Medicated mineralized micro-rods, methods of making and using the same

Abstract

The present application relates to the technical field of medical polymer materials, and particularly relates to a drug mineralized microstick, a preparation method and application thereof. The preparation method of the drug mineralized microstick provided by the present application comprises the following steps: A) mixing rhubarb acid and alkali liquor to obtain a first solution; B) mixing the first solution with a degradable high polymer compound to obtain a second solution; the degradable high polymer compound contains carboxylate; C) mixing the second solution with a metal salt solution to obtain a third solution; D) incubating the third solution under constant temperature and humidity to obtain the drug mineralized microstick. The drug mineralized microstick prepared by the present application has a highly crystalline structure and the ability to effectively release small molecule drugs; the drug mineralized microstick has the characteristics of low hemolysis and low cytotoxicity; in an environment of excessive inflammatory response, the drug mineralized microstick can effectively reduce the expression of proinflammatory mediators, and exhibits high anti-inflammatory activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical polymer materials, in particular to a drug mineralized micro-rod, a preparation method and application thereof. BACKGROUND

[0002] Inflammation is an emergency response of the organism when suffering from cell damage, tissue destruction or infection. This response protects the organism from bacteria, viruses or infection by resisting harmful stimuli, eliminating pathogens and promoting tissue regeneration. However, excessive inflammatory response can lead to tissue damage, organ dysfunction or imbalance of homeostasis in the body, eventually causing the development of various diseases, including systemic inflammatory response syndrome, autoimmune and neurodegenerative diseases, and can further cause cardiovascular diseases, cancer, diabetes and chronic kidney disease. These inflammation-related diseases are the main causes of global disability and death (more than 50%). Therefore, developing various therapeutic strategies to regulate excessive inflammatory response and meet the clinical needs to the maximum extent not only has scientific and clinical significance, but also has great social significance.

[0003] Drug therapy as a traditional treatment method is widely used to regulate inflammatory response. In drug therapy, natural small molecule drugs have become potential high-efficiency therapeutic drugs due to their wide range of pharmacophore types and high stereochemical structures. However, low stability, low bioavailability and adverse side effects still limit their wide application in clinical treatment. At present, researchers have designed various carrier structures to encapsulate drugs in order to achieve targeted drug delivery and controlled release, so as to achieve high-efficiency treatment and reduce drug toxicity. However, low drug loading rate, rapid release rate, complex carrier synthesis and potential side effects are still obstacles to its clinical application. Therefore, designing a natural small molecule drug delivery system with high drug loading rate, long-acting slow-release small molecule drugs, excellent biological safety and improved drug treatment activity has become one of the focuses of researchers in the field. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a drug mineralized micro-rod, a preparation method and application thereof. The drug mineralized micro-rod prepared by the present application has a high crystalline structure and the ability to effectively slow-release small molecule drugs. This drug mineralized micro-rod has the characteristics of low hemolysis and low cytotoxicity. In the environment of excessive inflammatory response, the drug mineralized micro-rod can effectively reduce the expression of pro-inflammatory mediators, and exhibits high-efficiency anti-inflammatory activity.

[0005] The present application provides a preparation method of a drug mineralized micro-rod, comprising the following steps:

[0006] A) mixing rhubarb acid with alkali solution to obtain a first solution;

[0007] B) mixing the first solution with a degradable high molecular compound to obtain a second solution; the degradable high molecular compound contains carboxylate;

[0008] C) mixing the second solution with a metal salt solution to obtain a third solution;

[0009] D) incubating the third solution under constant temperature and humidity to obtain the drug mineralized micro-rods.

[0010] Preferably, in step A), the alkali solution comprises at least one of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate-sodium bicarbonate buffer solution and triethylamine;

[0011] The pH value of the alkali solution is 7.5-9;

[0012] The molar ratio of the rhein to the alkali in the alkali solution is 1:0.2-2.

[0013] Preferably, in step B), the degradable high molecular compound comprises at least one of sodium hyaluronate, sodium alginate, polyamino acid salt, carboxymethyl chitosan and carboxymethyl cellulose;

[0014] The molecular weight Mn of the sodium hyaluronate is preferably 3-3000 kDa;

[0015] The molecular weight Mn of the sodium alginate is preferably 3-3000 kDa;

[0016] The molecular weight Mn of the polyamino acid salt is preferably 3-1000 kDa;

[0017] The viscosity of the carboxymethyl chitosan is 10-80 mPa·s;

[0018] The viscosity of the carboxymethyl cellulose is 300-15000 mPa·s.

[0019] Preferably, the molar ratio of the carboxylate in the degradable high molecular compound to the carboxylate in the rhein is 0.001-100:1.

[0020] Preferably, in step C), the metal salt solution comprises at least one of Ca 2+ , Fe 3+ , Zn 2+ and Mg 2+ ;

[0021] The metal salt is selected from at least one of calcium chloride, ferric chloride, zinc nitrate and magnesium chloride;

[0022] The concentration of the metal salt solution is 3-20 mg / mL.

[0023] Preferably, the molar ratio of the carboxylate in the rheum emic acid to the metal ion in the metal salt is 0.01-100:1.

[0024] Preferably, in step D), the temperature of the incubation is 10-50℃, the humidity is 30%-98% RH, and the time is 4-15d.

[0025] Preferably, in step D), after the incubation, further comprising: centrifugation, water washing, and freeze-drying.

[0026] The centrifugation is at a speed of 500-10000rpm for 5-30min.

[0027] The present application also provides a drug mineralized microstick prepared by the preparation method described above.

[0028] The present application also provides an application of the drug mineralized microstick described above in the biomedical field.

[0029] The present application provides a preparation method of a drug mineralized microstick, comprising the following steps: A) mixing rheum emic acid with an alkali solution to obtain a first solution; B) mixing the first solution with a degradable high molecular compound to obtain a second solution; the degradable high molecular compound contains carboxylate; C) mixing the second solution with a metal salt solution to obtain a third solution; D) incubating the third solution under constant temperature and humidity to obtain a drug mineralized microstick. In the present application, the drug mineralized microstick is obtained by aggregation of rheum emic acid / metal ion assembly with the degradable high molecular compound as a stabilizer; the rheum emic acid / metal ion assembly is formed by coordination of natural small molecule drug rheum emic acid and metal ion. The drug mineralized microstick prepared by the present application has a highly crystalline structure and the ability to effectively release small molecule drugs; this drug mineralized microstick has the characteristics of low hemolysis and low cytotoxicity; in an environment of excessive inflammatory response, the drug mineralized microstick can effectively reduce the expression of pro-inflammatory mediators, showing high anti-inflammatory activity. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The formation diagram of the drug mineralized microstick provided for an embodiment of the present application;

[0031] Figure 2 The SEM diagram of the free rheum emic acid small molecule in the sample after incubation of embodiment 2 of the present application;

[0032] Figure 3 The SEM diagram of the drug mineralized microstick of embodiment 2 of the present application;

[0033] Figure 4 The standard curve of rheum emic acid at 251nm of ultraviolet light;

[0034] Figure 5The content of rheum emodin in the drug mineralized micro-rods of different concentrations in Example 2 of the present application;

[0035] Figure 6 The modulus statistical diagram of the free rheum emodin small molecules and the drug mineralized micro-rods in Example 4 of the present application;

[0036] Figure 7 The UV spectrum of the free rheum emodin small molecules and the drug mineralized micro-rods in Example 7 of the present application;

[0037] Figure 8 The drug release curve of the drug mineralized micro-rods of Example 8 of the present application;

[0038] Figure 9 The hemolysis rate curve of the drug mineralized micro-rods of Example 9 of the present application under different concentrations;

[0039] Figure 10 The cytotoxicity test diagram of the drug mineralized micro-rods of Example 10 of the present application;

[0040] Figure 11 The aggregate structure test diagram of the drug mineralized micro-rods of Example 11 of the present application;

[0041] Figure 12 The result diagram of the drug mineralized micro-rods of Example 11 of the present application in down-regulating the intracellular ROS ability in excessive inflammatory response. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0043] The present application provides a preparation method of drug mineralized micro-rods, comprising the following steps:

[0044] A) mixing rheum emodin with alkali liquor to obtain a first solution;

[0045] B) mixing the first solution with a degradable high molecular compound to obtain a second solution; the degradable high molecular compound contains carboxylate;

[0046] C) mixing the second solution with a metal salt solution to obtain a third solution;

[0047] D) incubating the third solution under constant temperature and humidity conditions to obtain drug mineralized micro-rods.

[0048] In organisms, the biomineralization process encapsulates bioactive molecules such as proteins, polypeptides and enzymes through supramolecular co-assembly to form biological minerals. This active molecule-mediated mineralization can endow the biological minerals with special structures and functions. Inspired by this, in the present application, we directly use natural small molecule drugs as structural units to construct drug-mineralized materials by simulating the biological mineralization process, realize efficient drug encapsulation and sustained controllable drug release, and thus achieve the purposes of improving drug utilization, enhancing drug safety and strengthening drug efficacy.

[0049] In step A):

[0050] Mixing rhubarb acid with alkali liquor to obtain a first solution.

[0051] In the present application, the rhubarb acid has the structure shown in Formula I:

[0052]

[0053] In some embodiments of the present application, the alkali liquor is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate-sodium bicarbonate buffer solution and triethylamine. The solvent of the alkali liquor is deionized water. The pH value of the alkali liquor is 7.5-9, specifically 8.

[0054] The molar ratio of the rhubarb acid to the alkali in the alkali liquor is 1:0.2-2, such as 1:0.9, 1:0.75, 1:1.8, 1:0.7, 1:0.38, 1:0.39, 1:1.5, 1:0.84.

[0055] The mixing is carried out at room temperature. The mixing process is a process of complete dissolution.

[0056] In step B):

[0057] Mixing the first solution with a degradable high molecular compound to obtain a second solution.

[0058] In some embodiments of the present application, the degradable high molecular compound is selected from at least one of sodium hyaluronate, sodium alginate, polyamino acid salt, carboxymethyl chitosan and carboxymethyl cellulose.

[0059] The molecular weight Mn of the sodium hyaluronate is preferably 3-3000 kDa.

[0060] The molecular weight Mn of the sodium alginate is preferably 3-3000 kDa.

[0061] The polyamino acid salt has a molecular weight Mn of 3-1000 kDa. The polyamino acid salt can be selected from polyglutamic acid or polyaspartic acid. The polyglutamic acid has a molecular weight Mn of 3-100 kDa, and the polyaspartic acid has a molecular weight Mn of 3-70 kDa.

[0062] The present application further limits the molecular weight of the above-mentioned degradable high molecular compound. The high molecular compound with a molecular weight less than 3 kDa has low negative charge and cannot stabilize the rheum emic acid and metal ions. The high molecular compound system with a molecular weight greater than 3000 kDa has large viscosity, which results in low yield of the rheum emic acid mineralized micro rod when the system is washed with water and centrifuged to collect the rheum emic acid mineralized micro rod, and the rheum emic acid mineralized micro rod and unreacted high molecular compound cannot be successfully separated.

[0063] The carboxymethyl chitosan has a viscosity η of 10-80 mPa·s.

[0064] The carboxymethyl cellulose has a viscosity η of 300-15000 mPa·s. The carboxymethyl cellulose is sodium carboxymethyl cellulose.

[0065] The present application further limits the viscosity range of the carboxymethyl chitosan and carboxymethyl cellulose. The high molecular solution system with small viscosity cannot stabilize the suspension of rheum emic acid and metal ions, and the rheum emic acid and calcium ions are directly complexed and precipitated.

[0066] In the present application, the degradable high molecular compound containing carboxylate is a stabilizer, which drives the rheum emic acid / metal ion assembly to aggregate through various forces, and finally generates the mineralized micro rod with rheum emic acid small molecules as the structural unit.

[0067] The degradable high molecular compound provides a limited aggregation area for the rheum emic acid / metal ion assembly.

[0068] The molar ratio of the carboxylate in the degradable high molecular compound to the carboxylate in the rheum emic acid is 0.001-100:1, preferably 1-20:1, such as 1.4:1, 1.1:1, 3.4:1, 4.4:1, 2.52:1, 2.9:1, 2.8:1, 1.39:1.

[0069] The mixing is performed at room temperature. The mixing process is a complete dissolution process, which can be achieved by stirring.

[0070] In step C), the second solution is mixed with a metal salt solution to obtain a third solution.

[0071] The second solution is mixed with a metal salt solution to obtain a third solution.

[0072] In some embodiments of the present application, the metal salt solution comprises Ca 2+ , Fe 3+ , and Zn.2+ and Mg 2+ The metal salt is selected from at least one of calcium chloride, ferric chloride, zinc nitrate and magnesium chloride. The concentration of the metal salt solution is 3-20 mg / mL, specifically 5 mg / mL. The solvent of the metal salt solution is deionized water.

[0073] The molar ratio of the carboxylate in the rhein to the metal ion in the metal salt is 0.01-100:1, preferably 0.1-10:1, such as 5:1, 0.9:1, 1.2:1, 1.5:1, 1.1:1, 6.5:1, 1.9:1, 2.6:1, 1.75:1, 0.97:1.

[0074] The mixing is carried out at room temperature. The mixing is stirring to mix well. The time for stirring to mix well is 8-24 h, such as 8 h, 18 h, 10 h, 16 h, 12 h, 20 h, 21 h, 6 h, 23 h, 13 h.

[0075] In step D), the third solution is incubated under constant temperature and humidity to obtain the drug mineralized micro-rods.

[0076] The third solution is incubated under constant temperature and humidity to obtain the drug mineralized micro-rods.

[0077] In some embodiments of the present application, the temperature for incubation is 10-50℃, such as 28℃, 30℃, 37℃; the humidity is 30%-98% RH; the time is 4-15 d, such as 4 d, 5 d, 10 d, 9 d, 15 d, 6 d, 11 d. The incubation is carried out in a constant temperature and humidity incubator.

[0078] After the incubation, centrifugation, water washing and freeze-drying are further included. The centrifugation is carried out at a speed of 500-10000 rpm for 5-30 min. The centrifugation and water washing can be repeated 2-5 times. The freezing temperature is -80--10℃, and the drying temperature is -10-40℃; specifically, the freezing temperature is -20℃, -30℃, -40℃, -80℃, and the drying temperature is 4℃, 25℃, 18℃, 8℃, 7℃.

[0079] In the present application, the pH value of rhein is first adjusted by using alkali solution to change the ionic state of rhein, then the degradable high molecular compound is added and mixed uniformly with rhein, and then the metal ion is added. If the raw material components are added together, the alkali solution will change the ionization degree of the polyanion on one hand, and will directly form a precipitate with the metal ion on the other hand. If the metal ion is added before the degradable high molecular compound, it will also directly form a precipitate with the metal ion. It cannot slowly form a dense mineralized micro-rod under the regulation of the high molecular.

[0080] In the present application, the drug mineralized micro-rods are obtained by aggregation of rhubarb acid / metal ion assembly with degradable high polymer compound as stabilizer; the rhubarb acid / metal ion assembly is formed by coordination of natural small molecule drug rhubarb acid and metal ion.

[0081] Figure 1 The formation diagram of the drug mineralized micro-rods provided by an embodiment of the present application is shown.

[0082] The preparation method of the drug mineralized micro-rods provided by the present application is simple in operation and is conducive to industrialized production, and is expected to provide a new drug dosage form for clinical treatment of inflammatory diseases.

[0083] The present application further provides a drug mineralized micro-rod prepared by the above preparation method.

[0084] The rhubarb acid / metal ion assembly is formed by coordination of natural small molecule drug rhubarb acid and metal ion.

[0085] In some embodiments of the present application, the length of the drug mineralized micro-rods is 20.22±4.90 μm, and the length dimension of the cross section is 1.35±0.36 μm.

[0086] The present application further provides an application of the above drug mineralized micro-rods in the biomedical field.

[0087] The present application stabilizes the rhubarb acid / metal ion assembly with degradable high polymer compound, so that it is slowly aggregated and then crystallized in a limited area, and finally generates micro-rod-shaped crystalline with dense mineralized structure. The mineralized micro-rods prepared mainly with small molecule drugs as structural building units have high drug loading rate (drug loading amount is 75%). At the same time, due to the dense mineralized structure, the micro-rods can long-acting release small molecule drugs, thereby improving the drug stability, prolonging the drug action time and improving the drug biological safety, having higher treatment efficiency and more optimal anti-inflammatory activity. Therefore, the present application claims the application of the drug mineralized micro-rods as drug carriers, and the application of the drug mineralized micro-rods in preparation of anti-inflammatory drugs.

[0088] In an environment of excessive inflammatory response, the drug mineralized micro-rods prepared by the application can effectively regulate the expression of inflammatory mediators, improve the inflammatory microenvironment, and have high anti-inflammatory capacity. The technical scheme of the application not only perfectly solves the problems of low bioavailability and poor biological stability of traditional rhein, but also effectively improves the biological safety and biological activity. Therefore, the drug mineralized micro-rods prepared by the application are expected to be applied to clinical disease treatment as a new generation of rhein drug preparation.

[0089] The experimental results show that the drug mineralized micro-rods prepared by the application have a highly crystalline structure and the ability to effectively release small molecule drugs. The drug mineralized micro-rods have the characteristics of low hemolysis and low cytotoxicity. Finally, in an environment of excessive inflammatory response, the drug mineralized micro-rods can effectively reduce the expression of pro-inflammatory mediators, and exhibit high anti-inflammatory activity.

[0090] The raw materials used in the above are not particularly limited in the application, and can be generally commercially available.

[0091] In order to further illustrate the application, the drug mineralized micro-rods, the preparation method and the application thereof provided by the application are described in detail below, but they should not be understood as limiting the protection scope of the application.

[0092] Example 1

[0093] 1) 20 mg of rhein was mixed with 1 mL of an aqueous sodium hydroxide solution (pH = 8) at room temperature, and was fully dissolved to obtain a first solution; the molar ratio of the rhein to the sodium hydroxide was 1:0.9;

[0094] 2) 20 mg of sodium alginate (Mn = 100 kDa) was mixed with the first solution, and was fully dissolved by stirring at room temperature to obtain a second solution;

[0095] The molar ratio of the carboxylate in the sodium alginate to the carboxylate in the rhein was 1.4:1;

[0096] 3) 31 μL of a calcium chloride solution (50 mg / mL) was mixed with the second solution, and was uniformly mixed by stirring for 8 h to obtain a third solution;

[0097] The molar ratio of the carboxylate in the rhein to the calcium ion was 5:1;

[0098] 4) The third solution was placed in a constant-temperature and constant-humidity incubator (humidity 30% to 98% RH) at 28°C for incubation for 4 d, and after centrifugation at 2000 rpm for 5 min, the obtained sample was washed with water for 3 times, was frozen at -20°C, and was dried at 4°C to obtain the drug mineralized micro-rods.

[0099] Example 2

[0100] 1) 25 mg rhein was mixed with 1 mL aqueous sodium hydroxide solution (pH = 8.5) at room temperature to dissolve completely to obtain a first solution; the molar ratio of rhein to sodium hydroxide was 1:0.75;

[0101] 2) 20 mg sodium alginate (Mn = 10 kDa) was mixed with the first solution, and stirred at room temperature to dissolve completely to obtain a second solution;

[0102] The molar ratio of carboxylate in the sodium alginate to carboxylate in the rhein was 1.1:1;

[0103] 3) 23 μL of calcium chloride solution (500 mg / mL) was mixed with the second solution, and stirred for 18 h to mix uniformly to obtain a third solution;

[0104] The molar ratio of carboxylate in the rhein to calcium ions was 0.9:1;

[0105] 4) The third solution was placed in a constant temperature and humidity incubator at 30°C (humidity 30% to 98% RH) for incubation for 5 d, and after centrifugation at 2000 rpm for 5 min, the obtained sample was washed with water for 3 times, frozen at -20°C, and dried at 25°C to obtain the drug mineralized microrods.

[0106] The free rhein small molecules in the centrifugal liquid obtained by centrifugation of Example 2 were analyzed by field emission electron microscopy. Specifically, deionized water was used to prepare a solution of the centrifugal liquid with a free rhein concentration of 100 μg / mL, 10 μL of the solution was dropped on a clean silicon wafer surface, and after drying at room temperature, the microstructure of the sample was observed by field emission electron microscopy, as shown in Figure 2 . Figure 2 The SEM image of the free rhein small molecules in the sample after incubation of Example 2 of the application can be known from Figure 2 .

[0107] The drug mineralized microrods obtained in Example 2 were analyzed by field emission electron microscopy. Specifically, deionized water was used to prepare a solution of the drug mineralized microrods with a concentration of 100 μg / mL, 10 μL of the solution was dropped on a clean silicon wafer surface, and after drying at room temperature, the microstructure of the sample was observed by field emission electron microscopy, as shown in Figure 3 . Figure 3 The SEM image of the drug mineralized microrods of Example 2 of the application can be known from Figure 3 .

[0108] The absorbance of different mass concentrations of rhein (2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and the solvent is deionized water) at 251 nm of ultraviolet light was tested by the ultraviolet spectrophotometer method, and a standard curve was drawn as shown in Figure 4 . Figure 4 The standard curve of rhein at 251 nm of ultraviolet light was drawn. Meanwhile, the absorbance of different mass concentrations of the above-mentioned drug mineralized micro-rods (4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and the solvent is deionized water) at 251 nm of ultraviolet light was measured, and the mass content of rhein in the drug mineralized micro-rods was calculated according to the standard curve, which was 75%, and the results are shown in Figure 5 , Figure 5 The rhein content in the drug mineralized micro-rods of different concentrations in Example 2 of the present application was proved to have a high drug loading rate.

[0109] Example 3

[0110] 1) 25 mg of rhein was mixed with 1 mL of an aqueous solution of sodium hydroxide (pH = 8.5) at room temperature, and was fully dissolved to obtain a first solution; the molar ratio of the rhein to sodium hydroxide was 1:0.75;

[0111] 2) 50 mg of sodium hyaluronate (Mn = 10 kDa) was mixed with the first solution, and was fully dissolved by stirring at room temperature to obtain a second solution;

[0112] The molar ratio of the carboxylate in the sodium hyaluronate to the carboxylate in the rhein was 1.4:1;

[0113] 3) 26 μL of a calcium chloride solution (300 mg / mL) was mixed with the second solution, and was uniformly mixed by stirring for 10 h to obtain a third solution;

[0114] The molar ratio of the carboxylate in the rhein to the calcium ion was 1.2:1;

[0115] 4) The third solution was placed in a constant-temperature and constant-humidity incubator (humidity 30% to 98% RH) at 37°C for incubation for 10 d, and after centrifugation at 5000 rpm for 6 min, the obtained sample was washed with water for 3 times, was frozen at -30°C, and was dried at 25°C to obtain the drug mineralized micro-rods.

[0116] Example 4

[0117] 1) 10 mg of rhein was mixed with 1 mL of an aqueous solution of sodium hydroxide (pH = 8) at room temperature, and was fully dissolved to obtain a first solution; the molar ratio of the rhein to sodium hydroxide was 1:1.8;

[0118] 2) mix 50 mg sodium hyaluronate (Mn = 1000 kDa) with the first solution, stir at room temperature until it is fully dissolved to obtain a second solution;

[0119] The molar ratio of carboxylate in the sodium hyaluronate to carboxylate in rhein is 3.4:1;

[0120] 3) mix 26 μL of calcium chloride solution (100 mg / mL) with the second solution, stir for 16 h until it is uniformly mixed to obtain a third solution;

[0121] The molar ratio of carboxylate in the rhein to calcium ions is 1.5:1;

[0122] 4) place the third solution in a constant temperature and humidity incubator at 30°C (humidity 30% ~ 98% RH) for 9 d, centrifuge at 5000 rpm for 5 min, then wash the obtained sample with water for 3 times, freeze at -20°C, and dry at 25°C to obtain the drug mineralized microrods.

[0123] The mechanical properties of the free rhein small molecules (centrifuged to obtain the centrifugal liquid) and the drug mineralized microrods in Example 4 at the nanoscale are tested by atomic force microscopy (AFM). Specifically, deionized water is used to prepare a solution of the free rhein small molecules with a concentration of 50 μg / mL, 10 μL of the solution is dropped on a clean silicon wafer surface, dried at room temperature, and then the surface elastic modulus of the sample is tested by atomic force microscopy (AFM); deionized water is used to prepare a solution of the drug mineralized microrods with a concentration of 50 μg / mL, 10 μL of the solution is dropped on a clean silicon wafer surface, dried at room temperature, and then the surface elastic modulus of the sample is tested by atomic force microscopy (AFM). The results are shown in Figure 6 . Figure 6 The modulus statistical diagram of the free rhein small molecules and the drug mineralized microrods in Example 4. Figure 6 In the figure, Free-drug represents the free rhein small molecules drug, and M-microrod represents the drug mineralized microrods. It can be seen from Figure 6 that compared with the elastic modulus (191.9 ± 13.9 MPa) of the free rhein small molecules (Free drug), the elastic modulus of the drug mineralized microrods (M-microrod) is significantly improved, which is 654.2 ± 49.5 MPa, indicating that the molecules in the drug mineralized microrods are more closely packed.

[0124] Example 5

[0125] 1) mix 25 mg of rhein with 1 mL of aqueous sodium hydroxide solution (pH = 8) at room temperature until it is fully dissolved to obtain a first solution; the molar ratio of the rhein to sodium hydroxide is 1:0.7;

[0126] 2) 50 mg of polyglutamic acid (Mn = 70 kDa) was mixed with the first solution, and stirred at room temperature until it was completely dissolved to obtain a second solution;

[0127] The molar ratio of carboxylate in the polyglutamic acid to carboxylate in the rhein was 4.4:1;

[0128] 3) 36 μL of a calcium chloride solution (300 mg / mL) was mixed with the second solution, and stirred for 12 h until it was uniformly mixed to obtain a third solution;

[0129] The molar ratio of carboxylate in the rhein to calcium ions was 1.1:1;

[0130] 4) The third solution was placed in a constant-temperature and constant-humidity incubator at 37°C (humidity 30%-98% RH) for incubation for 15 d, and after centrifugation at 1000 rpm for 5 min, the obtained sample was washed with water for 3 times, frozen at -80°C, and dried at 4°C to obtain the drug-mineralized microrods.

[0131] Example 6

[0132] 1) 25 mg of rhein was mixed with 1 mL of an aqueous sodium hydroxide solution (pH = 8.5) at room temperature until it was completely dissolved to obtain a first solution; the molar ratio of rhein to sodium hydroxide was 1:0.75;

[0133] 2) 50 mg of sodium hyaluronate (Mn = 100 kDa) was mixed with the first solution, and stirred at room temperature until it was completely dissolved to obtain a second solution;

[0134] The molar ratio of carboxylate in the sodium hyaluronate to carboxylate in the rhein was 1.4:1;

[0135] 3) 26 μL of a calcium chloride solution (300 mg / mL) was mixed with the second solution, and stirred for 20 h until it was uniformly mixed to obtain a third solution;

[0136] The molar ratio of carboxylate in the rhein to calcium ions was 1.2:1;

[0137] 4) The third solution was placed in a constant-temperature and constant-humidity incubator at 37°C (humidity 30%-98% RH) for incubation for 10 d, and after centrifugation at 5000 rpm for 5 min, the obtained sample was washed with water for 3 times, frozen at -20°C, and dried at 4°C to obtain the drug-mineralized microrods.

[0138] Example 7

[0139] 1) 50 mg of rhein was mixed with 1 mL of an aqueous solution of sodium hydroxide (pH = 8.5) at room temperature, and was fully dissolved to obtain a first solution; the molar ratio of rhein to sodium hydroxide was 1:0.38;

[0140] 2) 100 mg of carboxymethyl chitosan (viscosity of 500 mPa-s) was mixed with the first solution, and was fully dissolved by stirring at room temperature to obtain a second solution;

[0141] The molar ratio of carboxylate in the carboxymethyl chitosan to carboxylate in the rhein was 1.4:1;

[0142] 3) 26 μL of a zinc nitrate solution (300 mg / mL) was mixed with the second solution, and was uniformly mixed by stirring for 21 h to obtain a third solution;

[0143] The molar ratio of carboxylate in the rhein to zinc ions was 6.5:1;

[0144] 4) The third solution was placed in a constant-temperature and constant-humidity incubator (humidity of 30% to 98% RH) at 37°C, and was incubated for 6 d. After centrifugation at 3500 rpm for 5 min, the obtained sample was washed with water for 3 times, was frozen at -20°C, and was dried at 25°C to obtain the drug-mineralized micro-rods.

[0145] The change in intermolecular force of the free rhein small molecule (centrifuged to obtain a centrifugal liquid) and the drug-mineralized micro-rods in Example 7 was tested by ultraviolet spectrophotometry (UV). Specifically, methanol was used to prepare a solution of the centrifugal liquid with a free rhein concentration of 124 μg / mL, and the absorption value at a wavelength of 200 to 800 nm was tested by UV. Methanol was used to prepare a solution of the drug-mineralized micro-rods with a concentration of 124 μg / mL, and the absorption value at a wavelength of 200 to 800 nm was tested by UV. The results are shown in Figure 7 Figure 7 The UV spectrum of the free rhein small molecule and the drug-mineralized micro-rods in Example 7 of the present application. Figure 7 Figure 7 As can be seen, compared with the free rhein small molecule, the drug-mineralized micro-rods have an absorption peak at 520 nm, indicating that there is π-π interaction between the molecules in the drug-mineralized micro-rods.

[0146] Example 8

[0147] 1) 50 mg of rhein was mixed with 1 mL of an aqueous solution of sodium hydroxide (pH = 8.5) at room temperature, and was fully dissolved to obtain a first solution; the molar ratio of rhein to sodium hydroxide was 1:0.38;

[0148] ​​2) Mix 180 mg of sodium hyaluronate (Mn = 200 kDa) with the first solution, stir at room temperature until it is fully dissolved, to obtain a second solution;

[0149] The molar ratio of carboxylate in the sodium hyaluronate to carboxylate in rhein is 2.52: 1;

[0150] 3) Mix 50 μΐ of ferric chloride solution (300 mg / mL) with the second solution, stir for 16 h until it is well mixed, to obtain a third solution;

[0151] The molar ratio of carboxylate in the rhein to iron ions is 1.9: 1;

[0152] 4) Place the third solution in a constant temperature and humidity incubator at 37°C (humidity 30% ~ 98% RH) for 10 d, after centrifugation at 5000 rpm for 4 min, the obtained sample is washed with water for 3 times, frozen at -20°C, and dried at 18°C, to obtain the drug mineralized microrods.

[0153] The drug release test is performed on the drug mineralized microrods of Example 8: specifically, the drug mineralized microrods are prepared into a solution with a concentration of 2 mg / mL using PBS, 1 mL of the solution is taken and added into a 1000 Da dialysis bag for sealing. Then, the sealed dialysis bag is soaked in 9 mL of clean PBS at 37°C, and 1 mL of liquid outside the dialysis bag is taken at intervals (24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h) and fresh 1 mL of PBS is added at the same time. The mixed liquid is tested by UV for absorbance value, and the release curve is calculated based on the absorbance value of the taken liquid. Figure 8 The drug release curve of the drug mineralized microrods of Example 8 of the present application is shown in Figure 6. Figure 8 It can be seen that the drug mineralized microrods can release about 55% of rhein after being soaked in PBS for 24 h, then maintain a slow rhein release behavior, and finally the rhein is completely released after being soaked for 192 h. Therefore, the drug mineralized microrods exhibit long-acting and slow-release characteristics.

[0154] Example 9

[0155] 1) Mix 48 mg of rhein with 1 mL of aqueous sodium hydroxide solution (pH = 8.5) at room temperature until it is fully dissolved, to obtain a first solution; the molar ratio of rhein to sodium hydroxide is 1:0.39;

[0156] 2) Mix 64 mg of sodium hyaluronate (Mn = 200 kDa) with the first solution, stir at room temperature until it is fully dissolved, to obtain a second solution;

[0157] the molar ratio of carboxylate in the sodium hyaluronate to carboxylate in the rhein is 1.1:1;

[0158] 3) 24 μL of calcium chloride solution (300 mg / mL) was mixed with the second solution, and stirred for 16 h to make it uniformly mixed to obtain a third solution;

[0159] the molar ratio of carboxylate in the rhein to calcium ion is 2.6:1;

[0160] 4) the third solution was placed in a constant temperature and humidity incubator at 30°C (humidity 30%-98% RH) for 11 d, and after centrifugation at 5000 rpm for 5 min, the obtained sample was washed with water for 3 times, frozen at -20°C, and dried at 8°C to obtain the drug-mineralized microrods.

[0161] The blood compatibility of the drug-mineralized microrods of Example 9 was evaluated by hemolysis test: specifically, first, a fresh rabbit blood cell suspension with a volume concentration of 2% was incubated with different concentrations of drug-mineralized microrods at 37°C, the negative control group was PBS group, and the positive control group was sterile water. After 3 h, the supernatant was taken out, and the ultraviolet spectrophotometer was used to test the absorption light at 545 nm, and finally the hemolysis rate was calculated by formula (1).

[0162] Hemolysis rate % = (Abs sample group - Abs negative group) / (Abs positive group - Abs negative group) x 100% (1)

[0163] The results are shown in Figure 9 . Figure 9 The hemolysis rate curve of the drug-mineralized microrods of Example 9 at different concentrations. Figure 9 In the figure, Free-drug represents free rhein small molecule drug, and M-microrod represents drug-mineralized microrods. It can be seen from Figure 9 that the hemolysis rate of free rhein small molecule drug is basically 0%, and the hemolysis rate of the drug-mineralized microrods is not more than 5%, which proves that the drug-mineralized microrods have excellent blood compatibility.

[0164] Example 10

[0165] 1) 12 mg of rhein was mixed with 1 mL of aqueous sodium hydroxide solution (pH = 8.5) at room temperature to fully dissolve to obtain a first solution; the molar ratio of rhein to sodium hydroxide is 1:1.5;

[0166] 2) 50 mg of sodium hyaluronate (Mn = 50 kDa) was mixed with the first solution, and stirred at room temperature to fully dissolve to obtain a second solution;

[0167] the molar ratio of carboxylate in the sodium hyaluronate to carboxylate in the rhein is 2.9:1;

[0168] 3) 24 μL of zinc nitrate solution (300 mg / mL) was mixed with the second solution, and stirred for 6 h to obtain a third solution;

[0169] The molar ratio of carboxylate to zinc ion in the rhein was 1.75:1;

[0170] 4) The third solution was incubated in a constant temperature and humidity incubator (humidity 30% to 98% RH) at 37°C for 5 d, and then centrifuged at 2500 rpm for 5 min. The obtained sample was washed with water for 3 times, frozen at -40°C, and dried at 4°C to obtain the drug-mineralized microrods.

[0171] The cell compatibility of the drug-mineralized microrods of Example 10 was evaluated by solution test. Specifically, 10000 L929 fibroblasts (commonly commercially available) were first planted in each well of a 96-well plate, and then incubated at 37°C using cell culture solution (DMEM + 10% fetal bovine serum + 1% penicillin and streptomycin) for 24 h. The original culture solution was discarded, and cell culture solution containing different concentrations of free rhein small molecules or different concentrations of drug-mineralized microrods was added. The positive control group was pure cell culture solution, and the negative control group was sterile water. After 24 h, the culture solution was discarded, 100 μL of CCK8 with a volume concentration of 10% was added to each well, and the incubation was continued for 2 h. The absorption spectrum at 450 nm was tested using an enzyme marker, and the cell survival rate was finally calculated by formula (2).

[0172] Cell survival rate % = (OD sample group - OD positive group) / (OD negative group - OD positive group) x 100% (2)

[0173] The results are shown in Figure 10 . Figure 10 The figure of the cytotoxicity test of the drug-mineralized microrods of Example 10 of the application. Figure 10 In the figure, Free-drug represents free rhein small molecule drug, and M-microrod represents drug-mineralized microrods. It can be seen from Figure 10 that free rhein small molecules exhibit a cell survival rate of less than 70% at a high concentration, while the cell survival rate of drug-mineralized microrods is greater than 95% in the entire concentration range, indicating that the microrods prepared in this mineralized form greatly improve the cell compatibility of small molecule drugs, and have excellent biological safety.

[0174] Example 11

[0175] 1) 25 mg of rhein was mixed with 1 mL of aqueous sodium hydroxide solution (pH = 8.5) at room temperature to obtain a first solution; the molar ratio of rhein to sodium hydroxide was 1:0.75;

[0176] 2) 32 mg of polyglutamic acid (Mn= 50 kDa) was mixed with the first solution, and stirred at room temperature until it was fully dissolved to obtain a second solution;

[0177] The molar ratio of carboxylate in the polyglutamic acid to carboxylate in rhein was 2.8:1;

[0178] 3) 22 μL of a magnesium chloride solution (400 mg / mL) was mixed with the second solution, and stirred for 23 h until it was uniformly mixed to obtain a third solution;

[0179] The molar ratio of carboxylate in rhein to magnesium ions was 0.97:1;

[0180] 4) The third solution was incubated in a constant temperature and humidity incubator at 37°C (humidity 30% to 98% RH) for 11 d, and after centrifugation at 3000 rpm for 5 min, the obtained sample was washed with water 3 times, frozen at -20°C, and dried at 4°C to obtain the drug-mineralized microrods.

[0181] The aggregate composition of the drug-mineralized microrods in Example 11 was determined by mass spectrometry (ESI):

[0182] Specifically, the drug-mineralized microrods obtained in Example 11 were prepared into a solution of 10 μg / mL using methanol, and the aggregates of the drug-mineralized microrods were tested by ESI. The results are shown in Figure 11 Figure 11 The figure is the test of the aggregate structure in the drug-mineralized microrods of Example 11 of the present application. From Figure 11 It can be seen that the drug-mineralized microrods exhibit obvious 239.0214 peaks and 589.0148 peaks, indicating that the drug-mineralized microrods mainly exist in the form of rhein small molecule / metal ion aggregates.

[0183] The anti-inflammatory properties of the drug-mineralized microrods in Example 11 were evaluated by a lipopolysaccharide-stimulated macrophage model:

[0184] Specifically, 12 x 10 4 ​One macrophage (commercially available) was incubated in a 37°C cell culture incubator for 24 h. Subsequently, the macrophages were pretreated with free rhein small molecules or drug-mineralized microrods for 2 h, and then lipopolysaccharide (1 μg / mL) was added to each well for another 24 h. Pure culture medium or pure lipopolysaccharide (1 μg / mL) was used as the control group or lipopolysaccharide group, respectively. After 24 h, the culture medium was removed and the cells were washed twice with PBS. Then, 200 μL of LCFH-DA (10 μmol / L) was added to each well. After co-culturing for 20 min, the culture medium was discarded, the cells were washed twice with PBS, and then 100 μL of PBS was added to each well. Finally, the fluorescence intensity of the samples at excitation light 488 nm and emission light 525 nm was measured using a fluorescence spectrometer. Intracellular ROS capacity was calculated using formula (3).

[0185] ROS level (% of control) = Values ​​of sample / Values ​​of control group × 100% (3)

[0186] The results are as follows Figure 12 As shown. Figure 12 This is a graph showing the results of the drug mineralization microrods of Example 11 of the present invention downregulating the intracellular ROS capacity in excessive inflammatory responses. Figure 12 In this context, LPS represents lipopolysaccharide, Free-drug represents free rhein (a small molecule drug), and M-microrod represents drug-mineralized microrods. From... Figure 12 It was found that, compared to the control group (macrophages without any treatment), LPS stimulation of macrophages (LPS group) significantly increased the expression of pro-inflammatory mediators reactive oxygen species (ROS) in cells. Further treatment with free-drug on LPS-stimulated macrophages (Free-drug group) did not significantly decrease ROS levels compared to the LPS group. However, treatment with M-microrod on LPS-stimulated macrophages (M-microrod group) significantly decreased ROS expression compared to the LPS group, and its ROS expression was comparable to the control group. Therefore, lipopolysaccharide (LPS) activation of macrophages significantly enhances their ability to secrete ROS. Co-incubation of free rhein small molecules or drug-mineralized microrods with LPS-activated macrophages for 24 hours both demonstrated the ability to eliminate intracellular ROS. Compared to free small molecule drugs, drug-mineralized microrods more effectively reduced intracellular ROS levels and exhibited superior anti-inflammatory activity.

[0187] Example 12

[0188] 1) 16 mg of rhein was mixed with 1 mL of an aqueous solution of potassium hydroxide (pH = 8.5) at room temperature, and dissolved completely to obtain a first solution; the molar ratio of rhein to sodium hydroxide was 1:0.84;

[0189] 2) 32 mg of sodium carboxymethylcellulose (100 mPa-s) was mixed with the first solution, and stirred at room temperature to dissolve completely to obtain a second solution;

[0190] The molar ratio of carboxylate in the sodium carboxymethylcellulose to carboxylate in the rhein was 1.39:1;

[0191] 3) 22 μL of a ferric chloride solution (400 mg / mL) was mixed with the second solution, and stirred for 13 h to mix uniformly to obtain a third solution;

[0192] The molar ratio of carboxylate in the rhein to iron ions was 1.1:1;

[0193] 4) The third solution was placed in a constant temperature and humidity incubator (30% to 98% RH) at 37°C, and incubated for 11 d; after centrifugation at 3500 rpm for 6 min, the obtained sample was washed with water for 3 times, frozen at -20°C, and dried at 7°C to obtain the drug mineralized micro-rods.

[0194] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a drug-mineralized microstick in the preparation of an anti-inflammatory drug, wherein the drug-mineralized microstick is prepared by the following steps: A) mixing rhein with an alkali solution to obtain a first solution, wherein the molar ratio of rhein to alkali in the alkali solution is 1:0.2-2; B) mixing the first solution with a degradable polymer compound to obtain a second solution; the degradable polymer compound comprises at least one of sodium hyaluronate, sodium alginate, polyamino acid salt, carboxymethyl chitosan and carboxymethyl cellulose; the molar ratio of carboxylate in the degradable polymer compound to carboxylate in the rhein is 1-20:1; the molecular weight Mn of the sodium hyaluronate is 3-3000 kDa; the molecular weight Mn of the sodium alginate is 3-3000 kDa; the molecular weight Mn of the polyamino acid salt is 3-1000 kDa; the viscosity of the carboxymethyl chitosan is 10-80 mPa·s; the viscosity of the carboxymethyl cellulose is 300-15000 mPa·s; C) mixing the second solution with a metal salt solution to obtain a third solution; the molar ratio of carboxylate in the rhein to metal ions in the metal salt is 0.1-10:1; D) after incubation of the third solution under constant temperature and humidity, centrifugation, water washing and freeze-drying to obtain a drug-mineralized microstick; the temperature of the incubation is 10-50°C, the humidity is 30%-98% RH, and the time is 4-15 days.

2. Use according to claim 1, characterized in that, In step A), the alkali solution comprises at least one of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate-sodium bicarbonate buffer solution and triethylamine; the pH value of the alkali solution is 7.5-9.

3. Use according to claim 1, characterized in that, In step C), the metal salt solution includes at least one of Ca 2+ , Fe 3 + , Zn 2+ , and Mg 2+ . the metal salt is selected from at least one of calcium chloride, ferric chloride, zinc nitrate and magnesium chloride; the concentration of the metal salt solution is 3-20 mg / mL.

4. Use according to claim 1, characterized in that, In step D), the centrifugation speed is 500-10000 rpm, and the time is 5-30 min.

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