PH and temperature dual-responsive drug-loaded hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a pH and temperature dual-responsive drug-loaded hydrogel and a preparation method and application thereof, and the preparation method comprises the following steps: adding methyl cellulose into a PBS solution for incubation, cooling to room temperature, adding a trace amount of PBS, and activating in a low-temperature environment; adding a sodium chloride solution and continuously activating in a low-temperature environment; adding epsilon-polylysine and fully stirring in a low-temperature environment; and adding oxidized hyaluronic acid and fully stirring to obtain MOE hydrogel, mixing the MOE hydrogel with the functional compound molecules or the nanoparticles loaded with the functional compound molecules, and fully stirring in a low-temperature environment to obtain the hydrogel. The hydrogel has the characteristics of in-situ injectability and pH and temperature dual response, can quickly form gel at the temperature of a human body, realizes direct injection filling and stable adhesion of a cartilage wear interface in an articular cavity, is used as a functional articular cavity injection medicine for local treatment of osteoarthritis, and has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a pH and temperature dual-responsive drug-loaded hydrogel, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is the most common chronic, degenerative joint disease with a poor prognosis and is the main cause of joint pain and disability in the elderly. According to epidemiological statistics, the incidence of OA in people over 65 years old can reach 50%, and the incidence in people over 80 years old is as high as 80%, which has brought huge economic and medical burdens to society, especially in countries with aging populations and rising obesity rates. The treatment of OA has always been a difficult problem in clinical practice, and there is still a lack of effective treatment options that can reverse the disease progression of OA and promote cartilage repair.

[0003] At present, the pathogenesis of OA has not been fully explored. It is generally believed that long-term chronic intra-articular inflammation and continuous cartilage destruction and loss mediated by secondary chondrocyte apoptosis are the basic characteristics of OA. Due to the limited inherent regenerative capacity of articular cartilage, conservative therapies such as drug therapy and hyaluronic acid injection and surgical treatment have been tried for intra-articular tissue repair and pain relief. Specifically, drug therapy includes oral and intra-articular injection of drugs, aiming to relieve pain and inflammation. However, most drugs are mainly used for temporary analgesia or lubrication, and the long-term efficacy is not ideal. In addition, long-term systemic administration usually brings inevitable side effects, such as adverse reactions such as gastrointestinal ulcers, bleeding and hepato-renal toxicity caused by long-term use of non-steroidal anti-inflammatory drugs. In contrast, intra-articular injection of drugs usually has the problem of too fast drug clearance and short half-life, which makes it difficult to maximize the therapeutic effect of the drug. Hyaluronic acid injection therapy can lubricate the joints and effectively relieve symptoms, but these inactive materials usually lack long-term therapeutic effects. Patients with end-stage OA who have poor conservative treatment can only undergo joint replacement surgery to improve joint function, but the surgery is traumatic, has a long recovery period and high cost, and there is also a risk of complications that cannot be ignored. In short, conventional treatments can usually only relieve local symptoms, are difficult to change the progression of OA, and have limited long-term efficacy. Therefore, despite the high incidence of OA, there is still a lack of effective treatment options, especially the lack of fundamental treatments for the progression mechanism of OA and safe and effective drug delivery systems, which is an important reason for the current limitations of OA treatment.

[0004] Tissue in situ regeneration technology with biomaterials as the core has broad application prospects in the field of cartilage repair and represents an important development direction of regenerative medicine. This technology uses bioactive scaffold materials with good performance to release stimulation signals to the tissue after implantation in the damaged area to induce and activate the body's own regeneration ability, thereby achieving in situ regeneration of damaged tissue. However, most of the existing intra-articular injections can only temporarily lubricate the bone-cartilage interface and cannot completely solve the problem. In fact, due to the release of various inflammatory mediators, the OA joint cavity usually exhibits a characteristic acidic environment. This unique acidic pH value makes it possible to develop an intelligent drug delivery system that can regulate drug release according to the degree of OA disease progression. However, how to achieve an intelligent response to the characteristic microenvironment in the joint cavity to release therapeutic factors on demand, long-term and stably, thereby improving treatment efficiency and minimizing drug side effects, there is currently a lack of reasonable and feasible methods. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies in the prior art, the main purpose of the present invention is to provide a pH and temperature dual-responsive drug-loaded hydrogel, which can achieve direct injection filling and stable adhesion at the cartilage wear interface in the joint cavity, and slow down the continuous wear of the bone-cartilage interface in the joint cavity through the local lubrication effect. At the same time, it can respond to changes in the characteristic microenvironment in the joint cavity, and release therapeutic factors on demand, long-term and stably, thereby reversing the progression of osteoarthritis and enhancing the repair and regeneration of cartilage tissue.

[0006] Another object of the present invention is to provide a method for preparing the pH and temperature dual-responsive drug-loaded hydrogel.

[0007] Another object of the present invention is to provide the use of the pH and temperature dual-responsive drug-loaded hydrogel in the preparation of drugs or products for treating osteoarthritis.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a method for preparing a pH and temperature dual-responsive drug-loaded hydrogel, comprising the following steps:

[0010] (1) preparing a pH and temperature dual-responsive hydrogel (MOE), comprising: first adding methyl cellulose (MC) to a PBS solution for incubation, cooling the resulting solution to room temperature, adding a trace amount of PBS and stirring it thoroughly, and placing the mixed solution in a low temperature environment for activation; then adding a sodium chloride solution to the activated MC solution, and continuing to place it in a low temperature environment for activation; then adding ε-polylysine (ε-EPL) to the mixed solution, and stirring it thoroughly at a low temperature; finally, adding oxidized hyaluronic acid (OHA) to the mixed solution, and stirring it thoroughly to obtain a MOE hydrogel;

[0011] (2) Preparing a pH and temperature dual-responsive drug-loaded hydrogel, comprising: mixing functional compound molecules or nanoparticles loaded with the functional compound molecules with the MOE hydrogel prepared in step (1), and fully stirring under a low temperature environment.

[0012] Preferably, in step (1), the incubation conditions of the MC solution are: incubation temperature is 60 to 90° C., and incubation time is 0.5 to 2 hours.

[0013] Preferably, in step (1), the conditions for activating the mixed solution are: activation temperature is 2 to 10° C., and activation time is 16 to 48 hours.

[0014] Preferably, in step (1), the mass concentration of the sodium chloride solution is 20%, and the volume ratio of the activated MC solution to the sodium chloride solution is 1:2-6.

[0015] Preferably, in step (1), the usage ratio of methylcellulose, ε-polylysine and oxidized hyaluronic acid is 1-2:1:1-3.

[0016] Preferably, in step (2), the functional compound molecule is selected from one of a bone-promoting or cartilage-forming compound molecule, an anti-inflammatory compound molecule and an antioxidant compound molecule.

[0017] Preferably, in step (2), the particle size of the nanoparticles loaded with the functional compound molecules is 100 to 800 nm, wherein the nanoparticles are selected from one of bioactive glass, silica and its derivative nanoparticles, metal and its derivative nanoparticles, polymer nanoparticles and liposomes, and the mass ratio of the functional compound molecules to the nanoparticles is 1:2 to 6.

[0018] Preferably, in step (2), the mass ratio of the functional compound molecules to the MOE hydrogel solution is 0.1 to 1:100.

[0019] The present invention also provides a pH and temperature dual-responsive drug-loaded hydrogel, which is obtained by the preparation method of the pH and temperature dual-responsive drug-loaded hydrogel, can quickly gel at human body temperature and is injectable in situ.

[0020] The present invention also provides application of the pH and temperature dual-responsive drug-loaded hydrogel in preparing a medicine or product for treating osteoarthritis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention proposes a pH and temperature dual-responsive drug-loaded hydrogel, which has the characteristics of in-situ injectability and dual response to pH and temperature, and can be transformed from liquid to solid at human body temperature, thereby achieving direct injection filling and stable adhesion at the cartilage wear interface in the joint cavity. At the same time, the local acidic environment in the OA joint cavity can lead to the rupture of the Schiff base bond in the hydrogel and accelerate the release of therapeutic factors, improve the treatment efficiency through the release of drugs on demand, and minimize the side effects of drugs.

[0023] 2. The process for preparing the pH and temperature dual-responsive drug-loaded hydrogel in the present invention is simple and efficient, with stable properties, easy application, good biological activity and compatibility, and strong flexibility. The release rate of the drug can be controlled by adjusting the ratio between the nanoparticles and the functional compound molecules. It is used as a functionalized joint cavity injection drug for the local treatment of osteoarthritis and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the stimulation-response principle of the pH and temperature dual-responsive drug-loaded hydrogel in the embodiment: (a) and (b) are temperature response and pH response principle diagrams of the hydrogel, respectively; (c) is a stimulation-response principle diagram of the drug-loaded hydrogel.

[0025] Figure 2 The macroscopic morphology and temperature responsiveness test of the pH and temperature dual-responsive drug-loaded hydrogel in the example: (a) and (b) show the state transition of the dual-responsive hydrogel loaded with TA@MBG at different temperatures; (c) shows that the hydrogel solution has good injectability; (d) shows that the hydrogel solution gels quickly at human body temperature.

[0026] Figure 3 The results of the drug release experiment of the pH and temperature dual-responsive drug-loaded hydrogel in the example.

[0027] Figure 4 The cell compatibility test results of the pH and temperature dual-responsive hydrogel in the example: (a) culture medium control; (b) live / dead cell fluorescence staining image after incubation in MOE hydrogel extract for 24 hours. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with embodiments.

[0029] Example 1

[0030] Methylcellulose was added to a PBS solution (pH = 7.4) and incubated at 80°C for 1 hour. The solution was then cooled to room temperature and a trace amount of PBS was added. After sufficient stirring, the mixed solution was placed in a 4°C refrigerator for activation for 36 hours. Sodium chloride solution was added to the activated methylcellulose solution at a volume ratio of 1:4 and placed in a 4°C refrigerator for activation again for 24 hours. Then, ε-polylysine was added to the mixed solution and stirred at 4°C for 1 hour. Oxidized hyaluronic acid was then added to the mixed solution and stirred thoroughly. Finally, triamcinolone acetonide (TA) was mixed with the aforementioned solution and stirred thoroughly at 4°C to obtain a dual-responsive hydrogel loaded with TA.

[0031] Example 2

[0032] Methylcellulose was added to PBS solution (pH=7.4) and incubated at 80℃ for 1 hour. After that, the solution was cooled to room temperature and a trace amount of PBS was added. After sufficient stirring, the mixed solution was placed in a 4℃ refrigerator for activation for 36 hours. Sodium chloride solution was added to the activated methylcellulose solution at a volume ratio of 1:4 and placed in a 4℃ refrigerator for activation for 24 hours. Then, ε-polylysine was added to the mixed solution and stirred at 4℃ for 1 hour. Oxidized hyaluronic acid was added to the mixed solution and stirred thoroughly. Triamcinolone acetonide and mesoporous bioactive glass (MBG) were added to anhydrous ethanol at a mass ratio of 1:4. After sufficient stirring for 24 hours, the mixture was centrifuged (10000rpm / min, 10min) and the supernatant was discarded. The mixture was freeze-dried to obtain mesoporous bioactive glass loaded with triamcinolone acetonide (TA@MBG). Finally, the mesoporous bioactive glass loaded with triamcinolone acetonide was mixed with the above solution and stirred thoroughly at 4℃ to obtain a dual responsive hydrogel loaded with TA@MBG.

[0033] Verification Example 1

[0034] The TA-loaded mesoporous bioactive glass was mixed with the prepared MOE hydrogel, stirred thoroughly in a low temperature environment, and its macroscopic morphology was recorded. Afterwards, the mixed hydrogel solution was transferred to a 37°C environment and allowed to stand for 2 minutes, and its macroscopic morphology was recorded again.

[0035] Taking Example 2 as an example, the experimental results are as follows Figure 2 As shown in a and 2b, Figure 2 a shows that the hybrid hydrogel loaded with TA@MBG is in liquid state at low temperature. Figure 2 b shows that the hybrid hydrogel loaded with TA@MBG can gel quickly at 37 °C.

[0036] Verification Example 2

[0037] The TA-loaded mesoporous bioactive glass was mixed with the prepared MOE hydrogel, fully stirred under low temperature and transferred into a 10 mL syringe, and its macroscopic morphology was recorded. Afterwards, the mixed hydrogel solution was injected into the human skin surface and left to stand for 2 minutes, and its macroscopic morphology was recorded again.

[0038] Taking Example 2 as an example, the experimental results are as follows Figure 2 c and 2d, Figure 2 c shows that the hybrid hydrogel loaded with TA@MBG is in liquid state at low temperature and has good injectability. Figure 2 d shows that the hybrid hydrogel loaded with TA@MBG can gel quickly at human body temperature.

[0039] Verification Example 3

[0040] TA was mixed with the prepared MOE hydrogel, stirred thoroughly at 4°C, and then transferred to 37°C and allowed to stand until complete gelation. After that, the hydrogel was placed in 10 mL of PBS solution and incubated at 37°C. The supernatant was taken out at a set time point, and the concentration of TA in the supernatant was determined by UV-visible absorption spectroscopy. The cumulative release rate was calculated by the following formula:

[0041]

[0042] Wherein, C represents the concentration of triamcinolone acetonide; V represents the volume of the supernatant removed; and M represents the total mass of TA contained in the hydrogel.

[0043] Taking Example 1 as an example, the experimental results are as follows Figure 3 As shown, in vitro drug release experiments showed that TA was able to be continuously and uniformly released from the hydrogel over time within two weeks. Importantly, at relatively low pH, the drug-loaded hydrogel showed a significantly increased drug release rate, which could achieve pH-responsive drug release to improve the local inflammatory microenvironment, thereby reversing the progression of osteoarthritis mediated by persistent inflammation.

[0044] Verification Example 4

[0045] Human skin fibroblasts (HSF-1) were cultured in a complete medium consisting of high glucose medium, 10% fetal bovine serum and 1% penicillin / streptomycin. According to the ISO 10993-5:2009 guideline, the prepared MOE hydrogel was sterilized by ultraviolet irradiation, and then the sterilized hydrogel was placed in a complete medium at 37°C for 24 hours to obtain an extract (0.1 g / mL). HSF-1 was uniformly inoculated in a 24-well plate (2.0×10 4 Cells were cultured with the above extracts or complete culture medium. After 24 hours of incubation, the cell viability of HSF-1 was analyzed by live / dead cell staining.

[0046] Taking MOE hydrogel as an example, the experimental results are as follows Figure 4 As shown, the live / dead cell staining results showed that MOE hydrogel exhibited good cell compatibility, and its extract had no obvious toxicity to human dermal fibroblasts, suggesting that the strategy of using MOE hydrogel for intra-articular injection treatment of osteoarthritis is feasible.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the protection scope of the present invention.

Claims

1. A method for preparing a pH and temperature dual-responsive drug-loaded hydrogel, characterized in that: The following steps are involved: (1) preparing MOE hydrogel, comprising: First, methylcellulose was added to PBS solution for incubation. After the obtained solution cooled to room temperature, a small amount of PBS was added and stirred thoroughly. The mixed solution was activated in a low temperature environment. Then, sodium chloride solution is added to the activated methyl cellulose solution, and activation is continued in a low temperature environment; Then, ε-polylysine is added to the mixed solution and stirred thoroughly under low temperature; Finally, oxidized hyaluronic acid was added to the mixed solution and stirred thoroughly to obtain MOE hydrogel; (2) Preparing a pH and temperature dual-responsive drug-loaded hydrogel, comprising: The functional compound molecules or nanoparticles loaded with the functional compound molecules are mixed with the MOE hydrogel prepared in step (1), and fully stirred under low temperature environment to obtain the product.

2. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (1), the incubation conditions of the methylcellulose solution are: incubation temperature is 60 to 90° C., and incubation time is 0.5 to 2 hours.

3. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (1), the conditions for activating the mixed solution are: activation temperature is 2 to 10° C., and activation time is 16 to 48 hours.

4. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (1), the mass concentration of the sodium chloride solution is 20%, and the volume ratio of the activated methyl cellulose solution to the sodium chloride solution is 1:2-6.

5. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (1), the usage ratio of methylcellulose, ε-polylysine and oxidized hyaluronic acid is 1-2:1:1-3.

6. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (2), the functional compound molecule is selected from one of a bone or cartilage-promoting compound molecule, an anti-inflammatory compound molecule and an antioxidant compound molecule.

7. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (2), the particle size of the nanoparticles loaded with the functional compound molecules is 100 to 800 nm, wherein: The nanoparticles are selected from one of bioactive glass, silicon dioxide and its derivative nanoparticles, metal and its derivative nanoparticles, polymer nanoparticles and liposomes; The mass ratio of the functional compound molecules to the nanoparticles is 1:2-6.

8. The method for preparing the pH and temperature dual-responsive drug-loaded hydrogel according to claim 1, characterized in that: In step (2), the mass ratio of the functional compound molecules to the MOE hydrogel solution is 0.1 to 1:

100.

9. A pH and temperature dual-responsive drug-loaded hydrogel, characterized in that: The drug-loaded hydrogel is obtained by the preparation method of the pH and temperature dual-responsiveness drug-loaded hydrogel according to any one of claims 1 to 8, and can quickly gel at human body temperature and can be injected in situ.

10. Use of the pH and temperature dual-responsive drug-loaded hydrogel according to claim 9 in the preparation of a drug or product for treating osteoarthritis.

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