CN115068442A - Nanoparticle compositions of nucleic acids, methods of making and uses thereof - Google Patents

Abstract

A nanoparticle composition is provided, the nanoparticle composition including an amphiphilic degradable block copolymer and a lipid-like polymer; the amphiphilic degradable block copolymer comprises polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid (PLA), Polycaprolactone (PCL), polyorthoester, polyanhydride, or an amphiphilic block copolymer of poly (beta-amino ester) (PBAE) and polyethylene glycol (PEG), or a combination thereof; the lipoid polymer is prepared by mixing and reacting Polyamidoamine (PAMAM) dendrimer with 1, 2-epoxy tetradecane in a ratio of 1:4-1: 7; further comprising an active substance embedded therein; the nanoparticles are prepared by: mixing the amphiphilic degradable block copolymer, the lipid polymer and the active substance in an organic solvent, and then uniformly dispersing the mixture into an aqueous phase containing a stabilizer. Also disclosed are methods for their preparation and use for preparing a medicament for the treatment of fibrosis by administration by inhalation. Nucleic acids for use in treating fibrosis are also disclosed.

Description

Nanoparticle compositions of nucleic acids, methods of making and uses thereof

(1) Field of the invention

The present invention relates to the field of medicine, in particular to nanoparticle compositions for use as pulmonary delivery systems for nucleic acids, in particular siRNA and mRNA, for the treatment of respiratory and pulmonary diseases including pulmonary fibrosis by preparing inhalable nucleic acid formulations (nucleic acid pharmaceutical compositions); also provides a gene therapy means targeting IL11, aiming at treating fibrosis of each organ (anti-fibrosis treatment).

(2) Background of the invention

Pulmonary fibrosis, including IPF (idiopathic pulmonary fibrosis), is a progressive interstitial lung disease with a persistent impairment of lung function. Currently, about 500 million people worldwide suffer from IPF with an average median survival time of 3-5 years. Factors causing pulmonary fibrosis are many, including environmental, drug side effects, genetic factors, etc., and furthermore, it is reported that patients infected with SARS coronavirus or new coronavirus (2019-nCoV) in 2019 are also at risk of developing pulmonary fibrosis. To date, however, the only FDA-approved drugs for IPF treatment are pirfenidone and nintedanib, and they only delay the progression of the disease and do not reverse existing fibrosis. The discovery of new targets and the development of corresponding therapies is therefore of great urgency and importance.

The pathological mechanism of IPF can be summarized as follows: repeatedly damaged alveolar epithelial cells and recruited inflammatory cells secrete a variety of pro-fibrotic growth factors, cytokines, and procoagulants, resulting in recruitment, proliferation, and activation of fibroblasts. Fibroblasts exhibit resistance to apoptosis after differentiation into myofibroblasts and accumulate at the foci, deposit excessive collagen and other extracellular matrix, cause changes in mechanical stiffness and scarring, reduce lung volume, and further lead to activation of fibroblasts through a positive feedback loop. Interleukins have a variety of roles in fibroblast-associated pulmonary fibrosis. Recent studies have shown that IL-11 is a potent pro-fibrotic cytokine associated with fibrosis in a variety of organs. IL-11 binds to the heterodimeric receptor complex of IL-11RA and glycoprotein 130(gp130), triggering a profibrotic response through the extracellular signal-regulated kinase (ERK) signaling pathway. The experimental result shows that the down regulation of the IL-11 has a therapeutic effect on relieving various organ fibrosis diseases including pulmonary fibrosis.

Small interfering RNA (siRNA) has shown great promise for treating various diseases, but its electronegativity is strong, molecular weight is large, and stability is poor, making it challenging for clinical application. The safe and efficient nano-carrier is the key to realize the in vivo and in vitro delivery of siRNA therapeutic molecules, and can greatly promote the process of gene therapy in clinical application.

However, systemically injected nanoparticles are quickly cleared by the Mononuclear Phagocyte System (MPS) in human blood, most of the particles are concentrated in the liver and spleen, and only less than 5% of the particles eventually reach the target tissue or cell. Aerosols deliver therapeutic agents centrally to the lung by non-invasive inhalation delivery and allow drug deposition throughout the bronchioles and alveolar epithelium, thereby improving compliance and reducing systemic exposure, providing a great opportunity for the treatment of a range of respiratory diseases, including Cystic Fibrosis (CF), asthma, and the global pandemic caused by SARS-CoV-2. It is noteworthy that the shear force generated during the atomization process may destroy the structure of the lipid nanoparticle, affect the stability of the entrapped nucleic acid molecule, and thus lose the activity of regulating gene expression. The lipid nanoparticles still keep the particle size and transfection activity after the atomization process, which shows that the lipid nanoparticles can bear the violent shearing force generated in the atomization process, protect the stability of the encapsulated nucleic acid molecules, efficiently deliver siRNA and mRNA to the lung, regulate and control the expression of target genes and achieve the treatment purpose.

(3) Summary of the invention

It is therefore an object of the present invention to provide a nanoparticle capable of delivering an active substance, in particular a nucleic acid, by inhalation administration.

In one aspect of the present invention, there is provided a nanoparticle composition comprising an amphiphilic degradable block copolymer and a lipoidal polymer;

the amphiphilic degradable block copolymer comprises polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), Polycaprolactone (PCL), polyorthoester, polyanhydride, or an amphiphilic block copolymer of poly (beta-amino ester) (PBAE) and polyethylene glycol (PEG), or a combination thereof, preferably PLGA-PEG or PLA-PEG;

the lipoidal polymer is prepared from Polyamidoamine (PAMAM) dendrimer and 1, 2-epoxy tetradecane in a ratio of 1:4-1:7, preferably 1:5 (G0-C14), and the PAMAM dendrimer is preferably low-generation PAMAM, particularly preferably G0-PAMAM;

further comprising an active substance embedded therein, said active substance being selected from the group consisting of nucleic acids, small molecule drugs, polypeptides, proteins, antibodies, detection groups,

the nanoparticles are prepared by: mixing the amphiphilic degradable block copolymer, the lipid polymer and the active substance in an organic solvent, and then uniformly dispersing the mixture into an aqueous phase containing a stabilizer. The mass ratio of the amphiphilic degradable block copolymer to the lipoid polymer is 1: 0.5-1: 4, preferably 1:1, the ratio of the active substance to the sum of the masses of the amphiphilic degradable block copolymer and the lipoid polymer is 1:2-1: 180.

In a preferred embodiment of this aspect, the lipoidal polymer is prepared by reacting G0-PAMAM and 1, 2-epoxytetradecane in a ratio of 1:4 to 1:7, preferably 1:5 and reacted at preferably 90 c for 2 days.

In one embodiment of this aspect, the nanoparticle composition further comprises an active agent entrapped therein, said active agent being selected from the group consisting of nucleic acids, small molecule drugs, polypeptides, proteins, antibodies, detection groups, preferably said nucleic acids being selected from the group consisting of siRNA, messenger RNA (mRNA), DNA, miRNA, antisense oligonucleotides (ASO) and non-coding RNA, more preferably siRNA and mRNA, said nucleic acids having a ratio to the sum of the masses of amphiphilic degradable block copolymer and lipid polymer of 1:2-1:180, preferably selected from 1:2, 1: 10,1: 30,1: 60,1: 120,1: 180, more preferably 1: 60.

in another embodiment of this aspect, the stabilizing agent comprises one or more of ceramide-PEG, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid esters, tween 80, tween 20, Span80, Span60, sodium dodecyl sulfonate, preferably polyvinyl alcohol and DSPE-PEG; wherein, the molecular weight range of the polyvinyl alcohol is 10,000-250,000 kDa, preferably 13,000-23,000 kDa; among them, polyvinyl alcohol is preferably used in a concentration range of 0.1% to 10% (w/v), more preferably 0.25% (w/v); among them, it is also preferable that the stabilizer is DSPE-PEG, preferably 0.005% to 1% (w/v), more preferably 0.01% (w/v).

In yet another embodiment of this aspect, the organic solvent comprises one or more of dichloromethane, chloroform, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, dioxane, diethyl ether, tetrahydrofuran, acetonitrile, methanol, propylene glycol, dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), or acetone, preferably DMF and DMSO.

In a preferred mode of this aspect, the sequence of the siRNA is selected from the group consisting of any one of the following:

in another aspect of the present invention, there is provided a method of preparing a nanoparticle composition of the present invention, comprising the steps of:

a) uniformly mixing the amphiphilic degradable block copolymer, the lipid polymer, and the active material, preferably a nucleic acid, in an organic solvent;

b) adding the mixture obtained in the step a) into an aqueous solution containing a stabilizer, and uniformly mixing;

c) the nanoparticles obtained are collected, purified and concentrated by means of ultrafiltration.

In one embodiment of this aspect, the organic solvent comprises one or more of dichloromethane, chloroform, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, dioxane, diethyl ether, tetrahydrofuran, acetonitrile, methanol, propylene glycol, dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), or acetone, preferably DMF and DMSO.

In another embodiment of this aspect, the stabilizing agent comprises one or more of ceramide-PEG, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid esters, tween 80, tween 20, Span80, Span60, sodium dodecyl sulfonate, preferably polyvinyl alcohol and DSPE-PEG; wherein, the molecular weight range of the polyvinyl alcohol is 10,000-250,000 kDa, preferably 13,000-23,000 kDa; among them, polyvinyl alcohol is preferably used in a concentration range of 0.1% to 10% (w/v), more preferably 0.25% (w/v); among them, it is also preferable that the stabilizer is DSPE-PEG, preferably 0.005% to 1% (w/v), more preferably 0.01% (w/v).

In another embodiment of this aspect, the nucleic acid is selected from the group consisting of siRNA, mRNA, DNA, miRNA, antisense oligonucleotide (ASO) and non-coding RNA, more preferably siRNA and mRNA; wherein the ratio of the nucleic acid to the sum of the masses of the amphiphilic degradable block copolymer and the lipoid polymer is 1:2-1:180, and is more preferably selected from 1:2, 1: 10,1: 30,1: 60,1: 120,1: 180, most preferably 1: 60.

in a preferred embodiment of this aspect, the method comprises the steps of:

in another aspect of the present invention, there is provided a method of making the nanoparticles of the present invention, comprising the steps of:

respectively dissolving the lipoid polymer, the amphiphilic degradable block copolymer (the amphiphilic degradable block copolymer comprises polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), Polycaprolactone (PCL), polyorthoester, polyanhydride, poly (beta-amino ester) (PBAE), the amphiphilic block copolymer with polyethylene glycol (PEG) or one or more of the polymers or the amphiphilic block copolymer, wherein the preferable material is PLGA-PEG or PLA-PEG) in an organic solvent (the organic solvent comprises one or more of dichloromethane, trichloromethane, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, dioxane, diethyl ether, tetrahydrofuran, acetonitrile, methanol, propylene glycol, dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF) or acetone, preferably DMF and DMSO); adding siRNA or mRNA mother liquor (without DNAase/RNase) into the solution; siRNA or mRNA, lipoidal polymer, PLGA-PEG or PLA-PEG are mixed in a mass ratio of 1:30: 30. The mixed solution is added into an aqueous solution containing a stabilizer (the stabilizer comprises one or more of ceramide-PEG, 1, 2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid ester, Tween 80, Tween 20, Span80, Span60 and sodium dodecyl sulfate, preferably polyvinyl alcohol and DSPE-PEG, wherein the molecular weight of the polyvinyl alcohol is in the range of 10,000-250,000 kDa, preferably 13,000-23,000kDa, the use concentration of the polyvinyl alcohol is preferably in the range of 0.1-10% (w/v), more preferably 0.25% (w/v), the stabilizer is also preferably DSPE-PEG, preferably 0.005-1% (w/v), more preferably 0.01% (w/v), the mixture is stirred and mixed at room temperature, preferably, the stirring and mixing time is 5 to 20 minutes, more preferably 10 minutes; purifying and concentrating the nanoparticles.

In this aspect, a preferred embodiment of the preparation of a particular nanoparticle composition is as follows:

step 1: dissolving the lipoid polymer G0-C14 in an organic solvent at a concentration of 10 mg/ml;

step 2: sequentially adding the lipoid polymer G0-C14, the amphiphilic degradable block copolymer PLGA-PEG or PLA-PEG and the mRNA or siRNA in a mass ratio of G0-C14: PLGA-PEG: nucleic acid 30: 30: 1, uniformly mixing the solvent which is the same as the solvent in the step 1;

and step 3: adding the stabilizer into sterile water (without DNAase/RNAase), and uniformly mixing;

and 4, step 4: adding the mixture of the step 2 into the mixture of the step 3, wherein the volume ratio is 1: 10, uniformly mixing;

and 5: standing for 20 minutes, and purifying and concentrating the obtained nanoparticles by an ultrafiltration method;

step 6: before cell experiments and animal experiments, the nanoparticles obtained in step 5 were diluted to the use concentration using sterile physiological saline for injection (without dnase/RNAase).

In another aspect of the present invention, there is provided an siRNA whose sequence is selected from a combination of any one row of table 1 below:

TABLE 1 sequences of siRNA

In a further aspect of the invention there is provided the use of a nanoparticle composition of the invention for the preparation of a medicament for the treatment of fibrosis by inhalation administration, preferably fibrosis selected from pulmonary fibrosis, liver fibrosis, cardiac fibrosis, kidney fibrosis, more preferably pulmonary fibrosis; most preferred is idiopathic pulmonary fibrosis.

In a further aspect of this aspect, wherein the active agent is a nucleic acid selected from the group consisting of siRNA, mRNA, DNA, miRNA, antisense oligonucleotide (ASO) and non-coding RNA, more preferably siRNA and mRNA, most preferably siRNA described in table 1 above.

In various embodiments of the present invention, the active agent may be selected from proteins (e.g., tumor necrosis factor inhibitor Etanercept, cytokines, immunogens, antibodies, fusion proteins, recombinases, recombinant proteins, etc.); a drug for treating a disease or condition selected from the group consisting of STING agonists, Lenalidomide, inhibitors of viral synthesis and assembly Ledipasvir, statin lipid lowering drugs, curcumin and its analogs, Tofacitinib (Tofacitinib) and its salts, narcotic analgesics, anti-inflammatory agents, Liver X receptor agonists (Liver X receptor agonists), anti-cancer agents, drugs for treating diabetes, drugs for treating obesity, and the like; antibodies (e.g., Adalilimumab, Rituximab, vascular endothelial growth factor inhibitor Bevacizumab, Trastuzumab, Infliximab, etc.); insulin (e.g., Insulin glargine, etc.), polypeptides, glucagon-like peptide-1 (GLP-1) and analogs thereof, immunogenic compositions, antigens, Exosomes (Exosomes), nucleic acids (e.g., DNA, siRNA, mRNA, miRNA, antisense oligonucleotides ASO, non-coding RNA, etc.), ribonucleoprotein complexes, vaccines (e.g., inactivated viral particles, pseudoviral-like particles, mRNA nanoparticles, etc.), or combinations thereof.

The invention utilizes the discovery that the expression increase of IL-11 in pulmonary fibrosis can be used as a potential therapeutic target, successfully develops siRNA molecules for inhibiting IL-11 for the first time, and realizes high-efficiency inhalation type pulmonary delivery through the lipid polymer of PAMAM and 1, 2-epoxy tetradecane and the nanoparticles thereof in a specific ratio; the invention also realizes the siRNA encapsulated by the block copolymer and the lipoid polymer for the first time, effectively realizes the high-efficiency inhalation type pulmonary delivery of nucleic acid therapeutic molecules and obtains good anti-fibrosis therapeutic effect.

Additional features and advantages of various embodiments will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of various embodiments. The objectives and other advantages of the various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.

(4) Description of the drawings

Fig. 1 provides the mechanism of action of inhaled nanoparticles of the invention for the treatment of respiratory diseases such as IPF.

FIG. 2. after activation by TGF-. beta.1 stimulation, the level of IL11 secreted by MLFs (mouse lung fibroblasts) increased and activated downstream pathways. a, IL11 mRNA levels in MLFs changed following TGF-. beta.1 (10ng/mL) stimulation. b, immunoblot analysis of ACTA2 and phosphorylated SMAD2 and total protein in TGF- β 1 treated MLF. GAPDH was used as the internal parameter (n-2).

FIG. 3 immunohistochemical staining of pulmonary fibrosis mouse lung tissue. a, b, representative images of lung sections from normal mice (n ═ 3) and bleomycin-induced fibrosis mice (n ═ 6) immunohistochemistry at different magnifications for IL11(a) and ACTA2 (b). Scale bar, upper: 50 μm, below: 20 μm.

FIG. 4 immunohistochemical staining of lung samples from IPF patients. Representative images of IL11 and ACTA2 immunohistochemical staining in lung tissue of IPF patients (n ═ 8) and healthy controls (n ═ 3). DAB positive area is indicated by arrows. Scale bar, 100 μm. b, c, quantification of IL11 positive region (b) and ACTA2 positive region (c). Data are presented as mean ± s.d. P <0.05, P <0.001, student's t assay. d, correlation analysis between IL11 and ACTA2 positive regions.

FIG. 5 characterization of hybrid nanoparticles of PLGA-PEG amphiphilic Polymer/G0-C14 lipoidal polymers (PPGC-NPs) and their property of penetrating lung mucus. a, optimizing the ratio between G0-C14 and siRNA by agarose gel electrophoresis. And b, analyzing the stability of naked siRNA and siRNA wrapped by nanoparticles within a specified time period after exposure to RNase. c, the influence of the pH value of the buffer solution on the particle size and the surface potential of the nanoparticles. Data are presented as mean ± s.d (n ═ 3). And d, measuring the particle size of the nanoparticles before and after atomization. And e, characterizing the morphology of the nanoparticles before and after atomization. Scale bar, 50 μm. f, g, and the uptake of Cy5.5 labeled nanoparticles in MLFs and A549 is analyzed. Red, blue and green fluorescence are indicative of nanoparticles, nuclei and ACTA2, respectively. Scale bar, 25 μm. h, quantitative analysis of uptake of FAM-siRNA @ NPs in MLFs. Data are presented as mean ± s.d (n ═ 3). i, research on the in vitro biocompatibility of the nanoparticles. j, sequence screening of siIL 11. Data are presented as mean ± s.d (n ═ 4). k, nanoparticles labeled by Cy5.5, and behavior analysis of in vitro lung mucus layer penetration. The behavior of PLGA-PEG-entrapped Cy7-siRNA penetrating the mouse lung mucus layer after pulmonary administration was analyzed using PLGA-entrapped FAM-siRNA as a reference (n ═ 3).

FIG. 6.siIL11@ NPs inhibit the activation and migration of mouse lung fibroblasts. a, ACTA2 and COL1A1 immunofluorescence in TGF-. beta.1 activated MLFs after treatment with PBS (phosphate buffer saline), siScr @ NPs or siIL11@ NPs. Scale bar, 100 μm. b, c, PBS, siScr @ NPs and siIL11@ NPs, ACTA2+ (b) and COL1A1 immunofluorescence intensity (c) of TGF-. beta.1-activated mouse lung fibroblasts. Data are presented as mean ± s.d (n ═ 4). P <0.0001, P <0.001, student's t assay. d, e, PBS, siScr @ NPs and siIL11@ NPs treatment, scratch experimental analysis of MLFs (n ═ 3). P <0.0001, student's t assay. f, schematic representation of the Transwell migration experiment. Cells were seeded in the upper transwell chamber and serum-free DMEM was added, DMEM containing 2% FBS and 10ng/mL TGF-. beta.1 was added to the lower chamber. g, h, crystal violet staining images and quantification of cell migration after treatment with siScr @ NPs and siIL11@ NPs (n ═ 3). P <0.0001, student's t assay. Phosphorylation of COL1a1, ACTA2, IL11, SMAD2, ERK and STAT3 in TGF- β 1 stimulated MLFs and immunoblot analysis of total protein after i, j, PBS, siScr @ NPs and siIL11@ NPs treatment with GAPDH as an internal reference (n ═ 2).

FIG. 7 bioluminescence imaging of Luciferase expressed by Luciferase mRNA in the mouse lung following nebulization of mLuc @ NPs. a, distribution schematic diagram of five lobules in lung. b, bioluminescence imaging of the lung lobes of the mice 24 hours after inhalation (n-3). And c, quantitative analysis of bioluminescence intensity in five lung leaves. Data are presented as mean ± s.d (n ═ 3). d, quantitative analysis of relative luciferase expression in mouse lung. Data are presented as mean ± s.d (n-3). P < 0.01.

FIG. 8 shows the distribution of Cy5.5 labeled nanoparticles in mouse lung after aerosol inhalation. and a, performing fluorescence imaging on each lung lobe of the lung of the mouse by using the Cy5.5 labeled nanoparticles 24 hours after inhalation administration, and referring to a lung lobe distribution schematic diagram in the figure. b, quantitative analysis of fluorescence intensity of cy5.5-labeled nanoparticles in five lung lobes, data are expressed as mean ± s.d (n ═ 3). c, d, quantitative analysis of the uptake condition of different cell subsets of lung tissue after the nano-particles marked by Cy5.5 are inhaled and administered. Data are presented as mean ± s.d (n ═ 3). P <0.01, P <0.001, student's t assay. E, representative picture of H & E staining of lung and liver 24 hours after nanoparticle inhalation administration. Scale bar, 50 μm.

FIG. 9. evaluation of the therapeutic efficacy of bleomycin-induced mouse fibrosis model following inhalation administration of siIL11@ NPs. a, animal experiment design schematic diagram. b, representative images of lung tissue of different groups of mice after dosing. And c, quantifying the change condition of the expression of the fibrosis related genes before and after the modeling by utilizing qPCR. Data are presented as mean ± s.d (n ═ 3). P<0.05，***P<0.001, student's t test. d, representative immunofluorescence images of COL1a1 and ACTA2 of lung sections of mice treated in different groups. Scale bar, 100 μm. e, f, COL1A1 immunofluorescence intensity (e) and ACTA2 corresponding to the immunofluorescence image described above ⁺ Quantitative analysis of area (f). Data are presented as mean ± s.d. P<0.05，***P<0.001, student's t test. g, different groups of treated mice, immunoblot analysis of fibronectin, COL1a1, ACTA2, IL11 in lung tissue. GAPDH was used as the internal reference (n ═ 3).

FIG. 10 histological analysis of lung sections from different groups of treated mice. a-c, representative images of lung section staining of mice from the treatment groups of saline, bleomycin + PBS, bleomycin + siIL11@ NPs (low) and bleomycin + siIL11@ NPs (high), including H & E staining (a), Masson staining (b) and picrosirius staining (c). Scale bar, 50 μm. d, total protein content in bronchoalveolar lavage fluid (BALF) was determined by BCA. Data are presented as mean ± s.d (n ═ 5). P <0.05, P <0.01, student's t assay. e, quantifying TGF- β 1 levels in BALF using an ELISA kit. Data are presented as mean ± s.d (n ═ 5). P <0.05, P <0.01, student's t assay. And f, measuring the content of hydroxyproline in lung tissues of mice treated by different groups. Data are presented as mean ± s.d (n ═ 5). P <0.05, P <0.01, student's t assay. g, survival curves of fibrotic mice treated with PBS or sil 11@ NPs, respectively, under high dose bleomycin molding conditions (n ═ 5). h, phosphorylation of SMAD2, ERK and STAT3 in lung tissue collected from different groups and immunoblot analysis of total protein. GAPDH was used as the internal parameter (n-3).

Figure 11 safety assessment of nanoparticles. analysis of a, b, serum ALT (a) and AST (b). Data are presented as mean ± s.d (n ═ 5). P <0.05, P <0.01, student's t assay. And c, organ coefficients of mice treated by different groups. Data are presented as mean ± s.d (n ═ 5). P >0.05 means no statistical significance. student's t test.

FIG. 12 Effect of aerosolized inhalation of siIL11@ NPs on lung function in mice. a-h, measured lung function parameters include deep inspiratory capacity (a), respiratory resistance (b), compliance (c), elasticity (d), forced vital capacity (e), forced expiratory capacity at 0.2s (f), hysteresis area (g), and static compliance (h). i, pressure-volume (PV) loop study of a fibrotic mouse model treated with PBS and siIL11@ NPs after bleomycin modelling. j, k, correlation analysis between hydroxyproline and static compliance (j) and respiratory elasticity (k). Data are presented as mean ± s.d (n ═ 5). P <0.05, P <0.01, student's t assay.

(5) Detailed description of the preferred embodiments

For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims are to be understood as being modified in all instances by the term "about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "1 to 10" includes any and all subranges between a minimum value of 1 and a maximum value of 10 (including 1 and 10), that is, any and all subranges having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10, e.g., 5.5 to 10.

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. Thus, for example, reference to "a pharmaceutical agent" includes one, two, three or more pharmaceutical agents.

Interpretation of professional terms:

G0-PAMAM refers to the 0 th generation polyamidoamine dendrimer;

pbs (phosphate buffer saline) refers to phosphate buffer solution;

FAM-siRNA refers to FAM-labeled siRNA;

cy7-siRNA refers to Cy7 labeled siRNA;

NPs refer to nanoparticles (nanoparticles);

Cy5.5-NPs refer to Cy5.5-labeled nanoparticles;

siIL11 refers to siRNA targeting IL 11;

siIL11@ NPs refer to nanoparticles encapsulating siRNA targeting IL 11;

siScr @ NPs refer to nanoparticles encapsulating a reference siRNA, where the reference siRNA refers to siRNA without specific targeting mRNA;

mLuc @ NPs refer to nanoparticles encapsulating Luciferase mRNA; PPGC-NPs refer to PLGA-PEG amphiphilic polymer/G0-C14 lipoid polymer hybrid nanoparticles of the present invention; the full chemical name of DSPE-PEG refers to 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol.

In one aspect, the PAMAM (polyamidoamine) dendrimers used in the preparation of the lipoidal polymers of the present invention are characterized by high degree of branching, high cationic functional group density and internal cavities. The PAMAM polymer used in the present invention is preferably a low generation PAMAM molecule, such as G0/G1 generation, preferably G0, having the formula I:

the reactions and structural formulas of the G0-C14 lipoid polymers of the invention are as follows:

in another aspect, the amphiphilic degradable block copolymer used in the preparation of the nanoparticle according to the present invention includes polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid (PLA), Polycaprolactone (PCL), polyorthoester, polyanhydride, poly (β -amino ester) (PBAE), amphiphilic block copolymer with polyethylene glycol (PEG), or one or more of the above polymers or amphiphilic block copolymers. Preferred materials among these are PLGA-PEG and PLA-PEG.

Wherein the PLGA is preferably a 50:50 copolymer of polylactic acid and polyglycolic acid having a molecular weight in the range of 5,000-200,000, preferably 40,000; the PEG molecular weight range is 1,000-100,000, preferably 3,000.

The nanoparticles of the invention are specifically prepared using the following method:

activation of PLGA with activating agents (preferably carbodiimides and succinimides, particularly preferably EDC and NHS), preferably for 2 hours at room temperature);

precipitating the resulting PLGA in a pre-cooled polar organic solvent, preferably an alcohol and an ether, particularly preferably methanol/diethyl ether (preferably 50/50v/v), preferably repeated 2 times;

mixing the obtained PLGA-NHS and heterobifunctional PEG (preferably NH) ₂ -PEG-OCH ₃ And NH ₂ PEG-SH, particularly preferably NH ₂ -PEG-OCH ₃ ) In a basic organic solvent (preferably an amine, particularly preferably N, N-diisopropylethylamine) (preferably 20 to 55 ℃ C., particularly preferably 25 ℃ C.; preferably 1 to 72 hours, more preferably 48 hours),removing residual organic solvent by rotary evaporation to obtain amphiphilic degradable block polymer PLGA-PEG;

PLGA-PEG was mixed with the lipid polymer obtained above, pharmaceutically active substance (preferably nucleic acid, particularly preferably siRNA and mRNA) in a ratio of 1: 1:1,5: 5: 1,15: 15: 1,30: 30: 1,60: 60: 1,90: 90: 1,120: 120: 1, preferably 30: 30: 1, adding the mixture into an aqueous solution containing a stabilizing agent (the stabilizing agent comprises one or more of ceramide-PEG, 1, 2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid ester, Tween 80, Tween 20, Span80, Span60 and sodium dodecyl sulfonate, preferably polyvinyl alcohol and DSPE-PEG), wherein the molecular weight of the polyvinyl alcohol is 10,000-250,000 kDa, preferably 13,000-23,000kDa, the use concentration of the polyvinyl alcohol is preferably 0.1-10% (w/v), more preferably 0.25% (w/v), and the stabilizing agent is DSPE-PEG, preferably 0.005-1% (w/v), more preferably 0.01% (w/v)). Stirring and mixing at room temperature, preferably stirring and mixing for 5-20 minutes, more preferably 10 minutes;

purifying and concentrating the nanoparticles.

The matrix of the nanoparticle obtained by the present invention is amphiphilic degradable block polymer (preferably PLGA-PEG or PLA-PEG) and lipoid polymer (preferably G0-C14 mentioned above), active substance is encapsulated in the nanoparticle, which is released after entering target cell or tissue, the active substance is preferably drug and nucleic acid molecule, preferably small molecule drug, STING agonist, polypeptide, protein, antibody, small interfering RNA, mRNA, DNA, miRNA, antisense oligonucleotide ASO and non-coding RNA, etc., more preferably DNA, siRNA, mRNA, miRNA, antisense oligonucleotide ASO and non-coding RNA, especially preferably siRNA and mRNA in the present invention. Wherein the siRNA sequence comprises any one of the row combinations in Table 1:

TABLE 1 sequences of siRNA

These sequences are listed in the sequence listing. Wherein the siRNA sense strand SEQ ID NO is 2x (SEQ ID NO) — 1 and the siRNA antisense strand SEQ ID NO is 2x (SEQ ID NO) — 1. The nucleotide sequences can be modified by those skilled in the art in accordance with the prior art, for example, tt at the end of the sequence serves only as a stabilizing sequence, and it is expected that the sequence obtained by removing the base will be equivalent to these sequences.

Preferably, the combination is the sense strand (5 '-3') GCUGUUCUCCUAACCCGAUTT of the sequence 1, the antisense strand (5 '-3') AUCGGGUUAGGAGAACAGCTT thereof; and sequence 2 sense strand (5 '-3') GCUGGGACAUUGGGAUCUUTT, its antisense strand (5 '-3') AAGAUCCCAAUGUCCCAGCTT. The siRNA, upon release, down-regulated IL-11 expression (FIG. 5 j). The nucleic acid may be a relatively short siRNA, miRNA, ASO, non-coding nucleic acid, and may be 2-200 nucleotides in length, preferably about 5-50, more preferably 8-30, etc.; the nucleic acid may be relatively long DNA, mRNA, long non-coding RNA (lncRNA), etc., and the length may be 200-12,000; derivatives thereof, such as chemically modified nucleotides, capping structures, tailing structures, 3 'UTR, 5' UTR, ORFs, signal peptides, nuclear localization sequences, membrane localization sequences, mitochondrial localization sequences, linker peptides, and the like are included within the scope of the present invention.

The nanoparticles of the present invention may also carry labeling groups such as chromophores, fluorophores, isotopic labels, etc., and in the present invention, preferred fluorescent dyes are Cy5, Cy5.5, Cy7, FAM, FITC, Cy3, rhodamine B, etc., are commercially available and incorporated, entrapped, or chemically attached to the nanoparticles by methods known to those skilled in the art.

The nano-particles have the particle size of 60-180nm, preferably 80-150nm, good dispersibility (PDI <0.2), uniform size, nucleic acid entrapment efficiency of more than 80 percent, and good stability within a certain pH range (4-8) and a certain temperature range (-20 ℃ -2 ℃). After nanoparticles are formed, good protection is provided for nucleic acid molecules (siRNA and mRNA) from nuclease degradation. The nanoparticles have good biocompatibility in vivo. The nanoparticles of the invention are mucus permeable and can pass through the mucus layer to the deep lung tissue and into the target cells. In addition, after the nano-particles enter target cells, nucleic acid molecules are released, target gene expression is regulated, and the purpose of safely and effectively treating diseases is achieved.

The term "inhibition of a target gene" as used in the present invention is to be understood as the amount of protein, which reduces or impairs the expression or transcription of the mRNA of the target gene, as a percentage or fold, compared to that observed without the use of nanoparticles.

The term "expression of a target gene" as used herein is to be understood as the amount of protein that upregulates expression or transcription of the target gene mRNA, as a percentage or fold, compared to that observed in the absence of the use of nanoparticles.

The shearing force generated in the atomization process generally destroys the structure of the lipid nanoparticle and affects the stability of the nucleic acid molecule carried by the lipid nanoparticle, thereby losing the activity of the nucleic acid molecule in regulating gene expression. The nanoparticles of the invention retain their particle size and transfection activity after the aerosolization process, are suitable for administration by inhalation, and can be conveniently delivered using conventional pressurized packaging, nebulizers and suitable propellant means, preferably in the form of an aerosol spray. The choice of delivery device and delivery protocol is within the routine discretion of those skilled in the art and the physician of clinical treatment.

In the nanoparticle of the present invention, the dose of siRNA and mRNA is 0.5-100. mu.g, preferably 0.5,1,2,3,4,5,10,15,20,30,40,50, 60. mu.g of siRNA and mRNA delivered to the target organ or tissue. The dosage range can be determined by one skilled in the art, with a preferred dosage range of 1-30 μ g, and the dosing regimen can be 5,10,15,20 μ g.

The present invention is based on the discovery that: TGF-. beta.1 stimulated upregulation of IL11 expression levels in mouse lung fibroblasts (FIG. 2). In both IPF patients (fig. 4a-c) and the bleomycin-induced mouse pulmonary fibrosis model (fig. 3), upregulation of IL11 expression was detected, along with upregulation of the expression level of the key marker for fibrosis, ACTA 2. A high correlation between ACTA2 and IL11 was found by semi-quantitative studies of immunohistochemical staining (fig. 4 d). Thus, the inventors identified IL11 as a potential target for treatment of IPF.

To study the delivery efficiency of the nanoparticles of the present invention to lung, we used the nanoparticles of the present invention to efficiently encapsulate mRNA (mluc) encoding luciferase and locally deliver the mRNA to lung of mouse by inhalation, and the results show that luciferase has significant expression in lung (fig. 7b-d), which indicates that the nanoparticles of the present invention can penetrate mucus layer to reach target cells deep in lung tissue and effectively mediate mRNA expression, and is a safe and effective mRNA delivery vehicle for inhalation.

In the invention, the nanoparticles of the invention are used for efficiently encapsulating siRNA (siIL11) targeting IL 11. In vitro studies show that the nanoparticle of the invention effectively down-regulates the expression of IL11 in mouse lung fibroblasts, remarkably inhibits the migration and activation of the fibroblasts, and reduces the deposition of collagen. On the other hand, the nanoparticles of the present invention were delivered locally to the mouse lungs by inhalation using a bleomycin-induced pulmonary fibrosis model in mice. The in vivo results show that the pulmonary fibrosis process of the mice is obviously inhibited, and the lung function is also obviously improved. In conclusion, the invention not only establishes the therapeutic effect of the nanoparticles in pulmonary fibrosis, but also establishes a delivery technology platform of the inhalation nucleic acid (siRNA, mRNA, DNA, miRNA, antisense oligonucleotide ASO and non-coding RNA), can be used for treating a series of respiratory diseases and lung diseases including IPF, COVID-19 and the like, and has important clinical application value.

While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents as may be included within the invention as defined by the appended claims.

The following headings are not meant to limit the disclosure in any way; embodiments under any one heading may be used in combination with embodiments under any other heading.

Examples

Materials:

50:50 polylactic-co-glycolic acid (PLGA) was purchased from latex Absorbable Polymers.

Heterobifunctional PEG polymers NH ₂ -PEG-OCH ₃ (MW ═ 3kDa) from KeyKa technology, NH ₂ PEG-SH (MW 3.4kDa) was purchased from Layson Bio.

Sulfo-cy 5-maleimide was purchased from Lumiprobe. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and N, N-Diisopropylethylamine (DIPEA) were purchased from YinuoKa, Beijing.

Polyamide dendrimer G0(PAMAM), 1, 2-epoxytetradecane (C14) and polyvinyl alcohol (PVA, MW 13,000-23,000kDa) were purchased from Sigma-Aldrich.

Example 1 in vitro expression study of mouse Lung fibroblasts

FIG. 1 illustrates the mechanism of action of inhaled nanoparticles for the treatment of respiratory diseases such as IPF. The nanoparticles are administrated by inhalation, delivered to the lung of a mouse, release the entrapped nucleic acid molecules, act on fibroblasts, reduce the expression of a target protein IL11, and reduce the transformation of the fibroblasts to myofibroblasts and the deposition of extracellular matrix through two pathways of ERK and SMAD2, thereby realizing the treatment of pulmonary fibrosis.

As shown in FIG. 2, the gene and protein expression of IL-11 was significantly up-regulated on average by using TGF-. beta.1 to stimulate mouse lung fibroblasts. Increased levels of IL11 and ACTA2, a marker of myofibroblasts, were detected in lung tissue sections from Bleomycin (Bleomycin) -induced mouse pulmonary fibrosis model (figure 3) and clinical IPF patients (figures 4 a-c). We further found a high correlation between ACTA2 and IL11 by semi-quantitative studies of immunohistochemical staining (fig. 4 d). Therefore, the invention identifies that IL11 is a potential target for treating IPF, and inhibition of the expression of IL11 can effectively reduce and reverse the progress of pulmonary fibrosis, thereby achieving the therapeutic effects of resisting fibrosis and recovering lung function. This is further confirmed by subsequent experiments with the nanoparticles of the present invention.

Example 2 preparation of nanoparticles

(1) Synthesis of PLGA-PEG (PP) and G0-C14

The synthetic method of PLGA-PEG (PP) comprises the following steps: PLGA was activated for 2 hours with EDC and NHS and precipitated twice in pre-cooled methanol/ether (50/50 v/v). Then, the obtained PLGA-NHS and NH are mixed ₂ -PEG-OCH ₃ DIPEA is reacted for another 48 hours, residual organic solvent is removed, and the product is purified and utilized ¹ H NMR was carried out for characterization. ¹ H NMR(CDCl3,400MHz):(5.30-5.08)ppm(m,-OCH(CH ₃ )CONH-),(4.90-4.56)ppm(m,-OCH ₂ COO-),3.62ppm(s,-CH ₂ CH ₂ O-),(1.81-1.37)ppm(m,-OCH(CH ₃ ) CONH-). The synthesis method of G0-C14 comprises the following steps: mixing PAMAM dendrimer and 1, 2-epoxy tetradecane at a molar ratio of 1:5, reacting at 90 deg.C for 2 days, purifying the product, and recovering ¹ H NMR was carried out for characterization. ¹ H NMR(CDCl ₃ ,400MHz):(3.65-2.20)ppm(m,-NHCH ₂ CH ₂ NH ₂ ,-NCH ₂ CH ₂ CONH-,-CH(OH)-),1.22ppm(s,-CH ₂ -),0.85ppm(t,J＝6.7Hz,-CH ₃ )。

(2) Preparation of nanoparticles (PPGC-NPs)

Adding the mixed solution of amphiphilic block polymer PLGA-PEG, lipoid polymer G0-C14, siRNA or mRNA into the water solution containing 0.25% (w/v) PVA or DSPE-PEG, and stirring at room temperature for about 15-30 minutes. The nanoparticles were purified and concentrated using an ultrafiltration system (MWCO of 100 kDa).

Example 3 characterization of PPGC-NPs (including methods and results)

(1) Characterization of physicochemical Properties

Analyzing the interaction between the siRNA and G0-C14 by agarose gel electrophoresis, optimizing the proportion according to the result, and determining the optimal proportion to be 30: 1(w/w) (FIG. 5 a). Meanwhile, the protection effect of the nanoparticles on siRNA is detected through agarose gel electrophoresis and RNase analysis experiments. The results show that the siRNA carried by the nanoparticle showed significantly improved stability even when exposed to RNase environment, compared to free siRNA (fig. 5 b).

At the indicated time points, the particle size and potential of the nanoparticles in PBS at different pH (pH 7.4, 7.0, 6.8) were measured. The results show that there is no significant change in both particle size and potential of the nanoparticles over the pH range examined (figure 5 c). In addition, before and after atomization, the particle size of the nanoparticles was not significantly changed, the hydration diameter was about 100-110nm (fig. 5d), and Transmission Electron Microscopy (TEM) showed that the nanoparticles were spherical in structure, uniform in size, and 50-60nm in diameter (fig. 5 e).

(2) Isolation of MLFs

MLFs were isolated from the lungs of 8-week-old male C57BL/6 mice. The extraction steps are briefly described as follows: the mouse lungs were removed, minced, immersed in serum-free DMEM containing 1mg/mL collagenase I and 1% penicillin-streptomycin, and digested for 30 minutes at 37 ℃. Then neutralized with DMEM containing 10% FBS and centrifuged. The resulting tissue pellet was washed with PBS and finally resuspended in complete medium, which was recorded as day 0. After the cells grew out on the fourth day, the cells were digested with 0.25% trypsin-EDTA and passaged. TGF-. beta.1 (10ng/mL) was used to induce the differentiation of the above MLFs into myofibroblasts.

(3) In vitro cellular uptake

The uptake of nanoparticles by two cells (MLFs and A549) was analyzed by laser scanning confocal microscope (Leica). As shown in fig. 5f, g, nanoparticles were able to be efficiently endocytosed into MLFs and a 549. In addition, the uptake of NPs in MLFs was dose-dependent as shown by flow cytometry (FACS) detection using PPGC-NPs encapsulating FAM-labeled siRNA (FIG. 5 h).

(4) Cell viability assay

The biocompatibility of the nanoparticles was evaluated. The MLFs were scaled to 1 × 10 ⁴ Individual cells/well were seeded in 96-well plates and after overnight culture, incubated with different doses of nanoparticles. After 24 hours, the medium was replaced with fresh serum-free medium containing 10. mu.L of CCK8 solution per well, incubated for 2 hours, and the absorbance value was measured at 450 nm. The results show that the nanoparticles exhibit good biocompatibility even at the highest dose (FIG. 5i)

(5) sequence screening of siIL11

The sequence of siIL11 was designed using software (table 1). Nanoparticles were used to entrap siIL11 of different sequences and deliver it into MLFs. After 24h, the silencing efficiency of the target gene is evaluated by qPCR, and the siIL11 sequence with the highest silencing efficiency is selected from the silencing efficiency. The qPCR primer sequences used in the above experiments were as follows: forward direction: 5'-TGTTCTCCTAACCCGATCCCT-3', respectively; and reverse, 5'-CAGGAAGCTGCAAAGATCCCA-3'. The results showed that the siRNA numbered 1 had the best silencing efficiency of the target gene, and the sequence of the sense strand (5 '-3') was GCUGUUCUCCUAACCCGAUTT; the sequence of the antisense strand (5 '-3') was AUCGGGUUAGGAGAACAGCTT (FIG. 5j, where 1,2,3 correspond to the siRNAs on rows 1,2 and 3 of Table 1, with the remaining siRNAs also being seen to have excellent results).

TABLE 1 sequences of siRNA

(6) Mucus penetration study

An experiment was designed (as shown in fig. 5 k) to assess the ability of nanoparticles to penetrate the mucus layer using artificial mucus to mimic lung mucus. The artificial mucus is prepared as follows: 500mg DNA, 250mg mucin, 250. mu.L sterile egg yolk emulsion, 0.295mg DTPA, 250mg NaCl, 110mg KCl and 1mL RPMI were dispersed in 50mL water and stirred overnight. 1mL of 10% (w/v) gelatin solution was placed in a 24-well plate, 1mL of artificial mucus was added after room temperature solidification, and 500. mu.L of Cy5.5-labeled nanoparticles of different concentrations were added dropwise. After 24 hours at room temperature, the artificial mucus was discarded, washed with PBS, and the nanoparticles permeated into gelatin were quantified using a microplate reader (Tecan, Switzerland). As shown in fig. 5k, the nanoparticles penetrate the mucus layer in a concentration-dependent manner.

FAM-siRNA is entrapped by PLGA, Cy7-siRNA is entrapped by PLGA-PEG, and the capability of PEG penetrating lung mucus layer in the nanoparticle is analyzed by trachea instillation comparison. As shown in fig. 5l, PEG modification of the surface promoted the nanoparticles to pass through the mucus layer to the bronchioles and alveoli (fig. 5 l).

Example 4 cell assay study

(1) Immunofluorescence analysis of ACTA2 and COL1A1

The activation of fibroblasts is an important stage in the development of pulmonary fibrosis. Fibroblasts can differentiate into myofibroblasts under in vitro TGF- β 1 stimulation, while type I collagen α 1(COL1a1) and ACTA2 are two major markers of myofibroblasts. Here we used PPGC-NPs to encapsulate the best siIL11 selected in the above experiments and to evaluate its ability to modulate the differentiation of MLFs. MLFs were inoculated onto the cell-plates for overnight culture, and after incubation for 4 hours with serum-free DMEM diluted NPs, the medium was replaced with complete medium and the culture was continued for 20 hours. Serum-free medium starved overnight, TGF-. beta.1 (10ng/mL) stimulated for 24 hours, 4% paraformaldehyde fixed for 20 minutes, 0.5% Triton X-100 permeabilized for 20 minutes, 3% BSA blocked, primary antibody incubated overnight, Alexa

The conjugated secondary antibodies were incubated for 1 hour at room temperature, stained nuclei for 10 minutes with DAPI, and finally observed with an upright fluorescence microscope (Olympus). Immunofluorescence results showed that intracellular expression of ACTA2 and COL1A1 was significantly increased after TGF- β 1 treatment, while levels of ACTA2 and COL1A1 were significantly reduced after siIL11@ NPs treatment (FIGS. 6 a-c).

(2) Cell migration ability assay

The development of pulmonary fibrosis also involves the migration of fibroblasts and myofibroblasts to the foci and the production of large amounts of extracellular matrix. We assessed the effect of siIL11@ NPs on fibroblast migration behavior by cell scratching and transwell experiments. As shown in FIG. 6d, e, significant differences in cell healing area occurred 24h post-scratch for the siScr @ NPs and siIL11@ NPs treated groups.

Furthermore, we also simulated the cell migration process triggered by profibrotic cytokines in vitro using a transwell experiment (as shown in figure 6 f). The results show that the migration capacity of cells treated with siIL11@ NPs is significantly reduced, further confirming that IL11 has an important role in the migration of fibroblasts (fig. 6g, h).

(3) Immunoblot analysis

MLFs were seeded in 24-well plates and the effect of IL11 knock-down on fibrosis-associated gene expression was investigated by immunoblot analysis. As shown in fig. 6i, sil 11 was able to significantly down-regulate the expression of IL11 protein in MLFs, with a concomitant significant decrease in the expression levels of ACTA2 and COL1a 1.

Meanwhile, relevant pathways of siIL11@ NPs inhibiting fibroblast activation are researched, and comprise classical signal transduction and transcriptional activator 3(STAT3), non-classical extracellular signal-regulated kinase (ERK) and SMAD2 signaling pathways. Immunoblot results showed that the activation of p-SMAD2 and p-ERK was significantly inhibited after treatment of cells with siIL11@ NPs, while the level of p-STAT3 did not change significantly (FIG. 6 j). The above results indicate that activation of fibroblasts by IL11 is associated with SMAD2 and ERK pathway, but not STAT 3.

Example 5 results of animal experiments

(1) Delivery of mRNA to the lungs of PPGC-NPs by aerosolized inhalation administration

mRNA encoding luciferase (mLuc @ NPs) was entrapped using PPGC-NPs and delivered to the mouse lungs by nebulization inhalation. The results showed that luciferase was efficiently expressed in each leaf of the lung 24 hours after nebulization (fig. 7a, b, c). The lung tissue luciferase protein quantification result shows that the luciferase content in the mouse lung tissue is increased by nearly 60 times after the mLuc @ NPs are subjected to aerosol inhalation (fig. 7d), which indicates that the PPGC-NPs can efficiently deliver the mRNA to the lung by an aerosol inhalation administration mode to express the target protein.

(2) Cy5.5-NPs distribution in mouse tissue after aerosol inhalation

Utilizing Cy5.5 marked nanoparticles (Cy5.5-NPs), delivering siRNA to the lung of a mouse through an atomization inhalation administration mode, and observing the tissue distribution and subcellular localization condition of the nanoparticles. Fluorescence imaging and quantitative analysis of five lobes using IVIS, cy5.5-NPs appeared evenly distributed in five lobes (fig. 8a, b). In addition, the subcellular localization of the nanoparticles was analyzed by FACS, and epithelial cells (EpCAM), endothelial cells (CD31) and immune cells (CD45) were labeled with the corresponding antibodies, respectively. Mice without nanoparticle inhalation were used as a control group. The results of the analysis showed that Cy5.5-NPs were mainly concentrated on epithelial cells (35.5%), followed by immune cells (30.1%) and endothelial cells (22.8%) by nebulization inhalation (FIG. 8c, d). Hematoxylin and eosin (H & E) staining of mouse lung and liver tissues showed that the nanoparticles after aerosol inhalation did not produce significant toxicity to lung and liver (fig. 8E).

(3) Establishment of pulmonary fibrosis mouse model

Male C57BL/6 mice, 8-10 weeks old, were purchased from Weitongli, Beijing. The mouse pulmonary fibrosis model is established by single intratracheal instillation of bleomycin sulfate. All animal experiments in the present invention were performed with approval from the committee for ethics and use of laboratory animals at the university of shanghai transportation.

(4) Therapeutic role of siIL11@ NPs in bleomycin-induced pulmonary fibrosis mouse model

Inhalation therapy was given after bleomycin instillation, with a low dose of 15 μ g siRNA per mouse and a high dose of 30 μ g siRNA per mouse. On day 21, blood and alveolar lavage fluid were collected (see FIG. 9a for experimental design). The left lung and other lung lobes of the mice were then harvested for RNA extraction and western blot analysis.

After bleomycin modeling, the lung of the mouse shows hemorrhagic necrosis (morphological examination of fig. 9b), and the expression content of fibrosis-related genes of lung tissues is remarkably increased (fig. 9 c). Following treatment with siIL11@ NPs, the necrotic fraction was significantly reduced (fig. 9 b).

Immunofluorescence analysis showed that after siIL11@ NPs treatment, the fluorescence intensity of ACTA2 and COL1A1 in lung tissue was significantly reduced, especially at high doses (FIG. 9 d-f). Immunoblotting results also further indicate that the levels of IL11 can be significantly inhibited after siIL11@ NPs are administered by aerosol inhalation. In addition, the expression of fibronectin, COL1a1, and ACTA2 was also significantly down-regulated (fig. 9 g).

(5) Histological analysis of mouse lung tissue

Histological analysis of mouse lung tissue samples included H & E staining, Masson staining, and sirius red (Picrosirius) staining. H & E staining results showed significant reduction in fibrotic area in the siIL11@ NPs treated group, with high dose groups being particularly evident (fig. 10 a). Masson and Picrosirius staining also showed that both low and high dose nanoparticles could alleviate fibrosis after aerosol inhalation administration (fig. 10b, c).

(6) Determination of Total protein and TGF-. beta.1 content in bronchoalveolar lavage fluid (BALF)

BALF was collected and the total protein and TGF-. beta.1 content was determined using the kit. As shown in fig. 10d, total protein mass in alveolar lavage fluid was dramatically increased after bleomycin molding, while the treated group was significantly decreased. TGF-beta 1 is a key medium involved in the development of pulmonary fibrosis, can be released by epithelial cells, macrophages and the like, and has important functions in apoptosis, fibroblast proliferation, myofibroblast differentiation and collagen synthesis. Quantitative analysis of TGF-. beta.1 in BALF by ELISA kits revealed that treatment with siiL11@ NPs reduced TGF-. beta.1 from 150pg/mL to 100pg/mL (FIG. 10 e).

(7) Determination of hydroxyproline content in mouse lung

The hydroxyproline content in the lung of the mouse is determined by an alkaline hydrolysis method. As shown in FIG. 10f, the hydroxyproline content in lung tissue was up-regulated from 250. mu.g/g to 410. mu.g/g after bleomycin molding, whereas the hydroxyproline content was reduced to 200. mu.g/g after administration of siIL11@ NPs by aerosol inhalation, which is almost close to the normal control.

(8) Survival analysis of mice with pulmonary fibrosis under bleomycin molding conditions

And (3) analyzing the survival rate of the mice with pulmonary fibrosis under the molding condition of bleomycin. As shown in fig. 10g, the survival rate of the molded mice was significantly improved after aerosol inhalation administration of siIL11@ NPs.

(9) Immunoblot analysis

Lung tissues were analyzed for protein expression of p-SMAD2, p-ERK and p-STAT3 using immunoblotting. The results show that the expression of p-SMAD2, p-ERK and p-STAT3 in lung tissues is remarkably increased after bleomycin molding. After the siIL11@ NPs atomized inhalation administration, the expression of p-SMAD2 and p-ERK is obviously inhibited, and the p-STAT3 has no obvious change (figure 10 h). This indicates that IL11 activation of fibroblasts is associated with SMAD2 and ERK pathway, but not STAT3, and animal results are consistent with the above cell results.

(10) Evaluation of safety

In addition to verifying the therapeutic effect of siIL11@ NPs, we also evaluated the safety of the nanoparticles. There was no hepatotoxicity observed in the serum as a result of AST and ALT biochemical assays (FIGS. 11a, b). In addition, organ coefficients of the animals in each group were not changed (fig. 11 c).

(11) Pulmonary Function Test (PFT)

PFT is a routine examination of clinical IPF diagnosis. The lung function of the mice was followed with the FlexiVent System (SCIREQ). After the model is made by bleomycin, the mouse deep Inspiratory Capacity (IC) is obviously inhibited, the respiratory system resistance (Rrs) and the elasticity (Ers) are obviously increased, and the lung compliance (Crs), the Forced Vital Capacity (FVC), the forced expiratory capacity (FEV) and the lung static compliance (Cst) are reduced. Both of the above parameters were significantly improved upon administration of siIL11@ NPs by aerosol inhalation (fig. 12a-f, h).

After bleomycin molding, the mouse pressure-volume loop (PV loop) showed a characteristic downward shift; following aerosol inhalation administration of siIL11@ NPs, the PV ring moved up significantly, nearly close to unmodeled healthy mice, and the area in the middle of the PV ring (hysteresis area) rose significantly (fig. 12g, i). Fig. 12j, k shows that there is a high correlation between hydroxyproline and static compliance (Cst) and respiratory elasticity (Ers).

Sequence listing

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gcuuauuuau acuuauuuat t 21

<210> 38

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

uaaauaagua uaaauaagct t 21

<210> 39

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cgaaaggauc ggagucuaat t 21

<210> 40

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

uuagacuccg auccuuucgt t 21

Claims (15)

1. A nanoparticle composition comprising an amphiphilic degradable block copolymer and a lipoid polymer;

the amphiphilic degradable block copolymer comprises polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), Polycaprolactone (PCL), polyorthoester, polyanhydride, or an amphiphilic block copolymer of poly (beta-amino ester) (PBAE) and polyethylene glycol (PEG), or a combination thereof, preferably PLGA-PEG or PLA-PEG;

the lipid polymer is prepared from Polyamidoamine (PAMAM) dendrimer and 1, 2-epoxytetradecane in a ratio of 1:4-1:7, preferably 1:5, the PAMAM dendritic polymer is preferably low-generation PAMAM, particularly preferably G0-PAMAM;

further comprising an active substance embedded therein, said active substance being selected from the group consisting of nucleic acids, small molecule drugs, polypeptides, proteins, antibodies, detection groups,

the nanoparticles are prepared by: mixing the amphiphilic degradable block copolymer, the lipid polymer and the active substance in an organic solvent, and then uniformly dispersing the mixture into an aqueous phase containing a stabilizer. The mass ratio of the amphiphilic degradable copolymer to the lipoid polymer is 1: 0.5-1: 4, preferably 1:1, the ratio of the active substance to the sum of the masses of the amphiphilic degradable block copolymer and the lipoid polymer is 1:2-1: 180.

2. The nanoparticle composition according to claim 1, further comprising an active substance embedded therein, said active substance being selected from the group consisting of nucleic acids, small molecule drugs, polypeptides, proteins, antibodies, detection groups, preferably said nucleic acids being selected from the group consisting of siRNA, messenger RNA (mRNA), DNA, miRNA, antisense oligonucleotides (ASO) and non-coding RNA, more preferably siRNA and mRNA, said nucleic acids having a ratio to the sum of the masses of amphiphilic degradable block copolymer and lipid polymer of 1:2-1:180, preferably selected from 1:2, 1: 10,1: 30,1: 60,1: 120,1: 180, more preferably 1: 60.

3. the nanoparticle composition of claim 1, wherein the stabilizer comprises one or more of ceramide-PEG, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid ester, tween 80, tween 20, Span80, Span60, sodium dodecyl sulfate, preferably polyvinyl alcohol and DSPE-PEG; wherein, the molecular weight range of the polyvinyl alcohol is 10,000-250,000 kDa, preferably 13,000-23,000 kDa; among them, polyvinyl alcohol is preferably used in a concentration range of 0.1% to 10% (w/v), more preferably 0.25% (w/v); among them, it is also preferable that the stabilizer is DSPE-PEG, preferably 0.005% to 1% (w/v), more preferably 0.01% (w/v).

4. The nanoparticle composition of claim 1, the organic solvent comprising one or more of dichloromethane, trichloromethane, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, dioxane, diethyl ether, tetrahydrofuran, acetonitrile, methanol, propylene glycol, dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), or acetone.

5. The nanoparticle composition of claim 4, wherein the organic solvent is selected from DMF and DMSO.

6. The nanoparticle composition of claim 2, wherein the sequence of the siRNA is selected from the group consisting of any one of the following rows in combination:

7. a method of preparing the nanoparticle composition of claim 1, comprising the steps of:

a) uniformly mixing the amphiphilic degradable block copolymer, the lipid polymer, and the active material, preferably a nucleic acid, in an organic solvent;

b) adding the mixture obtained in the step a) into an aqueous solution containing a stabilizer, and uniformly mixing;

c) the nanoparticles obtained are collected, purified and concentrated by means of ultrafiltration.

8. The method of claim 7, wherein the organic solvent comprises one or more of dichloromethane, trichloromethane, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, dioxane, diethyl ether, tetrahydrofuran, acetonitrile, methanol, propylene glycol, dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), or acetone, preferably DMF and DMSO.

9. The method of claim 7, wherein the stabilizer comprises one or more of ceramide-PEG, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), polyvinyl alcohol, polyglycerol fatty acid esters, Tween 80, Tween 20, Span80, Span60, sodium dodecylsulfonate, preferably polyvinyl alcohol and DSPE-PEG; wherein, the molecular weight range of the polyvinyl alcohol is 10,000-250,000 kDa, preferably 13,000-23,000 kDa; among them, polyvinyl alcohol is preferably used in a concentration range of 0.1% to 10% (w/v), more preferably 0.25% (w/v); among them, it is also preferable that the stabilizer is DSPE-PEG, preferably 0.005% to 1% (w/v), more preferably 0.01% (w/v).

10. The method of claim 7, wherein the nucleic acid is selected from the group consisting of siRNA, mRNA, DNA, miRNA, antisense oligonucleotide (ASO) and non-coding RNA, more preferably siRNA and mRNA; wherein the ratio of the nucleic acid to the sum of the masses of the amphiphilic degradable block copolymer and the lipoid polymer is 1:2-1:180, and is more preferably selected from 1:2, 1: 10,1: 30,1: 60,1: 120,1: 180, most preferably 1: 60.

11. an siRNA whose sequence is selected from the group consisting of any of the following:

12. use of a nanoparticle composition as claimed in claim 1 for the manufacture of a medicament for the treatment of fibrosis by administration by inhalation.

13. Use according to claim 12, wherein the fibrosis is selected from pulmonary fibrosis, liver fibrosis, cardiac fibrosis, kidney fibrosis, preferably pulmonary fibrosis.

14. Use according to claim 12, wherein the active substance is a nucleic acid selected from the group consisting of siRNA, mRNA, DNA, miRNA, antisense oligonucleotide (ASO) and non-coding RNA, more preferably siRNA and mRNA, most preferably siRNA as described in claim 11.

15. Use of the siRNA of claim 11, for the preparation of a medicament for the treatment of pulmonary fibrosis by administration by inhalation.

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