Daptomycin nanoformulations, their preparation methods and applications

Abstract

The present invention relates to a daptomycin nanoformulation and a preparation method and application thereof, belonging to the field of pharmaceutical technology. The preparation method of the daptomycin nanoformulation comprises: A. polylactic-co-glycolic acid copolymer is wrapped with daptomycin to obtain nanoparticles NPs; B. cell membrane vesicles and nanoparticles NPs overexpressing RANK are ultrasonicated for 3 minutes under water bath ultrasonic conditions, and then sequentially squeezed through 800nm, 400nm and 200nm polycarbonate membranes to obtain daptomycin nanoformulations; the weight ratio of the membrane protein to PLGA is 0.25. The preparation method of the present invention successfully prepares a daptomycin nanoformulation targeting rheumatoid arthritis. The daptomycin nanoformulation prepared by the present invention has good stability. The daptomycin nanoformulation of the present invention has good anti-RA efficacy.

Description

Technical Field

[0001] The invention relates to a daptomycin nano preparation and a preparation method and application thereof, belonging to the technical field of medicines. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease with erosive arthritis as the main clinical manifestation. Its pathological features are synovial hyperplasia, joint structure destruction, immune cell infiltration and pannus formation. The site of disease may involve synovial joints throughout the body. The prevalence of RA is 0.5-1% of the general population. The prevalence of RA in women is 2-3 times that of men. It can occur at any age, but the age of patients is mostly concentrated between 22 and 55 years old. It can also be seen in children. The incidence of RA in my country is about 0.42% of the total population, and there are about 5 million RA patients in the country. In the early and middle stages of RA patients, joints are often red, swollen and painful with functional impairment. In the late stage, joints will have varying degrees of stiffness and deformity, and may even cause disability. RA is a disease caused by multiple factors, including genetics, environment, infection, endocrine disorders, and immune regulation imbalance. Among them, immune regulation imbalance is the main cause of RA.

[0003] Currently, RA cannot be cured. The goal of clinical RA treatment is to achieve disease remission or low disease activity, that is, to achieve target treatment. The ultimate goal is to control the disease, reduce disability, and improve the patient's quality of life. Therefore, the development of safer and more effective drugs can address unmet clinical needs and benefit patients.

[0004] Daptomycin (DAP) is a secondary metabolite produced by Streptomyces roseosporus. It was approved by the U.S. Food and Drug Administration in 2003 for the treatment of severe skin infections. Studies have shown that DAP has excellent antibacterial activity and immunomodulatory effects. We first conducted a study on the therapeutic effect of DAP on RA through in vitro and in vivo inflammatory models. The results showed that DAP had a good therapeutic effect on collagen-induced RA mice. The relevant research results are recorded in CN110604808A on the application of daptomycin in the preparation of drugs for the treatment of rheumatoid arthritis. Given that the main pathological damage of RA occurs in the joints, especially cartilage destruction and bone erosion occur in the late stage of the disease, which seriously affects the patient's mobility. Therefore, targeted delivery of drugs to the joint lesions can produce better anti-RA efficacy, but there is currently no DAP targeted nanoformulation.

[0005] Conventional drugs for the treatment of RA are distributed throughout the body, and increased doses cause damage to normal tissues and organs, as well as treatment tolerance. The emergence of nanomedicines has improved the shortcomings of conventional drugs. Nanocarriers load conventional drugs for the treatment of RA and deliver them to the lesion site. However, the immune clearance of nanoparticles is still a huge obstacle to drug delivery. How to avoid the clearance of the immune system has always been a problem that researchers need to solve. In recent years, there has been an increasing number of studies on bionic functionalized nanoparticles, because bionic nanoparticles combine the characteristics of the variable flexibility of nanomaterials and the biological activity of natural materials, have long-term circulation, active targeting functions, and avoid immune clearance, making them a hot topic of research. Cell membranes are the most popular type of bionic nanocarriers. In RA, there have been studies that have made cell membranes of red blood cells, platelets, macrophages, neutrophils, etc. into nanovesicles, encapsulated nanoparticles, and evaluated their efficacy in RA animal models.

[0006] Joint bone destruction is an important pathological feature of RA patients in the late stage. Osteoclasts play an important role in bone absorption in bone metabolism. The two most important factors involved in the differentiation, maturation and activation of osteoclasts are macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). RANKL binds to its receptor RANK to activate downstream signaling pathways, ultimately leading to the formation of osteoclasts. RANKL is highly expressed in RA. Summary of the invention

[0007] The first object of the present invention is to provide a method for preparing a daptomycin nanoformulation.

[0008] To achieve the first object of the present invention, the preparation method of the daptomycin nanoformulation comprises:

[0009] A. Poly(lactic acid-co-glycolic acid) was used to encapsulate daptomycin to obtain nanoparticles NPs;

[0010] B. mixing the cell membrane vesicles overexpressing RANK and the nanoparticles NPs, sonicating them for 3 to 5 minutes under water bath sonication conditions, and then sequentially extruding them through 800 nm, 400 nm and 200 nm polycarbonate membranes to obtain daptomycin nanoformulations;

[0011] The weight ratio of the membrane protein of the cell membrane vesicles overexpressing RANK to the poly(lactic acid-glycolic acid) copolymer is 0.25.

[0012] In a specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer to daptomycin in step A is 7.5-15:1.

[0013] In a specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer and daptomycin in step A is 10:1.

[0014] In a specific embodiment, the preparation method of the nanoparticles NPs in step A includes: dissolving polylactic acid-glycolic acid copolymer in chloroform to obtain solution A, dissolving daptomycin in methanol to obtain solution B, mixing solution A and solution B as an oil phase, adding a PVA aqueous solution thereto as an external aqueous phase, ultrasonically emulsifying at a power of 20% to 30% to form an emulsion with a uniform appearance, then immediately performing reduced pressure rotary evaporation to evaporate the organic solvent to obtain a drug-loaded emulsion, and then centrifuging to collect the precipitated nanoparticles; the volume ratio of the external aqueous phase to the oil phase is 3 to 6:1, and the mass volume ratio of PVA to water in the PVA aqueous solution is 0.25g to 1g:100ml.

[0015] In a specific embodiment, the mass volume ratio of PVA to water in the PVA aqueous solution is 0.5g:100ml, the ultrasonic power is 25%, and the volume ratio of the external water phase and the oil phase is 4:1.

[0016] In a specific embodiment, the centrifugation condition is 12000-13000 rpm, 2-4°C low temperature centrifugation for 50-60 minutes.

[0017] In a specific embodiment, the method for preparing cell membrane vesicles overexpressing RANK includes: transfecting 293T cells with a RANK recombinant plasmid to establish a 293T cell line overexpressing RANK, and then extracting cell membrane vesicles overexpressing RANK; the RANK recombinant plasmid is a plasmid with an inserted RANK gene fragment.

[0018] Plasmids can be purchased commercially, such as pFV155-GFP-PURO plasmid, and the RANK gene is inserted between the EcoRI and XbaI restriction sites of the pFV155-GFP-PURO plasmid to construct the pFV155-GFP-PURO-RANK recombinant plasmid.

[0019] The RANK gene fragment is a gene that can express the nuclear factor κB receptor activating protein receptor. For example, the gene fragment with GI number: 1519243924 can express the nuclear factor κB receptor activating protein receptor and can be used as the RANK gene fragment.

[0020] In a specific embodiment, the method for establishing a RANK-overexpressing 293T cell line includes: transfecting 293T cells with a recombinant plasmid, a packaging plasmid psPAX2 and pMD2.G in a mass ratio of 2:2:1 using a PEI transfection reagent, collecting the culture supernatant containing the lentivirus 48 to 72 hours after transfection, filtering with a 0.45 μm filter, adding the supernatant containing the lentivirus to new 293T cells, changing the medium after 48 hours of infection, and adding 1 μg / mL puromycin for screening, and finally obtaining a stable RANK-overexpressing 293T cell line.

[0021] In a specific embodiment, the method for extracting cell membrane vesicles overexpressing RANK comprises:

[0022] Wash the cells with 4°C precooled PBS, centrifuge and discard the supernatant, add cell membrane protein extraction reagent with phenylmethylsulfonyl fluoride to the cells, mix and suspend the cells thoroughly, place in an ice bath for 10-15 minutes, homogenize 30-50 times, centrifuge the cell suspension at 2-4°C, 700g for 10 minutes, collect the supernatant and centrifuge it at 2-4°C, 14000-15000g for 35-40 minutes;

[0023] The ratio of membrane protein extraction reagent added with phenylmethylsulfonyl fluoride to cells is 1 mL: 20 to 50 million cells.

[0024] The second object of the present invention is to provide a daptomycin nanoformulation.

[0025] To achieve the second object of the present invention, the daptomycin nanoformulation is composed of daptomycin-polylactic-co-glycolic acid copolymer-cell membrane vesicles overexpressing RANK from the inside to the outside.

[0026] In a specific embodiment, the weight ratio of membrane protein to poly(lactic-co-glycolic acid) in the cell membrane vesicles overexpressing RANK is 0.25.

[0027] In a specific embodiment, the daptomycin nanoformulation is prepared by the above-mentioned method for preparing the daptomycin nanoformulation.

[0028] In one specific embodiment, the daptomycin nanoformulation has a particle size range of 159.9±1.5 nm and a potential of -32.43±2 mV.

[0029] In a specific embodiment, the daptomycin nanoformulation is an intravenous administration preparation or an intra-articular injection preparation.

[0030] The third object of the present invention is to provide a use of a daptomycin nanoformulation in the preparation of a drug for treating rheumatoid arthritis, wherein the daptomycin nanoformulation is the above-mentioned daptomycin nanoformulation.

[0031] Beneficial effects:

[0032] 1. The preparation method of the present invention successfully prepares a daptomycin nanoformulation targeting rheumatoid arthritis.

[0033] 2. The daptomycin nanoformulation prepared by the present invention has good stability.

[0034] 3. The daptomycin nanoformulation of the present invention has good anti-RA efficacy. Compared with DAP treatment alone, the bionic nanoformulation improves the efficacy, reduces the number of dosing times, shortens the dosing cycle, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the preparation of PLGA-encapsulated DAP nanoparticles of Example 1.

[0036] Figure 2 This is the RANK recombinant plasmid map of Example 1.

[0037] Figure 3 This is the RANK stable cell line verification of Example 1.

[0038] Figure 4 This is a transmission electron microscopy image of the nanoparticles of Example 1.

[0039] Figure 5 The nanoparticle size and potential diagram of Example 1.

[0040] Figure 6 is the average thickness of the mouse paw in Example 1.

[0041] Figure 7 is the ankle circumference of the mouse in Example 1.

[0042] Figure 8 The stability test results of daptomycin nanoformulations of Example 1 and Comparative Examples 1-7 are shown. DETAILED DESCRIPTION

[0043] To achieve the first object of the present invention, the preparation method of the daptomycin nanoformulation comprises:

[0044] A. Poly(lactic acid-co-glycolic acid) was used to encapsulate daptomycin to obtain nanoparticles NPs;

[0045] B. mixing the cell membrane vesicles overexpressing RANK and the nanoparticles NPs, sonicating them for 3 to 5 minutes under water bath sonication conditions, and then sequentially extruding them through 800 nm, 400 nm and 200 nm polycarbonate membranes to obtain daptomycin nanoformulations;

[0046] The weight ratio of the membrane protein of the cell membrane vesicles overexpressing RANK to the poly(lactic acid-glycolic acid) copolymer is 0.25.

[0047] In a specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer to daptomycin in step A is 7.5-15:1.

[0048] In a specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer and daptomycin in step A is 10:1.

[0049] In a specific embodiment, the preparation method of the nanoparticles NPs in step A includes: dissolving polylactic acid-glycolic acid copolymer in chloroform to obtain solution A, dissolving daptomycin in methanol to obtain solution B, mixing solution A and solution B as an oil phase, adding a PVA aqueous solution thereto as an external aqueous phase, ultrasonically emulsifying at a power of 20% to 30% to form an emulsion with a uniform appearance, then immediately performing reduced pressure rotary evaporation to evaporate the organic solvent to obtain a drug-loaded emulsion, and then centrifuging to collect the precipitated nanoparticles; the volume ratio of the external aqueous phase to the oil phase is 3 to 6:1, and the mass volume ratio of PVA to water in the PVA aqueous solution is 0.25g to 1g:100ml.

[0050] In a specific embodiment, the mass volume ratio of PVA to water in the PVA aqueous solution is 0.5g:100ml, the ultrasonic power is 25%, and the volume ratio of the external water phase and the oil phase is 4:1.

[0051] In a specific embodiment, the centrifugation condition is 12000-13000 rpm, 2-4°C low temperature centrifugation for 50-60 minutes.

[0052] In a specific embodiment, the method for preparing cell membrane vesicles overexpressing RANK includes: transfecting 293T cells with a RANK recombinant plasmid to establish a 293T cell line overexpressing RANK, and then extracting cell membrane vesicles overexpressing RANK; the RANK recombinant plasmid is a plasmid with an inserted RANK gene fragment.

[0053] Plasmids can be purchased commercially, such as pFV155-GFP-PURO plasmid, and the RANK gene is inserted between the EcoRI and XbaI restriction sites of the pFV155-GFP-PURO plasmid to construct the pFV155-GFP-PURO-RANK recombinant plasmid.

[0054] The RANK gene fragment is a gene that can express the nuclear factor κB receptor activating protein receptor. For example, the gene fragment with GI number: 1519243924 can express the nuclear factor κB receptor activating protein receptor and can be used as the RANK gene fragment.

[0055] In a specific embodiment, the method for establishing a RANK-overexpressing 293T cell line includes: transfecting 293T cells with a recombinant plasmid, a packaging plasmid psPAX2 and pMD2.G in a mass ratio of 2:2:1 using a PEI transfection reagent, collecting the culture supernatant containing the lentivirus 48 to 72 hours after transfection, filtering with a 0.45 μm filter, adding the supernatant containing the lentivirus to new 293T cells, changing the medium after 48 hours of infection, and adding 1 μg / mL puromycin for screening, and finally obtaining a stable RANK-overexpressing 293T cell line.

[0056] In a specific embodiment, the method for extracting cell membrane vesicles overexpressing RANK comprises:

[0057] Wash the cells with 4°C precooled PBS, centrifuge and discard the supernatant, add cell membrane protein extraction reagent with phenylmethylsulfonyl fluoride to the cells, mix and suspend the cells thoroughly, place in an ice bath for 10-15 minutes, homogenize 30-50 times, centrifuge the cell suspension at 2-4°C, 700g for 10 minutes, collect the supernatant and centrifuge it at 2-4°C, 14000-15000g for 35-40 minutes;

[0058] The ratio of membrane protein extraction reagent added with phenylmethylsulfonyl fluoride to cells is 1 mL: 20 to 50 million cells.

[0059] The second object of the present invention is to provide a daptomycin nanoformulation.

[0060] To achieve the second object of the present invention, the daptomycin nanoformulation is composed of daptomycin-polylactic-co-glycolic acid copolymer-cell membrane vesicles overexpressing RANK from the inside to the outside.

[0061] In a specific embodiment, the weight ratio of membrane protein to poly(lactic-co-glycolic acid) in the cell membrane vesicles overexpressing RANK is 0.25.

[0062] In a specific embodiment, the daptomycin nanoformulation is prepared by the above-mentioned method for preparing the daptomycin nanoformulation.

[0063] In one specific embodiment, the daptomycin nanoformulation has a particle size range of 159.9±1.5 nm and a potential of -32.43±2 mV.

[0064] In a specific embodiment, the daptomycin nanoformulation is an intravenous administration preparation or an intra-articular injection preparation.

[0065] The third object of the present invention is to provide a use of a daptomycin nanoformulation in the preparation of a drug for treating rheumatoid arthritis, wherein the daptomycin nanoformulation is the above-mentioned daptomycin nanoformulation.

[0066] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0067] Example 1

[0068] (1) Preparation of PLGA-coated DAP nanoparticles (NPs)

[0069] PLGA was dissolved in chloroform, DAP was dissolved in methanol, chloroform solution containing 20 mg PLGA and methanol solution containing 2 mg DAP were mixed as the oil phase, and 3 ml of PVA aqueous solution was added as the external aqueous phase. Probe-type ultrasonic emulsification was used to form an emulsion with a uniform appearance, with a power of 25%, an ultrasonic time of 3 minutes, 3 seconds on, and 3 seconds off. Subsequently, the organic solvent was evaporated immediately under reduced pressure to obtain a drug-loaded emulsion. Then, centrifuged at 13,000 rpm and 4°C for 60 minutes, and the precipitated nanoparticles were collected. The drug loading capacity (DLC) and encapsulation efficiency (EE) of DAP were determined by ultraviolet spectrophotometry, as shown in FIG. Figure 1 .

[0070] The following experiments selected the optimal conditions: power 25%, PVA concentration 0.5%, water-oil ratio 4:1, PLGA to DAP ratio 10:1, and the resulting drug encapsulation efficiency was 71.5% and drug loading was 7.1%.

[0071] (2) Construction of a cell line stably overexpressing RANK

[0072] First, construct a recombinant plasmid containing the target gene sequence. The plasmid map is as follows Figure 2 As shown, 293T cells were then plated and cultured overnight, and the recombinant plasmid, packaging plasmid psPAX2 and pMD2.G were transfected into 293T cells with a mass ratio of 2:2:1 using PEI transfection reagent. After transfection for 48 hours, the culture supernatant containing the lentivirus was collected and filtered with a 0.45 μm filter. The supernatant containing the lentivirus was added to new 293T cells and the medium was changed after 48 hours of infection. Puromycin (1 μg / mL) was added for screening, and finally a cell line stably overexpressing RANK was obtained, which was verified by Western Blot (WB). The WB results are shown in FIG. Figure 3 As shown, it was shown that the RANK overexpression 293T stable strain was successfully established.

[0073] (3) Extraction of Nanovesicles (NVs) from cells overexpressing RANK

[0074] Cell membrane extraction was performed using the Cell Membrane Protein and Cytoplasm Protein Extraction Kit (cat #P0033) from Bio-Tech. The cultured cells were collected, washed with pre-cooled PBS, centrifuged and the supernatant was discarded. 1 mL of membrane protein extraction reagent A with PMSF added was added to 20-50 million cells, mixed thoroughly and suspended, and placed in an ice bath for 10-15 minutes. The cell suspension was then transferred to a pre-cooled glass homogenizer and homogenized for about 30-50 minutes. The cell suspension was centrifuged at 4°C, 700g for 10 minutes, the supernatant was collected into a new centrifuge tube, and then centrifuged at 4°C, 14000g for 35 minutes to precipitate cell membrane fragments.

[0075] (4) Preparation and characterization of membrane-coated nanoparticles

[0076] Cell membrane vesicles and PLGA-wrapped DAP nanoparticles were obtained. The mass ratio of membrane protein to PLGA in the cell membrane vesicles was 0.25. They were ultrasonicated for 3 minutes under water bath ultrasonic conditions, and then sequentially squeezed through 800nm, 400nm and 200nm polycarbonate membranes on a membrane squeezer to obtain cell membrane-wrapped nanoparticles. After the membrane-wrapped nanoparticles were diluted, their morphology was observed under an electron microscope, and the particle size and potential of the nanoparticles were measured using a particle size analyzer.

[0077] The results showed that the cell membrane vesicles were teacup-shaped, and the membrane-wrapped nanoparticles were spherical with a complete membrane-wrapped structure, which was consistent with the characteristics of nanoparticles. Figure 4 The particle size and potential test results show that the average particle size of NPs is 147.1nm, the average potential is -10mV, the average particle size of NVs is 196.1nm, the average potential is -33.52mV, the average particle size of RANK-NVs is 191.0nm, the average potential is -31.44mV, the average particle size of NVs-NPs is 163.8nm, the average potential is -33.09mV, and the average particle size of RANK-NVs-NPs is 159.9nm, the average potential is -32.43mV, see for details. Figure 5 .

[0078] (5) In vivo efficacy test

[0079] Establishment of collagen-induced arthritis (CIA) model: 6-8 week-old male DBA1 / J mice were selected for the experiment. Bovine type II collagen and complete Freund's adjuvant were mixed in equal volumes and emulsified at 4°C to prepare the modeling agent. A total of 100 μL of the modeling agent was injected intradermally at multiple points at the base of the tail starting on day 0. On day 21, bovine type II collagen and incomplete Freund's adjuvant were mixed in equal volumes, emulsified at low temperature, and 100 μL was injected at the base of the tail for the second booster immunization. Clinical scoring of arthritis was performed. Arthritis scoring criteria: 0 points for no symptoms, 1 point for obvious swelling and redness of one toe, 2 points for mild swelling of the limbs or swelling of more than two toes and erythema, 3 points for obvious swelling and erythema of the limbs, 4 points for severe swelling and erythema of the limbs, obvious joint dysfunction, and ankylosis. Each paw needs to be scored, and the total score of a mouse is 16 points. When the sum of the scores of the four paws is ≥4, the modeling is considered successful.

[0080] Drug treatment and grouping: The animals were divided into healthy mice group (Normal group), CIA model group, DAP group, NPs group, NV group (37.5 mg / kg), RANK-NVs group, NVs-NPs group, and RANK-NVs-NPs group. Intravenous administration began on the 22nd day after model induction. The Normal group and the CIA group were given the same amount of PBS, once every 3 days, for a total of 9 times, and the animals were killed on the 62nd day.

[0081] The results showed that compared with the CIA group, each treatment group could significantly reduce arthritis in CIA mice and reduce the swelling of the paws. Figure 6 , improve ankle swelling, reduce ankle circumference, see Figure 7 Among them, the RANK-NVs-NPs group had the best effect.

[0082] In summary, based on the previous research on DAP anti-RA, this study further developed an engineered cell membrane-wrapped DAP bionic nanoformulation for the treatment of RA. The prepared DAP bionic nanoformulation has the characteristics of nanomedicine. The efficacy of the nanoformulation was further confirmed by animal experiments. The results showed that the DAP bionic nanoformulation has good anti-RA efficacy. Compared with DAP alone, the bionic nanoformulation improves the efficacy, reduces the number of dosing, shortens the dosing cycle, and has good application prospects.

[0083] Comparative Examples 1-7

[0084] The rest is consistent with Example 1. The weight ratios of membrane protein to PLGA in Comparative Examples 1-7 are 4, 3, 2, 1, 0.5, 0.33, and 0.2, respectively. The stability of the nanoparticles was evaluated by measuring the changes in particle size after synthesis, adjustment to PBS (adding the synthesized nanodrug to new PBS), and storage in PBS for 14 days. The results show that the nanoparticles of Example 1 are the most stable, and the particle size increase is the smallest after storage for 14 days. For details, see Figure 8 .

Claims

1. A method for preparing a daptomycin nanoformulation, characterized in that: The preparation method of the daptomycin nanoformulation comprises: A. Poly(lactic acid-co-glycolic acid) was used to encapsulate daptomycin to obtain nanoparticles NPs; B. The cell membrane vesicles overexpressing RANK and the nanoparticles NPs were mixed, sonicated for 3 to 5 minutes under water bath sonication conditions, and then sequentially extruded through 800 nm, 400 nm and 200 nm polycarbonate membranes to obtain daptomycin nanoformulations; The weight ratio of the membrane protein of the cell membrane vesicles overexpressing RANK to the poly(lactic acid-glycolic acid) copolymer is 0.

25.

2. The method for preparing the daptomycin nanoformulation according to claim 1, characterized in that: The mass ratio of the polylactic acid-glycolic acid copolymer to daptomycin in step A is 7.5-15:

1.

3. The method for preparing the daptomycin nanoformulation according to claim 1 or 2, characterized in that: The preparation method of the nanoparticles NPs in step A comprises: dissolving polylactic acid-glycolic acid copolymer in chloroform to obtain solution A, dissolving daptomycin in methanol to obtain solution B, mixing solution A and solution B as an oil phase, adding a PVA aqueous solution thereto as an external aqueous phase, ultrasonically emulsifying at a power of 20% to 30% to form an emulsion with a uniform appearance, then immediately performing reduced pressure rotary evaporation to evaporate the organic solvent to obtain a drug-loaded emulsion, and then collecting and precipitating nanoparticles by centrifugation; the volume ratio of the external aqueous phase to the oil phase is 3 to 6:1, and the mass volume ratio of PVA to water in the PVA aqueous solution is 0.25 g to 1 g: 100 ml.

4. The method for preparing the daptomycin nanoformulation according to claim 1 or 2, characterized in that: The method for preparing the cell membrane vesicles overexpressing RANK comprises: transfecting 293T cells with a RANK recombinant plasmid to establish a RANK overexpressing 293T cell line, and then extracting the cell membrane vesicles overexpressing RANK; the RANK recombinant plasmid is a recombinant plasmid with a RANK gene fragment inserted therein.

5. The method for preparing the daptomycin nanoformulation according to claim 4, characterized in that: The method for establishing a RANK-overexpressing 293T cell line comprises: transfecting 293T cells with a recombinant plasmid, a packaging plasmid psPAX2 and pMD2.G at a mass ratio of 2:2:1 using a PEI transfection reagent, collecting a culture supernatant containing a lentivirus 48 to 72 hours after transfection, filtering with a 0.45 μm filter, adding the supernatant containing the lentivirus to new 293T cells, changing the medium after infection for 48 hours, and adding 1 μg / mL puromycin for screening, and finally obtaining a stable RANK-overexpressing 293T cell line.

6. A daptomycin nanoformulation, characterized in that: The daptomycin nanoformulation is composed of daptomycin-polylactic acid-glycolic acid copolymer-cell membrane vesicles overexpressing RANK from the inside to the outside, and the weight ratio of membrane protein of the cell membrane vesicles overexpressing RANK to the polylactic acid-glycolic acid copolymer is 0.

25.

7. The daptomycin nanoformulation according to claim 6, characterized in that: The daptomycin nanoformulation is prepared by the preparation method of the daptomycin nanoformulation according to any one of claims 1 to 5.

8. The daptomycin nanoformulation according to claim 6, characterized in that: The particle size range of the daptomycin nanoformulation is 159.9±1.5 nm and the potential is -32.43±2 mV.

9. The daptomycin nanoformulation according to claim 6, characterized in that: The daptomycin nanoformulation is an intravenous administration preparation or an intra-articular injection preparation.

10. Use of daptomycin nanoformulation in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The daptomycin nanoformulation is the daptomycin nanoformulation according to any one of claims 6 to 9.

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