Dual-modular targeted delivery system and application thereof in targeted anti-inflammatory drugs

Abstract

The invention discloses a dual-modular targeted delivery system and application of the dual-modular targeted delivery system in targeted anti-inflammatory drugs. The dual-modular targeted delivery system comprises an intravenous injection module and a respiratory tract inhalation module, the intravenous injection module is mesenchymal stem cells a-MSCs functionally modified by a first click chemical reaction group, and the respiratory tract inhalation module is drug-loaded nanoparticles b-NPs functionally modified by a second click chemical reaction group; according to the dual-modular targeted delivery system, the drug removal barrier is broken through through conjugate immune camouflage, the alveolar residence time of nanoparticles is prolonged, the drug concentration of a pneumonia focus area is increased, and the treatment effective rate is increased; due to the modular design, the whole-body exposure of NPs is reduced, and the liver and kidney toxicity is reduced; the single-time inhalation dosage is reduced, the patient compliance is improved, and the application prospect is great.

Description

Technical Field

[0001] The present invention relates to a targeted delivery system, in particular to a dual-modular targeted delivery system, and also to an application of the dual-modular targeted delivery system in the preparation of an anti-inflammatory drug targeted to the lungs. Background Art

[0002] Pulmonary inflammatory diseases are airway or lung parenchymal inflammatory responses triggered by infection (bacteria, viruses, fungi, etc.), allergies, or environmental irritants. Common types include bacterial pneumonia, viral pneumonia, asthma, and chronic obstructive pulmonary disease (COPD). Their pathological characteristics include increased mucus secretion, airway spasm, and tissue damage, with typical symptoms including cough, wheezing, and dyspnea.

[0003] Traditional respiratory drug delivery systems (such as nebulization therapy and inhalation therapy) face significant challenges in the treatment of inflammatory lung diseases due to factors such as the physical barriers and particle deposition mechanisms of the respiratory tract, the defensive role of the mucociliary clearance system (MCC), the phagocytosis of alveolar macrophages, the influence of lung anatomy and disease state, respiratory dynamics and drug delivery technology limitations. Specifically, they include: ① Low drug deposition efficiency: particles reaching the alveolar area are easily phagocytosed and cleared by alveolar macrophages, resulting in decreased drug bioavailability; ② Poor particle stability: easily discharged by the exhaled airflow or enter the systemic circulation through the tracheal mucosa, causing off-target toxicity; ③ Insufficient targeting: particles >5 microns are easily retained in the upper respiratory tract and cannot be accurately delivered to the lung lesions. The current main solution is to balance the particle deposition efficiency (particle size range of 1-5μm) with avoiding problems such as alveolar macrophage clearance and respiratory airflow discharge. Existing technical solutions mainly improve the delivery effect through two aspects: particle engineering optimization and active targeting design.

[0004] In terms of particle engineering optimization, for example, BreztriAerosphere (Chinese trade name: Budesonide inhalation aerosol is used for the maintenance treatment of patients with moderate to severe COPD. It is the world's first triple therapy drug administered via a pressurized metered-dose inhaler (pMDI) using an innovative co-suspension delivery technology (Aerosphere™). Co-suspension delivery technology enables the sustained delivery of one or more different drugs from a single pressurized metered-dose inhaler. AstraZeneca is using this technology platform to develop a range of inhaled combination therapies. The related product, Bevespi Aerosphere (glycopyrrolate / formoterol fumarate) inhalation aerosol, was approved by the FDA as early as 2016 for the long-term maintenance treatment of COPD patients. Breztri Aerosphere is a suspension of micronized budesonide, micronized glycopyrrolate, and micronized formoterol fumarate crystals, blended with spray-dried porous particles (composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and calcium chloride) in a hydrofluoroalkane (HFA) propellant. Aerosphere co-suspension delivery technology (AerosphereTM) can improve the stability and homogeneity of suspended drugs, and the dosage delivery of each component drug is consistent with the target dose during the service life. At the same time, it greatly improves the problem of inconsistent drug delivery caused by poor pMDI device operation technology, and improves the lung deposition rate of pMDI. However, these particle engineering optimized products require complex preparation technology, and may have problems such as difficulty in quality control, poor storage stability, high cost, large individual differences, and low bioavailability. In terms of active targeting design, for example, Chinese patent CN202410607516.6 relates to an amino acid lipid targeting the lungs and a lipid nano-delivery system formed therefrom. The delivery system prepared by the amino acid lipid of this invention can efficiently and specifically deliver nucleic acid drugs to the lungs, but these drug-carrying systems achieve lung targeting by modifying active targeting ligands or adjusting the composition of lipid components. There are problems such as low targeting efficiency, complex prescription, difficulty in preparation, poor stability, large individual differences, and low bioavailability. It is urgent to develop drugs with stronger targeting capabilities and higher therapeutic efficiency. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a dual-modular targeted delivery system that achieves internal and external targeted enhancement through bioorthogonal chemical bonds, and to provide the application of the above dual-modular targeted delivery system in targeted anti-inflammatory drugs.

[0006] Technical solution: The present invention discloses a dual-modular targeted delivery system, which includes an intravenous injection module and a respiratory inhalation module. The intravenous injection module is a mesenchymal stem cell a-MSCs functionalized with a first click chemistry reaction group, which provides covalent binding sites while maintaining cell viability (>70%) and migration ability, and is directedly recruited to the lung inflammation area through its inherent homing ability; the respiratory inhalation module is a drug-loaded nanoparticle b-NPs functionalized with a second click chemistry reaction group, with a particle size of 10 nanometers to 15 microns to optimize alveolar deposition; wherein, the first click chemistry reaction group and the second click chemistry reaction group are specific covalent binding groups.

[0007] Among them, the specific covalent binding groups include the following combinations: azide (N3) and dibenzocyclooctyne (DBCO); azide (N3) and bicyclononyne (BCN); tetrazine (Tetrazine) and trans-cyclooctene (TCO); tetrazine (Tetrazine) and cyclopropene (Cyclopropene); dibenzocyclooctyne-azide (DBCO-N3) and tetrazine-trans-cyclooctene (tetrazine-TCO); thiol (-SH) and maleimide; thiol and acrylate; thiol and alkyne; thiol and halogenated hydrocarbon; thiol and epoxide; thiol and isocyanate; N-hydroxysuccinimide ester (NHS) and primary amine (-NH2).

[0008] Among them, the functional modification of the intravenous injection module adopts DSPE-PEG amphiphilic lipid molecules, and the functional group containing the first click chemical reaction group is integrated into the surface of the mesenchymal stem cell membrane through lipid membrane fusion technology or covalent coupling technology.

[0009] The intravenous injection module is prepared by adding 10 μg-1000 μg of lipid molecules per 1 million cells, incubating the functionalized DSPE-PEG amphiphilic lipid molecules with mesenchymal stem cells in culture medium or other isotonic liquid for 0.5-48 hours, and forming mesenchymal stem cells a-MSCs functionalized with the first click chemistry reaction group through lipid membrane fusion technology;

[0010] Alternatively, 10 μg-1000 μg of functionalized modified molecules are added per 1 million cells, and the functionalized modified molecules containing two different active functional groups are co-incubated with mesenchymal stem cells in culture medium or other isotonic liquid for 0.5-48 hours. Through the covalent coupling reaction of one of the functional molecules with the cell membrane surface group, mesenchymal stem cells a-MSCs functionalized with the first click chemistry reaction group are formed.

[0011] Among them, the respiratory inhalation module is obtained by combining liposomes, lipid nanoparticles, polymer micelles, polymer vesicles, exosomes, polylactic acid-co-glycolic acid (PLGA) nanoparticles, albumin nanoparticles and therapeutic agents, and the therapeutic agents include one or more combinations of chemical small molecule drugs, siRNA / mRNA nucleic acid drugs, monoclonal antibodies or cytokine protein drugs; the drug loading of the therapeutic agent is 1%-60% (w / w) by mass percentage; the particle size polydispersity index (PDI) of the b-NPs is ≤0.2 or the particle size distribution coefficient of variation (CV) is less than 30%.

[0012] The respiratory inhalation module is prepared by: preparing liposomes or lipid nanoparticles or polymer micelles or polymer vesicles by solvent injection, thin film hydration, reverse evaporation, double emulsion, microfluidics, or ultrasonic dispersion; preparing polylactic acid-co-glycolic acid nanoparticles by emulsification-solvent evaporation, nanoprecipitation, microfluidization, or double emulsion; and preparing albumin nanoparticles by self-assembly, emulsion crosslinking, high-pressure homogenization, or precipitation. The formulation comprises a material containing a second click chemistry reactive group, and these second click chemistry reactive groups can be exposed on the surface of the nanoparticles during the nanoparticle assembly process.

[0013] Alternatively, cells can be metabolized and labeled to produce exosomes containing a second click chemistry reactive group by cellular uptake of non-natural sugars, and then purified by ultracentrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, or immunomagnetic bead methods to obtain exosomes containing a second click chemistry reactive group; or lipid membrane fusion technology can be used to introduce a second click chemistry reactive group on the surface of exosomes.

[0014] The present invention also discloses the use of the dual-modular targeted delivery system in the preparation of anti-inflammatory drugs targeting the lungs and / or respiratory tract.

[0015] Among them, the inflammations suitable for application include bacterial pneumonia, viral pneumonia, fungal pneumonia, atypical pathogen pneumonia, tuberculosis, hypersensitivity pneumonitis, idiopathic interstitial pneumonia, radiation pneumonia, chemical pneumonia, chronic bronchitis or acute bronchitis.

[0016] The application is specifically:

[0017] (1) The intravenous injection module of the dual modular targeted delivery system is administered via intravenous injection, targeting the pneumonia area by utilizing the inflammatory tropism of MSC cells and simultaneously implanting click chemistry groups;

[0018] (2) At intervals of 0.1 hours to 24 hours, the respiratory inhalation module of the dual modular targeted delivery system is administered via respiratory atomization inhalation. Drug-loaded microspheres or nanoparticles modified with another corresponding click chemical group are delivered via the respiratory route. In the lungs, the two are covalently bound through a click chemical reaction, thereby solving the problem of drug-loaded particles being cleared by alveolar macrophages and discharged by respiratory airflow.

[0019] The intravenous injection module is a sterile injection preparation containing a concentration of 1×10 5 to 2×10 7 cells / mL of a-MSCs suspension, and the auxiliary material is cell isotonic buffer; the respiratory tract inhalation module is an atomized inhalation preparation: containing a b-NPs suspension or dry powder preparation with a drug loading concentration of 0.1-30 mg / mL.

[0020] Principle of the invention: The dual-modular targeted delivery system of the present invention is constructed into two parts, including an intravenous injection module and a respiratory inhalation module. Through the intravenous injection route, the inflammatory tropism of MSC cells is utilized to target the pneumonia area, and click chemical groups are implanted at the same time; at intervals of 0.1 hours to 24 hours, drug-loaded microspheres or nanoparticles modified with another corresponding click chemical group are delivered through the respiratory tract atomization inhalation route. In the lungs, the covalent bonding of the two is achieved through the click chemical reaction, thereby overcoming the problems of inhaled drug particles having too short a residence time in the alveoli, low targeting efficiency of systemic administration, and premature drug leakage in the single module design, and solving the problem of drug-loaded particles being cleared by alveolar macrophages and discharged by respiratory airflow.

[0021] The drug delivery system of the present invention adopts a modular design, which achieves the following improved effects: reduced systemic toxicity, a step-by-step delivery strategy (intravenous administration followed by inhalation) avoids systemic exposure of pre-assembled conjugates, and reduces the risk of premature leakage and off-target effects of anti-inflammatory agents; enhanced anti-clearance and targeting, covalent binding enables NPs to anchor to the surface of MSCs in the lesion area, reducing respiratory airflow clearance and reducing the phagocytic rate of alveolar macrophages; simplified process and universality, functionalized nanoparticles only require simple surface modification (such as liposome insertion of N3-DSPE-PEG), without complex engineering transformation, and are suitable for industrial production; easy replacement of therapeutic drugs, compatibility with multiple therapeutic agents (small molecules, nucleic acids, proteins), and support for multi-drug combination (such as anti-inflammatory + antioxidant combination regimens in acute lung injury); dynamic response and precise treatment, the two-step delivery interval can be adjusted according to disease progression (such as shortened to 1 hour in the acute phase) to achieve time-sequential drug release; by monitoring the homing signal of DBCO-MSCs (such as the concentration of inflammatory factors), the dose and frequency of inhaled NPs can be dynamically controlled.

[0022] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: the dual-modular targeted delivery system of the present invention breaks through the drug clearance barrier through conjugate immune camouflage, prolongs the alveolar retention time of nanoparticles, increases the drug concentration in the pneumonia lesion area, and improves the treatment efficiency; the modular design reduces the systemic exposure of NPs and reduces liver and kidney toxicity; it reduces the single inhalation dose and improves patient compliance, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The construction and evaluation of DBCO-functionalized mesenchymal stem cells, wherein a is a schematic diagram of the construction process of DBCO-functionalized mesenchymal stem cells; b is the analysis of the expression of mesenchymal stem cell surface markers by flow cytometry; c and d are the time-dependent optimization and quantitative analysis of the modification efficiency of DSPE-PEG-DBCO (80 μg / mL, Pengshuo) by flow cytometry (n=3); e is the optimization of the concentration-dependent modification efficiency of different concentrations of DSPE-PEG-DBCO after co-incubation with mesenchymal stem cells for 1 hour by flow cytometry; f is the quantitative evaluation of the modification efficiency (n=3); g is the use of Calce The effect of DSPE-PEG-DBCO modification on the viability of mesenchymal stem cells was assessed by in-AM / PI staining; h was the Transwell assay to assess the in vitro migration ability of mesenchymal stem cells after DSPE-PEG-DBCO modification (n=3); i was the CCK-8 assay to examine the effect of DSPE-PEG-DBCO modification on the viability of mesenchymal stem cells (n=5); j was the in vivo chemotactic properties of mesenchymal stem cells labeled with DSPE-PEG-Cy5 (Carbon Water Technology) in pneumonia lesions in mice with acute lung injury studied using small animal imaging technology; k was the semi-quantitative analysis of fluorescence signals (n=3);

[0024] Figure 2 Evaluation of enhanced retention of N3-liposomes in the lungs of mice with acute lung injury after conjugation with DBCO-MSCs, wherein a is a schematic diagram of the in vivo administration and analysis process, with unmodified MSCs serving as the control group; b is a fluorescence microscopy image of Cy5-labeled N3-liposomes conjugated to DBCO-MSCs in vitro; c is a scanning electron microscopy image of cell surface conjugation after 1 or 3 hours of co-incubation; d is an in vivo fluorescence imaging showing the accumulation of Cy5 liposomes in the lungs 1 or 3 hours after administration; e is a semi-quantitative analysis by fluorescence signal (n=3); f is an in vitro frozen section analysis of Cy5 red fluorescence in lung tissue (f); g is a semi-quantitative analysis by fluorescence signal (n=3), with PF488 used to label actin filaments and DAPI used to stain cell nuclei;

[0025] Figure 3The construction, characterization, and in vitro evaluation of N3-functionalized andrographolide-loaded liposomes. (a) The composition of the liposomes; (b) Dynamic light scattering analysis; (c) Transmission electron microscopy observation; (d) Particle size stability assessment (n = 3); The inset is a photograph of the liposome suspension.

[0026] Figure 4 The dual modular targeted delivery system of the present invention was used to evaluate the in vivo efficacy of N3-Lip@A, wherein a is the in vivo administration and detection scheme for evaluating the therapeutic effect, n=8; b is the lung morphology, Evans blue staining images and pathological analysis (HE, COX2, iNOS) of mice with acute lung injury after treatment in different experimental groups; c is the animal survival curve; d is the animal weight monitoring; e is the lung wet-to-dry weight ratio, n=3; f is the lung injury score, n=3; g is the quantitative Evans blue staining intensity in the resected lung tissue, n=3; h is the quantification of inflammatory factors and total protein in the bronchoalveolar lavage fluid (BALF);

[0027] Figure 5 Preparation and in vitro evaluation of N3-Exo@G exosomes, where a is a schematic diagram of the preparation process; b is a transmission electron microscopy image of exosome morphology; c is a flow cytometer analysis for exosome size analysis; d is a Western blotting method for confirming exosome markers;

[0028] Figure 6 The dual modular targeted delivery system of the present invention is used to evaluate the in vivo efficacy of N3-Exo@G, wherein a is the in vivo administration and detection scheme for evaluating the therapeutic effect, n=8; b is the lung morphology, Evans blue staining images and pathological analysis (HE, NE, MPO and CitH3) of mice with acute lung injury after treatment in different experimental groups; c is the animal survival curve; d is the average weight monitoring of surviving animals during treatment; e is a heat map of the levels of total protein and inflammatory cytokines (IL-1β, IL-6 and TNF-α) in bronchoalveolar lavage fluid (BALF); f is a radar chart of the quantitative analysis of lung injury index, lung wet-to-dry weight ratio, lung tissue Evans blue value, animal survival rate and average weight at the experimental endpoint;

[0029] Figure 7 This is the preparation process and application process of the dual-modular targeted delivery system of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below with reference to the examples. The test materials used in the examples can all be purchased through conventional channels.

[0031] Example 1

[0032] The dual-modular targeted delivery system of the present invention comprises an intravenous injection module and a respiratory inhalation module, wherein the intravenous injection module is DBCO-MSC and the respiratory inhalation module is N3-Lip@Cy5 liposome.

[0033] Construction of intravenous injection module DBCO-MSC Figure 7 As shown, the following steps are included:

[0034] (1) The isolation, culture and identification of adipose-derived mesenchymal stem cells (AD-MSCs) were as follows: First, SD rats were euthanized under sterile conditions and shaved and disinfected. Adipose tissue was then removed from the inguinal or epididymal region, minced, suspended in PBS, and centrifuged at 2000 rpm for 10 minutes (repeated once). The upper adipose tissue was collected, mixed with type I collagenase, and digested at 37°C for 1 hour. After filtering through a 75 μm filter, centrifuged at 2000 rpm for 10 minutes, the precipitate was resuspended in red blood cell lysis buffer for 2 minutes, and then centrifuged at 1600 rpm for 5 minutes. The obtained cells were resuspended in complete culture medium, filtered again through a 75 μm filter, and inoculated into T25 culture flasks and cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium was replaced every 3 days, and the cells were passaged when the confluence reached 80%-90%. Cells from passages 1 to 3 were used for subsequent experiments. To characterize the phenotype of AD-MSCs, single-cell suspensions were incubated with APC-CD29, FITC-CD90, PE-CD45, and PE-CD11b / c antibodies (Biolegend, 1:1000) for 30 minutes at 4°C. Unbound antibodies were removed by washing, and the cells were resuspended in PBS and analyzed using a flow cytometer (BD).

[0035] (2) The preparation and optimization process of DBCO surface-modified AD-MSCs are as follows: DSPE-PEG2000-DBCO was used to modify the cell membrane of AD-MSCs to prepare DBCO-functionalized mesenchymal stem cells. To optimize the modification conditions, DSPE-PEG2000-FITC (Pengshuo) was used as a substitute to conduct time- and concentration-dependent experiments. Specifically, in the time-dependent analysis, AD-MSCs (2×10 5Cells were seeded in 6-well plates (100 cells / well) and incubated with DSPE-PEG2000-FITC for 12 hours before incubation for 15 minutes, 30 minutes, 1 hour, 1.5 hours, or 2 hours. The cells were then harvested, washed to remove unbound DSPE-PEG2000-FITC, and analyzed by flow cytometry. For concentration-dependent analysis, DSPE-PEG2000-FITC was added at concentrations of 10, 20, 50, 80, or 100 μg for the same incubation time. The cells were then harvested, washed, and analyzed by flow cytometry.

[0036] The respiratory inhalation module is constructed as N3-Lip@Cy5 liposomes and includes the following steps:

[0037] N3-Lip@Cy5 liposomes were prepared as follows: 30 mg of HSPC (Aladdin), 10 mg of cholesterol (Aladdin), 0.5 mg of hydrophobic Cy5 (Aladdin), 8 mg of DSPE-PEG2000 (Pengshuo), and 2 mg of DSPE-PEG2000-N3 (Pengshuo) were dissolved in 2 mL of dichloromethane. The solvent was blown dry with nitrogen to form a dry lipid film. 2 mL of PBS (pH 7.4) was then added and sonicated in an ice-water bath for 30 minutes to hydrate the film. The dispersion was centrifuged at 3000 × g for 15 minutes and washed using a 100 kDa ultrafiltration tube (Millipore Sigma) to obtain a liposome dispersion. Liposome morphology was observed using a transmission electron microscope (JEM-1400plus, JEOL), and particle size and zeta potential were characterized using a dynamic light scattering instrument (Zetasizer ZS90, Malvern).

[0038] Example 2

[0039] Verification that DSPE-PEG2000-DBCO modification does not affect the activity and migration properties of MSCs:

[0040] The activity evaluation method of MSCs modified with DSPE-PEG2000-DBCO was as follows: For CCK-8 assay, MSCs (2×10 4 10 cells / well) were seeded in a 96-well plate. After 24 hours of culture, the medium was replaced with serum-free medium and DSPE-PEG2000-DBCO was added at a final concentration of 10, 20, 50, 80, or 100 μmol / L. After another 24 hours of culture, 10 μL of CCK-8 solution (Biyuntian) was added and incubated for another hour. The absorbance was measured at 450 nm, and the cell viability was calculated. For the Calcein-AM / PI staining assay, AD-MSCs (2 × 10 5Cells were seeded in confocal microplates (100 cells / well). After 24 hours of culture, the medium was replaced with DSPE-PEG2000-DBCO at final concentrations of 10, 20, 50, 80, or 100 μmol / L. After 24 hours of treatment, the cells were washed with PBS and stained using a Calcein-AM / PI fluorescent staining kit (Beyotime). Cell viability and live / dead cell ratios were analyzed using a fluorescence microscope (Axio Observer Z1, Carl Zeiss).

[0041] The in vitro Transwell-based DBCO-MSC migration assay is performed as follows: DBCO-MSCs or unmodified MSCs are seeded in the upper chamber of a 24-well Transwell plate. The lower chamber is filled with serum-free medium containing 100 ng / mL CXCL12. After 24 hours of culture, non-migrated cells are gently removed from the membrane using a cotton swab. Migrated cells are fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 20 minutes, washed three times with PBS, and observed and photographed under an inverted microscope.

[0042] Example 3

[0043] Study on the dual-binding targeting properties of the dual modular targeted delivery system of the present invention in vivo and in vitro:

[0044] The in vitro analysis method of DBCO-MSCs binding to N3-Lip@Cy5 was as follows: For fluorescence analysis, MSCs were plated at 2×10 5 Cells were seeded at a density of 100 μg per 1 million cells in confocal culture dishes. DSPE-PEG2000-DBCO (50 μg per 1 million cells) was added and incubated for 1.5 hours. The cells were then washed with PBS and incubated for 25 minutes at 4°C with serum-free medium containing an equal amount of fluorescein-labeled N3-Lip@Cy5 or Lip@Cy5. Cell nuclei were stained with Hoechst 33258 and fixed with 4% paraformaldehyde. The cells were then imaged under a fluorescence microscope for analysis. For electron microscopy, cells treated as described above were fixed with 2.5% glutaraldehyde for 12 hours at 4°C. Samples were prepared and observed using a biological scanning electron microscope (QUANTA 250FEG, Thermo Fisher).

[0045] Lipopolysaccharide (LPS)-induced acute lung injury (ALI) model and lung targeting study. Male C57BL / 6 mice (8-10 weeks old) received a single intratracheal LPS instillation (5 mg / kg) under anesthesia to establish an experimental ALI model. To analyze the lung distribution in vivo, 4 hours after LPS administration, the mice were divided into three groups: saline + N3-Lips@Cy5, mesenchymal stem cells (MSC) + N3-Lips@Cy5, and DBCO-MSC + N3-Lips@Cy5 ( Figure 3 a). Physiological saline, MSCs, or DBCO-MSCs (5×10^5 cells / mouse) were injected through the tail vein. Two hours later, N3-Lips@Cy5 (50 μL) was administered via endotracheal intubation. Mice were sacrificed 1 and 3 hours after administration, and the heart, liver, spleen, lungs, and kidneys were removed for small animal in vivo imaging (In-VivoXtreme FX-pro, Bruker) and semi-quantitative fluorescence analysis. Lung tissue was fixed in 4% paraformaldehyde for 24 hours, and frozen sections were prepared. Phalloidin-iFluor488 (PF488) was used to label the cytoskeleton, and DAPI was used to label the nuclei, followed by fluorescence scanning (Pannoramic250Flash, 3DHistech) and semi-quantitative analysis.

[0046] Example 4

[0047] Liposomes were used as an example to verify the in vivo efficacy of the dual modular targeted delivery system of the present invention:

[0048] N3-Lip@A liposomes were prepared as follows: 30 mg of HSPC, 10 mg of cholesterol, 5 mg of andrographolide (Aladdin), 8 mg of DSPE-PEG2000, and 2 mg of DSPE-PEG2000-N3 were dissolved in 2 mL of dichloromethane and dried with nitrogen to form a dry lipid film. 2 mL of PBS (pH 7.4) was added and the mixture was sonicated in an ice-water bath for 30 minutes for hydration. The dispersion was centrifuged at 3000 × g for 15 minutes and washed using a 100 kDa ultrafiltration tube to obtain a liposome dispersion. Liposome morphology was observed using a transmission electron microscope (JEM-1400plus, JEOL), and particle size and zeta potential were measured using a dynamic light scattering instrument (Zetasizer ZS90, Malvern). Furthermore, the unencapsulated free drug liquid in the ultrafiltration tube was collected and analyzed for andrographolide content by high-performance liquid chromatography (HPLC). Encapsulation efficiency (EE, %) and drug loading (DLC, %) were calculated using the following formulas: Control liposomes, Lip@A, were prepared identically, except that an equal amount of DSPE-PEG2000 was used in place of DSPE-PEG2000-N3. EE (%) = (total andrographolide input weight - free andrographolide weight) / total andrographolide input weight × 100%; DLC (%) = (total andrographolide input weight - free andrographolide weight) / total liposome weight × 100%.

[0049] In vivo pharmacodynamics of N3-Lip@A. An acute lung injury (ALI) mouse model was established according to previously described methods. The sham-operated group received saline instead of LPS. ALI mice were randomly divided into five groups (n = 8 per group): saline, DBCO-MSC, N3-Lip@A, MSC+N3-Lip@A, and DBCO-MSC+N3-Lip@A. Four hours after LPS administration, the mice were intravenously injected with DBCO-MSC or MSC cells (1 million cells per mouse). Two hours after LPS administration, N3-Lip@A (10 mg / kg, calculated as andrographolide) was administered intratracheally. Drug treatment was repeated every other day for a total of three doses. Body weight and survival were monitored during this period. Two days after the last dose, lung tissue was stained with Evans blue, bronchoalveolar lavage fluid (BALF) was analyzed, lung wet-to-dry weight ratio was measured, and ex vivo histopathological analysis was performed. For lung Evans blue staining, mice were injected with 1% Evans blue dye (40 mg / kg) via the tail vein at the end of treatment. Two hours later, the lungs were removed and photographed. After incubation in formamide (1 ml per 100 mg of tissue) at 37°C for 24 hours, the samples were centrifuged at 12,000 × g for 30 minutes. The supernatant was filtered and its optical density (OD) was measured. For BALF analysis, the trachea was exposed and the BALF was collected by injection with pre-chilled PBS. The fluid was centrifuged at 12,000 × g for 30 minutes at 4°C, and the supernatant was used to determine the total protein content (BCA method) and cytokine concentrations (IL-6, TNF-α, IL-1β) using ELISA kits (1210602, 1217202, 1210122, Dako). For pathological staining, heart, liver, spleen, lung, and kidney tissues were collected, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E). Immunohistochemistry was used to analyze the protein expression levels of COX2 and iNOS in lung tissue using specific antibodies. For the determination of lung wet-to-dry weight ratio, the lungs were removed, the surface moisture was blotted with filter paper, the wet weight was recorded, and then the lungs were dried at 60°C for 24 hours to measure the dry weight, and the wet-to-dry weight ratio was subsequently calculated.

[0050] Example 5

[0051] Using exosomes as an example to verify the in vivo efficacy of the dual modular targeted delivery system of the present invention:

[0052] Isolation and characterization of N3-exosomes. Adipose-derived mesenchymal stem cells (AD-MSCs) were isolated as described above. Subsequently, AD-MSCs were cultured in serum-free medium containing Ac4ManNAz (100 μM, Aladdin). N3-functionalized exosomes were isolated from the cell culture supernatant by sequential centrifugation steps (300 × g, 10 min; 2000 × g, 10 min; 10,000 × g, 30 min) and ultracentrifuged twice at 120,000 × g (70 min each). The purified exosomes were resuspended in PBS. To characterize the exosomes, their morphology was observed using transmission electron microscopy, and the particle size distribution was measured using a Flow NanoAnalyzer (Nanofcm). The expression of marker proteins (CD9, CD63, CD81) and a negative control protein (Calnexin) was verified by Western blot analysis. Whole-cell lysates were used as positive controls.

[0053] Preparation and characterization of N3-Exo@G. The small molecule drug GSK484 was loaded into exosomes by ultrasound. Specifically, 1 mg of exosomes (based on protein content) was mixed with 1 mg of GSK484 and intermittently sonicated at 60% power for 5 minutes in a cold water bath. Subsequently, the mixture was incubated at 37°C for 1 hour to restore the integrity of the exosome membrane. 3 ml of PBS was then added and ultrafiltration was performed using a 100 kDa ultrafiltration tube at 4°C for 10 minutes to remove free drugs. To quantify the drug loading efficiency, GSK484 in the drug-loaded exosomes was extracted with methanol and measured by high-performance liquid chromatography (HPLC).

[0054] In vivo pharmacodynamic studies. An acute lung injury (ALI) mouse model was established as described previously. The sham group was treated with saline instead of lipopolysaccharide (LPS). ALI mice were randomly divided into five groups (n = 8 per group): saline, DBCO-MSC, N3-Exo@G, MSC+N3-Exo@G, and DBCO-MSC+N3-Exo@G. Four hours after LPS stimulation, DBCO-MSC or MSC cells (1 × 10^6 cells per mouse) were administered via tail vein injection. Two hours later, N3-Exo@G (0.4 mg / kg, calculated as GSK484) was administered intratracheally. Treatment was administered every other day for three times. During this period, mouse body weight and survival were monitored. Two days after the last administration, lung tissue was stained with Evans blue, bronchoalveolar lavage fluid (BALF) was analyzed, lung wet-to-dry weight ratio was measured, and hematoxylin-eosin (HE) staining of major organs was performed according to established methods. In addition, the expression levels of neutrophil extracellular network (NETs)-related proteins (myeloperoxidase MPO, neutrophil elastase NE, and citH3) in lung tissues were detected by immunohistochemical analysis (antibody source: Abcam).

[0055] The results of the above experiments are as follows Figures 1 to 6 As shown:

[0056] like Figure 1Figure 2 shows the construction and evaluation of DBCO-functionalized mesenchymal stem cells, where a is a schematic diagram of the construction process of DBCO-functionalized mesenchymal stem cells; b is the analysis of the expression of mesenchymal stem cell surface markers by flow cytometry, and the results show that MSC has typical surface protein marker characteristics; c and d are the time-dependent optimization and quantitative analysis of DSPE-PEG-FITC (80 μg / mL) modification efficiency by flow cytometry (n=3), and the results show that 1-2h is the optimized incubation time; e is the concentration-dependent modification efficiency optimization of different concentrations of DSPE-PEG-FITC after co-incubation with mesenchymal stem cells for 1 hour by flow cytometry, and the results show that 50-100 μg / mL is the optimized concentration; f is the quantitative evaluation of modification efficiency (n=3); g is the evaluation of the effect of DSPE-PEG-DBCO modification on the viability of mesenchymal stem cells by Calcein-AM / PI staining. , the results showed that DSPE-PEG-DBCO concentration below 100μM still had high cell viability; h was evaluated by Transwell experiment on the in vitro migration ability of mesenchymal stem cells after DSPE-PEG-DBCO modification (n=3), and the results showed that cell migration was not affected by DSPE-PEG-DBCO modification; i was detected by CCK-8 method to detect the effect of DSPE-PEG-DBCO modification on the viability of mesenchymal stem cells (n=5), and the results showed that DSPE-PEG-DBCO concentration below 80μM maintained more than 70% cell viability; j was studied by small animal imaging technology to study the in vivo chemotactic properties of DSPE-PEG-Cy5-labeled mesenchymal stem cells in pneumonia lesions of mice with acute lung injury; k was semi-quantitative analysis by fluorescence signal (n=3), and the results showed that MSC cells modified with DSPE-PEG-DBCO still had lung inflammation chemotaxis;

[0057] like Figure 2Figure 2 shows the evaluation of enhanced retention of N3-liposomes in the lungs of mice with acute lung injury after combination with DBCO-MSCs, wherein a is a schematic diagram of the in vivo administration and analysis process, with unmodified MSCs serving as the control group; b is a fluorescence microscopy image of Cy5-labeled N3-liposomes conjugated to DBCO-MSCs in vitro, showing that the liposomes can rapidly couple to the cells; c is a scanning electron microscopy image of cell surface conjugation after 1 or 3 hours of co-incubation, showing that the liposomes can rapidly couple to the cells; d is an in vivo fluorescence imaging showing the accumulation of Cy5 liposomes in the lungs 1 or 3 hours after administration, showing that the dual-modular targeted delivery system has higher fluorescence retention in the lungs; e is a semi-quantitative analysis by fluorescence signal (n=3); f is an in vitro frozen section analysis of Cy5 red fluorescence in lung tissue (f); g is a semi-quantitative analysis by fluorescence signal (n=3), with PF488 used to label actin filaments and DAPI used to stain cell nuclei, showing that the dual-modular targeted delivery system has higher fluorescence retention in the lungs;

[0058] like Figure 3 Figure 2 shows the construction, characterization, and in vitro evaluation of N3 functionalized andrographolide-loaded liposomes, where a is the composition of the liposomes; b is dynamic light scattering analysis; c is transmission electron microscopy observation; and d is particle size stability assessment, n=3. The results indicate that the liposomes were successfully constructed.

[0059] Figure 4 The dual modular targeted delivery system of the present invention was used to evaluate the in vivo efficacy of N3-Lip@A, wherein a is the in vivo administration and detection scheme for evaluating the therapeutic effect, n=8; b is the lung morphology, Evans blue staining images and pathological analysis (HE, COX2, iNOS) of mice with acute lung injury after treatment in different experimental groups. The results showed that the alveolar integrity was better, the vascular permeability was lower, and the expression of inflammation-related proteins was lower after treatment with the dual modular targeted delivery system; c is the animal survival curve, and the results showed that more mice survived after treatment with the dual modular targeted delivery system; d is the animal weight monitoring, and the results showed that the weight remained stable after treatment with the dual modular targeted delivery system; e is the lung wet-to-dry weight ratio, n=3, and the results showed that the degree of pulmonary edema was lower after treatment with the dual modular targeted delivery system; f is the lung injury score, n=3, and the results showed that the degree of lung injury was lower after treatment with the dual modular targeted delivery system; g is the quantitative Evans blue staining intensity in the resected lung tissue, n=3, and the results showed that the degree of pulmonary vascular injury was lower after treatment with the dual modular targeted delivery system; h is the detection of inflammatory factors and total protein in BALF, and the results showed that the inflammatory factors in BALF were relatively lower after treatment with the dual modular targeted delivery system.

[0060] like Figure 5Figure 2 shows the preparation and in vitro evaluation of N3-Exo@G exosomes containing a second click chemistry group. Figure A is a schematic diagram of the preparation process; Figure B is a transmission electron microscopy image of exosome morphology; Figure D is a flow cytometer analysis of exosome size; and Figure E is a Western blotting analysis of exosome markers. Exo represents a control exosome without the second click chemistry group. The results demonstrate successful exosome preparation and purification.

[0061] like Figure 6 Figure 3 shows the in vivo efficacy of N3-Exo@G evaluated by a sequential two-step delivery strategy, where a is the in vivo administration and detection scheme used to evaluate the therapeutic effect, n=8; b is the lung morphology, Evans blue staining images and pathological analysis (HE, NE, MPO and CitH3) of mice with acute lung injury after treatment in different experimental groups. The results showed that the dual modular targeted delivery system had better alveolar integrity, lower vascular permeability, and lower expression of neutrophil extracellular trap-related proteins after treatment; c is the animal survival curve, showing that more mice survived after treatment with the dual modular targeted delivery system; d is the survival rate during treatment. The average weight of the animals was monitored, and the results showed that the weight remained stable after treatment with the dual modular targeted delivery system; e is a heat map of the levels of total protein and inflammatory cytokines (IL-1β, IL-6 and TNF-α) in bronchoalveolar lavage fluid (BALF), and the results showed that the inflammatory factors in BALF were relatively lower after treatment with the dual modular targeted delivery system; f is a quantitative analysis radar chart of the lung injury index, lung wet-to-dry weight ratio, lung tissue Evans blue value, animal survival rate and average weight at the end of the experiment, and the results showed that the degree of lung injury, pulmonary edema and pulmonary vascular permeability were lower after treatment with the dual modular targeted delivery system.

[0062] like Figure 7 As shown, the dual modular targeted delivery system of the present invention is composed of mesenchymal stem cells a-MSCs functionally modified with a first click chemistry reaction group, and drug-loaded nanoparticles b-NPs functionally modified with a second click chemistry reaction group. Among them, a-MSCs are administered via intravenous route, and use their inflammatory chemotaxis to reach the inflammatory lesions of the lungs and implant the click chemistry a group; b-NPs are administered via the respiratory route. In the lungs, MSC-NPs conjugates are formed through the click chemistry reaction between the a group and the b group, thereby greatly prolonging the retention time of the nanoparticles in the alveoli, increasing the drug concentration and duration in the pneumonia lesion area, and improving the effectiveness of treatment; the modular design of the present invention reduces the systemic exposure of NPs and reduces liver and kidney toxicity; reduces the single inhalation dose, improves patient compliance, and has great prospects in clinical application.

Claims

1. A dual modular targeted delivery system, characterized in that: The dual-modular targeted delivery system includes an intravenous injection module and a respiratory inhalation module. The intravenous injection module is mesenchymal stem cells a-MSCs functionally modified with a first click chemistry reaction group, and the respiratory inhalation module is drug-loaded nanoparticles b-NPs functionally modified with a second click chemistry reaction group, wherein the first click chemistry reaction group and the second click chemistry reaction group are specific covalent binding groups.

2. The dual modular targeted delivery system according to claim 1, characterized in that: The specific covalent binding groups include the following combinations: azide and dibenzocyclooctyne; azide and bicyclononyne; tetrazine and trans-cyclooctene; tetrazine and cyclopropene; dibenzocyclooctyne-azide and tetrazine-trans-cyclooctene; thiol and maleimide; thiol and acrylate; thiol and alkyne; thiol and halogenated hydrocarbon; thiol and epoxide; thiol and isocyanate; N-hydroxysuccinimide ester and primary amine.

3. The dual modular targeted delivery system according to claim 1, characterized in that: The intravenous injection module is functionally modified using DSPE-PEG amphiphilic lipid molecules, and the functional group containing the first click chemistry reaction group is integrated into the surface of the mesenchymal stem cell membrane through lipid membrane fusion technology or covalent coupling reaction.

4. The dual modular targeted delivery system according to claim 3, characterized in that: The intravenous injection module is prepared by adding 10-1000 μg of functionalized modified molecules per 1 million cells, co-incubating the functionalized modified DSPE-PEG amphiphilic lipid molecules with mesenchymal stem cells in culture medium or other isotonic liquid for 0.5-48 hours, and forming mesenchymal stem cells (a-MSCs) functionalized with the first click chemistry reaction group through lipid membrane fusion technology; Alternatively, 10 to 1000 μg of functionalized modified molecules are added per 1 million cells, and the functionalized modified molecules containing two different active functional groups are co-incubated with mesenchymal stem cells in culture medium or other isotonic liquid for 0.5 to 48 hours. Through the covalent coupling reaction of one of the functional molecules with the cell membrane surface group, mesenchymal stem cells a-MSCs functionalized with the first click chemistry reaction group are formed.

5. The dual modular targeted delivery system according to claim 1, characterized in that: The respiratory inhalation module is obtained by combining liposomes, lipid nanoparticles, polymer micelles, exosomes, polylactic-co-glycolic acid nanoparticles, albumin nanoparticles and therapeutic agents. The therapeutic agents include one or more combinations of chemical small molecule drugs, siRNA / mRNA nucleic acid drugs, monoclonal antibodies or cytokine protein drugs; the drug loading amount of the therapeutic agent is 1%-60% (w / w) by mass percentage; the carrier of the respiratory inhalation module has a particle size of 10nm-15μm.

6. The dual modular targeted delivery system according to claim 5, characterized in that: The respiratory inhalation module is prepared by: preparing liposomes, lipid nanoparticles, polymer micelles or polymer vesicles by solvent injection, thin film hydration, reverse evaporation, double emulsion, microfluidics or ultrasonic dispersion; preparing polylactic acid-glycolic acid copolymer nanoparticles by emulsification-solvent volatilization, nanoprecipitation, microjet homogenization or double emulsion; preparing albumin nanoparticles by self-assembly, emulsion crosslinking, high-pressure homogenization or precipitation; the formula contains compound molecules with second click chemical reaction groups, and these second click chemical reaction groups can be exposed on the surface of the drug-loaded particles during the assembly process of the drug-loaded particles. Alternatively, cells can be metabolized and labeled to produce exosomes containing a second click chemistry reactive group by cellular uptake of non-natural sugars, and then purified by ultracentrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, or immunomagnetic bead methods to obtain exosomes containing a second click chemistry reactive group; or functionalized DSPE-PEG amphiphilic lipid molecules can be co-incubated with exosomes in isotonic fluid for 0.5 to 48 hours, and lipid membrane fusion technology can be used to introduce a second click chemistry reactive group on the surface of the exosomes.

7. Use of the dual modular targeted delivery system according to claim 1 in the preparation of an anti-inflammatory drug targeting the lungs and / or respiratory tract.

8. The use according to claim 7, characterized in that The applicable inflammations include bacterial pneumonia, viral pneumonia, fungal pneumonia, atypical pathogen pneumonia, tuberculosis, hypersensitivity pneumonitis, idiopathic interstitial pneumonia, radiation pneumonia, chemical pneumonia, chronic bronchitis or acute bronchitis.

9. The use according to claim 7, characterized in that The application specifically includes the following steps: (1) administering the intravenous injection module of the dual modular targeted delivery system via intravenous injection; (2) The respiratory inhalation module of the dual modular targeted delivery system is administered via respiratory aerosol inhalation at intervals of 0.1 to 24 hours.

10. The use according to claim 9, characterized in that The intravenous injection module is a sterile injection preparation containing a concentration of 1×10 5 to 2×10 7 cells / mL of a-MSCs suspension, and the auxiliary material is cell isotonic buffer; the respiratory tract inhalation module is an atomized inhalation preparation: containing a b-NPs suspension or dry powder preparation with a drug loading concentration of 0.1-30 mg / mL.

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