Arginine-loaded albumin-based sustained-release microspheres, and preparation method and application thereof

Abstract

This invention relates to an albumin-based sustained-release microsphere loaded with arginine, its preparation method, and its application. The albumin-based sustained-release microspheres loaded with arginine are prepared as follows: First, the active pharmaceutical ingredient and albumin are dissolved in water to obtain an inner aqueous phase W1; a polymer is dissolved in an organic solvent to obtain an oil phase O1; an emulsifier and sodium chloride are dissolved in water to obtain an outer aqueous phase W2; wherein the active pharmaceutical ingredient is arginine; then, the inner aqueous phase W1 is slowly added dropwise to the oil phase O1, and after sufficient emulsification, a W1 / O1 type emulsion is obtained; then, the W1 / O1 type emulsion is slowly added dropwise to the outer aqueous phase W2, and after sufficient emulsification, a W1-O1-W2 type double emulsion is obtained, thus yielding the albumin-based sustained-release microspheres loaded with arginine. These sustained-release microspheres have the characteristics of small particle size and high drug loading capacity; and after the addition of albumin, they can continuously release arginine in vitro and in vivo for a long time, showing good therapeutic effects on osteoarthritis, and are expected to be further applied in osteoarthritis.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of sustained-release microspheres of medicinal type, and specifically relates to a kind of albumin-based sustained-release microspheres loaded with arginine and a preparation method and application in the treatment of osteoarthritis drugs. BACKGROUND

[0002] L-arginine (L-arg) is a semi-essential amino acid, which is involved in many biochemical processes. Studies have shown that there are three pathways for L-arginine metabolism in the body. One is that arginine is metabolized to produce NO and citrulline under the catalysis of nitric oxide synthase (NOS). Two is that arginine is decomposed into ornithine and urea under the action of arginine decomposing enzyme, and can also be decomposed into ornithine and creatinine acid by glycine transamidase. Three is that polyamine is generated from ornithine, and polyamine is the general term of putrescine, spermidine and spermine, which plays an important role in regulating cell growth and development. Arginine degradation is mainly completed in the small intestine. Therefore, through further reactions downstream of arginine, it can synthesize many essential substances for life activities.

[0003] At present, arginine has therapeutic effects in preventing cardiovascular diseases, diabetes, autoimmune diseases, wound healing and tumors in clinical practice. For example, L-arginine is a precursor of nitric oxide (NO) synthesis. In the brain, NO acts as a neurotransmitter; in the immune system, it acts as a mediator of host defense; in the cardiovascular system, it mediates the protective effect of the intact endothelium, acts as a vasodilator and an endogenous anti-atherosclerotic molecule (Altern Ther Health Med. 2014 May-Jun; 20(3): 48-54). And the intracellular L-arginine concentration directly affects the metabolic adaptability and survival ability of T cells, which are essential for anti-tumor response (Cell. 2016 Oct 20; 167(3): 829-842.e13). At the same time, the increased metabolism of L-arginine by bone marrow cells leads to impaired antigen response by lymphocytes during immune response and tumor growth (Nat Rev Immunol. 2005 Aug; 5(8): 641-54). In terms of wound healing, arginine is involved in many regulatory mechanisms related to wound healing by inducing isomers (iNOS) (Curr Opin Clin Nutr Metab Care. 2000 3: 197-204). In recent years, some scholars have also found that in vitro studies have shown that arginine has strong abilities to scavenge DPPH free radicals, ABTS free radicals, superoxide free radicals and certain reducing power (Acta Poloniae Pharmaceutica, 2015, 72(2): 245-252). And further studies have shown that the free radical scavenging activity of arginine is mainly related to the guanidino group (Molecular Pharmacology, 2002, 61(5): 1081-1088). The pathogenesis of osteoarthritis is related to factors such as cartilage degeneration, trauma, metabolism, etc., among which oxidative stress is one of the key factors in the occurrence and development of osteoarthritis (Free Radic Biol Med, 2019, 132: 73-82). Therefore, as a drug for regulating oxidative stress and inflammation, arginine is expected to be a new drug for the treatment of osteoarthritis and provide a new idea for the treatment of osteoarthritis in clinical practice.

[0004] In summary, L-arginine has great value in clinical applications, and its application in osteoarthritis has not been reported. Currently, arginine supplements mainly rely on oral administration, while there are still many shortcomings in local administration. The disadvantages of oral administration include slow efficacy, large dose, and adverse reactions on the gastrointestinal tract. Local administration can improve the efficacy and play a long-term role to improve the prognosis of patients. In recent years, microparticle dispersion systems such as liposomes, microemulsions, microspheres, nanocapsules, and nanoparticles have been prepared by emulsification encapsulation and other dispersion technologies using polymers as materials for drug sustained and controlled release, enhancing the safety and effectiveness of drugs, and improving patient compliance. However, there are still few reports on the dosage forms of L-arginine drugs. Chinese patent 202210494016 proposed a preparation method of arginine multi-capsule liposomes. However, due to the short half-life and poor stability of liposomes, they are not suitable for the treatment of osteoarthritis. Microspheres (MS) are a new dosage form developed in recent years and can be used for injection, oral administration, nasal drops, subcutaneous implantation, or intra-articular administration. After loading drugs, microspheres have targeting properties for specific organs and tissues and sustained-release properties of drugs in microparticles, so they only need to be injected once every few days or even months, which can significantly reduce the frequency of drug administration. Such preparations have become a hot topic in the research of sustained and controlled release dosage forms in recent years. Microspheres can also be used to load amino acid drugs, but due to the strong diffusivity of water-soluble small molecule amino acid drugs, the sustained-release effect in microspheres is poor. SUMMARY

[0005] Therefore, the present application develops, for the first time, an albumin-based sustained-release microsphere loaded with arginine through a large number of searches and creative experiments. The sustained-release microsphere has the characteristics of small particle size, high drug loading capacity, and sustained-release drug, and can achieve drug sustained-release in the joint cavity. The sustained-release microsphere provided by the present application can be widely used in the preparation of osteoarticular drugs, especially in the preparation of intra-articular injections. At the same time, through experiments, we also report for the first time the therapeutic effect of arginine on osteoarthritis.

[0006] The sustained-release microsphere provided by the present application is prepared from a drug active ingredient, albumin, an emulsifying agent, and a sustained-release material (carrier material). The drug active ingredient is arginine.

[0007] In the present application, albumin is used as a stabilizer for arginine. Under physiological conditions, albumin and arginine produce certain electrostatic interactions to improve the sustained-release effect of the microsphere. In addition, the addition of sodium chloride to the outer water phase can inhibit the exchange of substances between the inner and outer water phases, improve the drug encapsulation efficiency of the microsphere, reduce the drug burst release rate of the microsphere, and long-term play the role of arginine.

[0008] Specifically, the preparation method of the albumin-based sustained-release microsphere loaded with arginine includes the following steps:

[0009] 1) dissolving the pharmaceutical active ingredient and albumin in water to obtain an inner water phase W1; dissolving the high molecular polymer in an organic solvent to obtain an oil phase O1; dissolving the emulsifier and sodium chloride in water to obtain an outer water phase W2;

[0010] wherein the pharmaceutical active ingredient is arginine;

[0011] 2) slowly adding the inner water phase W1 into the oil phase O1, and after sufficient emulsification, obtaining a W1 / O1 emulsion;

[0012] 3) slowly adding the W1 / O1 emulsion into the outer water phase W2, and after sufficient emulsification, obtaining a W1-O1-W2 multiple emulsion, and then washing with ultrapure water to obtain the arginine-loaded albumin-based sustained-release microspheres.

[0013] wherein the albumin is one or more of bovine serum albumin, ovalbumin, human serum albumin, and urinary microalbumin.

[0014] Preferably, the mass ratio of arginine to albumin is 5:1 to 1:5, preferably 3:1 to 1:3.

[0015] Preferably, the high molecular polymer is one or more of polylactic glycolic acid copolymer (PLGA), polycaprolactone, polylactic acid, polytrimethylene carbonate, polyglycolic acid, polyhydroxybutyrate, polyhydroxybutyric acid-hydroxyvaleric acid copolymer, polyortho ester, and polyanhydride.

[0016] Preferably, the organic solvent is one or more of trichloromethane, dichloromethane, n-hexane, toluene, isopropyl alcohol, acetone, ethyl acetate, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0017] Preferably, the emulsifier is one or more of polyvinyl alcohol (PVA), polyoxyethylene ether, polyoxypropylene ether, ethylene oxide and propylene oxide block copolymer, and polyol fatty acid ester.

[0018] wherein the concentration of polylactic glycolic acid copolymer (PLGA) in the organic solvent is 3% to 10% w / v, preferably 4.5% to 5.5% w / v, and particularly 5% w / v.

[0019] wherein the mass concentration ratio of polyvinyl alcohol to sodium chloride is 3:1 to 1:3, and preferably 1:1.

[0020] Further, the inner water phase W1 is slowly added dropwise into the oil phase O1 at a volume ratio of 1:4 to 1:6, and preferably at a volume ratio of 1:5.

[0021] Further, the W1 / O1 emulsion is added dropwise into the external water phase W2 at a volume ratio of 1:8-12; preferably at a volume ratio of 1:10.

[0022] Further, the ultrasonic emulsification power in steps 2) and 3) is 180-540 W, and the emulsification time is 2-5 min; preferably, the emulsification power is 320-380 W, and the emulsification time is 2-5 min; in particular, the power is 360 W, and the emulsification time is 3 min, which is optimal for the emulsification of the above-mentioned raw materials.

[0023] In the preparation process, it is found that the concentration of polylactic acid-glycolic acid copolymer, the amount of bovine serum albumin, the mass ratio of polyvinyl alcohol to sodium chloride, and the ultrasonic power have a significant influence on the subsequent preparation of the sustained-release microspheres.

[0024] In the actual preparation process, the addition amount of the active pharmaceutical ingredient has little effect on the subsequent loading amount. Even if the addition amount is different, under the same preparation conditions, the final loading amount is basically the same. However, the amount of albumin added has a greater impact on the process; therefore, the feed ratio of the active pharmaceutical ingredient such as arginine to albumin still has a very obvious effect on the final product.

[0025] Based on this, the present application provides a preferred scheme, which is as follows:

[0026] A preparation method of albumin-based sustained-release microspheres loaded with arginine, comprising the following steps:

[0027] 1) Dissolve the active pharmaceutical ingredient arginine and bovine serum albumin in water as the internal water phase W1; the feed mass ratio of arginine to the albumin is 3:1-1:3;

[0028] 2) Dissolve polylactic acid-glycolic acid copolymer in chloroform at a concentration of 4.5%-5.5% w / v as the oil phase O1;

[0029] 3) Mix polyvinyl alcohol and sodium chloride at a mass ratio of 1:1 and dissolve in water as the external water phase W2;

[0030] 4) Slowly drop the internal water phase W1 into the oil phase O1; after sufficient emulsification, a W1 / O1 emulsion is obtained; the ultrasonic emulsification power is 320-380 W, and the emulsification time is 2-5 min;

[0031] 5) Slowly drop the W1 / O1 emulsion into the external water phase W2, and after sufficient emulsification, a W1-O1-W2 multiple emulsion is obtained, and then the sustained-release microspheres loaded with arginine are obtained by washing with ultrapure water; the ultrasonic emulsification power is 320-380 W, and the emulsification time is preferably 2-5 min.

[0032] Yet another object of the present application is to provide the albumin-based sustained-release microspheres for arginine prepared by any of the above preparation methods.

[0033] The prepared albumin-based sustained-release microspheres for arginine have a particle size of 0.2-2 μm.

[0034] Compared with conventional sustained-release particles, the sustained-release microspheres of the present application are prepared by using albumin, a high-molecular-weight biodegradable polymer and a PVA emulsifier as raw materials by a W / O / W double emulsion method. The albumin and the high-molecular-weight polymer can play a good drug sustained-release and controlled-release role. Experimental results show that the nanoparticles have good particle size and dispersibility, and have good anti-inflammatory therapeutic effect and protective properties for the cartilage cells mainly involved in osteoarthritis, and have no obvious cytotoxicity to the cartilage cells. Moreover, the sustained-release microspheres have a small volume, obvious effect, can effectively relieve and treat osteoarthritis, have good sustained-release effect, reduce the cost of multiple drug administration, reduce the pain and irritation of patients, and reduce adverse reactions and safety accidents caused by organic solvents.

[0035] The preparation method of the sustained-release microspheres of the present application is simple in operation, uniform in particle size, good in stability, can release arginine for a long time, and is expected to be further clinically applied to osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The morphological characterization diagram of the optimized sustained-release microspheres obtained in Example 1 in the preparation process; wherein A is the morphological diagram of the white powder of the freeze-dried sustained-release microspheres; B is the morphological diagram of the uniform white suspension after resuspension; C is the particle size distribution diagram of the microspheres; D is the potential distribution diagram of the microspheres;

[0037] Figure 2 The morphological diagram of the optimized sustained-release microspheres obtained in Example 1 under an optical microscope;

[0038] Figure 3 The TEM morphological characterization diagram of the optimized sustained-release microspheres obtained in Example 1;

[0039] Figure 4 The liquid chromatogram of the albumin microspheres without drug loading, different concentrations of small-molecule arginine and the albumin-based sustained-release microspheres loaded with arginine;

[0040] Figure 5 The in-vitro release curve of the albumin-based sustained-release microspheres loaded with arginine obtained in Example 1 and the microspheres loaded with arginine (without albumin);

[0041] Figure 6 The Western blot experiment of the in-vitro cartilage cells; wherein A is the experimental result diagram, and B is the statistical diagram of the experimental result data.

[0042] Figure 7 Histological evaluation of articular cartilage and synovium for hematoxylin-eosin (H&E) staining and fast-green-orange staining (F-O); wherein A is a graph of experimental results, and B is a graph of statistical data of experimental results. DETAILED DESCRIPTION

[0043] The following examples are intended to illustrate the present application but not to limit the scope of the present application.

[0044] Example 1

[0045] The present embodiment provides a preparation method of arginine albumin-based sustained-release microspheres, which is specifically as follows:

[0046] (1) The active pharmaceutical ingredient arginine and bovine serum albumin are dissolved in ultrapure water at the same time as the inner water phase W1; wherein the mass ratio of arginine to albumin is 1:1.

[0047] (2) The carrier material ester-terminated polylactic glycolic acid copolymer (PLGA) is weighed and dissolved in dichloromethane as the oil phase O1; wherein the concentration of polylactic glycolic acid copolymer (PLGA) in the organic solvent is 50 mg / mL.

[0048] (3) A mixed solution of polyvinyl alcohol (PVA) and sodium chloride is prepared as the outer water phase W2; wherein the mass concentration of polyvinyl alcohol (PVA) in the outer water phase W2 is 20 mg / mL. The mass ratio of PVA to sodium chloride is 1:1.

[0049] (4) The inner water phase W1 is slowly added dropwise into the oil phase O1 containing the carrier material at a volume ratio of 1:5; the W1 / O1 type emulsion formed is emulsified by an ultrasonic emulsifier at a power of 360 W for 3 min;

[0050] (5) Then the W1 / O1 type emulsion is slowly added dropwise into the outer water phase W2 at a volume ratio of 1:5, and then emulsified by an ultrasonic emulsifier at a power of 360 W for 3 min to obtain a W1-O1-W2 type multiple emulsion;

[0051] (6) The W1-O1-W2 type multiple emulsion is placed on a magnetic stirrer and stirred at a speed of 500 rpm / min for 6 h to volatilize the organic solvent. The liquid is collected and washed with ultrapure water for centrifugation 3 times to remove excess PVA solution, and the supernatant is discarded. The lower layer of the precipitate is the microspheres. The centrifuged microspheres are resuspended with ultrapure water, then mannitol is added as a freeze-drying protective agent, and then freeze-dried for 48 h to obtain dried microspheres.

[0052] Examples 2-9

[0053] This embodiment provides a method for preparing albumin-based sustained-release microspheres containing arginine. The main differences from Example 1 are the mass concentration of PLGA, the mass ratio of arginine (arg) to albumin (bsa), the mass ratio of PVA to sodium chloride, and the ultrasonic power.

[0054] The specific details are shown in the table below (No. 6 is Example 1):

[0055] Table 1

[0056] No. Example PLGA (mg / mL) m(arg):m(bsa) C(PVA):C(NaCl) Ultrasonic power (W) 1 Example 2 30 3:1 1:1 180 2 Example 3 30 1:3 1:3 360 3 Example 4 30 1:1 3:1 540 4 Example 5 50 3:1 1:3 540 5 Example 6 50 1:3 3:1 180 6 Example 1 50 1:1 1:1 360 7 Example 7 100 3:1 3:1 360 8 Example 8 100 1:3 1:1 540 9 Example 9 100 1:1 1:3 180

[0057] Comparative Example 1

[0058] This comparative example uses a method for preparing arginine-loaded microspheres. The only difference from Example 1 is that bovine serum albumin is not included in step (1).

[0059] Specifically, (1) the active ingredient arginine was dissolved in ultrapure water as the inner aqueous phase W1;

[0060] That is, following the same preparation steps as in Example 1, blank PLGA microspheres without drug loading and PLGA microspheres loaded with arginine were also prepared (the internal aqueous phase did not contain bovine serum albumin, but was an aqueous solution containing only arginine).

[0061] Experimental Example 1

[0062] The morphology of the arginine-loaded albumin-based sustained-release microspheres prepared in Example 1 was analyzed.

[0063] like Figure 1 The diagram shows the morphological characterization of the sustained-release microspheres during the preparation process;

[0064] 1) such as Figure 1 As shown in A, the obtained microspheres, after freeze-drying, appear as a white, loose powder.

[0065] 2) Take an appropriate amount of the sustained-release microsphere lyophilized powder prepared in Example 1 and resuspend it in deionized water. The dissolution of the microsphere lyophilized powder in water can be observed. Figure 1 As shown in Figure B, the resuspended suspension is relatively uniform and has good fluidity.

[0066] 3) Use a Malvern laser particle size analyzer to measure its particle size distribution and potential distribution for quantitative analysis, such as... Figure 1 As shown in C and D. The microspheres have a particle size of 612±30.52 nm, PDI=0.11, and a potential of -31.38±0.267 mV.

[0067] Experimental Example 2

[0068] Morphological examination of albumin-based sustained-release microspheres loaded with arginine prepared in Example 1

[0069] Take an appropriate amount of microspheres lyophilized powder resuspended in deionized water, drop on the glass slide, using a microscope to observe the morphology of the resuspended microspheres, as shown (A and B magnification of 10 x 40, 10 x 100). The powder after freeze-dried microspheres drop on the copper mesh, by transmission electron microscopy TEM observation of the morphology of the microspheres, as shown (A and B scale of 1.0 μm, 500 nm). Freeze-dried microspheres under a microscope and transmission electron microscope, drug-loaded microspheres appearance round, uniform size, microspheres particle size of 674 ± 18.67 nm. Figure 2 Figure 3

[0070] Test Example 3

[0071] The drug active ingredient arginine (free small molecule arginine) provided in Example 1, the drug loading and encapsulation efficiency of the arginine-loaded albumin-based sustained-release microspheres prepared in Example 1 were determined.

[0072] The treatment was divided into three groups, namely the blank microspheres group (not containing the drug active ingredient, the preparation method was the same as the microspheres prepared in Example 1), the standard group (free small molecule arginine), and the arginine microspheres group (sustained-release microspheres prepared in Example 1).

[0073] 1 mg of arginine standard was dissolved in 10 ml of deionized water to prepare a solution of 100 μg / ml, and then diluted by volume to obtain arginine standard solutions of different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL). At the same time, an appropriate amount of microspheres lyophilized powder (including blank microspheres and arginine-loaded microspheres) was ultrasonically dissolved in dichloromethane and vortexed, and then an appropriate amount of deionized PBS with pH = 7.4 was added to extract arginine. After centrifugation, the lower organic solution was discarded, and the above operation was repeated three times to collect the upper arginine solution. All samples were sampled through a 0.22 μm water filter membrane, and the encapsulated arginine content was determined by high performance liquid chromatography (HPLC). The total arginine amount was determined by the amount of arginine weighed in Example 1, and the drug loading (EE%) and encapsulation efficiency (DL%) of arginine were calculated by the formula. The liquid chromatogram is shown in Figure 4

[0074] EE% = Wencapsulated drug amount / Wtotal drug amount x 100%

[0075] DL% = Wencapsulated drug amount / Wtotal drug and material amount x 100%

[0076] ​​​The drug loading of arginine in the arginine sustained release microspheres prepared in Example 1 was 1.76 ± 0.29%, and the encapsulation efficiency was 79.73 ± 6.44%.

[0077] The HPLC conditions used were as follows:

[0078] Mobile phase: acetonitrile: 0.05M potassium dihydrogen phosphate solution = (0.1:99.9), 0.5 ml / min;

[0079] Detection wavelength: 206 nm;

[0080] Chromatographic column: Agilent ZORBAX C18 column (4.6 x 250 mm, 5 um).

[0081] Test Example 4

[0082] In vitro release of the arginine-loaded albumin-based sustained release microspheres prepared in Comparative Example 1 and Example 1 was investigated

[0083] Using a pH = 7.4 phosphate buffer as the dissolution medium, 100 mg of the microspheres prepared in Comparative Example 1 (arginine microspheres without bovine serum albumin) and Example 1 (arginine-albumin microspheres) were weighed into centrifuge tubes containing 10 mL of the release medium, and the dissolution bottles were placed in a constant temperature shaker at 37 ± 0.5°C and a rotation speed of 80 rpm. The above experiment was repeated three times. Then, samples were taken through a 0.22 μm water system filter at 1 h, 2 h, 4 h, 8 h, 16 h, 24 h; 2 d, 4 d, 8 d, 16 d, 20 d, and the same volume of the isothermal release medium was immediately supplemented, the filtrate was injected through a syringe, and the drug content was determined by HPLC, and the chromatographic conditions were as follows:

[0084] Mobile phase: acetonitrile: 0.05M potassium dihydrogen phosphate solution = (0.1:99.9), 0.5 ml / min;

[0085] Detection wavelength: 206 nm;

[0086] Chromatographic column: Agilent ZORBAX C18 column (4.6 x 250 mm, 5 um).

[0087] The cumulative release percentage was calculated, and the release curve is shown in Figure 5 The arginine release amount of the microspheres without albumin in Comparative Example 1 was 32.70% within the first 24 h, and almost completely released within 12 d; while the arginine release amount of the arginine microspheres with albumin was 20.50% within the first 24 h, and slowly released within the following time, and almost completely released within 20 d, which indicates that the addition of albumin in the internal water phase enables the arginine microspheres to achieve better sustained release effect.

[0088] Test Example 5

[0089] Process investigation of arginine-loaded sustained-release microspheres prepared in Examples 1-9

[0090] During the research, the inventors found that the following factors had a greater impact on the preparation process in the above experimental preparation steps: PLGA concentration, mass ratio of arginine (arg) and albumin (bsa) in the feed, concentration ratio (mass ratio) of PVA and NaCl, and ultrasonic power.

[0091] Table 2 Microsphere preparation process investigation

[0092]

[0093] According to the previous preparation prescription screening and process investigation, and the subsequent preparation process step optimization, the preparation method provided in Example 1 has the advantages of uniform diameter, high drug loading and encapsulation efficiency, and low 24h burst release rate.

[0094] Test Example 6

[0095] The same drug active ingredient arginine in Example 1 was compared with the arginine-loaded albumin-based sustained-release microspheres prepared in Example 1 for in vitro detection of cartilage protection

[0096] 1. Effect of arginine on osteoarthritis

[0097] Primary chondrocytes were taken for in vitro experimental verification. Lipopolysaccharide LPS was used to simulate an in vitro osteoarthritis model, and arginine was added for intervention. The results of Western blotting are shown in Figure 6 Compared with the untreated control group, the expression of col II, a protein synthesized by chondrocytes, was reduced, and the expression of MMP13, a protein related to catabolism, was increased, indicating that LPS caused metabolic damage to chondrocytes. After the addition of arginine drug intervention, the expression of col II was increased, and the expression of MMP13 was decreased, indicating that arginine drug reversed the metabolic damage of chondrocytes stimulated by LPS, providing preliminary data support for the treatment of arginine in osteoarthritis.

[0098] The test procedure is as follows: healthy male rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital. The skin surface was disinfected with iodophor, and a sterile drape was laid. An incision was made on the medial side of the knee, and the joint capsule was opened layer by layer with blunt tissue dissection forceps. The patella was everted, and the medial meniscus was removed in the flexion position. After the patella was reduced and the penicillin normal saline was washed (to keep the tissue moist), the joint capsule was closed, the surgical incision was sutured, and a small amount of bleeding during the operation was pressed with sterile gauze to stop bleeding. The rest of the treatment was the same as the operation group.

[0099] One week after modeling, the treatment was divided into 4 groups, respectively:

[0100] Blank control: sham operation group (sham)

[0101] Surgery blank control: surgery group (DMM+NaCl)

[0102] Small molecule arginine group: postoperative arginine treatment group (DMM+Arginine)

[0103] Example 1 slow-release microspheres group: postoperative arginine-loaded albumin-based slow-release microspheres treatment group (DMM+Arg-MB)

[0104] The administration frequency was 1 time / week, and each time 50 mg / kg was injected into the joint cavity at one time.

[0105] To determine the different treatment effects of small molecule arginine and arginine-loaded albumin-based slow-release microspheres on osteoarthritis, at the 10th week of treatment, all mice were sacrificed after drawing the joint fluid of the treatment side and the control healthy side. The tibial plateau and femoral condyle joint surface of the right knee joint of the rats in each group were taken out. The tissues were fixed in 4% paraformaldehyde and then decalcified with EDTA solution gradient. The decalcified tissues were stained with hematoxylin and eosin and safranin-fast green, respectively.

[0106] The histopathological H&E and F-O staining analysis is shown in Figure 7 , A is the experimental result graph; there are four groups, and the left graph in each group is S-O staining, and the right graph is HE staining. B is the statistical graph of the experimental result data.

[0107] As can be seen from Figure 7 , compared with the sham operation group (sham), the cartilage thickness in the osteoarthritis cartilage of the surgery group (DMM+NaCl) rats was significantly reduced, and the typical osteoarthritis characteristics such as surface irregularity, erosion crack, and osteoarthritis OARSI score were significantly increased. The arginine-loaded albumin-based slow-release microspheres after surgery (DMM+Arg-MB) inhibited the progression of osteoarthritis to different degrees, which was manifested as an increase in cartilage thickness and inhibition of chondrocyte apoptosis, and a significant decrease in OARSI score. In summary, the anti-inflammatory and cartilage repair treatment showed good anti-inflammatory effect and cartilage protection activity in the knee joints of rats with osteoarthritis.

[0108] Although the present application has been described in detail in the foregoing description with general general description, specific embodiments and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present application. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.

Claims

1. A method for preparing arginine-loaded albumin-based sustained-release microspheres, characterized by, Comprising the following steps: 1) dissolving the pharmaceutical active ingredient and albumin in water to obtain an inner aqueous phase W1; dissolving the high molecular polymer in an organic solvent to obtain an oil phase O1; dissolving the emulsifier and sodium chloride in water to obtain an outer aqueous phase W2; wherein the pharmaceutical active ingredient is arginine; the mass ratio of arginine to albumin is 3:1-1:3; Wherein, the albumin is one or several of bovine serum albumin, ovalbumin, human serum albumin, and urinary microalbumin; Wherein, the high molecular polymer is one or more of polylactic acid-glycolic acid copolymer, polycaprolactone, polylactic acid, polytrimethylene carbonate, polyglycolic acid, polyhydroxybutyric acid, polyhydroxybutyric acid-hydroxyvaleric acid copolymer, polyortho ester, and polyanhydride; the concentration of the high molecular polymer in the organic solvent is 4.5%-5.5% w / v; Wherein, the emulsifier is one or several of polyvinyl alcohol, polyoxyethylene ether, polyoxypropylene ether, ethylene oxide and propylene oxide block copolymer, and polyol fatty acid ester; In the aqueous phase, the concentration of the emulsifier is 0.5%-5% w / v; Wherein, the mass concentration ratio of sodium chloride to emulsifier is 3:1-1:3; 2) slowly dripping the inner aqueous phase W1 into the oil phase O1, and obtaining a W1 / O1 emulsion after sufficient ultrasonic emulsification; 3) slowly dripping the W1 / O1 emulsion into the outer aqueous phase W2, and obtaining a W1-O1-W2 multiple emulsion after sufficient ultrasonic emulsification, and then washing with ultrapure water to obtain the arginine-loaded albumin-based sustained-release microspheres; The ultrasonic power is 180-540 W; The emulsification time is 2-5 min.

2. The production method according to claim 1, characterized by, The organic solvent is one or several of trichloromethane, dichloromethane, n-hexane, toluene, isopropyl alcohol, acetone, ethyl acetate, N,N-dimethylformamide, and N,N-dimethylacetamide.

3. The preparation method according to claim 1, characterized in that, The mass concentration ratio of sodium chloride to emulsifier is 1:

1.

4. The method of claim 1, wherein, The ultrasonic power is 320-380 W.

5. The preparation method according to claim 1, characterized in that, Comprising the following steps: 1) dissolving the pharmaceutical active ingredient arginine and bovine serum albumin in water as an inner aqueous phase W1; the mass ratio of arginine to albumin is 3:1-1:3; 2) dissolving polylactic acid-glycolic acid copolymer in dichloromethane at a concentration of 4.5%-5.5% w / v as an oil phase O1; 3) mixing polyvinyl alcohol and sodium chloride in water at a mass ratio of 1:1 as an outer aqueous phase W2; 4) slowly adding the inner aqueous phase W1 dropwise into the oil phase O1; obtaining a W1 / O1 emulsion after sufficient emulsification; wherein the ultrasonic power for emulsification is 320-380 W; the emulsification time is 2-5 min; 5) slowly dripping the W1 / O1 emulsion into the outer aqueous phase W2, and obtaining a W1-O1-W2 multiple emulsion after sufficient emulsification, and then washing with ultrapure water to obtain the arginine-loaded sustained-release microspheres; wherein the ultrasonic power for emulsification is 320-380 W; the emulsification time is 2-5 min.

6. The arginine-loaded albumin-based sustained-release microspheres prepared by the preparation method of any one of claims 1-5.

7. The arginine-loaded albumin-based sustained-release microspheres according to claim 6, wherein the particle size is 0.2 μm to 2 μm.

8. Use of the arginine-loaded albumin-based sustained-release microspheres prepared by the method of any one of claims 1 to 5 in the preparation of a medicament for treating osteoarthritis.

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