Daptomycin nano-formulation, method for preparing same, and use thereof

Abstract

The present invention relates to the field of pharmaceutical technology, and in particular, to a daptomycin nano-FORMULATION, a method for preparing same, and use thereof. The method comprises: A, encapsulating daptomycin with a copolymer poly(lactic-co-glycolic acid) to give a nanoparticle (NP); and B, sonicating a cell membrane vesicle overexpressing RANK and the nanoparticle (NP) in a water bath for 3 min, and then sequentially extruding the mixture through polycarbonate membranes of 800 nm, 400 nm, and 200 nm to give the daptomycin nano-formulation. The weight ratio of the membrane protein to PLGA is 0.25. By the method of the present invention, a daptomycin nano-formulation targeting rheumatoid arthritis is successfully prepared. The daptomycin nano-formulation prepared by the present invention has good stability. The daptomycin nano-formulation of the present invention has good therapeutic effects against RA.

Description

Daptomycin nanoformulations, their preparation methods and applications

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of Chinese patent application CN 202411455669X, filed on October 18, 2024, which is incorporated herein by reference in its entirety and for all other purposes. Technical Field

[0003] This invention relates to a daptomycin nanoformulation, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0004] Rheumatoid arthritis (RA) is an autoimmune disease characterized by erosive arthritis. Its main pathological features include synovial hyperplasia, joint structural destruction, immune cell infiltration, and pannus formation. It can affect synovial joints throughout the body. The prevalence of RA in the general population is 0.5-1%, with women being 2-3 times more likely to be affected than men. It can occur at any age, but is most common between 22 and 55 years of age, although it can also occur in children. In my country, the incidence of RA is approximately 0.42% of the total population, with about 5 million RA patients nationwide. Early and mid-stage RA patients often experience redness, swelling, pain, and functional impairment in the joints. Late-stage RA patients develop varying degrees of stiffness and deformity, which can even lead to disability. RA is a multifactorial disease, including genetic, environmental, infectious, endocrine disorders, and immune dysregulation factors, with immune dysregulation being the primary cause.

[0005] Currently, rheumatoid arthritis (RA) cannot be cured. The clinical treatment goal for RA is to achieve disease remission or low disease activity, i.e., to achieve treatment targets. The ultimate goal is to control the disease, reduce disability rates, and improve patients' quality of life. Therefore, developing safer and more effective drugs can address unmet clinical needs and thus benefit patients.

[0006] Daptomycin (DAP) is a secondary metabolite produced by *Streptomyces roseosus* and was approved by the U.S. Food and Drug Administration in 2003 for the treatment of severe skin infections. Studies have shown that DAP, in addition to its excellent antibacterial activity, also possesses immunomodulatory effects. We were among the first to conduct research on the therapeutic effect of DAP on rheumatoid arthritis (RA) using in vitro and in vivo inflammation models. Results showed that DAP had a good therapeutic effect on collagen-induced RA in mice, and related research findings are documented in CN110604808A regarding the application of daptomycin in the preparation of drugs for treating rheumatoid arthritis. Given that the main pathological damage in RA occurs in the joints, especially in the later stages where cartilage destruction and bone erosion occur, severely affecting patients' mobility, targeted drug delivery to the affected joints could produce better anti-RA efficacy. However, there are currently no DAP-targeted nanoformulations.

[0007] Conventional treatments for rheumatoid arthritis (RA) often lead to systemic distribution, increased dosages, damage to normal tissues and organs, and treatment tolerance. Nanomedicines have addressed these shortcomings by loading conventional RA drugs onto nanocarriers and delivering them directly to the lesion site. However, immune clearance of nanoparticles remains a significant obstacle to drug delivery, and how to avoid immune clearance has been a persistent challenge for researchers. In recent years, research on biomimetic functionalized nanoparticles has increased significantly. These nanoparticles combine the variability and flexibility of nanomaterials with the bioactivity of natural materials, exhibiting long-lasting circulation, active targeting, and the ability to avoid immune clearance, making them a hot research topic. Cell membranes are among the most studied biomimetic nanocarriers. Current research has involved fabricating nanovesicles from the cell membranes of erythrocytes, platelets, macrophages, and neutrophils to encapsulate nanoparticles and evaluating their efficacy in animal models of RA.

[0008] Joint bone destruction is an important pathological feature in the later stages of RA patients. Osteoclasts play an important role in bone resorption during bone metabolism. The two most important factors involved in osteoclast differentiation, maturation and activation are macrophage-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). RANKL binds to its receptor RANK and activates downstream signaling pathways, ultimately leading to osteoclast formation. RANKL is highly expressed in RA. Summary of the Invention

[0009] The first objective of this invention is to provide a method for preparing daptomycin nanoparticles.

[0010] To achieve the first objective of this invention, the method for preparing the daptomycin nanoparticle formulation includes:

[0011] A. Encapsulating daptomycin with polylactic acid-glycolic acid copolymer to obtain nanoparticles (NPs);

[0012] B. Mix the cell membrane vesicles overexpressing RANK and the nanoparticles NPs, sonicate them in a water bath for 3 to 5 minutes, and then squeeze them through polycarbonate membranes of 800 nm, 400 nm and 200 nm in sequence to obtain daptomycin nanoformulation.

[0013] The weight ratio of the membrane protein of the RANK-overexpressing cell membrane vesicles to the polylactic acid-glycolic acid copolymer is 0.25.

[0014] In one specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer and daptomycin in step A is 7.5 to 15:1.

[0015] In one specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer and daptomycin in step A is 10:1.

[0016] In one 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 PVA aqueous solution as an external aqueous phase, ultrasonically emulsifying at 20%–30% power to form a uniform emulsion, then immediately rotary evaporating under reduced pressure to remove 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 3–6:1, and the mass-to-volume ratio of PVA to water in the PVA aqueous solution is 0.25 g–1 g:100 ml.

[0017] In one specific embodiment, the PVA aqueous solution has a PVA to water mass-to-volume ratio of 0.5g:100ml, an ultrasonic power of 25%, and an external aqueous phase to oil phase volume ratio of 4:1.

[0018] In one specific embodiment, the centrifugation conditions are 12000-13000 rpm and centrifugation at a low temperature of 2-4°C for 50-60 minutes.

[0019] In one specific embodiment, the method for preparing the RANK-overexpressing cell membrane vesicles includes: transfecting 293T cells with a RANK recombinant plasmid to establish a RANK-overexpressing 293T cell line, and then extracting RANK-overexpressing cell membrane vesicles; wherein the RANK recombinant plasmid is a plasmid with an inserted RANK gene fragment.

[0020] Commercially available plasmids, such as pFV155-GFP-PURO plasmid, can be purchased. The RANK gene is inserted between the EcoRI and XbaI restriction sites of the pFV155-GFP-PURO vector to construct the pFV155-GFP-PURO-RANK recombinant plasmid.

[0021] RANK gene fragments are genes that can express nuclear factor κB receptor activator protein receptor. For example, the gene fragment with GI number 1519243924 can express nuclear factor κB receptor activator protein receptor and can be used as RANK gene fragments.

[0022] In one specific embodiment, the method for establishing a RANK-overexpressing 293T cell line includes: transfecting 293T cells with recombinant plasmid, packaging plasmid psPAX2, and pMD2.G in a mass ratio of 2:2:1 using PEI transfection reagent. After 48–72 h of transfection, the culture supernatant containing lentivirus is collected, filtered through a 0.45 μm filter, and the lentivirus-containing supernatant is added to new 293T cells for infection. After 48 h of infection, the medium is changed, and 1 μg / mL puromycin is added for selection. Finally, a stable RANK-overexpressing 293T cell line is obtained.

[0023] In one specific embodiment, the method for extracting RANK-overexpressing cell membrane vesicles includes:

[0024] Wash cells with PBS pre-cooled to 4°C, centrifuge and discard the supernatant, add cell membrane protein extraction reagent with added benzyl sulfonyl fluoride to the cells, mix thoroughly and suspend the cells, place on ice for 10-15 minutes, homogenize 30-50 times, centrifuge the cell suspension at 700g for 10 minutes at 2-4°C, collect the supernatant and centrifuge at 14000-15000g for 35-40 minutes at 2-4°C.

[0025] The ratio of membrane protein extraction reagent to cells containing benzyl sulfonyl fluoride is 1 mL: 20 million to 50 million cells.

[0026] A second objective of this invention is to provide a daptomycin nanoformulation.

[0027] To achieve the second objective of this invention, the daptomycin nanoformulation consists, from the inside out, daptomycin-polylactic acid-glycolic acid copolymer-cell membrane vesicles overexpressing RANK.

[0028] In one specific embodiment, the weight ratio of the membrane protein of the RANK-overexpressing cell membrane vesicles to the polylactic acid-glycolic acid copolymer is 0.25.

[0029] In one specific embodiment, the daptomycin nanoparticle formulation is prepared using the above-described method for preparing daptomycin nanoparticle formulations.

[0030] In one specific embodiment, the daptomycin nanoparticle formulation has a particle size range of 159.9±1.5nm and a potential of -32.43±2mV.

[0031] In one specific embodiment, the daptomycin nanoformulation is an intravenous administration formulation or an intra-articular injection formulation.

[0032] A third objective of this invention is to provide the application of a daptomycin nanoformulation in the preparation of a drug for treating rheumatoid arthritis, wherein the daptomycin nanoformulation is the aforementioned daptomycin nanoformulation. Beneficial effects:

[0033] 1. The preparation method of the present invention successfully prepared daptomycin nanoparticles targeting rheumatoid arthritis.

[0034] 2. The daptomycin nano-formulation prepared by this invention has good stability.

[0035] 3. The daptomycin nanoformulation of the present invention has good anti-RA efficacy. Compared with DAP monotherapy, the biomimetic nanoformulation improves efficacy, reduces the number of administrations, and shortens the administration cycle, showing good application prospects. Attached Figure Description

[0036] Figure 1 shows the preparation of PLGA-encapsulated DAP nanoparticles in Example 1.

[0037] Figure 2 shows the RANK recombinant plasmid map of Example 1.

[0038] Figure 3 shows the validation of the RANK stable cell line in Example 1.

[0039] Figure 4 is a transmission electron microscope image of the nanoparticles in Example 1.

[0040] Figure 5 shows the particle size and potential of the nanoparticles in Example 1.

[0041] Figure 6 shows the average thickness of the mouse paw in Example 1.

[0042] Figure 7 shows the ankle circumference of mice in Example 1.

[0043] Figure 8 shows the stability test results of daptomycin nanoformulations in Example 1 and Comparative Examples 1-7. Detailed Implementation

[0044] To achieve the first objective of this invention, the method for preparing the daptomycin nanoparticle formulation includes:

[0045] A. Encapsulating daptomycin with polylactic acid-glycolic acid copolymer to obtain nanoparticles (NPs);

[0046] B. Mix the cell membrane vesicles overexpressing RANK and the nanoparticles NPs, sonicate them in a water bath for 3 to 5 minutes, and then squeeze them through polycarbonate membranes of 800 nm, 400 nm and 200 nm in sequence to obtain daptomycin nanoformulation.

[0047] The weight ratio of the membrane protein of the RANK-overexpressing cell membrane vesicles to the polylactic acid-glycolic acid copolymer is 0.25.

[0048] In one specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer and daptomycin in step A is 7.5 to 15:1.

[0049] In one specific embodiment, the mass ratio of the polylactic acid-glycolic acid copolymer and daptomycin in step A is 10:1.

[0050] In one 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 PVA aqueous solution as an external aqueous phase, ultrasonically emulsifying at 20%–30% power to form a uniform emulsion, then immediately rotary evaporating under reduced pressure to remove 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 3–6:1, and the mass-to-volume ratio of PVA to water in the PVA aqueous solution is 0.25 g–1 g:100 ml.

[0051] In one specific embodiment, the PVA aqueous solution has a PVA to water mass-to-volume ratio of 0.5g:100ml, an ultrasonic power of 25%, and an external aqueous phase to oil phase volume ratio of 4:1.

[0052] In one specific embodiment, the centrifugation conditions are 12000-13000 rpm and centrifugation at a low temperature of 2-4°C for 50-60 minutes.

[0053] In one specific embodiment, the method for preparing the RANK-overexpressing cell membrane vesicles includes: transfecting 293T cells with a RANK recombinant plasmid to establish a RANK-overexpressing 293T cell line, and then extracting RANK-overexpressing cell membrane vesicles; wherein the RANK recombinant plasmid is a plasmid with an inserted RANK gene fragment.

[0054] Commercially available plasmids, such as pFV155-GFP-PURO plasmid, can be purchased. The RANK gene is inserted between the EcoRI and XbaI restriction sites of the pFV155-GFP-PURO vector to construct the pFV155-GFP-PURO-RANK recombinant plasmid.

[0055] RANK gene fragments are genes that can express nuclear factor κB receptor activator protein receptor. For example, the gene fragment with GI number 1519243924 can express nuclear factor κB receptor activator protein receptor and can be used as RANK gene fragments.

[0056] In one specific embodiment, the method for establishing a RANK-overexpressing 293T cell line includes: transfecting 293T cells with recombinant plasmid, packaging plasmid psPAX2, and pMD2.G in a mass ratio of 2:2:1 using PEI transfection reagent. After 48–72 h of transfection, the culture supernatant containing lentivirus is collected, filtered through a 0.45 μm filter, and the lentivirus-containing supernatant is added to new 293T cells for infection. After 48 h of infection, the medium is changed, and 1 μg / mL puromycin is added for selection. Finally, a stable RANK-overexpressing 293T cell line is obtained.

[0057] In one specific embodiment, the method for extracting RANK-overexpressing cell membrane vesicles includes:

[0058] Wash cells with PBS pre-cooled to 4°C, centrifuge and discard the supernatant, add cell membrane protein extraction reagent with added benzyl sulfonyl fluoride to the cells, mix thoroughly and suspend the cells, place on ice for 10-15 minutes, homogenize 30-50 times, centrifuge the cell suspension at 700g for 10 minutes at 2-4°C, collect the supernatant and centrifuge at 14000-15000g for 35-40 minutes at 2-4°C.

[0059] The ratio of membrane protein extraction reagent to cells containing benzyl sulfonyl fluoride is 1 mL: 20 million to 50 million cells.

[0060] A second objective of this invention is to provide a daptomycin nanoformulation.

[0061] To achieve the second objective of this invention, the daptomycin nanoformulation consists, from the inside out, daptomycin-polylactic acid-glycolic acid copolymer-cell membrane vesicles overexpressing RANK.

[0062] In one specific embodiment, the weight ratio of the membrane protein of the RANK-overexpressing cell membrane vesicles to the polylactic acid-glycolic acid copolymer is 0.25.

[0063] In one specific embodiment, the daptomycin nanoparticle formulation is prepared using the above-described method for preparing daptomycin nanoparticle formulations.

[0064] In one specific embodiment, the daptomycin nanoparticle formulation has a particle size range of 159.9±1.5nm and a potential of -32.43±2mV.

[0065] In one specific embodiment, the daptomycin nanoformulation is an intravenous administration formulation or an intra-articular injection formulation.

[0066] A third objective of this invention is to provide the application of a daptomycin nanoformulation in the preparation of a drug for treating rheumatoid arthritis, wherein the daptomycin nanoformulation is the aforementioned daptomycin nanoformulation.

[0067] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0068] Example 1

[0069] (1) Preparation of PLGA-encapsulated DAP nanoparticles (NPs)

[0070] PLGA was dissolved in chloroform, and DAP was dissolved in methanol. A chloroform solution containing 20 mg PLGA and a methanol solution containing 2 mg DAP were mixed to form the oil phase. 3 ml of PVA aqueous solution was added to form the aqueous phase. The mixture was then ultrasonically emulsified using a probe-type ultrasonic emulsion at 25% power for 3 minutes, with an on-off cycle of 3 seconds. Immediately afterwards, the organic solvent was evaporated under reduced pressure to obtain the drug-loaded emulsion. The emulsion was then centrifuged at 13000 rpm at 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 Figure 1.

[0071] The following experiments selected the optimal conditions: power 25%, PVA concentration 0.5%, water-oil ratio 4:1, PLGA to DAP ratio 10:1, resulting in a drug encapsulation efficiency of 71.5% and a drug loading of 7.1%.

[0072] (2) Construction of a stable RANK overexpression cell line

[0073] First, a recombinant plasmid containing the target gene sequence was constructed, and the plasmid map is shown in Figure 2. Then, 293T cells were plated and cultured overnight. The recombinant plasmid, packaging plasmid psPAX2, and pMD2.G were transfected into 293T cells at a mass ratio of 2:2:1 using PEI transfection reagent. After 48 hours of transfection, the culture supernatant containing lentivirus was collected, filtered through a 0.45 μm filter, and added to new 293T cells for infection. After 48 hours, the medium was changed, and puromycin (1 μg / mL) was added for selection. Finally, a stable RANK overexpressing cell line was obtained, and Western Blot (WB) was used for verification. The WB results are shown in Figure 3, indicating that a stable RANK overexpressing 293T cell line was successfully established.

[0074] (3) Extraction of RANK-overexpressing cell membrane vesicles (NVs)

[0075] Cell membrane extraction was performed using the Cell Membrane and Cytoplasmic Protein Extraction Kit (CAT#P0033) from Beyotime Biotechnology. Cultured cells were collected, washed with pre-chilled PBS, centrifuged, and the supernatant discarded. 1 mL of membrane protein extraction reagent A supplemented with PMSF was added to 20-50 million cells, thoroughly mixed, and the cells were resuspended. The mixture was then incubated on ice for 10-15 minutes. The cell suspension was then transferred to a pre-chilled glass homogenizer and homogenized for approximately 30-50 cycles. The cell suspension was centrifuged at 700g for 10 minutes at 4°C, and the supernatant was collected into a new centrifuge tube. The tube was then centrifuged at 14000g for 35 minutes at 4°C to precipitate cell membrane debris.

[0076] (4) Preparation and characterization of membrane-coated nanoparticles

[0077] Cell membrane vesicles and PLGA-encapsulated DAP nanoparticles were used, with a vesicle protein to PLGA mass ratio of 0.25. The nanoparticles were sonicated in a water bath for 3 minutes, then sequentially extruded through polycarbonate membranes of 800 nm, 400 nm, and 200 nm using a membrane extruder to obtain cell membrane-encapsulated nanoparticles. The nanoparticles were diluted and their morphology was observed under an electron microscope. The particle size and potential of the nanoparticles were measured using a particle size analyzer.

[0078] The results showed that the cell membrane vesicles were teacup-shaped, and the membrane-encapsulated nanoparticles were spherical with a complete membrane-encapsulated structure, consistent with the characteristics of nanoparticles (see Figure 4). Particle size and potential measurements showed that the average particle size of NPs was 147.1 nm with an average potential of -10 mV; the average particle size of NVs was 196.1 nm with an average potential of -33.52 mV; the average particle size of RANK-NVs was 191.0 nm with an average potential of -31.44 mV; the average particle size of NVs-NPs was 163.8 nm with an average potential of -33.09 mV; and the average particle size of RANK-NVs-NPs was 159.9 nm with an average potential of -32.43 mV (see Figure 5).

[0079] (5) In vivo efficacy test

[0080] Establishment of a collagen-induced arthritis (CIA) model: Male DBA1 / J mice aged 6-8 weeks were used. Bovine type II collagen and complete Freund's adjuvant were mixed in equal volumes and emulsified at 4°C to prepare a modeling agent. Starting on day 0, 100 μL of the modeling agent was injected intradermally at multiple points at the base of the tail. 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 into the base of the tail for a second booster immunization. Clinical arthritis scoring was then performed. Arthritis scoring criteria: 0 points for asymptomatic; 1 point for significant swelling and redness of one toe; 2 points for mild swelling of the limb or swelling of two or more toes with erythema; 3 points for significant swelling and erythema of the limb; 4 points for severe swelling and erythema of the limb, significant joint dysfunction, and joint ankylosis. Each paw was scored, with a total score of 16 points per mouse. Successful modeling was considered achieved when the sum of the scores for all four paws was ≥4.

[0081] Drug treatment and grouping: Animals were divided into four groups: healthy mice (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 treatment began on day 22 after model induction. The Normal group and CIA group received the same amount of PBS, administered once every 3 days for a total of 9 doses. Animals were sacrificed on day 62.

[0082] The results showed that, compared with the CIA group, all treatment groups could significantly reduce arthritis in CIA mice, reduce paw swelling (see Figure 6 for details), improve ankle swelling, and reduce ankle circumference (see Figure 7 for details). Among them, the RANK-NVs-NPs group showed the best effect.

[0083] In summary, building upon previous research on DAP's anti-RA effects, this study further developed engineered, cell-membrane-encapsulated DAP biomimetic nanoparticles for RA treatment. The prepared DAP biomimetic nanoparticles exhibit the characteristics of nanomedicines. Animal experiments further confirmed the efficacy of the nanoparticles. The results showed that the DAP biomimetic nanoparticles have good anti-RA efficacy. Compared with DAP monotherapy, the biomimetic nanoparticles improved efficacy, reduced the frequency of administration, and shortened the treatment cycle, demonstrating promising application prospects.

[0084] Comparative Examples 1-7

[0085] Other parameters were consistent with Example 1. The weight ratios of membrane protein to PLGA in Comparative Examples 1-7 were 4, 3, 2, 1, 0.5, 0.33, and 0.2, respectively. The stability of the nanoparticles was assessed by measuring the particle size changes after synthesis, after adjustment to PBS (adding the synthesized nanomedicine to new PBS), and after storage in PBS for 14 days. The results showed that the nanoparticles of Example 1 were the most stable, exhibiting the smallest increase in particle size after 14 days of storage (see Figure 8).

Claims

1. A method for the preparation of a daptomycin nanoformulation, characterized in that, The preparation method of the daptomycin nanoparticle formulation includes: A. Encapsulating daptomycin with polylactic acid-glycolic acid copolymer to obtain nanoparticles (NPs); B. Mix cell membrane vesicles overexpressing RANK with nanoparticles NPs, sonicate under water bath sonication for 3-5 min, and then extrude them sequentially through polycarbonate membranes of 800 nm, 400 nm and 200 nm to obtain daptomycin nanoformulation. The weight ratio of the membrane protein of the RANK-overexpressing cell membrane vesicles to the polylactic acid-glycolic acid copolymer is 0.

25.

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

1.

3. The method for preparing a daptomycin nanoformulation according to claim 1 or 2, characterized in that, 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 the oil phase, adding PVA aqueous solution as the external aqueous phase, ultrasonically emulsifying at 20% to 30% power to form a uniform emulsion, and then immediately rotary evaporating under reduced pressure to remove the organic solvent to obtain the 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-to-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 a daptomycin nanoformulation according to claim 1 or 2, characterized in that, The method for preparing RANK-overexpressing cell membrane vesicles includes: transfecting 293T cells with a RANK recombinant plasmid to establish a RANK-overexpressing 293T cell line, and then extracting RANK-overexpressing cell membrane vesicles; wherein the RANK recombinant plasmid is a recombinant plasmid with an inserted RANK gene fragment.

5. The method for preparing daptomycin nanoformulation according to claim 4, characterized in that, The method for establishing a RANK-overexpressing 293T cell line includes: transfecting 293T cells with recombinant plasmid, packaging plasmid psPAX2, and pMD2.G in a mass ratio of 2:2:1 using PEI transfection reagent. After 48–72 h of transfection, the culture supernatant containing lentivirus is collected, filtered through a 0.45 μm filter, and then added to new 293T cells for infection. After 48 h of infection, the medium is changed, and 1 μg / mL puromycin is added for selection. Finally, a stable RANK-overexpressing 293T cell line is obtained.

6. A daptomycin nanofonnulation characterized in that, The daptomycin nanoformulation consists of, from the inside out, daptomycin-polylactic acid-glycolic acid copolymer-overexpressing RANK cell membrane vesicles, with the weight ratio of the membrane protein of the overexpressing RANK cell membrane vesicles to the polylactic acid-glycolic acid copolymer being 0.

25.

7. The daptomycin nanoformulation of claim 6, wherein, The daptomycin nanoparticle formulation is prepared using the method described in any one of claims 1 to 5.

8. The daptomycin nanoformulation of claim 6, wherein, The daptomycin nanoparticle formulation has a particle size range of 159.9±1.5nm and a potential of -32.43±2mV.

9. The daptomycin nanoformulation of claim 6, wherein, The daptomycin nanoformulation is an intravenous administration formulation or an intra-articular injection formulation.

10. Use of daptomycin nanoformulation for the preparation of a medicament for the treatment of 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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