Application of MgAl-LDH in the preparation of drugs for treating joint inflammation

Abstract

The present invention is applicable to the field of medical technology, and provides an application of MgAl-LDH in the preparation of a drug for treating joint inflammation. The present invention utilizes the advantages of intra-articular injection administration, uses MgAl-LDH (magnesium aluminum hydrotalcite) as a carrier, carries rhein (RH) with anti-inflammatory and cartilage repair activity, and combines with thermosensitive gel (Gel) to prepare LDH / RH-Gel for intra-articular injection. The preparation has good biological safety, significantly increases the solubility of RH, and prolongs the retention time of RH in the joint cavity; significantly reduces inflammatory response and cartilage loss, delays the progression of the disease, and effectively treats OA. The above scheme is also applicable to the delivery of non-steroidal anti-inflammatory drugs, hormone drugs, biological factors, monoclonal antibodies and other drugs for treating joint inflammation. And MgAl-LDH can also form a composite carrier with other gels. The present invention is expected to be used for the treatment of joint inflammation diseases such as rheumatism, rheumatoid arthritis, osteoarthritis and synovitis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to application of MgAl-LDH in preparing a drug for treating joint inflammation. Background Art

[0002] Arthritis is a common joint disease that involves inflammation and damage to the joints. Common arthritis includes osteoarthritis, rheumatoid arthritis, and osteoarthritis. The incidence of arthritis in my country has increased year by year, especially knee arthritis and osteoarthritis. At present, about 120 million people in the country suffer from pain in the knee joint and osteoarthritis, accounting for nearly 10% of the total population. Among people over 65 years old, the incidence rate is over 80%, which has seriously affected the daily activities of the elderly. At present, most arthritis treatment drugs have a single target, and long-term oral administration may cause a series of gastrointestinal and cardiovascular adverse reactions, and the drug concentration distributed to the local joint is limited. Therefore, intra-articular injection (IA) is considered to be the most suitable way to treat arthritis. Currently, IA drugs used in clinical practice include hyaluronic acid, glucocorticoids, and platelet-rich plasma, all of which are aqueous solutions or hydrogels.

[0003] Layered double hydroxides (LDHs) are a new type of inorganic layered nanomaterials with a two-dimensional structure. The general structural formula is: 2+ 1-x M 3+ x(OH)2] x+ A n- x / n ·mH2O, where A is the anion, m is the number of moles of crystal water per mole of LDHs, M 2+ is a divalent metal ion, M 3+ It is a trivalent metal ion. The most widely studied LDHs is MgAl-LDH, which has good safety and biocompatibility, high anion exchange capacity and good buffering capacity. MgAl-LDH has been used clinically to treat gastric diseases (Daxi). MgAl-LDH can inactivate the IKK / NF-κB signaling pathway when LPS stimulates inflammation, and can also induce macrophage polarization to M2 phenotype, exerting an anti-inflammatory effect. At the same time, our research found that MgAl-LDH has a certain effect on promoting the proliferation of chondrocytes ( Figure 4 ).

[0004] Rhein (RH) is an IL-1 inhibitor. It can effectively reduce the expression of inflammatory cytokines such as IL-1, NO and TNF-α, and exert anti-inflammatory and analgesic effects, which are similar to non-steroidal anti-inflammatory drugs; it can also reduce the secretion of matrix metalloproteinases and protect cartilage; it can also promote the synthesis of hyaluronic acid and collagen, and play a role in repairing cartilage. The mechanism of RH in treating joint inflammation is very clear, and it has the dual effects of anti-inflammatory and cartilage repair. However, due to the poor solubility of RH, its prodrug diacerein is used clinically. After oral administration, it is metabolized into the effective active ingredient RH in the liver to exert its pharmacological effect. The water-insoluble and slightly lipid-soluble properties of RH itself limit its IA administration and the choice of IA administration delivery carriers. There are reports of using PLGA microspheres / nanoparticles, liposomes, etc. to increase the solubility of RH, but the drug loading of the carrier is generally low (2%-10%), and the acid produced by PLGA degradation can also promote the development of inflammation. At the same time, because the injection volume of the joint cavity is limited, it is difficult to achieve an effective drug concentration with a low drug loading. Therefore, finding a suitable delivery vehicle for RH is of great significance for achieving IA administration of RH, and it also provides a reference for IA administration of other joint inflammation therapeutic drugs. Summary of the invention

[0005] The purpose of the present invention is to provide an application of MgAl-LDH in the preparation of a drug for treating joint inflammation, aiming to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention discloses an application of MgAl-LDH in preparing a drug for treating joint inflammation. The MgAl-LDH has magnesium ions and aluminum ions as a basic framework and can be doped with other divalent and trivalent metal ions.

[0008] Furthermore, the MgAl-LDH is used as a drug carrier to carry RH and is combined with a temperature-sensitive gel (Gel) to form a dual sustained-release drug delivery system.

[0009] Furthermore, the RH is replaced with joint inflammation treatment drugs such as non-steroidal anti-inflammatory drugs, hormone drugs, biological factors or monoclonal antibodies.

[0010] Furthermore, the thermosensitive gel is replaced by other gels except the thermosensitive gel.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. The present invention has found a suitable delivery carrier for RH, which is of great significance for achieving IA administration of RH, and also provides a reference method for IA administration of other drugs for treating joint inflammation.

[0013] 2. The present invention has practical significance in the treatment of joint inflammation by IA administration of drug delivery carriers. MgAl-LDH with magnesium ions and aluminum ions as the basic skeleton has good biosafety as a drug carrier, which can not only promote the proliferation of chondrocytes ATDC5 to a certain extent, but also promote the uptake of drugs by ATDC5 cells. MgAl-LDH can carry RH, increase the solubility of RH in water by nearly 500 times, and the drug loading amount reaches 25%, and the loss rate of drugs in the joint cavity is reduced by sustained release. Further combined with thermosensitive gel, low viscosity at room temperature is conducive to injection, and hydrogel is formed under body temperature conditions, thereby forming a dual sustained release drug delivery system. The present invention is expected to be used for the treatment of joint inflammation diseases through IA administration, including but not limited to the treatment of intervertebral disc herniation, scapulohumeral periarthritis, shoulder impingement syndrome, carpal tunnel syndrome, deformed joint disease, tennis / golf elbow, complex regional pain syndrome, other tenosynovitis and chronic joint rheumatism. In addition, MgAl-LDH is not only suitable for carrying RH, but also suitable for the delivery of other drugs for treating joint inflammatory diseases, including but not limited to non-steroidal anti-inflammatory drugs, hormone drugs, as well as biological factors and monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a flow chart for the preparation of LDH / RH-Gel.

[0015] Figure 2 TEM image of LDH / RH.

[0016] Figure 3 The appearance of LDH / RH-Gel at different temperatures.

[0017] Figure 4 In the figure, AD are MgAl-LDH (A), Al 3+ (B) Mg 2+ (C) and LDH / RH-Gel (D) on the cell proliferation activity of ATDC5 (group containing RH, the horizontal axis is the RH concentration).

[0018] Figure 5 HE staining of the knee joint after injection of MgAl-LDH, LDH / RH and LDH / RH-Gel.

[0019] Figure 6 These are graphs showing changes in mouse weight and knee joint diameter, where A: graph showing changes in mouse weight; B: graph showing changes in knee joint diameter.

[0020] Figure 7 Pathological sections and scores of knee joints after injection of MgAl-LDH, LDH / RH and LDH / RH-Gel; A: HE staining and safranin fast green staining of knee joints; B: Mankin's score.

[0021] Figure 8 These are the results of immunohistochemistry of mouse knee joints; A: Effects of MgAl-LDH, LDH / RH and LDH / RH-Gel on IL-1β inflammatory cytokines; B: Effects of MgAl-LDH, LDH / RH and LDH / RH-Gel on IL-6 inflammatory cytokines; C: Effects of MgAl-LDH, LDH / RH and LDH / RH-Gel on the synthesis of cartilage degradation-related protease MMP-13.

[0022] Fig. 9 These are in vivo fluorescence imaging images of the knee joint after injection of CY7, Gel-CY7, MgAl-LDH-CY7 and LDH-Gel-CY7. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0025] An embodiment of the present invention provides a use of MgAl-LDH in preparing a drug for treating joint inflammation.

[0026] As a preferred embodiment of the present invention, the MgAl-LDH is used as a drug carrier to carry RH and is combined with a thermosensitive gel to form a dual sustained-release drug delivery system, namely LDH / RH-Gel. The preparation process of the LDH / RH-Gel is as follows:

[0027] Step 1, weighing the prescribed amount of glycerol and carboxymethyl chitosan, adding deionized water to prepare solution A;

[0028] Step 2, under magnetic stirring, slowly add Poloxamer 407 powder to solution A to obtain solution B;

[0029] Step 3, stirring solution B at 4°C overnight;

[0030] Step 4: Slowly add LDH / RH solution to solution B, continue magnetic stirring, and finally place in a 4°C refrigerator to remove bubbles to obtain LDH / RH-Gel.

[0031] The preparation process of LDH / RH solution is as follows:

[0032] Step 1, preparing alkaline solution C;

[0033] Step 2, dissolving RH in alkaline solution C to prepare solution D with a pH of 10-12 (RH deprotonates carboxylates to generate carboxylate ions under alkaline conditions. If the pH is too high, the stability of RH will be destroyed; if the pH is too low, the degree of deprotonation of carboxylates in RH will be weakened, less carboxylate ions will be generated, RH will not be completely dissolved, and the drug loading will be low);

[0034] Step 3: Slowly add MgAl-LDH dropwise into solution D and mix well, and resuspend by centrifugation to obtain LDH / RH (the mass ratio of MgAl-LDH to RH in solution D is 0.1-40).

[0035] The preparation process of MgAl-LDH is as follows:

[0036] Step 1, preparing solution E by mixing magnesium salt, aluminum salt and water;

[0037] Step 2, preparing alkaline solution F;

[0038] Step 3, after mixing solution E and solution F, co-precipitate for a certain period of time, centrifuge and wash three times, and resuspend with deionized water to obtain a precursor solution;

[0039] Step 4: hydroheat the precursor solution at 40-120° C. for 2-24 h to obtain MgAl-LDH.

[0040] In the embodiment of the present invention, the thermosensitive gel matrix is ​​a mixture of poloxamer, carboxymethyl chitosan and glycerol. LDH / RH-Gel is a liquid with low viscosity at room temperature, which is convenient for injection. When the temperature rises to 28-30°C, a phase transition occurs to a semi-solid gel state with high viscosity.

[0041] As a preferred embodiment of the present invention, MgAl-LDH is not only suitable for delivering drugs such as rhein, but also suitable for delivering drugs such as non-steroidal anti-inflammatory drugs, hormone drugs, biological factors and monoclonal antibodies.

[0042] As a preferred embodiment of the present invention, MgAl-LDH as a drug carrier is not limited to being combined with thermosensitive gel to form a composite carrier, but can also be combined with other gels to form a composite carrier.

[0043] As a preferred embodiment of the present invention, the magnesium salt in MgAl-LDH can be magnesium carbonate, magnesium nitrate, or magnesium chloride, and the aluminum salt can be aluminum nitrate, aluminum chloride, or aluminum sulfate.

[0044] Example 1, preparation of LDH / RH-Gel (see flow chart Figure 1 );

[0045] (1) Weigh 923.04 mg Mg(NO3)2·6H2O (purchased from Merck, USA, CAS number: 13446-18-9) and 450.16 mg Al(NO3)3·9H2O (purchased from Merck, USA, CAS number: 7784-27-2) and dissolve them in 10 mL of deionized water to prepare solution A.

[0046] (2) Weigh 240.06 mg of NaOH (purchased from Aladdin (Shanghai) Co., Ltd., CAS number: 1310-73-2) and dissolve it in 40 mL of deionized water to prepare solution B.

[0047] (3) Solution A was quickly added to solution B and co-precipitated at 25°C for 4 h.

[0048] (4) Centrifuge at 4000 rpm for 10 min, wash three times with deionized water, and suspend the precipitate in 30 mL of deionized water.

[0049] (5) The suspension was transferred to a reactor and subjected to hydrothermal reaction at 100 °C for 16 h to obtain a MgAl-LDH solution.

[0050] (6) Weigh 50 mg of RH (purchased from Dalian Meilun Biotechnology Co., Ltd., CAS number: 478-43-3) and dissolve it in 10 mL of 0.15 M NaOH solution to prepare solution C.

[0051] (7) Slowly add 10 mL of MgAl-LDH nanoparticle solution into solution C and stir magnetically for 30 min.

[0052] (8) Centrifuge at 1000 rpm for 10 min and wash three times to obtain LDH / RH solution.

[0053] (9) Weigh 50 mg of glycerol and 70 mg of carboxymethyl chitosan and add 3 mL of deionized water.

[0054] (10) Add 950 mg of Poloxamer 407 under magnetic stirring and stir at 4 °C overnight.

[0055] (11) Slowly add 2 mL of LDH / RH solution and continue magnetic stirring for 4 h.

[0056] (12) Place the mixture in a 4°C refrigerator for 4 h and remove bubbles to obtain the LDH / RH-Gel solution.

[0057] The corresponding test results of the preparation of MgAl-LDH, LDH / RH and LDH / RH-Gel are as follows:

[0058] Figure 2 This is the TEM image of LDH / RH. It can be seen from the figure that LDH / RH are both typical regular hexagonal lamellar structures with complete morphological structure and uniform dispersion, indicating that the carrying of RH will not change the morphology of MgAl-LDH.

[0059] Figure 3 The appearance of LDH / RH-Gel at different temperatures (scale: 1 cm). LDH / RH-Gel is in a sol state at 25°C, red and fluid, but forms a gel at 37°C, showing temperature-sensitive properties.

[0060] Example 2, MgAl-LDH, Al 3+ Mg 2+ and LDH / RH-Gel on the cell proliferation activity of ATDC5;

[0061] (1) Add 1×10 4 ATDC5 cells (chondrocyte ATDC5, purchased from Shanghai Fuheng Biotechnology Co., Ltd., Shanghai, China) were cultured in a cell culture incubator at 37°C with 5% CO2 for 24 h.

[0062] (2) Remove the old culture medium, add different treatment groups to the cells, and continue to culture for 48 h. Set up 5 replicate wells for each group. The groups are: blank control (Control), MgAl-LDH, Al 3+ Mg 2+ and LDH / RH-Gel.

[0063] (3) Incubate the 96-well plate in a cell culture incubator at 37°C with 5% CO2 for 48 h.

[0064] (4) Add 20 μL of 5 mg / mL MTT to each well and incubate at 37°C in the dark for 4 h.

[0065] (5) Aspirate the supernatant, add 100 μL DMSO to each well, and shake for 10 min.

[0066] (6) Use an ELISA reader to measure the absorbance of each well at a wavelength of 490 nm.

[0067] Figure 4 For MgAl-LDH( Figure 4 A), Al 3+ ( Figure 4 B), Mg 2+ ( Figure 4 C) and LDH / RH-Gel ( Figure 4 The MTT assay results (D) show that MgAl-LDH concentrations of 50-100 μg / mL can promote ATDC5 cell proliferation to a certain extent. 3+ and Mg 2+ The results of separate tests show that the main 3+ Promote the proliferation of ATDC5 cells. LDH / RH-Gel also has the effect of promoting the proliferation of ATDC5 cells.

[0068] Example 3, in vivo biosafety of MgAl-LDH, LDH / RH and LDH / RH-Gel;

[0069] The specific steps are as follows:

[0070] (1) 80 C57BL / 6 male 8-week-old mice (C57BL / 6 mice, purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SCXK(Liao)2020-0001, approval number of Jilin University Animal Research Ethics Committee: 2022-YNPZSY046) were collected and pre-raised in an SPF environment in accordance with animal ethics for one week to allow the experimental animals to adapt to the animal room environment, and the physical condition of the mice was observed every day.

[0071] (2) The mice were randomly divided into 4 groups: blank control group (Control), MgAl-LDH group (50 μg), LDH / RH group (RH: 10 μg), and LDH / RH-Gel (RH: 10 μg). 10 μL of the above drugs were injected into the knee joints of the mice, and then the mice were killed on days 1, 7, 14, and 21, and the knee joints were taken for HE staining.

[0072] Figure 5 The results of HE staining of the knee joints after injection of MgAl-LDH, LDH / RH and LDH / RH-Gel. It can be seen from the figure that from 1 day to 21 days, the surface of the knee cartilage of each group of mice was smooth, the cartilage structure was intact, the tide line was clear, the chondrocytes were arranged regularly, the tide line was clear, and no chondrocyte clustering was observed, indicating that the biological safety of each group of drugs was good.

[0073] Example 4. Effectiveness of MgAl-LDH, LDH / RH and LDH / RH-Gel;

[0074] The specific steps are as follows:

[0075] (1) Twenty-five C57BL / 6 male mice aged 8 weeks were pre-raised in an SPF environment in accordance with animal ethics for one week to allow the experimental animals to adapt to the animal room environment. The physical condition of the mice was observed every day.

[0076] (2) The mice were randomly divided into 5 groups. One group served as a blank control, and the remaining 4 groups were injected with 20 μL of 4% Papain (0.03 M cysteine) solution on days 1, 3, and 5 to establish the mouse OA model.

[0077] (3) After successful modeling, 10 μL of PBS, MgAl-LDH (50 μg), LDH / RH (RH: 10 μg), and LDH / RH-Gel (RH: 10 μg) were injected into the knee joints of OA model mice. The injections were performed once a week for a total of 6 weeks. The changes in the knee joint diameter and body weight of the mice were recorded once a week. After the experiment, the animals were killed and the knee joints were removed for HE staining, safranin fast green staining, and immunohistochemistry. The treatment score was given in combination with the modified Mankin's scoring system.

[0078] Figure 6 is the change diagram of mouse body weight and knee joint diameter, Figure 6 As shown in Figure A, each group of drugs had no significant effect on the weight gain of mice. Figure 6 As shown in Figure B, compared with the OA group, the knee joint diameter of the mice in the LDH / RH group was significantly reduced after 3 weeks of administration; while the knee joint diameter of the mice in the LDH / RH-Gel group was significantly reduced after 2 weeks of administration, and returned to the level of the Control group after 6 weeks of administration, indicating that LDH / RH and LDH / RH-Gel have a good therapeutic effect on osteoarthritis.

[0079] Figure 7 Pathological sections and scores of knee joints after MgAl-LDH, LDH / RH and LDH / RH-Gel injection. Figure 7 Middle A and Figure 7 As shown in Figure B, compared with the OA group, the cartilage surface of the LDH / RH and LDH / RH-Gel groups was smoother, the cartilage structure was basically intact, the tide line was clearer, there was no inflammatory cell infiltration and vascular proliferation in the synovial tissue, and the cartilage loss was significantly reduced, and the Mankin's score was lowered, indicating that LDH / RH and LDH / RH-Gel can effectively treat OA, reduce inflammatory response, reduce cartilage loss, and delay disease progression.

[0080] Figure 8 The results of immunohistochemistry of mouse knee joints are shown in Figure 2. Figure 8 As shown in Figure A, MgAl-LDH, LDH / RH and LDH / RH-Gel groups can significantly reduce the production of IL-1β inflammatory cytokine. Figure 8 Medium B and Figure 8As shown in Figure C, the LDH / RH-Gel group can significantly reduce the synthesis of IL-6 inflammatory cytokines and cartilage degradation-related proteases MMP-13, indicating that LDH / RH-Gel mainly plays an anti-inflammatory and cartilage-protective role by inhibiting the synthesis of inflammatory factors and cartilage degradation-related proteases.

[0081] Example 5, in vivo living imaging;

[0082] The specific steps are as follows:

[0083] (1) Twelve 8-week-old C57BL / 6 male mice were pre-raised in an SPF environment in accordance with animal ethics for one week to allow the experimental animals to adapt to the animal room environment, and the physical condition of the mice was observed every day.

[0084] (2) The mice were randomly divided into 4 groups, namely, CY7 group (mouse in vivo imaging fluorescent dye), Gel-CY7 group, LDH-CY7 group and LDH-Gel-CY7 group. 10 μL of the above drugs were injected into the knee joints of the mice, and then the images were taken using an in vivo fluorescence imaging system at 0, 8, 24, 72, 168 and 336 h.

[0085] Fig. 9 The following are the in vivo fluorescence images of the knee joint after injection of CY7, Gel-CY7, MgAl-LDH-CY7 and LDH-Gel-CY7. It can be seen from the figure that the fluorescence of free CY7 disappears after 8 hours, while Gel and MgAl-LDH can significantly increase the retention time of CY7 in the body, which is 24 hours and 72 hours respectively. The nanocomposite composed of MgAl-LDH and Gel makes the drug stay in the body for the longest time, up to 168 hours, indicating that MgAl-LDH, Gel and LDH-Gel can significantly increase the retention time of the drug in the body.

[0086] In summary, the present invention takes advantage of the administration of intra-articular injection, uses MgAl-LDH as a delivery carrier, carries RH with multiple pharmacological activities, and combines with thermosensitive gel (Gel) to prepare LDH / RH-Gel for intra-articular injection. The preparation has good biosafety, significantly increases the solubility of RH, improves the bioavailability of RH, and prolongs the retention time of RH in the joint cavity; effectively reduces inflammatory response and cartilage loss, and delays the progression of joint inflammation through treatment.

[0087] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. An application of MgAl-LDH in the preparation of a drug for treating joint inflammation, characterized in that: The MgAl-LDH is based on magnesium ions and aluminum ions as a basic framework; The MgAl-LDH is used as a drug carrier to carry rhein and is combined with the thermosensitive gel to form a dual sustained-release drug delivery system.

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