WO2023036345A1 - 一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法 - Google Patents

一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法 Download PDF

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WO2023036345A1
WO2023036345A1 PCT/CN2022/118532 CN2022118532W WO2023036345A1 WO 2023036345 A1 WO2023036345 A1 WO 2023036345A1 CN 2022118532 W CN2022118532 W CN 2022118532W WO 2023036345 A1 WO2023036345 A1 WO 2023036345A1
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drug
peg
loaded
nanoparticles
group
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French (fr)
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章雪晴
汪开振
柏欣
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荣灿生物医药技术(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the field of medicine, in particular to a drug-loaded nano particle and an siRNA group sequence for treating pulmonary fibrosis and a design method thereof.
  • Pulmonary fibrosis including IPF (idiopathic pulmonary fibrosis), is a progressive interstitial lung disease with persistent impairment of lung function. At present, about 5 million people in the world suffer from IPF, and the average median survival period is 3-5 years. There are many factors that can cause pulmonary fibrosis, including environment, drug side effects, and genetic factors. In addition, it has been reported that patients infected with SARS coronavirus or 2019 novel coronavirus (2019-nCoV) are also at risk of developing pulmonary fibrosis. However, so far, only pirfenidone and nintedanib are FDA-approved drugs for the treatment of IPF, and they can only delay the progression of the disease, but cannot reverse the existing fibrosis. Therefore, the discovery of new targets and the development of corresponding therapies are particularly urgent and important.
  • the pathological mechanism of IPF can be summarized as follows: repeatedly injured alveolar epithelial cells and recruited inflammatory cells secrete a variety of pro-fibrotic growth factors, cytokines and coagulants, resulting in the recruitment, proliferation and activation of fibroblasts. Fibroblasts exhibit resistance to apoptosis after differentiation into myofibroblasts and accumulate at fibrous foci, depositing excess collagen and other extracellular matrix, causing changes in mechanical stiffness and scarring, reducing lung volume, And further lead to the activation of fibroblasts through a positive feedback loop. Interleukins have multiple roles in fibroblast-associated pulmonary fibrosis.
  • IL-11 is a potent pro-fibrotic cytokine associated with fibrosis in various organs.
  • IL-11 binds to a heterodimeric receptor complex composed of IL-11RA and glycoprotein 130 (gp130), triggering a pro-fibrotic response through extracellular signal-regulated kinase (ERK) signaling.
  • gp130 glycoprotein 130
  • ERK extracellular signal-regulated kinase
  • siRNA Small interfering RNA
  • the systemically injected nanoparticles are quickly cleared by the mononuclear phagocyte system (MPS) in human blood, most of the particles are enriched in the liver and spleen, and less than 5% of the particles finally reach the target tissues or cells .
  • Aerosols deliver therapeutic agents centrally to the lungs in a non-invasive inhalational manner and allow drug deposition throughout the bronchiole and alveolar epithelium, thereby improving compliance and reducing systemic exposure.
  • the treatment of a range of respiratory diseases such as asthma and the global pandemic caused by SARS-CoV-2 presents a huge opportunity.
  • the market is still unable to achieve inhalable nucleic acid delivery.
  • the reason is that the shear force generated during the nebulization process will destroy the structure of lipid nanoparticles, affect the stability of the nucleic acid molecules carried, and thus lose the activity of regulating gene expression.
  • the lipid nanoparticles in the present invention still maintain their particle size and transfection activity after the atomization process, indicating that they can withstand the severe shear force generated in the atomization process and protect the nucleic acid molecules contained in them. Stability, and can penetrate the mucus layer to reach the target cells deep in the lung tissue, efficiently deliver siRNA or mRNA to the lungs, regulate the expression of target genes, and achieve therapeutic purposes.
  • the invention discloses a drug-loaded nanoparticle for atomization inhalation, an siRNA sequence group and a design method for treating pulmonary fibrosis, and belongs to the field of biomedicine.
  • the drug-loaded nanoparticle includes: biodegradable amphiphilic block copolymerization drug and cationic lipid compound; the molar ratio of amphiphilic block copolymer and cationic lipid compound is 1:0.5-80; the drug-loaded nanoparticles can withstand the severe shear force generated by the nebulization process and can penetrate mucus The layer reaches the target cells deep in the lung tissue, breaking through the technical barriers of drug atomization delivery; the invention effectively improves the silencing efficiency of target gene siRNA by setting the screening conditions for siRNA design, and has unexpected technical effects in the treatment of pulmonary fibrosis.
  • the present invention aims to provide a drug-loaded nanoparticle for nebulization inhalation, an siRNA sequence group and its design method for treating pulmonary fibrosis, and the drug-loaded nanoparticle can withstand the severe shear force generated during the nebulization process And it can penetrate the mucus layer to reach the target cells deep in the lung tissue, achieving safe and effective drug delivery.
  • a drug-loaded nanoparticle for nebulization inhalation comprising: a biodegradable amphiphilic block copolymer and a cationic lipid compound; the molar ratio of the amphiphilic block copolymer to the cationic lipid compound is 1:0.5-80 ; As a preference, the molar ratio is 1:40.
  • the amphiphilic block copolymer is a block copolymer of PEG and one or more of the following polymer components, the polymer components include: : One of polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyorthoester, polyanhydride, poly( ⁇ -amino ester) (PBAE) or Various. It should be noted that: the types, numbers, and rules of alternate polymerization of polymer chain segments are not limited.
  • the amphiphilic block copolymer is PLGA-PEG or PLA-PEG.
  • PLGA in PLGA-PEG is a 50:50 copolymer of GA component and LA component, and its molecular weight range is 5000-200000; PLGA- PEG in PEG has a molecular weight in the range of 1,000-100,000.
  • the cationic lipid compound is polyamidoamine type (PAMAM) dendritic molecule and 1,2-epoxytetradecane mixed with a molar ratio of 1:4-1:7
  • PAMAM polyamidoamine type
  • 1,2-epoxytetradecane mixed with a molar ratio of 1:4-1:7
  • the reaction is made.
  • the preferred reaction conditions are 90 ° C for 2 days.
  • the PAMAM dendrimers are preferably low-generation PAMAMs, and as a preference, the cationic lipid compounds are G0-C14.
  • the aforementioned aerosol inhaled drug-loaded nanoparticles also include: loaded pharmaceutical reagents; as an example, the loaded pharmaceutical reagents include: nucleic acids, proteins, drugs for treating diseases or conditions, antibodies, insulin, One or more of polypeptides, glucagon-like peptide-1 (GLP-1) and its analogs, immunogenic compositions, antigens, exosomes (Exosomes), ribonucleic acid protein complexes or vaccines Combination; Nucleic acid is preferably siRNA, messenger RNA (mRNA), DNA, miRNA, antisense oligonucleotide (ASO) and non-coding RNA, more preferably siRNA and mRNA, most preferably siRNA; Nucleic acid and amphiphilic degradable insert The ratio of the mass sum of segment copolymers and lipid polymers is 1:2-1:180, preferably selected from 1:2, 1:10, 1:30, 1:60, 1:120, 1:180, more It is preferably 1:60; as an example, the
  • antibodies include: Adalimumab (Adalimumab), Rituximab (rituximab), vascular endothelial growth factor inhibitor Bevacizumab (bevacizumab), Trastuzumab (trastuzumab), Infliximab (infliximab), etc.;
  • examples of insulin include: Insulin glargine (insulin glargine), etc.;
  • vaccines include: inactivated virus particles, inactivated virus particles, pseudovirus-like particles, mRNA nanoparticles, etc.; it should be noted that these examples are not exhaustive, as long as they can be wrapped by nanoparticles , and agents with pharmaceutical properties are all within the protection scope of the present invention.
  • the aforesaid aerosol inhalation of drug-loaded nanoparticles also includes: a drug auxiliary; as an example, the drug auxiliary includes: an organic solvent or a stabilizer.
  • the stabilizer includes: ceramide-PEG, 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N- One or more of polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid ester, Tween 80, Tween 20, Span80, Span60, sodium dodecylsulfonate; need to explain Yes:
  • DSPE-PEG polyethylene glycol
  • polyvinyl alcohol polyvinyl alcohol
  • polyglycerol fatty acid ester polyglycerol fatty acid ester
  • Tween 80, Tween 20, Span80, Span60 sodium dodecylsulfonate
  • the stabilizer is polyvinyl alcohol or DSPE-PEG in one or more combination, as an example, the stabilizer is polyethylene Alcohol, polyvinyl alcohol molecular weight range is 10,000-250,000kDa, preferably 13,000-23,000kDa; The use concentration range of polyvinyl alcohol is 0.1% ⁇ 10% (w/v), preferably 0.25% (w/v ); As another embodiment, the stabilizer is DSPE-PEG, and the concentration range is 0.005%-1% (w/v), preferably 0.01% (w/v).
  • the aforementioned aerosolized drug-loaded nanoparticles, organic solvents include: dichloromethane, chloroform, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, dioxane, ether, tetrahydrofuran , acetonitrile, methanol, propylene glycol, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF) or a combination of one or more of acetone; preferred: DMF or DMSO; it should be noted that: These examples are not exhaustive, as long as they are organic solvents that can dissolve the composition of the present invention, they all fall within the protection scope of the present invention.
  • the aforementioned aerosol inhalation of drug-loaded nanoparticles, the preparation method of drug-loaded nanoparticles, comprises the following steps:
  • step b) Add the mixture obtained in step a) into an aqueous solution containing a stabilizer, stir and mix evenly at room temperature, preferably the stirring and mixing time is 5-20 minutes, more preferably 10 minutes;
  • step a) is: uniformly mix PLGA-PEG or PLA-PEG, G0-C14 and siRNA or mRNA in DMSO or DMF; siRNA or mRNA, G0-C14, PLGA-PEG or PLA-PEG were mixed at a mass ratio of 1:30:30.
  • the drug-loaded nanoparticle inhaled by atomization, can deliver the drug-loaded nanoparticle to the lung, the drug-loaded nanoparticle is used to deliver the nanoparticle carrying nucleic acid molecules to the lung, the drug-loaded nanoparticle
  • the purpose of the invention is to prepare the medicine for treating fibrosis through inhalation administration.
  • the fibrosis includes pulmonary fibrosis, liver fibrosis, myocardial fibrosis or renal fibrosis; preferably pulmonary fibrosis.
  • siRNA sequence group for treating pulmonary fibrosis is selected from any of the following groups:
  • Group 1 sense (5'-3'): GCUGUUCUCCUAACCCGAUTT SEQ01, antisense (5'-3'): AUCGGGUUAGGAGAACAGCTT SEQ02;
  • Group 3 sense (5'-3'): GCCGUUUACAGCUCUUGAUTT SEQ05, antisense (5'-3'): AUCAAGAGCUGUAAACGGCTT SEQ06;
  • Group 8 sense (5'-3'): UGCACAGAUGAGAGACAAATT SEQ15, antisense (5'-3'): UUUGUCUUCUCAUCUGUGCATT SEQ16;
  • the above sequence group is only a preference, as long as the pharmaceutical reagents using the drug-loaded delivery carrier of the present invention are within the protection scope of the present invention, as long as they are siRNAs screened out by the screening conditions of the siRNA of the present invention
  • siRNAs screened out by the screening conditions of the siRNA of the present invention
  • One group or combinations of several groups are within the protection scope of the present invention.
  • a method for designing an siRNA sequence group for treating pulmonary fibrosis comprising the steps of:
  • Step 1 design the siRNA sequence group complementary to IL11DNA by siRNA sequence design software, set the following screening conditions to obtain the primary screening siRNA sequence group;
  • the filter criteria are:
  • the first base at the 5' end of the antisense strand is A or U, and the first base at the 5' end of the sense strand is G or C;
  • Step 2 preparing the primary screening siRNA sequence groups into drug-loaded nanoparticles
  • Step 3 After co-incubating the drug-loaded nanoparticles containing the primary screening siRNA sequence group with the mouse lung fibroblasts, after amplification and culture, the total RNA was extracted, and the target gene silencing efficiency was evaluated by real-time fluorescent quantitative PCR. Select the target gene with high silencing efficiency to obtain the fine screening siRNA sequence group.
  • the siRNA sequence design software includes: Oligowalk, siDirect, i-Score or DSIR; the examples of the above software are not exhaustive, as long as the siRNA sequence design software is used
  • the siRNA groups obtained under the screening conditions of the present invention are all within the protection scope of the present invention.
  • the PEG-modified amphiphilic copolymer in the nucleic acid delivery carrier of the present invention can maintain the structure of nanoparticles, and the cationic lipid compound G0-C14 encapsulates and protects the nucleic acid inside the nanoparticles through electrostatic interaction; the PEG-modified amphiphilic copolymer
  • the nanoparticle obtained by the combination of the substance and G0-C14 can realize atomization without the influence of shear force, and it is surprisingly found that the nanoparticle can penetrate the mucus layer and reach the target cells in the deep lung tissue (Example 3(5) mucus penetration Research), and effectively mediate mRNA expression, it is a safe and effective mRNA delivery carrier for inhalation administration, breaking through the technical barriers of drug atomization delivery, and has unexpected technical effects;
  • the stabilizer disperses the formed nanoparticles in the water phase without aggregation and sedimentation
  • the present invention identifies IL11 as a potential target for the treatment of IPF, and inhibiting the expression of IL11 can effectively reduce and reverse the process of pulmonary fibrosis, and can achieve the therapeutic effect of anti-fibrosis and recovery of lung function; the nanoparticles of the present invention can effectively down-regulate lung fibrosis in mice
  • the expression of IL11 in the cells significantly inhibited the migration and activation of fibroblasts, and reduced the deposition of collagen; the experimental results showed that the process of lung fibrosis in mice was significantly inhibited, and the lung function was also significantly improved;
  • the stability of the siRNA double strands is enhanced to increase the probability and effect of inhibition, and it is verified by experiments that the four screening conditions have a synergistic effect on improving the efficiency of target gene silencing. It has unexpected technical effects in the treatment of pulmonary fibrosis;
  • the nanoparticle of the present invention has a particle size of 80-150nm, good dispersion (PDI ⁇ 0.2), uniform size, nucleic acid loading efficiency of more than 80%, and good performance in the range of 4-8pH and -20°C-2°C. stability.
  • Fig. 1 is the mechanism of action of the inhaled nanoparticle of the present invention for the treatment of respiratory diseases such as IPF;
  • Figure 6 is a schematic diagram of the experimental results of siIL11@NPs inhibiting the activation and migration of mouse lung fibroblasts; a, PBS (phosphate buffer saline, phosphate buffer solution), siScr@NPs or siIL11@NPs treatment, TGF- ⁇ 1 activation Representative images of ACTA2 and COL1A1 immunofluorescence in MLFs.
  • PBS phosphate buffer saline, phosphate buffer solution
  • siScr@NPs or siIL11@NPs treatment TGF- ⁇ 1 activation
  • TGF- ⁇ 1 activation Representative images of ACTA2 and COL1A1 immunofluorescence in MLFs.
  • a-h the measured lung function parameters include deep inspiratory volume (a), respiratory system resistance (b), compliance (c), elasticity ( d), forced vital capacity (e), forced expiratory volume at 0.2s (f),
  • Amphiphilic block copolymer refers to a macromolecule that contains both a hydrophilic segment and a hydrophobic segment in the same polymer.
  • the amphiphilic block copolymer is: a block copolymer formed of PEG and one or more of the following polymer components, and the polymer components include: polylactic acid-polyglycolic acid copolymer (PLGA), poly One or more of lactic acid (PLA), polycaprolactone (PCL), polyorthoester, polyanhydride, poly( ⁇ -amino ester) (PBAE).
  • Nucleic acid is the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is a biological macromolecule composed of multiple nucleotide monomers; base, nitrogenous base, or any modifying group. If the five-carbon sugar is ribose, the polymer formed is RNA; if the five-carbon sugar is deoxyribose, the polymer formed is DNA.
  • Nucleic acid molecules include single-stranded DNA, double-stranded DNA, short isomers, mRNA, non-coding RNA (microRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA), Telomerase RNA (Telomerase RNA Component) , scRNA, circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense DNA, antisense RNA, antisense oligonucleotide (ASO), ribozyme (ribozyme), asymmetric interference RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), gRNA, sgRNA, crRNA or tracrRNA, locked nucleic acid (LNA), peptide nucleic acid ( PNA), morpholino antisense
  • PAMAM refers to polyamidoamine-type dendritic molecules, which have the characteristics of high branching, high electropositive functional group density and internal cavity;
  • G0-PAMAM refers to the 0th generation polyamidoamine dendrimers, and its chemical structure is as follows: Formula I:
  • IL11 refers to interleukin-11, which is a cytokine of the chemokine family
  • TGF- ⁇ 1 refers to transforming growth factor- ⁇ , transforming growth factor- ⁇ , which belongs to a group of newly discovered TGF- ⁇ superfamily that regulates cell growth and differentiation;
  • ERK is the English abbreviation of extracellular regulated protein kinases, which refers to extracellular regulatory protein kinases, including ERK1 and ERK2, which are the key to transmitting signals from surface receptors to the nucleus;
  • p-ERK refers to phosphorylated extracellular regulated protein kinase
  • the protein encoded by the SMAD2 gene belongs to the SMAD protein family, and the SMAD protein is a regulator of signal transduction and transcription that mediates various signaling pathways;
  • p-SMAD2 refers to the protein encoded by the phosphorylated SMAD2 gene
  • GAPDH is the English abbreviation of glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase);
  • DAPI refers to: 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), a fluorescent dye that can strongly bind to DNA;
  • Cy5.5 refers to: Cyanine5.5NHS ester is a reactive dye for labeling peptides, proteins and oligonucleotides containing primary amines.
  • ACTA2 is the abbreviation of actin alpha 2, which is a kind of actin;
  • STAT3 is an abbreviation for STAT3 protein expressed by a gene located on chromosome 17: Signal transducer and activator of transcription 3.
  • p-STAT3 refers to phosphorylated STAT3 protein
  • COL1A1 refers to: type I collagen ⁇ 1 (COL1A1) recombinant protein, full name: Recombinant Collagen Type I Alpha 1;
  • PBS phosphate buffer saline
  • MLFs refers to mouse lung fibroblasts
  • DSPE-PEG is: 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-polyethylene glycol;
  • PLGA Polylactic acid-polyglycolic acid copolymer
  • PLA is: polylactic acid (polylactic acid), also known as polylactide, is a polyester polymer obtained by polymerization of lactic acid as the main raw material;
  • PCL is: Polycaprolactone (polycaprolactone);
  • PBAE Poly ⁇ -amino acid, poly( ⁇ -amino ester);
  • PEG is: Polyethylene glycol (polyethylene glycol);
  • DMSO Dimethyl sulfoxide (dimethyl sulfoxide);
  • DMF is: N,N-Dimethylformamide (N,N-dimethylformamide);
  • FAM-siRNA refers to FAM-labeled siRNA
  • Cy7-siRNA refers to Cy7-labeled siRNA
  • NPs refers to nanoparticles (nanoparticles);
  • Cy5.5-NPs refers to Cy5.5-labeled nanoparticles
  • BALF refers to bronchoalveolar lavage fluid.
  • siIL11 refers to siRNA targeting IL11
  • siIL11@NPs refers to nanoparticles loaded with siRNA targeting IL11
  • siScr@NPs refers to nanoparticles loaded with reference siRNA, where reference siRNA refers to siRNA that does not specifically target mRNA;
  • mLuc@NPs refers to nanoparticles loaded with Luciferase mRNA
  • PPGC-NPs refers to the hybrid nanoparticles of PLGA-PEG amphiphilic polymer/GO-C14 cationic lipid compound of the present invention.
  • the components in the composition of the present invention can be replaced on the same function, and the consumption of each component in the composition of the present invention can be any within the scope of the claims.
  • the formulations can have widely different embodiments and applications. There can be many changes in the selection of sequences, as long as the sequences obtained according to the design and screening idea of the present invention should be within the protection scope of the present invention.
  • the disclosures and descriptions herein are illustrative only and not limiting in any sense.
  • Figure 1 illustrates the mechanism of action of inhaled nanoparticles for the treatment of respiratory diseases such as IPF.
  • Nanoparticles are administered by inhalation, delivered to the lungs of mice, release the entrapped nucleic acid molecules, act on fibroblasts, reduce the expression of the target protein IL11, and reduce the myogenesis of fibroblasts through the two pathways of ERK and SMAD2. Transformation of fibroblasts and deposition of extracellular matrix to achieve the treatment of pulmonary fibrosis.
  • IL-11 and ACTA2 a marker of myofibroblasts
  • Co-incubate with slices wash with PBS, incubate with enzyme-labeled secondary antibody, and observe under a microscope after color development by the substrate.
  • the present invention identifies IL11 as a potential target for the treatment of IPF, and inhibiting the expression of IL11 can effectively alleviate and reverse the process of pulmonary fibrosis, and achieve the therapeutic effects of anti-fibrosis and recovery of lung function. Subsequent experiments using the nanoparticles of the present invention further confirmed this point.
  • siIL11 sequence The design method of siIL11 sequence is:
  • Step 1 Design the siRNA sequence group complementary to IL11DNA by four siRNA sequence design software Oligowalk, siDirect, i-Score, and DSIR, and set the following screening conditions to obtain the primary screening siRNA sequence group, as shown in Table 1;
  • the filter criteria are:
  • the first base at the 5' end of the antisense strand is A or U, and the first base at the 5' end of the sense strand is G or C.
  • step 2 the primary screening siRNA sequence group was mixed with PLGA-PEG and G0-C14 respectively to prepare nanoparticles (NPs) loaded with different siIL11 (the preparation of nanoparticles (NPs) is shown in Example 2).
  • each nanoparticle (NPs) loaded with siIL11 was co-incubated with MLFs for 4 hours, and after 4 hours, it was replaced with a complete medium containing 10% FBS fetal bovine serum to continue culturing for 20 hours.
  • Total RNA was extracted using Trizol kit, and the efficiency of target gene silencing was evaluated by qPCR.
  • the sequences of qPCR primers used in the experiment are as follows: Forward: 5'-TGTTCTCCTAACCCGATCCCT-3'; Reverse, 5'-CAGGAAGCTGCAAAGATCCCA-3'.
  • the selected sequences numbered 1, 6, and 10 are respectively represented by siIL11-1, siIL11-2, and siIL11-3.
  • the results of the target gene silencing experiment data are shown in Figure 5j. It can be seen that the siRNA numbered 1 has the best target gene silencing efficiency , the sequence of the sense strand (5'-3') is GCUGUUCUCCUAACCCGAUTT SEQ01; the sequence of the antisense strand (5'-3') is AUCGGGUUAGGAGAACAGCTT SEQ02.
  • NPs nanoparticles
  • the test sample can significantly inhibit the amount of IL11mRNA, and the comparison sample 1-3 can also inhibit, but the effect is not as significant as the test sample, indicating that these four screening conditions can produce a synergistic effect on improving the efficiency of target gene silencing, lack of any one None of the conditions can screen to obtain siRNA that can significantly inhibit IL11; the reason may be that such four screening conditions enhance the stability of the double strand and the targeting effect on the target gene, thereby improving the inhibition efficiency.
  • NPs nanoparticles
  • poly(lactic-co-polyglycolic acid) (PLGA) was purchased from Lactel Absorbable Polymers.
  • Sulfo-cy5-maleimide was purchased from Lumiprobe. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and N,N-diisopropylethylamine (DIPEA ) was purchased from Beijing Yinuokai.
  • EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • DIPEA N,N-diisopropylethylamine
  • Polyamide dendrimer GO PAMAM
  • 1,2-epoxytetradecane C14
  • the synthetic method of PLGA-PEG PLGA is activated by EDC and NHS, and the time is 2 hours, and the temperature is room temperature, and the PLGA obtained is precipitated twice in precooled methanol/ether (50/50v/v);
  • the PLGA-NHS reacted with heterobifunctional PEG (NH 2 -PEG-OCH 3 ) and DIPEA in a basic organic solvent (N,N-diisopropylethylamine) at 25°C for 48 hours, and removed by rotary evaporation
  • the residual organic solvent was used to obtain the amphiphilic degradable block polymer PLGA-PEG, and the product was characterized by 1 H NMR after purification.
  • composition of GO-C14 (cationic lipid compound) and amphiphilic block copolymer is prepared into the nanoparticle sample of embodiment 1-3 according to the formula:
  • Nanoparticles were prepared according to the following preparation method of the following formula
  • Step 1 Dissolve G0-C14 in an organic solvent at a concentration of 10 mg/ml
  • Step 2 Add G0-C14, PLGA-PEG or PLA-PEG, siRNA in sequence, the solvent is the same as step 1, and mix evenly to obtain a mixture;
  • Step 3 Add 0.25% (w/v) polyvinyl alcohol (stabilizer) into sterile water (no DNAase/RNAase), and mix evenly to obtain a mixture;
  • Step 4 Add the mixture obtained in step 2 to the mixture obtained in step 3, the volume ratio is 1:10, and mix well;
  • Step 5 standing for 20 minutes, purifying and concentrating the obtained nanoparticles by ultrafiltration;
  • Step 6 Before the cell experiment and animal experiment, dilute the nanoparticles obtained in step 5 to the use concentration with sterile saline for injection (no DNAase/RNAase).
  • NPs nanoparticles
  • Nanoparticles were prepared according to the following preparation method of the following formula
  • Step 1 dissolving the cationic lipid in an organic solvent at a concentration of 10 mg/ml
  • Step 2 Add cationic lipid, PLGA-PEG or PLA-PEG, siRNA in sequence, the solvent is the same as step 1, and mix evenly to obtain a mixture;
  • Step 3 Add 0.25% (w/v) polyvinyl alcohol (stabilizer) into sterile water (no DNAase/RNAase), and mix evenly to obtain a mixture;
  • Step 4 Add the mixture obtained in step 2 to the mixture obtained in step 3, the volume ratio is 1:10, and mix well;
  • Step 5 leave it to stand for 20 minutes, and purify and concentrate the obtained nanoparticles by ultrafiltration; obtain nanoparticles.
  • NPs nanoparticles
  • the samples of Examples 1-7 were taken for nebulization.
  • the nebulizer device was constructed as follows: the inhalation chamber was connected to a custom-made nose cone designed in such a way that only the nose of the mouse was exposed to the aerosol.
  • the upstream of the inhalation chamber was connected to a nebulizer (Aerogen, Ireland), and the required volume of nanoparticles (NPs) was added to the nebulizer to generate an aerosol through a vibrating mesh screen.
  • siRNA, cationic lipid compound, and PLGA-PEG are mixed to obtain a mixed solution, wherein the molar ratio of cationic lipid compound to PLGA-PEG is 40:1.
  • the siRNA combination adopted is: sequence 1: sense strand (5'-3') GCUGUUCUCCUAACCCGAUTT SEQ01, its antisense strand (5'-3') AUCGGGUUAGGAGAACAGCTT SEQ02; and sequence 2: sense strand (5'-3') GCUGGGACAUUGGGAUCUUTT SEQ03 , antisense strand (5'-3') AAGAUCCCAAUGUCCCAGCTT SEQ04.
  • the mixed solution was added to an aqueous solution containing 0.25% polyvinyl alcohol (molecular weight: 13,000-23,000 kDa), and stirred and mixed at room temperature for 10 minutes.
  • NPs Nanoparticles
  • MWCO ultrafiltration device
  • the nebulizer setup was built as follows: The inhalation chamber was connected to a custom-made nose cone designed in such a way that only the nose of the mouse was exposed to the aerosol. The upstream of the inhalation chamber was connected to a nebulizer (Aerogen, Ireland), and the required volume of nanoparticles (NPs) was added to the nebulizer to generate an aerosol through a vibrating mesh screen. Connect downstream to a vacuum pump to maintain a continuous flow of aerosol in the system. Constantly monitor the pressure gauge connected to the chamber and adjust the valve to ensure that the chamber is consistent with the outside atmospheric pressure.
  • NPs nanoparticles
  • NPs Nanoparticles
  • MWCO ultrafiltration device
  • Scramble siRNA (siScr, sense strand (5'-3') UUCUCCGAACGUGUCACGUTT SEQ27, its antisense strand (5'-3') ACGUGACACGUUCGGAGAATT SEQ28), cationic lipid compound, and PLGA-PEG were mixed to obtain a mixed solution, in which the cationic lipid compound The molar ratio with PLGA-PEG is 40:1.
  • the mixed solution was added to an aqueous solution containing 0.25% polyvinyl alcohol (molecular weight: 13,000-23,000 kDa), and stirred and mixed at room temperature for 10 minutes.
  • Nanoparticles were purified and concentrated by an ultrafiltration device (MWCO of 100 kDa) to obtain siScr@NPs.
  • the siScr@NPs after nebulization were obtained by the method in 2.1.
  • siRNA (sense strand (5'-3') GCUGUUCUCCUAACCCGAUTT SEQ01, its antisense strand (5'-3') AUCGGGUUAGGAGAACAGCTT SEQ02), cationic lipid compound, PLGA-PEG (10% PLGA-PEG coupled by Cy5.5 PLGA-PEG instead) mixed, wherein the molar ratio of cationic lipid compound to PLGA-PEG is 40:1.
  • the above mixed solution was added into an aqueous solution containing 0.25% polyvinyl alcohol (molecular weight: 13,000-23,000 kDa), and stirred and mixed at room temperature for 10 minutes.
  • Cy5.5-NPs were obtained by purifying and concentrating the nanoparticles (NPs) by an ultrafiltration device (MWCO: 100kDa). Use the method in 2.1 to obtain Cy5.5-NPs after nebulization.
  • FAM-labeled siRNA FAM-labeled siRNA
  • sense strand 5'-3'
  • GCUGUUCUCCUAACCCGAUTT sense strand
  • antisense strand 5'-3'
  • AUCGGGUUAGGAGAACAGCTT cationic lipid compound
  • PLGA mixed wherein cationic lipid compound and PLGA
  • the molar ratio is 40:1.
  • the above mixed solution was added into an aqueous solution containing 0.25% polyvinyl alcohol (molecular weight: 13,000-23,000 kDa), and stirred and mixed at room temperature for 10 minutes.
  • Nanoparticles Nanoparticles (NPs) were purified and concentrated by an ultrafiltration device (MWCO: 100kDa) to obtain FAM-siRNA@NPs.
  • Cy7-labeled siRNA (Cy7-siRNA, sense strand (5'-3') GCUGUUCUCCUAACCCGAUTT, its antisense strand (5'-3') AUCGGGUUAGGAGAACAGCTT), cationic lipid compound, PLGA-PEG mixed, wherein cationic lipid compound and The molar ratio of PLGA-PEG is 40:1.
  • the above mixed solution was added into an aqueous solution containing 0.25% polyvinyl alcohol (molecular weight: 13,000-23,000 kDa), and stirred and mixed at room temperature for 10 minutes.
  • Nanoparticles (NPs) were purified and concentrated by an ultrafiltration device (MWCO 100kDa) to obtain Cy7-siRNA@NPs.
  • nanoparticles were incubated with RNase (final concentration: 10 ng/mL) respectively, and the protective effect of nanoparticles (NPs) on siRNA was detected by agarose gel electrophoresis.
  • RNase final concentration: 10 ng/mL
  • the particle size and potential of siIL11@NPs in PBS at various pH were measured.
  • the results showed that neither the particle size nor the electric potential of the nanoparticles (NPs) changed significantly within the tested pH range (Fig. 5c).
  • the particle size of siIL11@NPs did not change significantly before and after atomization, and the hydrated diameter was about 100-110 nm (Fig. 5d). -60nm (Fig. 5e).
  • MLFs were isolated from the lungs of 8-week-old male C57BL/6 mice. The extraction steps are briefly described as follows: mouse lungs were removed, chopped, immersed in serum-free DMEM containing 1 mg/mL collagenase I and 1% penicillin-streptomycin, and digested at 37°C for 30 minutes. It was then neutralized with DMEM containing 10% FBS and centrifuged. The obtained tissue pellet was washed with PBS, and finally resuspended in complete medium (DMEM+10% FBS+1% penicillin/streptomycin), and this time was recorded as day 0. After the cells grew out on the fourth day, they were digested and passaged with 0.25% trypsin-EDTA. TGF- ⁇ 1 (10ng/mL) was used to induce the above MLFs to differentiate into myofibroblasts.
  • Cy5.5-NPs The preparation method of Cy5.5-NPs is shown in 2.4.
  • MLFs and A549 purchased from the Chinese Academy of Sciences
  • Cy5.5-NPs were co-incubated with MLFs and A549 for 4 hours, and discarded after 4 hours.
  • FAM-siRNA@NPs were obtained by using PPGC-NPs to carry FAM-siRNA, as shown. After co-incubating FAM-siRNA@NPs and MLFs for 4 hours, the supernatant was discarded, washed with PBS, digested with trypsin, centrifuged to obtain the cell pellet, resuspended in PBS, and detected by flow cytometry (FACS). As shown in Figure 5h, it can be seen from Figure 5h that the greater the dose of nanoparticles (NPs), the greater the proportion of FAM-positive lung fibroblasts, so the uptake of NPs in MLFs is dose-dependent.
  • NPs nanoparticles
  • NPs nanoparticles
  • CCK8 detection kit The biocompatibility of nanoparticles (NPs) was evaluated using CCK8 detection kit. MLFs were seeded in a 96-well plate at 1 ⁇ 104 cells/well, incubated overnight with different doses (0, 6, 12, 30, 60, 90, 120 ⁇ g/mL) of siScr@NPs for 4 h, and then replaced. into a complete culture medium. After 24 hours, the medium was replaced with fresh serum-free medium containing 10 ⁇ L of CCK8 solution per well, incubated for 2 hours, and the absorbance value was measured at 450 nm. The results are shown in Figure 5i, the cell viability is above 80%, and the nanoparticles (NPs) exhibit good biocompatibility even at the highest dose.
  • the artificial mucus was prepared as follows: 500 mg DNA, 250 mg mucin, 250 ⁇ L sterile egg yolk emulsion, 0.295 mg DTPA, 250 mg NaCl, 110 mg KCl, and 1 mL RPMI were dispersed into 50 mL water and stirred overnight.
  • Example 2 Using the preparation method of Example 2, prepare the FAM-siRNA@NPs sample (control group) entrapped by G0-C14 and PLGA, and the Cy7-siRNA sample (test group) entrapped by G0-C14 and PLGA-PEG , instilled through the trachea at the same time, and the lungs of the mice were taken out 30 minutes later, fixed with paraformaldehyde and sucrose gradient sedimentation, then embedded in OCT, sliced with a Leica microtome, and stained with DAPI for 5 minutes. The effect of PEG on the ability of nanoparticles to penetrate the lung mucus layer in vivo was compared using laser scanning confocal microscopy.
  • fibroblasts Under the stimulation of TGF- ⁇ 1 in vitro, fibroblasts can differentiate into myofibroblasts, and type I collagen ⁇ 1 (COL1A1) and ACTA2 are the two main markers of myofibroblasts.
  • PPGC-NPs to carry the best siIL11 (Example 2.1) screened in the above experiments, and evaluated its ability to regulate the differentiation of MLFs.
  • MLFs were inoculated on the slides for overnight culture, and NPs diluted in serum-free DMEM were added to incubate for 4 hours, then replaced with complete medium and continued to culture for 20 hours.
  • Serum-free medium was starved overnight, stimulated with TGF- ⁇ 1 (10ng/mL) for 24 hours, fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.5% Triton X-100 for 20 minutes, blocked with 3% BSA, incubated overnight with Alexa
  • Alexa Alexa
  • the conjugated secondary antibody was incubated at room temperature for 1 hour, DAPI stained the nucleus for 10 minutes, and finally observed with an upright fluorescent microscope (Olympus).
  • pulmonary fibrosis also involves the migration of fibroblasts and myofibroblasts to the fibrous focus and the production of a large amount of extracellular matrix.
  • siScr@NPs obtained in Example 2 as a control to evaluate the effect of the siIL11@NPs samples obtained in Example 2 on the migration behavior of fibroblasts through cell scratch and transwell experiments.
  • Cell scratch test mouse lung fibroblasts were inoculated in 24-well plates in advance and cultured overnight, and the cells adhered to the wall. The cells were incubated with PBS, siScr@NPs, and siIL11@NPs for 4 h, and then replaced with serum-free medium for 20 h. After 24 hours of starvation in the serum-free medium, scratch with a 10 ⁇ l gun tip and continue to culture, and count the healing area at 0 and 24 hours respectively. As shown in Figure 6d and 6e, 24h after scratching, there was a significant difference in the cell healing area between the siScr@NPs and siIL11@NPs treatment groups.
  • Transwell experiment The experimental method is shown in Figure 6f, in vitro simulation of cell migration process triggered by pro-fibrotic cytokines.
  • Mouse lung fibroblasts were seeded on the upper side of the transwell membrane (porous membrane in the figure) and cultured overnight until the cells adhered to the wall.
  • the cells were incubated with PBS, siScr@NPs, and siIL11@NPs for 4 h, and then replaced with complete medium for 20 h. After 24 hours of serum-free starvation culture, the culture medium was replaced with 10ng/mL TGF- ⁇ 1 and 1% FBS to continue the culture, and PBS treated cells without 10ng/mL TGF-beita1 treatment was used as a control.
  • MLFs were seeded in 24-well plates and cultured overnight.
  • the cells were incubated with PBS, siScr@NPs, and siIL11@NPs for 4 h, and then replaced with complete medium for 20 h.
  • the culture medium was replaced with 10ng/mL TGF-beita1 and 1% FBS to continue the culture, and cells incubated with PBS without 10ng/mL TGF-beita1 treatment were used as controls.
  • the cells were lysed for Western blot analysis to study the effect of IL11 knockdown on the expression of fibrosis-related genes.
  • the analysis results showed in Figure 6i that siIL11 could significantly down-regulate the expression of IL11 protein in MLFs, and the expression levels of ACTA2 and COL1A1 were also significantly reduced.
  • siIL11@NPs inhibited fibroblast activation, including canonical signal transducer and activator of transcription 3 (STAT3), non-canonical extracellular signal-regulated kinase (ERK) and SMAD2 signaling pathways.
  • STAT3 canonical signal transducer and activator of transcription 3
  • ERK extracellular signal-regulated kinase
  • SMAD2 signaling pathways including canonical signal transducer and activator of transcription 3 (STAT3), non-canonical extracellular signal-regulated kinase (ERK) and SMAD2 signaling pathways.
  • STAT3 canonical signal transducer and activator of transcription 3
  • ERK extracellular signal-regulated kinase
  • SMAD2 extracellular signal-regulated kinase
  • PPGC-NPs deliver mRNA to the lungs by aerosol inhalation
  • PPGC-NPs were used to carry luciferase-encoding mRNA (the sample mLuc@NPs in Example 2), and delivered to the lungs of mice by aerosol inhalation. After 24 hours, the mice were dissected, and the hearts, liver, spleen, lungs and kidneys were removed and imaged with the IVIS system. The results showed that 24 hours after aerosol inhalation, luciferase was effectively expressed in each lobe of the lung (Fig. 7a, b, c). The No. 2 lung lobe was lysed with the lysate, and detected with the luciferase detection kit.
  • Cy5.5-labeled nanoparticles (Cy5.5-NPs sample in Example 2) were used to deliver siRNA to the lungs of mice through aerosol inhalation, and the tissue distribution and subcellular localization of nanoparticles were observed. After 24 hours, the fluorescence imaging and quantitative analysis of the five lung lobes were performed using IVIS, and Cy5.5-NPs were evenly distributed in the five lung lobes (Fig. 8a, b). In addition, the No.
  • mice Male C57BL/6 mice aged 8-10 weeks were purchased from Beijing Weitong Lihua. A mouse model of pulmonary fibrosis was established by a single intratracheal instillation of bleomycin sulfate. All animal experiments in the present invention were carried out under the approval of the Experimental Animal Ethics and Use Committee of Shanghai Jiaotong University.
  • Inhalation therapy was administered after bleomycin instillation, wherein the low dose was 15 ⁇ g siRNA(siIL11@NPs) per mouse, and the high dose was 30 ⁇ g siRNA(siIL11@NPs) per mouse.
  • blood and alveolar lavage fluid were collected (see Figure 9a for a diagram of the experimental design).
  • the mouse left lung and other lung lobes were then collected for RNA extraction and Western blot analysis.
  • TGF- ⁇ 1 is a key mediator involved in the development of pulmonary fibrosis, which can be released by epithelial cells, macrophages, etc., and plays an important role in cell apoptosis, fibroblast proliferation, myofibroblast differentiation and collagen synthesis.
  • Determination of hydroxyproline content in mouse lung by alkaline hydrolysis method Take the No. 3 lung lobe of the mouse, alkaline hydrolyze it in a boiling water bath for 20 minutes, add a pH indicator, and adjust the pH to about 6.0-6.8. After being adsorbed by activated carbon, centrifuge to take the supernatant, add detection reagents for detection, and measure the absorbance value at a wavelength of 550 nm after incubation at 60°C.
  • the software analyzes the survival rate of mice with pulmonary fibrosis treated by different treatment groups. As shown in Figure 10g, after aerosol inhalation of siIL11@NPs, the survival rate of model mice was significantly improved.
  • Orbital blood was collected from the mice, and the blood was collected in a centrifuge tube at 37°C (or room temperature) for 1 hour for coagulation and stratification. Then centrifuge at room temperature for 10 min at 3000 rpm, and transfer the supernatant to a clean centrifuge tube. According to the instructions of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits (Nanjing Jiancheng), the serum was analyzed for AST and ALT biochemical detection results.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • PFT is a routine examination for the diagnosis of clinical IPF. Mice were given PBS and siIL11@NPs after tracheal instillation of bleomycin, and mice instilled with normal saline were used as healthy controls. On the 21st day, the mice in each group were anesthetized and intubated, and the lung function of the mice was tracked and evaluated with the FlexiVent system (SCIREQ).
  • SCIREQ FlexiVent system

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Abstract

一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法，具体地，提供了一种雾化吸入的载药纳米颗粒，其特征在于，包括：生物可降解两亲性嵌段共聚物和阳离子脂质化合物；所述两亲性嵌段共聚物和阳离子脂质化合物的摩尔比为1：0.5-80。

Description

一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法
优先权声明
本申请要求向中国国家知识产权局提交的专利申请号为：2021110677808的优先权，本发明将特此引用该专利的内容。
技术领域
本发明涉及医药领域，具体涉及载药纳米颗粒和治疗肺纤维化的siRNA组序列及其设计方法。
技术背景
肺纤维化，包括IPF(特发性肺纤维化)，是进行性的间质性肺病并伴有持续的肺功能损伤。目前全球约有500万人患有IPF，平均中位生存期为3-5年。引起肺纤维化的因素很多，包括环境、药物副作用和遗传因素等，此外，据报道，SARS冠状病毒或2019年新型冠状病毒(2019-nCoV)感染的患者也有发生肺纤维化的风险。然而迄今为止，FDA批准的用于IPF治疗的药物仅有吡非尼酮和尼达尼布，且他们仅能延迟疾病的进展，而不能逆转已有的纤维化。因此新的靶标发现以及对应性疗法的开发显得尤为迫切和重要。
IPF的病理机制可归纳如下：反复损伤的肺泡上皮细胞和募集而来的炎性细胞分泌多种促纤维化生长因子、细胞因子和促凝剂，从而导致成纤维细胞的募集、增殖和活化。成纤维细胞在分化成为肌成纤维细胞之后表现出凋亡抗性，并在纤维灶处积聚，沉积过多的胶原蛋白和其他细胞外基质，引起机械刚度改变和瘢痕形成，减少了肺体积，并通过正反馈回路进一步导致成纤维细胞的激活。白介素在成纤维细胞相关的肺纤维化中具有多种作用。最新研究表明，IL-11是一种有效的促纤维化细胞因子，与多种器官的纤维化相关。IL-11与IL-11RA和糖蛋白130(gp130)组成的异二聚体受体复合物结合，通过细胞外信号调节激酶(ERK)信号通路触发促纤维化反应。本发明实验结果表明，IL-11的下调对减轻包括肺纤维化在内的多种器官纤维化疾病均具有治疗作用。
小干扰RNA(siRNA)已经显示出具有治疗各种疾病的巨大前景，但是其负电性强、分子量大、稳定性差，使其在临床应用方面仍存在挑战。安全高效的纳米载体是实现体内、外递送siRNA治疗分子的关键，将极大促进基因治疗在临床应用中的进程。
然而系统注射的纳米粒子很快即被人体血液中的单核吞噬细胞系统(mononuclear phagocyte system,MPS)清除，多数粒子富集到肝脏和脾脏，仅不到5％的粒子最终到达靶组织或细胞。气雾剂以无创吸入的递药方式将治疗剂集中递送到肺部，并允许药物沉积在整个细支气管和肺泡上皮中，从而提高依从性并减少全身暴露，为囊性纤维化 (CF)、哮喘和由SARS-CoV-2引起的全球大流行等一系列呼吸系统疾病的治疗提供了巨大契机。
但是市场仍然无法实现可吸入式核酸递送，原因在于：雾化过程中产生的剪切力，会破坏脂质纳米粒子的结构，影响包载的核酸分子的稳定性，从而丧失调控基因表达的活性。而本发明中的脂质纳米粒子，在经过雾化过程后，仍然保持其粒径和转染活性，表明其能够承受雾化过程中产生的剧烈剪切力，保护所包载的核酸分子的稳定性，并能穿透粘液层到达肺组织深部目标细胞，向肺部高效递送siRNA或mRNA，调控目标基因的表达，达到治疗目的。
说明书摘要
本发明公开了一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法，属于生物医药领域，载药纳米颗粒包括：生物可降解两亲性嵌段共聚物和阳离子脂质化合物；两亲性嵌段共聚物和阳离子脂质化合物的摩尔比为1：0.5-80；该载药纳米颗粒能够承受雾化过程产生的剧烈剪切力并能够穿透粘液层到达肺组织深部目标细胞，突破了药物雾化递送的技术壁垒；本发明通过设定siRNA设计的筛选条件有效提升目标基因siRNA的沉默效率，在治疗肺纤维化上具有意想不到的技术效果。
发明内容
发明目的：本发明旨在提供一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法，该载药纳米颗粒能够承受雾化过程产生的剧烈剪切力并能够穿透粘液层到达肺组织深部目标细胞，实现安全有效的递送药物。
技术方案：
一种雾化吸入的载药纳米颗粒，包括：生物可降解两亲性嵌段共聚物和阳离子脂质化合物；两亲性嵌段共聚物和阳离子脂质化合物的摩尔比为1：0.5-80；作为一种优选，摩尔比为1:40。
前述的一种雾化吸入的载药纳米颗粒，作为一种实施例，两亲性嵌段共聚物为PEG与下列一种或多种聚合物组分的嵌段共聚物，聚合物组分包括：聚乳酸-聚羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚原酸酯、聚酸酐、聚(β-氨基酯)(PBAE)中的一种或多种。需要说明的是：聚合物链段的种类、个数、交替聚合的规律均不受限制。
前述的一种雾化吸入的载药纳米颗粒，作为一种优选，两亲性嵌段共聚物为PLGA-PEG或PLA-PEG。
前述的一种雾化吸入的载药纳米颗粒，作为一种优选，PLGA-PEG中的PLGA为GA组分和LA组分为50:50的共聚物，其分子量范围为5000-200000；PLGA-PEG中的PEG为分子量范围为1000-100000。
前述的一种雾化吸入的载药纳米颗粒，阳离子脂质化合物为聚酰胺胺型(PAMAM)树枝状分子和1,2-环氧十四烷以1:4-1:7的摩尔比混合反应制得。作为一种优选为：1：5的摩尔比混合反应制得，优选的反应条件为90℃下反应2天。
前述的一种雾化吸入的载药纳米颗粒，PAMAM树枝状分子优选低代PAMAM，作为一种优选，阳离子脂质化合物为G0-C14。
前述的一种雾化吸入的载药纳米颗粒，还包括：所载的药物试剂；作为一种实施例，所载的药物试剂包括：核酸、蛋白、治疗疾病或病况的药物、抗体、胰岛素、多肽、胰高血糖素样肽-1(GLP-1)及其类似物、免疫原性组合物、抗原、外泌体(Exosomes)、核糖核酸蛋白复合物或疫苗中的一种或几种的组合；核酸优选siRNA、信使RNA(mRNA)、DNA、miRNA、反义寡核苷酸(ASO)和非编码RNA，更优选为siRNA和mRNA，最优选为siRNA；核酸与两亲性可降解嵌段共聚物以及类脂聚合物的质量总和之比为1：2-1:180，优选选自1:2，1：10，1：30，1：60，1：120，1：180，更优选为1：60；作为一种实施例，蛋白可以举例包括：肿瘤坏死因子抑制剂Etanercept、细胞因子、免疫原、抗体、融合蛋白、重组酶和重组蛋白等；治疗疾病或病况的药物举例包括：STING激动剂、Lenalidomide(来那度胺)、抑制病毒合成和装配的抑制剂Ledipasvir、statin类降脂药、姜黄素及其类似物、托法替布(Tofacitinib)及其盐、麻醉镇痛药、止痛药、消炎药、肝X受体激动剂(Liver X receptor agonists)、抗癌药、治疗糖尿病的药物、治疗肥胖症的药物等；抗体可以举例包括：Adalimumab(阿达木单抗)、Rituximab(利妥昔单克隆抗体)、血管内皮生长因子抑制剂Bevacizumab(贝伐珠单抗)、Trastuzumab(曲妥珠单抗)、Infliximab(英夫利西单抗)等；胰岛素可以举例包括：Insulin glargine(甘精胰岛素)等；疫苗可以举例包括：减活病毒颗粒、灭活病毒颗粒、假病毒样颗粒、mRNA纳米颗粒等；需要说明的是：这些举例并非穷举，只要是可以被纳米颗粒包裹，且具有药物属性的试剂均在本发明的保护范围内。
前述的一种雾化吸入的载药纳米颗粒，还包括：药物辅助剂；作为一种实施例，药物辅助剂包括：有机溶剂或稳定剂。
前述的一种雾化吸入的载药纳米颗粒，作为一种实施例，稳定剂包括：神经酰胺-PEG、1,2-二硬脂酰-sn-甘油基-3-磷酰乙醇胺-N-聚乙二醇(DSPE-PEG)、聚乙烯醇、聚甘油脂肪酸酯、吐温80、吐温20、Span80、Span60、十二烷基磺酸钠中的一种或多种；需要说明的是：这些举例并非穷举，只要是可以用于本发明组合物的辅助试剂均在本发明的保护范围内。
前述的一种雾化吸入的载药纳米颗粒，作为一种实施例，稳定剂为聚乙烯醇或DSPE- PEG中的一种或多种的组合，作为一种实施例，稳定剂为聚乙烯醇，聚乙烯醇分子量范围为10，000-250，000kDa，优选为13,000-23,000kDa；聚乙烯醇的使用浓度范围为0.1％～10％(w/v)，优选为0.25％(w/v)；作为另一种实施例，稳定剂为DSPE-PEG，使用浓度范围为0.005％～1％(w/v)，优选为0.01％(w/v)。
前述的一种雾化吸入的载药纳米颗粒，有机溶剂包括：二氯甲烷、三氯甲烷、乙酸乙酯、乙酸甲酯、丙酸乙酯、乙酸丙酯、二氧六环、乙醚、四氢呋喃、乙腈、甲醇、丙二醇、二甲亚砜(DMSO)、N,N-二甲基甲酰胺(DMF)或丙酮中的一种或多种的组合；优选：DMF或DMSO；需要说明的是：这些举例并非穷举，只要是可以溶解本发明组合物的有机溶剂均在本发明的保护范围内。
前述的一种雾化吸入的载药纳米颗粒，载药纳米颗粒的制备方法，包括如下步骤：
a)在有机溶剂中均匀混合所述两亲性嵌段共聚物、阳离子脂质化合物、和所载的药物试剂；
b)将步骤a)获得的混合物加入到含有稳定剂的水溶液中，室温搅拌混合均匀，优选搅拌混合时间为5-20分钟，更优选为10分钟；
c)收集获得的纳米颗粒，通过超滤的方法，进行纯化和浓缩。
前述的一种雾化吸入的载药纳米颗粒，作为一种优选，步骤a)为：在DMSO或DMF中均匀混合PLGA-PEG或PLA-PEG、G0-C14和siRNA或mRNA；siRNA或mRNA、G0-C14、PLGA-PEG或PLA-PEG以1:30:30的质量比混合。
前述的一种雾化吸入的载药纳米颗粒，载药纳米颗粒可以向肺部递送载药纳米颗粒，载药纳米颗粒用于向肺部递送携载有核酸分子的纳米颗粒，载药纳米颗粒的用途在于：用于制备通过吸入给药治疗纤维化的药物。
前述的一种雾化吸入的载药纳米颗粒，纤维化包括肺纤维化、肝纤维化、心肌纤维化或肾脏纤维化；优选肺纤维化。
一种用于治疗肺纤维化的siRNA序列组，siRNA的序列组选自如下的任一组：
组1:正义(5’-3’)：GCUGUUCUCCUAACCCGAUTT SEQ01，反义(5’-3’)：AUCGGGUUAGGAGAACAGCTT SEQ02；
组2：正义(5’-3’)：GCUGGGACAUUGGGAUCUUTT SEQ03，反义(5’-3’)：AAGAUCCCAAUGUCCCAGCTT SEQ04；
组3：正义(5’-3’)：GCCGUUUACAGCUCUUGAUTT SEQ05，反义(5’-3’)：AUCAAGAGCUGUAAACGGCTT SEQ06；
组4：正义(5’-3’)：GGCAACUAGCUGCACAGAUTT SEQ07，反义(5’-3’)： AUCUGUGCAGCUAGUUGCCTT SEQ08；
组5：正义(5’-3’)：GGCUUCGAGUAGACUUGAUTT SEQ09，反义(5’-3’)：AUCAAGUCUACUCGAAGCCTT SEQ10；
组6：正义(5’-3’)：GGCUUCGAGUAGACUUGAUTT SEQ11，反义(5’-3’)：AUCAAGUCUACUCGAAGCCTT SEQ12；
组7：正义(5’-3’)：GGCCUGCUGUUGUUAAAGATT SEQ13，反义(5’-3’)：UCUUUAACAACAGCAGGCCTT SEQ14；
组8：正义(5’-3’)：UGCACAGAUGAGAGACAAATT SEQ15，反义(5’-3’)：UUUGUCUCUCAUCUGUGCATT SEQ16；
组9：正义(5’-3’)：GCAGCAGAUGGUAGAACUATT SEQ17，反义(5’-3’)：UAGUUCUACCAUCUGCUGCTT SEQ18；
组10：正义(5’-3’)：CGAAAGGAUCGGAGUCUAATT SEQ19，反义(5’-3’)：UUAGACUCCGAUCCUUUCGTT SEQ20；
需要说明的是：以上序列组仅是一种优选，只要是采用本发明载药递送载体的药物试剂均在本发明的保护范围内，只要是采用本发明的siRNA的筛选条件筛选出来的siRNA的一组或几组的组合均在本发明的保护范围内。
一种用于治疗肺纤维化的siRNA序列组的设计方法，包括如下步骤：
步骤一，通过siRNA序列设计软件设计与IL11DNA互补的siRNA序列组，设置如下的筛选条件得到初筛siRNA序列组；
筛选条件为：
1)序列长度为21；
2)3‘端的碱基突出UU或dTdT；
3)设置GC的含量为：40-50％；
4)反义链5‘端的第一个碱基为A或U，正义链5‘端的第一个碱基为G或C；
步骤二，将初筛siRNA序列组分别制备成载药纳米颗粒；
步骤三，将各个包载有初筛siRNA序列组的载药纳米颗粒与小鼠肺成纤维细胞共孵育后，经过扩增培养后，提取总RNA，通过实时荧光定量PCR评估目标基因沉默效率，选取目标基因沉默效率高的得到精筛siRNA序列组。
前述的一种用于治疗肺纤维化的siRNA序列组的设计方法，作为一种优选，siRNA序列设计软件包括：Oligowalk,siDirect,i-Score或DSIR；以上软件的举例并非穷举，只 要是采用了本发明的筛选条件得到的siRNA组均在本发明的保护范围内。
本发明的有益之处在于：
本发明的核酸递送载体中的PEG修饰的两亲性共聚物能够保持纳米粒子的结构，阳离子脂质化合物G0-C14通过静电作用将核酸包裹和保护在纳米粒子内部；PEG修饰的两亲性共聚物和G0-C14组合得到的纳米颗粒能够实现雾化不受剪切力的影响，且令人惊讶的发现纳米颗粒可以穿透粘液层到达肺组织深部目标细胞(实施例3(5)粘液渗透研究)，并有效介导mRNA表达，是吸入给药的安全有效的mRNA递送载体，突破了药物雾化递送的技术壁垒，具有意想不到的技术效果；
稳定剂使形成的纳米粒子分散在水相中，不发生聚集沉降；
本发明鉴定IL11为治疗IPF的潜在靶标，抑制IL11的表达能够有效减轻和逆转肺纤维化的进程，能够达到抗纤维化和恢复肺功能的治疗效果；本发明纳米颗粒有效下调小鼠肺成纤维细胞中IL11表达，显著抑制了成纤维细胞的迁移与激活，并减少了胶原的沉积；实验结果显示，小鼠肺纤维化进程受到显著抑制，肺功能也得到显著改善；
本发明通过设定抑制IL11表达的siRNA筛选条件，增强了siRNA双链的稳定性从而提高了抑制的概率和抑制效果，通过实验验证了四个筛选条件在提高目标基因沉默效率上具有协同作用，在治疗肺纤维化上具有意想不到的技术效果；
本发明的纳米颗粒粒径为80-150nm，分散性良好(PDI<0.2)，大小均一，核酸包载效率达到80％以上，并在4-8pH和-20℃-2℃范围内具有良好的稳定性。
附图说明
尽管本发明结合图解的实施方式进行描述，但是应当理解，它们并非意图使本发明限于那些实施方式。相反，本发明意图覆盖可以被所附权利要求书限定的本发明包括在内所有替换、修改和等价物。
下面的标题并不意味着以任何方式限制本公开；在任一标题下的实施方式可以结合任何其它标题下的实施方式使用。
图1是本发明的吸入式纳米颗粒用于治疗IPF等呼吸系统疾病的作用机理；
图2是经TGF-β1刺激活化后，MLFs(小鼠肺成纤维细胞)分泌的IL11水平增加并激活下游通路的示意图；a，经TGF-β1(10ng/mL)刺激后，MLFs中IL11mRNA的水平变化；b，经过TGF-β1处理的MLF中ACTA2和磷酸化SMAD2及总蛋白的免疫印迹分析，分析以GAPDH作为内参(n＝2)；
图3是肺纤维化小鼠肺组织的免疫组化染色实验结果示意图；a，在不同放大倍数下正常小鼠(n＝3)和博来霉素诱导的纤维化小鼠(n＝6)肺切片的IL11；b，ACTA2免疫组化的代表性图像；比例尺，上：50μm，下：20μm；
图4是IPF患者肺样本的免疫组化染色实验结果示意图；a，IPF患者(n＝8)和健康对照组(n＝3)的肺组织中，IL11和ACTA2免疫组化染色的代表性图像；DAB阳性 区域由箭头指示；比例尺，100μm；b，IL11阳性区域的定量；c，ACTA2阳性区域的定量；数据表示为平均值±s.d；*P<0.05，***P<0.001，student’s t检验；d，IL11和ACTA2阳性区域之间的相关性分析；
图5是PLGA-PEG两亲性聚合物和G0-C14阳离子脂质化合物制备得到的杂化纳米颗粒(PPGC-NPs)的表征及其穿透肺粘液的性质实验结果示意图；a，通过琼脂糖凝胶电泳优化G0-C14和siRNA之间的比例；b，暴露于RNase条件下，指定时长内，裸露的siRNA和被纳米粒包裹的siRNA的稳定性分析；c，缓冲溶液pH值对纳米粒的粒径和表面电位的影响；数据表示为平均值±s.d(n＝3)；d，雾化前后纳米粒的粒径测量；e，雾化前后的纳米粒的形貌表征；比例尺，50μm；f，g，Cy5.5标记的纳米粒在MLFs和A549中的摄取情况分析；红色、蓝色和绿色荧光分别表示纳米粒，细胞核和ACTA2；比例尺，25μm；h，MLFs中，FAM-siRNA@NPs的摄取定量分析；数据表示为平均值±s.d(n＝3)；i，纳米粒的体外生物相容性研究；j,siIL11的序列筛选；数据表示为平均值±s.d(n＝4)；k，Cy5.5标记的纳米粒，体外穿透肺粘液层的行为分析；l，以PLGA包载FAM-siRNA为参照，分析PLGA-PEG包载的Cy7-siRNA经肺给药后，穿透小鼠肺粘液层的的行为(n＝3)；
图6是siIL11@NPs抑制小鼠肺成纤维细胞的激活与迁移实验结果示意图；a，PBS(phosphate buffer saline，磷酸盐缓冲溶液)，siScr@NPs或siIL11@NPs处理后，TGF-β1激活的MLFs中ACTA2和COL1A1免疫荧光的代表性图像。比例尺，100μm；b，PBS，siScr@NPs和siIL11@NPs处理后，TGF-β1激活的小鼠肺成纤维细胞的ACTA2+；c，PBS，siScr@NPs和siIL11@NPs处理后，COL1A1免疫荧光强度(c)的定量分析；数据表示为平均值±s.d(n＝4)；****P<0.0001，P***<0.001，student’s t检验；d，e，PBS，siScr@NPs和siIL11@NPs处理后，MLFs的划痕实验分析(n＝3)；****P<0.0001，student’s t检验；f，Transwell迁移实验的示意图，细胞接种于transwell上室，并添加无血清的DMEM，含有2％FBS和10ng/mL TGF-β1的DMEM添加到下室中；g，h，用siScr@NPs和siIL11@NPs处理后，细胞迁移的结晶紫染色图像和定量分析(n＝3)；****P<0.0001，student’s t检验；i，j，PBS，siScr@NPs和siIL11@NPs处理后，TGF-β1刺激的MLFs中COL1A1、ACTA2、IL11、SMAD2、ERK和STAT3的磷酸化和总蛋白的免疫印迹分析，以GAPDH作为内参(n＝2)；
图7是mLuc@NPs经雾化吸入后，Luciferase mRNA表达的荧光素酶在小鼠肺部的生物信号发光成像实验结果示意图；a，肺部五个小叶的分布示意图；b，吸入给药后24小时，小鼠肺部各肺叶的生物发光成像(n＝3)；c，五个肺叶中生物发光强度的定量分析；数据表示为平均值±s.d(n＝3)；d，小鼠肺中，相对荧光素酶表达的定量分析；数据表示为平均值±s.d(n＝3)；**P＜0.01；
图8是Cy5.5标记的纳米粒经雾化吸入后在小鼠肺中的分布情况实验结果示意图； a，吸入给药后24小时，Cy5.5标记的纳米粒在小鼠肺部各肺叶的荧光成像，参考上图中的肺叶分布示意图；b，Cy5.5标记的纳米粒在五个肺叶中的荧光强度定量分析，数据表示为平均值±s.d(n＝3)；c，d，Cy5.5标记的纳米粒经吸入给药后，在肺组织不同细胞亚群摄取情况的定量分析；数据表示为平均值±s.d(n＝3)；**P<0.01，***P<0.001，student’s t检验；e，纳米粒吸入给药24小时后，肺和肝的H&E染色的代表性图片；比例尺，50μm；
图9是博来霉素诱导的小鼠纤维化模型经吸入给药siIL11@NPs后，治疗效果评估示意图；a，动物实验设计示意图；b，给药后，不同组别的小鼠肺组织的代表性图像；c，利用qPCR定量造模前后，纤维化相关基因表达的变化情况；数据表示为平均值±s.d(n＝3)；*P<0.05，***P<0.001，student’s t检验；d，不同组别处理的小鼠肺切片的COL1A1和ACTA2的代表性免疫荧光图像；比例尺，100μm；e，对应于COL1A1和ACTA2的代表性免疫荧光图像的COL1A1免疫荧光强度；f，对应于COL1A1和ACTA2的代表性免疫荧光图像的ACTA2+面积的定量分析；数据表示为平均值±s.d；*P<0.05，***P<0.001，student’s t检验；g，不同组别处理的小鼠，肺组织中的纤连蛋白，COL1A1，ACTA2，IL11的免疫印迹分析，分析以GAPDH作为内参(n＝3)；
图10是不同组别处理的小鼠肺切片的组织学分析结果示意图；a-c，来自生理盐水，博来霉素+PBS，博来霉素+siIL11@NPs(低)和博来霉素+siIL11@NPs(高)的治疗组别的小鼠的肺切片染色的代表性图像，包括H&E染色(a)，Masson染色(b)和picrosirius染色(c)；比例尺，50μm；d，通过BCA测定支气管肺泡灌洗液(BALF)中的总蛋白含量；数据表示为平均值±s.d(n＝5)；*P<0.05，**P<0.01，student’s t检验；e，使用ELISA试剂盒定量BALF中的TGF-β1水平；数据表示为平均值±s.d(n＝5)；*P<0.05，**P<0.01，student’s t检验；f，不同组别处理后的小鼠肺组织中羟脯氨酸的含量测定；数据表示为平均值±s.d(n＝5)；*P<0.05，**P<0.01，student’s t检验；g，高剂量博来霉素造模条件下，分别用PBS或siIL11@NPs治疗的纤维化小鼠的存活曲线(n＝5)；h，从不同组收集的肺组织中的SMAD2，ERK和STAT3的磷酸化以及总蛋白的免疫印迹分析；以GAPDH作为内参(n＝3)；
图11是纳米粒的安全性评估结果示意图；a，b，血清ALT(a)和AST(b)的分析；数据表示为平均值±s.d(n＝5)；*P<0.05，**P<0.01，student’s t检验；c，不同组别处理后的小鼠的脏器系数；数据表示为平均值±s.d(n＝5)；P>0.05表示无统计学意义；student’s t检验；
图12是雾化吸入siIL11@NPs对小鼠肺功能的影响实验示意图；a-h，测量的肺功能参数包括深吸气量(a)，呼吸系统阻力(b)，顺应性(c)，弹性(d)，用力肺活量(e)，0.2s时的用力呼气量(f)，磁滞面积(g)和静态顺应性(h)；i，博莱霉素造模后，经PBS和siIL11@NPs治疗的纤维化小鼠模型的压力-体积(PV)环研究；j，k， 羟脯氨酸与静态顺应性(j)以及呼吸系统弹性(k)之间的相关性分析；数据表示为平均值±s.d(n＝5)；*P<0.05，**P<0.01，student’s t检验。
术语、缩写解释说明：
两亲性嵌段共聚物：是指同一高分子中既含有亲水链段又有疏水链段的大分子。本发明中两亲性嵌段共聚物为：PEG与下列一种或多种聚合物组分形成的嵌段共聚物，聚合物组分包括：聚乳酸-聚羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚原酸酯、聚酸酐、聚(β-氨基酯)(PBAE)中的一种或多种。
核酸是脱氧核糖核酸(DNA)和核糖核酸(RNA)的总称，是由多个核苷酸单体组成的生物大分子；核酸由核苷酸组成，核苷酸单体由五碳糖、磷酸基、含氮碱基、或任何修饰基团组成。如果五碳糖是核糖，则形成的聚合物是RNA；如果五碳糖是脱氧核糖，则形成的聚合物是DNA。
核酸分子包括单链DNA、双链DNA、短异构体、mRNA、非编码RNA(microRNA、siRNA、piRNA、rRNA、tRNA、snRNA、snoRNA、SLRNA、SRPRNA)、端粒酶RNA(Telomerase RNA Component)、scRNA、环状RNA(circRNA)、合成miRNA(miRNA mimics、miRNA agomir、miRNA antagomir)、反义DNA、反义RNA、反义寡核苷酸(ASO)、核酶(ribozyme)、不对称干扰RNA(aiRNA)、Dicer-substrate RNA(dsRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)、gRNA、sgRNA、crRNA或tracrRNA、锁核酸(LNA)、肽核酸(PNA)、吗啉反义寡核苷酸、吗啉代寡核苷酸或生物定制寡核苷酸等。这里的举例也并非穷举，只要是由核苷酸单体聚合成的都可以应用于本发明。
PAMAM指聚酰胺胺型树枝状分子，这类聚合物具有高度枝化、高阳电性官能团密度和内部空腔的特性；
G0-PAMAM指第0代聚酰胺胺树型分子，其化学结构式如下式I：
Figure PCTCN2022118532-appb-000001
G0-C14的化学结构式如下式II：
Figure PCTCN2022118532-appb-000002
G0-C14阳离子脂质化合物的反应式如下所示：
Figure PCTCN2022118532-appb-000003
IL11指白细胞介素-11，白细胞介素-11是趋化因子家族的一种细胞因子；
TGF-β1指转化生长因子-β，transforming growth factor-β，属于一组新近发现的调节细胞生长和分化的TGF-β超家族；
ERK是extracellular regulated protein kinases的英文缩写，指细胞外调节蛋白激酶，包括ERK1和ERK2，是将信号从表面受体传导至细胞核的关键；
p-ERK是指磷酸化细胞外调节蛋白激酶；
SMAD2基因编码的蛋白质属于SMAD蛋白家族，SMAD蛋白是介导多种信号通路的信号转导和转录的调节剂；
p-SMAD2是指磷酸化的SMAD2基因编码的蛋白；
GAPDH是甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase)的英文缩写；
DAPI指的是：4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)，是一种能够与DNA强力结合的荧光染料；
Cy5.5指：Cyanine5.5NHS ester是用于标记含有伯胺的多肽，蛋白和寡核苷酸的反 应性染料。
ACTA2是actin alpha 2的缩写，是一种肌动蛋白；
STAT3是位于17号染色体的基因表达的STAT3蛋白：Signal transducer and activator of transcription 3的缩写。
p-STAT3是指磷酸化的STAT3蛋白；
COL1A1指；Ⅰ型胶原Α1(COL1A1)重组蛋白，全称：Recombinant Collagen Type I Alpha 1；
PBS(phosphate buffer saline)指磷酸盐缓冲溶液；
MLFs指小鼠肺成纤维细胞；
DSPE-PEG为：1,2-二硬脂酰-sn-甘油基-3-磷酰乙醇胺-N-聚乙二醇；
PLGA：为聚乳酸-聚羟基乙酸共聚物；
PLA为：polylactic acid(聚乳酸)，又称聚丙交酯，是以乳酸为主要原料聚合得到的聚酯类聚合物；
PCL为：Polycaprolactone(聚己内酯)；
PBAE为：Polyβ-amino acid，聚(β-氨基酯)；
PEG为：Polyethylene glycol(聚乙二醇)；
DMSO为：Dimethyl sulfoxide(二甲亚砜)；
DMF为：N,N-Dimethylformamide(N,N-二甲基甲酰胺)；
FAM-siRNA指FAM标记的siRNA；
Cy7-siRNA指Cy7标记的siRNA；
NPs指纳米粒(nanoparticles)；
Cy5.5-NPs指Cy5.5标记的纳米粒；
BALF指支气管肺泡灌洗液。
siIL11指靶向IL11的siRNA；
siIL11@NPs指包载靶向IL11的siRNA的纳米粒；
siScr@NPs指包载参照siRNA的纳米粒，此处的参照siRNA指不具有特异性靶向mRNA的siRNA；
mLuc@NPs指包载Luciferase mRNA的纳米粒；
PPGC-NPs指本发明的PLGA-PEG两亲性聚合物/G0-C14阳离子脂质化合物的杂化纳米颗粒。
具体实施例
为了本说明书和所附权利要求的目的，除非另外指出，所有表达成分数量、材料的百分比或比例、反应条件的数字以及在说明书和权利要求书中使用的其它数值，都应当被理解为在所有情况下被术语“约”修饰。因此，除非相反指示，在下面的说明书和所附权利要求书中列举的数值参数是近似值，其可以取决于本发明要求获得的期望性质而变。丝毫且并没有试图使等价原则的应用限于权利要求书的范围，每个数值参数至少应当根据所报告的有效数字的数目并且通过应用常规舍去技术进行解释。
尽管列举本发明的宽范围的数值范围和参数是近似值，但是具体实施例中列举的数值尽可能精确地报告。然而，任何数值固有地含有不可避免地由在其各自试验测量中遇到的标准偏差产生的一定误差。此外，本文公开的所有范围应当被理解为包括其中的任何和所有子范围。例如，“1到10”的范围包括最小值1和最大值10(包括1和10)之间的任何和所有子范围，也就是说，包括具有最小值等于或大于1和最大值等于或小于10的任何和所有子范围，例如5.5到10。
对于本发明所涉及的领域的技术人员来说，本发明的组合物中的组份可以进行相同功能上的替换，本发明组合物中各个组分的用量可以是权利要求书要求范围内的任意配比，可以有广泛不同的实施例和应用。序列的选择可以有许多变化，只要是根据本发明的设计筛选思路得到的序列均应该在本发明的保护范围内。这里的公开和描述仅仅是说明性的，并不会在任何意义上进行限制。
实验1小鼠肺成纤维细胞的体外表达研究
图1阐明了吸入式纳米颗粒用于治疗IPF等呼吸系统疾病的作用机理。纳米颗粒经吸入给药，递送至小鼠肺部，释放包载的核酸分子，作用于成纤维细胞，降低目标蛋白IL11的表达，并通过ERK和SMAD2两个通路，减少成纤维细胞向肌成纤维细胞的转化和细胞外基质的沉积，从而实现肺纤维化的治疗。
利用TGF-β1(终浓度为10ng/mL,Peprotech)在无血清条件下刺激原代小鼠肺成纤维细胞24h，用Trizol裂解提取总RNA，另外用RIPA裂解液裂解细胞并离心后得到蛋白上清液。将蛋白上清液通过qPCR检测得到的实验结果如图2a所示，将蛋白上清液通过蛋白质印迹法(免疫印迹试验)，western blotting检测结果如图2b。由以上结果可知IL-11的基因和蛋白表达水平均显著上调。
经博莱霉素(Bleomycin)诱导得到小鼠肺纤维化模型，将肺纤维化小鼠肺组织的免疫组化染色，在不同放大倍数下观察正常小鼠(n＝3)肺纤维化小鼠(n＝6)肺切片的IL11(a)和ACTA2(b)免疫组化的代表性图像如图3a、3b所示。比例尺，上：50μm，下：20μm。
采集临床IPF患者的肺组织切片，经过IPF患者肺样本的免疫组化染色，具体免疫组化染色的方法为：利用针对IL-11和ACTA2(一种肌成纤维细胞的标记物)的一抗与切片进行共孵育，经PBS洗涤后用酶标二抗进行孵育，经底物显色后于显微镜下观察。在IPF患者(n＝8)和健康对照组(n＝3)的肺组织中，IL11和ACTA2免疫组化染色的代表性图像如图4a所示，染色阳性区域由箭头指示，比例尺，100μm。对临床IPF患者的肺组织切片，IL11阳性区域的定量结果分析如图4b所示；ACTA2阳性区域的定量结果如图4c所示；数据表示为平均值±s.d。*P<0.05，***P<0.001。由图4b、4c可知：肺纤维化组织IL11和ACTA2的表达水平明显上升。
通过免疫组化染色的半定量研究发现，IL11和ACTA2阳性区域之间的相关性分析如图4d所示。ACTA2和IL11之间具有高度相关性(图4d)；
因此，本发明鉴定IL11为治疗IPF的潜在靶标，抑制IL11的表达能够有效减轻和逆转肺纤维化的进程，达到抗纤维化和恢复肺功能的治疗效果。随后利用本发明中的纳米颗粒所进行的实验进一步证实了这一点。
siIL11的序列筛选
siIL11序列的设计方法是：
步骤一，通过Oligowalk,siDirect,i-Score,DSIR这四种siRNA序列设计软件设计与IL11DNA互补的siRNA序列组，设置如下的筛选条件得到初筛siRNA序列组，如表1所示；
筛选条件为：
1)序列长度为21；
2)3‘端的碱基突出UU或dTdT；
3)设置GC的含量为：40-50％；
4)反义链5‘端的第一个碱基为A或U，正义链5‘端的第一个碱基为G或C。
步骤二，将初筛siRNA序列组分别与PLGA-PEG和G0-C14混合并制备得到包载有不同siIL11的纳米粒(NPs)(纳米粒(NPs)的制备如实施例2所示)。
步骤三，将各个包载有siIL11的纳米粒(NPs)与MLFs共孵育4h，4h后换成含有10％FBS胎牛血清的完全培养基继续培养20h。利用Trizol试剂盒提取总RNA，通过qPCR评估目标基因沉默效率。实验中使用的qPCR引物序列如下：正向：5'-TGTTCTCCTAACCCGATCCCT-3'；反向，5'-CAGGAAGCTGCAAAGATCCCA-3'。挑选编号1、6、10的序列分别由siIL11-1、siIL11-2、siIL11-3代表，目标基因沉默实验数据结果如图5j所示可知：编号为1的siRNA具有最佳的目标基因沉默效率，其正义 链(5’-3’)的序列是GCUGUUCUCCUAACCCGAUTT SEQ01；反义链(5’-3’)的序列是AUCGGGUUAGGAGAACAGCTT SEQ02。
表1 siRNA的序列
Figure PCTCN2022118532-appb-000004
各个筛选条件在提高目标基因沉默效率上具有协同作用的验证实验：
实验样品如表2所示：
表2验证实验siRNA的序列
Figure PCTCN2022118532-appb-000005
利用PPGC-NPs包载如表2的siRNA序列的样品，纳米粒(NPs)与MLFs共孵育4h，4h后换成含有10％FBS胎牛血清的完全培养基继续培养20h。利用Trizol试剂盒提取总RNA，通过qPCR评估目标基因沉默效率，实验结果如表3所示。实验中使用的qPCR引物序列如下：正向：5'-TGTTCTCCTAACCCGATCCCT-3'SEQ27；反向，5'-CAGGAAGCTGCAAAGATCCCA-3'SEQ28。
表3
样品名称 IL11mRNA倍数变化(倍)
试验样品 0.1
对照样品1 0.5
对照样品2 0.4
对照样品3 0.6
SiScr对照 1
实验结果分析：试验样品能够显著抑制IL11mRNA的数量，对比样品1-3也能抑制，但是效果不如试验样品显著，说明这四个筛选条件在提高目标基因沉默效率上能够产生协同作用，缺少任意一个条件都无法筛选得到能够显著抑制IL11的siRNA；原因可能在于：这样的四个筛选条件增强双链的稳定性及对目标基因的靶向作用，从而提高了抑制效率。
实验2纳米颗粒的制备
2.1为了验证组合物配方的雾化粒径稳定性，使用如下G0-C14与两亲性嵌段共聚物的配方制备得到纳米粒(NPs)样品：
材料：
50:50的聚乳酸聚乙醇酸共聚物(PLGA)购自Lactel Absorbable Polymers。
异双功能的PEG聚合物NH 2-PEG-OCH 3(MW＝3kDa)购自键凯科技，NH 2-PEG-SH(MW＝3.4kDa)购自Layson Bio。
磺基-cy5-马来酰亚胺购自Lumiprobe。1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)，N-羟基琥珀酰亚胺(NHS)和N，N-二异丙基乙胺(DIPEA)购自北京伊诺凯。
聚酰胺树枝状大分子G0(PAMAM)，1,2-环氧十四烷(C14)和聚乙烯醇(PVA，MW＝13,000-23,000kDa)购自Sigma-Aldrich。
2.1.1 PLGA-PEG(PP)和G0-C14的合成
PLGA-PEG(PP)的合成方法：PLGA经EDC和NHS活化，时间2小时，温度为室温，将得到的PLGA在预冷的甲醇/乙醚(50/50v/v)中沉淀两次；将获得的PLGA- NHS与异双功能性PEG(NH 2-PEG-OCH 3)、DIPEA，在碱性有机溶剂(N，N-二异丙基乙胺)，25℃下反应48小时，旋转蒸发去除残留的有机溶剂，获得两亲性可降解嵌段聚合物PLGA-PEG，产物纯化后利用 1H NMR进行表征。 1H NMR(CDCl3,400MHz):(5.30-5.08)ppm(m,-OCH(CH 3)CONH-),(4.90-4.56)ppm(m,-OCH 2COO-),3.62ppm(s,-CH 2CH 2O-),(1.81-1.37)ppm(m,-OCH(CH 3)CONH-)。G0-C14的合成方法：PAMAM树枝状大分子和1,2-环氧十四烷以1:5的摩尔比混合，并在90℃下反应2天，产物纯化后利用 1H NMR进行表征。 1H NMR(CDCl 3,400MHz):(3.65-2.20)ppm(m,-NHCH 2CH 2NH 2,-NCH 2CH 2CONH-,-CH(OH)-),1.22ppm(s,-CH 2-),0.85ppm(t,J＝6.7Hz,-CH 3)。
2.1.2 G0-C14(阳离子脂质化合物)和两亲性嵌段共聚物的组合物按照配方制备成实施例1-3的纳米粒样品：
按照如下配方如下制备方法制备纳米颗粒；
配方如下表4所示：
表4
Figure PCTCN2022118532-appb-000006
制备方法：
步骤1：将G0-C14溶于有机溶剂中，浓度为10mg/ml；
步骤2：依次加入G0-C14、PLGA-PEG或PLA-PEG、siRNA，溶剂与步骤1相同，混合均匀得到混合物；
步骤3：将0.25％(w/v)聚乙烯醇(稳定剂)加入无菌水(不含DNAase/RNAase)中，混合均匀得到混合物；
步骤4：将步骤2得到的混合物加入步骤3得到的混合物，体积比为1：10，混合均匀；
步骤5：静置20分钟，通过超滤的方法，对所获得的纳米颗粒进行纯化和浓缩；
步骤6：细胞实验和动物实验前，使用注射用无菌生理盐水(不含DNAase/RNAase)稀释步骤5所获得纳米颗粒至使用浓度。
2.2为了验证组合物配方的雾化粒径稳定性，使用如下阳离子脂质与两亲性嵌段共聚物的配方制备得到纳米粒(NPs)样品：
按照如下配方如下制备方法制备纳米颗粒；
配方如下表5所示：
表5
Figure PCTCN2022118532-appb-000007
制备方法：
步骤1：将阳离子脂质溶于有机溶剂中，浓度为10mg/ml；
步骤2：依次加入阳离子脂质、PLGA-PEG或PLA-PEG、siRNA，溶剂与步骤1相同，混合均匀得到混合物；
步骤3：将0.25％(w/v)聚乙烯醇(稳定剂)加入无菌水(不含DNAase/RNAase)中，混合均匀得到混合物；
步骤4：将步骤2得到的混合物加入步骤3得到的混合物，体积比为1：10，混合均匀；
步骤5：静置20分钟，通过超滤的方法，对所获得的纳米颗粒进行纯化和浓缩；得 到纳米颗粒。
2.3使用纳米粒(NPs)样品验证雾化前后的粒径，验证纳米粒的雾化稳定性：
雾化前取部分样品进行稀释，利用动态光散射仪(DLS)对纳米粒的粒径进行测量。
取实施例1-7的样品进行雾化，雾化器装置的搭建过程如下：吸入室与定制的鼻锥相连，该鼻锥的设计方式是只有小鼠的鼻部暴露在气溶胶中。吸入室的上游连接到雾化器(Aerogen，爱尔兰)，并在雾化器中添加所需体积的纳米粒(NPs)以通过振动网筛产生气溶胶。下游连接到真空泵，以保持系统中的气溶胶连续流动。持续监测连接到腔室的压力计并通过调整阀门以保证腔室内与外界大气压一致。雾化后取部分样品测算尺寸。
实验结果如表6所示：
表6
Figure PCTCN2022118532-appb-000008
实验结果分析：从雾化前后的纳米粒尺寸来看，雾化前后都形成了纳米粒子，但1:40的比例为最优，粒径增值都在20nm以内，显示雾化前后稳定的纳米结构，说明本发明的配方(两亲性嵌段共聚物和阳离子脂质化合物的摩尔比为1：0.5-80)制备得到的纳米粒均能够承受雾化过程中的剪切力。
2.4制备如下纳米颗粒(PPGC-NPs)用于以下实验：
2.4.1 siIL11@NPs的制备过程：
siRNA、阳离子脂质化合物、PLGA-PEG混合得到混合溶液，其中阳离子脂质化合物与PLGA-PEG的摩尔比为40:1。采用的siRNA组合为:序列1：正义链(5’-3’)GCUGUUCUCCUAACCCGAUTT SEQ01，其反义链(5’-3’)AUCGGGUUAGGAGAACAGCTT SEQ02；和序列2：正义链(5’-3’) GCUGGGACAUUGGGAUCUUTT SEQ03，反义链(5’-3’)AAGAUCCCAAUGUCCCAGCTT SEQ04。混合溶液加入到含有0.25％聚乙烯醇(分子量为13,000-23,000kDa)的水溶液中，室温搅拌混合10分钟。使用
Figure PCTCN2022118532-appb-000009
超滤装置(MWCO为100kDa)对纳米粒(NPs)进行纯化和浓缩得到siIL11@NPs。
雾化器装置的搭建过程如下：吸入室与定制的鼻锥相连，该鼻锥的设计方式是只有小鼠的鼻部暴露在气溶胶中。吸入室的上游连接到雾化器(Aerogen，爱尔兰)，并在雾化器中添加所需体积的纳米粒(NPs)以通过振动网筛产生气溶胶。下游连接到真空泵，以保持系统中的气溶胶连续流动。持续监测连接到腔室的压力计并通过调整阀门以保证腔室内与外界大气压一致。
2.4.2 mLuc@NPs的制备过程：
mLuc
(ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGCGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGG ATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAA SEQ26)、阳离子脂质化合物、PLGA-PEG混合，其中阳离子脂质化合物与PLGA-PEG的摩尔比为40:1。上述混合溶液加入到含有0.25％聚乙烯醇的水溶液中(分子量为13,000-23,000kDa)，室温搅拌混合10分钟。使用
Figure PCTCN2022118532-appb-000010
超滤装置(MWCO为100kDa)对纳米粒(NPs)进行纯化和浓缩得到mLuc@NPs。采用2.1中的方法得到雾化之后的mLuc@NPs。
2.4.3 siScr@NPs的制备过程：
Scramble siRNA(siScr，正义链(5’-3’)UUCUCCGAACGUGUCACGUTT SEQ27，其反义链(5’-3’)ACGUGACACGUUCGGAGAATT SEQ28)、阳离子脂质化合物、PLGA-PEG混合得到混合溶液，其中阳离子脂质化合物与PLGA-PEG的摩尔比为40:1。将混合溶液加入到含有0.25％聚乙烯醇的水溶液中(分子量为13,000-23,000kDa)，室温搅拌混合10分钟。使用
Figure PCTCN2022118532-appb-000011
超滤装置(MWCO为100kDa)对纳米粒(NPs)进行纯化和浓缩得到siScr@NPs。采用2.1中的方法得到雾化之后的siScr@NPs。
2.4.4 Cy5.5-NPs的制备过程：
siRNA(正义链(5’-3’)GCUGUUCUCCUAACCCGAUTT SEQ01，其反义链(5’-3’)AUCGGGUUAGGAGAACAGCTT SEQ02)、阳离子脂质化合物、PLGA-PEG(10％的PLGA-PEG由Cy5.5偶联的PLGA-PEG替代)混合，其中阳离子脂质化合物与PLGA-PEG的摩尔比为40:1。上述混合溶液加入到含有0.25％聚乙烯醇的水溶液中(分子量为13,000-23,000kDa)，室温搅拌混合10分钟。使用
Figure PCTCN2022118532-appb-000012
超滤装置(MWCO为100kDa)对纳米粒(NPs)进行纯化和浓缩得到Cy5.5-NPs。采用2.1中的方法得到雾化之后的Cy5.5-NPs。
2.4.5利用PLGA包载FAM-siRNA：
FAM标记的siRNA(FAM-siRNA，正义链(5’-3’) GCUGUUCUCCUAACCCGAUTT，其反义链(5’-3’)AUCGGGUUAGGAGAACAGCTT)、阳离子脂质化合物、PLGA混合，其中阳离子脂质化合物与PLGA的摩尔比为40:1。上述混合溶液加入到含有0.25％聚乙烯醇的水溶液中(分子量为13,000-23,000kDa)，室温搅拌混合10分钟。使用
Figure PCTCN2022118532-appb-000013
超滤装置(MWCO为100kDa)对纳米粒(NPs)进行纯化和浓缩得到FAM-siRNA@NPs。
2.4.6利用PLGA-PEG包载Cy7-siRNA：
Cy7标记的siRNA(Cy7-siRNA，正义链(5’-3’)GCUGUUCUCCUAACCCGAUTT，其反义链(5’-3’)AUCGGGUUAGGAGAACAGCTT)、阳离子脂质化合物、PLGA-PEG混合，其中阳离子脂质化合物与PLGA-PEG的摩尔比为40:1。上述混合溶液加入到含有0.25％聚乙烯醇的水溶液中(分子量为13,000-23,000kDa)，室温搅拌混合10分钟。使用
Figure PCTCN2022118532-appb-000014
超滤装置(MWCO为100kDa)对纳米粒(NPs)进行纯化和浓缩得到Cy7-siRNA@NPs。
实验3 PPGC-NPs的表征
(1)物化性质表征
将0.3g的琼脂糖溶解于30mL 1×TAE溶液中，微波炉加热煮沸至琼脂糖全部融化,摇匀后添加终浓度为1×的GelRed核酸染料得到1％琼脂糖胶。将不同比例的siRNA与G0-C14共孵育20min，与上样缓冲液混合后加入到每孔中，110V 50min。根据结果对其比例进行优化，确定优选比例为30：1(w/w)(图5a)。同时，将裸露的siRNA与纳米粒(NPs)包载的siRNA分别与RNase(终浓度为10ng/mL)孵育，通过琼脂糖凝胶电泳检测纳米粒(NPs)对siRNA的保护作用。结果显示，与游离的siRNA相比，纳米粒包载的siRNA即使暴露在RNase环境中，也表现出显著提高的稳定性(图5b)。
于指定时间点，测量siIL11@NPs在各个pH(pH 7.4、7.0、6.8)的PBS中的粒径和电势。结果显示，在检测pH范围内，纳米粒(NPs)的粒径和电势均无显著变化(图5c)。另外，雾化前后，siIL11@NPs的粒径无显著变化，水合直径约为100-110nm(图5d)，透射电子显微镜(TEM)显示纳米粒(NPs)为球形结构，大小均一，直径为50-60nm(图5e)。
(2)MLFs的分离
MLFs是从8周龄的雄性C57BL/6小鼠肺中分离获得。提取步骤简述如下：将小鼠肺取下、切碎、浸没在含有1mg/mL胶原酶I和1％青-链霉素的无血清DMEM中，37℃消化30分钟。然后用含10％FBS的DMEM中和，离心。将获得的组织沉淀用 PBS洗涤，最后重悬于完全培养基(DMEM+10％FBS+1％青/链霉素)中，此时记为第0天。待第四天细胞长出后，利用0.25％胰蛋白酶-EDTA消化、传代。使用TGF-β1(10ng/mL)诱导上述MLFs分化为肌成纤维细胞。
(3)体外细胞摄取
Cy5.5-NPs的制备方法如2.4所示。将MLFs和A549(购于中科院)分别接种在预先铺有24孔板的爬片上过夜培养，待细胞汇合度达到90％后将Cy5.5-NPs分别与MLFs和A549共孵育4h，4h后弃去上清，PBS洗涤后用多聚甲醛固定15min，PBS洗涤3min×3次，0.5％Triton X-100室温通透20min，PBS洗涤3min×3次，3％BSA室温封闭30min，滴加足够量的稀释好的ACTA2一抗并放入湿盒，4℃孵育过夜后PBST洗涤3min×3次，滴加稀释好的荧光二抗，湿盒中37℃孵育1h，PBST洗涤3min×3次，DAPI避光染核5min，PBST洗涤5min×4次后用含抗荧光淬灭剂的封片液封片，通过激光扫描共聚焦显微镜(Leica)分析两种细胞分别对纳米粒(NPs)的摄取情况。如图5f，5g所示，从红色荧光可知：纳米粒(NPs)能够被有效内吞到MLFs和A549中。
利用PPGC-NPs包载FAM-siRNA得到FAM-siRNA@NPs，所示。将FAM-siRNA@NPs与MLFs共孵育4h后，弃上清，经PBS洗涤后，利用胰酶消化、离心取细胞沉淀，经PBS重悬后用流式细胞仪(FACS)进行检测，检测结果如图5h所示，由图5h可知：纳米粒(NPs)剂量越大，FAM阳性的肺成纤维细胞的比例就越大，所以NPs在MLFs中的摄取呈现剂量依赖性。
(4)细胞活力测定
利用CCK8检测试剂盒对纳米粒(NPs)的生物相容性进行评估。将MLFs以1×10 4个细胞/孔接种在96孔板中，过夜培养后，与不同剂量(0、6、12、30、60、90、120μg/mL)的siScr@NPs孵育4h后换成完全培养基。24小时后，将培养基更换为每孔含10μL的CCK8溶液的新鲜无血清培养基，孵育2小时，于450nm处测量吸光度值。结果如图5i显示，细胞活力基本在80％以上，纳米粒(NPs)即使在最高剂量下均呈现出良好的生物相容性。
(5)粘液渗透研究
利用体外肺粘液模型，将荧光标记的纳米粒加入到模型粘液中，检测荧光信号以此来评估纳米粒穿透粘液层的能力。
人造粘液的配制如下：将500mg DNA、250mg粘蛋白、250μL无菌蛋黄乳剂、0.295mg DTPA、250mg NaCl、110mg KCl和1mL RPMI分散到50mL水中搅拌过夜。
实验过程如图5k所示，将1mL的10％(w/v)明胶溶液置于24孔板中，室温固化后加入1mL人造粘液，逐滴加入500μL不同浓度的Cy5.5标记的纳米粒(实施例2中的Cy5.5-NPs)。室温放置24小时后，弃去人造粘液，PBS洗涤，利用酶标仪(Tecan，Switzerland)对渗透到明胶中的纳米粒进行定量。定量结果如图5k所示，浓度越大，荧光强度越高，所以纳米粒穿透黏液层呈现浓度依赖性。
PEG修饰对于纳米粒粘液渗透的影响研究实验：
利用实施例2的制备方法，制备获得由G0-C14和PLGA包载的FAM-siRNA@NPs样品(对照组)，以及由G0-C14和PLGA-PEG包载的Cy7-siRNA样品(试验组)，经气管同时滴注，30min后取出小鼠肺部，经多聚甲醛固定及蔗糖梯度沉降后进行OCT包埋，并利用Leica切片机进行切片，DAPI染核5min。利用激光扫描共聚焦显微镜比较PEG对纳米粒体内穿透肺粘液层的能力的影响。实验结果如图5l所示，其中，蓝色为核信号，绿色为FAM信号，红色为Cy7信号，由共聚焦显微镜的观察结果可知，带有PEG组分的试验组的荧光强度显著高于没有PEG组分的对照组，所以表面的PEG修饰可以促进纳米粒穿过粘液层。
实验4细胞实验研究
(1)ACTA2和COL1A1的免疫荧光分析
成纤维细胞的激活是肺纤维化发展进程中的一个重要阶段。体外TGF-β1刺激下，成纤维细胞可以分化为肌成纤维细胞，而I型胶原蛋白α1(COL1A1)和ACTA2是肌成纤维细胞的两个主要标记物。在这里，我们利用PPGC-NPs包载上述实验中筛选出的最佳siIL11(实施例2.1)，对其调节MLFs分化的行为进行评估。
实验过程：将MLFs接种到爬片上过夜培养，加入无血清DMEM稀释的NPs共孵育4小时后，换回完全培养基继续培养20小时。无血清培养基饥饿过夜，TGF-β1(10ng/mL)刺激24小时，4％多聚甲醛固定20分钟，0.5％Triton X-100透化20分钟，3％BSA封闭，一抗孵育过夜，Alexa
Figure PCTCN2022118532-appb-000015
偶联的二抗室温孵育1小时，DAPI染核10分钟，最后用正置荧光显微镜(Olympus)进行观察。免疫荧光结果如图6a-c显示，经TGF-β1处理后，细胞内的ACTA2和COL1A1的表达明显升高，而siIL11@NPs处理后可显著降低ACTA2和COL1A1的水平。
(2)细胞迁移能力测定
肺纤维化发展过程中还涉及成纤维细胞和肌成纤维细胞向纤维灶迁移并产生大量的细胞外基质的过程。我们通过细胞划痕和transwell实验，用实施例2得到的siScr@NPs作为对照，评估实施例2得到的siIL11@NPs样品对成纤维细胞迁移行为的影响。
细胞划痕实验：将小鼠肺成纤维细胞提前接种在24孔板中过夜培养，细胞贴壁。将细胞分别与PBS、siScr@NPs、siIL11@NPs孵育4h后换成无血清培养基继续培养20h。无血清培养基饥饿24h后用10微升枪尖进行划痕后继续培养，分别于0h和24h统计愈合面积。如图6d、6e所示，划痕后24h，siScr@NPs与siIL11@NPs处理组的细胞愈合面积出现了显著性差异。
transwell实验：实验方法如图6f所示，体外模拟由促纤维化细胞因子触发的细胞迁移过程。将小鼠肺成纤维细胞接种在transwell膜(图中的多孔膜)的上侧过夜培养至细胞贴壁。将细胞分别与PBS、siScr@NPs、siIL11@NPs孵育4h后换成完全培养基继续培养20h。无血清饥饿培养24小时后，换成含有10ng/mL TGF-β1、1％FBS的培养基继续培养，PBS处理未经10ng/mL TGF-beita1处理的细胞作为对照。24h后用棉签擦去transwell膜上层中的细胞。将transwell膜置于多聚甲醛中固定后用结晶紫进行染色。结果如图6g、6h显示，siIL11@NPs处理后的细胞，迁移能力显著降低，进一步证实了IL11在成纤维细胞的迁移中具有重要作用。
(3)免疫印迹分析
将MLFs接种在24孔板中培养过夜。将细胞分别与PBS、siScr@NPs、siIL11@NPs孵育4h后换成完全培养基继续培养20h。无血清饥饿培养24小时后，换成含有10ng/mL TGF-beita1、1％FBS的培养基继续培养，与PBS共孵育、未经10ng/mL TGF-beita1处理的细胞作为对照。24h后裂解细胞进行免疫印迹分析，研究IL11的敲低对纤维化相关基因表达的影响。分析结果如图6i显示，siIL11能够显著下调MLFs中IL11蛋白的表达，ACTA2和COL1A1的表达水平也随之显著降低。
同时，我们还对siIL11@NPs抑制成纤维细胞激活的相关通路进行了研究，包括经典信号转导和转录激活因子3(STAT3)、非经典细胞外信号调节激酶(ERK)以及SMAD2信号通路。免疫印迹结果显示，细胞经siIL11@NPs处理后，p-SMAD2和p-ERK的激活受到明显的抑制，而p-STAT3的水平则没有明显变化(图6j)。上述结果表明，IL11对成纤维细胞的激活与SMAD2和ERK通路有关，而与STAT3无关。
实验5动物实验结果
(1)PPGC-NPs经雾化吸入给药方式向肺部递送mRNA
利用PPGC-NPs包载编码萤光素酶的mRNA(实施例2中的样品mLuc@NPs)，并经雾化吸入的方式递送至小鼠肺部。24小时后解剖小鼠，取出心肝脾肺肾并用IVIS系统进行成像。结果显示雾化吸入24小时后，荧光素酶在肺中各叶呈现有效表达(图7a,b，c)。取2号肺叶用裂解液进行裂解，并用荧光素酶检测试剂盒检测。肺组织荧光素 酶蛋白定量结果显示，mLuc@NPs经雾化吸入后，小鼠肺组织内荧光素酶含量增加了近60倍(图7d)，这表明PPGC-NPs可以通过雾化吸入给药方式向肺部高效递送mRNA，表达目标蛋白。
(2)Cy5.5-NPs经雾化吸入后在小鼠组织中的分布
利用Cy5.5标记的纳米粒(实施例2中的Cy5.5-NPs样品)，经过雾化吸入给药方式，递送siRNA到达小鼠肺部，观察纳米粒的组织分布和亚细胞定位情况。24h后，利用IVIS对五个肺叶进行荧光成像和定量分析，Cy5.5-NPs在五个肺叶中呈现均匀分布(图8a,b)。另外，取2号肺叶，用胶原酶进行消化、离心，经红细胞裂解液裂解后，离心、PBS重悬，分别使用相应的抗体对上皮细胞(EpCAM)，内皮细胞(CD31)和免疫细胞(CD45)进行标记利用FACS对纳米粒的亚细胞定位情况进行分析。使用未吸入纳米粒的小鼠作为对照组。分析结果如图8c、8d显示，Cy5.5-NPs经雾化吸入后主要集中在上皮细胞(35.5％)，其次是免疫细胞(30.1％)和内皮细胞(22.8％)。小鼠肺和肝组织的苏木精和曙红(H&E)染色结果表明，雾化吸入后的纳米粒对于肺和肝未产生明显毒性(图8e)。
(3)肺纤维化小鼠模型的建立
8-10周龄的雄性C57BL/6小鼠购自北京维通利华。小鼠肺纤维化模型通过单次气管内滴注硫酸博来霉素建立。本发明中的所有动物实验均在上海交通大学实验动物伦理与使用委员会的批准下进行。
(4)siIL11@NPs在博来霉素诱导的肺纤维化小鼠模型中的治疗作用
于博来霉素滴注后给予吸入治疗，其中，低剂量为每只小鼠15μg siRNA(siIL11@NPs)，高剂量为每只小鼠30μg siRNA(siIL11@NPs)。在第21天，收集血液和肺泡灌洗液(实验设计图示见图9a)。再收集小鼠左肺和其他肺叶，用于RNA提取和蛋白质免疫印迹分析。
博来霉素造模后，小鼠肺部表现为出血性坏死(图9b形态学检查)，肺组织的纤维化相关基因表达含量显著增加(图9c)。经siIL11@NPs治疗后，坏死部分显著减少(图9b)。
免疫荧光分析显示，siIL11@NPs治疗后，肺组织的ACTA2和COL1A1荧光强度明显降低，高剂量情况下尤为明显(图9d-f)。免疫印迹结果也进一步表明，siIL11@NPs经雾化吸入给药后，可以显著抑制IL11的水平。此外，纤连蛋白、COL1A1、ACTA2的表达也有显著下调(图9g)。
(5)小鼠肺组织的组织学分析
小鼠1号肺叶经多聚甲醛固定后用石蜡进行包埋、切片。小鼠肺组织样本的组织学分析包括H&E染色、Masson染色和天狼猩红(Picrosirius)染色。H&E染色结果如图10a显示，siIL11@NPs治疗组纤维化区域显著减少，高剂量组尤其明显。Masson和Picrosirius染色如图10b、10c也表明，低剂量和高剂量的纳米粒经雾化吸入给药后，均可以缓解纤维化发生。
(6)支气管肺泡灌洗液(BALF)中总蛋白和TGF-β1含量测定
使用2mL PBS分两次灌注小鼠肺中并利用注射器将液体抽出得到BALF，将两次BALF合并用于检测，利用BCA检测试剂盒和小鼠TGF-β1ELISA试剂盒分别对总蛋白和TGF-β1的含量进行测定。如图10d所示，博来霉素造模后，肺泡灌洗液中总蛋白质量急剧增加，而治疗组显著降低。TGF-β1是参与肺纤维化发展的关键介质，可由上皮细胞，巨噬细胞等释放，在细胞凋亡、成纤维细胞增殖、肌成纤维细胞分化和胶原合成中具有重要作用。将BALF加到每孔中孵育90min，洗涤后加入生物素化抗体孵育60min，洗涤后加入酶结合物工作液孵育30min，洗涤后加入底物溶液(TMB)避光孵育15min，终止液终止后利用酶标仪在450nm波长测量各孔的光密度(OD值)。通过ELISA试剂盒对BALF中的TGF-β1定量分析发现，siIL11@NPs治疗后，可以使TGF-β1从150pg/mL降低到100pg/mL，如图10e所示。
(7)小鼠肺中羟脯氨酸含量的测定
用碱性水解法测定小鼠肺中的羟脯氨酸含量：取小鼠3号肺叶，沸水浴中碱性水解20min，加入pH指示剂，调节pH至6.0-6.8左右。经活性炭吸附后离心取上清，加入检测试剂进行检测，60℃孵育后在波长550nm处测定吸光度值。如图10f所示，博来霉素造模后，肺组织中的羟脯氨酸含量从250μg/g上调至410μg/g，而经siIL11@NPs雾化吸入给药后，羟脯氨酸含量下降至200μg/g，几乎接近于正常对照。
(8)博来霉素造模条件下，肺纤维化小鼠的存活率分析
博来霉素造模条件下，软件对经不同治疗组处理的肺纤维化小鼠的存活率进行分析。如图10g所示，经siIL11@NPs雾化吸入给药后，造模小鼠的存活率显著提高。
(9)免疫印迹分析
取小鼠4号肺叶用裂解液进行裂解。利用免疫印迹，分析肺组织中p-SMAD2，p-ERK和p-STAT3的蛋白表达情况。
结果如图10h显示，经博来霉素造模后，肺组织中p-SMAD2，p-ERK和p-STAT3的表达均显著增加。而经siIL11@NPs雾化吸入给药后，p-SMAD2，p-ERK的表达被明显抑制，p-STAT3则无明显变化。这表明，IL11对成纤维细胞的激活与SMAD2和ERK 通路有关，而与STAT3无关，动物实验结果与上述细胞实验结果一致。
(10)安全性评价
除了验证siIL11@NPs的治疗作用外，我们还对纳米粒的安全性进行了评估。
对小鼠进行眼眶取血，血液收集在离心管中37℃(或室温)静置1h进行凝固分层。然后3000rpm室温离心10min，取上清转至干净的离心管中。根据谷丙转氨酶(ALT)和谷草转氨酶(AST)试剂盒(南京建成)的说明书要求，对血清进行AST和ALT生化检测结果分析。
实验结果如图11a、11b、11c所示，均未观察到肝毒性(图11a，b)。另外，各组动物的脏器系数也未发生变化(图11c)。
(11)肺功能测试(PFT)
PFT是临床IPF诊断的一项常规检查。小鼠气管滴注博来霉素造模后分别给予PBS和siIL11@NPs，以气管滴注生理盐水的小鼠作为健康对照。在第21天对各组小鼠麻醉并进行气管插管，用FlexiVent系统(SCIREQ)对小鼠的肺功能进行跟踪评估。
实验结果如图12a-f、12h所示，经博来霉素造模后，小鼠深吸气量(IC)受到显著抑制，呼吸系统阻力(Rrs)和弹性(Ers)显着增加，肺顺应性(Crs)、用力肺活量(FVC)、用力呼气量(FEV)、和肺静态顺应性(Cst)降低。经siIL11@NPs雾化吸入给药后，上述参数均得到显著改善。
实验结果如图12g、12i-k所示，经博来霉素造模后，小鼠压力-容积环(PV loop)表现出特征性下移；经siIL11@NPs雾化吸入给药后，PV环明显上移，几乎接近于未造模健康小鼠，且PV环中间的面积(磁滞面积)显著上升(图12g,i)。图12j,k表明，羟脯氨酸与静态顺应性(Cst)以及呼吸系统弹性(Ers)之间存在高度相关性。
虽然上文具体举例说明了本发明的优选实施例，但是并不意味着本发明的保护范围局限于这些实施例。需要强调的是：在不脱离权利要求书限定的技术范围和设计思路的情况下，可以对本发明进行任意的修改，这些修改都在本发明的保护范围之内。

Claims (20)

  1. 一种雾化吸入的载药纳米颗粒，其特征在于，包括：生物可降解两亲性嵌段共聚物和阳离子脂质化合物；所述两亲性嵌段共聚物和阳离子脂质化合物的摩尔比为1：0.5-80。
  2. 根据权利要求1所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述两亲性嵌段共聚物为PEG与下列一种或多种聚合物组分的嵌段共聚物，聚合物组分包括：聚乳酸-聚羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚原酸酯、聚酸酐、聚(β-氨基酯)(PBAE)中的一种或多种。
  3. 根据权利要求2所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述两亲性嵌段共聚物为PLGA-PEG或PLA-PEG。
  4. 根据权利要求3所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述PLGA-PEG中的PLGA为GA组分和LA组分为50:50的共聚物，其分子量范围为5000-200000；所述PLGA-PEG中的PEG为分子量范围为1000-100000。
  5. 根据权利要求4所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述阳离子脂质化合物为PAMAM树枝状分子和1,2-环氧十四烷以1:4-1:7的摩尔比混合反应制得。
  6. 根据权利要求5所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述PAMAM树枝状分子为低代PAMAM，所述阳离子脂质化合物为G0-C14。
  7. 根据权利要求1所述的一种雾化吸入的载药纳米颗粒，其特征在于，还包括：所载的药物试剂；所述所载的药物试剂包括：核酸、蛋白、治疗疾病或病况的药物、抗体、胰岛素、多肽、胰高血糖素样肽-1(GLP-1)及其类似物、免疫原性组合物、抗原、外泌体(Exosomes)、核糖核酸蛋白复合物或疫苗中的一种或几种的组合。
  8. 根据权利要求1所述的一种雾化吸入的载药纳米颗粒，其特征在于，还包括：药物辅助剂，所述药物辅助剂包括：稳定剂或有机溶剂。
  9. 根据权利要求8所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述稳定剂包括：神经酰胺-PEG、1,2-二硬脂酰-sn-甘油基-3-磷酰乙醇胺-N-聚乙二醇(DSPE-PEG)、聚乙烯醇、聚甘油脂肪酸酯、吐温80、吐温20、Span80、Span60、十二烷基磺酸钠中的一种或多种。
  10. 根据权利要求9所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述稳定剂为聚乙烯醇或DSPE-PEG中的一种或多种的组合，所述聚乙烯醇分子量范围为10，000-250，000kDa，聚乙烯醇的使用浓度范围为0.1％～10％(w/v)；所述DSPE-PEG的使用浓度范围为0.005％～1％(w/v)。
  11. 根据权利要求8所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述有 机溶剂包括：二氯甲烷、三氯甲烷、乙酸乙酯、乙酸甲酯、丙酸乙酯、乙酸丙酯、二氧六环、乙醚、四氢呋喃、乙腈、甲醇、丙二醇、二甲亚砜(DMSO)、N,N-二甲基甲酰胺(DMF)或丙酮中的一种或多种的组合。
  12. 根据权利要求1所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述载药纳米颗粒的制备方法，包括如下步骤：
    a)在有机溶剂中均匀混合所述两亲性嵌段共聚物、阳离子脂质化合物、和所载的药物试剂；
    b)将步骤a)获得的混合物加入到含有稳定剂的水溶液中，均匀混合；
    c)收集获得的纳米颗粒，通过超滤的方法，进行纯化和浓缩。
  13. 根据权利要求12所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述步骤a)为：在DMSO或DMF中均匀混合PLGA-PEG或PLA-PEG、G0-C14和siRNA或mRNA；所述siRNA或mRNA、G0-C14、PLGA-PEG或PLA-PEG以1:30:30的质量比混合。
  14. 根据权利要求1所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述载药纳米颗粒可以向肺部递送载药纳米颗粒。
  15. 根据权利要求14所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述载药纳米颗粒用于向肺部递送携载有核酸分子的纳米颗粒。
  16. 根据权利要求1所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述载药纳米颗粒的用途在于：用于制备通过吸入给药治疗纤维化的药物。
  17. 根据权利要求16所述的一种雾化吸入的载药纳米颗粒，其特征在于，所述纤维化包括肺纤维化、肝纤维化、心肌纤维化或肾脏纤维化。
  18. 一种用于治疗肺纤维化的siRNA序列组，其特征在于，所述siRNA的序列组选自如下的任一组：
    组1:正义(5’-3’)：GCUGUUCUCCUAACCCGAUTT SEQ01，反义(5’-3’)：AUCGGGUUAGGAGAACAGCTT SEQ02；
    组2：正义(5’-3’)：GCUGGGACAUUGGGAUCUUTT SEQ03，反义(5’-3’)：AAGAUCCCAAUGUCCCAGCTT SEQ04；
    组3：正义(5’-3’)：GCCGUUUACAGCUCUUGAUTT SEQ05，反义(5’-3’)：AUCAAGAGCUGUAAACGGCTT SEQ06；
    组4：正义(5’-3’)：GGCAACUAGCUGCACAGAUTT SEQ07，反义(5’-3’)：AUCUGUGCAGCUAGUUGCCTT SEQ08；
    组5：正义(5’-3’)：GGCUUCGAGUAGACUUGAUTT SEQ09，反义(5’-3’)：AUCAAGUCUACUCGAAGCCTT SEQ10；
    组6：正义(5’-3’)：GGCUUCGAGUAGACUUGAUTT SEQ11，反义(5’-3’)： AUCAAGUCUACUCGAAGCCTT SEQ12；
    组7：正义(5’-3’)：GGCCUGCUGUUGUUAAAGATT SEQ13，反义(5’-3’)：UCUUUAACAACAGCAGGCCTT SEQ14；
    组8：正义(5’-3’)：UGCACAGAUGAGAGACAAATT SEQ15，反义(5’-3’)：UUUGUCUCUCAUCUGUGCATT SEQ16；
    组9：正义(5’-3’)：GCAGCAGAUGGUAGAACUATT SEQ17，反义(5’-3’)：UAGUUCUACCAUCUGCUGCTT SEQ18；
    组10：正义(5’-3’)：CGAAAGGAUCGGAGUCUAATT SEQ19，反义(5’-3’)：UUAGACUCCGAUCCUUUCGTT SEQ20。
  19. 一种用于治疗肺纤维化的siRNA序列组的设计方法，其特征在于，包括如
    下步骤：
    步骤一，通过siRNA序列设计软件设计与IL11DNA互补的siRNA序列组，设置如下的筛选条件得到初筛siRNA序列组；
    筛选条件为：
    1)序列长度为21；
    2)3‘端的碱基突出UU或dTdT；
    3)设置GC的含量为：40-50％；
    4)反义链5‘端的第一个碱基为A或U，正义链5‘端的第一个碱基为G或C；
    步骤二，将初筛siRNA序列组分别制备成载药纳米颗粒；
    步骤三，将各个包载有初筛siRNA序列组的载药纳米颗粒与小鼠肺成纤维细胞共孵育后，经过扩增培养后，提取总RNA，通过实时荧光定量PCR评估目标基因沉默效率，选取目标基因沉默效率高的得到精筛siRNA序列组。
  20. 根据权利要求19所述的一种用于治疗肺纤维化的siRNA序列组的设计方法，其特征在于，所述siRNA序列设计软件包括：Oligowalk,siDirect,i-Score或DSIR。
PCT/CN2022/118532 2021-09-13 2022-09-13 一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法 WO2023036345A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008105773A2 (en) * 2006-03-31 2008-09-04 Massachusetts Institute Of Technology System for targeted delivery of therapeutic agents
CN103768606A (zh) * 2012-10-25 2014-05-07 中国医学科学院药用植物研究所 基于树枝状聚合物及其衍生物的新型纳米粒
WO2020152122A1 (en) * 2019-01-21 2020-07-30 Singapore Health Services Pte. Ltd. Treatment of hepatotoxicity
WO2020169783A1 (en) * 2019-02-22 2020-08-27 Singapore Health Services Pte. Ltd. Treatment of kidney injury
CN112638362A (zh) * 2018-07-23 2021-04-09 川斯勒佰尔公司 信使rna的干粉制剂

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10583091B2 (en) * 2014-10-23 2020-03-10 The Brigham And Women's Hospital, Inc. Amphiphile-polymer particles

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008105773A2 (en) * 2006-03-31 2008-09-04 Massachusetts Institute Of Technology System for targeted delivery of therapeutic agents
CN103768606A (zh) * 2012-10-25 2014-05-07 中国医学科学院药用植物研究所 基于树枝状聚合物及其衍生物的新型纳米粒
CN112638362A (zh) * 2018-07-23 2021-04-09 川斯勒佰尔公司 信使rna的干粉制剂
WO2020152122A1 (en) * 2019-01-21 2020-07-30 Singapore Health Services Pte. Ltd. Treatment of hepatotoxicity
WO2020169783A1 (en) * 2019-02-22 2020-08-27 Singapore Health Services Pte. Ltd. Treatment of kidney injury

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAI XIN, ZHAO GUOLIN, CHEN QIJING, LI ZHONGYU, GAO MINGZHU, HO WILLIAM, XU XIAOYANG, ZHANG XUE-QING: "Inhaled siRNA nanoparticles targeting IL11 inhibit lung fibrosis and improve pulmonary function post-bleomycin challenge", SCIENCE ADVANCES, vol. 8, no. 25, 24 June 2022 (2022-06-24), pages 7162, XP093046565, DOI: 10.1126/sciadv.abn7162 *
YANG ZONGZHAO, SHUAI JIANG-BING, MUDASSER HABIB: "Research Methods of RNA Interference", CHINESE JOURNAL OF VETERINARY DRUG, ZHONGGUO SHOUYI YAOPIN JIANCHASUO, CN, vol. 39, no. 11, 31 December 2005 (2005-12-31), CN , pages 23 - 28, XP093046543, ISSN: 1002-1280 *

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