WO2023246827A1 - 一种小分子药物-寡核苷酸偶联物及其应用 - Google Patents

一种小分子药物-寡核苷酸偶联物及其应用 Download PDF

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WO2023246827A1
WO2023246827A1 PCT/CN2023/101555 CN2023101555W WO2023246827A1 WO 2023246827 A1 WO2023246827 A1 WO 2023246827A1 CN 2023101555 W CN2023101555 W CN 2023101555W WO 2023246827 A1 WO2023246827 A1 WO 2023246827A1
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nfkbiz
aso
small molecule
oligonucleotide
nanomicelles
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French (fr)
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张川
祝瑞华
黄洋洋
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上海交通大学
浙江浥眸生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • inflammation may be psoriasis, Sjogren's syndrome, dry eye, uveitis, keratitis, conjunctivitis, atopic dermatitis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease
  • inflammation may be psoriasis, Sjogren's syndrome, dry eye, uveitis, keratitis, conjunctivitis, atopic dermatitis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease
  • a major driver of diseases such as Rohn's disease, heart disease, diabetes, cancer, asthma, inflammatory bowel disease, Alzheimer's disease and many more.
  • VCAM-1 vascular cell adhesion factor 1
  • E-selectin endothelial cell adhesion molecules in the blood circulation -1
  • ICM-1 intercellular adhesion molecule-1
  • vascular permeability will lead to local edema, allowing phagocytes, complement system proteins, eicosanoids, kinins, cytokines, platelet activating factor and other substances to enter the blood vessels (J Allergy Clin Immunol 2010,125,S3- S23), promotes the occurrence and development of inflammation.
  • cytokines such as tumor necrosis factor, interleukin, lymphokine, mononuclear factor, interferon, colony-stimulating factor, and transforming growth factor produced by monocytes, T cells, platelets, and endothelial cells are all involved in inflammation-related reactions.
  • the inflammatory response is enhanced when components of the innate immune system, monocytes (including macrophages and dendritic cells), and neutrophils are activated.
  • TLRs transmembrane Toll Like Receptors
  • LPS lipopolysaccharide
  • Circulating cytokines will further amplify the inflammatory response and trigger an adaptive immune response. Circulating cytokines interact with specific receptors on different cell types and activate Janus kinase/signal transduction and transcription protein (JAK-STAT), nuclear factor kappa B (NF- ⁇ B), and transforming growth factor beta (TGF- ⁇ ) signaling pathways, leading to inflammatory responses such as cell adhesion, increased permeability, apoptosis, and an increase in reactive oxygen species.
  • JAK-STAT Janus kinase/signal transduction and transcription protein
  • NF- ⁇ B nuclear factor kappa B
  • TGF- ⁇ transforming growth factor beta
  • inflammation-related diseases The common feature of inflammation-related diseases is the activation of relevant signaling pathways and the overexpression of a variety of inflammatory cytokines after the immune balance is disrupted, which in turn stimulates a sustained inflammatory response and has a destructive effect on the body.
  • inflammatory cytokines Based on their understanding of the mechanisms of inflammatory responses and immune activation processes that induce disease, researchers are committed to developing different types of drugs to regulate inflammation and ultimately cure the disease.
  • Existing inflammation-modulating drugs mainly include small molecule drugs with immunomodulatory functions, antibody drugs targeting cytokines and receptors involved in inflammatory response, and genetic drugs that regulate the expression of genes related to inflammatory signaling pathways.
  • small molecule drugs with immunomodulatory functions such as calcineurin inhibitors, mTOR inhibitors, glucocorticoids, vitamin D analogs, etc.
  • the above small molecule drugs can be produced on a large scale and at low cost.
  • Some small molecule drugs can also Made into oral preparations.
  • small molecule drugs generally have poor water solubility, low bioavailability, often have strong side effects, and are not suitable for long-term use.
  • Antibody drugs are usually highly targeted, quick-acting, and have excellent immunomodulatory effects.
  • these drugs generally require injection and are expensive to produce and treat.
  • long-term systemic administration can easily lead to drug resistance and other adverse reactions, with strong toxic and side effects, such as reduced immunity and susceptibility to infection and cancer.
  • RNA antisense oligonucleotides
  • target inflammatory response-related genes to achieve immune regulation are a new class of drugs that have low immunogenicity and a wide range of targets, and can target intracellular
  • the target can also target extracellular targets, is less likely to produce drug resistance, and can regulate more upstream signaling pathways, providing more possibilities for immune regulation and disease treatment.
  • nucleic acid drugs due to the large molecular weight and negative charge of nucleic acid drugs, it is difficult for individual nucleic acid drugs to cross the cell membrane barrier, thus facing delivery difficulties.
  • nucleic acid drugs In order to realize nucleic acid drugs For effective delivery and gene regulation function, it is often necessary to introduce specific vectors to help deliver functional nucleic acids to target tissues and cells.
  • viral vectors have high transfection efficiency, they still face a series of problems such as immunogenicity, insertional mutation, and complex preparation during practical application, which hinder their clinical transformation.
  • non-viral vectors such as cationic liposomes and cationic polymers are also commonly used to deliver nucleic acid drugs.
  • cationic delivery vectors generally face problems such as high cytotoxicity and difficult quality control.
  • each drug has its advantages, due to the complexity of the inflammatory response, using a single strategy to regulate a certain target or block a certain cytokine may lead to a compensatory increase in other pro-inflammatory cytokines, making it difficult to obtain satisfactory results. Satisfactory therapeutic effect.
  • the invention provides a small molecule drug-oligonucleotide conjugate, which is obtained by covalent coupling of a small molecule drug with immunomodulatory function and a functional oligonucleotide molecule that can regulate the expression of inflammation-related genes.
  • the small molecule drug and the functional oligonucleotide molecule are covalently coupled through a chemical linker.
  • the selected small molecule drugs are small molecule drugs that act on immune-related signaling pathways and can regulate immune responses; preferably, the small molecule drugs include calcineurin inhibitors, glucocorticoids, mTOR inhibitors and vitamins. One or more of D analogs.
  • the inflammation-related genes include one or more of the tumor necrosis factor-a gene, interleukin 1b gene, interleukin 17 gene, interleukin 23 gene, NFKBIZ gene, inflammasome NLRP3 gene, JAK gene and PDE4 gene. .
  • the functional oligonucleotide molecule includes one of double-stranded small interfering nucleic acid, micronucleic acid and single-stranded antisense oligonucleotide.
  • the small molecule drug is covalently coupled to the 3' end of the sense strand of the functional oligonucleotide molecule; when the When the functional oligonucleotide molecule is a single-stranded antisense oligonucleotide, the small molecule drug is covalently coupled to the 3' end or 5' end of the single-stranded antisense oligonucleotide.
  • the number of small molecule drugs covalently coupled to each functional oligonucleotide molecule is 1 to 40.
  • the small molecule drug is covalently coupled to the end of the functional oligonucleotide molecule or the small molecule drug is covalently coupled to the side of the 3' end or 5' end extension sequence of the functional oligonucleotide molecule. on chain bases or phosphate backbone;
  • the chemical structural formula of the small molecule drug-oligonucleotide conjugate is as shown in Formula 1 to Formula 14:
  • Q, Y, and Z are each independently absent, -O-, -S-, -C(O)-, -NH-, -CH 2 -, -C(O )NH-, -NHC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and One or more of them; (O) in the Q, Y, Z groups represents a carbonyl oxygen atom; the is the connection site;
  • G represents a small molecule immunomodulatory drug
  • m, n, and k are independently 1 to 15;
  • X means O or S
  • Y and Z are each independently absent, O, S, C(O), NH, CH 2 , C(O)NH, NHC(O), C(O)O , OC(O), OC(O)O, OC(O)NH, NHC(O)O and One or more of the above; (O) in the Y and Z groups represents a carbonyl oxygen atom; the is the connection site;
  • the present invention also provides the use of the small molecule drug-oligonucleotide conjugate described in the above scheme in the preparation of drugs for treating inflammation-related diseases.
  • the invention provides a small molecule drug-oligonucleotide conjugate, which is obtained by covalent coupling of a small molecule drug with immunomodulatory function and a functional oligonucleotide molecule that can regulate the expression of inflammation-related genes.
  • the present invention adopts a method of coupling with oligonucleotide drugs.
  • the excellent hydrophilicity of oligonucleotides can improve the dissolution of small molecule drugs. properties, improve its tissue distribution in the body, and promote delivery and absorption.
  • hydrophobic small molecule drugs can in turn promote the entry of oligonucleotides into cells, thereby improving their ability to regulate target genes.
  • the present invention achieves coordinated regulation of different targets of the inflammatory response through the co-delivery of small molecule drugs and functional oligonucleotide molecules, thereby achieving better disease treatment effects and achieving safe and efficient medication.
  • the two drug molecules assist each other to realize the preparation of a new drug delivery system without additional carriers, and simultaneously efficiently deliver functional oligonucleotide molecules and small molecule immunomodulatory drugs to achieve the purpose of collaboratively regulating the inflammatory response, thus providing a variety of Treatment of inflammation-related diseases offers better solutions.
  • Figure 1 is a schematic diagram of the preparation route of azide-modified cyclosporine A (CsA-N 3 ) and monocyclosporin A-NFKBIZ antisense oligonucleotide covalent conjugate (CsA-ASO NFKBIZ );
  • Figure 3 is the 13 C NMR spectrum of chloromethyl carbonate cyclosporine A (CsA-Cl);
  • Figure 7 shows the gel electrophoresis picture of the coupling product of CsA-N 3 and DBCO modified ASO. 10% denaturing polyacrylamide gel electrophoresis detection confirmed that the CsA-ASO NFKBIZ conjugate was efficiently synthesized;
  • Figure 8 shows the cellular uptake of CsA-ASO NFKBIZ-h conjugate
  • Figure 10 shows the evaluation of the effect of CsA-ASO NFKBIZ-m nanomicelles in treating dry eye;
  • A Fluorescein sodium staining of mouse eyes on days 0, 7, and 14 of treatment;
  • B On days 0 and 7 of treatment , Analysis of fluorescein sodium score data in the eyes of mice on the 14th day;
  • C Mouse phenol red cotton thread tear test results on the 0th, 7th, and 14th days of treatment;
  • Figure 14 shows the characterization of 3CsA-ASO NFKBIZ conjugates and their self-assembled nanomicelles.
  • the left picture shows the results of 10% denaturing polyacrylamide gel electrophoresis, and the right picture shows the results of 0.5% agarose gel electrophoresis;
  • Figure 16 shows the evaluation of the effect of 3CsA-ASO NFKBIZ-m nanomicelles in treating dry eyes;
  • A Fluorescein sodium staining of mouse eyes on days 0, 7, and 14 of treatment;
  • B On days 0 and 7 of treatment , Analysis of fluorescein sodium score data in the eyes of mice on the 14th day;
  • C Mouse phenol red cotton thread tear test results on the 0th, 7th, and 14th days of treatment;
  • Figure 17 is a schematic diagram of the synthesis of aminotris[(2-propynyloxy)methyl]methane
  • Figure 18 is the hydrogen nuclear magnetic resonance spectrum of Boc-aminotris[(2-propynyloxy)methyl]methane
  • Figure 19 is a schematic diagram of the synthesis of CsA 3 -ASO NFKBIZ conjugate
  • Figure 20 shows the 20% denaturing polyacrylamide gel electrophoresis characterization of the triynyl-modified ASO NFKBIZ conjugate
  • Figure 21 shows the 0.5% agarose gel electrophoresis characterization of CsA 3 -ASO NFKBIZ nanomicelles
  • Figure 22 shows the hydrated particle size (A) and morphology (B) of CsA 3 -ASO NFKBIZ nanomicelles
  • Figure 23 shows the real-time fluorescence quantitative PCR detection of target gene NFKBIZ mRNA expression levels after cells were treated with CsA 3 -ASO NFKBIZ-h nanomicelles and control samples;
  • Figure 24 is the evaluation of the effect of CsA 3 -ASO NFKBIZ-m nanomicelles in treating dry eyes;
  • A Fluorescein sodium staining of mouse eyes on the 0th and 14th days of treatment;
  • B On the 0th and 14th days of treatment Data analysis of fluorescein sodium score in mouse eyes;
  • C Mouse phenol red cotton thread tear test results on days 0 and 14 of treatment;
  • Figure 25 shows the 15% denaturing polyacrylamide gel electrophoresis results of CsA-siRNA NFKBIZ , 2CsA-siRNA NFKBIZ and 3CsA-siRNA NFKBIZ coupled molecules and the 0.5% agarose gelation of 2CsA-siRNA NFKBIZ and 3CsA-siRNA NFKBIZ nanomicelles. Gel electrophoresis results;
  • Figure 26 shows the expression level of I ⁇ B- ⁇ protein in human corneal epithelial cells after they were treated with CsA-siRNA NFKBIZ-h conjugate, 2CsA-siRNA NFKBIZ-h nanomicelles, 3CsA-siRNA NFKBIZ-h nanomicelles and control samples. ;
  • Figure 27 shows the real-time fluorescence quantitative PCR detection of CsA-siRNA NFKBIZ-h conjugates, 2CsA-siRNA NFKBIZ-h nanomicelles, 3CsA-siRNA NFKBIZ-h nanomicelles and control samples after treatment of human eye epithelial cells. ;
  • Figure 28 shows the evaluation of the effect of CsA-siRNA NFKBIZ-m conjugate, 2CsA-siRNA NFKBIZ-m nanomicelles and 3CsA-siRNA NFKBIZ-m nanomicelles in treating dry eyes;
  • A Mouse eyes at the 0th day of treatment , 7 and 14 days of fluorescein sodium staining;
  • B Analysis of fluorescein sodium score data in mouse eyes on days 0, 7 and 14 of treatment;
  • C Mouse phenol red cotton tears on days 0, 7 and 14 of treatment Test Results;
  • Figure 29 shows the synthesis steps of carbonyl ethyl bromide cyclosporine A
  • Figure 30 shows the LC-MS spectrum of carbonyl ethyl bromide cyclosporine A;
  • Figure 31 is a 10% denatured polyacrylamide gel image and a 1% agarose gel image of 20CsA-ASO NFKBIZ nanomicelles;
  • Figure 32 shows the real-time fluorescence quantitative PCR detection of NFKBIZ mRNA expression in cells after treatment with 20CsA-ASO NFKBIZ-h nanomicelles and control samples;
  • Figure 33 is the evaluation of the effect of 20CsA-ASO NFKBIZ-m nanomicelles in the treatment of dry eye;
  • A Data analysis of ocular fluorescein sodium scores in mice on the 0th and 14th days of treatment;
  • B In mice on the 0th and 14th days of treatment Phenol cotton thread tear test results;
  • Figure 34 is a real-time fluorescence quantitative PCR detection of the expression of various inflammatory mRNAs in human corneal epithelial cells after treatment with different target 3CsA-siRNA nanomicelles and control samples;
  • Figure 36 is the evaluation of the effect of 3CsA-siRNA nanomicelles on dry eye treatment for different targets;
  • A Data analysis of fluorescein sodium score in the eyes of mice on the 0th and 14th days of treatment;
  • B On the 0th and 14th days of treatment Mouse phenol red cotton thread tear test results;
  • Figure 37 is a real-time fluorescence quantitative PCR detection of the expression of various inflammatory target mRNAs in human corneal epithelial cells after treatment with different target 3CsA-siRNA nanomicelles and control samples;
  • Figure 38 is the evaluation of the effect of 3CsA-siRNA nanomicelles on dry eye treatment for different targets;
  • A Data analysis of fluorescein sodium score in the eyes of mice on the 0th and 14th days of treatment;
  • B On the 0th and 14th days of treatment Mouse phenol red cotton thread tear test results;
  • Figure 39 shows the real-time fluorescence quantitative PCR detection of the expression of different inflammatory mRNAs in human corneal epithelial cells after treatment with 3CsA-ASO nanomicelles of different targets and control samples;
  • Figure 40 shows the ELISA detection of the expression of different inflammatory factors in human corneal epithelial cells after treatment with 3CsA-ASO nanomicelles of different targets and control samples;
  • Figure 41 is the evaluation of the effect of different target 3CsA-ASO nanomicelles in treating dry eye;
  • A Data analysis of fluorescein sodium score in the eyes of mice on the 0th and 14th days of treatment;
  • B Mice on the 0th and 14th days of treatment Phenol cotton thread tear test results;
  • Figure 42 shows the real-time fluorescence quantitative PCR detection of inflammatory mRNA expression levels in human corneal epithelial cells after treatment with different target 20CsA-ASO nanomicelles and control samples;
  • Figure 43 shows the evaluation of the effect of different target 20CsA-ASO nanomicelles in treating dry eye;
  • A Analysis of ocular fluorescein sodium score data of mice on the 0th and 14th days of treatment;
  • B Mice on the 0th and 14th days of treatment Phenol cotton thread tear test results;
  • Figure 44 is a schematic diagram of the synthesis of RAP-Bz-Br
  • Figure 45 shows the hydrogen nuclear magnetic resonance spectrum (A) and carbon spectrum (B) of TK-Bz-Br;
  • Figure 46 shows the hydrogen nuclear magnetic resonance spectrum (A) and carbon spectrum (B) of RAP-31-O-TMS-42-Bz-Br;
  • Figure 47 shows 15% denaturing polyacrylamide gel electrophoresis; (A) and 1% agarose gel electrophoresis (B) characterization of the successful preparation of 10RAP-ASO conjugates and 10RAP-nanomicelles;
  • Figure 48 shows the hydrated particle size (left picture) and transmission electron microscope morphology (right picture) of 10RAP-ASO NFKBIZ nanomicelles
  • Figure 50 shows the evaluation of the effect of different target 10RAP-ASO NFKBIZ-m nanomicelles in the treatment of dry eye;
  • A Analysis of ocular fluorescein sodium score data of mice on the 0th, 7th and 14th days of treatment;
  • B On the 0th, 7th and 14th day of treatment Mouse phenol red cotton thread tear test results at 0, 7, and 14 days;
  • Figure 52 shows the ELISA detection of the secretion of different inflammatory factors in HaCaT cells after treatment with CsA 3 -ASO NFKBIZ-h nanomicelles and control samples;
  • Figure 56 shows the scores of erythema, scales, and skin thickening on the back skin lesions of psoriasis-like model mice after treatment with smear and drug administration, as well as the scores of the psoriasis lesion area severity index;
  • Figure 57 is a schematic diagram of the synthesis of carbonyl ethyl bromide triamcinolone acetonide (TA-Br);
  • Figure 58 is the hydrogen nuclear magnetic resonance spectrum of TA-Br
  • Figure 59 is the NMR carbon spectrum of TA-Br
  • Figure 62 shows the successful preparation of 10TA-ASO NFKBIZ nanomicelles characterized by 1% agarose gel electrophoresis
  • Figure 63 shows the RT-PCR detection of NFKBIZ mRNA expression in cells after co-incubation of 10TA-ASO NFKBIZ-h nanomicelles and control samples with HaCaT cells;
  • Figure 67 is a schematic diagram of the synthesis of Cal-Bz-Br
  • Figure 69 is the hydrogen nuclear magnetic resonance spectrum of Cal-Bz-Br
  • Figure 71 shows the successful preparation of 10Cal-ASO NFKBIZ conjugate characterized by 15% denaturing polyacrylamide gel electrophoresis
  • Figure 72 shows the successful preparation of 10Cal-ASO NFKBIZ nanomicelles characterized by 1% agarose gel electrophoresis
  • Figure 73 shows the hydrated particle size (left picture) and transmission electron microscope morphology (right picture) of 10Cal-ASO NFKBIZ nanomicelles
  • Figure 74 is the critical micelle concentration diagram of 10Cal-ASO NFKBIZ nanomicelles
  • Figure 76 shows the changes in skin thickness of psoriasis-like model mice during treatment
  • Figure 77 shows the scores of erythema, scales, and skin thickening on the back skin lesions of mice before and after treatment, as well as the psoriasis lesion area severity index scores;
  • Figure 78 shows photos of skin lesions on the back of psoriasis-like model mice on days 0 and 7 of treatment.
  • the invention provides a small molecule drug-oligonucleotide conjugate, which is obtained by covalent coupling of a small molecule drug with immunomodulatory function and a functional oligonucleotide molecule that can regulate the expression of inflammation-related genes.
  • the small molecule drug and the functional oligonucleotide molecule are preferably covalently coupled through a chemical linker.
  • the calcineurin inhibitor preferably includes cyclosporine A, tacrolimus, sirolimus, pimecrolimus, or cyclosporine A, tacrolimus, sirolimus, Limus or an analog of pimecrolimus;
  • the mTOR inhibitor preferably includes rapamycin or a derivative of rapamycin;
  • the derivative of rapamycin preferably includes Temsirolimus (CCI-779) Or Everolimus (RAD001);
  • the glucocorticoid preferably includes triamcinolone acetonide, dexamethasone, betamethasone, methylprednisolone, cortisone, hydrocortisone, prednisone acetate, prednisolone acetate, Prednisone or triamcinolone acetonide, dexamethasone, betamethasone, methylprednisolone, cortisone, hydrocortisone, prednisone acetate,
  • the inflammation-related genes include tumor necrosis factor-a (TNF-a) gene, interleukin 1b (IL-1b) gene, interleukin 17 (IL-17) gene, interleukin 23 (IL-23) gene, One or more of the NFKBIZ gene, inflammasome NLRP3 gene, JAK gene and PDE4 gene.
  • TNF-a tumor necrosis factor-a
  • IL-1b interleukin 1b
  • IL-17 interleukin 17
  • IL-23) gene interleukin 23
  • NFKBIZ gene NFKBIZ gene
  • JAK gene and PDE4 gene One or more of the NFKBIZ gene
  • Each gene has its corresponding functional oligonucleotide molecule that regulates the expression of the corresponding mRNA of the gene.
  • the functional oligonucleotide molecule includes one of double-stranded small interfering nucleic acid (siRNA), micronucleic acid (miRNA) and single-stranded antisense oligonucleotide (ASO).
  • the functional oligonucleotide molecules used can be unmodified functional oligonucleotide molecules, or functional oligonucleotide molecules with stability-enhancing modifications; the stability-enhancing modifications preferably include PS, One or more of the 2-position OMe, MOE and F generation modifications.
  • the small molecule drug when the functional oligonucleotide molecule is a double-stranded small interfering nucleic acid or a micronucleic acid, the small molecule drug is covalently coupled to the 3' end of the sense strand of the functional oligonucleotide molecule; when the functional oligonucleotide molecule is a single-stranded antisense oligonucleotide, the small molecule drug is covalently coupled to the 3' end or 5' end of the single-stranded antisense oligonucleotide.
  • the small molecule drug is covalently coupled to the end of the functional oligonucleotide molecule or the small molecule drug is covalently coupled to the 3' end or 5' end extension sequence of the functional oligonucleotide molecule. on the side chain base or phosphate backbone.
  • the number of small molecule drugs covalently coupled to each functional oligonucleotide molecule is preferably any integer value between 1 and 40.
  • the number of small molecule drugs covalently coupled to each functional oligonucleotide molecule is preferably any integer value between 1 and 6.
  • each functional oligonucleotide molecule is covalently covalently
  • the number of coupled small molecule drugs is preferably any integer value between 1 and 20.
  • the chemical structural formula of the small molecule drug-oligonucleotide conjugate is as shown in Formula 1 to Formula 14. Show:
  • L and T are each independently absent, and any one or more alkylene groups of -(CH 2 ) h - or -(CH 2 ) h - are replaced by the A group.
  • Substituted group; the h is 0 to 15; the A group includes: -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH -, -CH(R C )-, -C(R')(R”)-, -NH-, -N(R N )-, -SS-, -C(R') C(R”) -, -C ⁇ C-, One or more of them; RC , RN , R', R" in the A group means that any one or more hydrogen atoms on the specified atom are replaced by the B group, provided that it does not exceed the The normal valency of the specified atom and substitution generates a stable compound, the specified atom includes a
  • Q, Y, and Z are each independently absent, -O-, -S-, -C(O)-, -NH-, -CH 2 -, -C(O )NH-, -NHC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and One or more of them; (O) in the Q, Y, Z groups represents a carbonyl oxygen atom; the is the connection site;
  • X means O or S
  • the small molecule drug when the small molecule drug is covalently coupled to the side chain base or phosphate backbone of the 3' end or 5' end extension sequence of the functional oligonucleotide molecule, the small molecule drug-oligonucleotide
  • the chemical structural formulas of glycoside conjugates are shown in Formulas 15 to 20:
  • T is each independently absent, or any one or more alkylene groups in -(CH 2 ) h - or -(CH 2 ) h - are substituted by the A group.
  • Y and Z are each independently absent, O, S, C(O), NH, CH 2 , C(O)NH, NHC(O), C(O)O , OC(O), OC(O)O, OC(O)NH, NHC(O)O and One or more of the above; (O) in the Y and Z groups represents a carbonyl oxygen atom; the is the connection site;
  • G represents an immunomodulatory inhibitor
  • n is independently 1 to 15
  • m and i are independently 0 to 5
  • k and j are independently 1 to 20
  • R represents H
  • B represents a nucleic acid base.
  • the present invention also provides a method for preparing the small molecule drug-oligonucleotide conjugate described in the above scheme, which includes the following steps:
  • Small molecule drugs with immunomodulatory functions are covalently coupled with functional oligonucleotide molecules that can regulate the expression of inflammation-related genes to obtain small molecule drug-oligonucleotide conjugates.
  • the present invention has no special restrictions on the specific method of covalent coupling of the small molecule drug and the functional oligonucleotide molecule, and conventional coupling methods in this field can be used.
  • the reaction type involved in the covalent coupling preferably includes one or more of esterification, amidation, click chemical reaction, electrophilic substitution, nucleophilic substitution or Diels-Alder reaction. .
  • the present invention also provides the use of the small molecule drug-oligonucleotide conjugate described in the above scheme in the preparation of drugs for treating inflammation-related diseases.
  • the inflammation-related diseases include psoriasis, dry eye, Sjögren's syndrome, uveitis, keratitis, conjunctivitis, atopic dermatitis, rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease.
  • psoriasis dry eye
  • Sjögren's syndrome uveitis
  • keratitis conjunctivitis
  • atopic dermatitis atopic dermatitis
  • rheumatoid arthritis inflammatory bowel disease
  • Crohn's disease Crohn's disease.
  • Rohn's diseases include psoriasis, dry eye, Sjögren's syndrome, uveitis, keratitis, conjunctivitis, atopic dermatitis, rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease.
  • Rohn's diseases One or more of Rohn's diseases.
  • the small molecule drug-oligonucleotide conjugates can further assemble into micelles, thereby promoting Cellular uptake of small molecule drug-oligonucleotide conjugates overcomes the difficulty of nucleic acid drugs entering cells.
  • the small molecule drug-oligonucleotide conjugate of the present invention contains both small molecule drugs and functional oligonucleotide molecules that regulate immune responses. The combined use of the two can act on different inflammation-related signaling pathways at the same time, producing a synergistic effect. Solve the problem of poor efficacy of small molecule immunomodulatory drugs or oligonucleotide drugs alone, thereby achieving better therapeutic effects.
  • the present invention constructs an antisense oligonucleotide (ASO NFKBIZ ) consisting of a single cyclosporine A (CsA) molecule and targeting the nuclear factor kappa B inhibitory factor ⁇ (NFKBIZ) gene.
  • ASO NFKBIZ antisense oligonucleotide
  • CsA-NFKBIZ antisense oligonucleotide conjugate formed by covalent coupling.
  • the CsA-NFKBIZ antisense oligonucleotide conjugate can be used to prepare drugs for treating dry eye disease.
  • the present invention constructs a small molecule drug-antisense oligonucleotide conjugate (ASO NFKBIZ ) formed by covalent coupling of two CsA molecules and NFKBIZ antisense oligonucleotide (ASO NFKBIZ).
  • ASO NFKBIZ small molecule drug-antisense oligonucleotide conjugate
  • 2CsA-ASO NFKBIZ utilize its amphiphilicity to form a nanomicelle assembly structure through self-assembly for co-delivery of CsA and NFKBIZ antisense oligonucleotides
  • the 2CsA-ASO NFKBIZ can be used to prepare drugs for the treatment of dry eye disease .
  • the 2CsA-ASO NFKBIZ covalent conjugate formed after the coupling of two CsA small molecules exhibits amphiphilicity due to the increased number of hydrophobic CsA and can be dissolved in the solution.
  • Assembled into a nanomicelle structure the center of the nanomicelle is a hydrophobic CsA molecule and the outer shell is a water-soluble antisense oligonucleotide molecule.
  • the assembled nanomicelles can efficiently transfect cells without any transfection reagents, achieving efficient co-delivery of CsA and antisense oligonucleotides, thereby achieving a good synergistic therapeutic effect on dry eye.
  • the present invention uses small molecule drugs and nucleic acid drugs to act on different targets of the inflammatory signaling pathway at the same time to collaboratively reduce the level of cellular inflammation; it uses the hydrophilic characteristics of nucleic acids to give the immunomodulatory small molecule drugs better water solubility, and at the same time uses the hydrophobicity of CsA to enhance even...
  • the present invention constructs an antisense oligonucleotide (ASO NFKBIZ ) that targets the NFKBIZ gene by grafting multiple cyclosporine A molecules through phosphorothioate graft modification to construct a drug-antisense oligo.
  • the present invention constructs a small molecule drug prepared by covalently coupling three CsA molecules with siRNA NLRP3 targeting NLRP3 , siRNA JAK1 targeting JAK1 , and siRNA PDE4 targeting PDE4.
  • siRNA NLRP3 targeting NLRP3
  • siRNA JAK1 targeting JAK1
  • siRNA PDE4 targeting PDE4.
  • -Nucleic acid conjugates and their self-assembled nanomicelles for synergistic treatment of dry eye disease.
  • siRNA targeting key genes related to intracellular inflammation and cyclosporin A molecules was prepared, and its amphiphilicity was used to assemble into nanomicelles for the collaborative treatment of dry eye disease.
  • This example selects different inflammatory gene targets in cells to further verify the multi-target feasibility of this structure in treating dry eye disease.
  • the conjugate has strong amphiphilicity and can self-assemble in an aqueous solution to form a stable nanomicelle structure without a carrier.
  • the prepared spherical micellar nucleic acid nanoparticles The structure can be efficiently taken up by cells to achieve simultaneous co-delivery of CsA and ASO; cyclosporine A, as an immunomodulator and anti-inflammatory drug, can inhibit the production and release of pro-inflammatory cytokines and increase the levels of anti-inflammatory cytokines. Release level; at the same time, ASO can knock down the expression of the inflammation-related gene NFKBIZ protein and further inhibit the inflammatory response of psoriasis. Through coordinated regulation, it inhibits the inflammatory response and regulates immune balance to achieve better psoriasis treatment effects.
  • the present invention constructs a small molecule drug-antisense oligonucleotide prepared by covalent coupling of 10 calcipotriol molecules and an antisense oligonucleotide (ASO) targeting the NFKBIZ gene.
  • ASO antisense oligonucleotide
  • the glycolic acid conjugate (10Cal-ASO NFKBIZ ) and the nanomicelle formed by its assembly are used for the synergistic treatment of psoriasis.
  • the calcipotriol-antisense oligonucleotide conjugate constructed by grafting multiple calcipotriol molecules at the end of the ASO sequence of the present invention has a higher drug loading capacity.
  • the 13 C NMR spectrum of chloromethyl carbonate cyclosporine A is shown in Figure 3.
  • the solvent used in the nuclear magnetic spectrum test is CDCl 3.
  • the characteristic carbon contained in the product is The attribution is as follows: 173.7, 173.4, 173.1, 172.8, 171.6, 171.2, 170.9, 170.8, 170.0, 169.9, 167.6, 153.5.
  • the theoretical molecular weight of chloromethyl carbonate cyclosporine A is 1293.80, and the actual molecular weight measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) is 1294.80, which belongs to the molecular ion of chloromethyl carbonate cyclosporine A. Peak [M+1] + , as shown in Figure 4.
  • the antisense oligonucleotides targeting the NFKBIZ gene used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • dibenzocyclooctyne dibenzocyclooctyne (DBCO) modification enables efficient coupling with the azide group modified on CsA-N 3 through a click reaction.
  • DBCO dibenzocyclooctyne
  • the sequence of the antisense oligonucleotide (DBCO-ASO NFKBIZ-h ) targeting the human NFKBIZ gene is
  • the sequence of the antisense oligonucleotide (DBCO-ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is
  • CsA-ASO NFKBIZ covalent conjugate
  • HCEC Human corneal epithelial cells
  • HCEC cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well and cultured until they adhered, and then 100ng/mL lipopolysaccharide was used to (Lipopolysaccharides, LPS) was stimulated overnight. After removing the supernatant, 1 mL of Opti-MEM medium containing ASO NFKBIZ-h , ASO NFKBIZ-h + Lipofectamine2000 (Lipo2000), and CsA-ASO NFKBIZ-h (all containing 10 ⁇ M ASO), and the cells without any treatment were used as blank reference (Mock).
  • RT-qPCR real-time fluorescence quantitative PCR
  • the inhibitory effect is still about 1.25 times that of normal cells; after using Lipo2000 to transfect ASO NFKBIZ-h, its expression level is reduced to the expression level of normal cells, and after co-incubation with CsA-ASO NFKBIZ-h, the expression level of NFKBIZ mRNA is reduced to About 0.8 times that of normal cells. From this result, it can be seen that the knockdown ability of CsA-ASO NFKBIZ-h conjugate on NFKBIZ mRNA is better than that of unmodified ASO NFKBIZ-h .
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks.
  • hospital-prepared cyclosporine A eye drops 400 ⁇ M CsA, referred to as hospital-prepared CsA eye drops or CsA eye drops, provided by the Eye, Ear, Nose and Throat Specialist Hospital affiliated to Fudan University School of Medicine in Shanghai
  • CsA-ASO were used respectively.
  • NFKBIZ-m 400 ⁇ M CsA
  • fluorescein sodium staining evaluation and phenol red cotton thread tear secretion test were performed on the 0th, 7th, and 14th days of treatment to evaluate the treatment effect.
  • the experimental results are shown in Figure 10.
  • the results of sodium fluorescein staining showed that the eye scores of the mice in the hospital-prepared CsA eye drops treatment group declined on the 7th day. After the 14th day of treatment, the scores dropped from slightly more than 6 points to about 5 points.
  • the results of the phenol red cotton thread tear test increased from 1.5mm to about 3mm; the fluorescein sodium score in the eyes of mice in the CsA-ASO NFKBIZ-m treatment group dropped from the initial 5.5 to about 3 after 14 days, and the infiltration length of the phenol red cotton thread increased from 1.8mm to 3.2mm. about.
  • the antisense oligonucleotides targeting the NFKBIZ gene used in this example were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We introduced two dibenzo rings at the 3' end of the antisense oligonucleotides.
  • Octyne (dibenzocyclooctyne, DBCO) modification enables efficient coupling with the azide group modified on CsA-N 3 through a click reaction.
  • One of the DBCO modifications is the same as in Example 1 and is located at the 3' end of the nucleic acid sequence;
  • the second DBCO modification is close to the 3' end and is a modification of the T base in the middle of the nucleic acid sequence.
  • the T base containing the middle modification DBCO is represented by the /iDBCOdT/ symbol, and its structure is as follows:
  • nucleic acid sequences used in this example are as follows:
  • the sequence of the antisense oligonucleotide (2DBCO-ASO NFKBIZ-h ) targeting the human NFKBIZ gene is:
  • the sequence of the antisense oligonucleotide (2DBCO-ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is:
  • the synthesis method of 2CsA-ASO NFKBIZ conjugate is as follows: dissolve 0.4 mg of CsA-N 3 (260 nmol) prepared in Example 1 in 0.13 mL of dimethyl sulfoxide (DMSO), and add 5OD (Optical Density) amount of 2DBCO -ASO NFKBIZ (26nmol), then placed at 50°C for 24h shaking reaction. After adding 5 mL of water, extract with ethyl acetate to remove excess CsA-N 3 in the reaction. After concentration and evaporation to dryness, the 2CsA-ASO NFKBIZ conjugate molecule is obtained.
  • DMSO dimethyl sulfoxide
  • HCEC cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well and cultured until they adhered, and then stimulated with 100ng/mL LPS overnight. After removing the supernatant, ASO NFKBIZ-h +Lipo2000 complex and 2CsA-ASO NFKBIZ-h nanomicelles were mixed with 1 mL of Opti-MEM medium (both containing 10 ⁇ M ASO). After co-incubation and transfection at 37°C for 6 hours, the DMEM medium was replaced and the incubation continued for 48 hours. The cells were collected and RNA was extracted for RT-qPCR detection to analyze the target gene NFKBIZ mRNA expression level in HCEC cells. The experimental results are shown in Figure 12.
  • the NFKBIZ mRNA expression of HCEC cells stimulated by LPS increased to about 1.4 times that of normal cells.
  • ASO NFKBIZ-h after Lipo2000 transfection can downregulate it to normal cell levels.
  • 2CsA-ASO NFKBIZ- h After co-incubation with nanomicelles, the expression of NFKBIZ mRNA in LPS-stimulated HCEC cells decreased to about 0.8 times that of normal cells.
  • 2CsA-ASO NFKBIZ-h nanomicelles have a stronger ability to knock down the expression of NFKBIZ gene.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.). 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks. After successful modeling, the hospital-prepared cyclosporine A eye drops (400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Hospital affiliated to Fudan University School of Medicine, Shanghai) and 2CsA-ASO NFKBIZ-m (400 ⁇ M CsA) were used for eye drops. For treatment, drop 5 ⁇ L into each eye once a day in the morning and evening. At the same time, fluorescein sodium staining evaluation and phenol red cotton thread tear secretion test were performed on the 0th, 7th, and 14th days of treatment to evaluate the treatment effect. The experimental results are shown in Figure 13.
  • the formed spherical nucleic acid nanomicelles have better ability to enter cells.
  • small molecule drugs and nucleic acid drugs can achieve synergistic treatment, thereby better inhibiting inflammation of ocular tissue in mice and achieving the purpose of dry eye treatment.
  • the antisense oligonucleotides targeting the NFKBIZ gene used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We introduced three dibenzo rings at the 3' end of the antisense oligonucleotides.
  • Octyne (dibenzocyclooctyne, DBCO) modification enables efficient coupling with the azide group modified on CsA-N 3 through a click reaction.
  • One of the DBCO modifications is located at the 3' end of the nucleic acid sequence; in addition Both are DBCO modifications on the T base in the middle of the nucleic acid sequence, close to the 3' end. Their sequences are as follows:
  • sequence of antisense oligonucleotide (3DBCO-ASO NFKBIZ-h ) targeting the human NFKBIZ gene is:
  • the antisense oligonucleotide (3DBCO-ASO NFKBIZ-m ) sequence targeting the mouse NFKBIZ gene is:
  • the synthesis method of 3CsA-ASO NFKBIZ conjugate is as follows: dissolve 0.6 mg CsA-N 3 (390 nmol) in 0.13 mL DMSO, add 5OD DNA (26 nmol), and place it at 50°C for a shaking reaction for 24 hours. After adding 5 mL of water, extract with ethyl acetate to remove excess CsA-N 3 in the reaction. After concentration and evaporation to dryness, the 3CsA-ASO NFKBIZ conjugate molecule is obtained. Then it is redissolved with 50 ⁇ L of DMSO solution, and the resulting solution is added dropwise.
  • HCEC cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well and incubated until adherent. Stimulated with 100ng/mL LPS overnight, the supernatant was removed, and ASO NFKBIZ-h +Lipo2000 complex and 3CsA were added. -1 mL of Opti-MEM medium for ASO NFKBIZ-h nanomicelles (both containing 10 ⁇ M ASO), incubated at 37°C for 6 hours after transfection, then replaced with DMEM medium and continued incubation for 48 hours, and then collected cells and extracted RNA for RT-qPCR Tested to evaluate the expression of NFKBIZ mRNA in HCEC cells, the experimental results are shown in Figure 15.
  • the NFKBIZ mRNA expression of HCEC cells stimulated by LPS increased to about 1.4 times that of normal cells.
  • ASO NFKBIZ-h after Lipo2000 transfection can downregulate it to normal cell levels.
  • 3CsA-ASO NFKBIZ- h nanomicelles down-regulate the NFKBIZ gene to about 0.75 times that of normal cells. Therefore, compared with single ASO, 3CsA-ASO NFKBIZ-h nanomicelles have a better ability to down-regulate the expression of the NFKBIZ gene.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks.
  • hospital-prepared cyclosporine A eye drops 400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Hospital affiliated to Fudan University School of Medicine in Shanghai
  • 3CsA-ASO NFKBIZ-m nanomicelles 133 ⁇ M ASO concentration, 400 ⁇ M CsA
  • Cyclosporin A trimer (CsA 3 ) modified NFKBIZ antisense oligonucleotide covalent conjugate (CsA 3 -ASO NFKBIZ ) and its nanomicelle assembly structure for the treatment of dry eye disease
  • the antisense oligonucleotides targeting the NFKBIZ gene used in this example were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and we introduced N-hydroxysuccinimide at the 3' end of the antisense oligonucleotides.
  • Amine ester (N-Hydroxysuccinimide ester, NHS ester) functional group enables coupling reaction with aminotris[(2-propynyloxy)methyl]methane to obtain an antisense with three alkynyl groups at the 3' end.
  • the oligonucleotide is then efficiently coupled to the azide group modified on CsA-N 3 through a click reaction.
  • the 3' end NHS group modification is represented by -NHS-3' in all the following examples. Its modification The structure is as follows:
  • nucleic acid sequences used in this example are as follows:
  • the antisense oligonucleotide (NHS-ASO NFKBIZ-h ) sequence targeting the human NFKBIZ gene is:
  • the sequence of the antisense oligonucleotide (NHS-ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is:
  • CsA 3 -ASO NFKBIZ conjugate (10OD ASO) was dissolved in 100 ⁇ L DMSO and added dropwise to 300 ⁇ L of continuously stirred 1 ⁇ PBS buffer solution. The solution was then placed in a dialysis bag and dialyzed overnight to remove DMSO. After centrifugation at 3000 rpm for 5 min, CsA 3 -ASO NFKBIZ micellar nanoparticles can be obtained. Its diameter and morphology were characterized by DLS and TEM, as shown in Figure 22.
  • HCEC cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well and incubated until they adhered. They were stimulated with 100ng/mL LPS overnight. ASO NFKBIZ-h +Lipo2000 complex and CsA 3 -ASO NFKBIZ- were added respectively.
  • h nanomicelle Opti-MEM medium both containing 10 ⁇ M ASO, incubated at 37°C for 6 hours after transfection, then replaced the DMEM medium and continued incubation for 48 hours. The cells were then collected and RNA was extracted for RT-qPCR detection to evaluate the expression of NFKBIZ mRNA in HCEC cells. The experimental results are shown in Figure 23.
  • the expression level of NFKBIZ mRNA in HCEC cells stimulated by LPS is about 1.5 times that of normal cells.
  • the content of NFKBIZ mRNA is reduced to the expression level of normal cells.
  • Adding CsA 3 -ASO NFKBIZ After -h nanomicelles the expression of NFKBIZ mRNA dropped to about 0.5 times that of normal cells. It can be seen from the results that CsA 3 -ASO NFKBIZ-h nanomicelles have a significant ability to down-regulate NFKBIZ mRNA.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.). 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks. After successful modeling, the hospital-prepared cyclosporine A eye drops (400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Hospital affiliated to Fudan University School of Medicine, Shanghai) and CsA 3 -ASO NFKBIZ-m nanomicelles (400 ⁇ M CsA) were used for eye testing.
  • the small interfering RNA targeting the NFKBIZ gene used in this example was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • n 1, 2, or 3
  • DBCO dibenzocyclooctyne
  • siRNA sequence that modifies 1, 2, and 3 DBCOs and targets the human NFKBIZ gene is as follows:
  • Antisense 5’-rArUrCrArGrArCrArArCrGrArArUrCrGrGrCdTdT-3’ (SEQ ID NO.14).
  • mouse NFKBIZ siRNA modified with 1, 2, and 3 DBCOs is as follows:
  • Antisense 5 -rArArUrArCrUrGrGrUrArCrArUrUrGrArCrGrCdCdT-3’(SEQ ID NO.16)
  • Antisense 5 -rArArUrArCrUrGrGrUrArCrArUrUrGrArCrGrCdCdT-3’(SEQ ID NO.18)
  • cyclosporine A and siRNA conjugates are as follows: Dissolve 0.2mg, 0.4mg, 0.6mg CsA-N 3 (140nmol, 280nmol, 420nmol) in 0.14mL DMSO respectively, and add 5OD DBCO-siRNA NFKBIZ Sense chain respectively. , 2DBCO-siRNA NFKBIZ Sense chain, 3DBCO-siRNA NFKBIZ Sense chain (28 nmol each), and then placed at 37°C for shaking reaction for 24 hours. After adding 5 mL of water, extract with ethyl acetate to remove excess CsA-N 3 in the reaction.
  • CsA-siRNA NFKBIZ Sense chain After concentration and evaporation to dryness, CsA-siRNA NFKBIZ Sense chain, 2CsA-siRNA NFKBIZ Sense chain, and 3CsA-siRNA NFKBIZ Sense chain conjugates are obtained. molecular. Mix the prepared CsA-siRNA NFKBIZ Sense strand with equal amounts of siRNA NFKBIZ Antisense strand in 1 ⁇ PBS. The Sense strand and Antisense strand are complementary to form a soluble CsA-siRNA NFKBIZ conjugate double-stranded molecule.
  • nCsA-siRNA NFKBIZ-h nanomicelles are designed to reduce NFKBIZ mRNA levels in cells, thereby further reducing expression at the protein level.
  • western blot experiments were performed for characterization. HCEC cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well and adhered overnight. They were then stimulated with 100ng/mL LPS overnight.
  • siRNA NFKBIZ-h +Lipo2000 complex and 1 mL of Opti-MEM medium for nCsA-siRNA NFKBIZ-h (n 1, 2, 3) conjugates and nanomicelles (both containing 10 ⁇ M siRNA), incubated at 37°C for 6 hours after transfection, then changed to DMEM medium , continue to incubate for 48 hours, extract the proteins in the cells, and perform western blot experiments.
  • CsA-siRNA NFKBIZ-h has a significant reduction effect on the expression of NFKBIZ gene, which can be reduced to the level before LPS stimulation; at the same time, LPS
  • the expression of NFKBIZ in stimulated HCEC cells increased to about 1.5 times that of normal cells, and 1, 2 and 3 CsA siRNA NFKBIZ-h were grafted respectively.
  • the reducing effect increased, respectively reducing the expression of NFKBIZ in cells to about 0.5 to 0.7 times that of normal cells. This may be because the hydrophobic effect of CsA enhances the ability of siRNA NFKBIZ-h to enter cells, and the synergy between the two has better inflammation treatment capabilities.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks.
  • hospital-prepared cyclosporine A eye drops 400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Specialist Hospital affiliated to Fudan University School of Medicine, Shanghai
  • CsA were used respectively.
  • the results of fluorescein sodium staining showed that the eye score of mice after treatment with cyclosporine A eye drops dropped from 6 to about 4.5, and the phenol red cotton thread tear test results increased from 1.5mm to about 2.5mm; CsA-siRNA After NFKBIZ treatment, the eye score of mice dropped from 6 points to about 3 points, and the phenol red cotton thread tear test results increased from 2mm to about 2.5mm; after 2CsA-siRNA NFKBIZ nanomicelle treatment, the eye score of mice dropped from 6 points to about 3 points.
  • the phenol red cotton thread tear test result increased from 1.5 mm to about 2.5 mm; the mouse eye score after 3CsA-siRNA NFKBIZ nanomicelle treatment dropped from 6 points to about 2 points, and the phenol red cotton thread tear test result increased from 1.5 to 2.5 mm. mm increased to about 2.5mm; it can be seen from the animal experiment results that compared with the case group and the simple CsA eye drops group, CsA-siRNA NFKBIZ-m conjugate, 2CsA-siRNA NFKBIZ-m nanomicelles, The therapeutic effect of 3CsA-siRNA NFKBIZ-m nanomicelles on dry eyes in mice is more obvious. First, due to the synergistic treatment of small molecule drugs and nucleic acid drugs, it can better achieve the purpose of inhibiting inflammation of mouse eye cells. Second, The hydrophobic CsA gives siRNA better ability to enter cells.
  • Example 6 Grafting multiple cyclosporine A molecules with NFKBIZ antisense oligonucleotide conjugates and its self-assembled nanomicelles for the treatment of dry eye disease
  • Carbonyl ethyl bromide cyclosporin A has an isotopic ion peak due to the bromine element, and its theoretical m/z values are 1437.8098 and 1439.8077.
  • the m/z values measured by high performance liquid chromatography/quadrupole flight mass spectrometry are 1438.4549 and 1440.4506, which belong to the [M+H]+ peak of carbonyl ethyl bromide cyclosporin A, confirming that the target product was successfully synthesized. , as shown in Figure 30.
  • the antisense oligonucleotides targeting the NFKBIZ gene used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We chose to perform thio (PS) modification on the phosphate backbone of the antisense nucleotides. It can undergo a substitution reaction with the bromocarbonyl ethyl group modified on CsA-Br for efficient coupling. Its sequence is as follows:
  • the antisense oligonucleotide (20PS-ASO NFKBIZ-h ) sequence targeting the human NFKBIZ gene is:
  • * represents the phosphorothioate group modification site, indicating that the two nucleotide units before and after are connected through the phosphorothioate group.
  • the phosphorothioate modification in the nucleic acid sequence is represented by *
  • the underlined TTTTT is a plurality of T base spacer sequences connecting the 5' end ASO and the 3' end for drug modification sequences.
  • the underlined parts of the sequences listed in the following examples represent the connection between the functional small nucleic acid sequence and the drug modification sequence. The spacer sequence will not be repeated one by one) (SEQ ID NO. 21);
  • the sequence of the antisense oligonucleotide (20PS-ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is:
  • the synthesis method is: dissolve 7.5 mg CsA-Br (5 ⁇ mol) in 0.063 mL DMSO, add 5OD20PS-ASO NFKBIZ (12.5 nmol), and place it at 50°C for a shaking reaction for 24 hours. After adding 5 mL of water, extract with ethyl acetate to remove excess CsA-Br in the reaction, and then concentrate and evaporate to dryness to obtain 20CsA-ASO NFKBIZ conjugate molecules. Dissolve 20CsA-ASO NFKBIZ conjugate in 50 ⁇ L DMSO, add dropwise to 0.5 mL phosphate buffer saline (PBS), and stir for 30 min.
  • PBS phosphate buffer saline
  • HCEC cells were grafted into a 6-well plate at a density of 1 ⁇ 10 5 cells/well, then stimulated with 100ng/mL LPS for 12h, the supernatant was removed, and cells containing ASO NFKBIZ-h + Lipofectamine2000 (Lipo2000) and 20CsA were added.
  • Opti-MEM medium for ASO NFKBIZ-h nanomicelles all containing 10 ⁇ M ASO
  • incubate at 37°C for 6 hours after transfection, then change to DMEM medium, replace with new medium and continue to incubate for 48 hours, and then collect the cells RNA was extracted and RT-qPCR experiments were performed to examine the expression level of NFKBIZ mRNA in HCEC cells.
  • the experimental results are shown in Figure 32.
  • NFKBIZ mRNA in HCEC cells stimulated by LPS increased to about 1.5 times that of normal cells.
  • ASO NFKBIZ-h transfected with Lipo2000 can down-regulate it to the level of normal cells.
  • 20CsA-ASO NFKBIZ-h nanomicelles can down-regulate the NFKBIZ gene to about 0.75 times the expression level of normal cells. Therefore, compared with a single ASO NFKBIZ-h and 20CsA-ASO NFKBIZ-h nanomicelles have better ability to down-regulate the expression of NFKBIZ gene.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks.
  • the hospital-prepared cyclosporine A eye drops 400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Hospital affiliated to Fudan University School of Medicine, Shanghai
  • 20CsA-ASO NFKBIZ-m nanomicelles (20 ⁇ M, 400 ⁇ M CsA) were used respectively.
  • Cyclosporin A molecules are covalently conjugated with small interfering nucleic acids targeting IL-17, IL-1 ⁇ , IL-23 and TNF- ⁇ and their self-assembled nanomicelles for the treatment of dry eye disease. treat
  • the small interfering nucleic acids targeting different inflammatory factor genes used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • DBCO dibenzocyclooctyne
  • siRNA sequences targeting different human inflammatory factor genes are:
  • siRNA sequences targeting different inflammatory factor genes in mice are:
  • Antisense 5 -rUrUrCrArGrGrArCrCrArGrGrArUrCrUrCrUrUdGdC-3’(SEQ ID NO.32)
  • the synthesis method of 3CsA-siRNA conjugates for different targets is as follows: dissolve 30 times the molar equivalent of CsA-N 3 in DMSO, add 5OD of 3DBCO-siRNA Sense chains of different targets, and make the reaction concentration 200 ⁇ M. Then place it at 37°C for shaking reaction for 24h. After adding water, extract with ethyl acetate to remove excess CsA-N 3 in the reaction. After concentration and evaporation to dryness, 3CsA-siRNA IL-17 Sense chain, 3CsA-siRNA IL-1 ⁇ Sense chain, and 3CsA-siRNA IL-23 are obtained.
  • Sense chain and 3CsA-siRNA TNF- ⁇ Sense chain conjugate molecules Redissolve it with 50 ⁇ L DMSO solution and drop it into 500 ⁇ L PBS for assembly to form 3CsA-siRNA IL-17 Sense chain nanomicelles, 3CsA-siRNA IL-1 ⁇ Sense chain nanomicelles, and 3CsA-siRNA IL-23 Sense chain nanomicelles. micelles and 3CsA-siRNA TNF- ⁇ Sense chain nanomicelles, and then dialyzed in PBS to remove DMSO.
  • 3CsA-siRNA IL-17 nanomicelles 3CsA-siRNA IL-1 ⁇ nanomicelles, 3CsA-siRNA IL-23 nanomicelles and 3CsA-siRNA TNF- ⁇ nanomicelles dispersed in PBS solution were obtained. Micellar samples.
  • HCEC cells with a density of 1 ⁇ 10 5 cells/well were placed in a 6-well plate to adhere to the wall, and then stimulated with 100ng/mL LPS for 12h. After removing the supernatant, 1mL of IL-17-h nanogel containing 3CsA-siRNA was added.
  • Opti-MEM medium of bundles 3CsA-siRNA IL-1 ⁇ -h nanomicelles, 3CsA-siRNA IL-23-h nanomicelles and 3CsA-siRNA TNF- ⁇ -h nanomicelles (all containing 10 ⁇ M siRNA),
  • the control group was 1 mL of Opti-MEM medium containing different target siRNA and Lipo2000 complexes (siRNA concentrations were all 10 ⁇ M). After co-incubation and transfection for 6 hours at 37°C, it was changed to DMEM medium. After continuing to incubate for 48 hours, the cells were collected and RNA was extracted and PCR experiments were performed to examine the mRNA expression levels of different targets in HCEC cells. The experimental results are shown in Figure 34.
  • HCEC cells were seeded in a 12-well plate at a density of 5 ⁇ 10 4 cells/well, and then stimulated with 100ng/mL LPS for 12h. After removing the supernatant, 1mL of IL-17-h nanogel containing 3CsA-siRNA was added.
  • 3CsA-siRNA IL-1 ⁇ -h nanomicelles 3CsA-siRNA IL-1 ⁇ -h nanomicelles
  • Opti-MEM medium of 3CsA-siRNA IL-23-h nanomicelles and 3CsA-siRNA TNF- ⁇ -h nanomicelles both containing 10 ⁇ M siRNA
  • the control group is 1 mL containing different target siRNA and Lipo2000 complexes
  • Opti-MEM medium siRNA concentration is 10 ⁇ M
  • incubate at 37°C for 6 hours after transfection then change to DMEM medium, continue to incubate for 48 hours, collect the supernatant for ELISA to detect the expression of different inflammatory factors in HCEC cells, experiment The results are shown in Figure 35.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop it once a day in the morning and evening for two weeks.
  • mice in the untreated control group dropped from 13 to about 10
  • the phenol red cotton thread tear test results increased from 1mm to about 1.3mm, with no significant improvement
  • the mice treated with CsA eye drops The eye score dropped from 12 points to about 9 points, and the phenol red cotton thread tear test results increased from 1.5mm to about 3mm
  • the fluorescein sodium staining score of mice in the siRNA nanomicelle treatment groups with different targets dropped from about 13 points to about 9 points.
  • the infiltration length of the phenol red cotton thread increased from 1.5mm to about 4mm. It can be seen from this part of the data that 3CsA-siRNA conjugate nanomicelles targeting different inflammatory factor targets have certain efficacy in the treatment of dry eye in mice within two weeks.
  • Cyclosporine A molecules are covalently conjugated with small interfering nucleic acids targeting NLRP3, JAK1 and PDE4 and their self-assembled nanomicelles for the treatment of dry eye disease.
  • RNAs targeting different inflammatory genes in cells used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • DBCO dibenzocyclooctyne
  • siRNA sequences targeting different inflammation-related genes in humans are:
  • Antisense 5 -rUrUrGrArUrUrUrUrCrArGrArCrCrGrArCrUrCdTdT-3’(SEQ ID NO.44)
  • siRNA sequences targeting different inflammation-related genes in mice are:
  • the synthesis method of 3CsA-siRNA conjugates for different targets is as follows: dissolve 30 times the molar equivalent of CsA-N 3 in DMSO, add 5OD of 3DBCO-siRNA Sense chains of different targets, and make the reaction concentration 200 ⁇ M. Then place it at 37°C for shaking reaction for 24h. After adding water, extract with ethyl acetate to remove excess CsA-N 3 in the reaction. After concentration and evaporation to dryness, 3CsA-siRNA NLRP3 Sense chain, 3CsA-siRNA JAK1 Sense chain and 3CsA-siRNA PDE4 Sense chain conjugate molecules are obtained. .
  • HCEC cells with a density of 1 ⁇ 10 5 cells/well were placed in a 6-well plate to adhere to the wall, and then stimulated with 100ng/mL LPS for 12h. After removing the supernatant, 1mL of NLRP3-h nanomicelles containing 3CsA-siRNA, and Opti-MEM culture medium of 3CsA-siRNA JAK1-h nanomicelles and 3CsA-siRNA PDE4-h nanomicelles (both containing 10 ⁇ M siRNA). The control group was 1 mL Opti-MEM culture containing different target siRNA and Lipo2000 complexes.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop it once in the morning and evening for two consecutive weeks. After the modeling is successful, use it separately.
  • Example 9 Tricyclosporin A antisense oligonucleotide conjugate nanomicelles targeting different targets of IL-17, IL-1 ⁇ , IL-23 and TNF- ⁇ for the treatment of dry eye disease
  • the antisense oligonucleotides targeting different inflammatory factor genes used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We introduced three diphenyl oligonucleotides at the 3' end of the antisense oligonucleotides.
  • the dibenzocyclooctyne (DBCO) modification enables efficient coupling with the azide group modified on CsA-N 3 through a click reaction.
  • the detailed sequence information of the ASO used in this example is as follows:
  • antisense oligonucleotide sequences targeting different human inflammatory factor genes are:
  • antisense oligonucleotide sequences targeting different mouse inflammatory factor genes are:
  • the synthesis method of small molecule drugs and ASO conjugates targeting different targets is as follows: dissolve 30 times the molar equivalent of CsA-N 3 in DMSO, add 5OD of 3DBCO-ASO X with different targets, and allow it to react The concentration was 200 ⁇ M, and then placed at 50°C for 24h shaking reaction. After adding water, extract with ethyl acetate to remove excess CsA-N 3 in the reaction, concentrate and evaporate to dryness to obtain 3CsA-ASO IL-17 , 3CsA-ASO IL-1 ⁇ , 3CsA-ASO IL-23 and 3CsA-ASO.
  • the TNF- ⁇ conjugate molecules were then redissolved with 50 ⁇ L DMSO solution and dropped into 500 ⁇ L PBS for assembly to form 3CsA-ASO IL-17 nanomicelles, 3CsA-ASO IL-1 ⁇ nanomicelles, and 3CsA-ASO IL -23 nanomicelles and 3CsA-ASO TNF- ⁇ nanomicelles, and then dialyzed in PBS to remove DMSO to obtain fractions Different nanomicelle samples dispersed in PBS solution.
  • HCEC cells 1 ⁇ 10 5 cells/well HCEC cells were placed in a 6-well plate to adhere to the wall, and then stimulated with 100ng/mL LPS for 12h. The supernatant was removed, and 1mL containing 3CsA-ASO IL-17-h nanomicelles and Opti-MEM medium of 3CsA-ASO IL-1 ⁇ -h nanomicelles, 3CsA-ASO IL-23-h nanomicelles and 3CsA-ASO TNF- ⁇ -h nanomicelles (all containing 10 ⁇ M ASO), control group 1 mL of Opti-MEM medium containing different target ASO and Lipo2000 complexes (ASO concentration is 10 ⁇ M), incubated at 37°C for 6 hours after transfection, changed to DMEM medium, continued to incubate at 37°C for 48 hours, and then collected cells And RNA was extracted for RT-qPCR detection to examine the mRNA expression levels of different targets in HCEC cells. The experimental results are shown in Figure
  • HCEC cells were seeded in a 12-well plate at a density of 5 ⁇ 10 4 cells/well, then stimulated with 100ng/mL LPS for 12h, the supernatant was removed, and 1mL of 3CsA-ASO IL-17-h nanoparticles was added.
  • Opti-MEM medium for micelles 3CsA-ASO IL-1 ⁇ -h nanomicelles, 3CsA-ASO IL-23-h nanomicelles and 3CsA-ASO TNF- ⁇ -h nanomicelles (all containing 10 ⁇ M ASO)
  • the control group was 1 mL Opti-MEM medium containing different target ASO and Lipo2000 complexes (the ASO concentration was 10 ⁇ M), incubated at 37°C for 6 hours after transfection, then changed to DMEM medium, continued to incubate at 37°C for 48 hours, and then The supernatant was collected for ELISA to detect the expression of different inflammatory factors in HCEC cells.
  • the experimental results are shown in Figure 40.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening for two weeks.
  • hospital-prepared cyclosporine A eye drops 400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Hospital affiliated to Fudan University School of Medicine in Shanghai
  • 3CsA-ASO IL-17-m nanomicelles were used respectively.
  • the results of sodium fluorescein staining showed that the eye score of the mice in the untreated control group dropped from 11 points to about 10 points, and the phenol red cotton thread tear test results increased from 1.5mm to about 1.8mm, with no significant improvement; the results after CsA eye drops treatment The mouse eye score dropped from 11 points to about 8 points, and the phenol red cotton thread tear test results increased from 1.5mm to about 3mm; the mouse fluorescein sodium staining score of the 3CsA-ASO nanomicelle material group with different targets ranged from about The score dropped from 11 to about 5.5, and the infiltration length of the phenol red cotton thread increased from 1mm to about 3mm. It can be seen from this part of the data that drug-antisense oligonucleotide conjugate nanomicelles targeting different inflammatory factor targets have significant effects on the treatment of dry eye in mice within two weeks.
  • Example 10 Cyclosporine A-grafted antisense oligonucleotide self-assembled nanomicelles targeting different targets of NLRP3, JAK1 and PDE4 for the treatment of dry eye disease
  • the antisense oligonucleotide sequences used in this example to target different intracellular inflammatory pathway genes were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We chose to perform thio (PS) modification on the nucleic acid phosphate backbone. , enabling it to undergo a substitution reaction with the bromoacetyl bromide group modified on CsA-Br for efficient coupling. Its sequence is as follows:
  • antisense oligonucleotide sequences targeting different human inflammatory factor genes are:
  • antisense oligonucleotide sequences targeting different mouse inflammatory factor genes are:
  • the synthesis method is as follows: dissolve 400 times the molar equivalent of CsA-Br in DMSO, add 5OD of 20PS-ASO with different targets, make the reaction concentration 200 ⁇ M, and then place it at 50°C for a shaking reaction for 24 hours.
  • HCEC cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well, and then stimulated with 100ng/mL LPS for 12h. After removing the supernatant, 1mL of NLRP3-h nanomicelles containing 20CsA-ASO, Opti-MEM culture medium of 20CsA-ASO JAK1-h nanomicelles and 20CsA-ASO PDE4-h nanomicelles (both contain 10 ⁇ M ASO).
  • the control group is 1 mL Opti-MEM culture containing different target ASO and Lipo2000 complexes. base (ASO concentration is 10 ⁇ M), incubate the transfection medium at 37°C for 6 hours and then change to DMEM medium. After continuing to incubate for 48 hours, RNA is extracted for PCR experiments to test the mRNA expression levels of different targets in HCEC cells. The experimental results are shown in Figure 42.
  • 0.2% benzalkonium chloride was used to construct an ocular dry eye model in 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and 5 ⁇ L of 0.2% benzalkonium chloride was dropped into each eye of the mice. , drop once a day in the morning and evening.
  • mice in the untreated control group dropped from 13 points to about 12 points, and the phenol red cotton thread tear test results increased from 0.5mm to about 1mm, with no significant improvement; mice treated with CsA eye drops The eye score dropped from 13 points to about 10 points, and the phenol red cotton thread tear test results increased from 1.5mm to about 2.5mm; the mouse fluorescein sodium staining score of the 20CsA-ASO nanomicelle material group with different targets increased from about 13 The score dropped to about 7.5, and the infiltration length of the phenol red cotton thread increased from 1mm to about 3.5mm. From this part of the data, it can be seen that drug-antisense oligonucleotide conjugate nanomicelles targeting different intracellular inflammatory targets have significant effects on the treatment of dry eye in mice within two weeks.
  • the benzyl bromide-modified thioketal (compound TK-Bz-Br) is purified by silica gel column chromatography. During the chromatographic purification process, the The removing agent was petroleum ether/ethyl acetate mixed solution, and the final yield was 65%. The product was analyzed and detected by nuclear magnetic resonance spectrum. The nuclear magnetic resonance spectrum and the assignment of each peak are shown in Figure 45.
  • nucleotide sequences targeting the NFKBIZ gene used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We chose to carry out sulfate (PS) modification in the nucleic acid phosphate backbone so that it can be combined with the modification in The bromoacetyl bromide group on CsA-Br reacts to achieve efficient coupling. Its sequence is as follows:
  • the sequence of the antisense oligonucleotide (ASO NFKBIZ-h ) targeting the human NFKBIZ gene is
  • the sequence of the antisense oligonucleotide (ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is
  • the synthesis method of 10 rapamycin molecules-NFKBIZ antisense oligonucleotide conjugates (10RAP-ASO NFKBIZ ) is: dissolve 2.2mg compound RAP-Bz-Br (1.63 ⁇ mol) in 100 ⁇ L DMSO, and then add 5OD After ASO (20.55 nmol), place it at 50°C for shaking reaction for 1 hour. Then, after adding water, extract with ethyl acetate to remove excess RAP-Bz-Br in the reaction, and then concentrate and evaporate to dryness to obtain 10RAP-ASO NFKBIZ conjugate molecules. The obtained product was verified by 15% denaturing polyacrylamide gel electrophoresis that the antisense oligonucleotide was successfully grafted with rapamycin, see Figure 47.
  • HCEC Human corneal epithelial cells
  • mice Use 0.2% benzalkonium chloride to construct a dry eye model, select 6-8 week old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.), and add 5 ⁇ L of 0.2% benzalkonium chloride to each eye of the mice. Drop once a day in the morning and evening for two weeks. After successful modeling, different drugs were used for eye drop treatment, and the groups were as follows: untreated group (mock), hospital-prepared cyclosporine A eye drops (400 ⁇ M CsA, provided by the Eye, Ear, Nose and Throat Specialist Hospital affiliated to Fudan University School of Medicine) , and 10RAP-ASO NFKBIZ-m nanomicelles (400 ⁇ M rapamycin).
  • Example 12 Cyclosporine A trimer (CsA 3 ) and NFKBIZ antisense oligonucleotide covalent conjugate (CsA 3 -ASO NFKBIZ ) and its nanomicelle assembly structure for the treatment of psoriasis
  • the CsA 3 -ASO NFKBIZ covalent conjugate used is the same as the conjugate used in Example 5.
  • CsA 3 -ASO NFKBIZ nanomicelles For the preparation and characterization steps of cyclosporine A nanomicelles (CsA 3 -ASO NFKBIZ nanomicelles), please refer to section 5.3 in the Examples.
  • the NFKBIZ mRNA expression of HaCaT cells stimulated by IL-36 increased to about 2.1 times that of normal cells.
  • ASO NFKBIZ-h after Lipo2000 transfection can downregulate it to about 1.2, and CsA 3 -ASO NFKBIZ-h nanomicelles down-regulate the NFKBIZ gene to about 0.8. Therefore, compared to single ASO NFKBIZ-h , CsA 3 -ASO NFKBIZ-h nanomicelles have a better ability to down-regulate the expression of the NFKBIZ gene.
  • ⁇ 10 4 cells/well of human keratinocyte cell line HaCaT cells were placed in a 12-well plate to adhere to the wall, and then stimulated with 100ng/mL recombinant human interleukin-36 (IL-36) for 2 hours, buffered with 1 ⁇ PBS After cleaning the solution, 1 mL of Opti-MEM medium containing ASO NFKBIZ-h + Lipo2000 complex and CsA 3 -ASO NFKBIZ-h nanomicelles (the concentration of ASO NFKBIZ-h in each group of samples is 10 ⁇ M) was added to the well plate. , incubated with HaCaT cells for 6 hours after transfection and then replaced with fresh DMEM medium.
  • IL-36 human interleukin-36
  • the TNF ⁇ , IL-17A and IL-33 levels of HaCaT cells stimulated by IL-36 increased to about 2.1 times, 1.7 times and 2.0 times respectively of unstimulated cells.
  • ASO after Lipo2000 transfection NFKBIZ-h can reduce the levels of TNF ⁇ , IL-17A and IL-33 to about 1.1, 1.0 and 0.9 times, and CsA 3 -ASO NFKBIZ-h nanomicelles can reduce the levels of TNF ⁇ , IL-17A and IL-33 to 0.8- About 0.9 times, therefore compared to single ASO NFKBIZ-h , CsA 3 -ASO NFKBIZ-h nanomicelle has a stronger inhibitory effect on inflammatory factors such as TNF ⁇ , IL-17A and IL-33.
  • 5% imiquimod cream was used to construct psoriasis models on the back and ears of 6- to 8-week-old mice (C57BL/6, Suzhou Xishan Biotechnology Co., Ltd.). After removing the hair on the back of the mice, each The mice were smeared with 60 mg 5% imiquimod cream on their backs and ears for 6 consecutive days. After 6 days of modeling, as shown in Figure 53, psoriasis-like phenotypes such as obvious erythema, scales, and significantly thickened skin appeared on the back and ears of the mice, indicating that the model was successfully constructed. Mix CsA 3 -ASO NFKBIZ-m nanomicelles and carbomer gel, then apply to the skin lesions for treatment.
  • Carbopol gel without the addition of CsA 3 -ASO NFKBIZ-m nanomicelles was used as a control group for efficacy testing.
  • the skin thickness of the back lesions and the thickness of the ears were measured, and the erythema, scale, skin thickening, and psoriasis lesion area severity of the lesions were scored.
  • the experimental results are shown in Figure 54, Figure 55 and Figure 56.
  • the erythema and scales at the skin lesions of mice in the CsA 3 -ASO NFKBIZ-m nanomicelle group were significantly reduced, and the skin thickness at the ears and skin lesions was reduced. This result showed that CsA 3 - ASO NFKBIZ-m nanomicelles can effectively alleviate the psoriasis-like phenotype in diseased mice.
  • Triamcinolone acetonide molecule and NFKBIZ antisense oligonucleotide covalent conjugate (10TA-ASO NFKBIZ ) and its nanomicelle assembly structure are used for synergistic treatment of psoriasis
  • the 13 C NMR spectrum of carbonyl ethyl bromide triamcinolone acetonide is shown in Figure 59.
  • the test solvent is DMSO-d6.
  • the assignment of the characteristic carbons after coupling carbonyl ethyl bromide is as follows: 210.50, 185.68, 167.04, 153.19, 129.78,124.69,110.83,102.06,97.28,81.62,70.66,66.28,48.43,45.14,42.95,35.48,33.47,32.89,30.57,27.95,26.77,25.72,23.26,16.83 .
  • the theoretical molecular weight of carbonylethyl bromide triamcinolone acetonide is 555.43, and the molecular weights measured by high performance liquid chromatography/quadrupole flight mass spectrometry are 557.1391 and 1113.2782, which correspond to the [M+ of carbonylethyl bromide triamcinolone acetonide respectively. 1] + and [2M+1] + peaks, confirming that the target product was successfully synthesized, as shown in Figure 60.
  • nucleotide sequences targeting the NFKBIZ gene used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We chose to carry out sulfate (PS) modification in the nucleic acid phosphate backbone so that it can be combined with the modification in The carbonyl ethyl bromide group on TA-Br reacts to achieve efficient coupling. Its sequence is as follows:
  • the sequence of the antisense oligonucleotide (ASO NFKBIZ-h ) targeting the human NFKBIZ gene is
  • the sequence of the antisense oligonucleotide (ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is
  • the synthesis method of 10TA-ASO NFKBIZ covalent conjugate is as follows: dissolve the carbonyl ethyl bromide triamcinolone acetonide compound in 20 ⁇ L dimethyl sulfoxide solution, and add 2 ⁇ L phosphorothioate group-modified antisense oligonucleotide Solution, the oligonucleotide concentration is 200 ⁇ M (the ratio of phosphorothioate group to carbonyl ethyl bromide triamcinolone acetonide ranges from 1:1 to 1:50), place it at 55°C, and react with shaking overnight.
  • the NFKBIZ mRNA expression of HaCAT cells stimulated by IL-36 increased to about 2.2 times that of normal cells.
  • ASO NFKBIZ-h after Lipo2000 transfection can downregulate it to about 0.85, and 10TA-ASO NFKBIZ-h nanomicelles down-regulate the NFKBIZ gene to about 0.7. Therefore, compared with single ASO NFKBIZ-h , 10TA-ASO NFKBIZ-h nanomicelles have a lower effect on the NFKBIZ gene. Better expression down-regulation ability.
  • Carbopol gel without the addition of 10TA-ASO NFKBIZ-m nanomicelles was used as a control group for efficacy testing.
  • the skin thickness of the back lesions and the thickness of the ears were measured, and the erythema, scale, skin thickening, and psoriasis lesion area severity of the lesions were scored.
  • the experimental results are shown in Figure 64, Figure 65 and Figure 66.
  • mice in the 10TA-ASO NFKBIZ-m nanomicelle group were significantly reduced, and the skin lesions The skin thickness was reduced, indicating that 10TA-ASO NFKBIZ-m nanomicelles can effectively alleviate the psoriasis-like phenotype of diseased mice.
  • Example 14 Calcipotriol molecule and NFKBIZ antisense oligonucleotide covalent conjugate (10Cal-ASO NFKBIZ ) and its nanomicelle assembly structure are used for synergistic treatment of psoriasis
  • nucleotide sequences targeting the NFKBIZ gene used in this example were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd. We chose to carry out sulfate (PS) modification in the nucleic acid phosphate backbone so that it can be combined with the modification in The benzene bromide group on Cal-Br reacts to achieve efficient coupling. Its sequence is as follows:
  • the sequence of the antisense oligonucleotide (ASO NFKBIZ-h ) targeting the human NFKBIZ gene is
  • the sequence of the antisense oligonucleotide (ASO NFKBIZ-m ) targeting the mouse NFKBIZ gene is
  • the synthesis method of calcipotriol-NFKBIZ antisense oligonucleotide conjugate (10Cal-ASO NFKBIZ ) is as follows: dissolve the Cal-Bz-Br compound in 20 ⁇ L dimethyl sulfoxide solution, and add 2 ⁇ L phosphorothioate group Group modified antisense oligonucleotide solution, the oligonucleotide concentration is 200 ⁇ M (the ratio of phosphorothioate group to Cal-Bz-Br compound is 1:5), place it at 55°C, and react with shaking overnight.
  • Calcipotriol-antisense oligonucleotide conjugate The hydrated particle size of self-assembled nanomicelles is approximately 150nm.
  • the transmission electron microscope picture in Figure 73 reveals that the prepared nanomicelles have a spherical morphology.
  • the critical micelle concentration of the 10Cal-ASO NFKBIZ conjugate was determined to be 0.527 ⁇ M. The results are shown in Figure 74.
  • the NFKBIZ mRNA expression of HaCaT cells stimulated by IL-36 increased to about 1.9 times that of normal cells.
  • ASO NFKBIZ-h transfected with Lipo2000 can downregulate it to about 0.85 times, and 10Cal-ASO NFKBIZ-h nanomicelles down-regulate the NFKBIZ gene by about 0.65 times. Therefore, compared to single ASO NFKBIZ-h , 10Cal-ASO NFKBIZ-h nanomicelles have a better ability to down-regulate the expression of the NFKBIZ gene.
  • Carbopol gel without the addition of 10Cal-ASO NFKBIZ-m nanomicelles was used as a control group for efficacy testing.
  • the skin thickness of the back lesions and the thickness of the ears were measured, and the erythema, scale, skin thickening, and psoriasis lesion area severity of the lesions were scored.
  • the experimental results are shown in Figure 76, Figure 77 and Figure 78.

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Abstract

本发明提供了一种小分子药物-寡核苷酸偶联物及其应用，属于生物医药技术领域。本发明针对现有小分子免疫调节药物水溶性差、药物递送困难的问题，采用与寡核苷酸药物进行偶联的方法，一方面寡核苷酸优异的亲水性可改善小分子药物的溶解性质，改善其体内组织分布，促进递送和吸收。同时，疏水性小分子药物反过来也可促进寡核苷酸的入胞，从而提升其对靶标基因的调控能力。其次，本发明通过将小分子药物和功能寡核苷酸分子的共递送实现针对炎症反应不同靶点的协同调控，从而达到更好的疾病治疗效果，实现安全、高效用药。

Description

一种小分子药物-寡核苷酸偶联物及其应用
本申请要求于2022年06月21日提交中国专利局、申请号为“202210703857.4”、发明名称为“一种小分子药物-寡核苷酸偶联物及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药技术领域，具体涉及一种小分子药物-寡核苷酸偶联物及其应用。
背景技术
近年来，关于炎症反应机理的探讨以及炎症与疾病关系的研究呈现爆炸式增长。通过大量的研究，人们对炎症反应机制以及炎症如何影响众多疾病的发生发展也有了更深入的了解。基于所获得的研究结果，目前认为炎症可能是银屑病、干燥综合症、干眼症、葡萄膜炎、角膜炎、结膜炎、特应性皮炎、类风湿性关节炎、炎症性肠炎、克罗恩病、心脏病、糖尿病、癌症、哮喘、炎症性肠病、阿尔茨海默病等多种疾病的主要驱动因素。在常见的炎症反应过程中，在破裂或活化的肥大细胞、循环嗜碱性粒细胞以及血小板释放的组胺分子，血液循环中的血管细胞黏附因子1(VCAM-1)，内皮细胞粘附分子-1(E-selectin)以及细胞间粘附分子-1(ICAM-1)共同作用下会使血管发生舒张，从而导致血管通透性极大地增加(Cardiovasc Hematol Disord Drug Targets 2008,8,252-260)。而血管通透性的增加会导致局部水肿，使得吞噬细胞、补体系统的蛋白质、花生酸、激肽、细胞因子、血小板活化因子等物质进入到血管中(J Allergy Clin Immunol 2010,125,S3-S23)，促进炎症发生发展。其中，细胞因子如肿瘤坏死因子、白细胞介素、淋巴因子、单核因子、干扰素、集落刺激因子以及由单核细胞、T细胞、血小板和内皮细胞产生的转化生长因子均参与炎症相关反应。此外，先天免疫系统的组成部分、单核细胞(包括巨噬细胞和树突状细胞)以及中性粒细胞被激活时炎症反应也会被增强。
先天免疫系统通过跨膜Toll样受体(Toll Like Receptor,TLR)与特定配体的识别，实现对细菌和其他损伤的识别。例如，采用脂多糖(LPS)刺激巨噬细胞上的TLR4会触发炎症因子如TNF-α和IL-1β的合成，并刺激获得性免疫反应所必需的免疫调节细胞因子IL-12的产生。一旦巨噬细胞被炎症因子激活，其寿命将增长并产生大量促炎性细胞因子，包括TNF-a、IL-1、IL-6、IL-12/IL-23、IL-18和高迁移率族蛋白1(HMGB1)。这些细胞因子将进一步放大炎症反应并触发获得性免疫反应。循环细胞因子与不同种类细胞上的特定受体相互作用并激活Janus激酶/信号转导和转录蛋白(JAK-STAT)、核因子kappa B(NF-κB)以及转化生长因子β(TGF-β)信号通路，从而导致细胞粘附、通透性增加、细胞凋亡等炎症反应，以及活性氧的增加。
炎症相关疾病的共同特点是表现为免疫平衡破坏后相关信号通路的激活以及多种炎症细胞因子过量表达，进而激发持续的炎症反应并对机体产生破坏作用。基于对诱发疾病炎症反应机制以及免疫激活过程的了解，研究人员致力于开发不同种类的药物实现炎症调控从而达到最终治愈疾病的目的。现有的炎症调控药物主要包括具有免疫调节功能的小分子药物、靶向参与炎症反应细胞因子和受体的抗体类药物、以及调控炎症信号通路相关基因表达的基因药物。常用具有免疫调节功能的小分子药物如钙调磷酸酶抑制剂、mTOR抑制剂、糖皮质激素、维生素D类似物等，上述小分子药物能够大规模生产，成本较低，一些小分子药物还可以制成口服制剂。但是小分子药物普遍面临水溶性差，生物利用度较低，通常存在较强的副作用，不宜长期使用。抗体类药物通常针对性强，见效快，免疫调控效果优异，但这类药物一般需要注射给药，而且生产和治疗成本高。此外，长期系统给药容易导致耐药性及其他的不良反应，具有较强的毒副作用，如免疫力降低，容易发生感染和癌症。
靶向炎症反应相关基因进而实现免疫调控的功能寡核苷酸分子(siRNA，反义寡核苷酸)作为一类新型药物则具有免疫原性低，作用靶点广泛，既可以靶向胞内靶点也可靶向胞外靶点，不易产生耐药，可调控更上游的信号通路等优点，为免疫调控和疾病治疗提供更多可能。但由于核酸药物分子量大、本身带负电使得单独的核酸药物难以跨越细胞膜屏障，因而面临递送的难题。为了实现核酸药物 的有效递送并发挥基因调控功能，往往需要引入特定载体来帮助功能核酸递送至目标组织和细胞。其中病毒载体虽然转染效率高，但在实际应用过程当中还面临免疫原性、插入突变、制备复杂等一系列问题，阻碍其临床转化。除了病毒载体，非病毒载体如阳离子脂质体、阳离子聚合物等也常用于核酸药物的负载递送，但阳离子递送载体普遍面临着细胞毒性较大、质控困难等问题。虽然每种药物都有其优点，但由于炎症反应的复杂性，采用单一策略调控某一靶点或阻断某种细胞因子可能会导致其他促炎细胞因子的代偿式增加，难以取得令人满意的疗效。
发明内容
有鉴于此，本发明的目的在于提供一种小分子药物-寡核苷酸偶联物及其应用，本发明的小分子药物-寡核苷酸偶联物同时包含调节免疫反应的小分子药物和功能寡核苷酸分子，两者联合使用能够同时作用于不同的炎症相关信号通路，产生协同效应，从而达到更好的治疗效果。
本发明提供了一种小分子药物-寡核苷酸偶联物，由具有免疫调节功能的小分子药物和能够调控炎症相关基因表达的功能寡核苷酸分子共价偶联得到。
优选的，所述小分子药物和功能寡核苷酸分子之间通过化学连接子共价偶联。
优选的，所选小分子药物为作用于免疫相关信号通路并能够调控免疫反应的小分子药物；优选的，所述小分子药物包括钙调磷酸酶抑制剂、糖皮质激素、mTOR抑制剂和维生素D类似物中的一种或几种。
优选的，所述炎症相关基因包括肿瘤坏死因子-a基因、白介素1b基因、白介素17基因、白介素23基因、NFKBIZ基因、炎性小体NLRP3基因、JAK基因和PDE4基因中的一种或几种。
优选的，所述功能寡核苷酸分子包括双链小干扰核酸、微小核酸和单链反义寡核苷酸中的一种。
优选的，当所述功能寡核苷酸分子为双链小干扰核酸或微小核酸时，所述小分子药物共价偶联于功能寡核苷酸分子的正义链的3’端；当所述功能寡核苷酸分子为单链反义寡核苷酸时，所述小分子药物共价偶联于单链反义寡核苷酸的3’端或5’端。
优选的，每个功能寡核苷酸分子上共价偶联的小分子药物的数量为1～40个。
优选的，所述小分子药物共价偶联于功能寡核苷酸分子的末端或者所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上；
优选的，当所述小分子药物共价偶联于功能寡核苷酸分子的末端时，所述的小分子药物-寡核苷酸偶联物的化学结构式如式1～式14所示：


在所述式1～式14中，L、T各自独立地为不存在、-(CH2)h-或-(CH2)h-中的任意一个或多个亚烷基被A基团所取代后的基团；所述h为0～15；所述A基团包括：-O-、-S-、-C(O)-、-C(O)O-、-C(O)NH-、-CH(RC)-、-C(R’)(R”)-、-NH-、-N(RN)-、-S-S-、-C(R’)＝C(R”)-、-C≡C-、中的一种或几种；所述A基团中的RC、RN、R’、R”表示指定原子上的任何一个或多个氢原子被B基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物，所述指定原子包括碳原子或氮原子；所述B基团包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素；所述卤素包括F、C1、Br或I；所述A基团中(O)代表羰基氧原子；
在所述式1～式14中，Q、Y、Z各自独立地为不存在、-O-、-S-、-C(O)-、-NH-、-CH2-、-C(O)NH-、 -NHC(O)-、-C(O)O-、-OC(O)-、-OC(O)O-、-OC(O)NH-、-NHC(O)O-和中的一种或几种；所述Q、Y、Z基团中(O)代表羰基氧原子；所述为连接位点；
在所述式1～式14中，G表示小分子免疫调节药物；m、n、k独立地为1～15；
X表示O或S；
优选的，当所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上时，所述的小分子药物-寡核苷酸偶联物的化学结构式如式15～式20所示：

在所述式15～式20中，T各自独立地为不存在、-(CH2)h-或-(CH2)h-中的任意一个或多个亚烷基被A基团所取代后的基团；所述h为0～15；所述A基团包括：-O-、-S-、-C(O)-、-C(O)O-、-C(O)NH-、-CH(RC)-、-C(R’)(R”)-、-NH-、-N(RN)-、-S-S-、-C(R’)＝C(R”)-、-C≡C-、中的一种或几种；所述A基团中的RC、RN、R’、R”表示指定原子上的任何一个或多个氢原子被B基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物，所述指定原子包括碳原子或氮原子；所述B基团包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素；所述卤素包括F、C1、Br或I；所述A基团中(O)代表羰基氧原子；
在所述式15～式20中，Y、Z各自独立地为不存在、O、S、C(O)、NH、CH2、C(O)NH、NHC(O)、C(O)O、OC(O)、OC(O)O、OC(O)NH、NHC(O)O和中的一种或几种；所述Y、Z基团中(O)代表羰基氧原子；所述为连接位点；
在所述式15～式20中，G表式免疫调节抑制剂；n独立地为1～15；m、i独立地为0～5，k、j独立地为1～20；R表示H；B表示核酸碱基。
本发明还提供了上述方案所述的小分子药物-寡核苷酸偶联物在制备治疗炎症相关疾病的药物中的应用。
优选的，所述炎症相关疾病包括干眼症、银屑病、干燥综合症、葡萄膜炎、角膜炎、结膜炎、特应性皮炎、类风湿性关节炎、炎症性肠炎、和克罗恩病中的一种或几种。
本发明提供了一种小分子药物-寡核苷酸偶联物，由具有免疫调节功能的小分子药物和能够调控炎症相关基因表达的功能寡核苷酸分子共价偶联得到。本发明针对现有小分子免疫调节药物水溶性差、药物递送困难的问题，采用与寡核苷酸药物进行偶联的方法，一方面寡核苷酸优异的亲水性可改善小分子药物的溶解性质，改善其体内组织分布，促进递送和吸收。同时，疏水性小分子药物反过来也可促进寡核苷酸的入胞，从而提升其对靶标基因的调控能力。其次，本发明通过将小分子药物和功能寡核苷酸分子的共递送实现针对炎症反应不同靶点的协同调控，从而达到更好的疾病治疗效果，实现安全、高效用药。此外，两种药物分子相互协助，可实现无需额外载体的新型药递送系统的制备，同时高效递送功能寡核苷酸分子和小分子免疫调节药物，达到协同调控炎症反应的目的，从而为多种 炎症相关疾病的治疗提供更好的解决办法。
附图说明
图1为叠氮修饰环孢素A(CsA-N3)以及单环孢素A-NFKBIZ反义寡核苷酸共价偶联物(CsA-ASONFKBIZ)制备路线示意图；
图2为氯甲基碳酸酯环孢素A(CsA-Cl)的1H NMR谱图；
图3为氯甲基碳酸酯环孢素A(CsA-Cl)的13C NMR谱图；
图4氯甲基碳酸酯环孢素A(CsA-Cl)的MALDI-TOF质谱图；
图5为叠氮己酸碳酸酯修饰的环孢素A(CsA-N3)的1H NMR谱图；
图6为叠氮己酸碳酸酯修饰的环孢素A(CsA-N3)的MALDI-TOF质谱图；
图7为CsA-N3与DBCO修饰ASO偶联产物凝胶电泳图，10％变性聚丙烯酰胺凝胶电泳检测证实CsA-ASONFKBIZ偶联物高效合成；
图8为CsA-ASONFKBIZ-h偶联物的细胞摄取情况；
图9为实时荧光定量PCR检测CsA-ASONFKBIZ-h以及对照样品处理细胞后靶标基因NFKBIZ mRNA表达水平；
图10为CsA-ASONFKBIZ-m纳米胶束治疗干眼的效果评价；(A)小鼠眼部在治疗第0、7、14d的荧光素钠染色图；(B)在治疗第0、7、14d小鼠眼部荧光素钠评分数据分析；(C)在治疗第0、7、14d小鼠酚红棉线泪液测试结果；
图11为2CsA-ASONFKBIZ纳米胶束的10％变性聚丙烯酰胺凝胶电泳图与1％琼脂糖凝胶电泳图；
图12为实时荧光定量PCR检测2CsA-ASONFKBIZ-h以及对照样品处理细胞后靶标基因NFKBIZ mRNA表达水平；
图13为2CsA-ASONFKBIZ-m纳米胶束治疗干眼的效果评价；(A)小鼠眼部在治疗第0、7、14d的荧光素钠染色图；(B)在治疗第0、7、14d小鼠眼部荧光素钠评分数据分析；(C)在治疗第0、7、14d小鼠酚红棉线泪液测试结果；
图14为3CsA-ASONFKBIZ偶联物及其自组装纳米胶束的表征，左图为10％变性聚丙烯酰胺凝胶电泳结果，右图为0.5％琼脂糖凝胶电泳结果；
图15为实时荧光定量PCR检测3CsA-ASONFKBIZ-h以及对照样品处理细胞后靶标基因NFKBIZ mRNA表达水平；
图16为3CsA-ASONFKBIZ-m纳米胶束治疗干眼的效果评价；(A)小鼠眼部在治疗第0、7、14d的荧光素钠染色图；(B)在治疗第0、7、14d小鼠眼部荧光素钠评分数据分析；(C)在治疗第0、7、14d小鼠酚红棉线泪液测试结果；
图17为氨基三[(2-丙炔基氧)甲基]甲烷的合成示意图；
图18为Boc-氨基三[(2-丙炔基氧)甲基]甲烷的核磁共振氢谱；
图19为CsA3-ASONFKBIZ偶联物的合成示意图；
图20为20％变性聚丙烯酰胺凝胶电泳表征三炔基修饰ASONFKBIZ偶联物；
图21为0.5％琼脂糖凝胶电泳表征CsA3-ASONFKBIZ纳米胶束；
图22为CsA3-ASONFKBIZ纳米胶束的水合粒径(A)和形貌(B)；
图23为实时荧光定量PCR检测CsA3-ASONFKBIZ-h纳米胶束以及对照样品处理细胞后靶标基因NFKBIZ mRNA表达水平；
图24为CsA3-ASONFKBIZ-m纳米胶束治疗干眼的效果评价；(A)小鼠眼部在治疗第0、14d的荧光素钠染色图；(B)在治疗第0、14d小鼠眼部荧光素钠评分数据分析；(C)在治疗第0、14d小鼠酚红棉线泪液测试结果；
图25为CsA-siRNANFKBIZ、2CsA-siRNANFKBIZ与3CsA-siRNANFKBIZ偶联分子的15％变性聚丙烯酰胺凝胶电泳结果以及2CsA-siRNANFKBIZ与3CsA-siRNANFKBIZ纳米胶束的0.5％琼脂糖凝胶电泳结果；
图26为人眼角膜上皮细胞经CsA-siRNANFKBIZ-h偶联物、2CsA-siRNANFKBIZ-h纳米胶束与3CsA-siRNANFKBIZ-h纳米胶束以及对照样品处理后细胞中IκB-ζ蛋白表达水平；
图27为实时荧光定量PCR检测CsA-siRNANFKBIZ-h偶联物、2CsA-siRNANFKBIZ-h纳米胶束与3CsA-siRNANFKBIZ-h纳米胶束以及对照样品处理人眼上皮细胞后NFKBIZ mRNA表达量；
图28为CsA-siRNANFKBIZ-m偶联物、2CsA-siRNANFKBIZ-m纳米胶束以及3CsA-siRNANFKBIZ-m纳米胶束治疗干眼的效果评价；(A)小鼠眼部在治疗第0、7、14d的荧光素钠染色图；(B)在治疗第0、7、14d小鼠眼部荧光素钠评分数据分析；(C)在治疗第0、7、14d小鼠酚红棉线泪液测试结果；
图29为羰乙基溴代环孢素A的合成步骤；图30为羰乙基溴代环孢素A的LC-MS谱图；
图31为20CsA-ASONFKBIZ纳米胶束的10％变性聚丙烯酰胺凝胶图与1％琼脂糖凝胶图；
图32为实时荧光定量PCR检测20CsA-ASONFKBIZ-h纳米胶束以及对照样品处理后细胞中NFKBIZ mRNA的表达量；
图33为20CsA-ASONFKBIZ-m纳米胶束治疗干眼的效果评价；(A)在治疗第0、14d小鼠眼部荧光素钠评分数据分析；(B)在治疗第0、14d小鼠酚红棉线泪液测试结果；
图34为实时荧光定量PCR检测经过不同靶点3CsA-siRNA纳米胶束与对照样品处理后的人眼角膜上皮细胞中各炎症mRNA的表达量；
图35为ELISA检测经过不同靶点3CsA-siRNA纳米胶束与对照样品处理后的人眼角膜上皮细胞中各炎症因子表达量；
图36为针对不同靶点3CsA-siRNA纳米胶束治疗干眼的效果评价；(A)在治疗第0、14d小鼠眼部荧光素钠评分数据分析；(B)在治疗第0、14d小鼠酚红棉线泪液测试结果；
图37为实时荧光定量PCR检测经过不同靶点3CsA-siRNA纳米胶束与对照样品处理后的人眼角膜上皮细胞内各炎症靶点mRNA的表达量；
图38为针对不同靶点3CsA-siRNA纳米胶束治疗干眼的效果评价；(A)在治疗第0、14d小鼠眼部荧光素钠评分数据分析；(B)在治疗第0、14d小鼠酚红棉线泪液测试结果；
图39为实时荧光定量PCR检测经过不同靶点的3CsA-ASO纳米胶束以及对照样品处理后人眼角膜上皮细胞中不同炎症mRNA的表达情况；
图40为ELISA检测经过不同靶点的3CsA-ASO纳米胶束以及对照样品处理后人眼角膜上皮细胞中不同炎症因子的表达情况；
图41为不同靶点3CsA-ASO纳米胶束治疗干眼的效果评价；(A)在治疗第0、14d小鼠眼部荧光素钠评分数据分析；(B)在治疗第0、14d小鼠酚红棉线泪液测试结果；
图42为实时荧光定量PCR检测经过不同靶点20CsA-ASO纳米胶束与对照样品处理后人眼角膜上皮细胞各炎症mRNA表达量；
图43为不同靶点20CsA-ASO纳米胶束治疗干眼的效果评价；(A)在治疗第0、14d小鼠眼部荧光素钠评分数据分析；(B)在治疗第0、14d小鼠酚红棉线泪液测试结果；
图44为RAP-Bz-Br的合成示意图；
图45为TK-Bz-Br的核磁共振氢谱(A)和碳谱(B)；
图46为RAP-31-O-TMS-42-Bz-Br的核磁共振氢谱(A)和碳谱(B)；
图47为15％变性聚丙烯酰胺凝胶电泳；(A)和1％琼脂糖凝胶电泳(B)表征10RAP-ASO偶联物、10RAP-纳米胶束的成功制备；
图48为10RAP-ASONFKBIZ纳米胶束的水合粒径(左图)和透射电镜形貌(右图)；
图49为实时荧光定量PCR(RT-qPCR)检测NFKBIZ mRNA表达水平；
图50为不同靶点10RAP-ASONFKBIZ-m纳米胶束治疗干眼的效果评价；(A)在治疗第0、7、14d小鼠眼部荧光素钠评分数据分析；(B)在治疗第0、7、14d小鼠酚红棉线泪液测试结果；
图51为RT-PCR检测CsA3-ASONFKBIZ-h纳米胶束以及对照样品与HaCaT细胞共孵育后细胞NFKBIZ mRNA的表达量；
图52为ELISA检测经过CsA3-ASONFKBIZ-h纳米胶束以及对照样品处理后HaCaT细胞不同炎症因子分泌情况；
图53为小鼠背部在建模第7d的模型表型图；
图54为银屑病样模型小鼠背部经涂抹给药治疗后第0和7d的皮损照片；
图55为银屑病样模型小鼠经涂抹给药治疗后耳部和背部皮肤厚度变化；
图56为银屑病样模型小鼠经涂抹给药治疗后背部皮损处红斑、鳞屑、皮肤增厚的评分及银屑病皮损面积严重程度指数评分；
图57为羰乙基溴代曲安奈德(TA-Br)的合成示意图；
图58为TA-Br的核磁共振氢谱；
图59为TA-Br的核磁共振碳谱；
图60为TA-Br的LC-MS谱图；
图61为15％变性聚丙烯酰胺凝胶电泳表征10TA-ASONFKBIZ偶联物的成功制备；
图62为1％琼脂糖凝胶电泳表征10TA-ASONFKBIZ纳米胶束的成功制备；
图63为RT-PCR检测10TA-ASONFKBIZ-h纳米胶束以及对照样品与HaCaT细胞共孵育后细胞NFKBIZ mRNA的表达量；
图64为银屑病样模型小鼠经涂抹给药治疗后耳部和背部皮肤厚度变化；
图65为治疗前后小鼠背部皮损处红斑、鳞屑、皮肤增厚的评分及银屑病皮损面积严重程度指数评分；
图66为银屑病样模型小鼠背部在治疗第0和7d的皮损照片；
图67为Cal-Bz-Br的合成示意图；
图68为DTPA-Bz-Br的核磁共振氢谱；
图69为Cal-Bz-Br的核磁共振氢谱；
图70为Cal-Bz-Br的LC-MS谱图；
图71为15％变性聚丙烯酰胺凝胶电泳表征10Cal-ASONFKBIZ偶联物的成功制备；
图72为1％琼脂糖凝胶电泳表征10Cal-ASONFKBIZ纳米胶束的成功制备；
图73为10Cal-ASONFKBIZ纳米胶束的水合粒径(左图)和透射电镜形貌(右图)；
图74为10Cal-ASONFKBIZ纳米胶束的临界胶束浓度图；
图75为RT-PCR检测10Cal-ASONFKBIZ-h纳米胶束以及对照样品与HaCaT细胞共孵育后细胞NFKBIZ mRNA的表达量；
图76为银屑病样模型小鼠治疗过程皮肤厚度变化；
图77为治疗前后小鼠背部皮损处红斑、鳞屑、皮肤增厚的评分及银屑病皮损面积严重程度指数评分；
图78为银屑病样模型小鼠背部在治疗第0和7d的皮损照片。
具体实施方式
本发明提供了一种小分子药物-寡核苷酸偶联物，由具有免疫调节功能的小分子药物和能够调控炎症相关基因表达的功能寡核苷酸分子共价偶联得到。
在本发明中，所述小分子药物和功能寡核苷酸分子之间优选的通过化学连接子共价偶联。
在本发明中，所选小分子药物优选为作用于免疫相关信号通路并能够调控免疫反应的小分子药 物；在本发明中，所述小分子药物包括钙调磷酸酶抑制剂、糖皮质激素、mTOR抑制剂和维生素D类似物中的一种或几种。在本发明中，所述钙调磷酸酶抑制剂优选的包括环孢菌素A、他克莫司、西罗莫司、吡美莫司或者环孢菌素A、他克莫司、西罗莫司或吡美莫司的类似物；所述mTOR抑制剂优选的包括雷帕霉素或雷帕霉素的衍生物；所述雷帕霉素的衍生物优选的包括Temsirolimus(CCI-779)或Everolimus(RAD001)；所述糖皮质激素优选的包括曲安奈德、地塞米松、倍他米松、甲泼尼龙、可的松、氢化可的松、醋酸泼尼松、醋酸泼尼松龙、强的松或者曲安奈德、地塞米松、倍他米松、甲泼尼龙、可的松、氢化可的松、醋酸泼尼松、醋酸泼尼松龙、强的松的类似物；所述维生素D类似物优选的包括卡泊三醇、骨化三醇、骨化二醇、阿法骨化醇或帕立骨化醇。
在本发明中，所述炎症相关基因包括肿瘤坏死因子-a(TNF-a)基因、白介素1b(IL-1b)基因、白介素17(IL-17)基因、白介素23(IL-23)基因、NFKBIZ基因、炎性小体NLRP3基因、JAK基因和PDE4基因中的一种或几种。针对上述炎症反应关键基因设计对应调控其表达的功能寡核苷酸分子，每个基因有其对应的功能寡核苷酸分子调控基因对应mRNA表达。
在本发明中，所述功能寡核苷酸分子包括双链小干扰核酸(siRNA)、微小核酸(miRNA)和单链反义寡核苷酸(ASO)中的一种。在本发明中，所用功能寡核苷酸分子可以是未做修饰的功能寡核苷酸分子，也可以稳定性增强修饰的功能寡核苷酸分子；所述稳定性增强修饰优选的包括PS、2位OMe、MOE和F代修饰中的一种或几种。
在本发明中，所述小分子药物和功能寡核苷酸分子之间的组合多种多样，互不限制，一种小分子药物可与多种功能寡核苷酸分子进行偶联，而一种功能寡核苷酸分子也可与多种小分子药物进行偶联，以实现不同应用目的。
在本发明中，当所述功能寡核苷酸分子为双链小干扰核酸或微小核酸时，所述小分子药物共价偶联于功能寡核苷酸分子的正义链的3’端；当所述功能寡核苷酸分子为单链反义寡核苷酸时，所述小分子药物共价偶联于单链反义寡核苷酸的3’端或5’端。
在本发明中，所述小分子药物共价偶联于功能寡核苷酸分子的末端或者所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上。在本发明中，每个功能寡核苷酸分子上共价偶联的小分子药物的数量优选为1～40中取任意整数值。当所述小分子药物共价偶联于功能寡核苷酸分子的末端时，每个功能寡核苷酸分子上共价偶联的小分子药物的数量优选为1～6中取任意整数值；当所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上时，每个功能寡核苷酸分子上共价偶联的小分子药物的数量优选为1～20中取任意整数值。
在本发明中，当所述小分子药物共价偶联于功能寡核苷酸分子的末端时，所述的小分子药物-寡核苷酸偶联物的化学结构式如式1～式14所示：


在所述式1～式14中，L、T各自独立地为不存在、-(CH2)h-或-(CH2)h-中的任意一个或多个亚烷基被A基团所取代后的基团；所述h为0～15；所述A基团包括：-O-、-S-、-C(O)-、-C(O)O-、-C(O)NH-、-CH(RC)-、-C(R’)(R”)-、-NH-、-N(RN)-、-S-S-、-C(R’)＝C(R”)-、-C≡C-、中的一种或几种；所述A基团中的RC、RN、R’、R”表示指定原子上的任何一个或多个氢原子被B基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物，所述指定原子包括碳原子或氮原子；所述B基团包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素；所述卤素包括F、C1、Br或I；所述A基团中的(O)代表羰基氧原子；
在所述式1～式14中，Q、Y、Z各自独立地为不存在、-O-、-S-、-C(O)-、-NH-、-CH2-、-C(O)NH-、 -NHC(O)-、-C(O)O-、-OC(O)-、-OC(O)O-、-OC(O)NH-、-NHC(O)O-和中的一种或几种；所述Q、Y、Z基团中(O)代表羰基氧原子；所述为连接位点；
在所述式1～式14中，G表示小分子免疫调节药物；m、n、k独立地为1～15；
X表示O或S；
优选的，当所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上时，所述的小分子药物-寡核苷酸偶联物的化学结构式如式15～式20所示：

在所述式15～式20中，T各自独立地为不存在、-(CH2)h-或-(CH2)h-中的任意一个或多个亚烷基被A基团所取代后的基团；所述h为0～15；所述A基团中的(O)代表羰基氧原子；所述A基团包括：-O-、-S-、-C(O)-、-C(O)O-、-C(O)NH-、-CH(RC)-、-C(R’)(R”)-、-NH-、-N(RN)-、-S-S-、-C(R’)＝C(R”)-、-C≡C-、中的一种或几种；所述A基团中的RC、RN、R’、R”表示指定原子上的任何一个或多个氢原子被B基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物，所述指定原子包括碳原子或氮原子；所述B基团包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素；所述卤素包括F、C1、Br或I；所述A基团中的(O)代表羰基氧原子；
在所述式15～式20中，Y、Z各自独立地为不存在、O、S、C(O)、NH、CH2、C(O)NH、NHC(O)、C(O)O、OC(O)、OC(O)O、OC(O)NH、NHC(O)O和中的一种或几种；所述Y、Z基团中的(O)代表羰基氧原子；所述为连接位点；
在所述式15～式20中，G表式免疫调节抑制剂；n独立地为1～15；m、i独立地为0～5，k、j独立地为1～20；R表示H；B表示核酸碱基。
本发明还提供了上述方案所述的小分子药物-寡核苷酸偶联物的制备方法，包括以下步骤：
将具有免疫调节功能的小分子药物和能够调控炎症相关基因表达的功能寡核苷酸分子共价偶联，得到小分子药物-寡核苷酸偶联物。本发明对所述小分子药物和功能寡核苷酸分子共价偶联的具体方法没有特殊限制，采用本领域的常规偶联方法即可。
在本发明中，所述共价偶联涉及的反应类型优选的包括酯化、酰胺化、点击化学反应、亲电取代、和亲核取代或狄尔斯阿德耳反应中的一种或几种。
本发明还提供了上述方案所述的小分子药物-寡核苷酸偶联物在制备治疗炎症相关疾病的药物中的应用。
在本发明中，所述炎症相关疾病包括银屑病、干眼症、干燥综合症、葡萄膜炎、角膜炎、结膜炎、特应性皮炎、类风湿性关节炎、炎症性肠炎、和克罗恩病中的一种或几种。
本发明利用功能寡核苷酸分子亲水的特性促进具有免疫调节功能的疏水性的小分子药物在水溶液中的溶解，使其无需使用其他助溶剂即可很好地分散在缓冲溶液中并被细胞高效摄取。溶解性的增 加可减小给药时的使用浓度，降低药物分子的毒副作用。同时疏水性的小分子药物的修饰也使得功能寡核苷酸分子具备一定的疏水性质，在疏水药物较多时，小分子药物-寡核苷酸偶联物还能进一步组装成胶束，进而促进细胞对小分子药物-寡核苷酸偶联物的摄取，克服核酸药物入胞困难的问题。本发明的小分子药物-寡核苷酸偶联物同时包含调节免疫反应的小分子药物和功能寡核苷酸分子，两者联合使用能够同时作用于不同的炎症相关信号通路，产生协同效应，解决单独使用小分子免疫调节药物或寡核苷酸药物疗效不佳的问题，从而达到更好的治疗效果。
在本发明的一个实施例中，本发明构建了由单个环孢素A(Cyclosporine A，CsA)分子与靶向核因子κB抑制因子ζ(NFKBIZ)基因的反义寡核苷酸(ASONFKBIZ)共价偶联形成的CsA-NFKBIZ反义寡核苷酸偶联物(CsA-ASONFKBIZ)。在本发明中，所述CsA-NFKBIZ反义寡核苷酸偶联物能够用于制备治疗干眼症的药物。本发明通过构建CsA-ASONFKBIZ共价偶联物可实现小分子免疫调节药物与核酸药物协同治疗干眼症。CsA作为一种常用的免疫抑制剂，能够通过抑制钙调磷酸酶活性来抑制促炎细胞因子的生成与释放，在临床上已被用于干眼症的症状与体征。而靶向NFKBIZ基因的反义寡核苷酸(ASONFKBIZ)则能够有效敲低角膜上皮细胞内NFKBIZ基因的表达，进一步降低细胞炎症水平，调控免疫平衡。CsA-ASONFKBIZ共价偶联物中小分子药物和核酸药物作用于炎症信号通路不同靶点能够实现干眼症协同治疗，达到更佳的治疗效果。本发明利用核酸分子亲水性强的特点偶联后赋予小分子药物CsA良好的水溶性，可使其在没有其他助剂帮助的情况下能够高效地被细胞摄取，更快地发挥药效，降低治疗时给药浓度，从而减小药物可能的毒副作用。同时所制备的CsA-ASONFKBIZ共价偶联物由于疏水性CsA的存在能够增强其与细胞的相互作用，相比于未修饰反义寡核苷酸，CsA-ASONFKBIZ偶联物能够更好地被细胞内吞，从而更好地发挥基因调控作用。最终通过CsA与反义寡核苷酸双重炎症调控作用，达到良好的干眼症治疗效果。
在本发明的一个实施例中，本发明构建了由两个CsA分子与NFKBIZ反义寡核苷酸(ASONFKBIZ)共价偶联形成的小分子药物-反义寡核苷酸偶联物(2CsA-ASONFKBIZ)，并利用其双亲性通过自组装形成纳米胶束组装结构用CsA和NFKBIZ反义寡核苷酸的共递送；所述2CsA-ASONFKBIZ能够用于制备治疗干眼症的药物。本发明采用小分子药物CsA与反义寡核苷酸药物协同治疗干眼病，同时作用于炎症信号通路不同靶点协同降低细胞炎症水平；其次，本案名利用核酸亲水的特点赋予免疫调节小分子药物更好的水溶性，同时利用CsA疏水性增强偶联产物与细胞相互作用，提高药物递送效率；再者，相比单个CsA分子与靶向NFKBIZ基因的ASO偶联制备的CsA-ASONFKBIZ共价偶联物可以单分子形式溶解于水溶液中，两个CsA小分子偶联后形成的2CsA-ASONFKBIZ共价偶联物由于其疏水性CsA数量增加使其表现出双亲性，在溶液中能够组装成为纳米胶束结构，此纳米胶束中心为疏水的CsA分子，外壳为水溶性的反义寡核苷酸分子。组装的纳米胶束无需任何转染试剂即可高效地转染细胞，实现CsA和反义寡核苷酸的高效共递送，进而达到良好的干眼协同治疗效果。
在本发明的一个实施例中，本发明构建了由三个环孢素A分子修饰靶向NFKBIZ基因的反义寡核苷酸从而制备了三环孢素A-NFKBIZ反义寡核苷酸偶联物(3CsA-ASONFKBIZ)，并利用其双亲性通过自组装形成3CsA-ASONFKBIZ纳米胶束组装结构用于CsA和靶向NFKBIZ基因的反义寡核苷酸的共递送，进而用于干眼症的协同治疗。本发明采用小分子药物与核酸药物同时作用于炎症信号通路的不同靶点协同降低细胞炎症水平；利用核酸亲水的特点赋予免疫调节小分子药物更好的水溶性，同时利用CsA疏水性增强偶联产物与细胞的相互作用，提高药物的利用效率；相比于单个或两个CsA分子与靶向NFKBIZ基因的ASO偶联物，3个CsA小分子偶联后的药物-核酸偶联物3CsA-ASONFKBIZ，其与核酸偶联后所表现的双亲性更强，在溶液中更易组装为纳米胶束，从而使得3CsA-ASONFKBIZ纳米胶束更易与细胞相互作用，从而达到更好的干眼协同治疗效果。
在本发明的一个实施例中，本发明构建了环孢素A三聚体分子与靶向NFKBIZ的反义寡核苷酸(ASONFKBIZ)共价偶联形成的环孢素A-反义寡核苷酸偶联物(CsA3-ASONFKBIZ)，并探索由其自组装形成 的球形纳米胶束用于干眼症的协同治疗。本发明通过点击化学反应在ASO末端接枝三个环孢素A，其纯化及制备方法简单可控。利用三个环孢素A分子较强的疏水性使得偶联物具有较强的两亲性，所制备偶联产物在水溶液中可自组装形成稳定的纳米胶束结构，其临界胶束浓度低，球形核酸胶束纳米结构能够高效地被细胞摄取，实现CsA和ASO的同时共递送；环孢素A作为一种免疫调节剂与抗炎药，能抑制促炎症细胞因子的生成与释放，并提高抗炎细胞因子的释放水平，临床上可有效地改善由长期炎症所致的干眼症状与体征；同时ASO可以敲低炎症相关基因NFKBIZ蛋白的表达，进一步抑制干眼症的炎症反应。通过协同调控作用抑制炎症反应，调控免疫平衡，实现更好的干眼症治疗效果。
在本发明的一个实施例中，本发明构建了单个、两个、以及三个环孢素A分别与靶向NFKBIZ基因的小干扰RNA(siRNANFKBIZ)共价偶联形成药物-核酸偶联物，利用所制备偶联物或其组装纳米结构进一步实现了干眼症的协同治疗。本发明采用小分子药物与核酸药物协同治疗干眼病，CsA是一种免疫调节剂与抗炎药，能抑制促炎症细胞因子的生成与释放水平，临床上可有效地改善由长期炎症所致的干眼症状与体征；同时NFKBIZ siRNA可以敲低角膜上皮细胞内NFKBIZ的表达，进一步降低细胞炎症水平；利用siRNA亲水的特点赋予CsA药物更好的水溶性，减小系统性应用时的使用浓度，从而减小毒副作用，与此同时疏水性的免疫调节小分子药物CsA使得二者的偶联物更易与细胞相互作用，改善RNA药物入胞难的问题，进而达到更好的干眼病治疗效果。本实施例意在扩展在干眼治疗方面的药物-核酸偶联物类别。
在本发明的一个实施例中，本发明构建了由多个环孢素A分子通过硫代磷酸酯接枝修饰靶向NFKBIZ基因的反义寡核苷酸(ASONFKBIZ)构建药物-反义寡核苷酸偶联物，并利用其双亲性自组装形成纳米胶束，用于干眼症的协同治疗。该结构不仅可以实现环孢素A与ASONFKBIZ协同治疗干眼病，同时可以增强环孢素A的水溶性与核酸药物的稳定性，孵育药物-核酸偶联物更好的与细胞相互作用的能力。与此同时，在本实施例中，20个环孢素A分子接枝的ASONFKBIZ结构，其组装为纳米胶束的CMC值更小，所携带的治疗性小分子药物量更多，可以达到更好的治疗效果。
在本发明的一个实施例中，本发明构建了三个CsA分子分别与靶向IL-17的siRNAIL-17、靶向IL-1β的siRNAIL-1β、靶向IL-23的siRNAIL-23、以及靶向TNF-α的siRNATNF-α共价偶联修饰制备的小分子药物-核酸偶联物及其自组装纳米胶束用于干眼症的协同治疗。本实施例制备了不同炎症因子靶点的三环孢素A与小干扰RNA药物-核酸偶联物纳米胶束用于协同治疗干眼病。本实施例选择的基因靶点为细胞分泌于胞外的不同炎症因子，进一步验证该结构在治疗干眼疾病中的多靶点可行性。
在本发明的一个实施例中，本发明构建了三个CsA分子分别与靶向NLRP3的siRNANLRP3、靶向JAK1的siRNAJAK1、以及靶向PDE4的siRNAPDE4共价偶联修饰制备的小分子药物-核酸偶联物及其自组装纳米胶束用于干眼症的协同治疗。本实施例制备了靶向细胞内炎症相关关键基因的siRNA与环孢素A分子的偶联物，利用其双亲性组装形成纳米胶束用于干眼症的协同治疗。本实施例选择细胞内不同炎症基因靶点，进一步验证该结构在治疗干眼疾病中的多靶点可行性。
在本发明的一个实施例中，本发明构建了3CsA与ASOIL-17、3CsA与ASOIL-1β、3CsA与ASOIL-23以及3CsA与ASOTNF-α共递送药物核酸偶联物自组装纳米胶束用于干眼病的协同治疗。本实施例制备了不同炎症因子靶点的三环孢素A与反义寡核苷酸药物-核酸偶联物纳米胶束用于协同治疗干眼病。本实施例选择的基因靶点为细胞分泌于胞外的不同炎症因子，进一步验证该结构在治疗干眼疾病中的多靶点可行性。
在本发明的一个实施例中，本发明构建了20CsA与ASONLRP3、20CsA与ASOJAK1和20CsA与ASOPDE4共递送药物核酸偶联物纳米胶束，用于干眼病的协同治疗。本实施例采用20PS修饰的ASO，与多个环孢素A小分子接枝，增大了载药量的同时可减小治疗剂量，提高了治疗安全性；与此同时，本实施例选取干眼疾病中的多个胞内炎症治疗靶点，进一步炎症药物-核酸偶联物治疗干眼病的多靶 点可行性。
在本发明的一个实施例中，本发明构建了由10个雷帕霉素(Rapamycin,RAP)修饰靶向NFKBIZ基因的反义寡核苷酸得到的小分子药物-反义寡核苷酸偶联物(10RAP-ASONFKBIZ)及其自组装形成的纳米胶束用于干眼症的协同治疗。本发明通过在ASO序列一端的核酸骨架上接枝多个雷帕霉素分子所构建的雷帕霉素-反义寡核苷酸偶联物具有较高载药量。利用雷帕霉素分子的疏水性和核酸分子的亲水性赋予雷帕霉素-反义寡核苷酸偶联物双亲性特征，其在水溶液中可自组装即可形成纳米胶束结构。该纳米胶束结构无需其他载体材料即能够被细胞有效摄取，从而实现雷帕霉素和反义寡核苷酸的高效共递送；雷帕霉素-反义寡核苷酸偶联物可通过调控不同炎症相关信号通路实现干眼症的联合治疗。其中雷帕霉素作为雷帕霉素靶蛋白(mTOR)抑制剂可有效抑制mTOR信号转导通路实现炎症抑制作用，同时靶向NFKBIZ基因的反义寡核苷酸可以有效敲低NFKBIZ基因表达，进一步抑制干眼症相关的炎症反应。通过两者协同作用联合抑制炎症反应，从而实现了良好的干眼治疗效果。
在本发明的一个实施例中，本发明构建了由环孢素A三聚体(CsA3)与靶向NFKBIZ的反义寡核苷酸(ASONFKBIZ)共价偶联形成的小分子药物-反义寡核苷酸偶联物及其自组装形成的球形纳米胶束用于银屑病的协同治疗。本发明通过点击化学反应在ASO末端接枝三个环孢素A，其纯化及制备方法简单可控。利用三个环孢素A分子较强的疏水性使得偶联物具有较强的两亲性，无需载体即可在水溶液中自组装形成稳定的纳米胶束结构，所制备的球形胶束核酸纳米结构能够高效地被细胞摄取，实现CsA和ASO的同时共递送；环孢素A作为一种免疫调节剂与抗炎药，能抑制促炎症细胞因子的生成与释放，并上升抗炎症细胞因子的释放水平；同时ASO可以敲低炎症相关基因NFKBIZ蛋白的表达，进一步抑制银屑病的炎症反应。通过协同调控作用抑制炎症反应，调控免疫平衡，实现更好的银屑病治疗效果。
在本发明的一个实施例中，本发明构建了由10个曲安奈德分子修饰靶向NFKBIZ基因的反义寡核苷酸(ASO)的小分子药物-反义寡核苷酸偶联物及其组装形成的纳米胶束纳米结构用于银屑病的协同治疗。本发明通过在ASO序列末端接枝多个曲安奈德分子构建的曲安奈德-反义寡核苷酸偶联物具有较高载药量。利用曲安奈德的疏水性和核酸分子的亲水性赋予曲安奈德-反义寡核苷酸偶联物两亲性，在水溶液中通过自组装即可形成纳米胶束结构。该纳米胶束结构无需其他载体材料即可实现曲安奈德和反义寡核苷酸的高效共递送，能够有效地被细胞摄取；通过调控不同炎症相关信号通路实现银屑病的联合治疗，其中曲安奈德作为一种糖皮质激素，具有抗炎、抗瘙痒和收缩血管等作用，其在皮肤疾病治疗中的抗炎和抗过敏作用较强且持久。同时靶向NFKBIZ基因的ASO可以有效敲低NFKBIZ蛋白表达，进一步抑制银屑病相关的炎症反应。通过两者协同作用联合抑制炎症反应，从而达到良好的银屑病治疗效果。
在本发明的一个实施例中，本发明构建了由10个卡泊三醇分子与靶向NFKBIZ基因的反义寡核苷酸(ASO)共价偶联制备的小分子药物-反义寡核苷酸偶联物(10Cal-ASONFKBIZ)及其组装形成的纳米胶束用于银屑病的协同治疗。本发明通过在ASO序列末端接枝多个卡泊三醇分子构建的卡泊三醇-反义寡核苷酸偶联物具有较高载药量。利用卡泊三醇的疏水性和核酸分子的亲水性赋予卡泊三醇-反义寡核苷酸偶联物双亲性，在水溶液中通过自组装即可形成纳米胶束结构。该纳米胶束结构无需其他载体材料即可实现卡泊三醇和反义寡核苷酸的高效共递送，能够有效地被细胞摄取；通过调控不同炎症相关信号通路实现银屑病的联合治疗，其中卡泊三醇是一种维生素D3的类似物，能够抑制皮肤细胞(角朊细胞)增殖和诱导其分化，从而纠正银屑病皮损的增生和分化异常。同时靶向NFKBIZ基因的ASO可以有效敲低NFKBIZ蛋白表达，进一步抑制银屑病相关的炎症反应。通过两者协同作用联合抑制炎症反应，从而达到良好的银屑病治疗效果。
如无特殊说明，本发明对所用原料的来源没有特殊要求，采用本领域技术人员所熟知的市售商品即可。
下面结合具体实施例，进一步阐述本发明。应该理解，这些实施例仅用于说明本发明，而不用于限定本发明的保护范围。在实际应用中本领域技术人员根据本发明做出的改进和调整，仍属于本发明的保护范围。
实施例1单环孢素A(Cyclosporine A,CsA)与靶向核因子κB抑制因子ζ(NFKBIZ)反义寡核苷酸偶联物用于干眼症的协同治疗
1.1叠氮己酸碳酸酯修饰环孢素A(CsA-N3)的合成
本实施例中叠氮己酸碳酸酯修饰环孢素A(CsA-N3)以及CsA-ASONFKBIZ共价偶联物的制备路线如图1所示。
1.1.1氯甲基碳酸酯环孢素A(CsA-Cl)合成
在氮气保护下，取环孢素A(1000mg)置于干燥的50mL圆底烧瓶中，加入10mL无水二氯甲烷搅拌至溶解，将反应瓶至于冰浴中，然后逐滴加入592μL氯甲酸氯甲酯，再逐滴加入336μL无水吡啶，自然恢复至室温，搅拌反应24h。反应完毕后，加入40mL无水二氯甲烷稀释反应液，依次用50mL饱和碳酸氢钠溶液洗涤3次，50mL饱和氯化钠溶液洗涤一次，收集有机相后用无水硫酸钠干燥，减压蒸馏浓缩有机相后再经硅胶柱层析分离纯化，洗脱液为石油醚/乙酸乙酯，最后得到白色固体550mg，即制得氯甲基碳酸酯环孢素A，产率：～60％。
氯甲基碳酸酯环孢素A的1H NMR谱图如图2所示，核磁波谱测试使用溶剂为CDCl3，各质子峰的归属如下：δ(ppm):8.46(1H,d),7.91(1H,d),7.46(1H,d),7.37(1H,d),5.72(1H,d),5.62(1H,dd),5.47(1H,d),5.31(2H,m),5.21(2H,m),5.06(2H,m),5.00(1H,q),4.88(1H,t),4.77(1H,t),4.70(1H,t),4.60(1H,d),4.33(1H,t),4.05(1H,q),3.39(3H,s),3.25(3H,s),3.16(3H,s),3.13(1H,s),3.01(3H,d),2.60(3H,d),2.34(1H,m),2.06(4H,m),2.00(4H,m),1.92(2H,m),1.81(3H,m),1.66(6H,m),1.53(4H,d),1.42(2H,m),1.34(1H,m),1.26(3H,d),1.20(3H,d),1.14(1H,dq),1.05(3H,d),1.04(1H,d),0.98(3H,t),0.94(3H,d),0.91(3H,d),0.89(3H,d),0.88(3H,d),0.86(3H,d),0.82(9H,m),0.81(3H,d),0.76(3H,d).氯甲基碳酸酯环孢素A的13C NMR谱图如图3所示，核磁波谱测试使用溶剂为CDCl3，产物所含特征碳的归属如下：173.7,173.4,173.1,172.8,171.6,171.2,170.9,170.8,170.0,169.9,167.6,153.5。氯甲基碳酸酯环孢素A的理论分子量为1293.80，基质辅助激光解析电离飞行时间质谱(MALDI-TOF)实际测得的分子量为1294.80，归属于氯甲基碳酸酯环孢素A的分子离子峰[M+1]+，如图4所示。
1.1.2碘甲基碳酸酯环孢素A(CsA-I)的合成
在氮气保护下，取氯甲基碳酸酯环孢素A(500mg)放置于干燥的25mL圆底烧瓶中，加入1mL无水二氯甲烷搅拌溶解，氮气流下减压蒸馏除去二氯甲烷，加5mL无水乙腈溶解，将碘化钠(500mL)加入反应溶液中，在40℃条件下反应24h，反应完毕后，减压蒸馏除去乙腈，然后加入50mL无水二氯甲烷，用50mL饱和氯化钠溶液洗涤2次，收集有机相，然后用无水硫酸钠干燥，减压蒸馏除去溶剂后置真空干燥箱内(40℃)真空干燥即制得碘甲基碳酸酯环孢素A，所得产物直接用于下一步反应。
1.1.3叠氮己酸碳酸酯键连接的环孢素A(CsA-N3)的合成
在氮气保护下，取上述所制备碘甲基碳酸酯环孢素A(200mg)，放置于10mL圆底烧瓶中，加入1mL无水N,N-二甲基甲酰胺(DMF)搅拌溶解，取40mg6-叠氮基己酸溶解在1mL无水DMF中，滴加至碘甲基碳酸酯环孢素A中，加入60μL无水N,N-二异丙基乙胺，在室温条件下反应24h。反应完毕后，减压蒸馏除去DMF，加50mL乙酸乙酯溶解，加入50mL10％焦亚硫酸钠溶液(w/v)洗涤2次，饱和氯化钠溶液洗涤1次，然后收集有机相，加入无水硫酸钠进行干燥，过滤后将有机相减压蒸馏浓缩，然后利用硅胶柱层析分离纯化，洗脱液为石油醚/乙酸乙酯(～1:2)混合溶液，最后收集得到白色固体产物约120mg(Rf:～0.2)，即制得叠氮己酸碳酸酯修饰的环孢素A。
叠氮己酸碳酸酯键修饰的环孢素A(CsA-N3)的1H NMR谱图如图5所示，核磁波谱测试使用溶 剂为CDCl3，各质子峰的归属如下：δ(ppm):8.54(1H,d),7.98(1H,d),7.52(1H,d),7.46(1H,d),5.82(1H,d),5.70(1H,dd),5.68(1H,d),5.54(1H,d),5.37(2H,m),5.25(2H,m),5.11(2H,m),5.06(1H,d),4.97(1H,t),4.85(1H,t),4.77(1H,t),4.66(1H,d),4.40(1H,t),3.46(3H,s),3.31(3H,s),3.28(2H,d),3.23(3H,s),3.20(3H,s),3.09(1H,s),2.67(3H,d),2.41(1H,t),2.12(4H,m),2.06(4H,m),1.99(2H,m),1.87(3H,m),1.66(6H,m),1.60(4H,d),1.42(2H,m),1.33(3H,d),1.26(3H,d),1.20(3H,d),1.14(1H,dq),1.05(7H,d),1.00(3H,d),0.96(3H,t),0.94(12H,m),0.91(3H,d),0.89(3H,d),0.88(3H,d),0.86(3H,d),0.82(9H,m),0.81(3H,d),0.76(3H,d)。叠氮己酸碳酸酯修饰的环孢素A的理论分子量为1415.89，实际测得的分子量为1416.90，归属为叠氮己酸碳酸酯修饰的环孢素A的分子离子峰[M+1]+，如图6所示。
1.2单环孢素A与NFKBIZ反义寡核苷酸共价偶联物(CsA-ASONFKBIZ)的合成
本实施例中所采用的靶向NFKBIZ基因反义寡核苷酸均购自生工生物工程(上海)股份有限公司，在反义寡核苷酸3’端我们引入了二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，3’端DBCO基团修饰在以下所有实施例中均以-DBCO-3’表示，其修饰结构如下所示：
本实施例中使用的核酸序列如下：
其中靶向人源NFKBIZ基因的反义寡核苷酸(DBCO-ASONFKBIZ-h)序列为
Human:5’-ATCAGACAACGAATCGGGC-DBCO-3’(SEQ ID NO.1)；
其中靶向小鼠NFKBIZ基因的反义寡核苷酸(DBCO-ASONFKBIZ-m)序列为
Mouse:5’-AATACTGGTACATTGACGCC-DBCO-3’(SEQ ID NO.2)。
单个环孢素A分子共价修饰NFKBIZ反义寡核苷酸制备共价偶联物(CsA-ASONFKBIZ)的合成方法为：将0.2mg CsA-N3(130nmol)溶于0.13mL DMSO中，加入5OD DBCO-ASONFKBIZ(26nmol)后，放置于50℃下震荡反应24h。加入5mL水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，然后将水溶液浓缩蒸干后，即得到CsA-ASONFKBIZ偶联物分子。通过10％变性聚丙烯酰胺凝胶图验证CsA与ASO成功偶联，结果如图7所示。从图中可以看出单个CsA与DBCO-ASONFKBIZ的偶联效率较高，约为95％。
1.3 CsA-ASONFKBIZ-h偶联物的细胞摄取情况
将人角膜上皮细胞(Human corneal epithelial cells,HCEC)按照5×104细胞/孔的密度接种于置于12孔板中培养过夜，去除培养基后分别加入含有10μM或100μM带有FITC荧光的CsA-ASONFKBIZ-h偶联物的新鲜培养基0.5mL继续与HCEC细胞进行共孵育6h，不加偶联物的孔板作为空白对照(Mock组)。孵育完成后弃掉上清培养基，然后用胰酶消化细胞后用磷酸盐缓冲溶液(PBS，pH＝7.2)洗涤2～3次，收集细胞后使用流式细胞仪进行流式细胞分析，实验结果如图8所示。结果表明，CsA-ASONFKBIZ-h偶联物能够有效地被细胞摄取，孵育后细胞呈现明显的荧光增强信号。同时100μM CsA-ASONFKBIZ-h偶联物孵育后细胞平均荧光强度约为10μM共同孵育后荧光强度的10倍，证明CsA-ASONFKBIZ-h偶联物具有良好的入胞效果。
1.4 CsA-ASONFKBIZ-h偶联物对靶标基因NFKBIZ的调控作用
将HCEC细胞按照1×105细胞/孔的密度接种于6孔板培养至贴壁，然后采用100ng/mL脂多糖 (Lipopolysaccharides,LPS)刺激过夜，去掉上清液后，分别加入含ASONFKBIZ-h、ASONFKBIZ-h+Lipofectamine2000(Lipo2000)，以及CsA-ASONFKBIZ-h的Opti-MEM培养基1mL(均含10μM ASO)，同时以不做任何处理的细胞作为空白参考(Mock),37℃共孵育转染6h后，更换DMEM培养基继续孵育48h。然后收集细胞并提取RNA进行实时荧光定量PCR(RT-qPCR)检测，以分析HCEC细胞中靶标基因NFKBIZ mRNA表达水平，实验结果如图9所示。从图中可以看出，LPS刺激过后的HCEC细胞中NFKBIZ mRNA表达水平约为正常细胞的1.3倍，无转染试剂下ASONFKBIZ-h对LPS刺激后人眼上皮细胞中NFKBIZ mRNA的表达有一定的抑制作用，但仍为正常细胞的1.25倍左右；使用Lipo2000转染ASONFKBIZ-h后其表达水平降低至正常细胞的表达水平，加入CsA-ASONFKBIZ-h共孵育后NFKBIZ mRNA表达量降至正常细胞的0.8倍左右。由此结果可以看出，CsA-ASONFKBIZ-h偶联物对NFKBIZ mRNA的敲低能力优于无修饰的ASONFKBIZ-h。
1.5 CsA-ASONFKBIZ-m偶联物对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后分别使用院配环孢素A滴眼液(400μM CsA，简称院配CsA滴眼液或CsA滴眼液，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)与CsA-ASONFKBIZ-m(400μM CsA)进行眼部滴眼治疗，每只眼睛每次滴5μL，每日早晚各滴一次。同时在治疗的第0，7，14d进行荧光素钠染色评估与酚红棉线泪液分泌量测试，以评估治疗效果，实验结果如图10所示。
荧光素钠染色结果显示，院配CsA滴眼液治疗组小鼠眼部评分在第7d有所下降，14d治疗结束后评分由略高于6分降至5分左右，酚红棉线泪液测试结果由1.5mm增至3mm左右；CsA-ASONFKBIZ-m治疗组的小鼠眼部荧光素钠评分14d后由最初的5.5分降至3分左右，酚红棉线浸润长度由1.8mm增至3.2mm左右。相比于病理组与单纯的院配CsA滴眼液组，CsA-ASONFKBIZ-m由于能够更好地实现小分子药物以及核酸药物的递送，两者能够协同抑制干眼小鼠眼表炎症，显示了对小鼠干眼更好的治疗效果。
实施例2.双环孢素A修饰NFKBIZ反义寡核苷酸的共价偶联物(2CsA-ASONFKBIZ)及其组装结构用于干眼症的协同治疗
2.1 2CsA-ASONFKBIZ偶联物及其自组装纳米胶束的制备与表征
本实施例中所采用的靶向NFKBIZ基因的反义寡核苷酸均购自生工生物工程(上海)股份有限公司，在反义寡核苷酸3’端我们引入了2个二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，其中一个DBCO修饰与实施例1中相同，位于核酸序列3’端；第二个DBCO修饰靠近3’端，为核酸序列中间T碱基修饰，在以下所有实施例中含中间修饰DBCO的T碱基均以/iDBCOdT/符号表示，其结构如下所示：
本实施例中使用的核酸序列如下：
其中靶向人源NFKBIZ基因的反义寡核苷酸(2DBCO-ASONFKBIZ-h)序列为：
Human:5’-ATCAGACAACGAATCGGGC/iDBCOdT/TT-DBCO-3’(SEQ ID NO.3)；
而靶向鼠源NFKBIZ基因的反义寡核苷酸(2DBCO-ASONFKBIZ-m)序列为：
Mouse:5’-AATACTGGTACATTGACGCC/iDBCOdT/TT-DBCO-3’(SEQ ID NO.4)。
2CsA-ASONFKBIZ偶联物的合成方法为：将0.4mg实施例1中制备的CsA-N3(260nmol)溶于0.13mL二甲亚砜(DMSO)中，加入5OD(Optical Density)量的2DBCO-ASONFKBIZ(26nmol)后，放置于50℃下震荡反应24h。加入5mL水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，浓缩蒸干后，即得到2CsA-ASONFKBIZ偶联物分子，然后将其用50μL DMSO溶液重新溶解，然后滴入500μL PBS中进行自组装形成2CsA-ASONFKBIZ纳米胶束，然后在PBS溶液中透析除去DMSO得到最终的自组装核酸纳米胶束。通过10％变性聚丙烯酰胺凝胶电泳以及1％琼脂糖凝胶电泳验证CsA与反义寡核苷酸的成功接枝偶联以及纳米胶束的成功组装，见图11。从图中可以看出，CsA接枝2DBCO-ASONFKBIZ的效率较高，产率95％以上，同时组装后形成的2CsA-ASONFKBIZ纳米胶束尺寸较为均一，在1％琼脂糖凝胶电泳图中表现为单一条带。
2.2 2CsA-ASONFKBIZ-h纳米胶束对靶标NFKBIZ基因的调控作用
将HCEC细胞按照1×105细胞/孔的密度接种于6孔板培养至贴壁，然后采用100ng/mL LPS刺激过夜，去掉上清液后，分别加入含ASONFKBIZ-h+Lipo2000复合物以及2CsA-ASONFKBIZ-h纳米胶束的Opti-MEM培养基1mL(均含10μM ASO)，37℃共孵育转染6h后，更换DMEM培养基继续孵育48h。收集细胞并提取RNA进行RT-qPCR检测，以分析HCEC细胞中靶标基因NFKBIZ mRNA表达水平，实验结果如图12所示。
从图中可以看出，LPS刺激后的HCEC细胞，其NFKBIZ mRNA表达增至正常细胞的1.4倍左右，Lipo2000转染后的ASONFKBIZ-h可将其下调至正常细胞水平，2CsA-ASONFKBIZ-h纳米胶束共孵育处理后LPS刺激的HCEC细胞中NFKBIZ mRNA表达量下降至正常细胞的0.8倍左右。综上，相比于单一靶向NFKBIZ的ASO，2CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因的表达具有更强的敲低能力。
2.3 2CsA-ASONFKBIZ-m纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后分别使用的院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)与2CsA-ASONFKBIZ-m(400μM CsA)进行眼部滴眼治疗，每只眼睛每次滴5μL，每日早晚各滴一次。同时在治疗的第0，7，14d进行荧光素钠染色评估与酚红棉线泪液分泌量测试，以评估治疗效果，实验结果如图13所示。
荧光素钠染色结果显示，院配环孢素A滴眼液治疗后小鼠眼部评分6分降至4分左右，其对应酚红棉线泪液测试结果由1.5mm增加至3mm左右。而2CsA-ASONFKBIZ-m纳米胶束治疗后小鼠眼部荧光素钠染色评分由6分降至3.5分左右，酚红棉线浸润长度由1.5mm增至3.5mm左右。从动物实验结果中可以看出，相比于病例组和院配CsA滴眼液治疗组，2CsA-ASONFKBIZ-m纳米胶束对小鼠干眼的治疗效果更为明显，一是由于自组装形成的球形核酸纳米胶束具有更好的入胞能力，二是小分子药物与核酸药物能够实现协同治疗，从而更好地实现抑制小鼠眼部组织炎症，达到干眼治疗的目的。
实施例3.三环孢素A修饰NFKBIZ反义寡核苷酸共价偶联物(3CsA-ASONFKBIZ)及其组装结构用于干眼病的协同治疗
3.1 3CsA-ASONFKBIZ偶联物及其自组装纳米胶束的制备与表征
本实施例中所采用的靶向NFKBIZ基因的反义寡核苷酸均购自生工生物工程(上海)股份有限公司，在反义寡核苷酸3’端我们引入了3个二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，其中一个DBCO修饰位于核酸序列3’端；另外 两个均为核酸序列中间T碱基DBCO修饰，靠近3’端，其序列如下：
靶向人源NFKBIZ基因的反义寡核苷酸(3DBCO-ASONFKBIZ-h)序列为：
Human:5’-ATCAGACAACGAATCGGGC/iDBCOdT/T/iDBCOdT/TT-DBCO-3’(SEQ ID NO.5)；
靶向鼠源NFKBIZ基因的反义寡核苷酸(3DBCO-ASONFKBIZ-m)序列为：
Mouse:5’-AATACTGGTACATTGACGCC/iDBCOdT/T/iDBCOdT/TT-DBCO-3’(SEQ ID NO.6)。
3CsA-ASONFKBIZ偶联物合成方法为：将0.6mg CsA-N3(390nmol)溶于0.13mL DMSO中，加入5OD DNA(26nmol)后，放置于50℃下震荡反应24h。加入5mL水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，浓缩蒸干后，即得到3CsA-ASONFKBIZ偶联物分子，然后将其用50μL DMSO溶液重新溶解，所得溶液滴入500μL PBS中进行组装形成3CsA-ASONFKBIZ纳米胶束，然后在PBS溶液中透析除去DMSO得到最终的纳米胶束组装结构。通过10％变性聚丙烯酰胺凝胶电泳与1％琼脂糖凝胶电泳验证其成功接枝与组装，见图14。从图中可以看出，CsA接枝3DBCO-ASONFKBIZ的效率较高，同时组装后的尺寸较为均一。
3.2 3CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因调控作用
将HCEC细胞按照1×105细胞/孔的密度接种于6孔板中孵育至贴壁，用100ng/mL LPS刺激过夜，去掉上清液，分别加入含ASONFKBIZ-h+Lipo2000复合物以及3CsA-ASONFKBIZ-h纳米胶束的Opti-MEM培养基1mL(均含10μM ASO)，37℃共孵育转染6h后，更换为DMEM培养基继续孵育48h，后收集细胞并提取RNA进行RT-qPCR测试，以评估HCEC细胞中NFKBIZ mRNA的表达，实验结果如图15所示。
从图中可以看出，LPS刺激后的HCEC细胞，其NFKBIZ mRNA表达增至正常细胞的1.4倍左右，Lipo2000转染后的ASONFKBIZ-h可将其下调至正常细胞水平，3CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因下调至正常细胞的0.75倍左右，因此相比于单一的ASO，3CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因的表达下调能力更佳。
3.3 3CsA-ASONFKBIZ-m纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后分别使用院配环孢素A滴眼液(400μM CsA，上海复旦大学医学院附属眼耳鼻喉专科医院提供)与3CsA-ASONFKBIZ-m纳米胶束(133μM ASO浓度，400μM CsA)进行眼部滴眼治疗，每只眼睛每次滴5μL，每日早晚各滴一次。同时在治疗的第0，7，14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图16所示。
荧光素钠染色结果显示，院配环孢素A滴眼液治疗后的小鼠眼部评分10分降至8分左右，酚红棉线泪液测试结果由1mm增至2.5mm左右；3CsA-ASONFKBIZ-m纳米胶束治疗后的小鼠眼部荧光素钠评分由10分降至4分左右，酚红棉线浸润长度由1.3mm增至4mm左右。从动物实验结果中可以看出，相比于病例组与单纯的CsA滴眼液组，3个CsA与靶向NFKBIZ的ASO偶联后组装形成的纳米胶束对小鼠干眼的治疗效果更为明显。
实施例4环孢素A三聚体(CsA3)修饰NFKBIZ反义寡核苷酸共价偶联物(CsA3-ASONFKBIZ)及其纳米胶束组装结构用于干眼症治疗
4.1连接子氨基三[(2-丙炔基氧)甲基]甲烷的合成，步骤见图17
a)Boc-氨基三[(2-丙炔基氧)甲基]甲烷的合成。将1.0g Boc-氨基三(羟甲基)甲烷(4.52mmol，1当量)溶于10mL无水N,N-二甲基甲酰胺(DMF)中。将混合液置于冰浴中搅拌，加入3.2g溴丙炔(27.12mmol，6当量)。然后在15min内分3个批次将1.5g氢氧化钾粉末(27.12mmol，6当量)加入混合液中，反应升温至35℃，在氮气保护氛围下继续反应24h。反应液中加入100mL乙酸乙酯后，用200mL去离子水分3次洗涤。萃取收集乙酸乙酯有机相，加入无水硫酸钠除水后旋蒸蒸干，产物通过硅 胶柱层析进行纯化得到Boc-氨基三[(2-丙炔基氧)甲基]甲烷。洗脱剂为正己烷/乙酸乙酯混合溶液。Boc-氨基三(炔丙基)甲烷产率为64％。产物核磁共振图谱及各峰归属见图18。
b)氨基三[(2-丙炔基氧)甲基]甲烷的合成。将0.5g Boc-氨基三[(2-丙炔基氧)甲基]甲烷溶于5mL无水二氯甲烷中。然后将混合液放置于冰水浴中并进行搅拌，缓慢滴加3mL三氟乙酸(TFA)至混合液中继续搅拌约2h。反应结束后，利用旋转蒸发仪将溶剂蒸干，然后加入15mL饱和碳酸氢钠溶液，同时加入50mL乙酸乙酯萃取3次，收集合并有机层后用15mL去离子水洗涤，然后再次收集有机相后加入无水硫酸钠除水并通过旋蒸蒸干溶剂，最后得到氨基三[(2-丙炔基氧)甲基]甲烷，其产率为98％。
4.2环孢素A三聚体-ASO偶联物的合成(CsA3-ASO)，步骤见图19
本实施例中所采用的靶向NFKBIZ基因的反义寡核苷酸均购自生工生物工程(上海)股份有限公司，在反义寡核苷酸3’端我们引入了N-羟基琥珀酰亚胺脂(N-Hydroxysuccinimide ester，NHS ester)功能基团，使其能够与氨基三[(2-丙炔基氧)甲基]甲烷发生偶联反应得到3’端了三个炔基的反义寡核苷酸，进而与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，3’端NHS基团修饰在以下所有实施例中均以-NHS-3’表示，其修饰结构如下所示：
本实施例中使用的核酸序列如下：
靶向人源NFKBIZ基因的反义寡核苷酸(NHS-ASONFKBIZ-h)序列为：
Human:5’-ATCAGACAACGAATCGGGC-NHS-3’(SEQ ID NO.7)；
靶向鼠源NFKBIZ基因的反义寡核苷酸(NHS-ASONFKBIZ-m)序列为：
Mouse:5’-AATACTGGTACATTGACGCC-NHS-3’(SEQ ID NO.8)。
CsA3-ASO偶联物的合成方法为：
a)分别将1.20mg氨基三[(2-丙炔基氧)甲基]甲烷(5.12μmol，50当量)溶于800μL DMSO中，20OD羟基琥珀酰亚胺修饰的反义寡核苷酸(NHS-ASO,102.40nmol，1当量)溶于200μL1×PBS缓冲溶液中，按照DMSO与1×PBS缓冲溶液体积比4:1将两相充分混合后，在室温条件下搅拌过夜。加入去离子水后，用乙酸乙酯萃取除去反应中过量的氨基三[(2-丙炔基氧)甲基]甲烷，浓缩蒸干后，即得到三炔丙基修饰的ASO偶联物分子。在1×TBE缓冲溶液中，600V电压条件下运行2h，通过20％变性聚丙烯酰胺凝胶电泳验证偶联物分子成功接枝，见图20。
b)分别将1.09mg叠氮己酸碳酸酯键连接的环孢素A(CsA-N3,0.77μmol，15当量)和0.56mg CuI·TBTA(0.77μmol，15当量)溶于475μL DMF中，10OD三炔丙基修饰ASO偶联物分子(51.20nmol，1当量)溶于25μL无菌水中，按照DMF与水体积比为95:5将两相充分混合后，反应在氮气保护条件下搅拌过夜。然后真空浓缩至微量体积，通过高效液相色谱(HPLC)纯化得到环孢素A三聚体-ASO偶联物(CsA3-ASONFKBIZ)。在1×TAE缓冲溶液中，80V电压条件下运行20min，通过0.5％琼脂糖凝胶电泳验证其成功接枝，见图21。
4.3 CsA3-ASONFKBIZ纳米胶束的制备和表征
将CsA3-ASONFKBIZ偶联物(10OD ASO)溶于100μL DMSO中，逐滴加入至300μL连续搅拌的1×PBS缓冲溶液中。随后将溶液放入透析袋中透析过夜除去DMSO。3000转离心5min后，即可得到CsA3-ASONFKBIZ胶束纳米粒子。通过DLS、TEM表征其直径和形貌，如图22所示。
4.4 CsA3-ASONFKBIZ-h纳米胶束对NFKBIZ基因的调控效果
将HCEC细胞按照1×105细胞/孔的密度接种于6孔板中孵育至贴壁，用100ng/mL LPS刺激过夜，分别加入含有ASONFKBIZ-h+Lipo2000复合物以及CsA3-ASONFKBIZ-h纳米胶束的Opti-MEM培养基1mL(均含10μM ASO)，在37℃共孵育转染6h后，更换DMEM培养基继续孵育48h。后收集细胞并提取RNA进行RT-qPCR检测，以评估HCEC细胞中NFKBIZ mRNA的表达量，实验结果如图23所示。
从图中可以看出，LPS刺激过后的HCEC细胞中NFKBIZ mRNA表达水平约为正常细胞的1.5倍，使用Lipo2000转染ASO后NFKBIZ mRNA的含量降低至正常细胞的表达水平，加入CsA3-ASONFKBIZ-h纳米胶束后NFKBIZ mRNA表达量降至正常细胞的0.5倍左右。由此结果可以看出，CsA3-ASONFKBIZ-h纳米胶束对NFKBIZ mRNA具有显著下调能力。
4.5 CsA3-ASONFKBIZ-m纳米胶束对干眼症治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后分别使用院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)与CsA3-ASONFKBIZ-m纳米胶束(400μM CsA)进行眼部滴眼治疗，每只眼睛每次滴5μL，每日早晚各滴一次。同时在治疗的第0，14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图24所示。
荧光素钠染色结果显示，院配环孢素A滴眼液治疗后的小鼠眼部评分10分降至7.5分左右，酚红棉线泪液测试结果由1.5mm增至2mm左右；CsA3-ASONFKBIZ-m纳米胶束治疗后的小鼠眼部荧光素钠评分由10分降至6分左右，酚红棉线浸润长度由1.5mm增至3.5mm左右。从动物实验结果中可以看出，相比于病例组与单纯的CsA滴眼液组，3个CsA与靶向NFKBIZ的ASO偶联后组装形成的纳米胶束对小鼠干眼的治疗效果更为明显。
实施例5.单个、两个、以及三个环孢素A分子与靶向NFKBIZ小干扰RNA偶联物(nCsA-siRNANFKBIZ，n＝1,2,或3)用于干眼病的协同治疗
5.1单个、两个以及三个环孢素A与siRNANFKBIZ偶联物(nCsA-siRNANFKBIZ，n＝1,2,或3)的制备与表征
本实施例中所采用的靶向NFKBIZ基因的小干扰RNA均购自生工生物工程(上海)股份有限公司，在小干扰RNA正义链(Sense链)3’端我们引入了n(n＝1,2,或3)个二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联。下面详细例举了本实施例中所选用的siRNA具体序列信息。
修饰1、2、3个DBCO并靶向人源NFKBIZ基因的siRNA序列如下：
DBCO-siRNANFKBIZ-h
Sense 5’-rGrCrCrCrGrArUrUrCrGrUrUrGrUrCrUrGrArUdTdT-DBCO-3’(SEQ ID NO.9)；
Antisense 5’-rArUrCrArGrArCrArArCrGrArArUrCrGrGrGrCdTdT-3’(SEQ ID NO.10)；
2DBCO-siRNANFKBIZ-h
Sense 5’-rGrCrCrCrGrArUrUrCrGrUrUrGrUrCrUrGrArUdTdT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.11)；
Antisense 5’-rArUrCrArGrArCrArArCrGrArArUrCrGrGrGrCdTdT-3’(SEQ ID NO.12)；
3DBCO-siRNANFKBIZ-h
Sense 5’-rGrCrCrCrGrArUrUrCrGrUrUrGrUrCrUrGrArUdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3(SEQ ID NO.13)
Antisense 5’-rArUrCrArGrArCrArArCrGrArArUrCrGrGrGrCdTdT-3’(SEQ ID NO.14)。
修饰1、2、3个DBCO的鼠源NFKBIZ siRNA序列如下：
DBCO-siRNANFKBIZ-m
Sense 5’-rGrCrGrUrCrArArUrGrUrArCrCrArGrUrArUrUdTdT-DBCO-3’(SEQ ID NO.15)
Antisense 5’-rArArUrArCrUrGrGrUrArCrArUrUrGrArCrGrCdCdT-3’(SEQ ID NO.16)
2DBCO-siRNANFKBIZ-m
Sense 5’-rGrCrGrUrCrArArUrGrUrArCrCrArGrUrArUrUdTdT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.17)
Antisense 5’-rArArUrArCrUrGrGrUrArCrArUrUrGrArCrGrCdCdT-3’(SEQ ID NO.18)
3DBCO-siRNANFKBIZ-m
Sense 5’-rGrCrGrUrCrArArUrGrUrArCrCrArGrUrArUrUdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.19)
Antisense 5’-rArArUrArCrUrGrGrUrArCrArUrUrGrArCrGrCrCdT-3’(SEQ ID NO.20)
环孢素A与siRNA偶联物合成方法为：分别将0.2mg、0.4mg、0.6mg CsA-N3(140nmol、280nmol、420nmol)溶于0.14mL DMSO中，分别加入5OD DBCO-siRNANFKBIZ Sense链、2DBCO-siRNANFKBIZ Sense链，3DBCO-siRNANFKBIZ Sense链(各28nmol)后，置于37℃下震荡反应24h。加入5mL水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，浓缩蒸干后，即得到CsA-siRNANFKBIZ Sense链、2CsA-siRNANFKBIZ Sense链、3CsA-siRNANFKBIZ Sense链偶联物分子。将所制备的CsA-siRNANFKBIZ Sense链与等物质量siRNANFKBIZ Antisense链在1×PBS中混合，Sense链和Antisense链互补配对即可形成可溶的CsA-siRNANFKBIZ偶联物双链分子。而对于修饰两个和三个CsA的CsA-siRNANFKBIZ Sense链，首先将2CsA-siRNANFKBIZ Sense链、3CsA-siRNANFKBIZ Sense链重新溶解于50μL DMSO中，然后滴入500μL1×PBS缓冲溶液中进行组装，通过在1×PBS中透析除去DMSO得到2CsA-siRNANFKBIZ Sense链纳米胶束、3CsA-siRNANFKBIZ Sense链纳米胶束，最后与加入Antisense链互补配对形成2CsA-siRNANFKBIZ、和2CsA-siRNANFKBIZ纳米胶束。利用聚丙烯酰胺凝胶电泳实验与琼脂糖凝胶电泳实验对所得产物进行表征，结果如图25所示。从图中可以看出，CsA-N3成功与siRNANFKBIZ Sense链偶联，且不影响后续与相应Antisense链的互补配对与组装。
5.2 nCsA-siRNANFKBIZ-h(n＝1,2,3)及其组装结构对IκB-ζ蛋白的调控作用
nCsA-siRNANFKBIZ-h(n＝1,2,3)纳米胶束旨在降低细胞中的NFKBIZ mRNA水平，从而进一步降低蛋白层面的表达。为了检验HCEC细胞中的IκB-ζ蛋白表达水平以及不同药物对其的下调效果，因此进行western blot实验进行表征。将HCEC细胞按照1×105细胞/孔的密度接种于于6孔板中贴壁过夜后用100ng/mL LPS刺激过夜，去掉上清液后，分别加入含有siRNANFKBIZ-h+Lipo2000复合物以及nCsA-siRNANFKBIZ-h(n＝1,2,3)偶联物和纳米胶束的Opti-MEM培养基1mL(均含有10μM siRNA)，在37℃共孵育转染6h后换为DMEM培养基，继续孵育48h后提取细胞中的蛋白，进行western blot实验。
实验结果如图26所示，从数据中可以看出，LPS刺激过后的HCEC细胞中IκB-ζ蛋白表达水平约为正常细胞的1.5倍，使用Lipo2000转染siRNANFKBIZ-h后的IκB-ζ蛋白表达水平约为正常细胞的1.2倍，加入CsA-siRNANFKBIZ-h偶联物、2CsA-siRNANFKBIZ纳米胶束和3CsA-siRNANFKBIZ纳米胶束后IκB-ζ蛋白表达量降至正常细胞的0.5-0.8倍左右。因此nCsA-siRNANFKBIZ-h可以有效降低LPS刺激后炎症 引起的IκB-ζ蛋白表达量，且随着CsA修饰个数的增加，其降低IκB-ζ蛋白的效果更佳；与此同时，nCsA-siRNANFKBIZ-h(n＝1,2,3)偶联物以及纳米胶束的效果均优于Lipo2000转染siRNANFKBIZ-h组。
5.3 nCsA-siRNANFKBIZ-h(n＝1,2,3)及其组装结构对NFKBIZ基因的调控作用
将HCEC细胞按照1×105细胞/孔的密度接种于6孔板中贴壁过夜后用100ng/mL LPS刺激12h，去掉上清液后，分别加入含有siRNANFKBIZ-h+Lipo2000复合物以及nCsA-siRNANFKBIZ-h(n＝1,2,3)偶联物和纳米胶束的Opti-MEM培养基1mL(均含有10μM siRNA)，在37℃共孵育转染6h后换为DMEM培养基，继续孵育48h后收集细胞并提取RNA进行RT-qPCR实验以评估HCEC细胞中NFKBIZ mRNA的表达，实验结果如图27所示。
从数据中可以看出，相比于空白组(Mock组)，CsA-siRNANFKBIZ-h对NFKBIZ基因的表达量有明显降低作用，可将其降低至LPS刺激前的水平；与此同时，LPS刺激过后的HCEC细胞中NFKBIZ表达量增加至正常细胞的1.5倍左右，分别接枝了1个、2个和3个CsA的siRNANFKBIZ-h，随着CsA修饰个数的增加，其对NFKBIZ基因的降低作用增加，分别将细胞中NFKBIZ的表达量降低至正常细胞的0.5～0.7倍左右。这可能是由于CsA的疏水作用增强了siRNANFKBIZ-h的入胞能力，同时二者协同作用具有更好的炎症治疗能力。
5.4 nCsA-siRNANFKBIZ-m(n＝1,2,3)及其组装结构对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周，建模成功后分别使用的院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)与CsA-siRNANFKBIZ-m(400μM CsA)、2CsA-siRNANFKBIZ-m纳米胶束(400μM CsA)、3CsA-siRNANFKBIZ-m纳米胶束(400μM CsA)进行眼部滴眼治疗，每只眼睛每次滴5μL，每日早晚各滴一次。在治疗的第0，7，14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图28所示。
荧光素钠染色结果显示，院配环孢素A滴眼液治疗后的小鼠眼部评分6分降至4.5分左右，酚红棉线泪液测试结果由1.5mm增至2.5mm左右；CsA-siRNANFKBIZ治疗后的小鼠眼部评分6分降至3分左右，酚红棉线泪液测试结果由2mm增至2.5mm左右；2CsA-siRNANFKBIZ纳米胶束治疗后的小鼠眼部评分6分降至2分左右，酚红棉线泪液测试结果由1.5mm增至2.5mm左右；3CsA-siRNANFKBIZ纳米胶束治疗后的小鼠眼部评分6分降至2分左右，酚红棉线泪液测试结果由1.5mm增至2.5mm左右；从动物实验结果中可以看出，相比于病例组与单纯的CsA滴眼液组，CsA-siRNANFKBIZ-m偶联物、2CsA-siRNANFKBIZ-m纳米胶束、3CsA-siRNANFKBIZ-m纳米胶束对小鼠干眼的治疗效果更为明显，一是由于小分子药物与核酸药物协同治疗，可以更好的达到抑制小鼠眼部细胞炎症的目的，二是疏水的CsA赋予了siRNA更好的入胞能力。
实施例6多个环孢素A分子接枝NFKBIZ反义寡核苷酸偶联物及其自组装纳米胶束用于干眼病的治疗
6.1羰乙基溴代环孢素A(CsA-Br)的合成与表征
如图29所示，首先将CsA(100mg)与三光气(9mg)置于烧瓶中，抽排空气三次后在氮气保护下加入16mL二氯甲烷进行溶解，然后将DMAP(31mg)用1mL二氯甲烷溶解后逐滴加入反应液中，室温反应30min，现象为溶液变为乳白色，随后将丁二醇(72mg)用1mL二氯甲烷溶解后逐滴加入反应液中，室温反应过夜，现象为溶液从乳白色变为无色透明。反应结束后根据DMAP的量，用稀盐酸溶液洗涤三次，饱和氯化钠溶液洗涤1次，无水硫酸钠粉末干燥超过2h。减压蒸馏浓缩有机相后再经硅胶柱层析分离纯化，洗脱液为石油醚/乙酸乙酯混合溶液，后得到白色固体，即丁二醇修饰的环孢素A分子。
将CsA-丁二醇(90mg)与DIPEA(8mg)置于烧瓶中，抽排空气三次后在氮气保护下加入16mL二氯甲烷，随后将溴乙酰溴(16mg)用1mL二氯甲烷溶解后逐滴加入反应液中，室温反应过夜。反应结 束后根据DIPEA的量，用稀盐酸溶液洗涤三次，饱和氯化钠溶液洗涤1次，无水硫酸钠粉末干燥超过2h，减压蒸馏浓缩有机相后再经硅胶柱层析分离纯化，洗脱液为石油醚/乙酸乙酯混合溶液，最后得到黄色固体，即制得羰乙基溴代环孢素A。1H NMR(700MHz,Chloroform-d)δ7.99(d,J＝9.7Hz,1H),7.64(d,J＝7.5Hz,1H),7.47(d,J＝8.3Hz,1H),7.41(s,1H),7.28(s,1H),7.16(d,J＝7.9Hz,2H),7.11(s,1H),5.70(d,J＝6.6Hz,1H),5.48(d,J＝6.3Hz,1H),5.36–5.32(m,3H),5.12(d,J＝10.9Hz,1H),5.08–5.02(m,3H),4.97(dd,J＝9.9,5.9Hz,1H),4.85–4.81(m,2H),4.72(d,J＝13.9Hz,1H),4.65(dd,J＝9.9,8.4Hz,2H),4.52(t,J＝7.3Hz,2H),3.80(t,J＝6.5Hz,2H),3.51(s,3H),3.40(s,3H),3.26(s,3H),3.20(s,1H),3.18(s,1H),3.11(d,J＝1.6Hz,5H),2.96(s,1H),2.89(s,1H),2.69(d,J＝5.8Hz,6H),2.41(dd,J＝12.1,7.7Hz,2H),2.13(d,J＝11.1Hz,2H),2.08–2.05(m,2H),1.99(s,1H),1.77(s,2H),1.73–1.69(m,2H),1.63(d,J＝7.4Hz,4H),1.58(s,6H),1.47(d,J＝6.5Hz,1H),1.43(s,1H),1.35(d,J＝7.3Hz,3H),1.25(d,J＝6.7Hz,6H),1.07(d,J＝6.5Hz,2H),1.02(dd,J＝14.8,6.6Hz,7H),0.97–0.93(m,8H),0.89–0.84(m,13H),0.83(d,J＝6.6Hz,3H),0.71(d,J＝6.1Hz,3H).
羰乙基溴代环孢素A由于溴元素存在等位离子峰，其理论m/z值为1437.8098和1439.8077。高效液相色谱/四极杆飞行质谱联用仪测得的m/z值为1438.4549和1440.4506，归属于羰乙基溴代环孢素A的[M+H]+峰，证实目标产物成功合成，如图30所示。
6.2 20CsA-ASONFKBIZ偶联物与20CsA-ASONFKBIZ纳米胶束的制备与表征
本实施例中所采用的靶向NFKBIZ基因的反义寡核苷酸均购自生工生物工程(上海)股份有限公司，我们选择在反义核苷酸磷酸骨架中进行硫代(PS)修饰，使其能够与修饰在CsA-Br上的溴代羰乙基基团发生取代反应从而高效偶联，其序列如下：
靶向人源NFKBIZ基因的反义寡核苷酸(20PS-ASONFKBIZ-h)序列为：
5’-ATCAGACAACGAATCGGGCTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3’(其中*表示硫代磷酸酯基团修饰位点，表示前后两个核苷酸单元通过硫代磷酸酯基团连接，以下实施例中如无特定说明核酸序列中硫代磷酸酯修饰均由*表示，下划线TTTTT为连接5’端ASO和3’端用于药物修饰序列的多个T碱基间隔序列，下述实施例中所列序列下划线部分均代表连接功能小核酸序列和药物修饰序列之间的间隔序列，不再一一赘述)(SEQ ID NO.21)；
其中靶向鼠源NFKBIZ基因的反义寡核苷酸(20PS-ASONFKBIZ-m)序列为：
5’-AATACTGGTACATTGACGCCTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.22)；
合成方法为：将7.5mg CsA-Br(5μmol)溶于0.063mL DMSO中，加入5OD20PS-ASONFKBIZ(12.5nmol)后，放置于50℃下震荡反应24h。加入5mL水后，用乙酸乙酯萃取除去反应中过量的CsA-Br，浓缩蒸干后，即得到20CsA-ASONFKBIZ偶联物分子。将20CsA-ASONFKBIZ偶联物溶于50μL DMSO中，逐滴加入至0.5mL磷酸盐缓冲液(PBS)中，搅拌30min。随后放入透析袋中并在PBS中透析过夜，除去DMSO，即可得到20CsA-ASONFKBIZ纳米胶束结构。通过10％变性聚丙烯酰胺凝胶电泳和1％琼脂糖凝胶电泳进行表征。从图31中可以看出，CsA-Br成功接枝20PS-ASONFKBIZ，并形成了尺寸较为均一的20PS-ASONFKBIZ纳米胶束组装体。
6.3 20CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因敲低作用
将HCEC细胞按照1×105细胞/孔的密度接于6孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液，分别加入含有ASONFKBIZ-h+Lipofectamine2000(Lipo2000)以及20CsA-ASONFKBIZ-h纳米胶束的Opti-MEM培养基1mL(均含有10μM ASO)，在37℃共孵育转染6h后换为DMEM培养基，更换新的培养基后继续孵育48h，后收集细胞并提取RNA进行RT-qPCR实验，以检验HCEC细胞中NFKBIZ mRNA表达水平，实验结果如图32所示。
从图中可以看出，LPS刺激后的HCEC细胞，其NFKBIZ mRNA表达增至正常细胞的1.5倍左 右，Lipo2000转染后的ASONFKBIZ-h可将其下调至与正常细胞水平，20CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因下调至正常细胞表达量的0.75倍左右，因此相比于单一的ASONFKBIZ-h，20CsA-ASONFKBIZ-h纳米胶束对NFKBIZ基因的表达下调能力更佳。
6.4 20CsA-ASONFKBIZ-m纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后分别使用院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)与20CsA-ASONFKBIZ-m纳米胶束(20μM，400μM CsA)进行眼部滴眼治疗，每只眼睛每次滴5μL，每日早晚各滴一次。同时在治疗的第0和14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图33所示。
从动物实验结果中可以看出，荧光素钠染色结果显示，院配环孢素A滴眼液治疗后的小鼠眼部评分9分升至11分左右，未取得明显改善，酚红棉线泪液测试结果维持2.5mm左右无明显变化；20CsA-ASONFKBIZ-m纳米胶束治疗后的小鼠眼部荧光素钠评分由9分降至7.5分左右，酚红棉线浸润长度由3mm增至5.5mm左右。因此可以看出相比于院配环孢素A滴眼液，在两周内20CsA-ASONFKBIZ-m纳米胶束对小鼠干眼病的治疗效果显著。
实施例7.环孢素A分子分别与靶向IL-17、IL-1β、IL-23以及TNF-α的小干扰核酸共价偶联物及其自组装纳米胶束用于干眼症的治疗
7.1环孢素A与不同靶点siRNA偶联物及其纳米胶束的制备
本实施例中所采用的靶向不同炎症因子基因的小干扰核酸均购自生工生物工程(上海)股份有限公司，在siRNA正义链的3’端我们引入了3个二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，其序列如下：
靶向人源不同炎症因子基因的siRNA序列为：
IL-17 human(3DBCO-siRNAIL-17-h)
Sense 5’-rCrUrCrUrArArUrGrArGrUrUrUrArGrUrCrCrGrAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.23)
Antisense 5’-rUrCrGrGrArCrUrArArArCrUrCrArUrUrArGrArGdTdT-3'(SEQ ID NO.24)
IL-1βhuman(3DBCO-siRNAIL-1β-h)
Sense 5’-rArGrGrCrUrGrArUrCrUrGrUrUrGrCrCrGrUrAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.25)
Antisense 5’-rUrArCrGrGrCrArArCrArGrArUrCrArGrCrCrUdTdG-3’(SEQ ID NO.26)
IL-23 human(3DBCO-siRNAIL-23-h)
Sense 5’-rCrArGrCrArArCrCrCrUrGrArGrUrCrCrCrUrAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.27)
Antisense 5’-rUrArGrGrGrArCrUrCrArGrGrGrUrUrGrCrUrGdTdT-3’(SEQ ID NO.28)
TNF-αhuman(3DBCO-siRNATNF-α-h)
Sense 5′-rGrArCrArArCrCrArArCrUrArGrUrGrGrUrGrCdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3′(SEQ ID NO.29)
Antisense 5'-rGrCrArCrCrArCrUrArGrUrUrGrGrUrUrGrUrCdTdT-3'(SEQ ID NO.30)
其中靶向鼠源不同炎症因子基因的siRNA序列为：
IL-17 mouse(3DBCO-siRNAIL-17-m)
Sense  5’-rArArGrArGrArUrCrCrUrGrGrUrCrCrUrGrArAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.31)
Antisense 5’-rUrUrCrArGrGrArCrCrArGrGrArUrCrUrCrUrUdGdC-3’(SEQ ID NO.32)
IL-1βmouse(3DBCO-siRNAIL-1β-m)
Sense 5′-rGrGrArArGrGrCrArGrUrGrUrCrArCrUrCrArUdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3′(SEQ ID NO.33)
Antisense 5′-rArUrGrArGrUrGrArCrArCrUrGrCrCrUrUrCrCdTdT-3′(SEQ ID NO.34)
IL-23 mouse(3DBCO-siRNAIL-23-m)
Sense 5'-rArCrArArCrCrArUrCrArCrCrArCrArCrUrGrGrArUrArCrGrGdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3'(SEQ ID NO.35)
Antisense 5’-rCrCrGrUrArUrCrCrArGrUrGrUrGrGrUrGrArUrGrGrUrUrGrUdTdT-3’(SEQ ID NO.36)
TNF-αmouse(3DBCO-siRNATNF-α-m)
Sense 5′-rGrUrCrUrCrArGrCrCrUrCrUrUrCrUrCrArUrUrCrCrUdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3′(SEQ ID NO.37)
Antisense 5’-rArGrGrArArUrGrArGrArArGrArGrGrCrUrGrArGrArCdTdT-3’(SEQ ID NO.38)
针对不同靶点3CsA-siRNA偶联物的合成方法为：将30倍摩尔当量的CsA-N3溶于DMSO中，加入5OD不同靶点的3DBCO-siRNA Sense链后，使其反应浓度为200μM，然后置于37℃下震荡反应24h。加入水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，浓缩蒸干后，即得到3CsA-siRNAIL-17Sense链、3CsA-siRNAIL-1βSense链、3CsA-siRNAIL-23Sense链以及3CsA-siRNATNF-αSense链偶联物分子。将其用50μL DMSO溶液重新溶解后滴入500μL PBS中进行组装形成3CsA-siRNAIL-17Sense链纳米胶束、3CsA-siRNAIL-1βSense链纳米胶束、3CsA-siRNAIL-23Sense链纳米胶束以及3CsA-siRNATNF-αSense链纳米胶束，后在PBS中透析除去DMSO。与Antisense链互补配对后得到分散于PBS溶液中的3CsA-siRNAIL-17纳米胶束、3CsA-siRNAIL-1β纳米胶束、3CsA-siRNAIL-23纳米胶束以及3CsA-siRNATNF-α纳米胶束样品。
7.2针对不同靶点3CsA-siRNA纳米胶束对靶向基因的调控作用
将密度为1×105细胞/孔的HCEC细胞置于6孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液后分别将1mL含有3CsA-siRNAIL-17-h纳米胶束、3CsA-siRNAIL-1β-h纳米胶束、3CsA-siRNAIL-23-h纳米胶束以及3CsA-siRNATNF-α-h纳米胶束的Opti-MEM培养基(均含有10μM siRNA)，对照组为1mL含有不同靶点siRNA与Lipo2000复合物的Opti-MEM培养基(siRNA浓度均为10μM)，在37℃下共孵育转染6h后换为DMEM培养基，继续孵育48h后收集细胞并提取RNA进行PCR实验，以检验HCEC细胞中不同靶点的mRNA表达水平。实验结果如图34所示。
从图中可以看出，LPS刺激过后，不同炎症因子的mRNA表达量均有上调，在正常细胞中各炎症mRNA表达水平的1.2-1.4倍左右。在加入Lipo2000转染不同靶点的siRNA后，不同炎症因子表达量降低至正常细胞水平的0.8-1.1倍之间；加入不同靶点的纳米胶束材料后，不同炎症因子表达量降低至正常细胞水平的0.75倍左右。此部分数据说明，不同靶点的3CsA-siRNA纳米胶束材料具有良好的炎症因子基因下调效果。
7.3针对不同靶点3CsA-siRNA纳米胶束对相关炎症因子表达的抑制作用
将HCEC细胞按照5×104细胞/孔的密度接种于12孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液后分别将1mL含有3CsA-siRNAIL-17-h纳米胶束、3CsA-siRNAIL-1β-h纳米胶束、 3CsA-siRNAIL-23-h纳米胶束以及3CsA-siRNATNF-α-h纳米胶束的Opti-MEM培养基(均含有10μM siRNA)，对照组为1mL含有不同靶点siRNA与Lipo2000复合物的Opti-MEM培养基(siRNA浓度均为10μM)，在37℃下共孵育转染6h后换为DMEM培养基，继续孵育48h后收集上清液进行ELISA检测HCEC细胞不同炎症因子的表达量，实验结果如图35所示。
从图中可以看出，LPS刺激过后，HCEC细胞中各炎症因子表达量均有明显上升，上升区间在正常细胞的1.5～2.5倍左右；在加入lipo2000转染的不同靶点siRNA后，其炎症因子表达量有所下降；经各纳米胶束材料处理后的HCEC细胞炎症因子表达量降低最多，恢复至正常细胞表达水平。这是由于组装成纳米胶束后的药物核酸偶联物具有更好的入胞能力，其次因为环孢素A与siRNA协同作用，达到了更好的炎症抑制效果。
7.4针对不同靶点3CsA-siRNA纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周，建模成功后分别使用院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)、3CsA-siRNAIL-17-m纳米胶束、3CsA-siRNAIL-1β-m纳米胶束、3CsA-siRNAIL-23-m纳米胶束以及3CsA-siRNATNF-α-m纳米胶束(每组材料中的CsA浓度均为400μM)进行眼部滴眼治疗，每只眼睛每日每次滴5μL，每日早晚各滴一次。在治疗的第0和14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图36所示。
荧光素钠染色结果显示，无治疗对照组小鼠眼部评分13分降至10分左右，酚红棉线泪液测试结果由1mm增至1.3mm左右，没有明显好转；CsA滴眼液治疗的小鼠眼部评分由12分降至9分左右，酚红棉线泪液测试结果由1.5mm增加至3mm左右；不同靶点的siRNA纳米胶束治疗组的小鼠荧光素钠染色评分约从13分降至8分左右，酚红棉线浸润长度从1.5mm升至4mm左右。由此部分数据结果可以看出，针对不同炎症因子靶点的3CsA-siRNA偶联物纳米胶束在两周时间内对小鼠干眼的治疗具有一定疗效。
实施例8.环孢素A分子分别与靶向NLRP3、JAK1和PDE4的小干扰核酸共价偶联物及其自组装纳米胶束用于干眼症的治疗
8.1环孢素A与不同靶点siRNA偶联物及其自组装纳米胶束的制备
本实施例中所采用的靶向细胞内不同炎症基因的小干扰RNA均购自生工生物工程(上海)股份有限公司，在siRNA正义链3’端我们引入了3个二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，其序列如下：
靶向人源不同炎症相关基因的siRNA序列为：
NLRP3 human(3DBCO-siRNANLRP3-h)
Sense 5’-rGrUrGrGrArCrUrUrGrArArGrArArArUrUrUrAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.39)
Antisense 5’-rUrArArArUrUrUrCrUrUrCrArArGrUrCrCrArCdTdT-3’(SEQ ID NO.40)
JAK1 human(3DBCO-siRNAJAK1-h)
Sense 5’-rGrGrArUrUrArCrArArGrGrArUrGrArCrGrArArGrGdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.41)
Antisense 5’-rUrUrCrGrUrCrArUrCrCrUrUrGrUrArArUrCrCrArUdTdT-3’(SEQ ID NO.42)
PDE4 human(3DBCO-siRNAPDE4-h)
Sense  5’-rGrArGrUrCrGrGrUrCrUrGrGrArArArUrCrArAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3’(SEQ ID NO.43)
Antisense 5’-rUrUrGrArUrUrUrCrCrArGrArCrCrGrArCrUrCdTdT-3’(SEQ ID NO.44)
其中靶向鼠源不同炎症相关基因的siRNA序列为：
NLRP3 mouse(3DBCO-siRNANLRP3-m)
Sense 5'-rGrGrUrGrArArArUrGrUrArCrUrUrArArArUrCdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3'(SEQ ID NO.45)
Antisense 5′-rGrArUrUrUrArArGrUrArCrArUrUrUrCrArCrCdTdT-3′(SEQ ID NO.46)
JAK1 mouse(3DBCO-siRNAJAK1-m)
Sense 5'-rCrUrGrUrArUrGrGrCrGrArCrArUrUrCrUrCrCrArAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3'(SEQ ID NO.47)
Antisense 5'-rUrUrGrGrArGrArArUrGrUrCrGrCrCrArUrArCrArGdTdT-3'(SEQ ID NO.48)
PDE4 mouse(3DBCO-siRNAPDE4-m)
Sense 5'-rArUrGrArGrCrGrUrGrUrArGrArGrArGrGrArCrArAdTdT/iDBCOdT/dT/iDBCOdT/dTdT-DBCO-3'(SEQ ID NO.49)
Antisense 5'-rUrUrGrUrCrCrUrCrUrCrUrArCrArCrGrCrUrCrArUdTdT-3'(SEQ ID NO.50)
针对不同靶点3CsA-siRNA偶联物的合成方法为：将30倍摩尔当量的CsA-N3溶于DMSO中，加入5OD不同靶点的3DBCO-siRNA Sense链后，使其反应浓度为200μM，然后置于37℃下震荡反应24h。加入水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，浓缩蒸干后，即得到3CsA-siRNANLRP3Sense链、3CsA-siRNAJAK1Sense链以及3CsA-siRNAPDE4Sense链偶联物分子。将其用50μL DMSO溶液重新溶解后滴入500μL PBS中进行组装形成3CsA-siRNANLRP3Sense链纳米胶束、3CsA-siRNAJAK1Sense链纳米胶束以及3CsA-siRNAPDE4Sense链纳米胶束，后在PBS中透析除去DMSO。与Antisense链互补配对后得到分散于PBS溶液中3CsA-siRNANLRP3纳米胶束、3CsA-siRNAJAK1纳米胶束以及3CsA-siRNAPDE4纳米胶束的样品。
8.2针对不同靶点3CsA-siRNA偶联物纳米胶束对靶向基因的调控作用
将密度为1×105细胞/孔的HCEC细胞置于6孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液后分别将1mL含有3CsA-siRNANLRP3-h纳米胶束、3CsA-siRNAJAK1-h纳米胶束以及3CsA-siRNAPDE4-h纳米胶束的Opti-MEM培养基(均含有10μM siRNA)，对照组为1mL含有不同靶点siRNA与Lipo2000复合物的Opti-MEM培养基(siRNA浓度均为10μM)，在37℃下共孵育转染6h后换为DMEM培养基，继续孵育48h后收集细胞并提取RNA进行PCR实验，以检验HCEC细胞中不同靶点的mRNA表达水平。实验结果如图37所示。
从图中可以看出，LPS刺激过后，不同胞内炎症靶点的mRNA表达量均有明显上调，在正常细胞中各炎症mRNA表达水平的1.4倍左右。在加入Lipo2000转染不同靶点的siRNA后，不同胞内炎症靶点基因表达量降低至正常细胞水平的1.1倍左右；加入不同靶点的纳米胶束材料后，不同胞内炎症靶点基因表达量降低至正常细胞水平的0.8倍左右。此部分数据说明，不同靶点的3CsA-siRNA纳米胶束材料具有良好的胞内炎症基因下调效果。
8.3针对不同靶点3CsA-siRNA偶联物纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周，建模成功后分别使 用院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)、3CsA-siRNANLRP3-m纳米胶束、3CsA-siRNAJAK1-m纳米胶束以及3CsA-siRNAPDE4-m纳米胶束(每组材料中的CsA浓度均为400μM)进行眼部滴眼治疗，每只眼睛每日每次滴5μL，每日早晚各滴一次。在治疗的第0和14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图38所示。
荧光素钠染色结果显示，无治疗对照组小鼠眼部评分12分降至10分左右，酚红棉线泪液测试结果都为1mm左右，没有明显好转；CsA滴眼液治疗后的小鼠眼部评分由10分降至8分左右，酚红棉线泪液测试结果由1mm增加至2mm左右；不同靶点的siRNA纳米胶束材料组的小鼠荧光素钠染色评分约从10分降至7分左右，酚红棉线浸润长度从1mm升至3～4.5mm之间。由此部分数据结果可以看出，针对不同胞内炎症靶点的药物-核酸偶联物纳米胶束在两周时间内对小鼠干眼的治疗具有明显疗效。
实施例9针对IL-17、IL-1β、IL-23和TNF-α不同靶点的三环孢素A反义寡核苷酸偶联物纳米胶束用于干眼病的治疗
9.1三个环孢素A分子与不同靶点ASO偶联物及其自组装纳米胶束的制备
本实施例中所采用的靶向不同炎症因子基因的反义寡核苷酸均购自生工生物工程(上海)股份有限公司，在反义寡核苷酸3’端我们引入了3个二苯并环辛炔(dibenzocyclooctyne,DBCO)修饰，使其能够与修饰在CsA-N3上的叠氮基团通过点击反应高效偶联，本实施例中所用ASO详细序列信息如下：
其中靶向人源不同炎症因子基因的反义寡核苷酸序列为：
IL-17 human(3DBCO-ASOIL-17-h)
5’-TCGGACTAAACTCATTAGAGTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3′(SEQ ID NO.51)
IL-1βhuman(3DBCO-ASOIL-1β-h)
5′-GGTACTTCTGCCATGGCTGCTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3′(SEQ ID NO.52)
IL-23 human(3DBCO-ASOIL-23-h)
5’-CATTACAGCTCTGCTCCCCAGCATCTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3’(SEQ ID NO.53)
TNF-αhuman(ASOTNF-α-h)
5‘-CAGTGCTCATGGTGTCTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3’(SEQ ID NO.54)
其中靶向鼠源不同炎症因子基因的反义寡核苷酸序列为：
IL-17 mouse(3DBCO-ASOIL-17-m)
5’-TTCAGGACCAGGATCTCTTGCTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3′(SEQ ID NO.55)
IL-1βmouse(3DBCO-ASOIL-1β-m)
5′-ATGAGTGACACTGCCTTCCTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3′(SEQ ID NO.56)
IL-23 mouse(3DBCO-ASOIL-23-m)
5’-CCGTATCCAGTGTGGTGATGGTTGTTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3’(SEQ ID NO.57)
TNF-αmouse(3DBCO-ASOTNF-α-m)
5’-AACCCATCGGCTGGCACCACTT/iDBCOdT/T/iDBCOdT/TT-DBCO-3’(SEQ ID NO.58)
本实施例中小分子药物与针对不同靶点的ASO偶联物合成方法为：将30倍摩尔当量的CsA-N3溶于DMSO中，加入5OD不同靶点的3DBCO-ASOX后，使其反应浓度为200μM，然后置于50℃下震荡反应24h。加入水后，用乙酸乙酯萃取除去反应中过量的CsA-N3，浓缩蒸干后，即得到3CsA-ASOIL-17、3CsA-ASOIL-1β、3CsA-ASOIL-23以及3CsA-ASOTNF-α偶联物分子，然后将其用50μL DMSO溶液重新溶解，滴入500μL PBS中进行组装形成3CsA-ASOIL-17纳米胶束、3CsA-ASOIL-1β纳米胶束、3CsA-ASOIL-23纳米胶束以及3CsA-ASOTNF-α纳米胶束，后在PBS中透析除去DMSO得到分 散于PBS溶液中的不同纳米胶束样品。
9.2针对不同靶点纳米胶束对相应基因的敲低效果
将1×105细胞/孔的HCEC细胞置于6孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液，分别加入1mL含有3CsA-ASOIL-17-h纳米胶束、3CsA-ASOIL-1β-h纳米胶束、3CsA-ASOIL-23-h纳米胶束以及3CsA-ASOTNF-α-h纳米胶束的Opti-MEM培养基(均含有10μM ASO)，对照组为1mL含有不同靶点ASO与Lipo2000复合物的Opti-MEM培养基(ASO浓度均为10μM)，在37℃共孵育转染6h后换为DMEM培养基，继续于37℃孵育48h，后收集细胞并提取RNA进行RT-qPCR检测，以检验HCEC细胞中不同靶点的mRNA表达水平。实验结果如图39所示。
从图中可以看出，LPS刺激过后，不同炎症因子的mRNA表达量均有上调，是正常细胞的1.3-1.5倍左右。在加入Lipo2000转染的不同靶点ASO后，不同炎症因子表达量降低至正常细胞中不同炎症因子表达量0.8-1.1倍之间；加入不同靶点的纳米胶束材料后，不同炎症因子表达量降低至正常细胞表达量的0.6-0.8倍之间。此部分数据说明，不同靶点的3CsA-ASO纳米胶束材料具有良好的炎症因子基因下调效果。
9.3不同靶点纳米胶束的炎症因子抑制作用
将HCEC细胞按照5×104细胞/孔的密度为接种于12孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液，分别将1mL含有3CsA-ASOIL-17-h纳米胶束、3CsA-ASOIL-1β-h纳米胶束、3CsA-ASOIL-23-h纳米胶束以及3CsA-ASOTNF-α-h纳米胶束的Opti-MEM培养基(均含有10μM ASO)，对照组为1mL含有不同靶点ASO与Lipo2000复合物的Opti-MEM培养基(ASO浓度均为10μM)，37℃共孵育转染6h后换为DMEM培养基，继续于37℃孵育48h，后收集上清液进行ELISA检测HCEC细胞不同炎症因子的表达量，实验结果如图40所示。
从图中可以看出，LPS刺激过后，HCEC细胞中各炎症因子表达量均有明显上升，上升区间在正常细胞的1.5-2.5倍左右；在加入Lipo2000转染不同靶点的ASO后，其炎症因子表达量有所下降；经各纳米胶束材料处理后的HCEC细胞炎症因子表达量降低最多，恢复至正常细胞水平。此部分数据说明，不同靶点的3CsA-ASO纳米胶束材料具有良好的炎症因子下调效果。
9.4不同靶点纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后分别使用院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)、3CsA-ASOIL-17-m纳米胶束、3CsA-ASOIL-1β-m纳米胶束、3CsA-ASOIL-23-m纳米胶束以及3CsA-ASOTNF-α-m纳米胶束材料(每个样品组中的CsA浓度均为400μM)进行眼部滴眼治疗，每只眼睛每日每次滴5μL，每日早晚各滴一次。在治疗的第0和14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图41所示。
荧光素钠染色结果显示，无治疗对照组小鼠眼部评分11分降至10分左右，酚红棉线泪液测试结果由1.5mm增至1.8mm左右，没有明显好转；CsA滴眼液治疗后的小鼠眼部评分由11分降至8分左右，酚红棉线泪液测试结果由1.5mm增加至3mm左右；不同靶点的3CsA-ASO纳米胶束材料组的小鼠荧光素钠染色评分约从11分降至5.5分左右，酚红棉线浸润长度从1mm升至3mm左右。由此部分数据结果可以看出，针对不同炎症因子靶点的药物-反义寡核苷酸偶联物纳米胶束在两周时间内对小鼠干眼的治疗具有明显疗效。
实施例10.针对NLRP3、JAK1和PDE4不同靶点的环孢素A接枝反义寡核苷酸自组装纳米胶束用于干眼病的治疗
10.1溴代环孢素A(CsA-Br)的合成与表征
溴代环孢素A(CsA-Br)的合成参照实施例6中相应合成步骤。
10.2溴代环孢素A与不同靶点PS-ASO接枝以及纳米胶束的制备与表征
本实施例中所采用的靶向胞内不同炎症通路基因的反义寡核苷酸序列均购自生工生物工程(上海)股份有限公司，我们选择在核酸磷酸骨架中进行硫代(PS)修饰，使其能够与修饰在CsA-Br上的溴乙酰溴基团发生取代反应从而高效偶联，其序列如下：
其中靶向人源不同炎症因子基因的反义寡核苷酸序列为：
NLRP3 human(20PS-ASONLRP3-h)
5’-AGCTGCTGCCCCGACCCAAACCTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.59)
JAK1 human(20PS-ASOJAK1-h)
5’-TTCGTCATCCTTGTAATCCATTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.60)
PDE4 human(20PS ASOPDE4-h)
5’-TTGATTTCCAGACCGACTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.61)
其中靶向鼠源不同炎症因子基因的反义寡核苷酸序列为：
NLRP3 mouse(20PS-ASONLRP3-m)
5′-GATTTAAGTACATTTCACCTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3′(SEQ ID NO.62)
JAK1mouse(20PS-ASOJAK1-m)
5'-TTGGAGAATGTCGCCATACAGTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3'(SEQ ID NO.63)
PDE4 mouse(20PS ASOPDE4-m)
5'-TTGTCCTCTCTACACGCTCATTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3'(SEQ ID NO.64)
合成方法为：将400倍摩尔当量的CsA-Br溶于DMSO中，加入5OD不同靶点的20PS-ASO后，使其反应浓度为200μM，然后置于50℃下震荡反应24h。加入水后，用乙酸乙酯萃取除去反应中过量的CsA-Br，浓缩蒸干后，即得到20CsA-ASONLRP3、20CsA-ASOJAK1和20CsA-ASOPDE4偶联物分子，然后将其用50μL DMSO溶液重新溶解并滴入500μL PBS中进行组装形成20CsA-ASONLRP3纳米胶束、20CsA-ASOJAK1纳米胶束以及20CsA-ASOPDE4纳米胶束，后在PBS中透析除去DMSO得到分散于PBS溶液中的不同纳米胶束材料。
10.3不同靶点纳米胶束对靶标基因的调控作用
将HCEC细胞按照1×105细胞/孔的密度接种于6孔板中贴壁，然后用100ng/mL LPS刺激12h，去掉上清液后分别加入1mL含有20CsA-ASONLRP3-h纳米胶束、20CsA-ASOJAK1-h纳米胶束以及20CsA-ASOPDE4-h纳米胶束的Opti-MEM培养基(均含有10μM ASO)，对照组为1mL含有不同靶点ASO与Lipo2000复合物的Opti-MEM培养基(ASO浓度均为10μM)，在37℃下共孵育转染6h后换为DMEM培养基，继续孵育48h后提取RNA进行PCR实验，以检验HCEC细胞中不同靶点的mRNA表达水平。实验结果如图42所示。
从图中可以看出，LPS刺激过后，不同炎症因子的mRNA表达量均有上调，在正常细胞表达量的1.3～1.5倍左右。在加入Lipo2000转染不同靶点的ASO后，不同炎症因子表达量降低至正常细胞表达量的1-1.2倍之间；加入不同靶点的纳米胶束材料后，不同炎症因子表达量降低至正常细胞表达量的0.8倍左右。此部分数据说明，不同靶点的20CsA-ASO纳米胶束材料具有良好的炎症因子基因下调效果。
10.4不同靶点纳米胶束对干眼症的治疗作用
使用0.2％苯扎氯氨对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行眼部干眼模型的构建，小鼠每只眼睛滴5μL0.2％苯扎氯氨，每日早晚各滴一次，建模两周后分别使用院配环孢素A滴眼液(400μM CsA，由上海复旦大学医学院附属眼耳鼻喉专科医院提供)、20CsA-ASONLRP3-m纳米胶束、20CsA-ASOJAK1-m纳米胶束以及20CsA-ASOPDE4-m纳米胶束材料(各样品组CsA浓度均为400μM)进行眼部滴眼治疗，每只眼睛每日每次滴5μL，每日早晚各滴一次。在治疗的第0和14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图43所示。
荧光素钠染色结果显示，无治疗对照组小鼠眼部评分13分降至12分左右，酚红棉线泪液测试结果由0.5mm增至1mm左右，没有明显好转；CsA滴眼液治疗后小鼠眼部评分由13分降至10分左右，酚红棉线泪液测试结果由1.5mm增至2.5mm左右；不同靶点的20CsA-ASO纳米胶束材料组的小鼠荧光素钠染色评分约从13分降至7.5分左右，酚红棉线浸润长度从1mm升至3.5mm左右。由此部分数据结果可以看出，针对不同胞内炎症靶点的药物-反义寡核苷酸偶联物纳米胶束在两周时间内对小鼠干眼的治疗具有明显疗效。
实施例11雷帕霉素-反义寡核苷酸偶联物及其纳米胶束组装体用于干眼症的治疗
11.1苄溴修饰的雷帕霉素(RAP-Bz-Br)合成，具体合成步骤见图44
a)苄溴修饰的酮缩硫醇(TK-Bz-Br)的合成。3-巯基丙酸(2.2当量)、丙酮(1当量)和催化量的三氟乙酸(TFA)在室温下搅拌过夜，然后将所得混合物置于冰浴中冷却结晶。对所制备晶体进行过滤并进一步用正己烷进行洗涤，然后在真空烘箱中保持45℃干燥一夜后得到白色产品，制备得到具有活性氧(ROS)响应裂解特性的酮缩硫醇化合物(化合物TK)。进一步地，将4-溴甲基苯甲醇(1当量)和化合物TK(3当量)溶于超干的二氯甲烷中，然后加入4-二甲氨基吡啶(DMAP，0.5当量)，室温条件下搅拌5min后滴加二环己基碳二亚胺(DCC，1.2当量，溶于超干的二氯甲烷中)，反应过程中利用硅胶薄层层析检测反应进度。反应结束后，经过滤除去杂质，利用旋转蒸发仪将滤液中溶剂蒸干，通过硅胶柱层析纯化得到苄溴修饰的酮缩硫醇(化合物TK-Bz-Br)，层析纯化过程中洗脱剂为石油醚/乙酸乙酯混合溶液，最终产率为65％。产物经核磁共振波谱进行分析检测，核磁共振波谱图及各峰归属见图45。
b)将1g雷帕霉素(RAP，1当量)溶于20mL乙酸乙酯中。将混合液置于冰浴中搅拌，加入咪唑(10当量)搅拌至溶解。同时将三甲基氯硅烷(TMS-Cl)溶于5mL乙酸乙酯后逐滴滴入上述反应液中，搅拌约2h，得到雷帕霉素31和42位均被TMS保护的中间产物(化合物RAP-31,42-bis-O-TMS)。然后将6mL0.5M硫酸(H2SO4)溶液逐滴滴入上述混合液中，冰浴过夜。反应液中加入60mL乙酸乙酯后，分别用饱和碳酸氢钠(NaHCO3，2次)和去离子水(3次)进行萃取，水溶液层pH控制在6.0～7.0之间。萃取收集乙酸乙酯溶液部分，然后加入无水硫酸钠除去水分后通过旋蒸蒸干，得到仅31位羟基被TMS保护的雷帕霉素(化合物RAP-31-O-TMS)。化合物RAP-31-O-TMS产率：90％，质谱检测得到其m/z：1008.61，可归属于[M+Na]+。将a)中所得的化合物TK-Bz-Br(1当量)和化合物RAP-31-O-TMS(0.8当量)溶解于无水二氯甲烷中，室温下加入DMAP(0.5当量)，搅拌至溶解。然后加入溶解于二氯甲烷的二异丙基碳二亚胺(DIC，2当量)，混合物室温搅拌过夜。产物通过硅胶柱层析得到化合物RAP-31-O-TMS-42-Bz-Br。化合物RAP-31-O-TMS-42-Bz-Br产率：35％；质谱检测得到其m/z：1419.64/1421.64,可归属于[M+NH4]+。核磁共振氢谱和核磁共振碳谱结果如图46。最后，0.4g化合物RAP-31-O-TMS-42-Bz-Br溶解于10mL乙腈和20mL乙酸乙酯混合溶液中，缓慢滴加3mL 1M H2SO4并连续搅拌2h，最后再加入50mL乙酸乙酯，加入50mL进行水洗两次并萃取。收集乙酸乙酯层后用无水硫酸钠除去水分然后旋蒸蒸干，得到最终产物苄溴修饰的雷帕霉素(化合物RAP-Bz-Br)，化合物RAP-Bz-Br产率：85％，质谱检测得到m/z：1347.59/1349.59，可归属于[M+NH4]+。
11.2 10个雷帕霉素分子-NFKBIZ反义寡核苷酸偶联物的合成(10RAP-ASONFKBIZ)
本实施例中所采用的靶向NFKBIZ基因的核苷酸序列均购自生工生物工程(上海)股份有限公司，我们选择在核酸磷酸骨架中进行硫代(PS)修饰，使其能够与修饰在CsA-Br上的溴乙酰溴基团反应从而高效偶联，其序列如下：
其中靶向人源NFKBIZ基因的反义寡核苷酸(ASONFKBIZ-h)序列为
Human:5’-ATCAGACAACGAATCGGGCTTTT*T*T*T*T*T*T*T*T*T*T-3’；(SEQ ID NO.65)
其中靶向小鼠的NFKBIZ基因的反义寡核苷酸(ASONFKBIZ-m)序列为
Mouse:5’-AATACTGGTACATTGACGCCTTTT*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.66)。
10个雷帕霉素分子-NFKBIZ反义寡核苷酸偶联物(10RAP-ASONFKBIZ)合成方法为：将2.2mg化合物RAP-Bz-Br(1.63μmol)溶于100μL DMSO中，然后加入5OD ASO(20.55nmol)后，置于50℃下震荡反应1h。然后加入水后，用乙酸乙酯萃取除去反应中过量的RAP-Bz-Br，浓缩蒸干后，即得到10RAP-ASONFKBIZ偶联物分子。所得产物通过15％变性聚丙烯酰胺凝胶电泳验证反义寡核苷酸成功接枝雷帕霉素，见图47。
11.3 10雷帕霉素-NFKBIZ反义寡核苷酸偶联物(10RAP-ASONFKBIZ)纳米胶束的制备和表征
将10RAP-ASONFKBIZ偶联物(10OD ASO)溶于100μL DMSO中，逐滴加入至300μL连续搅拌的磷酸盐缓冲液(PBS)中。随后将溶液放入透析袋中在PBS中透析过夜除去DMSO。3000rpm离心5min后，即可得到10RAP-ASONFKBIZ纳米胶束。通过动态光散射(Dynamic Light Scattering，DLS)、透射电子显微镜(Transmission Electron Microscope，TEM)表征其水合粒径和形貌，如图48所示。
11.4 10RAP-ASONFKBIZ-h纳米胶束的基因调控作用
将人角膜上皮细胞(HCEC)细胞按照1×105细胞/孔的密度接种于6孔板中培养过夜，加入100ng/mL LPS刺激过夜，分别加入1mL含有ASONFKBIZ-h+Lipo2000复合物以及10RAP-ASONFKBIZ-h的Opti-MEM培养基(ASO浓度均为10μM)，37℃下共孵育转染6h，更换为DMEM培养基后在37℃继续孵育48h，后收集细胞并提取RNA进行实时荧光定量PCR(RT-qPCR)检测，评估HCEC细胞中NFKBIZ mRNA的表达水平，实验结果如图49所示。从图中可以看出，LPS刺激过后的HCEC细胞中NFKBIZ mRNA表达水平约为正常细胞的1.4倍，使用Lipo2000转染ASONFKBIZ-h后NFKBIZ mRNA的表达水平约为正常细胞的0.75倍，加入10RAP-ASONFKBIZ-h纳米胶束共孵育后NFKBIZ mRNA表达量降至正常细胞的0.6倍左右。由此可见10RAP-ASONFKBIZ-h纳米胶束可有效调控HCEC细胞中NFKIBZ基因表达，显著敲低NFKBIZ mRNA表达。
11.5 10RAP-ASONFKBIZ-m纳米胶束对干眼症治疗作用
使用0.2％苯扎氯氨进行干眼模型构建，选择6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)，小鼠每只眼睛滴加5μL0.2％苯扎氯氨，每日早晚各滴一次，连续滴加两周。建模成功后使用不同药物进行眼部滴眼治疗，分组如下：未治疗组(mock)，院配环孢素A滴眼液(400μM CsA，由复旦大学医学院附属眼耳鼻喉专科医院提供)，以及10RAP-ASONFKBIZ-m纳米胶束(400μM雷帕霉素)。每只眼睛每次滴5μL，每日早晚各滴一次。在治疗的第0、7、14d进行荧光素钠染色评估与酚红棉线评估治疗效果，实验结果如图50所示。对于未治疗的对照组(control对照组)，治疗14d后，荧光素钠染色评分和酚红棉线实验结果与治疗前相差不大，说明未治疗的干眼小鼠的眼睛自身不会恢复正常。对于CsA滴眼液组，治疗后荧光素钠染色评分稍微降低、酚红棉线所测的泪液分泌均有所增加，并且伴随着治疗时间的增加效果更为明显。但治疗14d后，酚红棉线所测得泪液分泌平均～3mm，并未恢复到正常。对于10RAP-ASONFKBIZ-m纳米胶束实验组，治疗效果最为显著，其中，治疗14d后，荧光素钠染色评分最低，平均值为5，泪液分泌最多，平均值4mm。
实施例12环孢素A三聚体(CsA3)与NFKBIZ反义寡核苷酸共价偶联物(CsA3-ASONFKBIZ)及其纳米胶束组装结构用于银屑病治疗
12.1 CsA3-ASONFKBIZ共价偶联物的合成
所用CsA3-ASONFKBIZ共价偶联物与实施例5中所用偶联物相同，其具体合成步骤参见实施例中5.1-5.2部分。
12.2 CsA3-ASONFKBIZ自组装纳米胶束的制备和表征
环孢素A纳米胶束(CsA3-ASONFKBIZ纳米胶束)的制备和表征步骤参见实施例中5.3部分。
12.3 CsA3-ASONFKBIZ-h纳米胶束对靶标基因的调控作用
将1×105细胞/孔的人角质生成细胞系HaCaT细胞置于6孔板中贴壁，然后用100ng/mL重组人白细胞介素-36(IL-36)刺激2h，用1×PBS缓冲溶液清洗后分别将1mL包含了ASONFKBIZ-h+Lipo2000复合物、CsA3-ASONFKBIZ-h纳米胶束的Opti-MEM培养基(每组样品ASONFKBIZ-h的浓度为10μM)加入孔板中，与HaCaT细胞孵育转染6h后更换新鲜DMEM培养基，同时以不进行任何处理以及只添加IL-36进行刺激的细胞作为对照，37℃孵育48h后提取RNA进行RT-qPCR实验，以检验HaCaT细胞中NFKBIZ mRNA表达水平，实验结果如图51所示。
从图中可以看出，IL-36刺激后的HaCaT细胞，其NFKBIZ mRNA表达增至正常细胞的2.1倍左右，Lipo2000转染后的ASONFKBIZ-h可将其下调至1.2左右，CsA3-ASONFKBIZ-h纳米胶束对NFKBIZ基因下调至0.8左右，因此相比于单一的ASONFKBIZ-h，CsA3-ASONFKBIZ-h纳米胶束对NFKBIZ基因的表达下调能力更佳。
12.4 CsA3-ASONFKBIZ纳米胶束对炎症因子的调控作用
将5×104细胞/孔的人角质生成细胞系HaCaT细胞置于12孔板中贴壁，然后用100ng/mL重组人白细胞介素-36(IL-36)刺激2h，用1×PBS缓冲溶液清洗后分别将1mL包含了ASONFKBIZ-h+Lipo2000复合物、CsA3-ASONFKBIZ-h纳米胶束的Opti-MEM培养基(每组样品ASONFKBIZ-h的浓度为10μM)加入孔板中，与HaCaT细胞孵育转染6h后更换新鲜DMEM培养基，同时以不进行任何处理以及只添加IL-36进行刺激的细胞作为对照，37℃孵育48h后收集上清液，以ELISA试剂盒检测TNFα、IL-17A和IL-33分泌水平，实验结果如图52所示。
从图中可以看出，IL-36刺激后的HaCaT细胞，其TNFα、IL-17A和IL-33水平分别增至未刺激细胞的2.1倍、1.7倍和2.0倍左右，Lipo2000转染后的ASONFKBIZ-h可将TNFα、IL-17A和IL-33水平下调至1.1、1.0和0.9倍左右，CsA3-ASONFKBIZ-h纳米胶束对TNFα、IL-17A和IL-33水平下调至0.8-0.9倍左右，因此相比于单一的ASONFKBIZ-h，CsA3-ASONFKBIZ-h纳米胶束对TNFα、IL-17A和IL-33等炎症因子抑制作用更强。
12.5 CsA3-ASONFKBIZ-m纳米胶束对银屑病治疗作用
使用5％咪喹莫特乳膏对6～8周龄小鼠(C57BL/6，苏州西山生物技术有限公司)进行背部和双耳银屑病模型的构建，剔除小鼠背部毛发后，每只小鼠涂抹60mg5％咪喹莫特乳膏于背部和双耳，连续涂抹6d。建模6d后，如图53所示小鼠背部和耳朵出现明显的红斑、鳞屑、皮肤明显增厚等银屑病样表型，即模型构建成功。将CsA3-ASONFKBIZ-m纳米胶束与卡波姆凝胶混匀后，涂抹于皮损处进行治疗。以未添加CsA3-ASONFKBIZ-m纳米胶束的卡波姆凝胶作为对照组(control)进行疗效测试。在治疗的第7d对背部皮损处皮肤厚度及耳朵厚度进行测量，并对皮损处红斑、鳞屑、皮肤增厚程度及银屑病皮损面积严重程度进行评分。实验结果如图54，图55和图56所示。治疗7d后，相较于对照组，CsA3-ASONFKBIZ-m纳米胶束组小鼠皮损处的红斑和鳞屑明显减轻，耳部和皮损处皮肤厚度减小，该结果表明CsA3-ASONFKBIZ-m纳米胶束可有效缓解患病小鼠的银屑病样表型。
实施例13曲安奈德分子与NFKBIZ反义寡核苷酸共价偶联物(10TA-ASONFKBIZ)及其纳米胶束组装结构用于银屑病的协同治疗
13.1羰乙基溴代曲安奈德(TA-Br)合成
羰乙基溴代曲安奈德(TA-Br)具体合成步骤见图57，首先在氮气保护下，将曲安奈德(200mg)置于干燥的250mL圆底烧瓶中，加50mL无水二氯甲烷溶解，逐滴加入130μL N,N-二异丙基乙胺， 冰浴条件下逐滴加入105mg溴乙酰溴，转移至室温，搅拌反应12h，反应完毕后，加入50mL饱和碳酸氢钠溶液洗涤3次，50mL饱和氯化钠溶液洗涤一次，收集有机相，无水硫酸钠干燥，减压蒸馏浓缩有机相，经硅胶柱层析分离，洗脱液为石油醚/乙酸乙酯，得浅黄色色固体(140mg)，即制得羰乙基溴代曲安奈德。
羰乙基溴代曲安奈德的1H NMR谱图，如图58所示，测试溶剂DMSO-d6，各质子峰的归属如下：δ(ppm):7.30(1H,d),6.25(1H,dd),6.02(1H,s),5.50(1H,s),5.27(1H,d),4.86(1H,d),4.82(1H,d),4.32-4.15(2H,d),4.21(1H,s),3.78(6H,s),2.64(1H,m),2.34(1H,dd),2.05(1H,m),1.95(1H,q),1.82(1H,t),1.73(1H,d),1.55(2H,m),1.50(3H,s),1.35(4H,s),1.26(1H,t),1.16(3H,s),0.80(3H,s)。羰乙基溴代曲安奈德的13C NMR谱图，如图59所示，测试溶剂为DMSO-d6，偶联羰乙基溴后的特征碳的归属如下：210.50,185.68,167.04,153.19,129.78,124.69,110.83,102.06,97.28,81.62,70.66,66.28,48.43,45.14,42.95,35.48,33.47,32.89,30.57,27.95,26.77,25.72,23.26,16.83。羰乙基溴代曲安奈德的理论分子量为555.43，高效液相色谱/四极杆飞行质谱联用仪测得的分子量为557.1391和1113.2782，分别对应羰乙基溴代曲安奈德的[M+1]+和[2M+1]+峰，证实目标产物成功合成，如图60所示。
13.2曲安奈德-NFKBIZ反义寡核苷酸偶联物的合成(10TA-ASONFKBIZ)
本实施例中所采用的靶向NFKBIZ基因的核苷酸序列均购自生工生物工程(上海)股份有限公司，我们选择在核酸磷酸骨架中进行硫代(PS)修饰，使其能够与修饰在TA-Br上的羰乙基溴基团反应从而高效偶联，其序列如下：
其中靶向人源NFKBIZ基因的反义寡核苷酸(ASONFKBIZ-h)序列为
Human:5’-ATCAGACAACGAATCGGGCTTTT*T*T*T*T*T*T*T*T*T*T-3’；(SEQ ID NO.67)
其中靶向小鼠的NFKBIZ基因的反义寡核苷酸(ASONFKBIZ-m)序列为
Mouse:5’-AATACTGGTACATTGACGCCTTTT*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.68)。
10TA-ASONFKBIZ共价偶联物的合成方法为：将羰乙基溴代曲安奈德化合物溶于20μL二甲基亚砜溶液中，加入2μL硫代磷酸酯基团修饰反义寡核苷酸溶液，寡核苷酸浓度在200μM(硫代磷酸酯基团与羰乙基溴代曲安奈德的比例范围为1:1～1:50)，置于55℃下，震荡反应过夜。反应结束后，加入500μL超纯水，利用乙酸乙酯多次萃取除去反应中过量的羰乙基溴代曲安奈德，减压蒸馏除去溶剂后，重新溶于超纯水中，即得到曲安奈德-反义寡核苷酸偶联物。通过15％变性聚丙烯酰胺凝胶电泳验证羰乙基溴代曲安奈德成功接枝至硫代磷酸酯修饰寡核苷酸，见图61。
13.3 10TA-ASONFKBIZ纳米胶束的制备和表征
将10TA-ASONFKBIZ共价偶联物(10OD ASO)溶于100μL DMSO中，逐滴加入至300μL连续搅拌的1×PBS缓冲溶液中。随后将溶液放入透析袋中在PBS中透析过夜除去DMSO，即可得到10TA-ASONFKBIZ纳米胶束粒子。通过1％琼脂糖凝胶电泳表征10TA-ASONFKBIZ纳米胶束的成功制备，如图62所示。
13.4 10TA-ASONFKBIZ纳米胶束的基因调控作用
将1×105细胞/孔的人角质生成细胞系HaCaT细胞置于6孔板中贴壁，然后用100ng/mL重组人白细胞介素-36(IL-36)刺激2h，用1×PBS缓冲溶液清洗后分别将1mL包含了ASONFKBIZ-h+Lipo2000复合物、10TA-ASONFKBIZ-h纳米胶束的Opti-MEM培养基(每组样品ASONFKBIZ-h的浓度为10μM)加入孔板中，与HaCaT细胞孵育转染6h后更换新鲜DMEM培养基，同时以不进行任何处理以及只添加IL-36进行刺激的细胞作为对照，37℃孵育48h后提取RNA进行RT-qPCR实验，以检验HaCaT细胞中NFKBIZ mRNA表达水平，实验结果如图63所示。
从图63中可以看出，IL-36刺激后的HaCAT细胞，其NFKBIZ mRNA表达增至正常细胞的2.2倍左右，Lipo2000转染后的ASONFKBIZ-h可将其下调至0.85左右，10TA-ASONFKBIZ-h纳米胶束对NFKBIZ基因下调至0.7左右，因此相比于单一的ASONFKBIZ-h，10TA-ASONFKBIZ-h纳米胶束对NFKBIZ基因的 表达下调能力更佳。
13.5 10TA-ASONFKBIZ-m纳米胶束对银屑病治疗作用
使用5％咪喹莫特乳膏对6～8周龄小鼠进行背部和双耳银屑病模型的构建，剔除小鼠背部毛发后，每只小鼠涂抹60mg5％咪喹莫特乳膏于背部和双耳，连续涂抹6d。建模6d后，小鼠背部和耳朵出现明显的红斑、鳞屑、皮肤明显增厚等银屑病样表型，即模型构建成功。将10TA-ASONFKBIZ-m纳米胶束与卡波姆凝胶混匀后，涂抹于皮损处进行治疗。以未添加10TA-ASONFKBIZ-m纳米胶束的卡波姆凝胶作为对照组(control)进行疗效测试。在治疗的第7d对背部皮损处皮肤厚度及耳朵厚度进行测量，并对皮损处红斑、鳞屑、皮肤增厚程度及银屑病皮损面积严重程度进行评分。实验结果如图64、图65和图66所示，治疗7d后，相较于未治疗组，10TA-ASONFKBIZ-m纳米胶束组小鼠皮损处的红斑和鳞屑明显减轻，皮损处皮肤厚度减小，表明10TA-ASONFKBIZ-m纳米胶束可有效缓解患病小鼠的银屑病样表型。
实施例14卡泊三醇分子与NFKBIZ反义寡核苷酸共价偶联物(10Cal-ASONFKBIZ)及其纳米胶束组装结构用于银屑病的协同治疗
14.1苄溴修饰卡泊三醇(Cal-Bz-Br)的合成
苄溴修饰卡泊三醇(Cal-Bz-Br)的合成步骤见图67，包括a)苄溴修饰的二硫代二丙酸的合成(DTPA-Bz-Br)。取4-溴甲基苯甲醇500mg和3,3’-二硫代二丙酸(DTPA)2.6g溶于超干的二氯甲烷和四氢呋喃的混合溶液(1/1，v/v)；然后加入4-二甲氨基吡啶91mg，搅拌5min后滴加二环己基碳二亚胺615mg(溶于超干的二氯甲烷中)，室温反应过夜后，利用旋转蒸发仪将溶剂蒸干，通过硅胶柱层析分离得到含有二硫键的苄溴结构(DTPA-Bz-Br)，洗脱剂为石油醚/乙酸乙酯。产物1H NMR及归属见图68。
b)取卡泊三醇(Cal)135mg和二环己基碳二亚胺81mg，加入30mL无水二氯甲烷搅拌溶解，加入10mg4-二甲氨基吡啶(溶于无水二氯甲烷中)，滴加DTPA-Bz-Br10mg(溶于无水的二氯甲烷中)，室温搅拌反应3h，利用旋转蒸发仪将溶剂蒸干，通过硅胶柱层析分离得到苄溴修饰的卡泊三醇前药分子(Cal-Bz-Br)，洗脱剂为石油醚/乙酸乙酯体系。产物核磁图见图69，测试溶剂DMSO-d6，各质子峰的归属如下：δ(ppm):7.43,H3(2H,s),7.36,H2(2H,m),6.35,H10(1H,d),6.02H9(1H,d),5.52,H12(2H,m),5.35,H17(1H,d),5.14,H4(2H,s),5.06,H8(1H,d),4.51,H1(2H,s),4.15,H14(1H,m),3.48,H13(1H,m),2.94,H15(2H,m),2.91,H16(2H,m),0.58,H11(3H,d),0.52,H6(2H,m),0.33,H5(1H,m),0.25,H7(1H,m)。Cal-Bz-Br的理论分子量为785.2，高效液相色谱/四极杆飞行质谱联用仪测得的分子量为786.25，对应Cal-Bz-Br的[M+1]+峰，证实目标产物成功合成，如图70所示。
14.2卡泊三醇-NFKBIZ反义寡核苷酸偶联物的合成(10Cal-ASONFKBIZ)
本实施例中所采用的靶向NFKBIZ基因的核苷酸序列均购自生工生物工程(上海)股份有限公司，我们选择在核酸磷酸骨架中进行硫代(PS)修饰，使其能够与修饰在Cal-Br上的卞溴基团反应从而高效偶联，其序列如下：
其中靶向人源NFKBIZ基因的反义寡核苷酸(ASONFKBIZ-h)序列为
Human:5’-ATCAGACAACGAATCGGGCTTTT*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.69)；
其中靶向小鼠的NFKBIZ基因的反义寡核苷酸(ASONFKBIZ-m)序列为
Mouse:5’-AATACTGGTACATTGACGCCTTTT*T*T*T*T*T*T*T*T*T*T-3’(SEQ ID NO.70)。
卡泊三醇-NFKBIZ反义寡核苷酸偶联物(10Cal-ASONFKBIZ)合成方法为：取Cal-Bz-Br化合物溶于20μL二甲基亚砜溶液中，加入2μL硫代磷酸酯基团修饰反义寡核苷酸溶液，寡核苷酸浓度在200μM(硫代磷酸酯基团与Cal-Bz-Br化合物的比例为1:5)，置于55℃下，震荡反应过夜。反应结束后，加入500μL超纯水，利用乙酸乙酯多次萃取除去反应中过量的Cal-Bz-Br化合物，减压蒸馏除去溶剂后，即得到卡泊三醇-反义寡核苷酸结合物前药。通过15％变性聚丙烯酰胺(PAGE)凝胶电泳验证卡 泊三醇成功接枝至硫代磷酸酯修饰寡核苷酸，见图71。
14.3 10Cal-ASONFKBIZ纳米胶束的制备和表征
将10Cal-ASONFKBIZ偶联物(10OD ASO)溶于100μL DMSO中，逐滴加入至300μL连续搅拌的1×PBS缓冲溶液中。随后将溶液放入透析袋中在PBS中透析过夜除去DMSO，即可得到10Cal-ASONFKBIZ纳米胶束粒子。通过1％的琼脂糖凝胶电泳证明其组装成功，结果见图72。此外利用动态光散射法测定卡泊三醇-反义寡核苷酸偶联物自组装纳米胶束的水合粒径，如图73所示，卡泊三醇-反义寡核苷酸偶联物自组装纳米胶束的水合粒径约为150nm。图73中透射电子显微镜图片揭示所制备的纳米胶束为球形形貌。10Cal-ASONFKBIZ偶联物的临界胶束浓度测定为0.527μM，结果见图74。
14.4 10Cal-ASONFKBIZ纳米胶束的基因调控作用
将1×105细胞/孔的人角质生成细胞系HaCaT细胞置于6孔板中贴壁，然后用100ng/mL重组人白细胞介素-36(IL-36)刺激2h，用1×PBS缓冲溶液清洗后分别将1mL包含了ASONFKBIZ-h+Lipo2000复合物、10Cal-ASONFKBIZ-h纳米胶束的Opti-MEM培养基(每组样品ASONFKBIZ-h的浓度为10μM)加入孔板中，与HaCaT细胞孵育转染6h后更换新鲜DMEM培养基，同时以不进行任何处理以及只添加IL-36进行刺激的细胞作为对照，37℃孵育48h后提取RNA进行RT-qPCR实验，以检验HaCaT细胞中NFKBIZ mRNA表达水平，实验结果如图75所示。
从图中可以看出，IL-36刺激后的HaCaT细胞，其NFKBIZ mRNA表达增至正常细胞的1.9倍左右，Lipo2000转染后的ASONFKBIZ-h可将其下调至0.85倍左右，10Cal-ASONFKBIZ-h纳米胶束对NFKBIZ基因下调至0.65倍左右，因此相比于单一的ASONFKBIZ-h，10Cal-ASONFKBIZ-h纳米胶束对NFKBIZ基因的表达下调能力更佳。
14.5 10Cal-ASONFKBIZ-m纳米胶束对银屑病治疗作用
使用5％咪喹莫特乳膏(四川明欣药业有限责任公司)对6～8周龄小鼠进行背部和双耳银屑病模型的构建，剔除小鼠背部毛发后，每只小鼠涂抹60mg5％咪喹莫特乳膏于背部和双耳，连续涂抹6d。建模6d后，小鼠背部和耳朵出现明显的红斑、鳞屑、皮肤明显增厚等银屑病样表型，即模型构建成功。将10Cal-ASONFKBIZ-m纳米胶束与卡波姆凝胶混匀后，涂抹于皮损处进行治疗。以未添加10Cal-ASONFKBIZ-m纳米胶束的卡波姆凝胶作为对照组(control)进行疗效测试。在治疗的第7d对背部皮损处皮肤厚度及耳朵厚度进行测量，并对皮损处红斑、鳞屑、皮肤增厚程度及银屑病皮损面积严重程度进行评分。实验结果如图76、图77以及图78所示，治疗7d后，相较于未治疗组，10Cal-ASONFKBIZ-m纳米胶束组小鼠皮损处的红斑和鳞屑明显减轻，皮损处皮肤厚度减小，表明10Cal-ASONFKBIZ-m纳米胶束可有效缓解患病小鼠的银屑病样表型。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (11)

  1. 一种小分子药物-寡核苷酸偶联物，由具有免疫调节功能的小分子药物和能够调控炎症相关基因表达的功能寡核苷酸分子共价偶联得到。
  2. 根据权利要求1所述的小分子药物-寡核苷酸偶联物，其特征在于，所述小分子药物和功能寡核苷酸分子之间通过化学连接子共价偶联。
  3. 根据权利要求1所述的小分子药物-寡核苷酸偶联物，其特征在于，所选小分子药物为作用于免疫相关信号通路并能够调控免疫反应的小分子药物；优选的，所述小分子药物包括钙调磷酸酶抑制剂、糖皮质激素、mTOR抑制剂和维生素D类似物中的一种或几种。
  4. 根据权利要求1所述的小分子药物-寡核苷酸偶联物，其特征在于，所述炎症相关基因包括肿瘤坏死因子-a基因、白介素1b基因、白介素17基因、白介素23基因、NFKBIZ基因、炎性小体NLRP3基因、JAK基因和PDE4基因中的一种或几种。
  5. 根据权利要求1或4所述的小分子药物-寡核苷酸偶联物，其特征在于，所述功能寡核苷酸分子包括双链小干扰核酸、微小核酸和单链反义寡核苷酸中的一种。
  6. 根据权利要求5所述的小分子药物-寡核苷酸偶联物，其特征在于，当所述功能寡核苷酸分子为双链小干扰核酸或微小核酸时，所述小分子药物共价偶联于功能寡核苷酸分子的正义链的3’端；当所述功能寡核苷酸分子为单链反义寡核苷酸时，所述小分子药物共价偶联于单链反义寡核苷酸的3’端或5’端。
  7. 根据权利要求6所述的小分子药物-寡核苷酸偶联物，其特征在于，每个功能寡核苷酸分子上共价偶联的小分子药物的数量为1～40个。
  8. 根据权利要求7所述的小分子药物-寡核苷酸偶联物，其特征在于，所述小分子药物共价偶联于功能寡核苷酸分子的末端或者所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上；
    优选的，当所述小分子药物共价偶联于功能寡核苷酸分子的末端时，所述的小分子药物-寡核苷酸偶联物的化学结构式如式1～式14所示：


    在所述式1～式14中，L、T各自独立地为不存在、-(CH2)h-或-(CH2)h-中的任意一个或多个亚烷基被A基团所取代后的基团；所述h为0～15；所述A基团包括：-O-、-S-、-C(O)-、-C(O)O-、-C(O)NH-、-CH(RC)-、-C(R’)(R”)-、-NH-、-N(RN)-、-S-S-、-C(R’)＝C(R”)-、-C≡C-、中的一种或几种；所述A基团中的RC、RN、R’、R”表示指定原子上的任何一个或多个氢原子被B基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物，所述指定原子包括碳原子或氮原子；所述B基团包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素；所述卤素包括F、C1、Br或I；所述A基团中(O)代表羰基氧原子；
    在所述式1～式14中，Q、Y、Z各自独立地为不存在、-O-、-S-、-C(O)-、-NH-、-CH2-、-C(O)NH-、-NHC(O)-、-C(O)O-、-OC(O)-、-OC(O)O-、-OC(O)NH-、-NHC(O)O-和中的一种 或几种；所述Q、Y、Z基团中(O)代表羰基氧原子；所述为连接位点；
    在所述式1～式14中，G表示小分子免疫调节药物；m、n、k独立地为1～15；
    X表示O或S；
    优选的，当所述小分子药物共价偶联于功能寡核苷酸分子的3’端或5’端延伸序列的侧链碱基或者磷酸骨架上时，所述的小分子药物-寡核苷酸偶联物的化学结构式如式15～式20所示：
    在所述式15～式20中，T各自独立地为不存在、-(CH2)h-或-(CH2)h-中的任意一个或多个亚烷基被A基团所取代后的基团；所述h为0～15；所述A基团包括：-O-、-S-、-C(O)-、-C(O)O-、-C(O)NH-、-CH(RC)-、-C(R’)(R”)-、-NH-、-N(RN)-、-S-S-、-C(R’)＝C(R”)-、-C≡C-、中的一种或几种；所述A基团中的RC、RN、R’、R”表示指定原子上的任何一个或多个氢原子被B基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物，所述指定原子包括碳原子或氮原子；所述B基团包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素；所述卤素包括F、C1、Br或I；所述A基团中(O)代表羰基氧原子；
    在所述式15～式20中，Y、Z各自独立地为不存在、O、S、C(O)、NH、CH2、C(O)NH、NHC(O)、C(O)O、OC(O)、OC(O)O、OC(O)NH、NHC(O)O和中的一种或几种；所述Y、Z基团中(O)代表羰基氧原子；所述为连接位点；
    在所述式15～式20中，G表式免疫调节抑制剂；n独立地为1～15；m、i独立地为0～5，k、j独立地为1～20；R表示H；B表示核酸碱基。
  9. 权利要求1～8任意一项所述的小分子药物-寡核苷酸偶联物在制备治疗炎症相关疾病的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述炎症相关疾病包括干眼症、银屑病、干燥综合症、葡萄膜炎、角膜炎、结膜炎、特应性皮炎、类风湿性关节炎、炎症性肠炎和克罗恩病中的一种或几种。
  11. 权利要求1～8任意一项所述的小分子药物-寡核苷酸偶联物在治疗炎症相关疾病中的应用。
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