CN114099481B

CN114099481B - Atomizing inhalation type glucocorticoid nano-drug and preparation method and application thereof

Info

Publication number: CN114099481B
Application number: CN202210103788.3A
Authority: CN
Inventors: 饶浪
Original assignee: Shenzhen Bay Laboratory
Current assignee: Shenzhen Bay Laboratory
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-24
Anticipated expiration: 2042-01-28
Also published as: CN114099481A

Abstract

The invention discloses an aerosol inhalation type glucocorticoid nano-drug, a preparation method and application thereof. The nano-drug comprises: nanoscale cell membrane vesicles; and a glucocorticoid loaded in the nanoscale cell membrane vesicles. The aerosol inhalation type glucocorticoid nano-medicament can be used for enhancing retention in inflammatory lung and improving targeting property on activated macrophage and dendritic cell, thereby promoting the down-regulation of glucocorticoid cytokine, and inhibiting infiltration of new coronary pneumonia COVID-19 inflammatory cells and lung tissue injury caused by new coronary virus SARS-CoV-2 infection; the nanometer glucocorticoid depends on rich cell factor receptors on neutrophile granulocyte membrane vesicles to neutralize broad-spectrum cell factors, and the lung inflammation is effectively relieved. In addition, the neutrophil membrane vesicle shows better in-vivo safety, and the aerosol inhalation type glucocorticoid nano-drug can effectively relieve osteoporosis caused by glucocorticoid after inhalation delivery.

Description

Atomizing inhalation type glucocorticoid nano-drug and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to an aerosol inhalation type glucocorticoid nano-drug, and a preparation method and application thereof.

Background

It is estimated that up to 12% of codv-19 hospitalized patients require invasive mechanical ventilation, most of which progresses to Acute Respiratory Distress Syndrome (ARDS). Lung histological examination of patients with COVID-19 revealed diffuse alveolar damage with a clear membrane, characteristic of ARDS. In addition, uncontrolled inflammatory states characterized by sustainably assessed levels of inflammatory cytokines (referred to as "cytokine storm") are common in these COVID-19 patients, possibly leading to multiple organ failure. The safe and effective treatment strategy for COVID-19 critically ill patients remains challenging and critical.

Ledford, H. Coronavirus breakthrough: dexamethasone is first drug shown to save lives. Nature 582, 469 (2020) discloses that Dexamethasone (DEX) is the first drug to show life-saving effects on patients with COVID-19. In the largest covd-19 treatment randomized control trial in the world, the use of DEX reduced the number of covd-19 mediated deaths by 35% and by 20% in patients requiring mechanical ventilation and non-ventilated patients requiring oxygen therapy. In addition, DEX treatment shortens hospital stays and increases the likelihood of discharge within 28 days.

However, the use of DEX can lead to serious side effects, such as femoral head necrosis, which limits its use in systemic applications, especially in high dose long-term treatments. At the same time, DEX does not provide a satisfactory level of therapeutic effect on COVID-19.

Disclosure of Invention

The invention aims to provide an aerosol inhalation type glucocorticoid nano-drug, a preparation method and application thereof, which aim to solve the problem that the glucocorticoid such as DEX in the prior art has serious side effect on COVID-19 and improve the treatment effect.

In order to achieve the above object, according to one aspect of the present invention, there is provided an inhaled glucocorticosteroid nano-drug. The aerosol inhalation type glucocorticoid nano-drug comprises: nanoscale cell membrane vesicles; and a glucocorticoid loaded in the nanoscale cell membrane vesicles.

Further, the nano-scale cell membrane vesicle is a cell membrane vesicle of an immune cell; preferably, the immune cell is a neutrophil.

Further, the aerosol inhalation type glucocorticoid nano-drug glucocorticoid comprises one or more selected from the group consisting of methylprednisolone, cortisone, prednisolone, betamethasone and dexamethasone; preferably, the glucocorticoid is dexamethasone.

Furthermore, the aerosol inhalation type glucocorticoid nano-medicament also comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is one or more of nano-scale cell membrane vesicles, phospholipid nanoparticles, polymer nanoparticles and metal oxide nanoparticles; preferably, the pharmaceutically acceptable carrier is a nanoscale cell membrane vesicle.

According to another aspect of the invention, a preparation method of the aerosol inhalation type glucocorticoid nano-drug is provided. The preparation method comprises the following steps: preparing nano-scale cell membrane vesicles; and loading the glucocorticoid into the nano-scale cell membrane vesicles to obtain the aerosol inhalation type glucocorticoid nano-drug.

Further, preparing the nanoscale cell membrane vesicles includes: removing intracellular components of cells, then carrying out ultrasonic treatment, and then extruding by a micro extruder to prepare the nano cell membrane vesicles; preferably, the cell is an immune cell, more preferably, the immune cell is a neutrophil; preferably, the removal of intracellular components of neutrophils is a combined treatment of hypotonic lysis, mechanical disruption and gradient centrifugation to remove intracellular components of neutrophils.

Further, loading the glucocorticoid into the nanoscale cell membrane vesicles comprises loading the glucocorticoid into the nanoscale cell membrane vesicles by means of sonication, incubation, extrusion, or electroporation, preferably sonication or electroporation; preferably, the glucocorticoid comprises one or more selected from the group consisting of methylprednisolone, cortisone, prednisolone, betamethasone, and dexamethasone; more preferably, the glucocorticoid is dexamethasone.

Further, loading glucocorticoids into nanoscale cell membrane vesicles includes: the glucocorticoids were mixed with nanoscale cell membrane vesicles, sonicated, and the sonicated product was centrifuged and washed with PBS to remove free glucocorticoids.

Further, when the glucocorticoid is loaded into the nanoscale cell membrane vesicle, the mass ratio of the glucocorticoid to the nanoscale cell membrane vesicle is 1: 1-1: 10, and preferably 1: 5.

According to another aspect of the invention, the invention provides an aerosol inhalation type glucocorticoid nano-drug and an application of the aerosol inhalation type glucocorticoid nano-drug prepared by the preparation method of the aerosol inhalation type glucocorticoid nano-drug in preparing an aerosol inhalation type medicinal preparation for treating neocoronary pneumonia.

The aerosol inhalation type glucocorticoid nano-medicament comprises a nano-scale cell membrane vesicle, glucocorticoid loaded in the nano-scale cell membrane vesicle and a carrier. By applying the aerosol inhalation type glucocorticoid nano-medicament, on one hand, because a large amount of chemokine receptors and an aerosol inhalation delivery strategy are arranged on a cell membrane vesicle, particularly a neutrophil membrane vesicle, the aerosol inhalation type glucocorticoid nano-medicament can enhance the retention of inflammatory lung and improve the targeting property to activated macrophage and dendritic cells, thereby promoting the down-regulation effect of glucocorticoid cytokine and inhibiting the infiltration of new coronary pneumonia COVID-19 inflammatory cells and the damage of lung tissues caused by the infection of new coronary virus SARS-CoV-2; on the other hand, the nanometer glucocorticoid effectively relieves the lung inflammation by neutralizing broad-spectrum cytokines by relying on abundant cytokine receptors on the neutrophil membrane vesicles. The synchronous down-regulation and neutralization of inflammatory cytokines is effective against the COVID-19 cytokine storm. In addition, the neutrophil membrane vesicle shows better in-vivo safety, and the aerosol inhalation type glucocorticoid nano-drug can effectively relieve osteoporosis caused by glucocorticoid after inhalation delivery.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows the preparation and characterization of Neu-EVMs. Wherein, a shows the flow cytometric analysis results of neutrophil purity and apoptosis; b shows neutrophil Wright-Giemsa staining (scale bar 50 μm). c shows TEM images of the neutrophils (i, ii, iii) Neu-EVMs (scale bar of (i) 2 μm, (ii) 200 nm, (iii) 50 nm, respectively). d shows the size distribution of the Neu-EVMs. e shows the stability of Neu-EVMs in PBS and 10% fetal calf serum for 14 days. The SDS-PAGE protein analysis of the neutrophil lysates and the Neu-EVMs is shown. g show Western blot analysis of chemokine receptors and cytokine receptors including CCR2, CXCR2, CXCR4, IL-6R, IL-1. beta.R and TNF- α R, among others, in RBC-EVMs and Neu-EVMs. h shows the binding capacity analysis of RBC-EVMs, Neu-EVMs to TNF-alpha, IL-6 and IL-1 beta. All data are expressed as mean ± standard deviation (n = 3).

Figure 2 shows that nanoDEX has enhanced targeting of inflammatory cells in vitro, improving cytokine down-regulation. Wherein a shows a schematic of a nanoDEX consisting of Neu-EVMs loaded with DEX. b shows the drug loading content after sonication and the efficiency of loading DEX into Neu-EVMs. c shows the drug loading efficiency of different drug loading strategies with an initial Neu-EVMs of 200 μ g. d shows drug release from DEX-Neu-EVMs. e shows fluorescence images (scale bar 100 μm) of non-activated or LPS activated RAW 264.7 cells after incubation with DEX-RBC-EVMs or DEX-Neu-EVMs. DEX-RBC-EVMs and DEX-Neu-EVMs were labeled with DiO (green) before incubation. f and g show fluorescence intensity analysis of DiO (green) in RAW 264.7 (f in FIG. 2) and DC2.4 (g in FIG. 2) cells after treatment. h shows TNF- α, IL-6 levels in the supernatant of LPS-activated RAW 264.7 cells after treatment. i shows ROS levels. j shows the dose-dependent effect of DEX-Neu-EVMs on TNF- α, IL-6 and ROS expression in the supernatant of LPS-activated RAW 264.7 cells. All data are expressed as mean ± standard deviation (n = 3 (b-d), n = 4 (f-j)). Statistical significance was calculated using the common one-way analysis of variance Dunnett's test (c, h, i) or Tukey's test (f, g). P < 0.05, P < 0.01, P < 0.001.

Figure 3 shows the enhanced accumulation and retention of nanoDEX in the inflamed lung following inhalation delivery. Wherein a shows a schematic diagram of the inhalation delivery of DEX-Neu-EVMs in mice. b shows inhalation deliveryDroplet size distribution containing DEX-Neu-EVMs after delivery. c shows the hydrodynamic diameter and Zeta potential of the DEX-Neu-EVMs before and after inhalation delivery. d shows drug release of DEX-Neu-EVM after inhalation delivery. e shows fluorescence images (scale bar 50 μm) showing lung accumulation of RBC-EVMs or Neu-EVMs after intravenous injection or inhalation delivery. RBC-EVMs and Neu-EVMs were labeled with DiO (green) prior to inhalation delivery. f shows the biodistribution of RBC-EVMs and Neu-EVMs in major organs 24 hours after intravenous injection or inhalation delivery in healthy mice. g shows the pulmonary accumulation of RBC-EVMs or Neu-EVMs in healthy mice or in LPS-infected mice 24 hours after inhalation delivery. h shows the pulmonary accumulation of RBC-EVMs or Neu-EVMs at various time points after LPS-infected mice inhalation delivery. i shows the biodistribution of DEX-RBC-EVMs or DEX-Neu-EVMs in major organs in LPS infected mice 24 hours after intravenous injection or inhalation delivery. All data are expressed as mean ± standard deviation (n = 3 (c, d), n = 4 (f-i)). Statistical significance was calculated using the general one-way analysis of variance Tukey's test (f, g, i) or Dunnett's test (h). *P < 0.05; **P < 0.01; ***P <0.001. i.h. inhaled delivery, i.v.: and (4) performing intravenous injection.

Figure 4 shows that nanoDEX can reduce lung injury in a mouse model of acute pneumonia and inhibit the decrease in bone density in a rat model of osteoporosis. Wherein, a shows a schematic diagram of a treatment scheme of the acute pneumonia mouse model. b shows cytokine production in lung homogenates after treatment. c shows post-treatment H&E staining lung tissue (scale bar 50 μm). d shows a schematic representation of the GIO rat model treatment protocol. e shows the change in body weight after treatment. f shows BMD analysis of whole body, femoral shaft and femoral neck. g shows the analysis of the weight, dry weight, ash/dry ratio of the ashes after the treatment. h shows Ca in the bone after treatment²⁺And P⁵⁺And (4) content. All data are expressed as mean ± standard deviation (n = 6 (b) and n = 8 (e-h)). Statistical differences were calculated using the common one-way ANOVA Dunnet's test (b, f-h) or the two-way ANOVA Tukey's test (e). P< 0.05；**P < 0.01；***P <0.001. q.o.d.: every other day.

FIG. 5 shows that nanodEX can reduce lung inflammation and injury in K18-hACE2 transgenic mice infected with live SARS-CoV-2. Wherein a shows a schematic representation of the treatment plan for K18-hACE2 transgenic mice infected with live SARS-CoV-2. b shows qRT-PCR analysis of lung cytokine gene expression after treatment. c shows H & E staining of lung tissue after treatment (scale bar 100 μm). d transcriptome analysis showed modulation of the new coronavirus infection-associated pathways in the lungs after treatment. e shows a multiplex immunoassay of cytokine/chemokine levels in serum after treatment. All data are expressed as mean ± standard deviation (n = 5). Statistical significance was calculated using the general one-way analysis of variance of Dunnet's test. P < 0.05; p < 0.01; p < 0.001.

Figure 6 shows in vitro toxicity. Wherein (a) RAW 264.7 and (b) DC2.4, after incubation with RBC-EVMs or Neu-EVMs at the indicated concentrations, have a cell viability.

FIG. 7 shows the binding capacity of different EVMs derived from Red Blood Cells (RBC), Platelets (PLT), White Blood Cells (WBC), macrophages (macrophage), Dendritic Cells (DC), T cells and neutrophils to IL-6.

FIG. 8 shows HPLC analysis of Neu-EVMs, DEX and DEX-Neu-EVMs.

FIG. 9 shows fluorescence images (scale bar 100 μm) of non-activated or LPS-activated DC2.4 cells after incubation with DEX-RBC-EVMs or DEX-Neu-EVMs. Before incubation EX-RBC-EVMs and DEX-Neu-EVMs were labeled with DiO (green).

FIG. 10 shows the in vivo toxicity of Neu-EVMs. a shows a schematic of a treatment protocol. Mice were repeatedly given PBS or PBS containing Neu-EVMs by inhalation every other day, and blood samples and major organs were collected from mice 15 days after treatment for blood biochemical, whole blood and histological analysis. b shows the body weight change curve of the mice. c shows blood biochemistry and whole blood cytome analysis. d shows the H & E stained section image of the major organ (scale bar: 200 μm). ALT: alanine aminotransferase; AST: aspartate aminotransferase; high mountain: alkaline phosphatase; BUN: blood urea nitrogen; WBC: (ii) a leukocyte; RBC: red blood cells; PLT: a platelet; HGB: (ii) hemoglobin; HCT: a hematocrit; MCV: mean red blood cell volume; MCH: mean corpuscular hemoglobin; MCHC: mean hemoglobin concentration of red blood cells.

FIG. 11 shows fluorescence images showing the accumulation of RBC-EVMs or Neu-EVMs (scale bar 50 μm) in (a) spleen, (b) liver, (c) kidney and (d) heart following intravenous or inhalation administration. RBC-EVMs and Neu-EVMs were labeled with DiO (Green fluorescence) prior to delivery.

A in FIG. 12 shows the in vivo biodistribution of RBC-EVMs or Neu-EVMs in the major organs of healthy mice or LPS-infected mice within 24 hours after intravenous injection or inhalation administration. b shows the in vivo biodistribution of RBC-EVMs or Neu-EVMs in major organs at different time points after the administration of LPS-infected mice by inhalation.

Figure 13 shows flow cytometry analysis of immune cells in lung homogenates after treatment. a shows a gating strategy illustrating flow cytometry. b shows flow cytometry analysis of CD3⁺CD45⁺T cells. c shows CD14⁺CD45⁺Inflammatory infiltrating monocytes/macrophages. d shows CD11b^lowF4/80^hiResident macrophages.

Figure 14 shows qRT-PCR analysis of pulmonary cytokine/chemokine expression after treatment.

Figure 15 shows a Luminex analysis of pulmonary cytokine/chemokine expression after treatment.

FIG. 16 shows a comparison of Neu-EVMs versus loading efficiency of different glucocorticoids. All data are expressed as mean ± standard deviation (n = 3).

Figure 17 shows a parallel comparison of different EVMs of Red Blood Cells (RBC), Platelets (PLT), White Blood Cells (WBC), macrophages (Macrophage), Dendritic Cells (DC), T cells (T cell) and neutrophils (Neu) to DEX loading efficiency. All data are expressed as mean ± standard deviation (n = 3).

Note: in the drawings of the present application, the terms "left", "left one", "left two", "middle", "right", and the like are used for similar descriptions, and those skilled in the art can understand, in combination with specific experiments, the positions of different columns in a set of column diagrams, for example, f in fig. 3, the first set of column diagrams includes 4 columns, "left one" refers to the first column from the left, and "left two" refers to the second column from the left, and so on; in the second set of bar graphs from left to right, the same is true for the sequence, "left one" means the first bar graph from left, and "left two" means the second bar graph from left, and so on.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

Noun explanation

DEX: dexamethasone.

Neu-EVMs: a neutrophil membrane vesicle.

RBC-EVMs: erythrocyte membrane vesicles.

nanoDEX: also known as DEX-Neu-EVMs, inhalable dexamethasone nanoformulations, including neutrophile membrane vesicles (Neu-EVMs) and DEX loaded in the neutrophile membrane vesicles.

DEX-RBC-EVMs: an inhalable dexamethasone nano-preparation comprises erythrocyte membrane vesicles and DEX loaded in the erythrocyte membrane vesicles.

PBS: phosphate buffered saline.

The invention provides the following technical scheme aiming at the technical problems that the use of glucocorticoid in the prior art may cause serious side effects, such as femoral head necrosis, unsatisfactory treatment effect of glucocorticoid on COVID-19 and the like.

According to an exemplary embodiment of the present invention, an inhaled glucocorticosteroid nanomedicine is provided. The aerosol inhalation type glucocorticoid nano-medicament comprises: the composition comprises a nanoscale cell membrane vesicle, glucocorticoid loaded in the nanoscale cell membrane vesicle and a pharmaceutically acceptable carrier. Among them, preferably, the nanoscale cell membrane vesicle is a cell membrane vesicle of an immune cell, and more preferably, the immune cell is a neutrophil. The glucocorticoid comprises one or more selected from the group consisting of methylprednisolone, cortisone, prednisolone, betamethasone, and dexamethasone; preferably, the glucocorticoid is dexamethasone.

By applying the aerosol inhalation type glucocorticoid nano-medicament, on one hand, because a large amount of chemokine receptors and an aerosol inhalation delivery strategy are arranged on a cell membrane vesicle, particularly a neutrophil membrane vesicle, the aerosol inhalation type glucocorticoid nano-medicament can enhance the retention of inflammatory lung and improve the targeting property to activated macrophage and dendritic cells, thereby promoting the down-regulation effect of glucocorticoid cytokine and inhibiting inflammatory cell infiltration and lung injury caused by SARS-CoV-2 infection; on the other hand, the nano-glucocorticoid neutralizes a broad-spectrum cytokine by virtue of a cytokine receptor which is abundant on a neutrophil membrane vesicle. The synchronous down-regulation and neutralization of inflammatory cytokines is effective against the COVID-19 cytokine storm. In addition, the neutrophil membrane vesicle shows better in-vivo safety, and the aerosol inhalation type glucocorticoid nano-drug can effectively relieve osteoporosis caused by glucocorticoid after inhalation delivery.

The aerosol inhalation type glucocorticoid nano-drug also comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is one or more of nano-scale cell membrane vesicles, phospholipid nanoparticles, polymer nanoparticles and metal oxide nanoparticles; preferably, the pharmaceutically acceptable carrier is a nanoscale cell membrane vesicle.

According to an exemplary embodiment of the present invention, a method for preparing an inhaled glucocorticoid nanomedicine by nebulization is provided. The preparation method comprises the following steps: preparing nano-scale cell membrane vesicles; and loading the glucocorticoid into the nano-scale cell membrane vesicles to obtain the aerosol inhalation type glucocorticoid nano-drug.

Typically, preparing a nanoscale cell membrane vesicle includes: removing intracellular components of cells, then carrying out ultrasonic treatment, and then extruding by a micro extruder to prepare the nano cell membrane vesicles; preferably, the cell is an immune cell, more preferably, the immune cell is a neutrophil; preferably, the removal of intracellular components of neutrophils is a combined treatment of hypotonic lysis, mechanical disruption and gradient centrifugation to remove intracellular components of neutrophils.

In one embodiment of the present invention, loading the glucocorticoid into the nanoscale cellular membrane vesicles comprises loading the glucocorticoid into the nanoscale cellular membrane vesicles by means of sonication, incubation, extrusion, or electroporation, preferably sonication or electroporation; preferably, the glucocorticoid comprises one or more selected from the group consisting of methylprednisolone, cortisone, prednisolone, betamethasone, and dexamethasone; more preferably, the glucocorticoid is dexamethasone. In a preferred embodiment of the invention, loading the glucocorticoid into the nanoscale cell membrane vesicles comprises: the glucocorticoids were mixed with nanoscale cell membrane vesicles, sonicated, and the sonicated product was centrifuged and washed with PBS to remove free glucocorticoids. Preferably, when the glucocorticoid is loaded into the nanoscale cell membrane vesicle, the mass ratio of the glucocorticoid to the nanoscale cell membrane vesicle is 1: 1-1: 10, for example, 1:1, 1:2, 1:5, 1:10, preferably 1:5, and the drug loading efficiency is optimal.

According to an exemplary embodiment of the present invention, there is provided a use of the inhaled and nebulized glucocorticoid nanopharmaceutical of the present invention for the preparation of an inhaled and nebulized pharmaceutical formulation for the treatment of neocoronary pneumonia.

According to an exemplary embodiment of the present invention, there is provided a use of the aerosolized inhaled glucocorticoid nanomedicine of the present invention for treating neocoronary pneumonia.

The advantageous effects of the present invention will be further explained below in conjunction with the test data. The steps or reagents described in the following examples, if not described in detail, can be performed using methods or reagents conventional in the art.

Examples

1. Cell and animal preparation

Mouse cell lines of RAW 264.7 macrophage-like cells and DC2.4 dendritic cell-like cells were obtained from the American Type Culture Collection (ATCC) and cultured according to the guidelines provided by the ATCC. ICR mice (male, 6 weeks old) and SD rats (female, 3 months old) were purchased from huntinglidaceae experimental animals ltd. K18-hACE2 mice (14 weeks old) were purchased from Chinesota Biotech, Inc. (China). All animal care and experimental procedures were approved by the Institute for Animal Care and Use Committee (IACUC) and the institute for microbiology and epidemiology, the shenzhen bay laboratory facility, southern medical university, according to the guidelines for experimental animal protection.

2. Preparation of nanoDEX

The preparation of nanodEX (DEX-Neu-EVMs) comprises three steps: 1) isolation of neutrophils, 2) obtaining of Neu-EVMs, 3) loading of DEX into Neu-EVMs.

First, mature neutrophils were extracted from mouse bone marrow and purified by density gradient centrifugation. Specifically, first, fresh bone marrow was washed from bone with RPMI1640 (Gibco, USA), filtered with a 70 μm filter, centrifuged at 450 g for 10 min, continuously resuspended with erythrocyte lysate and PBS, and then neutrophils were separated from the mixture by Percoll gradient method. Briefly, PBS of the obtained cell microspheres was added to Percoll working solution at 78%, 70% and 58% (v/v) with a Percoll gradient. After centrifugation at 490 g for 30 minutes, neutrophils were recovered from the 58% and 70% interfaces and cultured in RPMI1640 containing 10% fetal bovine serum with exosomes removed. Purity of cell population (Ly 6G)⁺CD11b⁺) And apoptosis (Beyotime, china) were identified using the CytoFLEX flow cytometer (Beckman Coulter, usa) and the CytoExpert software (Beckman Coulter, usa). The isolated neutrophils were stained with Wright-Giemsa (Solarbio, China) and visualized under an optical microscope (IX 71, Olympus, Japan). Flow cytometric analysis showed that the purity of the isolated neutrophils (Ly 6G +/CD11b +) was 91.21% (see a in FIG. 1), and only a small fraction of the cells underwent early apoptosis (Annexin V)⁺/PI^-) And late apoptosis (Annexin V)⁺/PI⁺) (see a in fig. 1). Furthermore, Wright-Giemsa staining showed purple color, a typical manifestation of neutrals and neutrophil polymorphonuclear morphology (see FIG. 1, b).

To obtain Neu-EVMs, the intracellular components of the isolated neutrophils can be removed by a combination of hypotonic lysis, mechanical disruption and gradient centrifugation. Subsequently, Neu-EVMs were prepared by a series of sonications and extrusion through a multi-nanopore membrane using a micro-extruder. Specifically, in this example, to obtain Neu-EVMs, isolated neutrophils were disrupted with a hypotonic lysis buffer (pH = 7.4, 0.25 × concentration, Gibico, usa) and a Dounce homogenizer. Treatment with DNase and RNase (Invitrogen, USA) for 30 min at room temperature, centrifugation at 3,200 g for 5 min, and then 20000g Centrifuging for 30 min, 80000gThe supernatant was enriched by centrifugation for 1.5 h. Subsequently, minicell membrane vesicles were collected, washed 3 times with PBS (pH = 7.4, 1 × concentration) mixed protease inhibitor tablets (rotz), sonicated for 5 min, and finally gradually extruded through 400-, 200-, and 100 nm nanoporous polycarbonate membranes on a micro-extruder (Avanti Polar Lipids, usa). Dynamic Light Scattering (DLS) was used to characterize the hydrodynamic diameter and zeta potential of Neu-EVMs. The morphology of the Neu-EVMs was characterized by transmission electron microscopy, TEM. The transmission electron microscope samples were negatively stained with uranyl acetate. The stability of Neu-EVMs in PBS and 10% fetal calf serum was monitored for 2 weeks with DLS.

As a control, erythrocyte-derived extracellular vesicles (RBC-EVMs) and other blood cell-and immune cell-derived extracellular vesicles (platelets: PLT-EVMs, leukocytes: WBC-EVMs, macrophages: Macrophage-EVMs, dendritic cells: DC-EVMs, T cells: T cell-EVMs) were also prepared using the same method.

Transmission Electron Microscopy (TEM) visualization and Dynamic Light Scattering (DLS) analysis showed that the Neu-EVMs were circular lipid droplets with an average size of 90 nm (c and d in FIG. 1). Notably, Neu-EVMs were stable in PBS buffer for at least two weeks (e in fig. 1) and showed very little cytotoxicity (fig. 6), ensuring downstream in vitro and in vivo experiments.

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the neutrophile lysate from the Dounce homogenizer lysis and Neu-EVMs were added to the protein extraction buffer and the protein content was determined using the bicinchoninic acid (BCA) kit. The samples were heated at 95 ℃ for 5 min, and each 20 μ g sample was loaded into 10% sodium dodecyl sulfate-polyacrylamide gel. The samples were run at 120V for 2 hours and the gels were stained with coomassie brilliant blue for 4 hours and then destained overnight before observation. Western blotting protein samples of RBC-EVMs and Neu-EVMs were denatured and loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels. The isolated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% (w/v) skim milk at 25 ℃ for 1 hour, incubated with C-C motif chemokine receptor 2 (CCR 2), C-X-C motif chemokine receptor 2 (CXCR 2), CXCR4, IL-6R, IL-1 β R, and TNF- α R primary antibodies (all from Abcam) overnight at 4 ℃ and then incubated with HRP-conjugated secondary antibodies (Thermo Fisher, USA), and blots were prepared using the West Pico PLUS chemiluminescence substrate kit (Thermo Fisher, USA).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that Neu-EVMs inherited specific protein content from neutrophils (FIG. 1, f). Furthermore, Western blotting experiments confirmed the presence of key chemokine and cytokine receptors on Neu-EVMs, but not on erythroid EVMs (RBC-EVMs) (g in FIG. 1), including C-C motif chemokine receptor 2 (CCR 2), C-X-C motif chemokine receptor 2 (CXCR 2), CXCR4, IL-6 receptor (IL-6R), IL-1 beta receptor (IL-1 beta R), and TNF-alpha receptor (TNF-alpha R).

To determine the binding capacity of Neu-EVMs to cytokines, 100. mu.L of PBS containing different concentrations of RBC-EVMs and Neu-EVMs were mixed with 100. mu.L of PBS containing 2 ng of IL-6, IL-1. beta. and TNF-. alpha. and incubated at 37 ℃ for 2 h. 15,000gThe RBC-EVMs and Neu-EVMs were removed by centrifugation for 15 minutes, and the concentrations of IL-6, IL-1. beta. and TNF-. alpha. in the supernatant were determined using a corresponding enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, USA) (h in FIG. 1). The results demonstrate that Neu-EVMs effectively adsorb inflammatory factors, including IL-6, IL-1. beta. and TNF-. alpha., in a dose-dependent manner by virtue of the cytokine receptors present in the above Neu-EVMs, which is expected to effectively alleviate the cytokine storm of Neu-EVMs.

Different EVMs and IL-6 neutralization performance were tested using the same method described above. The inventors found that Neu-EVMs showed better IL-6 neutralizing performance compared to other EVMs derived from blood cells and immune cells (figure 7).

Subsequently, DEX was loaded into Neu-EVMs by sonication to prepare nanodEX (a in FIG. 2).

The procedure for loading DEX into Neu-EVMs is as follows: 80 μ g of DEX was mixed with the indicated amount of Neu-EVMs and sonicated for 20 minutes. DEX-Neu-EVMs were centrifuged and washed repeatedly with PBS to remove free DEX. All washes were collected and the concentration of DEX was measured using High Performance Liquid Chromatography (HPLC). The loading efficiency was calculated from the difference between the initial and the remaining DEX in the supernatant.

The loading efficiency of Neu-EVMs loaded with methylprednisolone, cortisone, prednisolone, betamethasone was measured in the same way (fig. 16). DEX was also loaded into RBC-EVMs as well as PLT-EVMs, WBC-EVMs, Macrophage-EVMs, DC-EVMs, Tcell-EVMs using the same method, and the drug loading efficiency was determined by High Performance Liquid Chromatography (HPLC) (FIG. 17).

The release of DEX from Neu-EVMs was studied by dialysis. Briefly, DEX-Neu-EVMs containing 100. mu.g of free DEX were loaded into dialysis bags of limited molecular weight to 10 kDa, which were immersed in 50 mL PBS and incubated at 37 ℃ for various periods of time with constant shaking at 70 rpm. At the indicated time points, 100. mu.L of external medium was removed and an equal amount of fresh PBS was added. The concentration of DEX released in the dialysis medium was determined by high performance liquid chromatography.

The drug loading efficiency for DEX was determined by High Performance Liquid Chromatography (HPLC) (fig. 8 and 17), and as a result, it was found that DEX-Neu-EVMs were prepared under the condition that the mass ratio of the initial DEX and Neu-EVMs was 1:5, and the optimal drug loading efficiency was about 9.71% (b in fig. 2). This example also tested the effect of loading strategies such as incubation, sonication, extrusion and electroporation on efficiency, and found that sonication and electroporation outperformed incubation and extrusion in the preparation of DEX-Neu-EVMs (c in fig. 2). In addition, about 71.16% of DEX leaked from the Neu-EVMs within 48 hours (d in FIG. 2), indicating a sustained release profile of DEX-Neu-EVMs.

3. In vitro experiments, the targeting of the nanodEX to inflammatory cells is enhanced, and the anti-inflammatory effect is enhanced

Macrophages and dendritic cells are known to be involved in the COVID-19 cytokine storm, while SARS-CoV-2 infection results in activation of macrophages and dendritic cytokines and excessive secretion of inflammatory cytokines. In this example, DEX-Neu-EVMs were fluorescently labeled and incubated with RAW 264.7 macrophage-like cells and DC2.4 DC-like cells. DEX-loaded RBC-EVMs (DEX-RBC-EVMs) were tested as controls because they have a similar particle structure to DEX-Neu-EVMs, but RBCs have fewer cytokine receptors than neutrophils.

Specifically, RAW 264.7 or DC2.4 cells were seeded at 50% confluence in 12-well tissue culture plates and cultured overnight. The cell culture medium was changed and 100 ng/mL Lipopolysaccharide (LPS) from E.coli (Sigma-Aldrich, USA) was added. After 4 hours of stimulation, cells were washed with PBS, fixed with 10% phosphate buffered formalin (Thermo Fisher, usa) for 10 minutes, and blocked with 1% bovine serum albumin for 1 hour. Then incubated with 0.2 mg/mL DIO-labeled DEX-RBC-EVMs or DEX-Neu-EVMs for 60 s in PBS at 4 ℃. After incubation, cells were washed 5 times with ice-cold PBS, stained with 4, 6-diamino-2-phenylindole (DAPI), and imaged with a confocal laser scanning microscope. For flow cytometry analysis, cells were scraped after washing with PBS and analyzed by flow cytometry.

The inventors found that after incubation and washing, significant fluorescence was observed on cells incubated with DEX-Neu-EVMs, while no significant fluorescence was observed on cells incubated with DEX-RBC-EVMs (e-g in fig. 2 and fig. 9). Notably, Lipopolysaccharide (LPS) -activated cells showed a significant increase in fluorescence intensity upon incubation with DEX-Neu-EVMs compared to non-incubated cells (f and g in fig. 2). These results indicate that DEX-Neu-EVMs are able to target inflammatory cells, which may be related to the specific interaction between cytokine receptors on Neu-EVMs and cytokine molecules over-expressed by activated macrophages and DCs.

To test the anti-inflammatory therapeutic effect of DEX-Neu-EVMs, RAW 264.7 cells were stimulated with LPS to mimic the inflammatory state induced by viral infection in vitro. Specifically, RAW 264.7 cells were first cultured with 100 ng/mL LPS for 1 h, then RBC-EVMs, Neu-EVMs, DEX or DEX-Neu-EVMs were added at the corresponding concentrations, followed by incubation for 30 min. The supernatant was collected, centrifuged at 15,000 g for 15 min, and the nanoparticles were removed. The supernatants were assayed for TNF- α and IL-6 by ELISA. Reactive Oxygen Species (ROS) production was detected using an ROS detection kit. After treatment with DEX-Neu-EVMs, they were soaked with 20 mM 2 ', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 minutes at 37 ℃ and then washed three times with serum-free medium. And (3) detecting the fluorescence signal intensity of DCFH-DA oxidation conversion fluorescent dichlorofluorescein by adopting flow cytometry, and detecting the change of intracellular ROS level.

The inventors found that after stimulation, the secretion of TNF-. alpha.and IL-6 was significantly improved (h, i in FIG. 2), confirming the activated inflammatory state. IL-1 β production was not significantly affected and may be associated with negative regulation of NF-. kappa.B in IL-1 β secretion. Based on this model, DEX-Neu-EVMs showed significant anti-inflammatory effects in a dose-dependent manner (h-j in fig. 2), probably due to a powerful two-step anti-inflammatory strategy: the first step is DEX down-regulation of cytokines and the second step is Neu-EVMs neutralization of cytokines. In addition, treatment of nanoDEX also reduced the production of Reactive Oxygen Species (ROS) (i, j in fig. 2), indicating effective relief of systemic inflammation.

4. Enhanced retention of nanoDEX in inflamed lungs following inhalation

Potential systemic toxicity has been a major problem with drug delivery systems. To investigate the in vivo toxicity of Neu-EVMs, ICR mice were inhaled PBS or PBS containing 200. mu.g Neu-EVMs every other day using a portable nebulizer (a in FIG. 3 and a in FIG. 10). Specifically, body weight of toxic mice in vivo was monitored and recorded over 15 days, and mice were euthanized on day 15 after the first inhalation delivery. Collecting blood samples, and carrying out whole blood and serum biochemical detection by adopting a blood biochemical automatic analyzer. The main tissue specimens were routinely divided into sections as described above, and histologically examined with hematoxylin and eosin staining (H & E).

The inventors found that no death or significant body weight difference was observed within 15 days in the PBS group and the Neu-EVMs group (b in FIG. 10), indicating that Neu-EVMs did not cause detectable side effects. Results of serum biochemical, whole blood and histological examinations further showed that inhalation delivery had no significant side effects on experimental animals (c and d in fig. 10), suggesting biosafety of Neu-EVMs during delivery.

After this, the in vivo performance of DEX-Neu-EVMs after inhalation delivery was further investigated (a in FIG. 3). After inhalation, the droplets containing DEX-Neu-EVMs were approximately 3 μm in diameter (b in FIG. 3), suitable for inhalation delivery of the particular drug to the alveoli. Furthermore, the inhalation process had no significant effect on both the physicochemical properties of Neu-EVMs and the drug release profile of DEX-Neu-EVMs (c, d in fig. 3), ensuring downstream in vivo experiments for inhalation delivery. Thereafter, mice were injected with fluorescently labeled Neu-EVMs by inhalation delivery and the in vivo biodistribution of Neu-EVMs was studied. Specifically, ICR mice were intravenously injected or inhaled using a commercial portable nebulizer to deliver Hank's Balanced Salt Solution (HBSS) containing 200 μ g of DIO-labeled RBC-EVMs or Neu-EVMs. After 24 hours of treatment, mice were euthanized, and all organs were carefully harvested and cryosectioned for further immunofluorescence analysis of the in vivo biodistribution of Neu-EVMs following inhalation delivery. In order to detect the inflammation targeting of Neu-EVMs, an acute pneumonia model is established. Mice were anesthetized and then placed in a supine position and HBSS containing 8 mg/kg LPS was administered by inhalation delivery. 24 h after LPS stimulation, mice with acute pneumonia were given HBSS containing 200. mu.g of DIO-labeled RBC-EVMs or Neu-EVMs by inhalation delivery. After 24 hours of treatment, mice were euthanized and all organs were carefully collected for immunofluorescence analysis. Acute pneumonia mice were also given HBSS containing 200. mu.g of DIO-labeled RBC-EVMs or Neu-EVMs. Mice were euthanized 24, 48 and 72 h post-treatment, and all organs were carefully harvested for immunofluorescence analysis. Acute pneumonia mice also received inhaled delivery of DEX-RBC-EVMs or DEX-Neu-EVMs containing 200 μ g DIO markers. After 24 h of treatment, mice were euthanized and all organs carefully collected for biodistribution analysis by high performance liquid chromatography.

The inventors found that the inhalation delivery strategy significantly improved the accumulation of Neu-EVMs in the lungs compared to intravenous injection (e, f in fig. 3). In addition to the lung, Neu-EVMs were also detected in spleen, liver and kidney, indicating clearance through the reticuloendothelial system and metabolism through the body (f in FIG. 3 and FIG. 11).

Given the abundance of cytokine receptors on Neu-EVMs (g in fig. 1), and the ability of Neu-EVMs to target inflammatory macrophages and DCs in vitro (f, g in fig. 2), Neu-EVMs were further tested in vivo for their ability to target inflammatory lung tissue. Specifically, the mice were anesthetized and placed in a supine position, and then HBSS containing 8 mg/kg LPS was delivered into the lungs by inhalation. 4 h after inhalation, mice were given HBSS or HBSS containing DEX (1 mg/kg) or equal amounts of DEX-Neu-EVMs and Neu-EVMs by inhalation delivery. After 24 hours of LPS stimulation, all mice were euthanized and lung tissue was routinely collected. A portion of lung tissue was weighed and homogenized with lung homogenate. IL-6, IL-1. beta. and TNF-. alpha. were detected in lung homogenates using the corresponding ELISA kits. The other lung tissue was fixed with 4% neutral buffered formalin, paraffin was prepared, and 4 μm sections were taken. The remaining lung tissue was used for flow cytometry detection.

Flow cytometry was performed using the following method: lung tissue was perfused and dissected into individual lung lobes. The resulting lung lobes were washed in PBS in a petri dish, placed in a C-tube containing mixed enzymes, cut into small pieces, digested for 1 hour in an incubator at 37 ℃, and then filtered with a 70 μm cell strainer. Staining cells with fluorescently labeled antibody: Live/Dead (AF 700, Becton Dickinson, United States), CD45 (APC-cy 7, clone 30-F11, Becton Dickinson, USA), CD3 (BV 510, clone 145-2C11, Becton Dickinson, USA), CD14 (APC, clone Sa14-2, USA), CD11b (BB 515, clone M1/70, USA), F4/80 (APC, clone BM8, USA), CD206 (PE, clone BM8, Becton Dickinson). Samples were run on a CytoFLEX flow cytometer (Beckman Coulter, usa) and cytoexpepert software.

The inventors found that Neu-EVMs showed increased accumulation in the inflamed lung in the LPS-induced acute pneumonia model (g in fig. 3 and a in fig. 12). Notably, Neu-EVMs were still found in inflamed lungs 72 hours after a single inhalation delivery (h in fig. 3 and b in fig. 12), indicating that Neu-EVMs were better retained. Furthermore, the biodistribution of DEX-Neu-EVMs in vivo was similar to that of Neu-EVMs (i in FIG. 3). These results indicate that the capacity of DEX-Neu-EVMs to enhance retention in inflammatory lung tissue is due to a specific interaction between cytokine receptors on Neu-EVMs and excessive cytokine secretion in inflammatory lung tissue.

5. The nanoDEX can weaken cytokine storm in an acute pneumonia mouse model and inhibit bone density reduction in an osteoporosis rat model.

The in vivo performance of DEX-Neu-EVMs was tested on a mouse model of acute pneumonia. 4 h after LPS treatment, mice delivered DEX-Neu-EVMs by inhalation (DEX dose 1 mg/kg); after 24 hours of treatment, all mice were euthanized and lung tissue was collected for cytokine detection (a in fig. 4). TNF-. alpha.IL-6 and IL-1. beta. levels were significantly elevated after LPS stimulation in lung homogenates (b in FIG. 4), indicating an inflammatory state in the lung. Notably, DEX-Neu-EVMs significantly reduced cytokine levels in lung homogenates (b in fig. 4), due to a powerful two-step anti-inflammatory strategy: the DEX down-regulates the cytokine in the first step, and the Neu-EVMs neutralize the cytokine in the second step. Lung tissue was analyzed for blood flow and examined histologically. LPS treatment significantly increased CD3 in lung tissue⁺CD45⁺T cells, CD14⁺CD45⁺Inflammatory infiltrating monocytes/macrophages and CD11b^lowF4/80^hiProportion of resident macrophages (figure 13), these cells play a key role in acute pneumonia. The proportion of these critical immune cells was significantly lower in DEX-Neu-EVMs treated mice than mice treated with DEX or Neu-EVMs alone (figure 13). Furthermore, histological results showed that DEX-Neu-EVMs significantly inhibited LPS-induced severe lung injury characterized by alveolar wall thickening, alveolar space disappearance, vasodilation hyperemia, alveolar inflammatory cell infiltration (fig. 4 c). These results indicate that DEX-Neu-EVMs have great potential in inhibiting COVID-19-associated immune disorders and lung injury.

Although DEX has a good anti-inflammatory effect, it, as a glucocorticoid, may cause serious side effects such as femoral head necrosis. To investigate the safety of DEX-Neu-EVMs, a glucocorticoid-induced osteoporosis (GIO) model was established in rats, which were low calcium fed and inhaled DEX or DEX-Neu-EVMs every other day (d in FIG. 4). Specifically, low calcium diet was given daily for 45 consecutive days with either DEX (1 mg/kg) or equal amounts of DEX-Neu-EVMs inhaled. Rat body weight was monitored to reflect its overall health. After 45 days of monitoring, all rats were euthanized, femoral tissues were collected, and bone density (BMD) was measured using a bone densitometry system. All bone tissue was weighed and heated in a muffle furnace for 6 h to further determine the bone tissue ash weight. A small amount of burned bone tissue was added to 10 mL of a perchloric acid concentrated sulfuric acid mixture (v/v, 1: 5). Digesting at high temperature for 4 h, and storing for later use. The contents of calcium and phosphorus in the bone are respectively measured by adopting a flame atomic absorption spectrophotometry and an ultraviolet spectrophotometry.

The inventors observed that the body weight of rats decreased rapidly after DEX treatment, whereas the DEX-Neu-EVMs treated rats stabilized in body weight and gradually remained healthy after 4 weeks (e in FIG. 4). After 6 weeks of monitoring, all rats were euthanized for bone health analysis. Bone density of femoral shaft and neck is obviously reduced after DEX treatment; in contrast, DEX-Neu-EVMs bound by inhalation delivery inhibited DEX-induced decrease in bone density (f in FIG. 4). Furthermore, DEX treatment was found to cause a decrease in bone ash/dry weight and a decrease in bone calcium/phosphorus content (g, h in fig. 4), representing a typical GIO status. Notably, inhalation delivery of DEX-Neu-EVMs significantly protected rats from GIO injury (g, h in fig. 4) due to a reduction in non-specific distribution of DEX.

6. Nanodex reduced lung inflammation and injury in K18-hACE2 transgenic mice infected with live SARS-CoV-2.

K18-hACE2 transgenic mice expressing the human SARS-CoV-2 receptor (e.g., hACE 2) under the cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 infection and infection can lead to a dose-dependent progression of pulmonary inflammatory disease. Therefore, K18-hACE2 mice were selected and tested for anti-COVID-19 efficacy against DEX-Neu-EVMs.

The K18-hACE2 transgenic mice were inoculated intranasally with SARS-CoV-21.5X 10³PFU. On

days

2 and 4 post-infection (one administration each), mice were administered either PBS or DEX (1 mg/kg) or equal amounts of DEX-Neu-EVMs by pulmonary inhalationPBS. Body weight was monitored daily. On day 6 post-infection, mice were euthanized and lung tissue and serum samples were collected for cytokine measurements and histopathological analysis (a in fig. 5). Before inoculation of virus and inhalation administration, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Lung tissues were fixed with 10% buffered formalin, and the fixed tissues were embedded in paraffin and sectioned at 3 μm thickness. Slicing H&E staining, and observing under an optical microscope.

Measurement of cytokines/chemokines in lung and serum: the cytokine/chemokine levels in mouse lung tissues were detected using qRT-PCR. Total RNA was isolated from lung tissue and reverse transcribed to cDNA. Quantitative real-time quantitative polymerase chain reaction (qRT-PCR) was performed on the target genome and qRT-PCR detection was performed (primer sequences are shown in Table 1). Levels of inflammatory factors were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using a commercial ProcartaPlex^TMMouse cytokine/chemokine multi-panel 1A kit (Thermo Fisher, usa) quantitated cytokine/chemokine levels in serum. Measurement of one-panel cytokines and chemokines, including measurement of ENA-78 (epithelial neutrophil activating protein 78; C-X-C motif chemokine ligand 5, CXCL 5), GRO-alpha (growth regulating oncogene; CXCL 1), G-CSF (granulocyte colony stimulating factor), IFN-G (interferon γ), IL-1 α, IL-1 β, IL-2, IL-4, IL-6, IL-9, TNF- α, IL-13, IL-15/IL-15R, IL-17 α, IL-22, IL-23, IL-27, IL-28, LIF (leukemia inhibitory factor), MCP-1 (monocyte chemotactic protein 1; C-C motif chemokine ligand 2, CCL2), MCP-3 (CCL 7), MIP-1 alpha (macrophage inflammatory protein 1 alpha; CCL 3).

At the genetic level, SARS-CoV-2 infection induces upregulation of most of the cytokines/chemokines detected within the lung, these cytokines/chemokines include IL-1 α, IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-12 α, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 α (MIP-1 α), gamma interferon-induced single cell factor (MIG), TNF- α, and stromal cell derived factor-1 (SDF-1) (b in FIG. 5 and a in FIG. 14), but had little down-regulation effect on specific cytokines/chemokines (b in fig. 14), suggesting a cytokine storm of COVID-19 in the lungs. Notably, treatment with DEX-Neu-EVMs effectively inhibited the SARS-CoV-2 infection-induced cytokine/chemokine upregulation in the lung (b in FIG. 5) compared to DEX, which may be associated with enhanced delivery of DEX to lung tissue and neutralization of Neu-EVMs in concert with cytokines. Furthermore, histological examination of the lung of SARS-CoV-2 infected mice showed a variety of inflammatory lesions such as alveolar edema, inflammatory cell infiltration, alveolar space disappearance and alveolar wall thickening (c in FIG. 5). Notably, inhalation delivery of DEX-Neu-EVMs significantly inhibited lung injury (c in fig. 5), suggesting that this inhalable nanoDEX has great potential in the treatment of COVID-19 related immune disorders and lung injury. The pulmonary transcriptome results showed that nanoDEX was effective in relieving pulmonary inflammation by modulating the pathways associated with new coronary pneumonia infection (d in fig. 5). The results of multiple immunoassays for cytokines/chemokines in serum were consistent with the results for local inflammation, further confirming that DEX-Neu-EVMs can effectively inhibit systemic inflammation caused by SARS-CoV-2 infection (FIG. 5, e and FIG. 15).

TABLE 1 qRT-PCR primer sequences

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:

the inhaled CODVID-19 cytokine storm resistant nanodEX has the advantages that the retention of the nanodEX in inflammatory lung is enhanced, the targeting to activated macrophages and DCs is improved, the down regulation of DEX cytokine is promoted, and inflammatory cell infiltration and lung injury caused by SARS-CoV-2 infection are inhibited, due to the large number of chemokine receptors on Neu-EVMs and the inhalation delivery strategy. In addition, nanoDEX relies on an abundant cytokine receptor on Neu-EVMs, neutralizing a broad spectrum of cytokines. The synchronous down-regulation and neutralization of inflammatory cytokines is effective against the COVID-19 cytokine storm. In addition, Neu-EVMs show better in vivo safety and DEX-Neu-EVMs are effective in alleviating DEX-induced osteoporosis after inhaled delivery. The simplicity, safety and potent inflammation inhibition of nanoDEX make it an attractive candidate for clinical development.

DEX is a very effective anti-edema and anti-fibrosis drug. Since pulmonary edema and fibrosis have recently become a key complication in the long-term follow-up management of covi-19, nanoDEX can further enhance the anti-edema and anti-fibrosis effects of DEX by increasing the drug availability and drug activity of the hyperactivated immune cell population in the inflamed lung. SARS-CoV-2 infection comprises a mixture of cells from monocytes, macrophages, dendritic cells and T cells, which together coordinate the COVID-19 process. Thus, EVMs of these cells can also be used for DEX delivery and in combination with other nanotherapeutics for targeted and synergistic treatment of COVID-19. Although the current design uses DEX, it is envisioned that this nano-drug platform can be generalized to many other types of glucocorticoids, such as cortisone, methylprednisolone, prednisolone, betamethasone.

In achieving clinical application of nanoDEX, one should be vigilant of its in vivo toxicity. The safety data from the preliminary toxicity studies of the present invention, as well as clinical studies on autologous EVs, demonstrate its safety.

In terms of source, bioprocesses produced in vitro on clinical scale neutrophils can provide large amounts of EVMs. At the same time, significant progress has been made in the production of human cells with general immune compatibility, which will also provide a cellular supply for clinical research. In addition, these cells can be collected from a blood sample of a patient, and can be engineered prior to injection of the EVMs into the same patient, thereby maximizing immune tolerance of the EVMs and potentially expanding the application of ongoing COVID-19 and other inflammatory disease treatment devices.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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gtgatggagg gggtcagga 19

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(20)

<223> primer m6Ckine (Ccl21a) -R

<400> 38

gggatgggac agcctaaact 20

<210> 39

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(21)

<223> primer mMIG (Ccccl 9) -F

<400> 39

ggagttcgag gaaccctagt g 21

<210> 40

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(21)

<223> primer mMIG (Ccccl 9) -R

<400> 40

gggatttgta gtggatcgtg c 21

<210> 41

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(24)

<223> primer mGAPDH-F

<400> 41

tcaacagcaa ctcccactct tcca 24

<210> 42

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(24)

<223> primer mGAPDH-R

<400> 42

accctgttgc tgtagccgta ttca 24

Claims

1. An aerosol inhalation glucocorticoid nano-drug, comprising:

nanoscale cell membrane vesicles; and

a glucocorticoid loaded in the nanoscale cellular membrane vesicle;

the nano-scale cell membrane vesicles are cell membrane vesicles of immune cells;

the immune cell is a neutrophil;

the glucocorticoid comprises one or more selected from the group consisting of methylprednisolone, cortisone, prednisolone, betamethasone, and dexamethasone;

when the glucocorticoid is loaded into the nanoscale cell membrane vesicle, the mass ratio of the glucocorticoid to the nanoscale cell membrane vesicle is 1: 1-1: 10.

2. The aerosolized glucocorticoid nanomedicine according to claim 1, characterized in that the glucocorticoid is dexamethasone.

3. The inhaled and atomized glucocorticoid prodrug of claim 1, further comprising a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is one or more of a nanoscale cellular membrane vesicle, a phospholipid nanoparticle, a polymeric nanoparticle, and a metal oxide nanoparticle.

4. The aerosolized inhaled glucocorticoid nanomedicine according to claim 3, wherein the pharmaceutically acceptable carrier is a nanoscale cell membrane vesicle.

5. A process for the preparation of an inhaled glucocorticosteroid nano-drug according to any one of claims 1 to 4, comprising the steps of:

preparing nano-scale cell membrane vesicles; and

loading glucocorticoid into the nanoscale cell membrane vesicle to obtain the aerosol inhalation type glucocorticoid nano-drug;

the cell is an immune cell, and the immune cell is a neutrophil;

6. The method for preparing according to claim 5, wherein the preparing of the nanoscale cell membrane vesicle comprises: removing intracellular components of cells, then carrying out ultrasonic treatment, and then extruding through a micro extruder to prepare the nanoscale cell membrane vesicles.

7. The method according to claim 6, wherein the cell-depleted intracellular component is a cell-depleted intracellular component of neutrophils by a combined treatment of hypotonic lysis, mechanical disruption and gradient centrifugation.

8. The method of claim 5, wherein the loading a glucocorticoid into the nanoscale cellular membrane vesicles comprises loading the glucocorticoid into the nanoscale cellular membrane vesicles by means of sonication, incubation, extrusion, or electroporation.

9. The method of claim 8, wherein the loading a glucocorticoid into the nanoscale cellular membrane vesicles comprises loading the glucocorticoid into the nanoscale cellular membrane vesicles by means of ultrasound or electroporation.

10. The method of claim 8, wherein the glucocorticoid comprises one or more selected from the group consisting of methylprednisolone, cortisone, prednisolone, betamethasone, and dexamethasone.

11. The method of claim 10, wherein the glucocorticoid is dexamethasone.

12. The method of claim 5, wherein the loading of the nanocellular membrane vesicle with a glucocorticoid comprises: mixing the glucocorticoid with the nanoscale cell membrane vesicles, sonicating, then centrifuging the sonicated product, and washing with phosphate buffered saline solution to remove free the glucocorticoid.

13. The method according to claim 5, wherein the mass ratio of the glucocorticoid to the nanoscale cell membrane vesicles is 1: 5.

14. Use of the aerosolized inhaled glucocorticoid nano-drug according to any one of claims 1 to 4 or the aerosolized inhaled glucocorticoid nano-drug prepared by the method for preparing the aerosolized inhaled glucocorticoid nano-drug according to any one of claims 5 to 13 for preparing an aerosolized inhaled drug formulation for treating neocoronary pneumonia COVID-19.