A nanoparticle for preventing endometriosis and a method of preparing the same

Abstract

The present application relates to a kind of nanoparticles for preventing endometrial heterotopia and preparation method thereof, belong to biological medicine technical field.The present application provides a kind of nanoparticles, this nanoparticle includes bacterial outer membrane vesicle (OMV) and polylactic acid-glycolic acid copolymer (PLGA), wherein, bacterial outer membrane vesicle is wrapped in polylactic acid-glycolic acid copolymer surface.The present application will be wrapped in the surface of PLGA core OMV, retains its biological characteristics, imitates bacteria and presents antigen to immune system.Simultaneously, PLGA core provides a series of controllable physical and chemical properties (such as particle size and formation), so that the size of synthesized OMV-PLGA is more uniform.In addition, PLGA provides a hard core for OMV membrane, so that OMV-PLGA can more effectively pass through cell membrane than OMV, to improve the uptake efficiency of cell.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of nanoparticles for preventing endometriosis and its preparation method, belong to biomedicine technical field. BACKGROUND

[0002] Endometriosis (endometriosis) refers to the endometrial tissue (glands and stroma) in the uterine cavity covered endometrium and other parts of the uterus appears, growth, infiltration, repeated bleeding, and then cause pain, infertility and nodules or mass, etc., seriously affect the quality of life and fertility of women of childbearing age. Endometriosis in the global incidence of women of childbearing age is about 10%, and shows an upward trend. Endometriosis treatment includes surgical treatment and drug treatment, conservative surgery is prone to recurrence, so postoperative drug treatment is needed. 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 to reduce estrogen levels has the side effects of reducing bone density and delaying patient fertility, limiting its application. Therefore, seeking new non-estrogen-dependent prevention and treatment drugs has important clinical significance for endometriosis.

[0003] Endometriosis is a complex chronic inflammatory disease, and the pathogenesis is complex. In addition to the retrograde menstruation theory, 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 the number and functional changes of various immune cells, such as reduced phagocytic ability of macrophages, reduced cytotoxicity of T cells, polyclonal activation of B cells, etc. Among them, macrophages are considered to play a key role in the formation of pelvic chronic inflammation and the growth of ectopic endometrium. As an important part of the immune system, macrophages not only participate in tissue repair and regulation of inflammatory response, but also can remove 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 it is the most significant immune cell type, which participates in the regulation of inflammatory response and angiogenesis of ectopic endometrial tissue. In addition to the number change, the macrophages in the body of patients with endometriosis also have functional disorders, and their reduced phagocytic ability leads to the ineffective removal of endometrial tissue flowing with menstrual blood, which promotes its implantation and growth in ectopic sites. In addition, macrophages can also increase the secretion of various pro-inflammatory factors such as insulin-like growth factor 1 (IGF-1), leading to the growth of endometrial ectopic tissue and the exacerbation of pain symptoms. Using low-dose lipopolysaccharide (LPS) to induce macrophages to produce innate immune memory can weaken the fibrous inflammatory characteristics of ectopic lesions in an IL-10-dependent manner, effectively inhibit the growth of lesions, suggesting that targeting macrophages is a new direction for the treatment of endometriosis.

[0004] The polarization state of macrophages has a significant impact on the progression of endometriosis. Under physiological conditions, macrophages can form two polarized states: M1 type of classical activation and M2 type of alternative activation, according to different signals in the local microenvironment. M1 type of macrophages are mainly involved in inflammatory response and pathogen clearance, while M2 type of macrophages are associated with tissue repair, healing and fibrosis process. Previous studies have found that the M2 / M1 ratio in the ectopic lesions and peritoneal fluid of endometriosis patients is significantly increased. In the pathological environment of endometriosis, the activation and increase of M2 type of macrophages are considered to be related to the fibrosis of the lesion tissue. Fibrosis is a pathological process in which abnormal tissue proliferation and scar formation lead to damage to the structure and function of organs. 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 type of macrophages promote the activation of myofibroblasts and the deposition of collagen by secreting a series of pro-fibrotic factors, such as transforming growth factor-β, thus leading to the fibrosis phenomenon in endometriosis. Therefore, the imbalance of M1 type and M2 type of macrophages in the polarization process of macrophages may play a promoting role in the pathogenesis of endometriosis, especially in the fibrosis process of the lesion tissue. Treatment targeting the polarization of macrophages may reduce the degree of fibrosis in endometriosis.

[0005] Bacterial outer membrane vesicles (OMVs) are natural nanoscale vesicles released by Gram-negative bacteria during growth, with a diameter of about 20-250 nm. These vesicles contain various bacterial components, such as lipopolysaccharide (LPS), proteins, enzymes, DNA, and RNA. Due to the inclusion of pathogen-associated molecular patterns (PAMPs), bacterial outer membrane vesicles have immunoadjuvant function, which can activate multiple Toll-like receptor (TLR) signaling pathways in the host, triggering immune response, and thus are widely used in immunotherapy. Studies have shown that bacterial outer membrane vesicles can interact with macrophages, promoting their polarization to M1 type, producing pro-inflammatory cytokines, and enhancing immune response. In terms of application, bacterial outer membrane vesicles have been widely studied and applied in the development of tumor vaccines. They can act as nanocarriers to effectively deliver tumor antigens to the host immune system, thereby activating specific immune response 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, so far, there is no research on bacterial outer membrane vesicles in the field of endometriosis. SUMMARY

[0006] To solve the above problems, the present application provides a kind of nanoparticle, the nanoparticle includes bacterial outer membrane vesicles and polylactic acid-glycolic acid copolymer;The bacterial outer membrane vesicles are wrapped in the surface of polylactic acid-glycolic acid copolymer.

[0007] In one embodiment of the present application, the preparation method of 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 respectively to synthesize the nanoparticles, so as to obtain the nanoparticles; and performing ultrasonic treatment on the microfluidic chip during the synthesis of the nanoparticles to break 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 application, the preparation method of the nanoparticles comprises: injecting a polylactic acid-glycolic acid copolymer solution and a bacterial outer membrane vesicle solution into a middle inlet and two side inlets of a microfluidic chip respectively to synthesize the nanoparticles, so as to obtain the nanoparticles.

[0009] In one embodiment of the present application, the injection flow rate of the bacterial outer membrane vesicle solution in the microfluidic chip is 60-100 mL / h; and 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 application, the concentration of the bacterial outer membrane vesicle solution is 0.05-0.2 mg / mL; and the concentration of the polylactic acid-glycolic acid copolymer solution is 5-10 mg / mL.

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

[0012] In one embodiment of the present application, the preparation method of the bacterial outer membrane vesicle solution comprises: inoculating a gram-negative bacterium into a culture medium to culture, so as to obtain a culture solution; and extracting bacterial outer membrane vesicles from the culture solution, so as to obtain the bacterial outer membrane vesicle solution.

[0013] In one embodiment of the present application, the preparation method of the bacterial outer membrane vesicle solution comprises: inoculating a gram-negative bacterium into a culture medium to culture, so as to obtain a culture solution; centrifuging the culture solution to obtain a supernatant; filtering the supernatant through a filter membrane, concentrating the supernatant through an ultrafiltration membrane, and collecting a precipitate after ultracentrifugation; resuspending the precipitate in a buffer solution to obtain a resuspension; and filtering the resuspension through a filter membrane, so as to obtain the bacterial outer membrane vesicle solution.

[0014] In one embodiment of the present application, the gram-negative bacterium comprises Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa, and / or Shigella.

[0015] In an embodiment of the present application, the preparation method of the polylactic acid-glycolic acid copolymer solution comprises: dissolving polylactic acid-glycolic acid copolymer in an organic solvent to obtain a dissolving solution; taking supernatant after oscillation, ultrasonic treatment and centrifugation of the dissolving solution; diluting the supernatant with an organic solvent to obtain the polylactic acid-glycolic acid copolymer solution.

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

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

[0018] The present application also provides a method for preparing the nanoparticles, which comprises: injecting the bacterial outer membrane vesicle solution and the polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip respectively to synthesize the nanoparticles, to obtain the nanoparticles; and performing ultrasonic treatment on the microfluidic chip during the synthesis of the nanoparticles to break the bacterial outer membrane vesicles and reassemble them on the surface of the polylactic acid-glycolic acid copolymer core.

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

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

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

[0022] In an embodiment of the present application, the ultrasonic treatment on the microfluidic chip is performed at a frequency of 60-100 kHz and a power of 80-120 W.

[0023] In an embodiment of the present application, the preparation method of the bacterial outer membrane vesicle solution comprises: inoculating gram-negative bacteria into a culture medium to culture, to obtain a culture solution; and extracting bacterial outer membrane vesicles from the culture solution to obtain the bacterial outer membrane vesicle solution.

[0024] In an embodiment of the present application, the method for preparing the bacterial outer membrane vesicle solution comprises inoculating gram-negative bacteria into a culture medium for culture to obtain a culture solution; centrifuging the culture solution to obtain a supernatant; filtering the supernatant through a filter membrane, concentrating the supernatant through an ultrafiltration membrane, and collecting a precipitate after ultracentrifugation; resuspending the precipitate in a buffer to obtain a resuspension; and filtering the resuspension through a filter membrane to obtain the bacterial outer membrane vesicle solution.

[0025] In an embodiment of the present application, the gram-negative bacteria comprise Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa, and / or Shigella.

[0026] In an embodiment of the present application, the method for preparing the polylactic acid-glycolic acid copolymer solution comprises dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a dissolution solution; treating the dissolution solution by oscillation, ultrasonic treatment, and centrifugation to obtain a supernatant; and diluting the supernatant with an organic solvent to obtain the polylactic acid-glycolic acid copolymer solution.

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

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

[0029] The present application also provides the use of the nanoparticles or the method described above in the preparation of a medicament for preventing and / or treating endometriosis.

[0030] In an embodiment of the present application, the prevention and / or treatment of endometriosis comprises reducing the size of ectopic lesions of endometriosis, reducing the number of lesions of endometriosis, reducing the weight of lesions of endometriosis, and / or reducing the degree of fibrosis of lesions of endometriosis.

[0031] In an embodiment of the present application, the reduction of the degree of fibrosis of lesions of endometriosis comprises promoting the transformation of M2-type macrophages into M1-type macrophages by adjusting the polarization state of macrophages in lesions of endometriosis, thereby inhibiting the secretion of the fibrosis-promoting factor TGF-β in M2-type macrophages, further inhibiting the activation of myofibroblasts, and finally inhibiting the fibrosis of lesions of endometriosis.

[0032] The present application also provides a medicament for preventing and / or treating endometriosis, characterized in that the components of the medicament comprise the nanoparticles described above.

[0033] In an embodiment of the present application, the prevention and / or treatment of endometriosis comprises reducing the size of ectopic lesions of endometriosis, reducing the number of lesions of endometriosis, reducing the weight of lesions of endometriosis and / or reducing the degree of fibrosis of lesions of endometriosis.

[0034] In an embodiment of the present application, the reduction of the degree of fibrosis of lesions of endometriosis comprises promoting the transformation of M2 type macrophages into M1 type macrophages by regulating the polarization state of macrophages in lesions of endometriosis, thereby inhibiting the secretion of fibrosis-promoting factor TGF-β in M2 type macrophages, further inhibiting the activation of myofibroblasts, and finally inhibiting the fibrosis of lesions of endometriosis.

[0035] In an embodiment of the present application, the components of the drug further comprise a pharmaceutically acceptable excipient.

[0036] In an embodiment of the present application, the pharmaceutically acceptable excipient comprises a solvent, a surfactant, a stabilizer and / or a hydrogel.

[0037] The technical scheme of the present application has the following advantages:

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

[0039] 2. The application provides application of OMV-PLGA nanoparticles in preparation of drugs for preventing and / or treating endometriosis, the OMV-PLGA nanoparticles comprising bacterial outer membrane vesicles (OMVs) 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 the 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 fibrosis degree of endometriosis lesions (here, reducing the fibrosis degree of endometriosis lesions includes promoting the transformation of M2 type macrophages into M1 type macrophages by regulating the polarization state of macrophages in endometriosis lesions, thereby inhibiting the secretion of fibrosis-promoting factor TGF-β in M2 type macrophages, further inhibiting the activation of myofibroblasts and finally inhibiting the fibrosis of endometriosis lesions). The application applies OMVs to the prevention and treatment of endometriosis, breaks away from the limitations of traditional hormone treatment approaches, shows the potential of macrophage polarization regulation in non-hormone treatment and provides a new idea for non-estrogen pathway treatment. Moreover, the application wraps OMVs on the surface of PLGA for application in the prevention and treatment of endometriosis, further improving the prevention and treatment effect.

[0040] 3. The application further provides a method for preparing the OMV-PLGA nanoparticles, which comprises: separately injecting a bacterial outer membrane vesicle solution and a polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip for the synthesis of nanoparticles to obtain OMV-PLGA nanoparticles; and performing ultrasonic treatment on the microfluidic chip in the synthesis process of the OMV-PLGA nanoparticles to make the bacterial outer membrane vesicles break and reassemble on the surface of the polylactic acid-glycolic acid copolymer core; the OMV-PLGA nanoparticles comprise bacterial outer membrane vesicles (OMVs) and polylactic acid-glycolic acid copolymer (PLGA); and the bacterial outer membrane vesicles are wrapped on the surface of the polylactic acid-glycolic acid copolymer. In the aspect of particle preparation, the microfluidic chip synthesis technology is used to complete the preparation of OMV-PLGA nanoparticles with good uniformity and high repeatability. At present, the main methods for preparing biomimetic membrane-polymer nanoparticles are extrusion and ultrasonic. Both of the two preparation methods have relatively complex synthesis processes and problems such as poor uniformity of synthesized particles and low biomimetic membrane wrapping rate. The extrusion method consumes time and energy and has poor controllability, and the ultrasonic method cannot completely coat the biomimetic membrane on the surface of the polymer particles, and the synthesis rate of the particles is low. The microfluidic synthesis technology has the advantages of accurate controllable reaction conditions, rapid heat and mass transfer and integration of multiple reactions, and provides a transformative platform for precise assembly of functional materials. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0044] Figure 4 : DLS analysis results of OMV-PLGA nanoparticles. Figure 4 , A: Particle size of OMV-PLGA nanoparticles; B: Zeta potential of OMV-PLGA nanoparticles.

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

[0046] Figure 6 : Uptake analysis results of THP-1 cells to OMVs and OMV-PLGA nanoparticles. Figure 6 , A: Representative flow cytometry diagram of DID-labeled nanoparticles taken up by THP-1 cells; B: Mean fluorescence intensity of THP-1 cells after taking up the particles.

[0047] Figure 7 : Effect of OMV-PLGA nanoparticles in endometriosis injection model. Figure 7 , A: Representative diagram of successful modeling of endometriosis injection model; B: Representative diagram of taking out lesions of endometriosis after treatment of PBS, OMVs and OMV-NPs; C: Number of lesions of endometriosis after treatment of PBS, OMVs and OMV-NPs; D: Weight of lesions of endometriosis after treatment of PBS, OMVs and OMV-NPs.

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

[0049] Figure 9 : IHC analysis results of lesions of endometriosis after treatment of PBS, OMVs and OMV-NPs. Figure 9Figure 6: The effect of OMV-NPs treatment on endometriosis mouse model. A: Representative images of IHC staining of lesions after 21 days of endometriosis mouse model establishment (Scale bar = 20 pm); B: The number of CD86-positive (Ml type) and CD163-positive (M2 type) macrophages, and IHC score of TGF- b and a-SMA in lesions after 21 days of endometriosis mouse model establishment.

[0050] Figure 10 Figure 7: The effect of OMV-NPs treatment on the body weight of mice.

[0051] Figure 11 Figure 8: The effect of OMV-NPs treatment on the levels of inflammatory factors in the serum of mice. Figure 11 A: The effect of OMV-NPs treatment on the level of IL-6 in the serum of mice; B: The effect of OMV-NPs treatment on the level of TNF-a in the serum of mice.

[0052] Figure 12 Figure 9: The effect of OMV-NPs treatment on the blood routine indexes of mice. Figure 12 A: The effect of OMV-NPs treatment on the level of white blood cells (WBC) in the whole blood of mice; B: The effect of OMV-NPs treatment on the level of neutrophils in the whole blood of mice; C: The effect of OMV-NPs treatment on the level of lymphocytes in the whole blood of mice; D: The effect of OMV-NPs treatment on the level of platelets in the whole blood of mice.

[0053] Figure 13 Figure 10: The effect of OMV-NPs treatment on the organs of mice (H&E staining results) (Scale bar = 50 pm).

[0054] Figure 14 Figure 11: The effect of OMV-NPs treatment on the serum biochemical indexes of mice. Figure 14 A: The effect of OMV-NPs treatment on the level of alanine aminotransferase (ALT) in the serum of mice; B: The effect of OMV-NPs treatment on the level of creatinine (CREA) in the serum of mice; C: The effect of OMV-NPs treatment on the level of lactate dehydrogenase (LDH) in the serum of mice; D: The effect of OMV-NPs treatment on the level of aspartate aminotransferase (AST) in the serum of mice; E: The effect of OMV-NPs treatment on the level of blood urea nitrogen (BUN) in the serum of mice.

[0055] Figure 15 Figure 12: The effect of OMV-NPs treatment on the endometrium of mice. Figure 15Figure 6. The effect of OMV-NPs treatment on the endometrial thickness of mice. A: H&E staining results of the endometrium of mice treated with OMV-NPs (Scale bar = 50 pm); B: The effect of OMV-NPs treatment on the endometrial thickness of mice.

[0056] Figure 16 Figure 7. The effect of OMV-NPs treatment on the serum reproductive hormone indicators of mice. Figure 16 Figure 7. The effect of OMV-NPs treatment on the serum reproductive hormone indicators of mice. DETAILED DESCRIPTION

[0057] The following examples are provided to better further understand the present application and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application. Any person under the inspiration of the present application or the combination of the present application with other prior art features will fall within the scope of protection of the present application.

[0058] In the following experimental examples, the specific experimental steps or conditions are not specified, and can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by purchase.

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

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

[0061] Preparation of OMV solution: The bacterial liquid of E. coli DH5a (purchased from Novozyme Biotech Co., Ltd.) was coated on LB plate (purchased from Solabio Technology Co., Ltd.) and cultured at 37℃ for 16 hours. After the culture, a single colony was picked and inoculated into 5 mL LB liquid medium (purchased from Solabio Technology Co., Ltd.) and cultured in a 37℃ constant temperature incubator at 180 rpm for about 8 hours to obtain a seed liquid. 50 μL of the seed liquid was inoculated into 500 mL of LB liquid medium and cultured in a 37℃ constant temperature incubator at 180 rpm for 12 hours until the OD value reached 1.5 to obtain a bacterial liquid. The bacterial liquid was centrifuged at 4℃ and 8,000g for 15 minutes to obtain the supernatant. The supernatant was first filtered through a 0.45 μm filter membrane, and then concentrated at a ratio of 1:3 (after concentration: before concentration = 1:3) using a 100 kDa ultrafiltration membrane to obtain a concentrated liquid. The concentrated liquid was first filtered through a 0.22 μm filter membrane, and then the precipitate was collected after ultracentrifugation at 4℃ and 150,000g for 3 hours. The precipitate was resuspended in 1 mL of PBS buffer (purchased from Sigma Company) to obtain a resuspension liquid. The resuspension liquid was filtered through a 0.22 μm filter membrane to obtain an OMV solution. The protein concentration of the OMV solution was determined by BCA method (the measured protein concentration was 0.5 mg / mL), and the OMV solution was stored at -80℃ after being divided into aliquots.

[0062] Preparation of PLGA solution: Polylactic acid-glycolic acid copolymer (PLGA, purchased from Sigma Company) 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℃ 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 12000g 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: The PLGA solution and the OMV solution were injected through the middle inlet (flow rate 7 mL / h) and the two side inlets (flow rate 80 mL / h) of the microfluidic chip (see patent application No. CN110560186A for the microfluidic chip) respectively to perform particle synthesis to 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., model KQ-200TDE) and the microfluidic chip was subjected to acoustic stress at a frequency of 80 kHz and a power of 100 W to break and reassemble the OMV membrane on the surface of the PLGA core in a short time (see patent application No. CN110560186A for the particle synthesis process). 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 explores the ratio of OMVs to PLGA during the synthesis of OMV-PLGA nanoparticles, and the experimental process is as follows:

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

[0067] On the basis of 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 technology (DLS), and the distribution of the detected particle size was evaluated to obtain the average particle size of the OMV-PLGA nanoparticles and the PDI (PDI is a dimensionless value reflecting the width of the particle size distribution, ranging from 0 to 1, and the smaller the value, the more uniform the particle size and the more concentrated the particle size distribution), and the experimental results are shown in Figures 2-3 . The OMV-PLGA nanoparticles prepared at an OMV solution flow rate of 80 mL / h and a concentration of 0.1 mg / mL were selected as controls, and the particle size and zeta potential of the OMV-PLGA nanoparticles were detected by dynamic light scattering technology (DLS), and the structure of the OMV-PLGA nanoparticles was observed using a transmission electron microscope (TEM), and the experimental results are shown in Figures 4-5 .

[0069] As can be seen from the results in Figures 2-3 , the OMV-PLGA nanoparticles with the highest degree of uniform particle size can be obtained when the flow rate is 80 mL / h and the concentration is 0.1 mg / mL.

[0070] Figure 4 The particle size analysis results in

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

[0072] Depend on Figure 5 The results show that OMVs themselves exhibit a vesicle structure of 30–50 nm with significant differences in particle size distribution. In contrast, OMV-PLGA nanoparticles demonstrate a clear core-shell structure and better particle uniformity. This structural feature further confirms that the OMV film successfully encapsulates the surface of PLGA particles, forming a stable core-shell structure.

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

[0074] This experiment verified the uptake efficiency of OMV-PLGA nanoparticles. The experimental procedure is as follows:

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

[0076] THP-1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were used at a rate of 5 × 10^ 5 THP-1 cells were seeded at a density of 1 / mL into 12-well plates containing 1 mL of RMPI medium (from Sigma) supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (from Sigma). 100 ng / mL of phorbol ester (PMA, from Sigma) was added to the culture system, and the plates were incubated at 5% (v / v) CO2 and 37°C for 48 hours to induce THP-1 cell differentiation into macrophages. After incubation, using an equal volume of PBS buffer as a blank control, fluorescently labeled OMV solution and fluorescently labeled OMV-PLGA nanoparticle solution were added to the culture system at a concentration of 5 μg / mL, and incubated at 5% (v / v) CO2 and 37°C for 4 hours. After incubation, the amount of particles taken up by the cells was quantitatively analyzed using flow cytometry. The results are shown in [Figure 1].Figure 6 .

[0077] Depend on Figure 6 The results showed that THP-1 cells uptake OMV-PLGA nanoparticles at approximately four times the efficiency of natural OMVs. This finding indicates that the design improvements of OMV-PLGA nanoparticles have successfully enhanced their uptake capacity in macrophages, potentially improving their bioavailability and therapeutic efficacy.

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

[0079] This experiment verified the therapeutic effect of OMV-PLGA nanoparticles on endometriosis. The experimental procedure is as follows:

[0080] Twenty-four BALB / c mice (purchased from Beijing Vital River Laboratory Technology Co., Ltd.) were randomly divided into three groups: a PBS control group, an OMVs intervention group, and an OMV-PLGA intervention group, with eight mice in each group. After grouping, OMV-PLGA nanoparticles prepared at a flow rate of 80 mL / h and a concentration of 0.1 mg / mL were used. Mice in the OMVs intervention group were intraperitoneally injected with 50 μg / kg of OMVs (solvent: 200 μL PBS buffer), and mice in the OMV-PLGA intervention group were intraperitoneally injected with 50 μg / kg of OMV-PLGA nanoparticles (solvent: 200 μL PBS buffer). Mice in the PBS control group were intraperitoneally injected with an equal volume of PBS buffer as a blank control. One week after injection, endometriosis was induced in all three groups to observe the therapeutic effect of OMV-PLGA nanoparticles on endometriosis. The experimental results are shown in the table below. Figure 7 ;

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

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

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

[0084] From the results of Figure 7 , compared with the PBS group, the size of ectopic lesions in the OMV-PLGA nanoparticle treatment group was significantly reduced (the size of ectopic lesions was reduced from 59.72 ± 14.01 mm 3 to 36.28 ± 9.31 mm 3 , a reduction of 40%), and the number and weight of lesions were also significantly reduced (the average number of lesions was reduced from 4 to 2, a reduction of 50%, and the weight of lesions was reduced from 77.65 ± 11.2 mg to 9.19 ± 1.49 mg, a reduction of 88%); while OMVs treatment also had some effect, but the effect was much worse than that of OMV-PLGA nanoparticles.

[0085] Figure 8 The H&E staining results in Figure 8 showed that the endometrial line of the ectopic lesion was clear and complete, indicating that the model was successfully constructed.

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

[0087] Experimental Example 4: Biological safety evaluation experiment of OMV-PLGA nanoparticles

[0088] This experimental example verified the biological safety evaluation of OMV-PLGA nanoparticles, and the experimental process was as follows:

[0089] Based on Experimental Example 3, biological safety evaluation was performed, and the specific evaluation process was as follows:

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

[0091] Heart, liver, spleen, lung, kidney and serum samples of mice were collected 7 days after injection of OMV-PLGA nanoparticles, and organ sections were observed by H&E staining, and biochemical detection was performed on serum, and the experimental results are shown in Figures 13-14 .

[0092] Figure 10 The results show that the body weight of the OMV-PLGA nanoparticle treatment group and the PBS treatment group showed stable growth, and no significant difference was observed between the two groups.

[0093] Figure 11 The results show that the IL-6 and TNF-α levels of the OMV-PLGA nanoparticle treatment group on the first day after administration were significantly increased, about 4 times of the control group, compared with the PBS treatment group. However, the levels of these inflammatory factors gradually decreased and returned to normal levels on the seventh day. Since the most significant inflammatory response occurred in the OMV-PLGA nanoparticle treatment group on the first day after administration, further blood routine tests were performed on the peripheral blood at this time point, and the experimental results are shown in Figure 12 .

[0094] Figure 12 The results show that the white blood cell, neutrophil, lymphocyte and platelet levels of the OMV-PLGA nanoparticle treatment group are comparable to the PBS treatment group, which indicates that OMV-PLGA nanoparticles only cause a controllable degree of inflammatory response and do not cause significant damage to the mouse body.

[0095] According to the H&E staining results of Figure 13 , no obvious pathological changes were found in the mouse organs.

[0096] Figure 14 The biochemical detection results show that the mouse's blood urea nitrogen (BUN), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) and other liver function, kidney function and heart function indicators have no significant changes.

[0097] According to the results of Figures 10-14 , the application of OMV-PLGA nanoparticles in vivo is relatively safe.

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

[0099] This experimental example investigates the effect of OMV-PLGA nanoparticles on the reproductive system, and the experimental process is as follows:

[0100] Based on Experimental Example 3, the effect of OMV-PLGA nanoparticles on the reproductive system was investigated, and the specific investigation process was as follows:

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

[0102] According to theFigure 15 The H&E staining results showed that the structure and thickness of the endometrium in mice did not change significantly after OMV-PLGA nanoparticle injection.

[0103] Figure 16 The results of reproductive hormone detection showed that the levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in mice did not change significantly.

[0104] In summary Figures 15-16 The results showed that OMV-PLGA nanoparticles did not affect the ovarian reserve function and hypothalamic-pituitary-ovarian (H-P-O) axis in mice.

[0105] Obviously, the above examples are only examples for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. The application of nanoparticles in the preparation of drugs for the prevention and / or treatment of endometriosis, characterized in that, The nanoparticles comprise bacterial outer membrane vesicles and polylactic acid-glycolic acid copolymer; the bacterial outer membrane vesicles are coated on the surface of the polylactic acid-glycolic acid copolymer. The method for preparing the nanoparticles includes: injecting bacterial outer membrane vesicle solution and polylactic acid-glycolic acid copolymer solution into different inlets of a microfluidic chip to synthesize nanoparticles; during the synthesis of nanoparticles, the microfluidic chip is subjected to ultrasonic treatment to break down the bacterial outer membrane vesicles and reassemble them onto the surface of the polylactic acid-glycolic acid copolymer core. The bacterial outer membrane vesicle solution was injected into the microfluidic chip at a flow rate of 60-100 mL / h; the polylactic acid-glycolic acid copolymer solution was injected into the microfluidic chip at a flow rate of 3-9 mL / h. 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.

2. The application as described in claim 1, characterized in that, The frequency of ultrasonic processing of the microfluidic chip is 60~100 kHz and the power is 80~120 W.

3. The application as described in claim 1, characterized in that, The method for preparing the bacterial outer membrane vesicle solution includes: inoculating Gram-negative bacteria into a culture medium for culture to obtain a culture solution; and extracting bacterial outer membrane vesicles from the culture solution to obtain a bacterial outer membrane vesicle solution.

4. The application as described in claim 1, characterized in that, The method for preparing the polylactic acid-glycolic acid copolymer solution includes: dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain a solution; taking the supernatant after shaking, sonicating and centrifuging the solution; and diluting the supernatant with a buffer solution to obtain the polylactic acid-glycolic acid copolymer solution.

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