CN113197880B - A kind of macrophage exosome membrane-coated biomimetic nanoparticle and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to a macrophage exosome membrane-coated bionic nanoparticle as well as a preparation method and application thereof, wherein the macrophage exosome membrane-coated bionic nanoparticle is formed by exosome membrane-coated nanoparticles; the exosome membrane is separated from M2 macrophage derived from bone marrow; the nano-particles are prepared from polylactic-co-glycolic acid (PLGA) and Dnmt3aos^{smart silencer}And (3) co-emulsification formation. The invention uses EM-PLGA @ Dnmt3aos^{smart silencer}The M2 macrophage polarization in allergic asthma is remarkably inhibited, and the bionic drug is effectively accumulated in the lung and promotes gene silencing, and simultaneously reduces the infiltration degree of inflammatory cells to the airway. PLGA NP based on M2 macrophage exosome membrane contributes to therapeutic Dnmt3aos^{smart silencer}Delivered to the airways, directing its more effective treatment in an AA mouse model.

Description

Macrophage exosome membrane-coated bionic nanoparticle and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to a macrophage exosome membrane coated bionic nanoparticle as well as a preparation method and application thereof.

Background

About 3 hundred million people worldwide suffer from asthma, and it is estimated that 4 hundred million will be affected by 2025, Allergic Asthma (AA) accounts for 90% of childhood asthma cases and 50% of adult asthma cases. Macrophages, the most abundant immune cells in the lung (accounting for approximately 70% of all immune cells), play an important role in airway inflammation caused by environmental allergens. Adoptive transfer of M2 macrophages exacerbates allergic airway inflammation in mice, suggesting that polarization of M2 macrophages plays an important role during AA onset. Therefore, the development of new strategies against M2 macrophages is considered a promising approach to treat AA.

In our previous studies, thousands of lncRNAs were differentially expressed in M1/M2 polarized bone marrow-derived macrophages (BMDM). Of these, Dnmt3aos (DNA methyltransferase 3A, opposite strand) is a known lncRNA, located on the antisense strand of Dnmt 3A. And we have demonstrated that Dnmt3aos is highly expressed in M2 macrophages and is involved in regulating expression of Dnmt3 a. The expression of Dnmt3aos and Dnmt3a is coordinated and plays a key role in macrophage polarization. Thus, Dnmt3aos in M2 macrophages may be a good therapeutic target for AA, but its role in allergic asthma is unclear.

A smart silencer composed of 3 small interfering RNAs (siRNAs) and 3 antisense oligonucleotides (ASOs) plays an important role in mediating the specific silencing of target genes. Free ASO/siRNA is nearly impossible to use in clinical therapy due to its instability, limited cellular uptake and inadequate blood circulation. To overcome the obstacles of ASO/siRNA delivery, more efficient and safer delivery vehicles are crucial for the study and production of ASO or siRNA therapeutics, such as lipid-based particles and Nanoparticles (NPs). Due to their good biodegradability and biocompatibility, the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) have approved polylactic-glycolic acid copolymer (PLGA) for drug and biomolecule delivery, bioabsorbable sutures, bioimaging and phototherapy and tissue regeneration. In particular, the siRNA loaded PLGA NPs exhibited sustained release characteristics, as previously reported, releasing 35% on the first day and showing sustained release over 10 days. Therefore, the sustained regulation of target cells by PLGA-encapsulated smart silencer of Dnmt3aos is worthy of study.

Due to the rapid development of nanocarriers, new drug/gene delivery vehicles are also emerging, such as cell-derived exosomes, especially membranes of exosomes, which exhibit excellent targeting. The organ tropism naturally inherited from the parental cells confers exosomes with a series of surface adhesion proteins and specific vector ligands to achieve precise cargo shuttling. As a natural vector, exosomes can deliver exogenous mirnas or sirnas in vivo to target cells or tissues to modulate gene expression and inhibit disease progression. However, conventional transfection of genes, expression of genes in exosomes is less, and recent studies have also demonstrated that sirnas are susceptible to degradation when electroporation induces siRNA into exosomes, suggesting a need to establish more reliable protocols. Studies have shown that certain NPs can form complexes with siRNA or ASO, and the formation of such nanocomplexes is important to fully protect the RNA, prevent its degradation and enable their cellular delivery by endocytosis and intracellular release. Therefore, in order to further improve the biodistribution, stability, efficacy and biocompatibility of NPs, we urgently need to establish an exosome modification-based PLGA system to form a complex combining the advantages of both systems.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a bionic nanoparticle coated with a macrophage exosome membrane, and a preparation method and application thereof.

In order to overcome the defects, the technical scheme adopted by the invention comprises the following steps: a macrophage exosome membrane-coated bionic nanoparticle is characterized in that the nanoparticle (i.e. PLGA @ Dnmt3 aos) is coated by an exosome membrane (i.e. M2 macrophage exosome membrane)^{smartsilencer}) Forming; the exosome membrane is separated from M2 macrophage derived from bone marrow; the nano-particle PLGA @ Dnmt3aos^{smartsilencer}From poly (lactic-co-glycolic acid) PLGA and Dnmt3aos inhibitor (i.e. Dnmt3 aos)^{smartsilencer}) And (3) co-emulsification formation.

The M2 macrophage exosome membrane-coated bionic nanoparticle is formed by exosome membrane-coated nanoparticles, and the nanoparticles are PLGA @ Dnmt3aos nanoparticles with surfaces coupled with polyetherimide PEI (polyetherimide)^{smartsilencer}(ii) a The exosome membrane is separated from M2 macrophage derived from bone marrow; the nano-particle PLGA @ Dnmt3aos^{smartsilencer}From poly (lactic-co-glycolic acid) PLGA and Dnmt3aos inhibitor (i.e. Dnmt3 aos)^{smartsilencer}) And (3) co-emulsification formation.

A preparation method of macrophage exosome membrane coated bionic nanoparticles comprises the following steps:

1) nanoparticle PLGA @ Dnmt3aos^{smartsilencer}Preparing;

2) preparing a bone marrow M2 macrophage exosome membrane;

3) in nanoparticles PLGA @ Dnmt3aos^{smartsilencer}Bone marrow M2 macrophage exosome membrane is covered on the surface of the substrate to prepare the bionic nano-particles coated by the macrophage exosome membrane.

Between steps 1) and 2), the method also comprises the step of enabling the nanoparticles PLGA @ Dnmt3aos^{smartsilencer}Coupling the surface with Polyetherimide (PEI).

The step 1) is specifically as follows: 60 μ g PLGA, 2ug Dnmt3aos^{smartsilencer}Respectively dissolving, mixing and emulsifying to generate primary emulsion; adding an emulsifier into the primary emulsion for further emulsification; collecting nanoparticles PLGA @ Dnmt3aos^{smartsilencer}。

Nanoparticles PLGA @ Dnmt3aos^{smartsilencer}The coupling of the surface and the polyetherimide PEI specifically comprises the following steps: nanoparticles PLGA @ Dnmt3aos^{smartsilencer}Mixing with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide solution (i.e., N-hydroxysuccinimide (NHS, bainwei technologies ltd. beijing)) to activate the nanoparticle surface; then adding Polyetherimide (PEI) dropwise and stirring for 1 hour; collecting the PEI-coupled nanoparticles.

The step 2) is specifically as follows: transforming the polarized bone marrow macrophage to M2 type, collecting supernatant of M2 macrophage, centrifuging, precipitating, and removing cell debris; centrifuging the final supernatant to obtain exosomes; suspending the harvested exosome in ice-cold TM buffer solution, and repeatedly freezing and thawing for 5 times; adding 0.25M sucrose for resuspension and centrifuging; the supernatant was collected and further centrifuged to collect the precipitate as an exosome membrane.

PLGA @ Dnmt3aos coupling exosome membranes and surface to PEI^{smartsilencer}Co-incubation and ultrasonic treatment are carried out to obtain M2 macrophage exosome membrane coated bionic nanoAnd (3) granules.

It is still another object of the present invention to provide a therapeutic drug or drug carrier for allergic asthma comprising the aforementioned M2 macrophage exosome membrane-coated biomimetic nanoparticles.

The invention also aims to provide application of the preparation method in preparing a medicament or a medicament carrier for treating allergic asthma.

Compared with the prior art, the invention uses EM-PLGA @ Dnmt3aos^{smartsilencer}The bionic drug has the advantages that M2 macrophage polarization in AA is remarkably inhibited, the bionic drug is effectively accumulated in the lung, gene silencing is promoted, and meanwhile, the infiltration degree of inflammatory cells to the air passage is reduced. Thus, M2 macrophage exosome membrane-based PLGA NPs contribute to the therapeutic Dnmt3aos^{smartsilencer}Delivered to the airways, directing its more effective treatment in an AA mouse model.

Drawings

FIG. 1 delivery of exosome membrane modified PLGA NPs Dnmt3aos^{smartsilencer}To ameliorate airway inflammation in AA.

FIG. 2 Assembly and characterization of biomimetic nanoparticles. (A) Representative TEM images. (B) And (4) size distribution. 1: PLGA @ Dnmt3aos^{smartsilencer},2: exosomes, 3: exosome membrane, 4: EM-PLGA @ Dnmt3aos^{smartsilencer}. (C) Zeta potential distribution. 1: PLGA, 2: PEI modified PLGA, 3: exosomes, 4: exosome membrane, 5: EM-PLGA @ Dnmt3aos^{smartsilencer}. (D) Phenotypic analysis was performed using confocal laser scanning microscopy (1,000 ×). (E) EM-PLGA @ Dnmt3aos^{smartsilencer}Stability of (2). (F) From EM-PLGA @ Dnmt3aos within 48 hours as measured by Nanodrop^{smartsilencer}Dnmt3aos of^{smartsilencer}The release profile of (1). (G) Phenotypic analysis by flow cytometry, representing EM-PLGA @ Dnmt3aos in the upper right quadrant^{smartsilencer}Percentage of (c).

FIG.3M 2 macrophages in vitro on EM-PLGA @ Dnmt3aos^{smartsilencer}The intake of (1). (A) By laser confocal microscopyFor free Cy5-Dnmt3aos^{smartsilencer}，Cy5-PLGA@Dnmt3aos^{smartsilencer}And Cy5-EM-PLGA @ Dnmt3aos^{smartsilencer}In vitro cellular uptake of (a). Cy5-Dnmt3aos^{smartsilencer}Shown as red, DAPI was used to stain nuclei (blue).Scale bar 25 μm. (B) Flow cytometry analysis of Cy5-Dnmt3aos in M2 macrophages^{smartsilencer}Fluorescence distribution and percentage of (c). (C) Dnmt3aos mRNA expression was analyzed by qRT-PCR. (D) Dnmt3a mRNA expression was analyzed by qRT-PCR. (E) Western blot analysis for Dnmt3a protein expression. Results represent mean ± SEM. (5 mice. ns per group, representing no significance,. P,<0.05，**P，<0.01，***P，<0.001)。

FIG. 4 EM-PLGA @ Dnmt3aos in AA mice^{smartsilencer}In vivo tracking and tissue distribution. (A) Whole body near infrared imaging for tracking EM-PLGA @ Dnmt3aos at designated time points post intravenous infusion^{smartsilencer}And (4) distribution in vivo. (B) At a time point of 2 hours after intravenous injection, AA mouse organs were taken for ex vivo near-infrared imaging. (C) Fluorescence signal intensities at seven time points. (D) Fluorescence signal intensity in 2 hours organ. Results represent mean ± SEM. (5 mice per group P,<0.05，**P，<0.01，***P，<0.001)。

FIG. 5 in vivo M2 macrophage pair EM-PLGA @ Dnmt3aos^{smartsilencer}The cellular uptake of (2). (A) Lungs were collected fromAA mice 4 hours after intravenous injection and frozen sections were prepared. Then, macrophages and CD4 were treated with FITC-labeled antibody (green) respectively⁺T cells were stained and observed by confocal imaging, using DAPI to stain the nuclei (blue). (B) 4 hours after injection, macrophages (M1 and M2) and CD4 were injected⁺T cells taken up Cy5 labeled biomimetic NPs for flow cytometry analysis. Results represent mean ± SEM. (5 mice. ns per group, no significance,. P,<0.05，**P，<0.01，***P，<0.001)。

FIG. 6 EM-PLGA @ Dnmt3aos^{smartsilencer}Treatment reduced Der f 1-induced allergic airway inflammation. (A-B) H&Histopathology with E and PAS stainingExamination (original magnification 200 times and 400 times, scale bar: 200 μm). (C) Total IgE levels in mouse plasma were detected by ELISA. (D) Allergen-specific IgG was detected by ELISA. (E) Statistical map of cell proportion in BALF, flow cytometry detection gating strategy: BALF neutrophils (Neu: CD 45)⁺Ly6G⁺) Eosinophils (Eos: CD45⁺Ly6G^-Siglec^-F⁺CD11c^-) Alveolar macrophages (AM: CD45⁺Ly6G^-SiglecF⁺). (F) Airway hyperresponsiveness.

FIG. 7 EM-PLGA @ Dnmt3aos^{smartsilencer}Treatment can reduce the inflammatory response by inhibiting M2 macrophage polarization. (A) Flow cytometric analysis of CD206 and iNOS in macrophages in BALF. (B) qRT-PCR analysis of M1-related genes. (C) qRT-PCR analysis of M2-related genes. mRNA expression levels were normalized to GAPDH, respectively, and 2 was used^-ΔΔCtAnd (4) calculating by using the method. Results represent mean ± SEM. (5 mice. ns per group, no significance,. P,<0.05，**P，<0.01，***P，<0.001)。

FIG. 8 EM-PLGA @ Dnmt3aos^{smartsilencer}The treatment does not have obvious side effects on the whole immune function. (A) Using free Dnmt3aos^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}And EM-PLGA @ Dnmt3aos^{smartSilencer treatment}Then, H is performed on the major organ&And E, dyeing. The scale bar is 200 μm. (B) Tumor size,day 1 after AA mouse Der f1 immunization, a549 cells (2 × 106 cells per mouse) were injected in the right groin. Then, the mice were randomized into three groups (seven mice per group) and given EM-PLGA @ Dnmt3aos through the tail vein on days 21-27^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}Or free Dnmt3aos^{smartsilencer}. Tumor growth curve (C) and survival (D). 7 mice per group were used for the experiment.

FIG. 9 asthmatic mice show aberrant lncRNA Dnmt3aos and Dnmt3a expression. (A) H&E staining (magnification 200X and 400X, scale bar: 200 μm). (B) Macrophages were sorted in BALF. (C)) Dnmt3aos and Dnmt3a expression were detected in macrophages in BALF. mRNA expression levels were normalized to GAPDH, respectively, and 2 was used^-ΔΔCtAnd (4) calculating by using the method. Results represent mean ± SEM. Each group had 5 mice. (ii) a value of P,<0.01，***P，<0.001)。

FIG. 10 exosome membranes of M2 macrophages have no polarizing capability. (A) Western blot analysis of CD9, CD63 and HSP70 from M2 macrophages and exosomes. (B) BMDM was treated with 20ng/mL IL-4(PeproTech) to generate M2 macrophages. Meanwhile, we added exosomes or exosome membranes (100 μ g/mL) of M2 macrophages to M0 macrophages. After 48 hours of stimulation, cells were harvested for identification by qRT-PCR. (C) Protein electrophoresis of exosomes and exosome membranes of M2 macrophages. Results represent mean ± SEM. (n-3 wells ns in each group, no significant difference,. P, < 0.001).

FIG. 11 use of EM-PLGA @ Dnmt3aos in vivo^{smartsilencer}Silencing expression of the Dnmt3aos gene in macrophages. (A) Expression of Dnmt3aos and Dnmt3a mRNA was detected in macrophages of BALF using qRT-PCR. (B) Dnmt3a protein expression was detected by Western blotting. Results represent mean ± SEM. (5 mice. ns per group, no significant differences,. P,<0.05，**P，<0.01，***P，<0.001)。

Detailed Description

The following describes embodiments of the present invention with reference to the drawings and examples.

In the present invention, we explored biomimetic nanoparticles (EM-PLGA @ Dnmt3 aos) coated by the membrane of M2 macrophage exosomes^{smartsilencer}) It is prepared from PLGA @ Dnmt3aos^{smartsilencer}Core and exosome membrane. Using EM-PLGA @ Dnmt3aos^{smartsilencer}The inhibition of M2 macrophage polarization in AA (allergic asthma) was very significant (fig. 1), and the biomimetic drug efficiently accumulated in the lung and promoted gene silencing while reducing the degree of infiltration of inflammatory cells into the airways. Therefore, we believe that exosome membrane-based PLGA NPs contribute to the therapeutic Dnmt3aos^{smartsilencer}Delivery to the airways, guidance for it in an AA mouse modelMore effective treatment.

1. Materials and methods

1.1 mice

C57BL/6(H-2d) female mice (6-8 weeks, 20-22g) were purchased from the Green Longshan laboratory animal center (Nanjing, China) and housed in a pathogen-free SPF-rated animal house. All animal experiments were performed according to the "guidelines for care and use of laboratory animals" (Ministry of health of the people's republic of China, 1998) and the guidelines of the ethical Committee for laboratory animals of the southern Anhui medical college. All experimental protocols were evaluated and approved by the animal ethics committee of southern anhui medical college (permit No.: 20130138).

1.2 allergic asthma

To induce allergic asthma, 100 μ g Der f1 was injected intraperitoneally into C57BL/6 mice ondays 0, 7, and 14 of the experiment. On days 21-28, mice were sensitized to Der f1 by nebulizer daily for 15 minutes and sacrificed 24 hours after the last nebulization.

1.3 PLGA@Dnmt3aos^{smartsilencer}Preparation of

PLGA NPs were prepared according to our previous double emulsion solvent evaporation method (W1/O/W2). To produce PLGA @ Dnmt3aos^{smartsilencer}PLGA (60. mu.g) was dissolved in chloroform (200uL) (PLGA is a polylactic-co-glycolic acid copolymer, PLGA50/50, Jinan Dai big Dipper bioengineering Co., Ltd.), and 2ug Dnmt3aos was added^{smartsilencer}Dissolved in RNase-free water (average molecular weight 13,300g/mol based on siRNA), both (mixture of PLGA and chloroform and 2ug Dnmt3aos^{smartsilencer}A mixture with water) to form a mixture. The mixture was emulsified using a microprobe sonicator on an ice bath for 40s to produce a primary (W1/O) emulsion. Next, 1mL of 1% PVA (i.e., polyvinyl alcohol) was added to the primary emulsion solution, which was then emulsified on an ice bath for 2 minutes at 40w (40w refers to the sonicator run set power). Finally, the organic solvent was evaporated by continuing stirring with a magnetic stirrer, and PLGA @ Dnmt3aos was collected using a centrifuge (10,000 Xg, 5 minutes)^{smartsilencer}(which are nanoparticles) and washed twice with RNase-free water.

To enhance the ability of NPs (i.e., the aforementioned nanoparticles) to bind proteins, including exosome membranes, we further modified nanospheres (i.e., PLGA @ Dnmt3 aos) with PEI (i.e., polyetherimide)^{smartsilencer}) Such that the surface is covered with positive amino groups, thereby crossing the exosome membrane. Briefly, 60 μ g of NPs were further mixed with 1mg 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 350 μ g of n-hydroxysuccinimide solution (Sigma-Aldrich, 350 μ g referring to the mass of n-hydroxysuccinimide (NHS)) to activate the NPs surface. Then 2mL of 1% PEI was added dropwise thereto and stirred with a magnetic stirrer at room temperature for 4 hours. Finally, after washing with RNase-free water, PEI-conjugated NPs were collected and stored at 4 ℃ for later use. Size distribution was measured using NTA. The Zeta potential was measured using a PALS Zeta instrument (brueck hafen instrument). PEI-conjugated PLGA @ Dnmt3aos were observed with TEM^{smartsilencer}Shape and surface morphology. In the present invention, all figures and hereinafter, reference is made to PLGA @ Dnmt3aos^{smartsilencer}All refer to PLGA @ Dnmt3aos coupled to PEI^{smartsilencer}。

TABLE 1 smart silencer sequences

The sequences are SEQ ID NO.1-6 in sequence.

Dnmt3aos^{smartsilencer}The composition is prepared by mixing 6 sequences in a molar ratio of 1:1:1:1:1:1 in Table 1. The siRNA is double-stranded, and the 6 sequences provided herein are all single-stranded when active. Dnmt3aos^{smartsilencer}The siRNA is composed of 3 siRNA of Dnmt3aos and 3 ASO sequences of Dnmt3aos, usually, one siRNA or ASO of the gene can be used for silencing the expression of the gene when the inhibition gene silencing is possible, and Smart silencer integrates 3 siRNA and 3 Dnmt3aos, so that the inhibition efficiency is more efficient.

TABLE 2 primer sequences

The sequences are SEQ ID NO.7-24 in sequence. The primers in Table 2 are primers for the genes detected in the fold change bar chart.

1.4 isolation of exosomes from bone marrow macrophages (BMDMs, M2)

Bone marrow-derived M2 macrophages were polarized (i.e., bone marrow macrophages were polarized to switch to M2 type) using the method previously described (Lv et al. FASEB J.2020; 34(4):5077-5091.PMID: 32052888). The supernatant of M2 macrophages was collected, pelleted by centrifugation at 1500rpm for 20 minutes, and then centrifuged at 10,000 Xg for 30 minutes to remove cell debris. The final supernatant (i.e., the supernatant obtained after the aforementioned cell precipitation and cell debris removal step) was ultracentrifuged at 100,000 Xg for 4h to obtain exosomes. Exosomes were suspended in PBS and stored at-80 ℃ prior to use.

Subsequently, the protein concentration of the purified exosomes was determined using the BCA protein assay kit. Western blot was used to detect the expression of TSG101, CD9 and HSP70 in exosomes. The size distribution was measured using a Particle tracking analyzer (Particle metric, germany). The Zeta potential was measured using a PALS Zeta instrument (brueck hafen instrument). The size and morphology of the exosomes were observed with TEM.

1.5 isolation of exosome membranes and detection of their polarizing capacity

Briefly, exosome membranes were isolated. The exosomes harvested in step 1.4 were suspended in ice-cold Tris-magnesium buffer (TM buffer, pH 7.4, 0.01M Tris and 0.001M MgCl₂) In (c), rapidly frozen at-80 ℃ and thawed at RT (i.e., room temperature). This freeze-thaw cycle is repeated up to five times to destroy the inclusion of exosomes. To these exosome preparations (i.e., exosomes obtained after the aforementioned repeated freeze-thaw cycles) was added 1M sucrose to a final concentration of 0.25M sucrose and centrifuged at 2,000 × g for 10 minutes to centrifugeThe content precipitate was removed. The supernatant was collected and centrifuged at 3,000 × g for a further 60 minutes to collect the exosome membrane (which was the pellet obtained this time by 3,000 × g centrifugation). The size distribution was measured using a Particle tracking analyzer (Particle metric, germany). The Zeta potential was measured using a PALS Zeta instrument (brueck hafen instrument). The morphology of the exosome membranes was observed by TEM.

To assess the safety of exosome membranes, we examined the polarizing capacity of exosome membranes, we first prepared mouse bone marrow-derived macrophages (BMDM, M0). BMDM was then treated with 20ng/mL IL-4 and we added exosomes or exosome membranes (100. mu.g/mL) of M2 macrophages to the other wells. After 48 hours of stimulation, the resulting cells were collected for qRT-PCR identification.

1.6 EM-PLGA@Dnmt3aos^{smartsilencer}Synthesis and characterization of

Macrophage exosome membrane coated biomimetic nanoparticles (i.e., EM-PLGA @ Dnmt3 aos)^{smartsilencer}) Is PLGA @ Dnmt3aos coupled with PEI by an incubation and ultrasonic method^{smartsilencer}Covering with exosome membrane. The Exosome Membrane (EM) and nanoparticles (i.e., PEI-conjugated PLGA @ Dnmt3 aos) were first contacted^{smartsilencer}) Incubate at 4 ℃ for 40 minutes and treat in the ultrasonic bath for 3 minutes. The size distribution was measured using a Particle tracking analyzer (Particle metric, germany). The Zeta potential was detected using a PALS Zeta instrument (Brookhaven Instruments Corporation). Morphology was observed by TEM.

Cy 5-labeled Dnmt3aos^{smartsilencer}Fluorescence value of (2) for measuring EM-PLGA @ Dnmt3aos^{smartsilencer}The encapsulation efficiency of (a). The prepared biological solution was centrifuged at 10000rpm for 1min, and the fluorescence value of the solution supernatant was analyzed by fluorescence spectroscopy to obtain Dnmt3aos^{smartsilencer}The encapsulation efficiency of (a). Encapsulation efficiency was equal to Cy 5-labeled EM-Dnmt3aos^{smartsilencer}The difference between the fluorescence and the supernatant divided by EM-PLGA @ Dnmt3aos^{smartsilencer}-Cy5 fluorescence value.

Prepared as described aboveEM-PLGA@Dnmt3aos^{smartsilencer}Subsequently, it was dispersed in 1mL RNase-free water at 37 ℃. At predetermined intervals, 5. mu.L of the solution was withdrawn and mixed with 20 volumes of DMSO. Cy5-EM-PLGA @ Dnmt3aos was measured using a microplate reader^{smartsilencer}The fluorescence intensity of (2).

1.7 Dnmt3aos^{smartsilencer}Stability of (2)

To test the stability of the nanoparticles, PLGA @ Dnmt3aos^{smartsilencer}And EM-PLGA @ Dnmt3aos^{smartsilencer}Incubate for 3 days at 37 ℃ in DMEM containing 10% FBS. And particle size was monitored using a particle tracking analyzer.

1.8 PLGA@Dnmt3aos^{smartsilencer}Co-localization with exosome membranes

PLGA @ Dnmt3aos were measured using a laser scanning confocal microscope (Zeiss LSM 800, Germany)^{smartsilencer}And exosome membrane co-localization for evaluation. PKH 26 fluorescently labeled exosome membrane (red) and 5. mu.g FAM (green) labeled Dnmt3aos^{smartsilencer}。

1.9 in vitro cellular uptake

BMDM (M2 macrophages) were seeded in 24-well plates for 6 hours. Next, exosome-modified PLGA @ Dnmt3aos was added^{smartsilencer}(cy5 labeled red) was added to the well plate and incubated at 37 ℃ for 4 hours. Cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min, nuclei were stained with DAPI, and then photographed using a laser scanning confocal microscope (Zeiss LSM 800, germany). For flow cytometry analysis, M2 macrophages were resuspended in PBS and then tested using a Beckman Cytoflex flow cytometer.

1.10 in vitro determination of Dnmt3aos/Dnmt3a expression

BMDM (M2 macrophages) were seeded in 6-well plates (1X 106 cells/well) at a dose of 200nM with EM-PLGA @ Dnmt3aos^{smartsilencer}And (4) incubating. Dose 200nM of free Dnmt3aos^{smartsilencer}Used as a negative control. Cells were harvested after 48 hours of incubation. Evaluation of Dnmt3aos mRNA tables Using quantitative real-time PCR (qRT-PCR)Dnmt3a mRNA and protein expression were assessed using qRT-PCR and Western blotting, respectively.

1.11 EM-PLGA@Dnmt3aos^{smartsilencer}In vivo therapeutic effect on AA

1.11.1 measurement of Total IgE and Der f 1-specific IgG

Mouse sera were collected according to the instructions for enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems) to detect total IgE levels.

1.11.2 airway hyperreactivity detection

Mice were anesthetized with pentobarbital (Sigma-Aldrich) at a dose of 25mg/kg body weight. Airway resistance was measured using the FinePointe RC system (Buxco Research Systems).

1.11.3 lung pathology

For further histological analysis, lung tissue from asthmatic mice was subsequently removed and fixed in 4% paraformaldehyde for 24 hours. Subsequently, hematoxylin and eosin (H & E) staining and Periodic Acid Schiff (PAS) staining were performed to assess lung inflammation.

1.11.4 evaluation of cytokines by ELISA

IL-4, IL-5 and IL-13 cytokine levels were quantified in BALF using an ELISA kit (MultiSciences (Lianke), Hangzhou, Biotech Co., Ltd.) according to the instructions.

1.11.5 flow cytometry analysis

Cells collected from BALF were incubated with different antibodies according to the instructions. Cells were analyzed on a Beckman FC500 flow cytometer and data was analyzed using the FlowJo software package.

1.11.6 in vivo expression of Dnmt3aos/Dnmt3a by qRT-PCR and Western blot

EM-PLGA @ Dnmt3aos^{smartsilencer}Injected intravenously into AA mice (6-8 weeks). EM-Dnmt3aos^{smartsilencer}The dose of (A) was 150nmol/kg body weight. Using free Dnmt3aos^{smartsilencer}As a negative control. 24 hours after the last injection, immune cells from BALF were cultured for 6 hours in 6-well plates in RPMI 1640 medium (10% FBS). Removing the cell suspension, andmacrophages were collected from the bottom of the plate. Expression of Dnmt3aos mRNA in macrophages was detected by qRT-PCR. Protein expression of Dnmt3aos was detected by Western blotting.

1.12 in vivo imaging and distribution analysis

Construction of asthmatic mice evaluation EM-PLGA @ Dnmt3aos^{smartsilencer}Distributed in its body. Mice were injected with Cy 5-labeled Dnmt3aos via tail vein^{smartsilencer}Cy 5-labeled PLGA @ Dnmt3aos^{smartsilencer}And Cy 5-labeled EM-PLGA @ Dnmt3aos^{smartsilencer}(200. mu.L, 200 nM). Fluorescence distribution was monitored by in vivo IVIS Spectrum (PerkinElmer, usa) at 1 hour, 2 hours, 6 hours, 12 hours, 24 hours and 48 hours post-administration. Mice were euthanized for 2 hours and lungs and major organs were imaged. All data were analyzed by the IVIS system.

1.13 cellular uptake in vivo

Equal volume (200. mu.L) of Cy 5-labeled EM-PLGA @ Dnmt3aos^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}And free Dnmt3aos^{smartsilencer}AA mice (female, 6-8 weeks old) were injected via tail vein and lungs were collected after injection. Lung tissue was collected from eachgroup 4 hours after injection, and frozen sections with a thickness of 7 μm were prepared. Lungs were fixed with acetone and isopropanol and blocked with 2% BSA for 4 hours at room temperature, followed by staining with FITC-anti-mouse F4/80 for 2 hours at 4 ℃. Finally, EM-PLGA @ Dnmt3aos was obtained on a laser scanning confocal microscope^{smartsilencer}And fluorescence images of macrophages.

On the other hand, lung cells collected at 1200 Xg for 10 minutes were centrifuged, and stained with PE-Cy 7-labeled anti-F4/80, APC-labeled anti-iNOS, APC-labeled anti-CD 206 and FITC-labeled anti-CD 4 for 30 minutes to remove excess antibody by centrifugation and washed and analyzed by Beckman Cytoflex flow cytometer.

1.14 evaluation of EM-PLGA @ Dnmt3aos^{smartsilencer}Treatment of side effects on overall immune function

1.14.1 PLGA@Dnmt3aos^{smartsilencer}In vivo biosafety of

Mice were randomly divided into 3 groups (n ═ 5), and were subjected to intravenous injection. Dnmt3aos were injected on days 21-27, respectively^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}And EM-PLGA @ Dnmt3aos^{smartsilencer}. Mice were anesthetized and euthanized 24 hours after the last dose. Taking major organs (heart, spleen, liver and kidney collected) for H&E stained paraffin sections.

1.14.2 detection of anti-tumor ability

AA mice were injected with a549 cells (2 x 10 per mouse) in the right groin one day after the first immunization of dust mite major allergen Der f1 in s.c. (2 x 10 per mouse)⁶A cell). Thereafter, the mice were randomized into three groups (seven mice per group) and given EM-PLGA @ Dnmt3aos through the tail vein on days 21-27^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}Or Dnmt3aos^{smartsilencer}. Measuring the size of the tumor with a size of 2500mm every day³We record it as dead. The product of the perpendicular diameters was determined and the tumor volume was calculated using the formula: (shortest diameter)²X (longest diameter) × 0.5.

1.15 statistical analysis

All measured parameters are expressed as mean ± SEM. Statistical software GraphPad Prism (version 6.0) was used for analysis. Statistical analysis was performed using GraphPad Prism software (version 6.0). A t-test was performed to determine the difference between the two independent groups. P values <0.05 were considered statistically significant.

2. Results

2.1 asthma mice lncRNA Dnmt3aos and Dnmt3a are expressed

We have previously found that the expression levels of lncRNA Dnmt3aos and Dnmt3a are coordinated, both of which are associated with macrophage polarization. Since macrophages are a key factor in the pathogenesis of asthma, we first isolated macrophages in alveolar lavage fluid and evaluated Dnmt3aos and Dnmt3a expression in allergic asthma mice. Dnmt3aos and Dnmt3a were expressed at high levels in macrophages from mice induced by Der f1, whereas low expression of Dnmt3aos and Dnmt3a was observed in PBS-induced mice (FIGS. 9B-C). Overall, these results indicate that lncRNA Dnmt3aos and Dnmt3a are involved in the progression of allergic asthma.

2.2 EM-PLGA@Dnmt3aos^{smartsilencer}Is characterized by

As shown in FIG. 1, for successful design of EM-PLGA @ Dnmt3aos^{smartsilencer}Nanocomposite, Dnmt3aos^{smartsilencer}First encapsulated with PLGA, TEM showed its smooth spherical surface (fig. 2A). To assemble PLGA @ Dnmt3aos^{smartsilencer}The exosome membranes were integrated around the nanocomplexes and exosomes were first isolated from the supernatant of cultured autologous M2 macrophages. TEM and western blot showed high expression of exosome-associated proteins CD63, HSP70 and CD9 (fig. 10A), indicating successful isolation of M2-derived exosomes.

Following isolation of the exosome membrane, EM-PLGA @ Dnmt3aos were observed by TEM^{smartsilencer}Has a typical core-shell structure in which spherical PLGA is covered with EM (fig. 2A). Size analysis showed 83% PLGA @ Dnmt3aos^{smartsilencer}In the range of 80 to 140nm, and an average diameter of 108. + -. 3.2 nm. Meanwhile, 91% of EM-PLGA @ Dnmt3aos^{smartsilencer}The particle size was 100 to 180nm and the average size was 137. + -. 4.5nm (FIG. 2B). PEI modified PLGA @ Dnmt3aos^{smartsilencer}The mean zeta potential of the inhibitor was 26.5. + -. 3.2mV, indicating that the NP surface was rich in cationic NH₂And has high capacity of integrating an exosome membrane. The exosome membranes showed negative zeta potentials (-24.0 ± 4.1 mV). After binding EM-PLGA @ Dnmt3aos^{smartsilencer}The zeta potential of the complex was-22.5 ± 3.6mV, confirming the binding of the two components and leading to the formation of novel nanoparticles with binding properties (fig. 2C).

2.3 EM-PLGA@Dnmt3aos^{smartsilencer}Phenotype and Performance of

Dnmt3aos with the aid of PLGA^{smartsilencer}Is made effectiveEncapsulated in PLGA, EM (PKH 26, red) and PLGA @ Dnmt3aos were confirmed by confocal images^{smartsilencer}Coexistence of (FAM, green, fig. 2D). Dnmt3aos^{smartsilencer}Encapsulation efficiency in PLGA was 70.70 ± 1.82% (data not shown). In addition, we obtained by combining EM-PLGA @ Dnmt3aos^{smartsilencer}The stability of smart silencer was tested by incubation with 10% FBS in DMEM for 48 hours. EM-PLGA @ Dnmt3aos, as shown in FIG. 2E^{smartsilencer}Is only slightly changed, indicating that it is fairly stable in blood. On the other hand, Dnmt3aos was studied in vitro^{smartsilencer}The release kinetics of (a). Dnmt3aos^{smartsilencer}Has sustained release properties, more than 24% of Dnmt3aos^{smartsilencer}Has been released within 24 hours (fig. 2F).

Flow cytometry analysis also confirmed EM-PLGA @ Dnmt3aos^{smartsilencer}Success of the complex. Histograms show that EM is efficiently coupled to PLGA @ Dnmt3aos^{smartsilencer}In the above, the fluorescence signal was significantly shifted (data not shown). Dot plot (FIG. 2G) indicates PLGA @Dnmt3aos^{smartsilencer}85% of them collectively displayed exosome membranes by double positive EM-PLGA @ Dnmt3aos in each two-color dot plot^{smartsilencer}To be determined.

2.4 exosome membranes derived from M2 macrophages have no polarizing capacity

To confirm the polarizing capacity of the exosome membranes derived from M2 macrophages, we co-cultured exosome membranes with M0 macrophages for 48 hours. The results (fig. 10B) confirm our hypothesis, indicating that the exosome membrane was unable to polarize M0 macrophages to M2. This is because the content of exosomes was released, which was also confirmed by the results of protein electrophoresis (fig. 10C), and thus could be safely used in our subsequent experiments.

2.5 binding of exosome membranes to PLGA NP effectively binds Dnmt3aos^{smartsilencer}In vitro delivery into macrophages

Macrophages are responsible for AA airway inflammationMajor factors, therefore, we first evaluated Dnmt3aos^{smartsilencer}Whether it can be delivered to M2 macrophages in vitro by EM modified PLGA NPs. EM-PLGA @ Dnmt3aos^{smartsilencer}Dnmt3aos^{smartsilencer}The ability to deliver into target cells is critical to the therapeutic efficacy of AA. Here, Cy 5-labeled Dnmt3aos^{smartsilencer}Used as a fluorescence indicator to monitor EM-PLGA @ Dnmt3aos^{smartsilencer}Internalization in M2 macrophages (fig. 3A). Our data indicate that EM-PLGA @ Dnmt3aos^{smartsilencer}There is intense red fluorescence in M2 macrophages. This clearly indicates EM-PLGA @ Dnmt3aos^{smartsilencer}High affinity for target cells, just to demonstrate that exosome membranes can promote cell binding. Although Dnmt3aos was used^{smartsilencer}Or PLGA @ Dnmt3aos^{smartsilencer}But the fluorescence signal is much lower. Furthermore, flow cytometry analysis provided quantitative evidence supporting conclusions drawn from fluorescence microscopy observations. And Dnmt3aos without Cy5^{smartsilencer}In contrast, the histogram (FIG. 3B) shows EM-PLGA @ Dnmt3aos in M2 macrophages^{smartsilencer}The fluorescence signal of (a) is significantly shifted beyond PLGA @ Dnmt3aos^{smartsilencer}. Dot plot shows 72% EM-PLGA @ Dnmt3aos^{smartsilencer}Absorbed by M2 macrophages. These results indicate that EM modified PLGA NP is a DNA encoding Dnmt3aos^{smartsilencer}An effective carrier for delivery into macrophages.

2.6 use of EM-PLGA @ Dnmt3aos^{smartsilencer}Silencing Dnmt3aos expression in macrophages in vitro

Our previous studies showed that lncRNA Dnmt3aos regulates the polarization of M0 macrophages to M2 and is involved in regulating the expression of Dnmt3 a. Since M2 macrophage polarization plays an important role in the pathogenesis of AA, inhibition of expression of Dnmt3aos in macrophages is expected to be an effective strategy for treating AA. To determine EM-PLGA @ Dnmt3aos^{smartsilencer}Whether to macrophage M2 in vitroSilencing of Dnmt3aos expression in cells, we combined BMDMs (M2) with EM-PLGA @ Dnmt3aos^{smartsilencer}After incubation for 6h, Dnmt3aos/Dnmt3a and protein mRNA expression were detected. Expression of Dnmt3a was measured 48 hours after transfection. As shown in FIG. 3C, by using EM-PLGA @ Dnmt3aos^{smartsilencer}Transfection silenced Dnmt3aos mRNA expression in M2, while free Dnmt3aos^{smartsilencer}No silencing effect was shown. EM-PLGA @ Dnmt3aos at a dose of 200nM^{smartsilencer}Resulting in a 43% and 46% decrease in Dnmt3aos and Dnmt3a mRNA expression, respectively (FIG. 3D). Furthermore, the expression of the Dnmt3a protein in M2 was also down-regulated (FIG. 3E). EM-PLGA @ Dnmt3aos^{smartsilencer}There was a greater silencing effect in M2 due to better cellular uptake by M2 macrophages. In summary, EM modified PLGA NP was able to convert Dnmt3aos^{smartsilencer}Delivered to macrophages to silence Dnmt3aos and Dnmt3a expression.

2.7 EM-PLGA @ Dnmt3aos in vivo^{smartsilencer}Distribution and M2 macrophage targeting ability

To further confirm EM-PLGA @ Dnmt3aos^{smartsilencer}Targeted ability in AA, mice were injected with Cy 5-labeled EM-PLGA @ Dnmt3aos^{smartsilencer}And imaged by IVIS. FIG. 4A shows EM-PLGA @ Dnmt3aos in AA by NIR fluorescence imaging^{smartsilencer}The in vivo kinetic process of (a). In vivo whole body fluorescence imaging showed that EM-PLGA @ Dnmt3aos was injected^{smartsilencer}From the last 1 to 36h, the fluorescence intensity of the mice was strongest and the whole body duration exceeded 48 h. As a control, Dnmt3aos^{smartsilencer}And PLGA @ Dnmt3aos^{smartsilencer}Show a similarity to EM-PLGA @ Dnmt3aos^{smartsilencer}In vivo tracking of (1). Free Dnmt3aos^{smartsilencer}Cleared rapidly after 6h, while PLGA @ Dnmt3aos^{smartsilencer}Less accumulation and shorter retention times occurred attime points 1 to 36 h. Fluorescence imaging results show that EM-PLGA @ Dnmt3aos^{smartsilencer}Is mainly distributed in the liver,spleen, kidney and lung. In the lung, EM-PLGA @ Dnmt3aos^{smartsilencer}The fluorescence signal of (2) was visible at 1h, and the fluorescence intensity peaked at 6 h. In contrast, PLGA @ Dnmt3aos^{smartsilencer}Decreases at 6h and 24 h. At 2h, EM-PLGA @ Dnmt3aos^{smartsilencer}Pulmonary fluorescence signal intensity ratio of free Dnmt3aos for groups^{smartsilencer}Group enhancement 5 fold over PLGA @ Dnmt3aos^{smartsilencer}The group was 1.3 times stronger. Thus, EM-PLGA @ Dnmt3aos in lung tissue^{smartsilencer}Distribution ratio of (PLGA @ Dnmt3 aos)^{smartsilencer}The increased distribution of (c) may be mainly due to the high affinity function of the exosome membrane. More importantly, EM-PLGA @ Dnmt3aos^{smartsilencer}The persistent and intense fluorescence indicates that they have excellent targeting ability.

On the other hand, lungs were collected 2 hours after injection, and frozen sections were prepared for immunofluorescent staining. As found in confocal images (FIG. 5A), Cy 5-labeled EM-PLGA @ Dnmt3aos^{smartsilencer}Mainly present with macrophages but with CD4⁺Co-localization of T cells is rare. In parallel, lung single cell suspensions were prepared and then immunofluorescent stained. As shown by flow cytometry (FIG. 5B), with Dnmt3aos^{smartsilencer}And PLGA @ Dnmt3aos^{smartsilencer}Group comparison, EM-PLGA @ Dnmt3aos^{smartsilencer}The percentage of Dnmt3 aos-M2 macrophage conjugate was shown to be significantly higher than M1, whereas Dnmt3 aos-CD 4⁺The percentage of T cell binders was significantly reduced.

Subsequently, we determined EM modified PLGA @ Dnmt3aos^{smartsilencer}Whether the expression of the Dnmt3aos gene can be downregulated in vivo. Seven injections of EM-PLGA @ Dnmt3aos^{smartsilencer}Thereafter, macrophages were isolated from BALF and the expression of Dnmt3aos and Dnmt3a genes were detected. As shown in FIG. 11A, EM-PLGA @ Dnmt3aos^{smartsilencer}Significantly down-regulated Dnmt3aos and Dnmt3a mRNA expression in macrophages, whereas in Dnmt3aos^{smartsilencer}No silencing effect was observed in the group. Furthermore, the expression of Dnmt3a protein in macrophages was also analyzed. As shown in FIG. 11B, EM-PLGA @ Dnmt3aos^{smartsilencer}Protein expression is also greatly reduced. From these results, we concluded that intravenous injection of EM-PLGA @ Dnmt3aos^{smartsilencer}Can silence the expression of Dnmt3aos in M2 macrophage in vivo.

2.8 EM-PLGA@Dnmt3aos^{smartsilencer}Inhibiting the development of Der f 1-induced AA inflammation

Next, we evaluated the use of EM-PLGA @ Dnmt3aos^{smartsilencer}Whether the development of allergic inflammation induced by Der f1 allergen can be inhibited. And Dnmt3aos^{smartsilencer}And PLGA @ Dnmt3aos^{smartsilencer}In contrast, EM-PLGA @ Dnmt3aos^{smartsilencer}Histological results of treated asthmatic mice showed less recruitment of inflammatory cells to the lung, less peribronchial infiltration and goblet cell proliferation (fig. 6A-B). Free Dnmt3aos in response to Der-f1 challenge^{smartsilencer}Total IgE in plasma of treated mice increased to 1150.37 + -103.9 ng/mL, PLGA @ Dnmt3aos^{smartsilencer}Total IgE in the plasma of treated mice increased to 813.67 + -53.9 ng/mL, while EM-PLGA @ Dnmt3aos^{smartsilencer}The treated mice were much lower, 598.32. + -. 63.17 ng/mL. At the same time, the levels of Der f 1-specific IgG1 and IgG2a were also reduced (fig. 6C-D). At the same time, with Dnmt3aos^{smartsilencer}EM-PLGA @ Dnmt3aos in treated mice^{smartsilencer}Eosinophils and macrophages were reduced by 28% and 50% in BAL fluid of treated mice, respectively (fig. 6E).

To further evaluate EM-PLGA @ Dnmt3aos^{smartsilencer}The effect on asthma pathogenesis, we studied AHR by measuring the lung resistance (Rn) response of mechanically ventilated mice to increasing concentrations of acetylcholine. As expected, with free Dnmt3aos^{smartsilencer}In contrast, EM-PLGA @ Dnmt3aos^{smartsilencer}The treated mice exhibited acetylcholineThe Rn value of the response of (a) is significantly reduced (fig. 6F).

Given the key role of M2 macrophages in mediating asthma pathogenesis, we used free Dnmt3aos in AA^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}Or EM-PLGA @ Dnmt3aos^{smartsilencer}Macrophages were detected after treatment. Flow cytometry revealed EM-PLGA @ Dnmt3aos^{smartsilencer}The number of M2 macrophages in BALF of treated mice was significantly reduced as shown by CD206 expression (fig. 7A). From EM-PLGA @ Dnmt3aos^{smartsilencer}PCR analysis of BALF samples from treated mice showed binding to free Dnmt3aos^{smartsilencer}The expression of M2-labeled Arginase-1 was reduced by 0.6-fold compared to treated mice (FIG. 7B). Similar results were also observed for the other M2-labeled FIZZ-1, although YM-1 expression did not differ significantly between them. EM-PLGA @ Dnmt3aos^{smartsilencer}Injection of (a) also affected the production of M1 marker (fig. 7C). Taken together, these data indicate that EM-PLGA @ Dnmt3aos^{smartsilencer}By inhibiting polarization of M2 macrophages in the airways, mice were protected from Der f 1-induced allergic airway inflammation.

2.9 EM-PLGA@Dnmt3aos^{smartsilencer}The treatment does not generate obvious side effect on the whole immune function

Briefly, mice were randomly divided into 4 groups (n ═ 5) and injected intravenously. Injection into PBS, Dnmt3aos^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}Or EM-PLGA @ Dnmt3aos^{smartsilencer}. Mice were anesthetized and euthanized 24 hours after the last dose. Paraffin sectioning of major organs (including heart, spleen, liver and kidney) was performed with H&And E, dyeing. The results (FIG. 8A) show that EM-PLGA @ Dnmt3aos compared to the PBS group^{smartsilencer}The group showed almost no obvious pathological abnormalities, indicating good biosafety in vivo.

In addition, the anti-tumor ability of mice is commonly used to monitor the overall immune function of humansIs one of the indexes of (1). AA mice were s.c. injected with a549 cells (1 × 106 cells per mouse) in theright groin 1 day after the first immunization withDer f 1. Then, the mice were randomized into three groups (seven mice per group) and given EM-PLGA @ Dnmt3aos through the tail vein on days 21-27^{smartsilencer}，PLGA@Dnmt3aos^{smartsilencer}Or Dnmt3aos^{smartsilencer}. As shown, tumor growth curves (FIG. 8B) and survival rates (FIG. 8C) of tumor-bearing AA mice did not show significant differences between the treatment groups.

The delivery of siRNA drugs in vivo is a non-negligible problem. Because of the stability and low off-target effects of ASO/siRNA, some ASO/siRNA-mediated gene silencing has successfully achieved clinical translation. However, as polyanions and hydrophilic molecules, naked ASO/siRNA cannot automatically penetrate negatively charged cell membranes to reach the intracellular space where the target mRNA is located. In addition, there is potential for off-target effects and immunogenicity. Thus, complex ASO delivery remains a significant challenge to the development of ASO-based therapeutic approaches. Therefore, the invention designs a biodegradable high-molecular organic compound and exosome membrane compound to realize high-efficiency Dnmt3aos inhibitor delivery, further improve the targeting of the nano-carrier to M2 macrophages in lung tissues and completely overcome the problem that the oligonucleotide nano-carrier falls off from target tissues.

Exosome membrane-modified PLGA NPs were derived from exosomes of M2 macrophages and the contents were removed by repeated freezing and thawing, repeated centrifugation, and then hybridization with PLGA NPs. Wherein the application of the exosome membrane can lead EM-PLGA @ Dnmt3aos^{smartsilencer}Inherits the natural nucleic acid delivery behavior of exosomes and their "homing" properties, while the addition of PLGA NPs improves the stability of the entire delivery system. M2 macrophage pair EM-PLGA @ Dnmt3aos^{smartsilencer}Has long circulation time in vivo and has the capacity of overcoming blood clearance. The tissue and organ distribution results of the EM-PLGA @ Dnmt3 aossmear silencer in mice indicate its good "homing" target in lung tissue. At the same time, we also evaluated EM-PLGA@Dnmt3aos^{smartsilencer}Gene silencing efficiency at cellular and animal levels, and anti-inflammatory effects of AA. The results show that EM-PLGA @ Dnmt3aos^{smartsilencer}Provided Dnmt3aos^{smartsilencer}Has biological activity at cell and animal level, can effectively silence the expression of Dnmt3aos, has obvious anti-inflammatory effect and is nontoxic to normal tissues.

In the present invention we used smart silencer for lncRNA Dnmt3aos silencing, rather than single ASO or siRNA. We used a lncRNA smart silencer inhibitor series consisting of 3 sirnas and 3 ASOs, which had higher sensitivity and inhibition efficiency than the normal sirnas. The results of in vivo and in vitro experiments also confirm that Dnmt3aos^{smartsilencer}The excellent therapeutic effect of (1).

In conclusion, the invention successfully designs EM-PLGA @ Dnmt3aos^{smartsilencer}It is a simple and natural nano-scale drug delivery platform, and can effectively treat AA (allergic asthma).

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Sequence listing

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Claims (8)

1. The macrophage exosome membrane-coated bionic nanoparticle is characterized by being formed by exosome membrane-coated nanoparticles, wherein the nanoparticles are PLGA @ Dnmt3aos nanoparticles with surfaces coupled with polyetherimide PEI (polyetherimide)^{smartsilencer}(ii) a The exosome membrane is separated from M2 macrophage derived from bone marrow; the nano-particle PLGA @ Dnmt3aos^{smartsilencer}From polylactic-co-glycolic acid PLGA and Dnmt3aos^{smartsilencer}And (3) co-emulsification formation.

2. The method for preparing the macrophage exosome membrane-coated biomimetic nanoparticle according to claim 1, comprising the following steps:

1) nanoparticle PLGA @ Dnmt3aos^{smartsilencer}Preparing;

2) preparing a bone marrow M2 macrophage exosome membrane;

3) in nanoparticles PLGA @ Dnmt3aos^{smartsilencer}Covering a bone marrow M2 macrophage exosome membrane to prepare a bionic nanoparticle coated with the macrophage exosome membrane;

in step 1) and2) also included are nanoparticles of PLGA @ Dnmt3aos^{smartsilencer}Coupling the surface with Polyetherimide (PEI).

3. The method for preparing the M2 macrophage exosome membrane-coated biomimetic nanoparticles according to claim 2, wherein the step 1) is specifically as follows: 60 μ g PLGA, 2ug Dnmt3aos^{smartsilencer}Respectively dissolving, mixing and emulsifying to generate primary emulsion; adding an emulsifier into the primary emulsion for further emulsification; collecting nanoparticles PLGA @ Dnmt3aos^{smartsilencer}。

4. The method for preparing the biomimetic nanoparticles coated with the M2 macrophage exosome membrane according to claim 2, wherein the nanoparticles are PLGA @ Dnmt3aos^{smartsilencer}The coupling of the surface and the polyetherimide PEI specifically comprises the following steps: nanoparticles PLGA @ Dnmt3aos^{smartsilencer}Mixing with a solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide to activate the nanoparticle surface; then adding Polyetherimide (PEI) dropwise and stirring for 1 hour; collecting the PEI-coupled nanoparticles.

5. The method for preparing the M2 macrophage exosome membrane-coated biomimetic nanoparticles according to claim 2, wherein the step 2) is specifically as follows: transforming the polarized bone marrow macrophage to M2 type, collecting supernatant of M2 macrophage, centrifuging, precipitating, and removing cell debris; centrifuging the final supernatant to obtain exosomes; suspending the harvested exosome in ice-cold TM buffer solution, and repeatedly freezing and thawing for 5 times; adding 0.25M sucrose for resuspension and centrifuging; the supernatant was collected and further centrifuged to collect the precipitate as an exosome membrane.

6. The method for preparing the M2 macrophage exosome membrane-coated bionic nanoparticle as claimed in claim 2, wherein the PLGA @ Dnmt3aos of which the exosome membrane and the surface are coupled with PEI is prepared by^{smartsilencer}Co-incubation and ultrasonic treatment are carried out to obtain the M2 macrophage exosome membrane coated bionic nano-particles.

7. A therapeutic drug or drug carrier for allergic asthma comprising the nanoparticle of claim 1.

8. Use of the preparation method according to any one of claims 2 to 6 for the preparation of a medicament or a pharmaceutical carrier for the treatment of allergic asthma.

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