ROS (reactive oxygen species) response type liposome hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ROS (reactive oxygen species) response type liposome hydrogel as well as a preparation method and application thereof, and the preparation method comprises the following steps: sequentially preparing drug-loaded liposome, phenylboronic acid modified hyaluronic acid and ROS response type hydrogel, and mixing the drug-loaded liposome and the ROS response type hydrogel to obtain the ROS response type liposome hydrogel. According to the preparation method disclosed by the invention, through surface modification of the cartilage affinity peptide and ROS responsive hydrogel encapsulation, the targeting capability and the retention capability of the medicines resveratrol and chondroitin sulfate are successfully improved, and the purpose of responsive release of the medicines is achieved. According to the ROS response type liposome hydrogel, accumulation of ROS in cells can be reduced, and the inflammatory mediator and matrix degrading enzyme level can be lowered; meanwhile, cartilage matrix degradation and subchondral bone change can be inhibited, development of osteoarthritis is delayed, and the novel delivery system for osteoarthritis treatment has huge potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a ROS-responsive liposome hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Osteoarthritis (OA) is a common chronic disease characterized by degeneration of articular cartilage and persistent inflammation of the joint capsule, which seriously endangers the health of the elderly worldwide. The goal of osteoarthritis treatment is to control pain and improve joint function through physical, drug or surgical treatment. Among them, drug therapy, such as anti-inflammatory drugs, analgesics, etc., is an effective way for early osteoarthritis treatment due to its high acceptance rate, low treatment cost, good treatment effect and high safety. However, frequent use of oral or intravenous drugs to relieve inflammation and pain can cause new problems, such as missed doses, overdoses, drug resistance, and even serious gastrointestinal reactions, especially for the elderly. In order to solve these problems, people have developed safer and more convenient drug delivery methods, among which microcarrier-based drug delivery systems using different types of polymers as elements have received extensive attention. Although many successes have been achieved, most drug delivery microcarriers use exogenous materials, which can cause side effects such as inflammatory reactions, local discomfort and tissue adhesion. In addition, common drug delivery microcarriers are usually prepared by expensive equipment or complex chemical synthesis, resulting in high cost, unsatisfactory biocompatibility and poor drug treatment effect. Therefore, drug release systems for osteoarthritis treatment are still worth looking forward to.

[0003] Liposomes have been widely used as drug carriers, with hydrophobic drugs encapsulated in the phospholipid shell and hydrophilic drugs encapsulated in the core. As a drug delivery carrier, liposomes have good biocompatibility, good drug stability and sustained release characteristics; and intra-articular administration can increase the local drug concentration while minimizing systemic adverse reactions, making it one of the most attractive methods for treating osteoarthritis. However, unmodified liposomes still have the following disadvantages: relatively easy to be removed by the reticuloendothelial system in vivo, leading to rapid drug elimination; easy to aggregate into large bilayer membranes, resulting in the loss of the advantages of liposome nanostructure; small size liposomes are easily cleared by synovial fluid, and to ensure the effective drug amount, it is inevitable to increase the injection frequency, which can easily increase the incidence of adverse events such as pain, joint bleeding and infection. Microhydrogels have the characteristics of biocompatibility, mechanical stability and small invasion through syringe needles, and using liposomes encapsulated in hydrogel matrix networks is an effective strategy to protect liposomes from being rapidly eliminated in vivo, thereby promoting long-term local administration of liposomes in a controlled and sustained manner. Recent studies have shown that composite carriers of liposomes and hydrogel microspheres can combine the advantages of different material forms, showing stronger plasticity and flexibility than traditional single carriers, and are expected to become a new type of local drug delivery system.

[0004] Normal levels of reactive oxygen species (ROS) can regulate physiological processes of chondrocytes as second messengers, such as cell cycle, gene expression, cell signal transduction, etc. However, oxidative stress caused by excessive production of ROS is closely related to the pathogenesis of OA, which is considered to be the potential culprit of OA because it can modify lipids and proteins in cells, damage DNA, and cause other adverse reactions. In addition, the increase of ROS level is closely related to the pathological manifestations of osteoarthritis, such as matrix metalloproteinases, degradation of extracellular cartilage matrix, synovitis, chondrocyte apoptosis, etc. In addition, long-term ROS stimulation not only activates inflammatory signaling pathways such as nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) in chondrocytes, but also leads to the sustained activation of inflammasomes in synoviocytes, resulting in the formation of local inflammatory cytokine storm. In view of the above, the excessive production of ROS may eventually hinder tissue repair and aggravate degradation. A number of studies have observed that reducing excessive ROS can reduce OA synovitis and cartilage damage. Therefore, how to obtain a ROS-responsive liposome hydrogel has a promising significance for drug delivery treatment of osteoarthritis. SUMMARY

[0005] The technical problem solved by the present application is to overcome the shortcomings of the prior art and provide a ROS-responsive liposome hydrogel for realizing response release of drugs and a preparation method and application thereof.

[0006] To solve the above technical problems, the present application adopts the following technical solutions.

[0007] The preparation method of the ROS-responsive liposome hydrogel comprises the following steps:

[0008] S1, dissolving WYRGRL polypeptide and tris (2-carboxyethyl) phosphine in a phosphate buffer solution, stirring, adding phospholipid polyethylene glycol maleimide, stirring again, dialysis, freeze-drying to obtain DSPE-PEG-WYRGRL;

[0009] S2, mixing DSPE-PEG-WYRGRL obtained in step S1, film-forming material, resveratrol and organic solvent, adding edge activator, evaporating to obtain liposome membrane; dissolving chondroitin sulfate in water and adding into the liposome membrane, rotating the membrane, ultrasonic crushing, dialysis to obtain drug-loaded liposome; the film-forming material is at least one of soybean lecithin, phosphatidylserine, dimyristoyl lecithin and dipalmitoyl phosphatidylcholine, and the edge activator is at least one of Tween 80 and Span 80;

[0010] S3, dissolving hyaluronic acid in water, adding an activating agent, stirring, adding 3-aminobenzene boronic acid, and reacting, and then dialyzing, freeze-drying to obtain benzene boronic acid modified hyaluronic acid; the activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

[0011] S4, mixing the benzene boronic acid modified hyaluronic acid obtained in step S3 and a polyvinyl alcohol solution, and adding a basic solution to obtain a ROS-responsive hydrogel;

[0012] S5, mixing the drug-loaded liposome obtained in step S2 and the ROS-responsive hydrogel obtained in step S4 to obtain a ROS-responsive liposome hydrogel.

[0013] The preparation method, preferably, in step S1, the mass ratio of the WYRGRL polypeptide, tris(2-carboxyethyl) phosphine, phospholipid polyethylene glycol maleimide is 1-10:10:10.

[0014] The preparation method, preferably, in step S2, the mass ratio of the DSPE-PEG-WYRGRL, film-forming material, edge activator, resveratrol, chondroitin sulfate is 1:100:20:5:1-10.

[0015] The preparation method, preferably, in step S3, the mass ratio of 3-aminobenzene boronic acid, hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide is 1-5:50:20:20.

[0016] The preparation method, preferably, in step S4, the volume ratio of the benzene boronic acid modified hyaluronic acid, polyvinyl alcohol solution, basic solution is 100:10:1, the concentration of the polyvinyl alcohol solution is 10mg / mL-30mg / mL, the concentration of the basic solution is ≤1mol / L, and the basic solution is sodium hydroxide solution.

[0017] The preparation method, preferably, in step S5, the volume ratio of the drug-loaded liposome and the ROS-responsive hydrogel is 1:80-120.

[0018] The preparation method, preferably, in step S1, the concentration of the phosphate buffer solution is ≤1mol / L, the stirring time is 1h-4h, the second stirring time is 2h-8h, and the dialysis time is 12h-36h.

[0019] Preferably, in the step S2, the organic solvent is at least one of ethanol and chloroform, the evaporation temperature is 35-50 DEG C, the particle size of the drug-loaded liposome is 80-110 nm, the polydispersity index of the drug-loaded liposome is 0.29-0.46, and the Zeta potential of the drug-loaded liposome is -36 mV to -21 mV.

[0020] Preferably, in the step S3, the stirring time is 2-8 h, the reaction time is 12-36 h, and the dialysis time is greater than or equal to 3 days.

[0021] As a general technical concept, the application further provides a ROS-responsive liposome hydrogel prepared by the preparation method of the ROS-responsive liposome hydrogel.

[0022] As a general technical concept, the application further provides an application of the ROS-responsive liposome hydrogel in preparation of an osteoarthritis drug.

[0023] Compared with the prior art, the application has the following advantages:

[0024] The application provides a preparation method of a ROS-responsive liposome hydrogel. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The nuclear magnetic resonance spectrum of WYRGRL polypeptide, phospholipid polyethylene glycol maleimide (DSPE-PEG-MAL) and DSPE-PEG-WYRGRL in Example 1 of the application.

[0026] Figure 2 The transmission electron microscope image of the drug-loaded liposome (TFs@Res+Chs) in Example 1 of the application.

[0027] Figure 3Particle size diagram of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), drug-loaded liposome (TFs@Res+Chs) in Example 1 of the present application.

[0028] Figure 4 Potential diagram of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), drug-loaded liposome (TFs@Res+Chs) in Example 1 of the present application.

[0029] Figure 5 NMR spectrum of hyaluronic acid (HA), 3-aminobenzoic acid (PBA), and benzene boronic acid-modified hyaluronic acid (HA-PBA) in Example 1 of the present application.

[0030] Figure 6 UV spectrum diagram of hyaluronic acid (HA), 3-aminobenzoic acid (PBA), benzene boronic acid-modified hyaluronic acid (HA-PBA), polyvinyl alcohol (PVA), and ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0031] Figure 7 FTIR spectrum diagram of hyaluronic acid (HA), 3-aminobenzoic acid (PBA), benzene boronic acid-modified hyaluronic acid (HA-PBA), polyvinyl alcohol (PVA), and ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0032] Figure 8 Frequency scanning G' and G" diagram of ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0033] Figure 9 Viscosity curve diagram of ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0034] Figure 10 Amplitude scanning curve diagram of ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0035] Figure 11 Time scanning diagram of ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0036] Figure 12 Actual diagram of ROS-responsive hydrogel (HA-PBA-PVA) in Example 1 of the present application.

[0037] Figure 13The SEM images of ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in Example 1 of the present application before and after H2O2 incubation treatment.

[0038] Figure 14 The release curve of Res of drug-loaded liposome (TFs@Res+Chs) and ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in Example 1 of the present application under different conditions (pH 7.4, pH 6.5, pH 6.5+1mM H2O2).

[0039] Figure 15 The release curve of Chs of drug-loaded liposome (TFs@Res+Chs) and ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in Example 1 of the present application under different conditions (pH 7.4, pH 6.5, pH 6.5+1mM H2O2).

[0040] Figure 16 The cumulative release amount graph of Res of ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in Example 1 of the present application under different H2O2 concentrations (0, 0.25, 0.50, 0.75, 1.00, 2.00mM).

[0041] Figure 17 The cumulative release amount graph of Chs of ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in Example 1 of the present application under different H2O2 concentrations (0, 0.25, 0.50, 0.75, 1.00, 2.00mM).

[0042] Figure 18 The in vitro clearance experiment result graph of ABTS and DPPH free radicals of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) in Example 1 of the present application.

[0043] Figure 19 The clearance rate data graph of ABTS and DPPH free radicals of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) in Example 1 of the present application.

[0044] Figure 20 The cytotoxicity effect graph of blank liposome (TFs) of different concentrations on chondrocytes in Example 1 of the present application.

[0045] Figure 21The cytotoxic effect diagram of ROS-responsive hydrogel (HA-PBA-PVA) of different concentrations in Example 1 of the present application on chondrocytes.

[0046] Figure 22 The repair ability effect diagram of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) on chondrocyte damage (H2O2) in Example 1 of the present application.

[0047] Figure 23 The repair ability effect diagram of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) on chondrocyte damage (LPS) in Example 1 of the present application.

[0048] Figure 24 The influence effect diagram of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) on ROS level in mouse ATDC5 cells in Example 1 of the present application.

[0049] Figure 25 The influence effect diagram of blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) on pro-inflammatory cytokine secretion and matrix synthesis in mouse ATDC5 cells in Example 1 of the present application.

[0050] Figure 26 The representative diagram of OA mouse knee joint at different time periods after injection of ROS-responsive liposome hydrogel in Example 1 of the present application.

[0051] Figure 27 The histopathology H&E staining diagram of mouse knee joint cartilage after treatment in Example 1 of the present application.

[0052] Figure 28 The H&E staining result diagram of main organs of mice after treatment in Example 1 of the present application. DETAILED DESCRIPTION

[0053] The present application will be further described below in conjunction with the drawings of the specification and specific preferred embodiments, but the protection scope of the present application is not limited thereby. The materials and instruments used in the following examples are all commercially available.

[0054] Example 1:

[0055] A preparation method of the ROS-responsive liposome hydrogel of the present application, comprising the following steps:

[0056] S1, preparation of DSPE-PEG-WYRGRL

[0057] 4 mg of WYRGRL polypeptide and 10 mg of tris(2-carboxyethyl)phosphine (TCEP) were dissolved in 5 mL of phosphate buffer solution (PBS solution) with a concentration of 0.01 M and pH = 7.4, and stirred at room temperature for 2 h; then, 10 mg of phospholipid polyethylene glycol maleimide (DSPE-PEG-MAL) was added and stirred at room temperature for 4 h; subsequently, it was placed in a MD44 (3500 MWCO) dialysis bag with PBS solution (0.01 M, pH = 7.4) and dialyzed for 24 h, with water change every 4 h; after dialysis, the liquid was freeze-dried to obtain DSPE-PEG-WYRGRL. The successful synthesis of DSPE-PEG-WYRGRL was confirmed by nuclear magnetic hydrogen spectrum, i.e. Figure 1

[0058] S2, preparation of drug-loaded liposomes

[0059] 1 mg of DSPE-PEG-WYRGRL, 100 mg of soybean lecithin, 20 mg of edge activator Tween 80 and 5 mg of resveratrol (Res) were dissolved in 25 mL of chloroform and evaporated at 40°C to obtain a dry liposome membrane; 4 mg of chondroitin sulfate (Chs) was dissolved in 10 mL of deionized water to obtain a chondroitin sulfate aqueous solution; subsequently, the chondroitin sulfate aqueous solution was added to the liposome membrane, and rotation washing (150 rpm, 40°C) was performed to achieve hydration, followed by ultrasonic crushing (180 Hz, 30 min), filtration (extruded through 0.45 μm and 0.22 μm filters in sequence), and dialysis (3500 MWCO, 24 h) to obtain drug-loaded liposomes, denoted as TFs@Res+Chs, and stored at 4°C.

[0060] S3, preparation of phenylboronic acid-modified hyaluronic acid

[0061] ​500 mg of hyaluronic acid (HA) was dissolved in 100 mL of deionized water, and the solution was stirred constantly during the dissolution process to obtain a hyaluronic acid solution. The pH value of the hyaluronic acid solution was adjusted to 6.5 using a 1 mol / L HCl solution. Then, 200 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 200 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at room temperature for 4 h. Then, 25 mg of 3-aminobenzoic acid (PBA) was added, and the mixture was continuously stirred and reacted for 24 h. After the reaction was completed, the mixture was transferred to a dialysis bag with a molecular weight cut-off (MWCO) of 3.5 kDa, and dialysis was performed against a PBS solution at room temperature for 3 days, with water being changed 4 times per day. After dialysis was completed, the dialyzed solution was freeze-dried using a benchtop freeze dryer to obtain phenylboronic acid-modified hyaluronic acid, which was denoted as HA-PBA.

[0062] S4, Preparation of Hydrogel Solution

[0063] The volume ratio of the phenylboronic acid-modified hyaluronic acid, the polyvinyl alcohol solution, and the sodium hydroxide solution was 100:10:1. The concentration of the polyvinyl alcohol solution was 25 mg / mL, and the concentration of the sodium hydroxide solution was 0.5 mol / L. The phenylboronic acid-modified hyaluronic acid obtained in step S3 was mixed with the polyvinyl alcohol (PVA) solution, and the sodium hydroxide solution was added to obtain a ROS-responsive hydrogel, which was denoted as HA-PBA-PVA.

[0064] S5, Preparation of ROS-Responsive Liposome Hydrogel

[0065] The volume ratio of the drug-loaded liposomes and the hydrogel solution was 1:100. The drug-loaded liposomes obtained in step S2 were mixed with the hydrogel solution obtained in step S4 to obtain a ROS-responsive liposome hydrogel, which was denoted as Gel-TFs@Res+Chs.

[0066] In this example, different liposomes were also prepared. The preparation method of the blank liposomes was basically the same as that of the drug-loaded liposomes (TFs@Res+Chs) in Example 1, except that no resveratrol and chondroitin sulfate were added in step S2. The blank liposomes obtained were denoted as TFs.

[0067] Resveratrol-loaded liposomes were prepared by the same method as that of the drug-loaded liposomes (TFs@Res+Chs) in Example 1, except that no chondroitin sulfate was added in step S2. The resveratrol-loaded liposomes obtained were denoted as TFs@Res.

[0068] Chondroitin sulfate-loaded liposomes, the preparation method is basically the same as that of the drug-loaded liposomes (TFs@Res+Chs) in example 1, the only difference is that in step S2, resveratrol is not added; the chondroitin sulfate-loaded liposomes prepared are denoted as TFs@Chs.

[0069] Indocyanine green-loaded liposomes, the preparation method is basically the same as that of the drug-loaded liposomes (TFs@Res+Chs) in example 1, the only difference is that in step S2, resveratrol is not added, and chondroitin sulfate is replaced by indocyanine green; the indocyanine green-loaded liposomes prepared are denoted as TFs@ICG.

[0070] In this embodiment, different liposome hydrogels are also prepared, and the preparation method of the ROS-responsive blank liposome hydrogel is basically the same as that of the ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in example 1, the only difference is that in step S5, the drug-loaded liposomes are replaced by blank liposomes (TFs); the ROS-responsive blank liposome hydrogel prepared is denoted as Gel-TFs.

[0071] ROS-responsive resveratrol-loaded liposome hydrogel, the preparation method is basically the same as that of the ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in example 1, the only difference is that in step S5, the drug-loaded liposomes are replaced by resveratrol-loaded liposomes (TFs@Res); the ROS-responsive resveratrol-loaded liposome hydrogel prepared is denoted as Gel-TFs@Res.

[0072] ROS-responsive chondroitin sulfate-loaded liposome hydrogel, the preparation method is basically the same as that of the ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in example 1, the only difference is that in step S5, the drug-loaded liposomes are replaced by chondroitin sulfate-loaded liposomes (TFs@Chs); the ROS-responsive chondroitin sulfate-loaded liposome hydrogel prepared is denoted as Gel-TFs@Chs.

[0073] ICG-loaded ROS-responsive hydrogel solution, the preparation method is basically the same as that of the ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in example 1, the only difference is that in step S5, the drug-loaded liposomes are replaced by indocyanine green (ICG); the ICG-loaded ROS-responsive hydrogel solution prepared is denoted as Gel@ICG.

[0074] The preparation method of the ROS-responsive liposome hydrogel solution loaded with ICG is basically the same as that of the ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) in Example 1, except that in step S5, the drug-loaded liposomes are replaced with liposomes loaded with indocyanine green (TFs@ICG); the resulting ROS-responsive liposome hydrogel solution loaded with ICG is denoted as Gel-TFs@ICG.

[0075] The morphology and size of liposomes were observed using transmission electron microscopy (FEI Talos L120C, USA), such as... Figure 2 As shown, the size and zeta potential of drug-loaded liposomes were analyzed using the Zetasizer (Malvern Nano-ZS, UK) dynamic light scattering method. Figure 3 , Figure 4 As shown. Combined with Figures 2-4 It can be seen that the particle size of the drug-loaded liposomes is about 90 nm, the polydispersity index is 0.30, and the potential is -35 mV.

[0076] The success of PBA grafting onto the HA molecule was determined using ultraviolet, infrared, and nuclear magnetic resonance (NMR) analyses. Figures 5-7 As shown. By Figure 5 As can be seen, NMR analysis confirmed the successful synthesis of HA-PBA, revealing that approximately 23.4% of the carboxylic acid in the HA backbone was partially amidated by PBA, meaning the degree of substitution of phenylboronic acid was 23.4%. Figure 6 It can be seen that HA does not exhibit a UV absorption peak in the 200–400 nm range, while PBA shows a benzene ring UV absorption peak at 236 nm and a distinct p-π conjugation system absorption peak between the amino group and the benzene ring in the 260–325 nm range. PBA-HA exhibits a benzene ring characteristic absorption peak similar to that of PBA at 242 nm, but the absorption peak in the 260–325 nm range almost disappears. This is because after the amino group of PBA is reactively grafted onto HA, the p-π conjugation effect between the amino group and the benzene ring in PBA-HA is weakened. This result proves that PBA was successfully grafted onto the HA chain segment. Figure 7 It can be seen that HA was successfully grafted with PBA. Comparing the FTIR spectra before and after modification, it was found that at 1375 cm⁻¹... -1 and 880cm -1 A new peak appeared, which is caused by the asymmetric and symmetric vibrations of the BO bond of the phenylboronic acid group after the formation of the boronic acid ester bond.

[0077] The rheological properties of the ROS-responsive hydrogel were investigated using a Thermo Haake rheometer (RS150L), and the viscoelastic properties were measured using a 25mm parallel flat cell. Figures 8-12 As shown. By Figure 8It can be seen that the storage modulus of the hydrogel modified by PBA is always greater than the loss modulus, the mechanical property is improved, and the shape retention ability is enhanced. Figure 9 It can be seen from the viscosity of HA-PBA-PVA that the ROS-responsive hydrogel has high viscosity and shear thinning performance. Figure 10 The results show that the ROS-responsive hydrogel modified by PBA has a shear strain of 25, and the storage modulus starts to be less than the loss modulus, and has the classical rheological behavior of gel. The injectability and self-healing performance of the ROS-responsive hydrogel are investigated, Figure 11 The results show that under low shear strain 0.1, the storage modulus of the ROS-responsive hydrogel is much greater than the loss modulus, and under high shear strain 100, the storage modulus of the ROS-responsive hydrogel is less than the loss modulus. Figures 8-12 The gel to sol transition of HA-PBA-PVA is embodied, which is suitable for injection.

[0078] Investigation of the ROS stimulation response ability of ROS-responsive liposome hydrogel

[0079] The ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs) was placed in a dialysis bag (MWCO, 3400 Da) and immersed in 40 mL PBS solution containing 0.2% (w / v) Tween 80 and different concentrations of H2O2 (0-2 mM) at 37°C and 300 rpm. The drug release amount at a certain time point was determined by ultraviolet spectrophotometry, and fresh solution was replaced with equal volume. The results are shown in Figures 13-17 The drug-loaded liposome (TFs@Res+Chs) was used as a control to evaluate the sustained release ability of the microgel under ROS stimulation, and the calculation formula is shown in formula (1).

[0080]

[0081] In formula (1), C n represents the concentration of the nth sample, V0 represents the total volume of the buffer, V1 represents the volume of each sample, and m0 represents the total amount of drug in the sample.

[0082] Figure 13 In the formula, the left graph is the SEM graph of the ROS-responsive liposome hydrogel (Gel-TFs@Res+Chs), and the right graph is the SEM graph of the ROS-responsive liposome hydrogel after incubation with 1 mM H2O2. From Figures 13-17It can be seen that the ROS-responsive liposome hydrogel is obviously larger in pore size and looser in structure after incubation with H2O2 (1 mM, 30 min). Meanwhile, the drug release response was detected, and the results showed that the ROS-responsive liposome hydrogel exhibited a gradient-dependent response to ROS.

[0083] Investigation of ROS-responsive liposome hydrogel on the in vitro scavenging effect of free radicals

[0084] In order to better simulate the in vivo drug release behavior, the drug-loaded liposome (TFs@Res+Chs) was first incubated with H2O2 (1 mM, 30 min), and the supernatant was obtained by centrifugation (3000 rpm, 10 min) for subsequent experiments. The antioxidant activity was evaluated by a multifunctional enzyme marker (SpectraFluor Plus, Tecan, Crailsheim, Germany). The scavenging capacity of drug-loaded liposome (TFs@Res+Chs) on ROS was detected by 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, respectively. The effective drug amount added was Res: 30 μg / mL, Chs: 30 μg / mL. The specific method of ABTS free radical detection: ABTS (0.007 mol / L) and potassium persulfate (0.005 mol / L) were mixed in a volume ratio of 1:1, and the mixture was reacted in the dark for 24 h to obtain ABTS working solution. Then, the blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) were immersed in the ABTS working solution, and the absorbance of the solution at 730 nm was recorded by a multifunctional enzyme marker after 1 h of reaction. The specific method of DPPH free radical detection: 0.002 g of DPPH was added to 50 mL of anhydrous ethanol to obtain DPPH working solution. Then, the blank liposome (TFs), resveratrol-loaded liposome (TFs@Res), chondroitin sulfate-loaded liposome (TFs@Chs), and drug-loaded liposome (TFs@Res+Chs) were immersed in the DPPH working solution for 1 h, and the absorbance at 520 nm was measured. The experimental results are shown in Figure 18 、 Figure 19 .

[0085] As can be seen from Figures 18-19 , the blank liposome (TFs) does not have free radical scavenging capacity, while the drug-loaded liposome (TFs@Res+Chs) has good scavenging capacity, with DPPH and ABTS free radical scavenging rates of 53.4% and 94.6%, respectively, which is mainly due to the drug resveratrol.

[0086] Investigation of ROS-responsive liposome hydrogel on the protection of cell damage

[0087] To test the biocompatibility of ROS-responsive hydrogels and blank liposomes, ATDC5 chondrocytes were seeded in culture flasks containing DMEM (Gibco, USA), 10% fetal bovine serum (Gibco, USA), and 1% penicillin / streptomycin (Solarbio, China) and cultured at 37°C with 5% CO2. When the cell confluence reached 70%–80%, the cells were passaged using 0.25% Trypsin-EDTA (Solarbio, China). Subsequently, ATDC5 chondrocytes (104 cells per well) were seeded in 96-well plates overnight. The cytotoxicity of ROS-responsive hydrogels (HA-PBA-PVA) and blank liposomes (TFs) to chondrocytes was assessed using the MTT assay based on the absorbance at 450 nm.

[0088] To evaluate the cytoprotective effects of TFs@Res, TFs@Chs, and TFs@Res+Chs, cells were soaked in culture medium for 2 days, and the supernatant (extract) was collected for subsequent cell experiments. Lipopolysaccharide (LPS) is widely used to induce osteoarthritis-like inflammatory responses, inducing the production of matrix metalloproteinases (MMPs) and inhibiting the synthesis of type II collagen (COL2). In this study, ATDC5 cells were exposed to 10 ng / mL LPS (Macklin, China) and cultured in normal culture medium or the above-mentioned extract. Untreated ATDC5 cells served as the control group (Ctrl), and LPS-treated ATDC5 cells served as the model group (Blank).

[0089] Depend on Figures 20-21 It was found that the ROS-responsive hydrogel (HA-PBA-PVA) and blank liposomes (TFs) showed good cell compatibility. Next, cell damage was induced by H2O2 or LPS, respectively, followed by incubation with the materials. Cell viability was used to assess the repair effect of the materials on cells. Figures 22-23 It can be seen that, compared with the control group, blank liposomes (TFs) did not have a protective effect, while liposomes loaded with resveratrol and chondroitin sulfate (TFs@Res+Chs) increased the viability of chondrocytes.

[0090] Investigating the effect of ROS-responsive liposome hydrogels on intracellular ROS levels in mouse chondrocytes

[0091] ATDC5 cells were seeded in 12-well plates containing 10 ng / mL LPS or H2O2 (200 μM) for 4 h, and then 20 mM TFs, TFs@Res, TFs@Chs, TFs@Res+Chs were added respectively for 24 h to detect the ROS production in chondrocytes; each well was gently washed twice with fresh culture medium, and then incubated with 10 μM dichlorofluorescein diacetate (DCFH-DA) in serum-free (FBS) medium for 30 min; after washing with serum-free medium for 3 times, the green fluorescence intensity in cells was observed at Ex / Em=488 / 525 nm, and then inverted fluorescence microscope was used for shooting and analysis.

[0092] It can be known that the ROS-responsive liposome hydrogel has a good scavenging effect on reactive oxygen species (ROS) in cells. Figure 24

[0093] Investigation of the influence of ROS-responsive liposome hydrogel on the secretion of pro-inflammatory cytokines and matrix synthesis in mouse ATDC5 cells

[0094] The ROS-responsive liposome hydrogel was soaked in the culture medium for 2 days, and the supernatant (leaching liquid) was collected for subsequent cell experiments. Lipolyaccharide (LPS) is widely used to induce osteoarthritis-like inflammatory response, in this experiment, ATDC5 cells were exposed to LPS (10 ng / mL) or H2O2 (200 μM) stimulation to induce damage, and then cultured with normal culture medium or the above leaching liquid. ATDC5 cells without any treatment were used as the control group (Ctrl), and ATDC5 cells treated with LPS or H2O2 were used as the model group (Blank).

[0095] ​24h later, the chondrocyte proteins were extracted using RIPA lysis buffer (Beyotime, China) containing 1 mM PMSF (phenylmethanesulfonyl fluoride, Beyotime, China). The cell lysate was centrifuged at 4°C for 15 min at 12000 rpm, and the protein concentration was determined using a BCA protein detection kit (Beyotime, China). The protein samples (20-30 pg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk at 37°C for 1 h, the primary antibody was incubated on the PVDF membrane overnight, followed by secondary antibody incubation. The diluted primary antibody was COL2 (1:1000), iNOS (1:1000), ADAMTS5 (1:1000), p-AKT (1:1000), and Actin (1:5000), respectively. The polyvinylidene fluoride membrane was washed with TBST for 3 times, and then incubated with HRP-secondary antibody (RiboBio, China) at 37°C for 1.5 h. After washing with TBST for 3 times, electrochemiluminescence and reagent were added for development. The intensity of these blots was shown by an imaging system (Bio-Rad, USA), and the results are shown in Fig. 6. Figure 25

[0096] As can be seen from Figure 25 compared with the model group (Blank), the expression of collagen was up-regulated in the ROS-responsive liposome hydrogel group, while the expression of catabolism-related enzymes was down-regulated, and the expression of inflammation-related factors was also down-regulated, indicating that ROS-responsive liposome hydrogel can indeed alleviate cell inflammation and promote matrix synthesis at the cellular level.

[0097] Investigation of the residence time of ROS-responsive liposome hydrogel in the joint of mice

[0098] The animals were purchased from Hunan Slayjendai Experimental Animal Co., Ltd. (Changsha, China) and were raised under pathogen-free conditions, with free access to food and water. The animal experiments were approved by the Hunan University Animal Ethics Committee.

[0099] The 12-week-old Kunming OA mice were randomly divided into 4 groups (n=3), and the mice were anesthetized with sodium pentobarbital. Indocyanine green (ICG) was used as a dye molecule to encapsulate liposome gel to simulate drug retention in the joint. Next, a near-infrared dye, indocyanine green (ICG), 775 Da, was selected, which can be dissolved in water. Free ICG (Free ICG), liposome loaded with indocyanine green (TFs@ICG), ROS-responsive hydrogel solution loaded with ICG (Gel@ICG), and ROS-responsive liposome hydrogel solution loaded with ICG (Gel-TFs@ICG) were prepared.

[0100] ​All experimental operations were performed under 2% sodium pentobarbital anesthesia, and different formulations were injected into the right joint of the mice (25 μL). At the set time interval, the retention of the injected material was determined by imaging at an excitation wavelength of 745 nm and an emission wavelength of 810 nm using the IVIS Spectrum Measurement System (Xenogen, Hopkinton, MA, USA), and the results are shown in Figure 26 It can be seen that, compared with free ICG, the imaging time can be extended to 96 h after being coated by ROS-responsive liposome hydrogel. Figure 26

[0101] Investigation of the in vivo therapeutic effect of ROS-responsive liposome hydrogel on OA mice

[0102] After 6 weeks of treatment, all mice were euthanized, and bone samples were collected, fixed with paraformaldehyde, decalcified, and paraffin-embedded. H&E histopathological analysis was performed on the sagittal section of the knee joint sample of the mice, and the results are shown in Figure 27 .

[0103] As can be seen from Figure 27 compared with the normal control group (Control), the model group (Saline) had severe joint cartilage surface damage, was not smooth, and had cracks. Although joint wear was observed in the carrier group (Gel-TFs) and the free drug group (Res+Chs), the cartilage surface was improved in different treatment groups. The joint damage of the ROS-responsive liposome hydrogel group (Gel-TFs@Res+Chs) was lighter, the cartilage surface was smooth, and the chondrocyte distribution was uniform.

[0104] Investigation of the in vivo biological safety of ROS-responsive liposome hydrogel

[0105] After the treatment, the hearts, livers, spleens, lungs, and kidneys of the mice in each group were taken for H&E staining histological analysis, and the results are shown in Figure 28 .

[0106] As can be seen from Figure 28 compared with the normal control group (Control), the hearts, livers, and kidneys of each group had almost no obvious pathological changes, indicating that the ROS-responsive liposome hydrogel had good biological safety.

[0107] ​The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art, without departing from the spirit and technical solutions of the present application, can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.

Claims

1. A method for preparing a ROS-responsive liposome hydrogel, characterized in that, Includes the following steps: S1. Dissolve WYRGRL peptide and tris(2-carboxyethyl)phosphine in phosphate buffer solution, stir, add phospholipid polyethylene glycol maleimide, stir again, dialyze and freeze dry to obtain DSPE-PEG-WYRGRL. S2. Mix the DSPE-PEG-WYRGRL obtained in step S1, the film-forming material, resveratrol, and an organic solvent, add an edge activator, and evaporate to obtain a liposome membrane; dissolve chondroitin sulfate in water, add it to the liposome membrane, and wash the membrane by rotation, sonicate, and dialyze to obtain drug-loaded liposomes; the film-forming material is at least one of soybean lecithin, phosphatidylserine, dimyristoyl lecithin, and dipalmitoyl phosphatidylcholine, and the edge activator is at least one of Tween 80 and Span 80; S3. Hyaluronic acid is dissolved in water, an activator is added, the mixture is stirred, and then 3-aminophenylboronic acid is added to carry out the reaction. After dialysis and freeze-drying, phenylboronic acid-modified hyaluronic acid is obtained. The activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. S4. Mix the phenylboronic acid-modified hyaluronic acid obtained in step S3 with a polyvinyl alcohol solution, and add an alkaline solution to obtain a ROS-responsive hydrogel. S5. Mix the drug-loaded liposomes obtained in step S2 with the ROS-responsive hydrogel obtained in step S4 to obtain a ROS-responsive liposome hydrogel.

2. The method for preparing ROS-responsive liposome hydrogel according to claim 1, characterized in that, In step S1, the mass ratio of the WYRGRL polypeptide, tris(2-carboxyethyl)phosphine, and phospholipid polyethylene glycol maleimide is 1 to 10:10:

10.

3. The method for preparing ROS-responsive liposome hydrogel according to claim 2, characterized in that, In step S2, the mass ratio of DSPE-PEG-WYRGRL, film-forming material, edge activator, resveratrol, and chondroitin sulfate is 1:100:20:5:1 to 10.

4. The method for preparing ROS-responsive liposome hydrogel according to claim 3, characterized in that, In step S3, the mass ratio of 3-aminophenylboronic acid, hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 1-5:50:20:

20.

5. The method for preparing ROS-responsive liposome hydrogel according to claim 4, characterized in that, In step S4, the volume ratio of the phenylboronic acid-modified hyaluronic acid, the polyvinyl alcohol solution, and the alkaline solution is 100:10:1, the concentration of the polyvinyl alcohol solution is 10 mg / mL to 30 mg / mL, the concentration of the alkaline solution is ≤1 mol / L, and the alkaline solution is a sodium hydroxide solution.

6. The method for preparing ROS-responsive liposome hydrogel according to claim 5, characterized in that, In step S5, the volume ratio of the drug-loaded liposomes to the ROS-responsive hydrogel is 1:80 to 120.

7. The method for preparing ROS-responsive liposome hydrogels according to any one of claims 1 to 6, characterized in that, In step S1, the concentration of the phosphate buffer solution is ≤1 mol / L, the stirring time is 1 h to 4 h, the second stirring time is 2 h to 8 h, and the dialysis time is 12 h to 36 h.

8. The method for preparing ROS-responsive liposome hydrogels according to any one of claims 1 to 6, characterized in that, In step S2, the organic solvent is at least one of ethanol and chloroform, the evaporation temperature is 35℃~50℃, the particle size of the drug-loaded liposomes is 80nm~110nm, the polydispersity index of the drug-loaded liposomes is 0.29~0.46, and the Zeta potential of the drug-loaded liposomes is -36mV~-21mV. And / or, in step S3, the stirring time is 2h to 8h, the reaction time is 12h to 36h, and the dialysis time is ≥3 days.

9. A ROS-responsive liposome hydrogel prepared by the method described in any one of claims 1 to 8.

10. The use of the ROS-responsive liposome hydrogel as described in claim 9 in the preparation of osteoarthritis drugs.

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