Application of hydrophobic modified polypeptide in preparation of lung targeted drug delivery system and lung targeted drug

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of hydrophobic modified polypeptide in preparation of a lung-targeted drug delivery system. The technical problem to be solved by the invention is that efficient and accurate lung targeting delivery of drugs is difficult to realize. The technical scheme for solving the technical problem is to provide the hydrophobic modified cationic polypeptide for preparing a lung-targeted drug delivery system or a drug composition. The hydrophobic modified cationic polypeptide as a drug delivery system efficiently delivers siRNA and other drugs to the lung through intravenous injection or aerosol inhalation, inhibits acute lung injury, lung fibrosis and lung tumors, is high in targeting property, low in toxicity, safe and short in preparation period, and has a very good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the use of a hydrophobic modified polypeptide in the preparation of a lung-targeted drug delivery system and a lung-targeted drug. Background Art

[0002] Targeted drug delivery system refers to a treatment method that can specifically direct therapeutic drugs or active substances to the organs or parts where they are needed to act, with little or no adverse effects on non-target tissues. Targeted preparations are widely valued by the medical community at home and abroad because they can improve drug efficacy, reduce toxicity, and improve the safety, effectiveness, reliability of drugs and patient compliance.

[0003] The lung is one of the most important organs in the human body, and its abnormality can cause various serious diseases, such as lung infection, lung inflammation, acute lung injury, lung fibrosis or chronic obstructive pulmonary disease (COPD). At the same time, lung cancer is also one of the malignant tumors that threaten human health and life. Primary tumors in other parts of the body will also have a certain chance of metastasizing to the lungs through the blood circulation, lymphatic system and direct infiltration in the late stage. Therefore, the effective treatment of various lung diseases, including lung tumors, is of great significance in clinical practice.

[0004] There are some problems in the current treatment of lung diseases: (1) Due to the lack of targeting, drugs have a killing effect on normal tissues, resulting in high toxic side effects and poor patient tolerance. (2) Many drug molecules are difficult to be taken up by cells because they are water-soluble, and it is difficult to reach effective drug treatment concentrations in cells. (3) The stability of drug molecules in the body. Before reaching the lesion, there will be many enzymes in the body to degrade them. Biomacromolecules have poor stability and are easily degraded in the body. Therefore, there is an urgent need for a drug delivery system that should have good biocompatibility, strong transmembrane delivery ability, lung tissue targeting and high stability.

[0005] There are some known targets for drugs to prevent and treat lung-related diseases, which can be used for targeted treatment, such as intercellular adhesion molecule-1 (ICMA1), transforming growth factor-β (TGFβ), cyclooxygenase 2 (COX2), receptor interacting protein 2 (RIP2), myeloid differentiation factor 88 (MYD88), chemokine receptor 4 (CXCR4), tumor necrosis factor-α (TNFα), high mobility group protein B1-A (HMGB1A), matrix metalloproteinase 7 (MMP7), stromal cell-derived factor-1 (CXCL12), etc., which can be used as prevention and treatment. The targets of acute lung injury and pulmonary fibrosis, while hedgehog interacting protein (HHIP), nicotinic choline receptor α-5 (CHRNA5), 5-hydroxytryptamine receptor 4 (HTR4), family sequence similarity gene 13A (FAM13A), Ras and Rab interacting factor 3 (RIN3), glutathione S-transferase C segment domain (GSTCD), human cytochrome P4502A6 (CYP2A6), interleukin-27 (IL27), coiled coil domain protein 101 (CCDC101) can be used as targets for preventing, treating or improving COPD. Signal transducer and activator of transcription 3 (STAT3), nuclear factor-E2 related factor 2 (NRF2), vascular endothelial growth factor (VEGF), mouse double minute 2 (MDM2), B lymphocyte tumor factor-2 (BCL2) or cell division cycle 20 (CDC20) can be used as effective therapeutic targets for lung cancer.

[0006] Acute lung injury is a critical illness of acute hypoxic respiratory failure caused by multiple injury factors other than cardiogenic factors. Its deterioration stage is acute respiratory distress syndrome. The pathological manifestations of acute lung injury are mainly divided into three stages: 1. Damage to the pulmonary vascular endothelium and pulmonary epithelial cells, diffuse infiltration of inflammatory cells in the lungs, and protein-rich pulmonary edema in the alveoli; 2. Repair and remodeling of lung tissue, proliferation of type II alveolar epithelial cells, accompanied by proliferation of fibroblasts in the lung interstitium; 3. Some patients develop pulmonary fibrosis due to excessive abnormal repair and remodeling.

[0007] ICAM1, also known as CD54, is one of the main members of the immunoglobulin superfamily. When inflammation occurs, local tissue cells secrete inflammatory stimulators to activate adhesion molecules on the surface of leukocytes. This leads to an increase in the expression of adhesion molecules on the surface of leukocytes, causing leukocytes to adhere rapidly and migrate to the site of inflammation through blood exudation through the endothelium to participate in the inflammatory response. Therefore, the adhesion function of ICAM1 is an important pathophysiological basis for its participation in many inflammatory diseases. Existing studies have found that the expression of ICAM1 mRNA and protein in the lung tissue of mice with acute lung injury is significantly higher than that of normal control mice, and the expression of ICAM1-positive cells in lung tissue is positively correlated with the total number of cells and neutrophil count in bronchoalveolar lavage fluid (BALF). Therefore, ICAM1 is a potential target for the treatment of acute lung injury, but there are currently few small nucleic acid drugs targeting ICAM1.

[0008] COX2 is an important rate-limiting enzyme in the synthesis of prostaglandins. It can metabolize arachidonic acid into various prostaglandin products, thereby maintaining various physiological and pathological processes of the body. COX2 is an inducible gene. It is expressed after being induced by intravascular and extravascular stimuli such as cytokines, growth factors, oncogenes, chemical carcinogens, etc., and is closely related to the occurrence of inflammation and tumors. Current studies have shown that COX2 expression is increased in patients with idiopathic pulmonary fibrosis, and the recruitment of HAT in the promoter region of the COX2 gene is reduced while the transcriptional co-inhibition complex increases, resulting in reduced acetylation of histones H3 and H4. In mice with pulmonary fibrosis models, the expression of COX2 is significantly increased. Therefore, the overexpression of COX2 promotes the increase of downstream prostaglandin E2 levels, leading to inflammatory damage and repair of alveoli, which may be one of the mechanisms of pulmonary fibrosis.

[0009] Pulmonary fibrosis is a chronic, progressive lung disease characterized by damage and scarring of lung tissue, which leads to a gradual decline in lung function and subsequent breathing difficulties. During the development of pulmonary fibrosis, normal lung tissue is gradually replaced by fibrosis or scar tissue, which affects gas exchange and respiratory function. Common causes of pulmonary fibrosis include long-term exposure to harmful substances, autoimmune diseases or infections. There is currently no cure, and treatment mainly focuses on drug control and supportive care to relieve symptoms, delay disease progression and improve quality of life.

[0010] MMP7 is an enzyme that can degrade extracellular matrix proteins and plays a key role in tissue remodeling and inflammatory response. MMP7 is not only involved in the degradation of extracellular matrix during fibrosis, but also activates TGF-β, promotes the proliferation and differentiation of fibroblasts, increases the deposition of collagen, and leads to fibrosis of lung tissue. In addition, MMP7 further promotes the development of fibrosis by affecting the migration and activation of inflammatory cells. Current studies have shown that the expression level of MMP7 is significantly increased in patients with idiopathic pulmonary fibrosis. Given the important role of MMP7 in pulmonary fibrosis, it is considered a potential therapeutic target.

[0011] CXCL12, also known as SDF-1, is a chemokine that mainly acts through its receptor CXCR4 and participates in processes such as cell migration, tissue repair and inflammatory response. CXCL12 can attract fibroblasts, fibroblasts and inflammatory cells to migrate to the site of injury. These cells secrete a large amount of extracellular matrix and collagen, leading to tissue fibrosis. CXCL12 promotes the transformation of epithelial cells to mesenchymal cells by activating the CXCR4 receptor, and regulates the migration and activation of various inflammatory cells (such as monocytes and macrophages), further aggravating the local inflammatory response and promoting the process of fibrosis. The expression level of CXCL12 is significantly increased in mice and patients with pulmonary fibrosis models. Due to the important role of the CXCL12 / CXCR4 axis in pulmonary fibrosis, therapeutic strategies targeting this pathway have received widespread attention.

[0012] TGFβ is considered to be a key factor in fibrosis and is produced by a variety of cells, including alveolar macrophages, alveolar epithelial cells, and monocytes. Studies have shown that in bleomycin-induced pulmonary fibrosis, the TGFβ content in lung tissue increases, thereby activating myofibroblasts, overproducing extracellular matrix, and inhibiting extracellular matrix degradation. Therefore, blocking TGFβ expression can effectively reduce the inflammatory response of the lung and inhibit the progression of pulmonary fibrosis.

[0013] Although clinical supportive treatment techniques and drug therapies for acute lung injury / pulmonary fibrosis have been greatly improved in recent years, the mortality rate is still as high as 30%-40%. So far, there is no effective treatment to curb the progression of acute lung injury.

[0014] Lung cancer is one of the most common causes of cancer-related deaths worldwide. The growth and metastasis of lung cancer is a multi-step and complex process, and the formation of tumor angiogenesis is an important pathological process for the growth and metastasis of malignant tumors. VEGF is an important factor that promotes the generation and proliferation of vascular endothelial cells and regulates angiogenesis. VEGF is expressed at a low level in normal tissues, but is overexpressed in many diseases and tumors. As a key factor in angiogenesis in tumors, VEGF expression can be upregulated under oncogene expression, multiple growth factors and hypoxic conditions. The role of VEGF in the occurrence and development of lung cancer mainly has two aspects: on the one hand, VEGF can act as a promoter of tumor angiogenesis, promote tumor blood supply and nutrition, thereby causing tumor proliferation and spread; on the other hand, VEGF can directly affect the migration and infiltration of tumor cells. The core role of VEGF in tumor angiogenesis makes it a reasonable target for anti-tumor therapy. By blocking VEGF, tumor angiogenesis is inhibited, tumor growth and metastasis are inhibited, thereby producing anti-tumor effects. Loading drugs with a drug delivery system with lung-targeted properties and inhaling them by intravenous injection or nebulization is a promising, safe and effective treatment for lung tumors. Summary of the invention

[0015] The technical problem to be solved by the present invention is that it is difficult to achieve efficient and accurate lung-targeted delivery of drugs. The technical solution of the present invention to solve the above technical problem is to provide a hydrophobic modified polypeptide for preparing a lung-targeted drug delivery system or pharmaceutical composition, wherein the sequence of the cationic polypeptide is VQWRIRVAVIRK (SEQ ID No. 1), and the hydrophobic modification is coupling a hydrophobic fragment to the nitrogen terminus of the cationic polypeptide.

[0016] Wherein, the polypeptide VQWRIRVAVIRK described in the above use is amidated and modified at the carbon terminus to VQWRIRVAVIRK-NH2.

[0017] Wherein, the hydrophobic fragment described in the above use is a sterol compound or a saturated straight-chain fatty acid or PEG or a PEG derivative.

[0018] Wherein, the sterol compound described in the above use is a cholesterol compound or a bile acid compound; or the saturated straight-chain fatty acid is at least one of C6-C20.

[0019] Wherein, the sterol compound described in the above use is at least one of succinylated cholesterol, bile acid or deoxycholic acid.

[0020] Wherein, the PEG derivative described in the above use is at least one of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol or dipalmitoylphosphatidylethanolamine-polyethylene glycol.

[0021] The coupling method between the nitrogen end of the polypeptide and the hydrophobic segment in the above-mentioned use is generated by amidation reaction between -CO-OH on the hydrophobic segment and -NH2 on the polypeptide.

[0022] Wherein, the hydrophobic modified polypeptide structure described in the above use is:

[0023]

[0024] Wherein, the R is a sterol compound, a saturated straight-chain fatty acid or a PEG derivative.

[0025] Wherein, R in the polypeptide structure described in the above use is:

[0026]

[0027]

[0028] At least one of .

[0029] Wherein, the drug described in the above use is at least one of small molecule drugs, nucleic acid drugs, polypeptide drugs or protein drugs.

[0030] Wherein, the drug described in the above use is a drug for preventing and / or treating tumors, or a drug for preventing and / or treating lung diseases.

[0031] Wherein, the tumor described in the above use includes at least one of lung cancer and / or a tumor producing lung metastases.

[0032] Wherein, the disease described in the above use is at least one of lung infection, lung inflammation, acute lung injury, lung fibrosis or COPD.

[0033] The lung infections mentioned in the above uses include: bacterial infections, viral infections or infections caused by other pathogens.

[0034] Wherein, the viral infection described in the above use includes: coronavirus infection, influenza virus infection or RSV virus infection.

[0035] Furthermore, the drug described in the above-mentioned use is a drug targeting at least one target among ICMA1, TGFβ, COX2, RIP2, MYD88, CXCR4, TNFα, HMGB1A, HHIP, CHRNA5, HTR4, FAM13A, RIN3, GSTCD, CYP2A6, IL27, CCDC101, STAT3, NRF2, VEGF, MDM2, BCL2, MMP7, CXCL12 or CDC20.

[0036] Wherein, the above-mentioned drug is a small nucleic acid drug. Further, the small nucleic acid drug is at least one of small interfering nucleic acid (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), micro RNA (miRNA), antisense nucleic acid (ASO) or nucleic acid aptamer (Aptamer).

[0037] Wherein, the drug delivery system or pharmaceutical composition described in the above-mentioned use also contains other pharmaceutically and / or immunologically acceptable auxiliary components.

[0038] Wherein, the pharmaceutically acceptable auxiliary component described in the above use is at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

[0039] Wherein, the lung-targeted drug delivery system or drug composition described in the above use is prepared by co-incubating the hydrophobic modified polypeptide with the drug.

[0040] Among them, the lung-targeted drug delivery system or drug composition described in the above-mentioned use is obtained by co-incubating the hydrophobic modified cationic polypeptide and the drug in water, buffer or liquid culture medium for 5 to 15 minutes; or, the buffer is at least one of phosphate buffer, Tris-HCl buffer or HEPES buffer, and the culture medium is at least one of RPMI1640, DMEM double-free culture medium or Optim culture medium.

[0041] On this basis, the present invention also provides a lung-targeted drug composition. The lung-targeted drug delivery system is composed of a hydrophobic Xiushui segment; the drug is a drug for preventing and / or treating lung tumors, or a drug for preventing and / or treating lung diseases.

[0042] Wherein, the polypeptide VQWRIRVAVIRK described in the above lung-targeted pharmaceutical composition is carbon-terminally amidated and modified to VQWRIRVAVIRK-NH2.

[0043] Wherein, the hydrophobic fragment in the above lung-targeted pharmaceutical composition is a sterol compound or a saturated straight-chain fatty acid or a PEG derivative.

[0044] Wherein, the sterol compound in the above-mentioned lung-targeted pharmaceutical composition is a cholesterol compound or a bile acid compound; or, the saturated straight-chain fatty acid is at least one of C6-C20.

[0045] Wherein, the sterol compound in the above-mentioned lung-targeted pharmaceutical composition is at least one of succinylated cholesterol, bile acid or deoxycholic acid.

[0046] Among them, the PEG derivative described in the above-mentioned lung-targeted pharmaceutical composition is at least one of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol or dipalmitoyl phosphatidylethanolamine-polyethylene glycol.

[0047] The nitrogen terminus of the polypeptide in the lung-targeted pharmaceutical composition is coupled to the hydrophobic fragment by an amidation reaction between -CO-OH on the hydrophobic fragment and -NH2 on the polypeptide.

[0048] Wherein, the hydrophobic modified polypeptide structure described in the above lung-targeted pharmaceutical composition is:

[0049]

[0050] Wherein, the R is a sterol compound, a saturated straight-chain fatty acid or a PEG derivative.

[0051] Wherein, R in the polypeptide structure of the lung-targeted pharmaceutical composition is:

[0052]

[0053]

[0054] At least one of .

[0055] Among them, the drug described in the above-mentioned lung-targeted pharmaceutical composition is a drug targeting at least one target among ICMA1, TGFβ, COX2, RIP2, MYD88, CXCR4, TNFα, HMGB1A, HHIP, CHRNA5, HTR4, FAM13A, RIN3, GSTCD, CYP2A6, IL27, CCDC101, STAT3, NRF2, VEGF, MDM2, BCL2, MMP7, CXCL12 or CDC20.

[0056] Wherein, the drug in the lung-targeted drug composition is at least one of a small molecule drug, a nucleic acid drug, a polypeptide drug or a protein drug. Further, the drug is a small nucleic acid drug.

[0057] Among them, the small nucleic acid drug described in the above-mentioned lung-targeted drug composition is at least one of small interfering nucleic acid (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), micro RNA (miRNA), antisense nucleic acid (ASO) or nucleic acid aptamer (Aptamer).

[0058] Furthermore, the small nucleic acid drug in the above-mentioned lung-targeted pharmaceutical composition is at least one of the following:

[0059] 1) siRNA for acute lung injury target ICAM1: sense strand: 5'-GCGGGAAAGUUCCUGUUUATT-3'; antisense strand: 5'-UAAACAGGAACUUUCCCGCTT-3'; or: sense strand: 5'-GCCAGCUUAUACACAAGAATT-3'; antisense strand: 5'-UUCUUGUGUAUAAGCUGGCTT-3';

[0060] 2) siRNA for the pulmonary fibrosis target COX2: sense strand: 5'-GCCAUGGAGUGGACUUAAATT-3'; antisense strand: 5'-UUUAAGUCCACUCCAUGGCTT-3';

[0061] 3) siRNA for the pulmonary fibrosis target MMP7: sense strand: 5'-GGCUCAGAAUUAUCUUAGATT-3'; antisense strand: 5'-UCUAAGAUAAUUCUGAGCCTT-3';

[0062] 4) siRNA for CXCL12, a target of pulmonary fibrosis: sense strand: 5'-CCAACGUCAAGCAUCUGAATT-3'; antisense strand: 5'-UUCAGAUGCUUGACGUUGGTT-3';

[0063] 5) siRNA for COPD target TGFβ: sense strand: 5'-CGGACUACUAUGCUAAAGATT-3'; antisense strand: 5'-UCUUUAGCAUAGUAGUCCGTT-3';

[0064] 6) siRNA for the tumor target VEGF: sense strand: 5'-GTCCGATTGAGACCCTGGTG-3'; antisense strand: 5'-ACCGGGATTTCTTGCGCTTT-3'.

[0065] Furthermore, the drug for inhibiting ICMA1 gene expression is siRNA for inhibiting ICMA1 gene expression.

[0066] Furthermore, the small nucleic acid drug in the lung-targeted pharmaceutical composition is a cocktail siRNA, and the cocktail siRNA includes MMP7 siRNA, CXCL12 siRNA and siTGFβ siRNA, that is, a combination of siRNAs targeting three targets: MMP7, CXCL12 and TGFβ.

[0067] Furthermore, the sequence of the MMP7 siRNA in the cocktail siRNA is: sense chain 5'-GGCUCAGAAUUAUCUUAGATT-3', antisense chain 5'-UCUAAGAUAAUUCUGAGCCTT-3'; the sequence of the CXCL12 siRNA is: sense chain 5'-CCAACGUCAAGCAUCUGAATT-3', antisense chain 5'-UUCAGAUGCUUGACGUUGGTT-3'; the sequence of the TGFβ siRNA is: sense chain 5'-CGGACUACUAUGCUAAAGATT-3', antisense chain: 5'-UCUUUAGCAUAGUAGUCCGTT-3'.

[0068] The polypeptide and siRNA in the lung-targeted pharmaceutical composition are prepared as raw materials at a mass ratio of 1 to 20:1.

[0069] Wherein, the hydrophobic modified polypeptide and siRNA in the lung-targeted pharmaceutical composition are prepared as raw materials at a mass ratio of 1 to 10: 1. Preferably, the hydrophobic modified polypeptide and siRNA are prepared as raw materials at a mass ratio of 5: 1.

[0070] The lung-targeted pharmaceutical composition is prepared by co-incubating the hydrophobic-modified polypeptide with siRNA.

[0071] Among them, the lung-targeted drug delivery system or drug composition described in the above-mentioned use is obtained by co-incubating the hydrophobic modified cationic polypeptide and the drug in water, buffer or liquid culture medium for 5 to 15 minutes; or, the buffer is at least one of phosphate buffer, Tris-HCl buffer or HEPES buffer, and the culture medium is at least one of RPMI1640, DMEM double-free culture medium or Optim culture medium.

[0072] The present invention also provides use of the lung-targeted pharmaceutical composition in preparing drugs for treating tumors and / or lung diseases.

[0073] Wherein, the tumor is at least one of lung cancer and / or a tumor producing lung metastases; or, the lung disease is at least one of lung infection, lung inflammation, acute lung injury, lung fibrosis or COPD.

[0074] Furthermore, the lung infection includes: at least one of bacterial infection, viral infection or infection caused by other pathogens; wherein, the viral infection includes: at least one of coronavirus infection, influenza virus infection or RSV virus infection.

[0075] Furthermore, the dosage form of the above-mentioned medicine is an injection or an inhalation preparation, wherein the inhalation preparation is at least one of a liquid preparation for atomization inhalation, an inhalation aerosol or an inhalation powder.

[0076] The beneficial effects of the present invention are: the present invention uses hydrophobic modified VQWRIRVAVIRK to make a drug delivery system, which can efficiently deliver drugs to lung tissue through intravenous injection or atomized inhalation. It does not require any special modification or treatment to be significantly enriched in the lungs, and has a good application prospect in the preparation of lung-targeted drugs. The hydrophobic modified polypeptide can be loaded with various active ingredients including nucleic acid drugs to achieve efficient and accurate targeted lung tissue administration. The drug delivery system of the present invention can be completed by self-assembly, and the preparation is also very simple and quick. The embodiments of the present invention prove that the hydrophobic modified polypeptide can protect nucleic acid molecules such as siRNA, avoid their degradation in plasma or mucosa, and deliver siRNA to the lungs in a targeted manner, significantly increase the concentration of drugs in the lungs, thereby improving the therapeutic effect of lung diseases and reducing systemic toxic side effects, and has a very good clinical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 .Nano-characterization of DP7-C. A. Particle size of DP7-C; B. Potential of DP7-C; C. Transmission electron microscopy image of DP7-C; D. Solubility of DP7-C in culture medium.

[0078] Figure 2 .Toxicity detection of different concentrations of DP7-C injected into the tail vein on mouse organs.

[0079] Figure 3 .Distribution diagram of DP7-C / Cy5-siRNA in different tissues after tail vein injection. A. In vivo distribution results of Cy5-siRNA injected through tail vein; B. Statistical results of fluorescence intensity in different tissues; C. In vivo fluorescence distribution of each group at 15 minutes after tail vein injection; D. Frozen sections of lung tissue of mice injected with DP7-C / Cy5-siRNA through tail vein at different time points.

[0080] Figure 4 .Distribution of DP7-C / Cy5-siRNA administered by aerosol in different tissues.

[0081] Figure 5.DP7-C / siICAM1 complex effectively inhibited ICAM1 expression in vitro. A. Detection results of ICAM1 mRNA level in RAW264.7 cells after DP7-C delivered different mouse ICAM1 siRNAs; B. Detection results of ICAM1 protein expression level in RAW264.7 cells after DP7-C delivered ICAM1-1234 siRNA; C. Detection results of ICAM1 mRNA level in HepG2 cells after DP7-C delivered different human ICAM1-Homo siRNAs; D. Detection results of ICAM1 protein expression level in HepG2 cells after DP7-C delivered ICAM1-Homo-1174 siRNA.

[0082] Figure 6 .DP7-C / siCOX2 complex effectively inhibited COX2 gene expression in vitro.

[0083] Figure 7 .DP7-C / siTGFβ complex effectively inhibits TGFβ gene expression in vitro.

[0084] Figure 8 .DP7-C / siMMP7 complex effectively inhibits MMP7 expression in vitro. A. Results of detection of MMP7 mRNA level in NIH3T3 cells after DP7-C delivered different MMP7 siRNAs; B. Results of detection of MMP7 protein expression level in NIH3T3 cells after DP7-C delivered different MMP7 siRNAs.

[0085] Fig. 9 .DP7-C / siCXCL12 complex effectively inhibits CXCL12 expression in vitro. A. Detection results of CXCL12 mRNA level in NIH3T3 cells after DP7-C delivered different CXCL12 siRNAs; B. Detection results of CXCL12 protein expression level in NIH3T3 cells after DP7-C delivered CXCL12-244 siRNAs.

[0086] Fig.10 .DP7-C / siVEGF complex effectively inhibits VEGF gene expression in vitro.

[0087] Fig.11 .In vitro detection of protein silencing efficiency of DP7-C / Cocktail at the cellular level.

[0088] Fig.12 .DP7-C / siICAM1 complex inhibits the secretion of inflammatory factors in vitro. A.TNF-α; B.IL1-β; C.IL-6.

[0089] Fig.13 Tail vein injection of DP7-C / siICAM1 complex effectively inhibited LPS-induced lung injury in mice.

[0090] Fig.14 Tail vein injection of DP7-C / siICAM1 complex effectively inhibited the determination of total protein concentration (Figure A) and total cell number (Figure B) in BALF of mice with LPS-induced lung injury.

[0091] Fig.15 Tail vein injection of DP7-C / siICAM1 complex effectively inhibited lung cell apoptosis in mice with LPS-induced lung injury.

[0092] Fig.16 Tail vein injection of DP7-C / Cocktail complex effectively inhibited the progression of bleomycin-induced pulmonary fibrosis in mice.

[0093] Fig.17 Tail vein injection of DP7-C / Cocktail complex effectively inhibited lung weight gain in mice with bleomycin-induced pulmonary fibrosis.

[0094] Fig.18 Tail vein injection of DP7-C / Cocktail complex effectively inhibited the total number of cells in BALF of mice with bleomycin-induced pulmonary fibrosis.

[0095] Fig.19 Tail vein injection of DP7-C / Cocktail complex effectively inhibited the formation of extracellular matrix in mice with bleomycin-induced pulmonary fibrosis.

[0096] Fig. 20 Tail vein injection of DP7-C / siVEGF for the treatment of B16F10 melanoma lung metastases in mice. A. Photographs and HE results of lung tissues in each group after tail vein injection of siRNA drugs; B. Statistical results of the number of lung nodules in each group; C. Statistical results of lung weight in each group; D. Immunofluorescence staining results of blood vessels (CD31 staining) in lung tumor tissues in each group. DETAILED DESCRIPTION

[0097] In the preliminary study of the present invention, a cationic polypeptide with antibacterial effect was obtained, whose sequence is VQWRIRVAVIRK (SEQ ID No. 1), and it was named DP7. In further studies, it was found that the DP7 polypeptide was hydrophobically modified to become an amphiphilic compound with the ability to self-assemble into micelles. On the one hand, it can reduce the cytotoxicity of the DP7 polypeptide and maintain the antibacterial activity; on the other hand, it can be used as a transport carrier for some drugs after being assembled into nanoparticles. The commonly used DP7 is a form in which its carbon end is amidated. The DP7 used in the embodiment of the present invention is QWRIRVAVIRK-NH2. Hydrophobic fragments (or hydrophobic compounds) such as cholesterol, bile acid, long-chain fatty acids, etc. are coupled with hydrophilic polypeptides, and may have the ability to self-assemble to form nanostructures. The antimicrobial peptide DP7 is nano-sized by coupling the hydrophobic fragment to increase its stability and reduce hemolytic toxicity, so that its intravenous and inhalation administration can be achieved. The nanomicelles formed by the hydrophobically modified DP7, especially the cholesterol-modified DP7-C conjugate, can achieve the effective delivery of multiple drugs.

[0098] Unexpectedly, the present invention also found that the derivative obtained by coupling the hydrophobic fragment with the antimicrobial peptide DP7 has lung targeting properties, and the micelles obtained by self-assembly can be used as a carrier for drug delivery, especially as a carrier for drug delivery targeted to the lung.

[0099] As a drug carrier targeting the lungs, the hydrophobically modified DP7 peptide can be loaded with various types of drugs due to its self-assembly function, such as small molecule drugs, nucleic acid drugs, peptide drugs and protein drugs.

[0100] The small molecule drugs can be cytokine inhibitors and chemokine receptor antagonists related to lung diseases, leukotriene B4 (LTB4) receptor antagonists and LTB4 synthesis regulators, etc.

[0101] The nucleic acid drug can be at least one of small nucleic acid drugs such as small interfering nucleic acid (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), micro RNA (miRNA), antisense nucleic acid (ASO) or nucleic acid aptamer for treating lung diseases.

[0102] The polypeptide and protein drugs can be various cytokines, antibodies, vaccines and other drugs for treating lung diseases.

[0103] According to the different types of drugs loaded therein, it can be used for various indications, including the prevention and treatment of various lung diseases. For example, it can be loaded with various drugs for preventing or treating lung infection, lung inflammation, acute lung injury, lung fibrosis or COPD lung disease.

[0104] Wherein, the lung infection described in the above use includes: bacterial infection, viral infection or infection caused by other pathogens. Wherein, the viral infection described in the above use includes: coronavirus infection or influenza virus infection. The coronavirus includes SARS coronavirus, new coronavirus, RSV virus, etc.

[0105] For example, the active ingredients for preventing and / or treating tumors can be loaded. The tumors can be lung cancer or lung metastases of other tumors.

[0106] Among them, the above-mentioned drugs may be drugs targeting at least one target among ICMA1, TGFβ, COX2, RIP2, MYD88, CXCR4, T NFα, HMGB1A, MMP7, CXCL2, HHIP, CHRNA5, HTR4, FAM13A, RIN3, GSTCD, CYP2A6, IL27, CCDC101, STAT3, NRF2, VEGF, MDM2, BCL2 or CDC20.

[0107] In one embodiment of the present invention, the cationic micelles formed by the DP7-C conjugate are used to successfully achieve efficient transfection of drugs targeting lung tissue, and the siRNA of the acute lung injury target ICAM1 (sense chain: 5'-GCGGGAAAGUUCCUGUUUATT-3'; antisense chain: 5'-UAAACAGGAACUUUCCCGCTT-3'; or: sense chain: 5'-GCCAGCUUAUACACAAGAATT-3'; antisense chain: 5'-UUCUUGUGUAUAAGCUGGCTT-3'), the siRNA of the pulmonary fibrosis target COX2 (sense chain: 5'-GCCAUGGAGUGGACUUAAATT-3'; antisense chain: 5'-UUUAAGUCCACUCCAUGGCTT-3'), and the siRNA of MMP7 (sense chain: 5'-GGCUCAGAAUUAUCUUAGATT-3'; antisense chain: 5'- After being transfected into the target cells, the acute lung injury target ICAM1, pulmonary fibrosis targets COX2, MMP7, CXCL12, COPD target TGFβ and tumor target VEGF were efficiently knocked out.

[0108] In another embodiment of the present invention, the cationic micelles formed by the DP7-C conjugate were also used to successfully achieve efficient transfection of Cocktail siRNA (MMP7 siRNA + CXCL12 siRNA + TGFβ siRNA). After the Cocktail siRNA was transferred into the target cells, the expression level of each target gene was significantly reduced; compared with the DP7-C / single siRNA group, there was no significant difference. This shows that DP7-C can effectively deliver a combination of multiple siRNAs to target cells.

[0109] In the examples of the present invention, the cationic micelles formed by the DP7-C conjugate were used to successfully achieve efficient targeted drug introduction into lung tissue and effectively inhibit lung diseases. In one embodiment, ICAM1 siRNA was delivered to treat acute lung injury. In another embodiment, Cocktail siRNA was delivered to treat pulmonary fibrosis. In another example, VEGF siRNA was delivered to treat melanoma lung metastasis, and significant effects were achieved.

[0110] For example, the drug used in the example of treating acute lung injury is a drug that inhibits the expression of ICAM1 gene, specifically, siRNA that inhibits the expression of ICAM1 gene. DP7-C / siRNA composite nanoparticles were obtained by simple preparation, and it was proved that both intravenous injection and inhalation can make the siRNA carried by DP7-C / siRNA composite nanoparticles accumulate rapidly in lung tissue, have good lung targeting, and effectively treat acute lung injury. Furthermore, the nucleotide sequence of the ICAM1 siRNA is preferably as follows: sense chain: 5'-GCGGGAAAGUUCCUGUUUATT-3; antisense chain: 5'-UAAACAGGAACUUUCCCGCTT-3'; or: sense chain: 5'-GCCAGCUUAUACACAAGAATT-3'; antisense chain: 5'-UUCUUGUGUAUAAGCUGGCTT-3'.

[0111] The DP7-C used in the examples of the present invention is a succinylated cholesterol-modified DP7 polypeptide (Chol-suc-VQWRIRVAVIRK-NH2), and its peptide structure is:

[0112]

[0113] Where R is:

[0114]

[0115] The synthesis of DP7-C and the preparation of DP7-C nanoparticles can be carried out by referring to the method described in Chinese patent document CN107446019 A.

[0116] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments are further descriptions of the technical solutions of the present invention and do not indicate that the present invention is limited to these embodiments.

[0117] The main experimental materials and equipment used in the examples are as follows: RAW264.7 (mouse mononuclear macrophage leukemia cells), HepG2 (human liver cancer cells), NIH3T3 (mouse embryonic fibroblasts), and B16F10 (mouse skin melanoma cells) were purchased from the American Type Culture Collection (ATCC). Cell culture was performed using DMEM (Gibco) or 1640 (Gibco) medium containing 10% fetal bovine serum (FBS). 6-8 week-old C57BL / 6J female mice and 8-week-old C57BL / 6J male mice were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd. and raised in an SPF environment.

[0118] Example 1. Physical characteristics of DP7-C micelles

[0119] Transmission electron microscopy and Malvern particle size analyzer were used to detect some physical characteristics of DP7-C micelles, such as particle size and Zeta potential.

[0120] 1. Transmission electron microscope photography: Prepare different concentrations of DP7-C solution, add it dropwise to the copper mesh, stain it with phosphotungstic acid, dry it naturally, and place the copper mesh coated with DP7-C on a transmission electron microscope for photography.

[0121] 2. Particle size and Zeta potential detection: DP7-C was dissolved in MilliQ water, and the particle size and Zeta potential were measured using a Malvern particle size analyzer. Each sample was tested 4 times and the average value was taken.

[0122] 3. DP7-C appearance and solution state: Observe the state of DP7-C after dissolving in the culture medium and take photos with a camera.

[0123] Results Figure 1 , the particle size of DP7-C micelles is about 42.2±2.7nm ( Figure 1 A), Zeta potential is about 52.4±1.5mV( Figure 1 B), DP7-C micelles are spherical or ellipsoidal in shape ( Figure 1 C). DP7-C freeze-dried powder is white and fluffy, easily soluble in water or culture medium, and the solution is transparent ( Figure 1 D).

[0124] Example 2. DP7-C in vivo lung toxicity detection

[0125] In order to detect the in vivo toxicity of DP7-C on lung tissue, three different dose groups were set up: 3 mg / kg, 10 mg / kg, and 25 mg / kg. 24 hours after injection through the tail vein, the mice were killed, and their hearts, livers, spleens, lungs, and kidneys were paraffin-embedded, sliced, and HE-stained.

[0126] The results are as follows Figure 2 The results showed that even a high dose of DP7-C (25 mg / kg) had no obvious tissue toxicity to the main organs of mice, especially to the lungs.

[0127] Example 3. Detection of lung targeting of DP7-C / siRNA composite nanoparticles

[0128] After incubation at room temperature for 10 minutes with DP7-C and Cy5-siRNA at a mass ratio of 5:1 (DP7-C: 100 μg, Cy3-siRNA: 20 μg), DP7-C / Cy5-siRNA composite nanoparticles were injected into the tail vein. After 15 minutes, 2 hours, 4 hours, and 24 hours, the heart, liver, spleen, lung, and kidney of the mice were harvested to detect the distribution of DP7-C / Cy5-siRNA composite nanoparticles in the major organs.

[0129] The experimental results show that:

[0130] In addition to the nonspecific distribution in the liver (most nanomaterials have a certain distribution in the liver after intravenous administration), after intravenous injection, DP7-C / Cy5-siRNA composite nanoparticles showed lung tissue targeting, while almost no fluorescence signal was observed in other tissues ( Figure 3 A). As the metabolic time increases, the fluorescence signal of lung tissue is still the strongest after the fluorescence signal of liver ( Figure 3 A, B). In particular, 15 minutes after intravenous injection, the strongest fluorescence signal was detected in the lungs, accounting for more than 42% of the total fluorescence ( Figure 3 C). At the same time, fluorescence microscopy was used to analyze the fluorescence in the frozen sections of lung tissues of each group of mice ( Figure 3 D). As can be seen from the results, there is still a clear fluorescent signal in the lung tissue 24 hours after intravenous injection of DP7-C / Cy5-siRNA. These results show that intravenous injection of DP7-C / siRNA composite nanoparticles allows siRNA to accumulate rapidly in lung tissue and has good lung targeting.

[0131] In addition, aerosol inhalation is a direct drug delivery method with the respiratory tract and lungs as target organs, which has the advantages of good drug efficacy, high safety, and few adverse reactions. We incubated DP7-C and Cy5-siRNA at a mass ratio of 5:1 (DP7-C: 100μg, Cy5-siRNA: 20μg) at room temperature for 10 minutes, then used an aerosol device to aerosolize the DP7-C / Cy5-siRNA composite nanoparticles, and placed the mice in the aerosol box for aerosol inhalation. After the mice were aerosolized for 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 24 hours, the distribution of DP7-C / Cy5-siRNA composite nanoparticles in various major organs was detected by in vitro imaging. The results showed that DP7-C / Cy5-siRNA had obvious and specific lung aggregation. At 5 minutes of inhalation, DP7-C was able to deliver siRNA to the lungs, Cy5-siRNA aggregation peaked at 30 minutes, and metabolism began 4 hours later ( Figure 4 ). In other tissues, almost no obvious fluorescence was observed. The experimental results showed that DP7-C / siRNA composite nanoparticles can quickly deliver siRNA to lung tissue in the aerosol administration route and have good lung targeting.

[0132] Example 4. Silencing effect of DP7-C / siICAM1 composite nanoparticles on ICAM1 in vitro

[0133] In lung tissue injury, the adhesion and activation of granulocytes to endothelial cells and the release of inflammatory mediators all require the participation of adhesion molecules. ICAM1 plays an important role in many immune and inflammatory responses by interacting with its ligand LFA-1. ICAM1 expressed on lung macrophages promotes leukocyte adhesion and recruits leukocytes to aggregate in the alveolar region, which is a key factor leading to lung parenchymal cell damage and sustained cytokine release. Therefore, the ICAM1 gene is selected to target in the present invention to verify the effectiveness of the composite nanoparticles.

[0134] First, siRNA targeting the ICAM1 gene was co-incubated with DP7-C to form a DP7-C / siICAM1 complex. We then verified the gene silencing effect of this composite nanoparticle in RAW264.7 cells in vitro.

[0135] The ICAM1 siRNA sequence screened in the early stage of the present invention is:

[0136] ICAM1-686: positive strand (SEQ ID No. 2): 5′-GGCUGGCAUUGUUCUCUAATT-3′;

[0137] Antisense strand (SEQ ID No. 3): 5′-UUAGAGAACAAUGCCAGCCTT-3′;

[0138] ICAM1-595: positive strand (SEQ ID No. 4): 5′-CCCAAGGAGAUCACAUUCATT-3′;

[0139] Antisense strand (SEQ ID No. 5): 5′-UGAAUGUGAUCUCCUUGGGTT-3′;

[0140] ICAM1-1234: positive strand (SEQ ID No. 6): 5′-GCGGGAAAGUUCCUGUUUATT-3′;

[0141] Antisense strand (SEQ ID No. 7): 5'-UAAACAGGAACUUUCCCGCTT-3'.

[0142] In order to select the best ICAM1 siRNA, based on the previous optimization, the selected ICAM1 siRNA was transfected with DP7-C, and a single material group (DP7-C) and an unrelated siRNA group (DP7-C / siScramble) were used as controls. First, RAW264.7 cells with good growth status were evenly spread into a six-well plate, and after the cells adhered to the wall, drug treatment was performed (10μg DP7-C and 2μg siRNA per well were incubated at room temperature for 15min). After 48 hours, the knockdown efficiency of the three siRNAs on the ICAM1 gene was compared by qPCR. Figure 5 As shown in A, the knockdown efficiency of ICAM1-1234 (0.27±0.02) sequence was better than that of ICAM1-595 (1.23±0.06) and ICAM1-686 (0.98±0.03). Based on the qPCR screening results, we used WB to detect the protein expression level of ICAM1 in RAW264.7 cells after transfection of ICAM1-1234 ( Figure 5 B). The results showed that compared with the control group, the DP7-C / siICAM1-1234 (hereinafter referred to as DP7-C / siICAM1) treatment group significantly downregulated the mRNA expression level of ICAM1 gene in RAW264.7 cells ( Figure 5 A) and protein expression levels ( Figure 5 B), indicating that the ICAM1 siRNA we selected by DP7-C can effectively silence ICAM1.

[0143] ICAM1-Homo-1174: positive strand (SEQ ID No. 8): 5′-GCCAGCUUAUACACAAGAATT-3′;

[0144] Antisense strand (SEQ ID No. 9): 5'-UUCUUGUGUAUAAGCUGGCTT-3'.

[0145] ICAM1-Homo-1554: positive strand (SEQ ID No. 10): 5′-CAGCGGAAGAUCAAGAAAUTT-3′;

[0146] Antisense strand (SEQ ID No. 11): 5'-AUUUCUUGAUCUUCCGCUGTT-3'.

[0147] ICAM1-Homo-1468: positive strand (SEQ ID No. 12): 5′-CCCGGUAUGAGAUUGUCAUTT-3′;

[0148] Antisense strand (SEQ ID No. 13): 5'-AUGACAAUCUCAUACCGGGTT-3'.

[0149] ICAM1-Homo-1318: positive strand (SEQ ID No. 14): 5′-CCGAGCUCAAGUGUCUAAATT-3′;

[0150] Antisense strand (SEQ ID No. 15): 5'-UUUAGACACUUGAGCUCGGTT-3'.

[0151] At the same time, we screened the best human ICAM1 siRNA. Based on the previous optimization, we transfected the selected ICAM1 siRNA with DP7-C, and used a single material group (DP7-C) and an unrelated siRNA group (DP7-C / si-Scramble) as controls. First, HepG2 cells with good growth status were evenly spread into a six-well plate, and after the cells adhered to the wall, they were treated with drugs (10μg DP7-C and 2μg siRNA per well, incubated at room temperature for 15 minutes). After 48 hours, the knockdown efficiency of the four siRNAs on the ICAM1 gene was compared by qPCR. Figure 5 As shown in C, the knockdown efficiency of ICAM1-homo-1174 (0.28±0.05) sequence is better than that of ICAM1-Homo-1554 (0.57±0.08), ICAM1-Homo-1468 (0.75±0.04) and ICAM1-homo-1318 (0.43±0.08). According to the qPCR screening results, we used WB to detect the protein expression level of ICAM1 gene in HepG2 cells after transfection of ICAM1-homo-1174 ( Figure 5 D) The results are as follows Figure 5As shown, in human and mouse cells, the DP7-C / siICAM1 treatment group significantly downregulated the mRNA level of ICAM1 gene compared with the control group ( Figure 5 A, C) and protein expression levels ( Figure 5 B, D), indicating that our DP7-C delivery of the ICAM1 siRNA we selected can effectively silence ICAM1. Example 5. In vitro DP7-C / siCOX2 complex effectively inhibits COX2 gene expression

[0152] The present invention aims at the COX2 gene, a target for the treatment of pulmonary fibrosis, and obtains siRNA (siCOX2) that targets and inhibits the COX2 gene by designing and screening, in order to obtain siCOX2 with good pulmonary fibrosis inhibition and anti-tumor effects.

[0153] In the early stage of the experiment, we selected and synthesized three siRNAs targeting the COX2 gene through software design and preliminary optimization. The three synthesized siRNAs targeting the COX2 gene were combined with DP7-C to form DP7-C / siCOX2 complexes.

[0154] According to the preferred ratio of siCOX2 and DP7-C obtained in previous experiments, siCOX2 was dissolved in DEPC water to 1 μg / μl, 2 μg siCOX2 was pipetted into 200 μl culture medium and mixed, and then 10 μg DP7-C was added. After incubation at room temperature for 10 minutes, the obtained DP7-C / siCOX2 complex was gently added to the cells.

[0155] By comparing NIH3T3 cell transfection and qPCR experiments, siCOX2-730 ( Figure 6 ), and its knockdown efficiency reached 70%. The sequence of siCOX2-730 is as follows:

[0156] Sense strand (SEQ ID No. 16): 5'-GCCAUGGAGUGGACUUAAATT-3'

[0157] Antisense strand (SEQ ID No. 17): 5'-UUUAAGUCCACUCCAUGGCTT-3'.

[0158] Example 6. DP7-C / siTGFβ complex effectively inhibits TGFβ gene expression in vitro

[0159] The present invention aims at treating TGFβ gene for pulmonary fibrosis, and obtains siRNA (siTGFβ) that targets and inhibits TGFβ gene by designing and screening, so as to obtain siTGFβ with good inhibitory and anti-tumor effects.

[0160] In the early stage of the experiment, we selected and synthesized three siRNAs targeting the TGFβ gene through software design and preliminary optimization. First, the three synthesized siRNAs targeting the TGFβ gene were combined with DP7-C to form a DP7-C / siTGFβ complex.

[0161] According to the optimal ratio of siTGFβ and DP7-C obtained in the previous experiment, siTGFβ was dissolved in DEPC water to 1μg / μl, 2μg siTGFβ was taken and mixed in 200μl culture medium, and then 10μg DP7-C was added. After incubation at room temperature for 10 minutes, it was gently added to the cells. NIH-3T3 cell transfection and qPCR experiments were compared to select siTGFβ-1167 ( Figure 7 ), and its knockdown efficiency reached 60%. The sequence of siTGFβ-1167 is as follows:

[0162] Sense strand (SEQ ID No. 18): 5'-CGGACUACUAUGCUAAAGATT-3'

[0163] Antisense strand (SEQ ID No. 19): 5'-UCUUUAGCAUAGUAGUCCGTT-3'.

[0164] Example 7. DP7-C / siMMP7 complex effectively inhibits MMP7 gene expression in vitro

[0165] The present invention targets the pulmonary fibrosis target MMP7 gene, and obtains siRNA (siMMP7) that targets and inhibits the MMP7 gene by designing and screening, in order to obtain siMMP7 that can effectively inhibit the progress of pulmonary fibrosis.

[0166] In the early stage of the experiment, we selected and synthesized four siRNAs targeting the MMP7 gene through software design. First, the four synthesized siRNAs targeting the MMP7 gene were combined with DP7-C to form a DP7-C / siMMP7 complex.

[0167] According to the preferred ratio of siMMP7 and DP7-C obtained in the previous experiment, siMMP7 was dissolved in DEPC water to 1μg / μl, 2μg siMMP7 was drawn into 200μl 1640 medium and mixed, and then 10μg DP7-C was added, gently mixed, incubated at room temperature for 10 minutes and then gently added to the cells. NIH3T3 cells were transfected, and qPCR and WB experiments were compared to select siMMP7-266 ( Figure 8), the knockdown efficiency of which reached 65%. The sequence of siMMP7-266 is as follows:

[0168] Sense strand (SEQ ID No. 20): 5'-GGCUCAGAAUUAUCUUAGATT-3'

[0169] Antisense strand (SEQ ID No. 21): 5'-UCUAAGAUAAUUCUGAGCCTT-3'.

[0170] Example 8. DP7-C / siCXCL12 complex effectively inhibits CXCL12 gene expression in vitro

[0171] The present invention targets the pulmonary fibrosis target CXCL12 gene, and obtains siRNA (siCXCL12) that targets and inhibits the CXCL12 gene by designing and screening, in order to obtain siCXCL12 that can effectively inhibit the progression of pulmonary fibrosis.

[0172] In the early stage of the experiment, we selected and synthesized three siRNAs targeting the CXCL12 gene through software design. First, the three synthesized siRNAs targeting the CXCL12 gene were combined with DP7-C to form a DP7-C / siCXCL12 complex.

[0173] According to the optimal ratio of siCXCL12 and DP7-C obtained in the previous experiment, siCXCL12 was dissolved in DEPC water to 1μg / μl, 2μg siCXCL12 was drawn into 200μl 1640 medium and mixed, and then 10μg DP7-C was added, gently mixed, incubated at room temperature for 10 minutes and then gently added to the cells. NIH3T3 cells were transfected, and qPCR and WB experiments were compared to select siCXCL12-244 (which had the highest knockdown efficiency after forming a complex with DP7-C) from the candidate siCXCL12. Fig. 9 ), the knockdown efficiency of which reached 70% siCXCL12-244 sequence is as follows:

[0174] Sense strand (SEQ ID No. 22): 5'-CCAACGUCAAGCAUCUGAATT-3'

[0175] Antisense strand (SEQ ID No. 23): 5'-UUCAGAUGCUUGACGUUGGTT-3'.

[0176] Example 9. DP7-C / siVEGF complex effectively inhibits VEGF gene expression in vitro

[0177] The present invention targets the tumor target VEGF gene, and obtains siRNA (siVEGF) that targets and inhibits the VEGF gene by designing and screening, in order to obtain siVEGF with a good effect of inhibiting lung tumor growth.

[0178] In the early stage of the experiment, we selected and synthesized three siRNAs targeting the VEGF gene through software design. First, the three synthesized siRNAs targeting the VEGF gene were combined with DP7-C to form a DP7-C / siVEGF complex.

[0179] According to the optimal ratio of siVEGF and DP7-C obtained in the previous experiment, siVEGF was dissolved in DEPC water to 1μg / μl, 2μg siVEGF was taken and mixed in 200μl culture medium, and then 10μg DP7-C was added. After incubation at room temperature for 10 minutes, it was gently added to the cells. Through B16F10 cell transfection and qPCR experiment comparison, siVEGF-952 ( Fig.10 ), the sequence of siVEGF-952 with a knockdown efficiency of 50% is as follows:

[0180] Sense strand (SEQ ID No. 24): 5'-CGAUGAAGCCCUGGAGUGCTT-3'

[0181] Antisense strand (SEQ ID No. 25): 5'-GCACUCCAGGGCUUCAUCGTT-3'.

[0182] Example 10. Knockdown efficiency of DP7-C transfected with cocktail siRNA

[0183] In this part of the experiment, cocktail siRNA (including siMMP7-266+siCXCL12-244+siTGFβ-1167) was transferred into NIH3T3 cells to detect the knockdown efficiency of DP7-C transfected cocktail siRNA. The specific method was to co-incubate 0.7μg siMMP7, 0.7μg siCXCL12, 0.7μg siTGFβ and 10.5μg DP7-C in DMEM medium, and then add them to the 2×10 5 In NIH3T3 cells, after 4 hours, the DMEM medium was replaced with DMEM+10% FBS+1% PS medium, and the transfection continued for 48 hours. After 48 hours, the protein of NIH3T3 cells was extracted, and the protein expression levels of MMP7, CXCL12, and TGFβ were detected by WB.

[0184] The experimental results showed that Cocktail could significantly reduce the expression level of target genes compared with the control group (Control), material group (DP7-C), and irrelevant siRNA group (DP7-C / si-Scramble); there was no significant difference compared with the DP7-C / single siRNA group. Therefore, it was confirmed that DP7-C can effectively deliver the combination of siRNAs to NIH3T3 cells ( Fig.11 ).

[0185] Example 11. In vitro DP7-C / siICAM1 composite nanoparticles inhibit the secretion of inflammatory factors

[0186] In order to detect the inhibitory effect of DP7-C / siICAM1 composite nanoparticles on inflammatory factors, PBS was used as the normal control group, and the stimulation group (LPS), the single material group (DP7-C), and the irrelevant siRNA group (DP7-C / siScramble) were used as the treatment control to compare the anti-inflammatory effect of the treatment group (DP7-C / siICAM1). We cultured RAW264.7 cells in good growth state at a rate of 1x10 5 The cells were inoculated in a 24-well plate at a density of 100 μg and cultured overnight. The next day, the culture medium in the wells was replaced with serum-free culture medium, and siRNA (0.5 μg) encapsulated with DP7-C (2.5 μg) was added. Three replicate wells were set up in each group. After 1 hour of transfection, bacterial lipopolysaccharide (LPS) was added for stimulation. After 24 hours, the cell supernatant was collected and centrifuged at 3000 rpm for 30 minutes, and the supernatant was retained for ELISA detection.

[0187] This example uses the aforementioned preferred ICAM1 siRNA, whose sequence is:

[0188] Sense strand: 5′-GCGGGAAAGUUCCUGUUUATT-3;

[0189] Antisense strand: 5′-UAAACAGGAACUUUCCCGCTT-3′.

[0190] The experimental results showed that the supernatant of each cell culture was collected, centrifuged and placed at -80℃ for later use. Taking the untreated PBS group as the control, the secretion of TNFα, IL-1β and IL-6 increased in cells stimulated by LPS (Control group). However, the group transfected with DP7-C / siICAM1 composite nanoparticles could inhibit the secretion of TNFα, IL-1β and IL-6, indicating that inhibiting the expression of ICAM1 can effectively inhibit the secretion of pro-inflammatory factors ( Fig.12 ).

[0191] Example 12. Evaluation of the therapeutic effect of DP7-C / siICAM1 composite nanoparticles on acute lung injury

[0192] In order to evaluate the therapeutic effect of DP7-C / siICAM1 on acute lung injury in mice, we induced acute lung injury in mice by LPS tracheal instillation, and mice instilled with PBS in the trachea were used as normal controls (PBS group). Six hours after modeling, the mice were randomly divided and immediately given drugs, with tail vein injection of PBS (LPS group), DP7-C (material group), DP7-C / siScramble (irrelevant siRNA group) and DP7-C / siICAM1 (experimental group), respectively, with injection doses of siRNA 12μg and DP7-C 60μg. After 24 hours, the same dose was given again. Healthy mice without modeling were used as normal controls. The experimental results showed that the lung injury caused by LPS group was the most severe, with obvious inflammatory cell infiltration, thickening of alveolar wall and interstitium, and congestion of lung tissue. The material group (DP7-C) and irrelevant siRNA group (DP7-C / siScramble) had no therapeutic effect on lung injury. The experimental group (DP7-C / siICAM1) showed a significant reduction in pulmonary edema, reduced bronchial and alveolar exudation, and significantly reduced inflammatory cell infiltration and pulmonary interstitial edema. The results suggest that DP7-C / siICAM1 has a good therapeutic effect on acute lung injury (see Fig.13 ).

[0193] In severe lung injury, the permeability of pulmonary capillaries increases, leading to the extravasation of large amounts of protein and other substances. Therefore, the total protein concentration in BALF is an important indicator of capillary permeability in lung injury. Under normal circumstances, there are no cells in the BALF of healthy mice. After acute lung injury, inflammatory cells and macrophages (especially neutrophils) will infiltrate the alveolar lavage fluid. After treatment with DP7-C / siICAM1, the total protein concentration in the BALF of mice during acute lung injury was significantly reduced ( Fig.14 A), the number of inflammatory cells decreased significantly ( Fig.14 B).

[0194] We used the TUNEL method to detect the apoptosis of lung cells in each group after treatment, and the green fluorescent marker indicated apoptotic cells. Using PBS as the control for healthy mice, the results showed that compared with the PBS group, each group had different degrees of apoptosis, but after DP7-C / siICAM1 treatment, apoptosis decreased ( Fig.15 ).

[0195] Example 13. Evaluation of the therapeutic effect of systemic delivery of DP7-C / Cocktail siRNA composite nanoparticles on bleomycin-induced pulmonary fibrosis in mice

[0196] In order to detect the potential application of DP7-C systemically delivered cocktail combination siRNA (siMMP7+siCXCL12+siTGFβ) in the treatment of pulmonary fibrosis, we established a BLM-induced mouse pulmonary fibrosis model to further verify its therapeutic effect.

[0197] (1) Eight-week-old C57 / BL6 male mice weighing approximately 24–26 g were selected. After anesthetization, the mice were fixed on the experimental table in a supine position. The neck was depilated and disinfected with alcohol. The skin was cut open and the trachea was exposed layer by layer.

[0198] (2) A 1 mL syringe was inserted into the trachea through the gap between the two tracheal cartilage rings toward the cardiac end. If there was no resistance when the syringe was withdrawn, BLM (at a dose of 5 mg / kg) was injected. After the operation, the animal was quickly placed upright and rotated to allow the drug solution to be evenly distributed in the lungs. Mice that were injected with PBS into the trachea served as normal controls. The animals were fed routinely after they woke up.

[0199] (3) On the 7th day after the bolus injection of bleomycin, the mice were randomly divided into 3 groups, 7 mice in each group, and marked.

[0200] (4) Three groups of mice were intravenously injected with normal saline (Untreated group), DP7-C / siScramble (unrelated siRNA group) and DP7-C / Cocktail siRNA (experimental group). The dose of each tail vein injection was 30μg of siRNA (10μg of each of the three siRNAs) and 150μg of DP7-C. The drug was administered once every five days, for a total of 2 times. HE staining results showed that tracheal instillation of BLM would lead to obvious lung pathological morphological changes, including thickening of the alveolar septum, destruction of alveolar structure, and infiltration of a large number of inflammatory cells in the alveolar space and interstitium; DP7-C / Cocktail siRNA treatment could restore the alveolar epithelial structure to a certain extent, alleviate lung inflammation in mice, and had a significant therapeutic effect ( Fig.16 ). At the same time, after the mice were killed, the lung tissues of each group of mice were weighed. Tracheal instillation of BLM induced lung tissue fibrosis and increased lung weight, while DP7-C / Cocktail siRNA treatment effectively inhibited the weight of lung tissue ( Fig.17 ). After BLM induction, the number of cells in the BALF of mice in the untreated and material groups increased significantly, while DP7-C / Cocktail siRNA treatment significantly reduced the number of cells in the BALF to a level close to that of healthy mice ( Fig.18 ).

[0201] Collagen I is the main component of the extracellular matrix of pulmonary fibrosis, and α-SMA is a marker for the transformation of fibroblasts into myofibroblasts. It is an important indicator of collagen deposition and damage in lung tissue of pulmonary fibrosis. WB results showed that the expression levels of α-SMA and Collagen I in the lung tissue of BLM-induced pulmonary fibrosis mice were increased, and the collagen deposition in lung tissue and the transformation of fibroblasts into myofibroblasts were reduced after DP7-C / Cocktail siRNA treatment, suggesting that DP7-C / Cocktail siRNA improves pulmonary fibrosis in the lung by inhibiting epithelial-mesenchymal transition and inflammatory response (see Fig.19 ).

[0202] Example 14 Systemic delivery of DP7-C / siVEGF complex to treat B16F10 mouse melanoma lung metastasis model

[0203] To characterize the potential of DP7-C systemically delivering VEGF siRNA in the treatment of lung cancer, a B16F10 mouse melanoma lung metastasis model was established to further verify its anti-tumor effect on lung metastasis.

[0204] (1) Establishment of lung metastasis model: On day 0, 0.2 mL of cell suspension (containing approximately 2.5×10 5 B16F10 cells).

[0205] (2) On the third day after inoculation, the mice were randomly divided into 4 groups, 7 mice in each group, and marked.

[0206] (3) Four groups of mice were intravenously injected with normal saline (NS group), DP7-C (material group, 60 μg), DP7-C / siScramble (irrelevant siRNA group) and DP7-C / VEGF siRNA (experimental group). The tail vein injection dose was 12 μg siRNA and 60 μg DP7-C per time. The drugs were administered once every other day for a total of 7 times.

[0207] (4) On the 21st day after inoculation, all mice were killed by cervical dislocation, and lung tissues as well as heart, liver, spleen, and kidney tissues were immediately collected, weighed, and analyzed. The number of lung tumor nodules in each group of mice was counted, and the tumor tissues of each experimental group were analyzed by CD31 fluorescence staining.

[0208] Fig. 20 The effect of intravenous treatment of the B16F10 lung metastasis model with DP7-C / siVEGF complex is shown. Fig. 20A is the photos of the lung tissues of the mice in each group and the representative tissue sections of the lung tissues of the mice in each group. Compared with the other three groups, the tumor burden of the lung tissues of the mice treated with DP7-C / siVEGF was significantly reduced, and the number of tumor nodules was reduced ( Fig. 20 B). The average lung tissue weight of the DP7-C / siVEGF complex treatment group was 1.66±0.59g, the NS group was 7.58±0.98g, the DP7-C material group was 6.43±0.85g, and the NC group was 3.86±0.74g ( Fig. 20 C). The number of lung metastatic nodules and lung weight of mice treated with DP7-C / siVEGF complex were significantly less than those of other treatment groups. Therefore, the lung tumor growth of mice treated with DP7-C / siVEGF complex was significantly inhibited.

[0209] In addition, CD31 immunofluorescence staining was performed on lung tissue sections, and the results showed that compared with the other three experimental groups, the number of new blood vessels in the tumor tissue of the DP7-C / siVEGF complex treatment group was less, proving that DP7-C can effectively inhibit the formation of blood vessels in lung tumor tissue by systemically delivering VEGF siRNA ( Fig. 20 D).

Claims

1. Use of a hydrophobic modified polypeptide in preparing a lung-targeted drug delivery system or a pharmaceutical composition, characterized in that The amino acid sequence of the polypeptide is VQWRIRVAVIRK, and the hydrophobic modification is coupling a hydrophobic fragment to the nitrogen terminal of the polypeptide.

2. The use according to claim 1, characterized in that The hydrophobic fragment is a sterol compound or a saturated straight-chain fatty acid or PEG or a PEG derivative; or the carbon terminal of the polypeptide VQWRIRVAVIRK is amidated and modified to VQWRIRVAVIRK-NH2; Wherein, the sterol compound is a cholesterol compound or a bile acid compound; or, the saturated straight-chain fatty acid is at least one of C6-C20.

3. The use according to claim 2, characterized in that The sterol compound is at least one of succinylated cholesterol, bile acid or deoxycholic acid; or the PEG derivative is at least one of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol or dipalmitoyl phosphatidylethanolamine-polyethylene glycol.

4. The use according to any one of claims 1 to 3, characterized in that The nitrogen end of the polypeptide is coupled to the hydrophobic segment by an amidation reaction between -CO-OH on the hydrophobic segment and -NH2 on the polypeptide; preferably, the hydrophobic modified polypeptide structure is: Wherein, the R is a sterol compound or a saturated straight-chain fatty acid or a PEG derivative; further, the R in the polypeptide structure is: At least one of .

5. The use according to claim 1, characterized in that The drug is at least one of a small molecule drug, a nucleic acid drug, a polypeptide drug or a protein drug; or, the drug is a drug for preventing and / or treating tumors, or a drug for preventing and / or treating lung diseases.

6. The use according to claim 5, characterized in that The tumor includes at least one of lung cancer and / or a tumor that produces lung metastases; or, the lung disease is at least one of lung infection, lung inflammation, acute lung injury, lung fibrosis or chronic obstructive pulmonary disease (COPD); further, the lung infection includes at least one of bacterial infection, viral infection or infection caused by other pathogens; or, the viral infection includes at least one of coronavirus infection, influenza virus infection or respiratory syncytial virus (RSV).

7. The use according to claim 5, characterized in that The nucleic acid drug is a small nucleic acid drug; further, the small nucleic acid drug is at least one of small interfering nucleic acid (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), micro RNA (miRNA), antisense nucleic acid (ASO) or nucleic acid aptamer.

8. The use according to claim 5, characterized in that The drug is a drug targeting at least one of ICMA1, TGFβ, COX2, RIP2, MYD88, CXCR4, TNFα, HMGB1A, HHIP, CHRNA5, HTR4, FAM13A, RIN3, GSTCD, CYP2A6, IL27, CCDC101, STAT3, NRF2, VEGF, MDM2, BCL2, MMP7, CXCL12 or CDC20.

9. The use according to any one of claims 1 to 8, characterized in that The drug delivery system or pharmaceutical composition further contains other pharmaceutically and / or immunologically acceptable auxiliary ingredients; preferably, the pharmaceutically acceptable auxiliary ingredients are at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

10. The use according to any one of claims 1 to 9, characterized in that The lung-targeted drug delivery system or drug composition is prepared by co-incubating the hydrophobic modified polypeptide with the drug; further, the lung-targeted drug delivery system or drug composition is obtained by co-incubating the hydrophobic modified cationic polypeptide with the drug in water, buffer or liquid culture medium for 5 to 15 minutes; preferably, the buffer is at least one of phosphate buffer, Tris-HCl buffer or HEPES buffer, and the liquid culture medium is at least one of RPMI 1640, DMEM double-free culture medium or Optim culture medium.

11. A lung-targeted pharmaceutical composition, characterized in that The invention is prepared by loading a hydrophobic modified polypeptide with a drug, wherein the sequence of the polypeptide is VQWRIRVAVIRK, and the hydrophobic modification is coupling a hydrophobic fragment to the nitrogen terminus of the polypeptide; the drug is a drug for preventing and / or treating lung tumors, or a drug for preventing and / or treating lung diseases.

12. The lung-targeted pharmaceutical composition according to claim 11, characterized in that The drug is a drug targeting at least one of ICMA1, TGFβ, COX2, RIP2, MYD88, CXCR4, TNFα, HMGB1A, HHIP, CHRNA5, HTR4, FAM13A, RIN3, GSTCD, CYP2A6, IL27, CCDC101, STAT3, NRF2, VEGF, MDM2, BCL2, MMP7, CXCL12 or CDC20.

13. The lung-targeted pharmaceutical composition according to claim 11, characterized in that The drug is at least one of a small molecule drug, a nucleic acid drug, a polypeptide drug or a protein drug; further, the drug is a small nucleic acid drug.

14. The lung-targeted pharmaceutical composition according to claim 13, characterized in that The small nucleic acid drug is at least one of small interfering nucleic acid (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), micro RNA (miRNA), antisense nucleic acid (ASO) or nucleic acid aptamer (Aptamer).

15. The lung-targeted pharmaceutical composition according to claim 14, characterized in that The small nucleic acid drug is at least one of the following: 1) siRNA for acute lung injury target ICAM1: sense strand: 5'-GCGGGAAAGUUCCUGUUUATT-3'; antisense strand: 5'-UAAACAGGAACUUUCCCGCTT-3'; or: sense strand: 5'-GCCAGCUUAUACACAAGAATT-3'; antisense strand: 5'-UUCUUGUGUAUAAGCUGGCTT-3'; 2) siRNA for the pulmonary fibrosis target COX2: sense strand: 5'-GCCAUGGAGUGGACUUAAATT-3'; antisense strand: 5'-UUUAAGUCCACUCCAUGGCTT-3'; 3) siRNA for the pulmonary fibrosis target MMP7: sense strand: 5'-GGCUCAGAAUUAUCUUAGATT-3'; antisense strand: 5'-UCUAAGAUAAUUCUGAGCCTT-3'; 4) siRNA for CXCL12, a target of pulmonary fibrosis: sense strand: 5'-CCAACGUCAAGCAUCUGAATT-3'; antisense strand: 5'-UUCAGAUGCUUGACGUUGGTT-3'; 5) siRNA for COPD target TGFβ: sense strand: 5'-CGGACUACUAUGCUAAAGATT-3'; antisense strand: 5'-UCUUUAGCAUAGUAGUCCGTT-3'; 6) siRNA for the tumor target VEGF: sense strand: 5'-GTCCGATTGAGACCCTGGTG-3'; antisense strand: 5'-ACCGGGATTTCTTGCGCTTT-3'.

16. The lung-targeted pharmaceutical composition according to claim 15, characterized in that The small nucleic acid drug is a cocktail siRNA, and the cocktail siRNA includes MMP7 siRNA, CXCL12 siRNA and TGFβ siRNA; Furthermore, the sequence of siMMP7 in the cocktail siRNA is: sense strand 5'-GGCUCAGAAUUAUCUUAGATT-3', antisense strand 5'-UCUAAGAUAAUUCUGAGCCTT-3'; the sequence of siCXCL12 is: siRNA sense strand 5'-CCAACGUCAAGCAUCUGAATT-3', antisense strand 5'-UUCAGAUGCUUGACGUUGGTT-3'; the sequence of siTGFβ is: sense strand 5'-CGGACUACUAUGCUAAAGATT-3', antisense strand: 5'-UCUUUAGCAUAGUAGUCCGTT-3'.

17. The lung-targeted pharmaceutical composition according to any one of claims 15 to 16, characterized in that The hydrophobic fragment is a sterol compound or a saturated straight-chain fatty acid or PEG or a PEG derivative; or, the carbon terminal of the polypeptide VQWRIRVAVIRK is amidated and modified to VQWRIRVAVIRK-NH2; Furthermore, the sterol compound is a cholesterol compound or a bile acid compound; or, the saturated straight-chain fatty acid is at least one of C6-C20.

18. The lung-targeted pharmaceutical composition according to claim 17, characterized in that The sterol compound is a cholesterol compound or a bile acid compound; or the saturated straight-chain fatty acid is at least one of C6-C20; Preferably, the sterol compound is at least one of succinylated cholesterol, bile acid or deoxycholic acid; or The PEG derivative is at least one of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol or dipalmitoyl phosphatidylethanolamine-polyethylene glycol.

19. The lung-targeted pharmaceutical composition according to claim 17, characterized in that The nitrogen end of the polypeptide is coupled to the hydrophobic segment by an amidation reaction between -CO-OH on the hydrophobic segment and -NH2 on the polypeptide; preferably, the hydrophobic modified polypeptide structure is: Wherein, the R is a sterol compound, a saturated straight-chain fatty acid or a PEG derivative.

20. The lung-targeted pharmaceutical composition according to claim 19, characterized in that R in the polypeptide structure is: At least one of .

21. The lung-targeted pharmaceutical composition according to any one of claims 14 to 20, characterized in that The polypeptide and siRNA are prepared as raw materials in a mass ratio of 1 to 20:1; further, the hydrophobic modified polypeptide and siRNA are prepared as raw materials in a mass ratio of 1 to 10:1; preferably, the hydrophobic modified polypeptide and siRNA are prepared as raw materials in a mass ratio of 5:

1.

22. The lung-targeted pharmaceutical composition according to any one of claims 14 to 21, characterized in that It is prepared by co-incubating a hydrophobic modified polypeptide with siRNA; further, the lung-targeted pharmaceutical composition is obtained by co-incubating a hydrophobic modified polypeptide with a nucleic acid in water, a buffer or a liquid culture medium for 5 to 15 minutes; further, the buffer is at least one of a phosphate buffer, a Tris-HCl buffer or a HEPES buffer, and the liquid culture medium is at least one of RPMI 1640, DMEM double-free culture medium or Optim culture medium.

23. The lung-targeted pharmaceutical composition according to any one of claims 14 to 22, characterized in that The dosage form of the pharmaceutical composition is an injection or an inhalation preparation; further, the inhalation preparation is at least one of a liquid preparation for atomized inhalation, an inhalation aerosol or an inhalation powder.

24. Use of the lung-targeted pharmaceutical composition according to any one of claims 11 to 23 in the preparation of a drug for treating tumors and / or lung diseases.

25. The use according to claim 24, characterized in that The dosage form of the drug is an injection or an inhalation preparation, or the tumor is at least one of lung cancer and / or a tumor that produces lung metastases; or the lung disease is at least one of lung infection, lung inflammation, acute lung injury, lung fibrosis or COPD.

26. The use according to claim 25, characterized in that The lung infection is at least one of bacterial infection, viral infection or infection caused by other pathogens; further, the viral infection is at least one of coronavirus infection, influenza virus infection or RSV virus infection.

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