Nano-drug for treating aseptic loosening of bone implant and preparation method thereof

Abstract

The invention relates to a nano-drug for treating aseptic loosening of a bone implant and a preparation method of the nano-drug. The nano-drug comprises an STING inhibitor C-176 and a layered double hydroxide LDH, and the C-176 is adsorbed to a layer gap or a surface of the LDH space structure through an intermolecular force. According to the invention, the occurrence of aseptic inflammation of the bone implant and the development of loosening of the implant can be obviously reduced, and the clinical prognosis of a patient is improved. Compared with the prior art, the invention has higher clinical adaptability, and is particularly suitable for patients who cannot be subjected to operative treatment. In addition, the use method is easy and convenient to operate, small in side effect and capable of effectively improving the living quality of the patient and reducing the treatment cost.

Description

Technical Field

[0001] The invention belongs to the field of bone implants, and particularly relates to a nano drug for treating aseptic loosening of bone implants and a preparation method thereof. Background Art

[0002] Bone implants have been widely used in clinical treatment fields such as fracture repair and joint replacement. However, the long-term stability of bone implants has always been an important issue in clinical practice. Aseptic loosening, as a major cause of implant failure, seriously affects the quality of life of patients and increases the need for further surgery and treatment. Aseptic loosening refers to the phenomenon that the implant gradually shifts or loosens due to the loss of stability or fixation at the interface between the bone and the implant in the absence of infection. This phenomenon usually occurs during the long-term use of bone implants, especially after joint replacement surgery, and manifests as joint pain, limited movement and loss of function.

[0003] The mechanism of aseptic loosening is complex and is usually closely related to factors such as osteoporosis, biomechanical stress at the implant-bone interface, poor bone healing, and individual differences among patients. Current clinical treatments mainly include surgical repair, bone reconstruction, and implant replacement. However, these traditional treatments are not only highly invasive, but also have a long postoperative recovery period and cannot completely solve the fundamental problem of aseptic loosening of orthopedic implants. For some patients, especially the elderly or those with underlying diseases, repeated surgeries may bring higher risks and further reduce their quality of life.

[0004] In this context, finding a drug treatment that can effectively treat aseptic loosening of bone implants has become an important direction of medical research. Existing drug treatments mainly focus on promoting bone healing, enhancing bone density, and improving the biomechanical contact force between implants and bones. However, these treatments can often only play an auxiliary role and cannot fundamentally solve the problem of aseptic loosening. For example, although common bone density enhancing drugs can improve bone strength to a certain extent, they have limited effects on improving the stability of orthopedic implants and bone interface adhesion. In addition, although traditional anti-inflammatory drugs can relieve local inflammatory responses, they cannot directly solve the mechanism of aseptic loosening. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a nano drug for treating aseptic loosening of bone implants and a preparation method thereof, wherein the nano drug can regulate the biological response around the bone implant, optimize the local bone metabolism and bone healing process, and alleviate the occurrence and development of aseptic inflammation.

[0006] The present invention provides a nano drug for treating aseptic loosening of bone implants, wherein the nano drug comprises a STING inhibitor C-176 and a layered double hydroxide LDH; the C-176 is adsorbed on the interlayer gap or surface of the LDH spatial structure through intermolecular forces.

[0007] Preferably, the LDH is a double metal hydroxide comprising Al and Co, and the molar ratio of Al to Co is 1:0.2-1:5. More preferably, the molar ratio of Al to Co is 1:3. The microstructure of the LDH is a single-layer or multi-layer two-dimensional nanosheet structure, the size of which is no greater than 10 nm in one dimension and the longest diameter of which is no greater than 100 nm in another two-dimensional scale.

[0008] Preferably, the weight ratio of C-176 to LDH is 0.01:1-0.1:1.

[0009] Furthermore, the nanomedicine also includes one or more of a stabilizer, a dispersion medium, and an osmotic pressure regulator.

[0010] Preferably, the stabilizer is selected from at least one of PEG2000-COOH, hyaluronic acid, and albumin. The weight ratio of the stabilizer to LDH is 0.01:1-0.1:1.

[0011] Preferably, the dispersion medium is selected from at least one of ultrapure water, physiological saline and PBS.

[0012] Preferably, the nano drug is a nano suspension, and the content of the nano drug in the nano suspension is 0.1%-10% (w / v), preferably 0.5%-2% (w / v).

[0013] Preferably, the osmotic pressure regulator is selected from at least one of glucose, sodium chloride, mannitol, glycine or its hydrochloride. The amount of the osmotic pressure regulator added is 0%-5% (w / v), preferably 0.2%-2% (w / v).

[0014] The present invention also provides a method for preparing a nano drug for treating aseptic loosening of bone implants, comprising the following steps:

[0015] (1) mixing a metal nitrate solution with a formamide solution for reaction, and centrifuging and washing the mixture after the reaction to obtain an LDH aqueous solution;

[0016] (2) The LDH aqueous solution and the C-176 solution are mixed and reacted, and then centrifuged and washed to obtain a nanomedicine for treating aseptic loosening of bone implants.

[0017] Preferably, the metal nitrate in step (1) comprises aluminum nitrate and cobalt nitrate, and the molar ratio of aluminum nitrate to cobalt nitrate is 1:0.2-1:5, preferably 1:3.

[0018] Preferably, the metal nitrate in step (1) may be an anhydrous compound or a hydrate thereof, for example: cobalt nitrate hexahydrate and aluminum nitrate nonahydrate.

[0019] Preferably, the volume ratio of the metal nitrate solution to the formamide solution in step (1) is 1:0.5-1:5, preferably 1:1.

[0020] Preferably, the volume fraction of the formamide solution in step (1) is 20%-25%, and the solvent is water.

[0021] Preferably, the mixing method in step (1) is to add dropwise using a syringe pump at a dropping speed of 1-2 mL / min.

[0022] Preferably, the pH value of the mixed solution in step (1) is 10-11.

[0023] Preferably, the pH environment of the mixed solution is maintained by adding alkali solution, which is selected from one or more of sodium hydroxide, potassium hydroxide and the like.

[0024] Preferably, the mixing reaction temperature of step (1) is 60°C-100°C, preferably 80°C.

[0025] Preferably, the amount of C-176 added in step (2) is 0.01-0.1 (w / w) of LDH.

[0026] Preferably, the cleaning solvents in step (2) are ethanol and deionized water, and different solvent combinations can be selected for multiple cleanings.

[0027] Preferably, the solvent of the C-176 solution in step (2) is a mixture of tetrahydrofuran and water, wherein the volume fraction of tetrahydrofuran in the mixture of tetrahydrofuran and water is 20% to 100%.

[0028] Preferably, the mixing method in step (2) is magnetic stirring, the mixing time is 12h-24h, and the ambient temperature is 10°C-60°C.

[0029] Preferably, the cleaning solvent in step (2) is deionized water.

[0030] Furthermore, the method also includes post-processing the obtained nano drug for treating aseptic loosening of bone implants, including adding a stabilizer and an osmotic pressure regulator to adjust the concentration of the nano drug.

[0031] C-176 is a highly effective small molecule STING (stimulator of interferon genes) inhibitor that covalently targets the transmembrane cysteine ​​residue Cys91 of the STING protein, blocking its palmitoylation and polymer formation, thereby inhibiting the production of type I interferon (IFN) and inflammatory cytokines. The drug has shown significant anti-inflammatory effects in a variety of inflammatory disease models, including inflammatory pain, rheumatoid arthritis, and acute kidney injury, and reduces tissue inflammation and damage by regulating the STING pathway.

[0032] On the other hand, layered double hydroxides (LDH) are anion intercalation materials, which are composed of positively charged metal hydroxide layers and interlayer anions. They have high specific surface area, good chemical stability and controllable composition. LDH nanosheets show excellent performance in anti-oxidation. They can effectively remove reactive oxygen free radicals and reduce oxidative stress by capturing free radicals and catalyzing the decomposition of peroxides. In addition, LDH can adsorb and remove surrounding free DNA and reduce the activation of the STING pathway to reduce inflammation. In addition, LDH nanosheets can also be used as nano-drug carriers to regulate the release of drugs and enhance the therapeutic effect.

[0033] In the present invention, C-176 exhibits excellent anti-inflammatory effects in a bone implant-induced aseptic inflammatory disease model, thereby regulating the local bone metabolism process. When it is used in combination with LDH, it is surprisingly found that the nanomedicine formed by assembling C-176 and LDH is beneficial to solving the above problems.

[0034] The term LDH refers to a class of nanosheets with a layered microstructure, usually composed of positively charged two-dimensional layers and exchangeable anions and water molecules between the layers. The general chemical formula is generally expressed as M(II) 1-x M(III) x (OH)2(A)·mH2O, where M(II) represents a divalent metal cation, such as Mg 2+ 、Zn 2+ etc.; M(III) represents a trivalent metal cation, such as Al 3+ , Fe 3+ etc.; A represents the interlayer anion, which can be CO3 2- 、NO3 - , Cl - or other organic or inorganic anions; x is the molar ratio of M(III) to total metal ions, and m represents the number of water molecules. By adjusting the synthesis conditions or post-treatment, the physicochemical properties of LDH can be regulated to meet specific application requirements, such as drug carriers, adsorbents, catalysts, etc.

[0035] The term single-layer 2D nanosheet refers to a structure whose thickness is limited to the nanometer scale (usually 1-100 nanometers) in one or two dimensions, while it can extend to the macroscopic scale in the other two dimensions. When "monolayer" is mentioned, it means that the thickness of these nanosheets is approximately equivalent to the thickness of one or a few atomic layers, which gives them a high specific surface area.

[0036] Beneficial Effects

[0037] (1) The nanomedicine of the present invention is applied via a local administration route. In the case of local application, the drug can be accurately delivered to the bone tissue around the implant through professional equipment, achieving a rapid and efficient therapeutic effect.

[0038] (2) The present invention can significantly reduce the occurrence of aseptic inflammation of bone implants and the development of implant loosening, and improve the clinical prognosis of patients. Compared with the prior art, the present invention has higher clinical adaptability and is particularly suitable for patients who cannot undergo surgical treatment. In addition, the method of use of the present invention is simple to operate, has few side effects, can effectively improve the quality of life of patients, and reduce treatment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the TEM microscopic morphology of the nanomedicine in Example 2.

[0040] Figure 2 This is the AFM morphology of the nanomedicine in Example 2.

[0041] Figure 3 This is the antioxidant capacity of the nanomedicine in Example 2.

[0042] Figure 4 This is the inhibitory effect of the nanodrug in Example 2 on the STING pathway.

[0043] Figure 5 This is the inflammatory response alleviating effect of the nano drug in Example 2.

[0044] Figure 6 This is the inhibitory effect of the nanomedicine in Example 2 on macrophage polarization.

[0045] Figure 7 This is the inhibitory effect of the nanomedicine in Example 2 on osteoclast activation.

[0046] Figure 8-10 This is the alleviating effect of the nanomedicine in Example 2 on osteolysis in an animal model. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0048] Example 1

[0049] 55 mg of cobalt nitrate hexahydrate and 24 mg of aluminum nitrate nonahydrate were dissolved in 10 mL of water and added dropwise to 20 mL of 23 vol% formamide solution at a rate of 1 mL / min using a syringe pump, while magnetic stirring was performed and the reaction environment was maintained at 80 °C. During the reaction, sodium hydroxide solution was added intermittently to maintain the pH value of the reaction system at 10. After the addition was completed, the reaction was continued for 10 minutes. After the reaction was completed, the mixed solution was collected and centrifuged at 12000 rpm for 10 minutes. The centrifuged product was washed twice with anhydrous ethanol and then washed three times with deionized water, and finally dispersed in 10 mL of water to obtain an LDH aqueous solution.

[0050] 1 mg of C-176 was dissolved in 2 mL of 50 vol% tetrahydrofuran aqueous solution, and then added to 20 mg of LDH aqueous solution, and mixed under magnetic stirring at room temperature for 12 hours. After the reaction was completed, the reaction solution was collected and centrifuged at 12000 rpm for 10 minutes. The centrifuged product was washed twice with deionized water and redispersed in deionized water to obtain a nanomedicine for treating aseptic loosening of bone implants.

[0051] Example 2

[0052] 109 mg of cobalt nitrate hexahydrate and 47 mg of aluminum nitrate nonahydrate were dissolved in 20 mL of water, and added dropwise to 20 mL of 23 vol% formamide solution at a rate of 1 mL / min using a syringe pump, while magnetic stirring was performed and the reaction was maintained in a water bath at 80°C. During the reaction, potassium hydroxide solution was added intermittently to maintain the pH value of the reaction system at 10. After the addition was completed, the reaction was continued for 10 minutes. After the reaction was completed, the reaction solution was collected and centrifuged at 12000 rpm for 10 minutes. The centrifuged product was washed twice with anhydrous ethanol and then washed three times with deionized water, and finally dispersed in 10 mL of water to obtain an LDH aqueous solution.

[0053] 1 mg of C-176 was dissolved in 2 mL of 60 vol% tetrahydrofuran aqueous solution, and then added to 10 mg of LDH aqueous solution, and mixed under magnetic stirring at room temperature for 6 hours. After the reaction was completed, the reaction solution was collected and centrifuged at 12000 rpm for 10 minutes. The centrifuged product was washed twice with deionized water and redispersed in deionized water to obtain a nanomedicine for treating aseptic loosening of bone implants.

[0054] Example 3

[0055] 221 mg of cobalt nitrate hexahydrate and 97 mg of aluminum nitrate nonahydrate were dissolved in 20 mL of water, and added dropwise to 20 mL of 23 vol% formamide solution at a rate of 1 mL / min using a syringe pump, while magnetic stirring was performed and the reaction was maintained in a water bath at 80°C. During the reaction, sodium hydroxide solution was added intermittently to maintain the pH value of the reaction system at 10. After the addition was completed, the reaction was continued for 10 minutes. After the reaction was completed, the reaction solution was collected and centrifuged at 12000 rpm for 10 minutes. The centrifuged product was washed twice with anhydrous ethanol and then washed three times with deionized water, and finally dispersed in 10 mL of water to obtain an LDH aqueous solution.

[0056] 10 mg of C-176 was dissolved in 2 mL of 50 vol% tetrahydrofuran aqueous solution, and then added to 20 mg of LDH aqueous solution, and mixed under magnetic stirring at room temperature for 24 hours. After the reaction was completed, the reaction solution was collected and centrifuged at 12000 rpm for 10 minutes. The centrifuged product was washed twice with deionized water and redispersed in deionized water to obtain a nano drug.

[0057] 5 mg of PEG2000-COOH was dissolved in 1 mL of deionized water, then added to 10 mL of nanomedicine and mixed evenly, and then 45 mg of sodium chloride was added to adjust the osmotic pressure to obtain the nanomedicine for treating aseptic loosening of bone implants.

[0058] Test Example 1

[0059] Microscopic morphology:

[0060] Take an appropriate amount of the finished product of Example 2 and drip it onto the copper mesh. After drying, use a FEI Talos F200X G2 transmission electron microscope to observe the morphology. Figure 1 As shown, LDH exhibits a nanosheet structure with a maximum diameter of 51 nm.

[0061] Take an appropriate amount of the finished product of Example 2 and drop it on a clean substrate and let it dry naturally. Then, install the substrate on the sample stage of the atomic force microscope and select a suitable probe for scanning. Figure 2 As shown, the thickness of LDH nanosheets is about 1.39 nm.

[0062] Test Example 2

[0063] Antioxidant capacity of nanomedicine:

[0064] Nano drug solutions of Example 2 with different concentrations were prepared, and the nano drug solutions were fully mixed with 1,2,3-trihydroxybenzene solution at 37°C for 0.5 hours. The changes in absorbance were measured by UV-visible spectrometer to evaluate the antioxidant properties of the nano drug. The antioxidant activity of the nano drug solutions with different concentrations was determined by comparing their absorbances. Figure 3 As shown, the nanodrug solution exhibited significant antioxidant activity.

[0065] Test Example 3

[0066] Example 2 Nanomedicine inhibits excessive activation of the STING pathway:

[0067] Ti particles were co-cultured with bone marrow-derived macrophages to induce the formation of sterile inflammation. PBS, nanomedicine without C-176 (0.1 mg / mL), and nanomedicine containing C-176 (0.1 mg / mL) were added to each group for intervention, and the inhibitory effect of nanomedicine on the STING pathway was evaluated by detecting the expression of IRF3, p-IRF3, TBK1, p-TBK1, and STING proteins. Figure 4 As shown, the nanomedicine containing C-176 exhibited the strongest inhibitory effect.

[0068] Test Example 4

[0069] Example 2 Nanomedicine relieves local inflammatory response:

[0070] Ti particles were co-cultured with bone marrow-derived macrophages to induce the formation of sterile inflammation. Each group was intervened by adding PBS, nanomedicine without C-176 (0.1 mg / ml), and nanomedicine containing C-176 (0.1 mg / ml). The expression of TNF-α and IL-6 pro-inflammatory factors was detected to evaluate the inhibitory effect of nanomedicine on the inflammatory response induced by Ti particles. Figure 5 As shown, nanomedicine containing C-176 can inhibit the expression of cellular proinflammatory factors.

[0071] Test Example 5

[0072] Nanomedicine regulates local bone metabolism:

[0073] Ti particles were co-cultured with bone marrow-derived macrophages to induce polarization of macrophages. PBS, nanodrugs without C-176 (0.1 mg / ml), and nanodrugs containing C-176 (0.1 mg / ml) were added to each group for intervention. The proportion of M1 macrophages was detected by flow cytometry to evaluate the mitigation effect of nanodrugs on macrophage polarization. Furthermore, after seven days of continuous culture, the degree of differentiation of macrophages into osteoclasts was detected by TRAP staining to evaluate the inhibitory effect of nanodrugs on osteoclast activation. Figure 6 As shown in Figure 2, nanomedicine containing C-176 can inhibit the polarization of macrophages. Figure 7 As shown, nanomedicine containing C-176 can inhibit the activation of osteoclasts.

[0074] Test Example 6

[0075] Nanomedicine inhibits the formation of skull osteolysis in mice:

[0076] Ti particles were locally injected into the cortex of the mouse skull to obtain a living model of mouse skull osteolysis. The animals were randomly divided into 5 groups, and each group was injected with PBS, C-176, nanomedicine without C-176, and nanomedicine containing C-176 locally in the skull for intervention treatment. The injection was once every two days, for a total of 5 injections. The skulls of each group of mice were scanned by a Micro-CT machine and 3D reconstruction was performed. The skull status was quantified by BMD and BV / TV indicators. Figure 8-10 As shown, nanomedicine containing C-176 can effectively alleviate the formation of osteolysis, with the highest bone density and bone volume fraction.

Claims

1. A nanomedicine for treating aseptic loosening of bone implants, characterized in that: The nano drug includes a STING inhibitor C-176 and a layered double metal hydroxide LDH; the C-176 is adsorbed on the interlayer gap or surface of the LDH spatial structure through intermolecular forces.

2. The nano drug according to claim 1, characterized in that: The LDH is a double metal hydroxide comprising Al and Co, and the molar ratio of Al to Co is 1:0.2-1:

5.

3. The nano drug according to claim 1, characterized in that: The weight ratio of C-176 to LDH is 0.01:1-0.1:

1.

4. The nano drug according to claim 1, characterized in that: The nano drug also includes one or more of a stabilizer, a dispersion medium, and an osmotic pressure regulator.

5. The nano drug according to claim 4, characterized in that: The stabilizer is selected from at least one of PEG2000-COOH, hyaluronic acid, and albumin; the dispersion medium is selected from at least one of ultrapure water, physiological saline, and PBS; and the osmotic pressure regulator is selected from at least one of glucose, sodium chloride, mannitol, glycine, or its hydrochloride.

6. A method for preparing a nanomedicine for treating aseptic loosening of bone implants, comprising the following steps: (1) mixing a metal nitrate solution with a formamide solution for reaction, and centrifuging and washing the mixture after the reaction to obtain an LDH aqueous solution; (2) The LDH aqueous solution and the C-176 solution are mixed and reacted, and then centrifuged and washed to obtain a nanomedicine for treating aseptic loosening of bone implants.

7. The preparation method according to claim 6, characterized in that: The metal nitrate in step (1) comprises aluminum nitrate and cobalt nitrate, and the molar ratio of aluminum nitrate to cobalt nitrate is 1:0.2-1:

5.

8. The preparation method according to claim 6, characterized in that: The volume ratio of the metal nitrate solution to the formamide solution in the step (1) is 1:0.5-1:

5.

9. The preparation method according to claim 6, characterized in that: The amount of C-176 added in step (2) is 0.01-0.1 (w / w) of LDH.

10. The preparation method according to claim 6, characterized in that: The invention also includes post-processing the obtained nanomedicine for treating aseptic loosening of bone implants.

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