Nanoparticles for preventing endometriosis and preparation method thereof

Abstract

The invention relates to nanoparticles for preventing endometriosis and a preparation method thereof, and belongs to the technical field of biological medicines. The invention provides a nanoparticle, the nanoparticle comprises bacterial outer membrane vesicles (OMV) and a polylactic acid-glycolic acid copolymer (PLGA), and the bacterial outer membrane vesicles wrap the surface of the polylactic acid-glycolic acid copolymer. According to the invention, the OMV is wrapped on the surface of the PLGA core, the biological characteristics of the PLGA core are retained, and bacteria are simulated to present antigens to an immune system. Meanwhile, the PLGA core provides a series of adjustable physical and chemical properties (such as particle size and formation), so that the size of the synthesized OMV-PLGA is more uniform. Besides, the PLGA provides a hard core for the OMV membrane, so that the OMV-PLGA can more effectively penetrate through a cell membrane compared with the OMV, and the uptake efficiency of cells is improved.

Description

Technical Field

[0001] The invention relates to a nanoparticle for preventing endometriosis and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0002] Endometriosis (endometriosis) refers to the appearance, growth, and infiltration of endometrial tissue (glands and stroma) in the lining of the uterine cavity and outside the uterus, repeated bleeding, and subsequent pain, infertility, and nodules or masses, which seriously affect the quality of life and fertility of women of childbearing age. The incidence of endometriosis in women of childbearing age worldwide is about 10%, and it is on the rise. The treatment of endometriosis includes surgical treatment and drug treatment. Conservative surgery is prone to recurrence, so drug treatment is required after surgery. At present, the drug treatment method for endometriosis is mainly to reduce estrogen levels by inhibiting the pituitary-ovarian axis. However, long-term use of drugs that reduce estrogen levels has side effects such as reduced bone density and delayed patient fertility, which limits its application. Therefore, seeking new non-estrogen-dependent preventive and therapeutic drugs for endometriosis is of great clinical significance.

[0003] Endometriosis is a complex chronic inflammatory disease with a complex etiology. In addition to the theory of retrograde menstruation, immune dysfunction, inflammatory environment, angiogenesis and endocrine imbalance are all involved in the occurrence and development of the disease. Studies have found that the occurrence of endometriosis is closely related to changes in the number and function of various immune cells, such as reduced macrophage phagocytosis, reduced T cell cytotoxicity, and polyclonal activation of B cells. Among them, macrophages are considered to play a core role in the formation of chronic pelvic inflammatory processes and the growth of ectopic endometrium. As an important component of the immune system, macrophages are not only involved in tissue repair and regulation of inflammatory responses, but also can clear foreign bodies and dead cells in the body through phagocytosis. In patients with endometriosis, the number of macrophages in peritoneal fluid and ectopic lesions increases, and they are the most significantly changed immune cell type, participating in the regulation of inflammatory responses and angiogenesis in ectopic endometrial tissues. In addition to changes in number, macrophages in patients with endometriosis also have dysfunctional functions. Their reduced phagocytic ability leads to the failure to effectively clear the endometrial tissue that flows back with menstrual blood, causing it to implant and grow in ectopic sites. In addition, macrophages can also increase the secretion of a variety of proinflammatory factors such as insulin-like growth factor 1 (IGF-1), leading to the growth of endometriotic tissue and aggravation of pain symptoms. Using low-dose lipopolysaccharide (LPS) to induce macrophages to produce innate immune memory can reduce the fibroinflammatory characteristics of ectopic lesions in an IL-10-dependent manner and effectively inhibit lesion growth, suggesting that targeting macrophages is a new direction for the treatment of endometriosis.

[0004] The polarization state of macrophages has an important impact on the progression of endometriosis. Under physiological conditions, macrophages can form two polarization states according to different signals from the local microenvironment: classically activated M1 and alternatively activated M2. M1 macrophages are mainly involved in inflammatory responses and pathogen clearance, while M2 macrophages are associated with tissue repair, healing, and fibrosis. Previous studies have found that the M2 / M1 ratio in ectopic lesions and peritoneal fluid of patients with endometriosis is significantly increased. In the pathological environment of endometriosis, the activation and increase of M2 macrophages are considered to be related to the fibrosis of lesion tissue. Fibrosis is a pathological process in which abnormal tissue proliferation and scar formation lead to damage to organ structure and function. Studies have confirmed that fibrosis is the terminal stage of the development of endometriosis lesions, and its degree is closely related to the progression of the disease. M2 macrophages promote the activation of myofibroblasts and the deposition of collagen by secreting a series of profibrotic factors, such as transforming growth factor-β, thereby leading to fibrosis in endometriosis. Therefore, the imbalance of M1 and M2 macrophages in the process of macrophage polarization may play a role in the pathogenesis of endometriosis, especially in the fibrosis of lesions. Treatment targeting macrophage polarization may reduce the degree of fibrosis in endometriosis.

[0005] Bacterial outer membrane vesicles (OMVs) are natural nanoscale vesicles released by Gram-negative bacteria during their growth, with a diameter of about 20 to 250 nm. These vesicles contain a variety of bacterial components, such as lipopolysaccharide (LPS), proteins, enzymes, DNA, and RNA. Due to the pathogen-associated molecular patterns (PAMPs) contained in them, bacterial outer membrane vesicles have immune adjuvant functions, can activate multiple Toll-like receptor (TLR) signaling pathways of the host, and trigger immune responses, so they are widely used in immunotherapy. Studies have shown that bacterial outer membrane vesicles can interact with macrophages, prompting them to polarize to the M1 type, produce proinflammatory cytokines, and enhance immune responses. In terms of application, bacterial outer membrane vesicles have been widely studied and applied to the development of tumor vaccines. They can be used as nanocarriers to effectively deliver tumor antigens to the host immune system, thereby activating specific immune responses against tumors. In addition, bacterial outer membrane vesicles also show great potential in the fields of infectious disease prevention and treatment, immune regulation, and bioengineering. However, currently, no studies have been found on bacterial outer membrane vesicles in the field of endometriosis. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a nanoparticle, which comprises bacterial outer membrane vesicles and polylactic acid-glycolic acid copolymer; the bacterial outer membrane vesicles are wrapped on the surface of the polylactic acid-glycolic acid copolymer.

[0007] In one embodiment of the present invention, the method for preparing the nanoparticles comprises: injecting a bacterial outer membrane vesicle solution and a polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip to synthesize the nanoparticles to obtain the nanoparticles; during the synthesis of the nanoparticles, ultrasonically treating the microfluidic chip to break up the bacterial outer membrane vesicles and reassemble them on the surface of the polylactic acid-glycolic acid copolymer core.

[0008] In one embodiment of the present invention, the method for preparing the nanoparticles comprises: injecting a polylactic acid-glycolic acid copolymer solution and a bacterial outer membrane vesicle solution into the middle inlet and two side inlets of a microfluidic chip respectively to synthesize the nanoparticles to obtain the nanoparticles.

[0009] In one embodiment of the present invention, the injection flow rate of the bacterial outer membrane vesicle solution in the microfluidic chip is 60-100 mL / h; the injection flow rate of the polylactic acid-glycolic acid copolymer solution in the microfluidic chip is 3-9 mL / h.

[0010] In one embodiment of the present invention, the concentration of the bacterial outer membrane vesicle solution is 0.05-0.2 mg / mL; the concentration of the polylactic acid-glycolic acid copolymer solution is 5-10 mg / mL.

[0011] In one embodiment of the present invention, the frequency of ultrasonic treatment of the microfluidic chip is 60-100 kHz and the power is 80-120W.

[0012] In one embodiment of the present invention, the method for preparing the bacterial outer membrane vesicle solution comprises: inoculating Gram-negative bacteria into a culture medium for culturing to obtain a culture solution; and extracting bacterial outer membrane vesicles from the culture solution to obtain a bacterial outer membrane vesicle solution.

[0013] In one embodiment of the present invention, the preparation method of the bacterial outer membrane vesicle solution includes: inoculating Gram-negative bacteria into a culture medium for culturing to obtain a culture solution; centrifuging the culture solution to obtain a supernatant; filtering the supernatant through a filter membrane, concentrating with an ultrafiltration membrane and collecting the precipitate after ultracentrifugation; resuspending the precipitate in a buffer solution to obtain a resuspension; filtering the resuspension through a filter membrane to obtain a bacterial outer membrane vesicle solution.

[0014] In one embodiment of the present invention, the Gram-negative bacteria include Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa and / or Shigella.

[0015] In one embodiment of the present invention, the preparation method of the polylactic acid-glycolic acid copolymer solution comprises: dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a solution; taking a supernatant after oscillating, ultrasonicating and centrifuging the solution; and diluting the supernatant with an organic solvent to obtain a polylactic acid-glycolic acid copolymer solution.

[0016] In one embodiment of the present invention, the organic solvent is a mixed solution of trifluoroethanol and dimethylamide.

[0017] In one embodiment of the present invention, in the mixed solution, the volume ratio of trifluoroethanol to dimethylamide is 2-4:5-10.

[0018] The present invention also provides a method for preparing the above-mentioned nanoparticles, which comprises: respectively injecting a bacterial outer membrane vesicle solution and a polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip to synthesize the nanoparticles, thereby obtaining the above-mentioned nanoparticles; during the synthesis of the nanoparticles, ultrasonically treating the microfluidic chip so that the bacterial outer membrane vesicles are broken and reassembled on the surface of the polylactic acid-glycolic acid copolymer core.

[0019] In one embodiment of the present invention, the method for preparing the nanoparticles comprises: injecting a polylactic acid-glycolic acid copolymer solution and a bacterial outer membrane vesicle solution into the middle inlet and two side inlets of a microfluidic chip respectively to synthesize the nanoparticles to obtain the nanoparticles.

[0020] In one embodiment of the present invention, the injection flow rate of the bacterial outer membrane vesicle solution in the microfluidic chip is 60-100 mL / h; the injection flow rate of the polylactic acid-glycolic acid copolymer solution in the microfluidic chip is 3-9 mL / h.

[0021] In one embodiment of the present invention, the concentration of the bacterial outer membrane vesicle solution is 0.05-0.2 mg / mL; the concentration of the polylactic acid-glycolic acid copolymer solution is 5-10 mg / mL.

[0022] In one embodiment of the present invention, the frequency of ultrasonic treatment of the microfluidic chip is 60-100 kHz and the power is 80-120W.

[0023] In one embodiment of the present invention, the method for preparing the bacterial outer membrane vesicle solution comprises: inoculating Gram-negative bacteria into a culture medium for culturing to obtain a culture solution; and extracting bacterial outer membrane vesicles from the culture solution to obtain a bacterial outer membrane vesicle solution.

[0024] In one embodiment of the present invention, the preparation method of the bacterial outer membrane vesicle solution includes: inoculating Gram-negative bacteria into a culture medium for culturing to obtain a culture solution; centrifuging the culture solution to obtain a supernatant; filtering the supernatant through a filter membrane, concentrating with an ultrafiltration membrane and collecting the precipitate after ultracentrifugation; resuspending the precipitate in a buffer solution to obtain a resuspension; filtering the resuspension through a filter membrane to obtain a bacterial outer membrane vesicle solution.

[0025] In one embodiment of the present invention, the Gram-negative bacteria include Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa and / or Shigella.

[0026] In one embodiment of the present invention, the preparation method of the polylactic acid-glycolic acid copolymer solution comprises: dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a solution; taking a supernatant after oscillating, ultrasonicating and centrifuging the solution; and diluting the supernatant with an organic solvent to obtain a polylactic acid-glycolic acid copolymer solution.

[0027] In one embodiment of the present invention, the organic solvent is a mixed solution of trifluoroethanol and dimethylamide.

[0028] In one embodiment of the present invention, in the mixed solution, the volume ratio of trifluoroethanol to dimethylamide is 2-4:5-10.

[0029] The present invention also provides the use of the nanoparticles or the method in preparing a drug for preventing and / or treating endometriosis.

[0030] In one embodiment of the present invention, the prevention and / or treatment of endometriosis includes reducing the size of endometriosis lesions, reducing the number of endometriosis lesions, reducing the weight of endometriosis lesions and / or reducing the degree of fibrosis of endometriosis lesions.

[0031] In one embodiment of the present invention, reducing the degree of fibrosis in endometriosis lesions includes regulating the polarization state of macrophages in endometriosis lesions, promoting the transformation of M2 macrophages to M1 macrophages, thereby inhibiting the secretion of pro-fibrotic factor TGF-β in M2 macrophages, and then inhibiting the activation of myofibroblasts, and ultimately inhibiting the fibrosis of endometriosis lesions.

[0032] The present invention also provides a drug for preventing and / or treating endometriosis, characterized in that the drug comprises the above-mentioned nanoparticles.

[0033] In one embodiment of the present invention, the prevention and / or treatment of endometriosis includes reducing the size of endometriosis lesions, reducing the number of endometriosis lesions, reducing the weight of endometriosis lesions and / or reducing the degree of fibrosis of endometriosis lesions.

[0034] In one embodiment of the present invention, reducing the degree of fibrosis in endometriosis lesions includes regulating the polarization state of macrophages in endometriosis lesions, promoting the transformation of M2 macrophages to M1 macrophages, thereby inhibiting the secretion of pro-fibrotic factor TGF-β in M2 macrophages, and then inhibiting the activation of myofibroblasts, and ultimately inhibiting the fibrosis of endometriosis lesions.

[0035] In one embodiment of the present invention, the composition of the drug further comprises a pharmaceutically acceptable excipient.

[0036] In one embodiment of the present invention, the pharmaceutically acceptable excipients include solvents, surfactants, stabilizers and / or hydrogels.

[0037] The technical solution of the present invention has the following advantages:

[0038] 1. The present invention provides an OMV-PLGA nanoparticle, wherein the OMV-PLGA nanoparticle comprises bacterial outer membrane vesicles (OMV) and polylactic acid-co-glycolic acid (PLGA); the bacterial outer membrane vesicles are wrapped on the surface of the polylactic acid-co-glycolic acid. The present invention wraps OMV on the surface of the PLGA core, retains its biological characteristics, and imitates bacteria to present antigens to the immune system. At the same time, the PLGA core provides a series of adjustable physicochemical properties (such as particle size and formation), making the size of the synthesized OMV-PLGA more uniform. In addition, PLGA provides a hard core for the OMV membrane, so that OMV-PLGA can pass through the cell membrane more effectively than OMV, thereby improving the cell uptake efficiency (cell experiments show that the uptake efficiency of THP-1 cells for OMV-PLGA nanoparticles is about 4 times that of natural OMVs, indicating that wrapping bacterial outer membrane vesicles on the surface of polylactic acid-co-glycolic acid can significantly enhance its ability to be taken up by macrophages, thereby improving its bioavailability and efficacy).

[0039] 2. The present invention provides the use of OMV-PLGA nanoparticles in the preparation of a drug for preventing and / or treating endometriosis, wherein the OMV-PLGA nanoparticles include bacterial outer membrane vesicles (OMV) and polylactic acid-glycolic acid copolymer (PLGA); the bacterial outer membrane vesicles are wrapped on the surface of the polylactic acid-glycolic acid copolymer. Animal experiments show that OMV-PLGA nanoparticles can reduce the size of ectopic lesions of endometriosis, reduce the number of lesions of endometriosis, reduce the weight of lesions of endometriosis, and reduce the degree of fibrosis of endometriosis lesions (here, reducing the degree of fibrosis of endometriosis lesions includes regulating the polarization state of macrophages in endometriosis lesions, promoting the transformation of M2 macrophages to M1 macrophages, thereby inhibiting the secretion of profibrotic factor TGF-β in M2 macrophages, thereby inhibiting the activation of myofibroblasts, and finally inhibiting the fibrosis of endometriosis lesions). The present invention applies OMVs to the prevention and treatment of endometriosis, breaking away from the limitations of traditional hormone therapy, demonstrating the potential of macrophage polarization regulation in non-hormonal therapy, and providing a new idea for non-estrogen pathway treatment. In addition, the present invention encapsulates OMVs on the surface of PLGA for the prevention and treatment of endometriosis, further improving its prevention and treatment effect.

[0040] 3. The present invention also provides a method for preparing the above-mentioned OMV-PLGA nanoparticles, the method comprising: injecting a bacterial outer membrane vesicle solution and a polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip respectively to synthesize nanoparticles to obtain OMV-PLGA nanoparticles; in the synthesis process of the OMV-PLGA nanoparticles, the microfluidic chip is ultrasonically treated to break the bacterial outer membrane vesicles and reassemble them on the surface of the polylactic acid-glycolic acid copolymer core; the OMV-PLGA nanoparticles include bacterial outer membrane vesicles (OMV) and polylactic acid-glycolic acid copolymer (PLGA); the bacterial outer membrane vesicles are wrapped on the surface of the polylactic acid-glycolic acid copolymer. In terms of particle preparation, the present invention uses microfluidic chip synthesis technology to complete the preparation of OMV-PLGA nanoparticles with good uniformity and high repeatability. At present, the methods for preparing biomimetic membrane-polymer nanoparticles mainly include extrusion method and ultrasonic method. Both preparation methods have relatively complex synthesis processes, and there are problems such as poor uniformity of synthetic particles and low biomimetic membrane wrapping rate. The extrusion method is time-consuming, labor-intensive and poorly controllable, and the ultrasound method cannot completely coat the biomimetic membrane on the surface of polymer particles, resulting in a low synthesis rate of particles. Microfluidic synthesis technology has the advantages of precise controllable reaction conditions, rapid heat and mass transfer, and the integration of multi-step reactions, providing a revolutionary platform for the precise assembly of functional materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Schematic diagram of the synthesis of OMV-PLGA nanoparticles.

[0042] Figure 2 : Effect of flow rate of OMV solution on synthesis of OMV-PLGA nanoparticles.

[0043] Figure 3 : Effect of OMV solution concentration on the synthesis of OMV-PLGA nanoparticles.

[0044] Figure 4 : DLS analysis results of OMV-PLGA nanoparticles. Figure 4 In the figure, A: particle size of OMV-PLGA nanoparticles; B: potential of OMV-PLGA nanoparticles.

[0045] Figure 5 : Electron microscopy structure of OMV-PLGA nanoparticles (Scale bar = 200 nm).

[0046] Figure 6 : Analysis results of THP-1 cells' uptake of OMVs and OMV-PLGA nanoparticles. Figure 6 In the figure, A: representative flow cytometric graph of DID-labeled nanoparticles taken up by THP-1 cells; B: mean fluorescence intensity after THP-1 cells took up particles.

[0047] Figure 7 : Effects of OMV-PLGA nanoparticles in an injection model of endometriosis. Figure 7 A: Representative image of successful establishment of endometriosis injection model; B: Representative image of endometriosis lesions after treatment with PBS, OMVs and OMV-NPs; C: Number of endometriosis lesions after treatment with PBS, OMVs and OMV-NPs; D: Weight of endometriosis lesions after treatment with PBS, OMVs and OMV-NPs.

[0048] Figure 8 : Staining results of endometriosis lesions after PBS, OMVs and OMV-NPs treatment. Figure 8 In the figure, A: Representative images of H&E and Masson staining of lesions obtained 21 days after the establishment of the endometriosis mouse model (Scale bar = 200 μm); B: Statistics of the degree of fibrosis of lesions obtained 21 days after the establishment of the endometriosis mouse model.

[0049] Fig. 9 : IHC analysis results of endometriosis lesions after PBS, OMVs and OMV-NPs treatment. Fig. 9A: Representative images of IHC staining of lesions obtained 21 days after the establishment of the endometriosis mouse model (Scale bar = 20 μm); B: The number of CD86-positive (M1 type) and CD163-positive (M2 type) macrophages in the lesions obtained 21 days after the establishment of the endometriosis mouse model, as well as the IHC scores of TGF-β and α-SMA.

[0050] Fig.10 : Effects of OMV-NPs treatment on body weight of mice.

[0051] Fig.11 : Effects of OMV-NPs treatment on the levels of inflammatory factors in mouse serum. Fig.11 In the figure, A: Effect of OMV-NPs treatment on IL-6 level in mouse serum; B: Effect of OMV-NPs treatment on TNF-α level in mouse serum.

[0052] Fig.12 :Effects of OMV-NPs treatment on routine blood parameters of mice. Fig.12 A: Effect of OMV-NPs treatment on the level of white blood cells (WBC) in the whole blood of mice; B: Effect of OMV-NPs treatment on the level of neutrophils in the whole blood of mice; C: Effect of OMV-NPs treatment on the level of lymphocytes in the whole blood of mice; D: Effect of OMV-NPs treatment on the level of platelets in the whole blood of mice.

[0053] Fig.13 : H&E staining results of mouse organs treated with OMV-NPs (Scalebar=50μm).

[0054] Fig.14 :Effects of OMV-NPs treatment on serum biochemical parameters in mice. Fig.14 A: Effect of OMV-NPs treatment on alanine aminotransferase (ALT) level in mouse serum; B: Effect of OMV-NPs treatment on creatinine (CREA) level in mouse serum; C: Effect of OMV-NPs treatment on lactate dehydrogenase (LDH) level in mouse serum; D: Effect of OMV-NPs treatment on aspartate aminotransferase (AST) level in mouse serum; E: Effect of OMV-NPs treatment on blood urea nitrogen (BUN) level in mouse serum.

[0055] Fig.15 : Effects of OMV-NPs treatment on mouse endometrium. Fig.15In the figure, A: H&E staining results of mouse endometrium treated with OMV-NPs (Scale bar = 50 μm); B: Effect of OMV-NPs treatment on the thickness of mouse endometrium.

[0056] Fig.16 :Effects of OMV-NPs treatment on serum reproductive hormone indexes in mice. Fig.16 A: Effect of OMV-NPs treatment on the level of anti-Mullerian hormone (AMH) in mouse serum; B: Effect of OMV-NPs treatment on the level of follicle-stimulating hormone (FSH) in mouse serum; C: Effect of OMV-NPs treatment on the level of luteinizing hormone (LH) in mouse serum. DETAILED DESCRIPTION

[0057] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0058] If no specific experimental steps or conditions are specified in the following experimental examples, the conventional experimental steps or conditions described in the literature in this field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0059] Example 1: OMV-PLGA nanoparticles and preparation method thereof

[0060] This embodiment provides an OMV-PLGA nanoparticle, wherein the OMV-PLGA nanoparticle comprises bacterial outer membrane vesicles (OMV) and polylactic acid-co-glycolic acid (PLGA), wherein the OMV is wrapped on the surface of the PLGA. The preparation method of the OMV-PLGA nanoparticle is as follows:

[0061] Preparation of OMV solution: Dip the bacterial solution of Escherichia coli DH5α (purchased from Novozyme Biotech Co., Ltd.) on an LB plate (purchased from Solebold Technology Co., Ltd.), and incubate it upside down at 37°C for 16 hours. After the incubation, pick a single clone colony and inoculate it into 5 mL LB liquid culture medium (purchased from Solebold Technology Co., Ltd.), and shake and culture it at 180 rpm in a 37°C constant temperature incubator for about 8 hours to obtain seed solution; take 50 μL of seed solution and inoculate it into 500 mL In LB liquid medium, shake and culture at 180 rpm in a 37°C constant temperature incubator for 12 hours until the OD value reaches 1.5 to obtain a bacterial solution; the bacterial solution is centrifuged at 4°C and 8,000g for 15 minutes and the supernatant is taken; the supernatant is first filtered through a 0.45μm filter membrane, and then concentrated using a 100kDa ultrafiltration membrane at a ratio of 1:3 (after concentration: before concentration = 1:3) to obtain a concentrated solution; the concentrated solution is first filtered through a 0.22μm filter membrane, and then ultracentrifuged at 4°C and 150,000g for 3 hours to collect the precipitate; the precipitate is resuspended in 1mL PBS buffer (purchased from Sigma) to obtain a resuspension; the resuspension is filtered through a 0.22μm filter membrane to obtain an OMV solution. The protein concentration of the OMV solution is determined by the BCA method (the measured protein concentration is 0.5mg / mL), and the solution is stored at -80°C after aliquoting.

[0062] Preparation of PLGA solution: Polylactic acid-co-glycolic acid (PLGA, purchased from Sigma) was dissolved in a mixed solution of trifluoroethanol and dimethylamide (trifluoroethanol: dimethylamide = 3:7, volume ratio) to prepare a PLGA solution with a concentration of 10 mg / mL; the PLGA solution was first oscillated at 37°C and 3000 rpm for 16 hours, then ultrasonically treated at a frequency of 80 kHz and a power of 100 W for 15 minutes, and then centrifuged at 12000 g for 10 minutes to obtain the supernatant; the supernatant was diluted to a concentration of 5 mg / mL using a mixed solution of trifluoroethanol and dimethylamide (trifluoroethanol: dimethylamide = 3:7, volume ratio) to obtain a PLGA solution.

[0063] Preparation of OMV-PLGA nanoparticles: PLGA solution and OMV solution were injected into the middle inlet (flow rate 7 mL / h) and the two side inlets (flow rate 80 mL / h) of the microfluidic chip (for the microfluidic chip, see the patent application with publication number CN110560186A) to synthesize particles and obtain OMV-PLGA nanoparticles (OMV-NPs); during the particle synthesis process, the microfluidic chip was placed in a water bath ultrasonic device (purchased from Kunshan Ultrasonic Co., Ltd., China, model KQ-200TDE) and acoustic wave stress was generated on the microfluidic chip at a frequency of 80 kHz and a power of 100 W to break the OMV membrane and reassemble it on the surface of the PLGA core in a short time (for the particle synthesis process, see Figure 1 ).

[0064] Experimental Example 1: Effect of the ratio of OMVs to PLGA on the synthesis of OMV-PLGA nanoparticles

[0065] This experimental example explored the ratio of OMVs to PLGA during the synthesis of OMV-PLGA nanoparticles. The experimental process is as follows:

[0066] On the basis of Example 1, the flow rates of the OMV solution injected from the inlets on both sides were set to 60 mL / h, 80 mL / h, and 100 mL / h, respectively, to obtain OMV-PLGA nanoparticles (OMV-NPs).

[0067] Based on Example 1, the OMV solution was diluted with PBS buffer to a protein concentration of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively, to obtain OMV-PLGA nanoparticles (OMV-NPs).

[0068] The particle size of the prepared OMV-PLGA nanoparticles was detected by dynamic light scattering (DLS) technology and the distribution of the measured particle size was evaluated to obtain the average particle size and PDI of the OMV-PLGA nanoparticles (PDI is a dimensionless value reflecting the width of the particle size distribution, ranging from 0 to 1. The smaller the value, the more uniform the particle size and the more concentrated the particle size distribution). The experimental results are shown in Figure 2-3 OMV-PLGA nanoparticles prepared at an OMV solution flow rate of 80 mL / h and a concentration of 0.1 mg / mL were selected, and PLGA and OMV were used as controls. The particle size and potential of OMV-PLGA nanoparticles were detected by dynamic light scattering (DLS), and the structure of OMV-PLGA nanoparticles was observed using a transmission electron microscope (TEM). The experimental results are shown in Figure 4-5 .

[0069] Depend on Figure 2-3 The results show that when the flow rate is 80 mL / h and the concentration is 0.1 mg / mL, the OMV-PLGA nanoparticles with the highest particle size uniformity can be obtained.

[0070] Figure 4 The particle size analysis results showed that naked PLGA easily aggregated in PBS buffer to form large particle precipitation. In contrast, the size of OMVs themselves was relatively small, ranging from about 30 to 50 nm, while the particle size of OMV-PLGA nanoparticles synthesized by microfluidic chip technology was significantly increased, averaging about 180 nm. This result indicates that OMVs were successfully encapsulated on the surface of PLGA particles.

[0071] Figure 4The results of potential analysis showed that the surface potential of PLGA particles was about -20 mV, while that of OMVs and OMV-PLGA nanoparticles was about -10 mV. This significant potential difference further confirmed that OMVs were successfully encapsulated on the surface of PLGA particles.

[0072] Depend on Figure 5 The results show that OMVs themselves present a vesicle structure of 30 to 50 nm, and there is a large difference in particle size distribution. In contrast, OMV-PLGA nanoparticles show a clear core-shell structure and the particles have good uniformity. This structural feature further confirms that the OMVs membrane is successfully wrapped on the surface of PLGA particles to form a stable core-shell structure.

[0073] Experimental Example 2: Verification of the uptake efficiency of OMV-PLGA nanoparticles

[0074] This experimental example verifies the uptake efficiency of OMV-PLGA nanoparticles. The experimental process is as follows:

[0075] OMV-PLGA nanoparticles prepared at an OMV solution flow rate of 80 mL / h and a concentration of 0.1 mg / mL were selected, and fluorescent dye DiD (purchased from Thermofisher) was added to the OMV solution and OMV-PLGA nanoparticles at an addition amount of 4 μM to obtain a mixed solution; the mixed solution was shaken and incubated at 37°C and 1500 rpm for 15 minutes for fluorescent labeling to obtain a reaction solution; the reaction solution was transferred to a 100 kD dialysis bag, and dialyzed in 2 L PBS buffer at 4°C for 16 hours to remove unbound DiD dye, to obtain a fluorescently labeled OMV solution and a fluorescently labeled OMV-PLGA nanoparticle solution.

[0076] THP-1 cells (purchased from the Chinese Academy of Sciences Cell Bank) were cultured at 5×10^ 5 After the cells were inoculated at a density of 100 cells / mL into a 12-well plate with 1 mL of RMPI medium (purchased from Sigma) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (purchased from Sigma) added to each well, 100 ng / mL of phorbol ester (PMA, purchased from Sigma) was added to the culture system and the cells were cultured in 5% (v / v) CO 2 The cells were incubated in a 37°C cell culture incubator for 48 hours to induce THP-1 cells to differentiate into macrophages. After the incubation, an equal amount of PBS buffer was used as a blank control, and the fluorescently labeled OMV solution and the fluorescently labeled OMV-PLGA nanoparticle solution were added to the culture system at an addition amount of 5 μg / mL, respectively, and incubated in a 5% (v / v) CO 2, incubated in a 37°C cell culture incubator for 4 hours; after the incubation, flow cytometry was used to quantitatively analyze the particles taken up by the cells. The experimental results are shown in Figure 6 .

[0077] Depend on Figure 6 The results showed that the uptake efficiency of OMV-PLGA nanoparticles by THP-1 cells was about 4 times that of natural OMVs. This finding suggests that by improving the design of OMV-PLGA nanoparticles, their ability to be taken up by macrophages was successfully enhanced, which may improve their bioavailability and efficacy.

[0078] Experimental Example 3: Verification of the therapeutic effect of OMV-PLGA nanoparticles on endometriosis

[0079] This experimental example verifies the therapeutic effect of OMV-PLGA nanoparticles on endometriosis. The experimental process is as follows:

[0080] Twenty-four BALB / c mice (purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd.) were divided into three groups: PBS control group, OMVs intervention group, and OMV-PLGA intervention group, with 8 mice in each group. After grouping, OMV-PLGA nanoparticles prepared at an OMV solution flow rate of 80 mL / h and a concentration of 0.1 mg / mL were selected, and 50 μg / kg of OMVs (solvent was 200 μL PBS buffer) were intraperitoneally injected into the mice in the OMVs intervention group, and 50 μg / kg of OMV-PLGA nanoparticles (solvent was 200 μL PBS buffer) were intraperitoneally injected into the mice in the OMV-PLGA intervention group. The same amount of PBS buffer was injected into the mice in the PBS control group as a blank control. One week after the injection, endometriosis models were established in the three groups to observe the therapeutic effect of OMV-PLGA nanoparticles on endometriosis. The experimental results are shown in Figure 7 ;

[0081] The modeling process of endometriosis is as follows: on the day of endometriosis modeling, the donor mice were euthanized and then immersed in 75% (v / v) alcohol solution for disinfection for 5 minutes; after disinfection, the Y-shaped uterine horns were separated and removed, and washed in sterile PBS buffer at 37°C; after washing, the uterine horns were cut into 1×1 mm 2 The fragments of 100 μg were then injected into the abdominal cavity of the recipient mice using a 1 ml syringe (the solvent was 200 μL PBS buffer). Each recipient mouse received 10 pieces of endometrial tissue fragments. After the injection, the recipient mice continued to receive subcutaneous injections of estradiol every other day, once a day, with a dose of 2 μg each time (the solvent was 200 μL PBS buffer) until 21 days after modeling.

[0082] In order to verify the effect of OMV-PLGA nanoparticles on fibrosis at the histological level, endometriosis lesions were obtained for H&E staining and Masson staining on day 21 after the establishment of the endometriosis model. The experimental results are shown in Figure 8 .

[0083] To further explore the effect of OMV-NPs on the polarization of macrophages in endometriosis lesions, on day 21 after the establishment of the endometriosis model, endometriosis lesions were harvested and IHC analysis was performed to evaluate the polarization state of macrophages. The experimental results are shown in Fig. 9 .

[0084] Depend on Figure 7 The results showed that the size of ectopic lesions in the OMV-PLGA nanoparticle-treated group was significantly reduced compared with the PBS group (the size of ectopic lesions was reduced from 59.72±14.01mm 3 Reduced to 36.28±9.31mm 3 , reduced by 40%), the number and weight of lesions were also significantly reduced (the average number of lesions decreased from 4 to 2, a decrease of 50%, and the weight of lesions decreased from 77.65±11.2mg to 9.19±1.49mg, a decrease of 88%); although OMVs treatment can also have a certain effect, the effect is far inferior to that of OMV-PLGA nanoparticles.

[0085] Figure 8 The H&E staining results in the images showed that the endometrial line of the ectopic lesions was clear and complete, indicating that the model was successfully constructed. Figure 8 The Masson staining results further showed that compared with the PBS group, OMV-PLGA nanoparticles treatment significantly reduced the fibrosis area (the fibrosis area decreased from 58.93±5.46% to 23.06±2.04%, a decrease of 61%), and this difference was statistically significant, indicating that OMV-PLGA nanoparticles can effectively reduce the degree of fibrosis of lesions; although OMVs treatment can also have a certain effect, the effect is far inferior to OMV-PLGA nanoparticles.

[0086] Fig. 9The results of IHC analysis showed that compared with the PBS control group, the number of M1 macrophages (CD86+) in endometriosis lesions treated with OMV-PLGA nanoparticles increased significantly (M1 macrophages increased from 5.67±1.05% to 43.50±2.66%, an increase of 6.7 times), while the number of M2 macrophages (CD163+) decreased accordingly (M2 macrophages decreased from 45.83±2.55% to 8.50±1.23%, a decrease of 81%). Further analysis showed that after OMV-PLGA nanoparticle treatment, the expression level of the profibrotic factor TGF-β secreted by M2 macrophages in endometriosis lesions was significantly decreased (the staining intensity score of TGF-β decreased from 34.61±2.20 to 5.93±0.82, a decrease of 83%). This finding suggests that OMV-PLGA nanoparticles may affect the secretion of TGF-β by regulating the polarization state of macrophages. In addition, it was observed that the content of α-SMA-positive myofibroblasts in the endometriosis lesions of mice in the OMV-PLGA intervention group was significantly decreased (the staining intensity score of α-SMA-positive myofibroblasts decreased from 67.95±4.06 to 16.61±5.06, a decrease of 76%). This suggests that OMV-PLGA nanoparticles may promote the differentiation of macrophages from M2 to M1 in endometriosis lesions, thereby inhibiting the secretion of TGF-β, and ultimately achieving the effect of inhibiting the activation of myofibroblasts.

[0087] Experimental Example 4: Biosafety Assessment Experiment of OMV-PLGA Nanoparticles

[0088] This experimental example verifies the biosafety assessment of OMV-PLGA nanoparticles. The experimental process is as follows:

[0089] A biosafety assessment was conducted based on Experimental Example 3. The specific assessment process was as follows:

[0090] The weight changes of mice were monitored within 7 days after the administration of OMV-PLGA nanoparticles. In addition, considering that the membrane surface substances of OMVs (such as LPS) may induce acute systemic inflammatory response and cytokine storm at high doses, the serum of mice was collected on the day of OMV-PLGA nanoparticle administration and on days 1, 3, 5, and 7 after administration, and the whole blood of mice was collected on day 1 after OMV-PLGA nanoparticle administration to evaluate the levels of systemic inflammatory factors. The experimental results are shown in Figure 10 to Figure 12 .

[0091] Seven days after OMV-PLGA nanoparticle injection, the heart, liver, spleen, lung, kidney and serum samples of mice were collected, the organ sections were observed by H&E staining, and the serum was subjected to biochemical testing. The experimental results are shown in Figure 13-14 .

[0092] Fig.10 The results showed that the body weight of both the OMV-PLGA nanoparticle-treated group and the PBS-treated group showed a steady increase, and no significant difference was observed between the two groups.

[0093] Fig.11 The results showed that compared with the PBS-treated group, the IL-6 and TNF-α levels in the OMV-PLGA nanoparticle-treated group were significantly increased on the first day after administration, which was about 4 times that of the control group. However, the levels of these inflammatory factors gradually decreased and returned to normal levels on the 7th day. Since the OMV-PLGA nanoparticle-treated group showed the most significant inflammatory response on the first day after administration, a blood routine test was performed on the peripheral blood at this time point. The experimental results are shown in Fig.12 .

[0094] Fig.12 The results showed that the levels of white blood cells, neutrophils, lymphocytes and platelets in the OMV-PLGA nanoparticle-treated group were comparable to those in the PBS-treated group, suggesting that the OMV-PLGA nanoparticles only caused a controllable degree of inflammatory response and did not cause significant damage to the mouse body.

[0095] according to Fig.13 The H&E staining results showed that no obvious pathological changes were found in the organs of the mice.

[0096] Fig.14 The biochemical test results showed that there were no significant changes in the liver function, kidney function and heart function indicators such as blood urea nitrogen (BUN), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) of mice.

[0097] comprehensive Figure 10 to Figure 14 The results show that the in vivo application of OMV-PLGA nanoparticles is relatively safe.

[0098] Experimental Example 5: Effect of OMV-PLGA Nanoparticles on the Reproductive System

[0099] This experimental example investigated the effect of OMV-PLGA nanoparticles on the reproductive system. The experimental process is as follows:

[0100] Based on Experimental Example 3, the effect on the reproductive system was investigated. The specific investigation process was as follows:

[0101] Considering the possible interaction between immune cells and estrogen pathway, uterus and serum samples of mice were collected 7 days after OMV-PLGA nanoparticle injection, organ sections were observed by H&E staining, and reproductive hormones were detected in serum. The experimental results are shown in Figure 15-16 .

[0102] according to Fig.15 The H&E staining results showed that the structure and thickness of the mouse endometrium did not change significantly after the injection of OMV-PLGA nanoparticles.

[0103] Fig.16 The results of reproductive hormone tests showed that there was no significant change in the levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in mice.

[0104] comprehensive Figure 15-16 The results showed that OMV-PLGA nanoparticles had no effect on the ovarian reserve function and hypothalamus-pituitary-ovary (HPO) axis of mice.

[0105] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A nanoparticle, characterized in that: The nanoparticles include bacterial outer membrane vesicles and polylactic acid-glycolic acid copolymer; the bacterial outer membrane vesicles are wrapped on the surface of the polylactic acid-glycolic acid copolymer.

2. The nanoparticle according to claim 1, characterized in that The preparation method of the nanoparticles comprises: respectively injecting a bacterial outer membrane vesicle solution and a polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip to synthesize the nanoparticles to obtain the nanoparticles; during the synthesis of the nanoparticles, ultrasonically treating the microfluidic chip to break up the bacterial outer membrane vesicles and reassemble them on the surface of the polylactic acid-glycolic acid copolymer core.

3. The nanoparticle according to claim 2, characterized in that The injection flow rate of the bacterial outer membrane vesicle solution in the microfluidic chip is 60-100 mL / h; the injection flow rate of the polylactic acid-glycolic acid copolymer solution in the microfluidic chip is 3-9 mL / h.

4. The nanoparticles according to claim 2 or 3, characterized in that The concentration of the bacterial outer membrane vesicle solution is 0.05-0.2 mg / mL; the concentration of the polylactic acid-glycolic acid copolymer solution is 5-10 mg / mL.

5. The nanoparticle according to any one of claims 2 to 4, characterized in that The frequency of ultrasonic treatment on the microfluidic chip is 60-100kHz and the power is 80-120W.

6. The nanoparticle according to any one of claims 2 to 5, characterized in that The preparation method of the bacterial outer membrane vesicle solution comprises: inoculating Gram-negative bacteria into a culture medium for culturing to obtain a culture solution; and extracting bacterial outer membrane vesicles from the culture solution to obtain a bacterial outer membrane vesicle solution.

7. The nanoparticle according to any one of claims 2 to 6, characterized in that The preparation method of the polylactic acid-glycolic acid copolymer solution comprises: dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a dissolving solution; taking a supernatant after vibrating, ultrasonically treating and centrifuging the dissolving solution; and diluting the supernatant with a buffer solution to obtain a polylactic acid-glycolic acid copolymer solution.

8. A method for preparing the nanoparticles according to any one of claims 1 to 7, characterized in that: The method comprises: respectively injecting a bacterial outer membrane vesicle solution and a polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip to synthesize nanoparticles, thereby obtaining the nanoparticles according to any one of claims 1 to 7; and during the synthesis of the nanoparticles, ultrasonically treating the microfluidic chip to break up the bacterial outer membrane vesicles and reassemble them on the surface of the polylactic acid-glycolic acid copolymer core.

9. Use of the nanoparticles according to any one of claims 1 to 7 or the method according to claim 8 in the preparation of a drug for preventing and / or treating endometriosis.

10. A drug for preventing and / or treating endometriosis, characterized in that: The drug comprises the nanoparticles according to any one of claims 1 to 7.

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