examples 1-2 and Comparative Example 1 and ARTZ (a commercial hyaluronic acid product, used as a control) were measured for rheological parameters modulus by using a Hake rotary rheometer.

[0070] The test temperature was 25°C, the vibration frequency was 1 Hz, a rotor with a diameter of 8 mm was used, and the measurement was carried out in the strain range of 0.1-1000%, and the results are shown in Figure 5 .

[0071] As shown in Figure 5 : the loss modulus (G") and the storage modulus (G') of ARTZ are both small, and G" is greater than G', indicating that ARTZ injection tends to be in a mucous state, has poor viscoelasticity, and has weak mechanical strength. Although the G" and G' of 0.5 n-HA have a certain improvement, G' is greater than G', but its G' is still small, and the difference between G" and G' is small, and the elasticity is poor. The G' of 0.8 n-HA and 1.0 n-HA of the present application is about 1000 pa, and the difference between G" and G' is one order of magnitude, which proves that the 0.8 n-HA and 1.0 n-HA prepared in Examples 1-2 of the present application have excellent mechanical strength and elasticity, and can better improve the viscoelasticity of synovial fluid in bone joints.

[0072] Enzymatic resistance of hyaluronic acid particle hydrogel of Example 5

[0073] In order to simulate the state of the human body, the obtained HA particle hydrogel was placed in physiological saline for 4 h to change the swelling material in the microspheres from pure water to physiological saline, and the following experiments were all carried out on the HA particle hydrogel treated with physiological saline.

[0074] 2g 0.5%n-HA, 0.8%n-HA, 1.0%n-HA or ARTZ was added to 2 mL PBS buffer, 300 U / mL hyaluronidase was added, the solution was placed in a 37 °C shaker at 150 rpm, and 100 uL of supernatant was taken at 2h, 4h, 8h, 12h, 24h, 2day, 3day, 4day, 5day, 6day, 7day, respectively, 20 uL of carbazole reagent was added, heated in a boiling water bath for 15 min, cooled to room temperature, and the absorbance was measured by UV spectrophotometry to obtain the concentration of glucuronic acid, and the concentration of hyaluronic acid in each time period was determined according to the carbazole sulfate formula, and then multiplied by the volume of the solution to obtain the weight of n-HA degradation; the time was taken as the abscissa and the degradation rate as the ordinate to plot a graph, as shown in Figure 6 .

[0075] Carbazole sulfate formula: hyaluronic acid concentration = glucuronic acid concentration x n (n is the ratio of the molecular weight of hyaluronic acid to glucuronic acid)

[0076] The results are shown in Figure 6 : ARTZ was almost completely degraded after 4 days, 0.5%n-HA was almost completely degraded after 24h, and 1%n-HA had a degradation rate of only 27.65% after 24h, and was completely degraded only after 7 days, indicating that 1%n-HA has good anti-enzymatic ability.

[0077] Example 6 Study on the in vivo treatment effect of hyaluronic acid particle hydrogel on mouse osteoarthritis

[0078] C57BL / 6 mice were randomly divided into 5 groups, 6 in each group, and a DMM surgical model was selected to construct an early OA model. For the treatment study, a microsyringe was used to inject 10 μl of saline (Saline), 1.0%n-HA or ARTZ into the joint each time, and the first injection was performed two weeks after the operation. Among them, saline and ARTZ were injected once a week, a total of 4 times, while n-HA was injected only once. The sham group (Sham) of mice did not have arthritis, only the knee joint of the mouse was exposed, and then sutured to recover. The OA group was an early OA model without injection of any substance.

[0079] The mouse tissues were observed after Safrani O / Fast Green staining and Toluidine Blue staining. Safrani O / Fast Green staining can reflect the condition of articular cartilage in the tissue, and the larger the red area, the better the cartilage structure; Toluidine Blue staining can reflect the content of proteoglycans in the tissue, and the deeper the blue color, the higher the content of proteoglycans.

[0080] As shown in Figure 7 :

[0081] The thickness of the articular cartilage was reduced and the articular surface was destroyed in the OA group and the saline group compared with the sham group. However, the morphology and structure of the articular cartilage were improved after the intra-articular injection of 1 n-HA and ARTZ, and the surface was smoother. The effect of the injection of 1 n-HA was more obvious.

[0082] The blue area was less and the content of proteoglycan was extremely low in the OA group and the saline group. However, the blue area was increased and the content of proteoglycan was significantly increased after the injection of ARTZ and 1 n-HA. The effect of the injection of 1 n-HA was similar to that of the injection of ARTZ four times, which proved that the effect of 1 n-HA was better.

[0083] The histopathology of each group of mice was evaluated by OARSI scoring, and the results are shown in Figure 8 Compared with the OARSI score of the OA group, the score of 1 n-HA was significantly decreased, and the degree of decrease was more than that of the injection of ARTZ four times, which proved that the treatment effect of 1 n-HA was the best.

[0084] The cartilage thickness and proteoglycan content of the knee joint sections of the mice treated in different groups were analyzed by software and statistically analyzed, as shown in Figure 9 The cartilage thickness data statistical results show (left graph) that compared with the sham group, the articular cartilage thickness of the OA group was reduced, indicating that the articular surface was destroyed. The articular cartilage thickness was significantly increased after the intra-articular injection of 1 n-HA and ARTZ, and the structure of the articular cartilage was significantly improved. The proteoglycan statistical results show (right graph) that after the treatment of 1 n-HA and ARTZ, more proteoglycan (blue) was retained in the articular cartilage matrix, and the content of proteoglycan was significantly increased compared with the OA group.

[0085] The mice treated in different groups were tested by MicroCT, as shown in Figure 10 There was no bone sclerosis phenomenon after the treatment of ARTZ and 1 n-HA, and the thickness of the subchondral bone plate was reduced, and the effect of 1 n-HA was better. The red arrow indicates the subchondral bone sclerosis around the medial tibial plateau; the red line indicates the thickness of the subchondral bone plate.

[0086] The content of cartilage-related substances was statistically analyzed by MicroCT related software, as shown in Figure 11As shown: compared with the sham operation group, the OA group and the saline group showed higher subchondral bone plate thickness, bone mineral density (BMD), bone volume density (BV / TV), subchondral bone trabecular thickness (Tb.Th) and trabecular number (Tb.N), and in addition, the bone trabecular separation (Tb.sp) of the OA group and the saline group was significantly lower than that of the sham operation group, and these OA-related subchondral bone abnormalities were alleviated after treatment with n-HA or ARTZ, while there was no significant difference in these histomorphometric parameters between the two treatment groups. In the experiment, ARTZ was injected 4 times, while 1 n-HA was injected only once, and the effects of the two were similar, proving that the 1 n-HA of the present application has better effect.

[0087] Example 7 Study on the in vivo safety of hyaluronic acid particle hydrogel

[0088] C57BL / 6 mice were randomly divided into 5 groups, 6 in each group, and an early OA model was constructed by selecting a DMM operation model. For the treatment study, 10 μl of saline, 1 n-HA or ARTZ was injected intra-articularly each time using a microsyringe, and the first injection was performed two weeks after the operation. Among them, the saline and ARTZ were injected once a week, a total of 4 times, while the 1 n-HA was injected only once. The organs of the heart (Heart), liver (Liver), spleen (Spleen), lung (Lung) and kidney (Kidney) of each group of mice were observed under a microscope after H&E staining section. The OA model group was not injected with anything as the control group (Control) group.

[0089] As shown: Figure 12 Each organ of each group had clear texture, and there were no obvious pathological features such as inflammation, edema and necrosis, indicating that 1 n-HA had ideal biocompatibility and no cytotoxicity in vivo.

[0090] Example 8 Effect of hyaluronic acid particle hydrogel on cell aging and cell inflammation

[0091] A tumor necrosis factor (TNF-α) of 20 ng / ml was used to culture mouse chondrocytes for 24 h, and normal chondrocytes were stimulated with TNF-α (100 ng / ml) for 48 h to induce chondrocyte aging, thereby constructing an in vitro cell aging model.

[0092] The cell aging condition was detected by the senescence-associated-β-galactosidase (SA-β-Gal) staining test. The senescence-associated-β-galactosidase (SA-β-Gal) staining test method was as follows: the chondrocytes were plated at a density of 5×10 3Cells were seeded at a density of 100 cells / well in 96-well plates and cultured overnight. After the addition of TNF-α, 1 mg / mL of 1% n-HA or ARTZ was added to the cell culture medium. After 24 h, chondrocytes were washed twice with PBS according to the manufacturer's protocol, then fixed with fixative for 15 min at room temperature. After washing, staining solution was added, and the cells were incubated at 37 °C. The number of SA-β-Gal positive cells in three random regions was recorded, and the average count was calculated. The blank group (Blank) was treated with only TNF-α, and the control group (Control) received no treatment for normal chondrocytes (no TNF-α stimulation or other substances were added). The results are as follows: Figure 13 .

[0093] like Figure 13 As shown: Compared with the control group, the number of SA-β-Gal positive chondrocytes was significantly increased in the blank group, while the addition of 1% n-HA or ARTZ reduced the number of SA-β-Gal positive cells, with 1% n-HA showing a more significant reduction. Figure 13 A) Statistical analysis of positive cells, such as... Figure 13 B, and Figure 13 The result for A is consistent.

[0094] The effect of n-HA on the expression level of the target gene was analyzed using real-time quantitative polymerase chain reaction (qRT-PCR), and the results are as follows: Figure 14 .

[0095] like Figure 14 As shown: the presence of 1% n-HA or ARTZ significantly reversed the increase in p21 expression levels in chondrocytes after TNF-α treatment. Figure 14 B) More importantly, compared to ARTZ, 1% n-HA can also inhibit p16. INK4a The expression ( Figure 14 A) promotes the expression of the Nrf2 gene in chondrocytes. Figure 14 C); Furthermore, compared with ARTZ, 1% n-HA significantly enhanced both its inhibitory effect on COX-2 and its ability to promote SOD expression levels. Figure 14 C). Subsequently, the expression levels of pro-inflammatory cytokines in chondrocytes after different treatments were measured. Compared to the control group, the expression levels of IL-6 and IL-1β decreased after treatment with 1% n-HA. Notably, compared to ARTZ, the expression levels of IL-6 and IL-1β in chondrocytes were also significantly decreased after n-HA treatment. Figure 14 D).

[0096] In summary, n-HA can significantly reduce oxidative stress in chondrocytes and inhibit inflammatory responses by preventing cell senescence.

[0097] Effect of hyaluronic acid particle hydrogel on TLR-2 / NF-KB pathway

[0098] Chondrocytes were seeded in 96-well plates at a density of 5 x 10 3 cells / well and cultured overnight. The group without TNF-α was the control group (Control), the group without treatment after the addition of TNF-α was the blank group (Blank), and the group with 1 n-HA at 1 mg / mL in the cell culture medium was the 1 n-HA group. After 24 h, the chondrocytes were washed twice with PBS, and the protein levels of p16 INK4a , TLR-2, NF-κB p-p65, NF-κB p65, and related proteins in the senescent cells were detected by Western blotting using GAPDH as an internal standard. The expression of genes related to cartilage synthesis and decomposition in the cells in each group was also detected, and the results are shown in Figure 15 .

[0099] As shown in Figure 15 , Figure 15 A shows that 1 n-HA significantly reduces the protein level of p16 INK4a in senescent cells, Figure 15 B shows that 1 n-HA significantly reduces the protein level of TLR-2 in senescent cells and inhibits the activation of NF-κB, indicating that n-HA plays an anti-inflammatory role by inhibiting the expression level of TLR-2.

[0100] The protein contents of p16 INK4a , TLR-2, NF-κB p-p65 / NF-κB p65 (p-p65 / p65) in the senescent cells after treatment in each group were statistically analyzed, and the results are shown in Figure 15 D, which is consistent with the above results.

[0101] TNF-α solutions with concentrations of 20, 100, and 150 ng / mL were also tested, and the results are shown in Figure 15 C: The expression level of TLR-2 in chondrocytes increases with the increase in the concentration of the pro-inflammatory cytokine TNF-α solution.

[0102] Total RNA was extracted from the cultured chondrocytes or articular cartilage using an RNA extraction kit (Qiagen, Germany), cDNA was transcribed from the RNA sample using a reverse transcription reagent (Roche, China), and qRT-PCR detection was performed to quantify the mRNA expression levels of these genes.

[0103] As shown in Figure 16After the treatment of TNF-α, the expression levels of MMP3, MMP13 and ADAMTS-5 in the senescent chondrocytes were significantly increased, and the expression levels of COL2A1 and Aggrecan were significantly decreased. After the addition of 1 n-HA, the expression levels of MMP3, MMP13 and ADAMTS-5 were effectively reduced, and the expression levels of COL2A1 and Aggrecan were effectively increased.

[0104] In summary, 1 n-HA can block the TLR-2 / NF-KB signaling pathway, alleviate the senescence of chondrocytes, maintain the balance of cartilage catabolism, and reduce the progression of OA.

[0105] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A method for preparing a hyaluronic acid particle hydrogel, characterized by, It comprises the following steps: The hyaluronic acid is dissolved in an alkaline reagent, 1,4-butanediol diglycidyl ether is added and dissolved, the obtained liquid is poured into an oil phase containing a fluorine surfactant as an aqueous phase, stirring is carried out at 35-45℃ until the reaction is completed, screening is carried out, and post-treatment is carried out to obtain the product. The ratio of the addition amount of the hyaluronic acid to 1,4-butanediol diglycidyl ether is 1g:(0.8-1.2)ml. The volume ratio of the aqueous phase to the oil phase is 1:10-15.

2. The preparation method according to claim 1, characterized in that, The oil phase is fluorine oil or vegetable oil.

3. The preparation method according to claim 1, characterized in that, The fluorine surfactant accounts for 2-6% of the mass of the oil phase.

4. The preparation method according to claim 3, characterized in that, The fluorine surfactant is C5-18-perfluoroalkane.

5. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. The hyaluronic acid is sodium hyaluronate or potassium hyaluronate.

6. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. The alkaline reagent is a 1-3% NaOH solution or a NaHCO3 solution.

7. A hyaluronic acid particle hydrogel, characterized in that, It is prepared by the method of any one of claims 1-6.

8. The hyaluronic acid particle hydrogel of claim 7, wherein The particle size of the hyaluronic acid particle hydrogel is 100-200μm.

9. The use of the hyaluronic acid particle hydrogel of claim 7 or 8 in the preparation of a drug for treating or preventing osteoarthritis.

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