WO2020064017A1 - 核酸纳米载体药物、其制备方法、药物组合物及其应用 - Google Patents

核酸纳米载体药物、其制备方法、药物组合物及其应用 Download PDF

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WO2020064017A1
WO2020064017A1 PCT/CN2019/109563 CN2019109563W WO2020064017A1 WO 2020064017 A1 WO2020064017 A1 WO 2020064017A1 CN 2019109563 W CN2019109563 W CN 2019109563W WO 2020064017 A1 WO2020064017 A1 WO 2020064017A1
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sequence
nucleic acid
drug
nanoparticles
seq
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PCT/CN2019/109563
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English (en)
French (fr)
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王力源
王萌
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百药智达（北京）纳米生物技术有限公司
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Priority to US17/281,247 priority Critical patent/US20220226473A1/en
Priority to EP19867901.1A priority patent/EP3858846A4/en
Publication of WO2020064017A1 publication Critical patent/WO2020064017A1/zh

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
    • A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • the present application relates to the field of medicine, and in particular, to a nucleic acid nanocarrier drug, a preparation method, a pharmaceutical composition, and an application thereof.
  • non-viral vectors especially biodegradable polymer materials to achieve targeted drug delivery.
  • the advantage of non-viral vectors is that under the conditions of ensuring the expected transfection activity, the immunogenicity and many inflammatory responses brought by viral vectors can be greatly reduced.
  • Polycationic gene carriers are currently relatively mature, however, it is difficult to ensure that the targeting group is on the surface of the structure in the structural design, and there is a self-design contradiction between toxicity and transfection activity. At the same time, its connection is difficult to achieve non-toxic degradation in vivo .
  • the main purpose of the present application is to provide a nucleic acid nanocarrier, a nucleic acid nanocarrier drug, a preparation method, a pharmaceutical composition, and an application thereof, so as to improve drug delivery reliability.
  • a nucleic acid nanocarrier drug which includes a nucleic acid nanoparticle and a drug mounted on the nucleic acid nanoparticle.
  • the drug includes tacroline, epirubicin, and methylamine Any one or more of pterin, pirarubicin, daunorubicin, pentafluorouracil, 10-hydroxycamptothecin, aspirin, and gemcitabine; nucleic acid nanoparticles include a nucleic acid domain, the nucleic acid domain contains an a sequence, b sequence and c sequence, a sequence contains a1 sequence or a1 sequence with at least one base insertion, deletion or replacement, b sequence contains b1 sequence or b1 sequence with at least one base insertion, deletion or replacement sequence, c sequence Contains a c1 sequence or a sequence in which at least one base has been inserted, deleted or replaced; wherein the a1 sequence is SEQ ID NO:
  • sequence a1 is SEQ ID NO: 1
  • sequence b1 is SEQ ID NO: 3
  • sequence c1 is SEQ ID NO: 5
  • at least one of the sequences a, b, and c includes at least one base insertion , Missing or replaced sequences.
  • WC represents Watson-Crick pairing
  • each position of WC is independently selected from CG or GC
  • the first N from the 5 ′ end is A
  • the second N is G
  • the third N is U or T
  • the fourth N is any of U, T, A, C, or G
  • the first from the 5 'end N ' is any one of U, T, A, C, or G
  • the second N' is U or T
  • the NNNN sequence is CAUA or CATA.
  • a sequence, the b sequence, and the c sequence are any of the following groups: (1) a sequence: 5'-GGAGCGUUGG-3 ', b sequence: 5'-CCUUCGCCG-3', and c sequence: 5'-CGGCCAUAGCCC- 3 '; (2) a sequence: 5'-GCAGCGUUCG-3', b sequence: 5'-CGUUCGCCG-3 ', c sequence: 5'-CGGCCAUAGCGC-3'; (3) a sequence: 5'-CGAGCGUUGC- 3 ', b sequence: 5'-GCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCG-3 '; (4) a sequence: 5'-GGAGCGUUGG-3', b sequence: 5'-CCUUCGGGG-3 ', c sequence: 5'-CCCCCAUAGCCC-3 '; (5) a sequence: 5'-GCAGCGUUCG-3', b sequence: 5'-CGUUC
  • the nucleic acid domain further includes a first extension, the first extension is a Watson-Crick paired extension, and the first extension is located at the 5 ′ end of any one of the a sequence, the b sequence, and the c sequence and / or 3 'end;
  • the first extension is selected from any one of the following: (1): 5' end of a chain: 5'-CCCA-3 ', 3' end of c chain: 5'-UGGG-3 '; (2): 3 'end of a chain: 5'-GGG-3', 5 'end of b chain: 5'-CCC-3'; (3): 3 'end of b chain: 5'-CCA-3', 5 'end of c-chain: 5'-UGG-3'; (4): 5 'end of a-chain: 5'-CCCG-3', 3 'end of c-chain: 5'-CGGG-3'; (5): 5 'end of a chain: 5'-CCCC-3', 3
  • the nucleic acid domain further includes a second extension, the second extension is located at the 5 'end and / or the 3' end of any of the a sequence, the b sequence, and the c sequence, and the second extension is a Watson-Crick pair
  • the second extension is an extension sequence of CG base pairs; more preferably, the second extension is an extension sequence of 1 to 10 CG base pairs.
  • the nucleic acid domain further includes at least one of the following second extensions: the first group: the 5 'end of the a chain: 5'-CGCGCG-3', the 3 'end of the c chain: 5'-CGCGCG-3'; Two groups: 3 'end of a chain: 5'-CGCCGC-3', 5 'end of b chain: 5'-GCGGCG-3'; third group: 3 'end of b chain: 5'-GGCGGC-3', c 5 'end of the strand: 5'-GCCGCC-3'.
  • the second extended segment is an extended sequence containing both CG base pairs and AT / AU base pairs, and the second extended segment is preferably an extended sequence of 2 to 50 base pairs.
  • the second extended segment is an extended sequence in which a continuous sequence of 2 to 8 CG base pairs is alternately arranged with a continuous 2 to 8 AT / AU base pair sequence; or the second extended segment is 1 CG bases.
  • bases, riboses, and phosphates in the a, b, and c sequences have at least one modifiable site, and any modifiable site is modified by any of the following modified linkers: -F, methyl, amino , Disulfide, carbonyl, carboxyl, thiol, and aldehyde groups; preferably, the C or U bases in the a sequence, the b sequence, and the c sequence have 2'-F modification.
  • the drug is mounted on the nucleic acid nanoparticle in the form of physical connection and / or covalent connection, and the molar ratio between the tacrine and the nucleic acid nanoparticle is 2 to 300: 1, preferably 10 to 50: 1. , More preferably 15 to 25: 1.
  • the nucleic acid nanoparticle also includes a biologically active substance, which is connected to a nucleic acid domain.
  • the biologically active substance is a target, fluorescein, interference nucleic acid siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein , Peptides, flavonoids, glucose, natural salicylic acid, monoclonal antibodies, vitamins, phenolic lecithin, and small molecule drugs.
  • the small molecule drugs are except tacrine, epirubicin, and methylamine. Small molecule drugs other than propterin, pirarubicin, daunorubicin, pentafluorouracil, 10-hydroxycamptothecin, aspirin and gemcitabine.
  • the relative molecular weight of the nucleic acid domain is denoted as N 1
  • the total relative molecular weight of the drug and the biologically active substance is denoted as N 2
  • the biologically active substance is one or more of a target, fluorescein, and miRNA, wherein the target is located on any one of the sequences a, b, and c, and preferably 5 of any one of the sequences a, b, and c.
  • miRNA is anti-miRNA
  • fluorescein is modified at the 5 'or 3' end of the anti-miRNA
  • miRNA is located at the 3 'end of the a sequence
  • c Any one or more of the 5 'and 3' ends of the sequence; preferably, the target is folic acid or biotin
  • fluorescein is any one or more of FAM, CY5, and CY3, and anti-miRNA is anti- miR-21.
  • the small molecule drug is a drug containing any one or more of the following groups: amino group, hydroxyl group, carboxyl group, mercapto group, benzene ring group, and acetamino group.
  • the protein is one or more of SOD, survivin, hTERT, EGFR, and PSMA; the vitamins are L-C and / or esterified C; the phenols are tea polyphenols and / or grape polyphenols.
  • the particle size of the nucleic acid nanoparticles is 1 to 100 nm, preferably 5 to 50 nm; more preferably 10 to 30 nm; and still more preferably 10 to 15 nm.
  • a method for preparing a nucleic acid nanocarrier drug includes the following steps: providing the above-mentioned nucleic acid nanoparticle; and mounting the drug on the nucleic acid by means of physical connection and / or covalent connection. On the nanoparticles, a nucleic acid nanocarrier drug is obtained.
  • the step of mounting the drug by means of physical connection includes: mixing and stirring the drug, the nucleic acid nanoparticles and the first solvent to obtain a premixed system; precipitating the premixed system to obtain a nucleic acid nanocarrier drug; preferably
  • the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid.
  • the step of obtaining a nucleic acid nanocarrier drug by precipitating the premixed system includes: The mixed system is subjected to precipitation and precipitation to obtain a precipitate; the precipitate is washed to remove impurities to obtain a nucleic acid nanocarrier drug; more preferably, the premixed system is mixed with anhydrous ethanol to precipitate at a temperature lower than 10 ° C. A precipitate is obtained; a nucleic acid nanocarrier drug; more preferably, the precipitate is precipitated at a temperature of 0 to 5 ° C. to obtain a precipitate. More preferably, the precipitate is washed with 6 to 12 times the volume of absolute ethanol to remove impurities to obtain a nucleic acid nanocarrier drug.
  • the step of mounting the drug by covalent connection includes: configuring a drug solution; reacting the drug solution with the G-ring amino group of the nucleic acid nanoparticle under the mediation of formaldehyde to obtain a reaction system; purifying the reaction system, A nucleic acid nanocarrier drug is obtained.
  • the reaction step includes: mixing a drug solution with a paraformaldehyde solution and nucleic acid nanoparticles, and performing a reaction under dark conditions to obtain a reaction system; preferably, the concentration of the paraformaldehyde solution is preferably 3.7 to 4 wt%, preferably the paraformaldehyde solution is a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid.
  • the preparation method further includes a step of preparing a nucleic acid nanoparticle, which comprises: self-assembling a single strand corresponding to the above-mentioned nucleic acid domain to obtain a nucleic acid domain; preferably, after obtaining the nucleic acid domain, the preparation method further comprises: The method includes: mounting the above-mentioned biologically active substance on a nucleic acid domain through physical connection and / or covalent connection, thereby obtaining nucleic acid nanoparticles.
  • the solvent is covalently connected, the linker is covalently connected, or the link is clicked to mount;
  • the third solvent used in the solvent covalent connection is used as the connection Medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid;
  • the linker is selected from disulfide bonds, p-phenylazide, Bromopropyne or PEG;
  • clicking the link is to perform alkynyl or azide modification on both the biologically active substance precursor and the nucleic acid domain, and then click on the link.
  • the alkynyl or azide modification site of the biologically active substance precursor is selected from 2 ′ hydroxyl, carboxyl, or amino
  • the nucleic acid domain is alkynyl or The azide-modified site is selected from the group consisting of an exo G ring amino group, a 2'-hydroxyl group, an A amino group, or a 2'-hydroxyl group.
  • a pharmaceutical composition is also provided, and the pharmaceutical composition includes any one of the foregoing nucleic acid nanocarrier drugs.
  • nucleic acid nanocarrier drugs in the preparation of a medicament for treating Alzheimer's disease, tumor, autoimmune disease, or heart disease.
  • the tumor is any one or more of the following: pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, cervical cancer, liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumor, lymphoma, mesothelioma, head and neck Cancer, gastric cancer, leukemia, colon cancer, rectal cancer, chorionic epithelial cancer, malignant hydatidiform mole, skin cancer, lung cancer, ureteral cancer, renal pelvis cancer, chorionic epithelial cancer, bone tumor, leukemia meningeal spinal invasion, nephroblastoma Soft tissue sarcoma and medullary thyroid carcinoma; autoimmune diseases are refractory psoriasis, systemic lupus erythematosus, mandatory spondylitis or dermatomyositis.
  • the leukemia is an acute leukemia
  • the acute leukemia is preferably an acute lymphocytic leukemia or a granulocytic leukemia.
  • lung cancer includes bronchial lung cancer or non-small cell lung cancer.
  • liver cancer includes primary hepatocellular carcinoma or metastatic liver cancer.
  • a method for preventing and / or treating Alzheimer's disease, tumor, autoimmune disease, or heart disease includes: providing any one of the nucleic acid nanocarrier drugs or drug combinations described above. A corresponding effective amount of the aforementioned nucleic acid nanocarrier drug or pharmaceutical composition to a patient.
  • the nucleic acid nanocarrier drugs provided in the present application include nucleic acid nanoparticles and drugs, and the drugs are mounted on the nucleic acid nanoparticles through physical and / or covalent connection.
  • the nucleic acid nanoparticle can not only self-assemble to form a nucleic acid domain by including the three sequences or variant sequences provided in the present application, but also can be used as a carrier to connect the drug at any 5 'end and / or 3' end of the three strands. Or, the drug can be stably inserted between the strands of the nucleic acid domain.
  • the present application utilizes the internal hydrophobicity, external hydrophilicity of the nucleic acid nanoparticle, and the base-stacking effect to play a "coating effect" on the drug.
  • the valence connection prevents the drug from being dissolved within a certain time, and improves the stability of delivery.
  • the nucleic acid domain is modified by the target, it can have better targeting, can stably deliver drugs, and has high reliability; meanwhile, it can reduce the chance of drug contact with non-target cells or tissues, and reduce toxic and side effects .
  • FIG. 1 shows the results of electrophoretic detection of RNA nanoparticles formed by self-assembly in Example 1 of the present application
  • Example 3 is a result of 2% agarose gel electrophoresis detection of 7 groups of short-sequence RNA nanoparticles formed by self-assembly in Example 2 of the present application;
  • FIG. 6 shows 4% agarose gel electrophoresis detection results of 7 groups of conventional sequence RNA nanoparticles formed by self-assembly in Example 3 of the present application;
  • FIG. 8 shows the results of 4% agarose gel electrophoresis of 7 groups of conventional sequence DNA nanoparticles formed by self-assembly in Example 4 of the present application
  • FIG. 10 shows a standard curve of the absorbance of taclaline during the mounting rate detection process in Example 5 of the present application
  • FIG. 11 shows microscope observation results of the binding and internalization of RNAh-Biotin-quasar670 nanoparticles and RNAh-Biotin-quasar670-tacrine nanoparticles with SH-SY5Y cells in Example 6 of the present application;
  • FIG. 12 shows the results of electrophoresis detection of RNAh-Biotin-quasar670-tacrine nanoparticles in Example 7 of the present application after different incubation times in serum under the Coomassie Blue program;
  • Example 13 shows the results of electrophoresis detection of RNAh-Biotin-quasar670-tacrine nanoparticles in Example 7 of the present application after incubating in serum for different times under the Stain Free Gel program;
  • FIG. 15 shows the detection results of the fluorescence targeting vector Bio-Cy5-RNAh inhibiting the proliferation of SH-SY5Y cells in Example 8 of the present application;
  • FIG. 16 shows the results of non-denaturing PAGE gel electrophoresis detection of 7 groups of extended deformation + core short sequence RNA self-assembly products in Example 9 of the present invention
  • FIG. 17 shows a dissolution curve of RNA nanoparticles R-15 in Example 9 of the present invention
  • FIG. 18 shows a dissolution curve of RNA nanoparticles R-16 in Example 9 of the present invention
  • FIG. 19 shows a dissolution curve of RNA nanoparticles R-17 in Example 9 of the present invention
  • FIG. 20 shows the dissolution profile of RNA nanoparticles R-18 in Example 9 of the present invention
  • FIG. 21 shows a dissolution curve of RNA nanoparticles R-19 in Example 9 of the present invention
  • FIG. 22 shows a dissolution curve of the RNA nanoparticle R-20 in Example 9 of the present invention
  • FIG. 23 shows the dissolution profile of RNA nanoparticles R-21 in Example 9 of the present invention
  • FIG. 24 shows the results of non-denaturing PAGE gel electrophoresis detection of 7 groups of extended deformation + core short sequence DNA self-assembly products in Example 10 of the present invention
  • FIG. 25 shows the dissolution profile of DNA nanoparticle D-8 in Example 10 of the present invention
  • FIG. 26 shows a dissolution curve of the DNA nanoparticle D-9 in Example 10 of the present invention
  • FIG. 27 shows a dissolution curve of the DNA nanoparticle D-10 in Example 10 of the present invention
  • FIG. 28 shows the dissolution profile of DNA nanoparticles D-11 in Example 10 of the present invention
  • FIG. 29 shows a dissolution curve of the DNA nanoparticle D-12 in Example 10 of the present invention
  • FIG. 30 shows a dissolution curve of the DNA nanoparticle D-13 in Example 10 of the present invention
  • FIG. 31 shows a dissolution curve of DNA nanoparticles D-14 in Example 10 of the present invention
  • FIG. 32 shows the results of electrophoretic detection of RNA nanoparticles R-15 in Example 11 of the present invention after different incubation in serum;
  • FIG. 33 shows the results of electrophoresis detection of RNA nanoparticles R-16 in Example 11 of the present invention after different incubations in serum;
  • FIG. 34 shows the results of electrophoresis detection of RNA nanoparticles R-17 in Example 11 of the present invention after different incubation in serum;
  • FIG. 35 shows the results of electrophoresis detection of RNA nanoparticles R-18 in Example 11 of the present invention after being incubated in serum for different times;
  • FIG. 36 shows the results of electrophoresis detection of RNA nanoparticles R-19 in Example 11 of the present invention after different incubation in serum;
  • FIG. 37 shows the results of electrophoresis detection of RNA nanoparticles R-20 in Example 11 of the present invention after being incubated in serum for different times;
  • FIG. 38 shows the results of electrophoretic detection of RNA nanoparticles R-21 in Example 11 of the present invention after different incubation in serum;
  • FIG. 39 shows the results of electrophoresis detection of DNA nanoparticles D-8 in Example 12 of the present invention after being incubated in serum for different times;
  • FIG. 40 shows the results of electrophoresis detection of DNA nanoparticle D-9 in Example 12 of the present invention after different incubation in serum;
  • FIG. 42 shows the results of electrophoresis detection of DNA nanoparticle D-11 in Example 12 of the present invention after being incubated in serum for different times;
  • FIG. 43 shows the results of electrophoresis detection of DNA nanoparticle D-12 in Example 12 of the present invention after different incubation in serum;
  • FIG. 44 shows the results of electrophoresis detection of DNA nanoparticle D-13 in Example 12 of the present invention after different incubation in serum;
  • FIG. 45 shows the results of electrophoresis detection of DNA nanoparticles D-14 in Example 12 of the present invention after different incubation in serum;
  • Figure 46a, Figure 46b, Figure 46c, Figure 46d, Figure 46e, Figure 46f, Figure 46g, and Figure 46h show the DMSO and the original drug doxorubicin, D-8 and D-8-A in Example 15 of the present invention, respectively.
  • Domycin, D-9 and D-9-doxorubicin, D-10 and D-10-doxorubicin, D-11 and D-11-doxorubicin, D-12 and D-12-doxorubicin Cell survival curves for D-13, D-13 and D-13-doxorubicin, D-14 and D-14-doxorubicin;
  • FIG. 47 shows a standard curve of daunorubicin absorbance used in the mounting rate detection process of Example 16;
  • FIGS. 48a and 48b, FIG. 49, FIG. 50a and FIG. 50b, FIG. 51, FIG. 52, FIG. 53, FIG. 54a and FIG. 54b, and FIG. 55 sequentially show the flexibility of the mounting rate detection process in Embodiment 17 of the present application.
  • Figures 64 to 81 show epirubicin (Figures 64 to 66), methotrexate (Figures 67 and 68), pirarubicin ( Figures 69, 70, and 71), respectively, Daunorubicin ( Figure 72 and Figure 73), Pentafluorouracil ( Figure 74 and Figure 75), 10-Hydroxycamptothecin ( Figure 76 and Figure 77), Aspirin ( Figure 78 and Figure 79), and Gemcitabine (Figure 80 and Figure 81) Stability of nucleic acid nanoparticles in serum;
  • Figures 82 to 101 show epirubicin (Figure 82, Figure 83, Figures 84a to 84d, and Figures 85a to 85d), methotrexate ( Figures 86 and 87), and pirarubicin ( Figures 88, 89, and 91a to 91d), daunorubicin ( Figures 92 and 93), pentafluorouracil ( Figures 94 and 95), 10-hydroxycamptothecin ( Figures 96 and 97), aspirin ( Figures 98 and 99) ) And Gemcitabine (Figures 100 and 101).
  • Blank carrier refers to a blank nucleic acid nanoparticle carrier that does not contain any biologically active substances, such as RNAh or DNAh.
  • Targeting carrier refers to a nucleic acid nanoparticle carrier that contains a target but does not contain a fluorescent substance, such as Biotin-RNAh or Biotin-DNAh.
  • Fluorescent carrier refers to a nucleic acid nanoparticle carrier that contains a fluorescent substance but does not contain a target, such as Cy5-RNAh or Cy5-DNAh.
  • Targeted fluorescent carrier refers to a nucleic acid nanoparticle carrier containing a target and a fluorescent substance, such as Biotin-Cy5-RNAh or Biotin-Cy5-DNAh.
  • Targeted drug refers to a nucleic acid nanoparticle carrier containing a target, a fluorescent substance and a chemical agent, such as taclaline-Biotin-Cy5-RNAh or taclaline-Biotin-Cy5-DNAh.
  • RNA nanoparticles Based on the analysis of the reported nanoparticles formed by self-assembly of DNA and RNA, it is found that compared to relatively rigid DNA nanoparticles, RNA nanoparticles have a larger number of stem-loop structures within or between molecules. Large flexibility and stronger tension make it more advantageous as a candidate drug carrier.
  • the stability of RNA nanoparticles in the natural state is relatively poor, and most of the current improvements based on the application of RNA nanocarriers are focused on improving their stability and reliability.
  • the current research results provide the possibility of mounting drugs to a certain extent, they focus more on the possibility and effectiveness of mounting nucleic acid drugs, especially siRNA drugs or miRNA drugs. Whether non-nucleic acid drugs are equally effective is rarely reported.
  • RNA nanoparticle carrier In order to provide a new RNA nanoparticle carrier with good reliability and self-assembly, the applicant compared and improved the existing RNA nanoparticles, and developed a series of new RNA nanoparticles. From the perspective of performance and cost reduction, further attempts were made to use pure DNA strands for self-assembly. It was unexpectedly discovered that these DNA single strands could not only achieve self-assembly into DNA nanoparticles, but also perform as well as RNA nanoparticles. Moreover, the self-assembly of DNA nanoparticles also has the advantages of being cheap and easy to operate.
  • RNA nanoparticles and DNA nanoparticles improved by the inventors can both mount drugs and stably exist in serum; further experiments have verified that they can carry these drugs into cells and separate nucleic acid nanoparticles
  • the carrier is non-toxic to cells.
  • the drug carrier can relieve and treat the corresponding diseases.
  • the application provides a nucleic acid nanocarrier drug.
  • the drug includes nucleic acid nanoparticles and a drug.
  • the drug is mounted on the nucleic acid nanoparticle.
  • the drug includes tacroline, epirubicin, methotrexate, and pirarubicin.
  • the nucleic acid nanoparticle includes a nucleic acid domain, the nucleic acid domain comprising a sequence, b sequence, and c sequence, a
  • the sequence contains a1 sequence or a sequence in which at least one base insertion, deletion or replacement occurs in a1 sequence
  • b sequence contains a sequence in which b1 sequence or at least one base insertion, deletion or replacement occurs in b1 sequence
  • c sequence contains c1 sequence or c1 sequence occurrence At least one base insertion, deletion or replacement sequence; wherein the a1 sequence is SEQ ID NO: 1: 5'-CCAGCGUUCC-3 'or SEQ ID NO: 2: 5'-CCAGCGTTCC-3';
  • b1 sequence is SEQ ID NO: 3: 5'-GGUUCGCCG-3 'or SEQ ID NO: 4: 5'-GGTTCGCCG-3';
  • the nucleic acid nanocarrier drugs provided in the present application include nucleic acid nanoparticles and drugs, and any one or more of the above drugs are mounted on the nucleic acid nanoparticles.
  • the nucleic acid nanoparticle not only can self-assemble to form a nucleic acid domain by including the above three sequences or variant sequences thereof, but also can be used as a carrier to connect drugs at any 5 'end and / or 3' end of the three strands, or can make The drug is stably intercalated between the strands of the nucleic acid domain.
  • the nucleic acid nanocarrier drug provided by the present application by mounting the above-mentioned drug on the nucleic acid nanoparticle, because the nucleic acid nanoparticle has internal hydrophobicity, external hydrophilicity, and base-stacking effect, it is equivalent to acting on the drug.
  • Coating effect while coating or covalent attachment prevents the drug from being dissolved within a certain period of time, improving the stability of delivery.
  • the nucleic acid domain is modified by the target, it can have better targeting, can stably deliver drugs, and has high reliability; meanwhile, it can reduce the chance of contacting the corresponding drug with non-target cells or tissues, and reduce toxicity side effect.
  • the above self-assembly refers to a technique in which the basic structural unit spontaneously forms an ordered structure.
  • the basic structural unit spontaneously organizes or aggregates into a stable structure with a certain regular geometric appearance under the interaction of non-covalent bonds.
  • the self-assembly process is not a simple superposition of a large number of weak interaction forces between atoms, ions, or molecules (where "weak interaction forces” refers to hydrogen bonds, van der Waals forces, electrostatic forces, hydrophobic forces, etc.), but rather between several individuals.
  • weak interaction forces refers to hydrogen bonds, van der Waals forces, electrostatic forces, hydrophobic forces, etc.
  • the generation of self-assembly requires two aspects: the power and guidance of self-assembly.
  • the power of self-assembly refers to the synergy of weak interaction forces between molecules, which provides energy for molecular self-assembly.
  • Self-assembly guidance refers to the complementarity of molecules in space, that is, self-assembly needs to meet the requirements of molecular rearrangement in the size and direction of space.
  • DNA nanotechnology is a bottom-up molecular self-assembly model that starts from a molecular structure and spontaneously forms a stable structure based on the physical and chemical properties of the nucleic acid molecule, following strict principles of nucleic acid base pairing. Multiple DNA fragments are ligated together in the correct order in vitro. Based on the principle of complementary base pairing, a sub-assembly structure is established, and finally a complex multi-level structure is formed. Unlike DNA, the structure of RNA can exceed the limits of the double helix. RNA can form a series of different base pairs with at least two hydrogen bonds between the base pairs.
  • RNA nanotechnology can take advantage of these naturally occurring 3D modules and their predictable interactions.
  • many biologically active RNA structures can have atomic resolution, such as ribosomes, various ribozymes, and ribose Natural RNA aptamer within the switch.
  • One of the advantages of RNA nanotechnology is that structures can be designed that are comparable in size and complexity to natural RNA materials. The unique assembly properties of RNA in natural RNA complexes can also be exploited.
  • nucleic acid nanoparticles of the present application include the three sequences shown in the sequences SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 or their mutated sequences, or the sequences SEQ ID NO: 2 and SEQ:
  • the three sequences shown in ID NO: 4 and SEQ ID NO: 6 or their mutated sequences are based on the ability to form nucleic acid nanoparticles through self-assembly.
  • the specific mutated sequence can be found in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 based on the reasonable selection of the mutation site and its type of mutation, or by extending the appropriate fragment.
  • the nanoparticles formed by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 are RNA nanoparticles, and SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 are self-assembled nanoparticles.
  • the particles are DNA nanoparticles.
  • at least one of the a sequence, the b sequence, and the c sequence includes a sequence of at least one base insertion, deletion, or replacement.
  • the specific position and base type of the mutated sequence in the RNA nanoparticle can be improved to a nanoparticle that can increase the amount of drug mounted or improve the stability under the premise that self-assembly can be realized.
  • the above-mentioned SEQ ID NO: 1/2, SEQ ID NO: 3/4, and / or SEQ ID NO: When performing the base insertion, deletion or replacement of the sequence shown in 5/6, it can be performed on the base at some specific positions of the above sequence.
  • the mutated sequence can be self-assembled into nanoparticles as the original sequence.
  • the mutation retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% homology to the original sequence, so that it is homologous to the above sequence.
  • the assembled nanoparticles have the same drug-loading characteristics and similar stability, and can well mount and deliver drugs.
  • the above-mentioned base insertion, deletion or replacement occurs at: (1) 1, 2, 4, and 5 starting from the 5 ′ end of the a sequence shown by SEQ ID NO: 1 or 2. Between bases; and / or (2) SEQ ID NO: 1 or 2 between the 8th and 10th bases from the 5 'end of the a sequence shown in 1 or 2; and / or (3) SEQ ID IDNO : Between the bases 1 to 3 of the 5 'end of the b sequence shown in 3 or 4; and / or (4) the 5' end of the b sequence shown in SEQ ID NO: 3 or 4 Between the 6th and 9th bases; and / or (5) between the 1st and 4th bases starting from the 5 'end of the c sequence shown in SEQ ID NO: 5 or 6; and / or ( 6) Between the 9th and 12th bases from the 5 ′ end of the c sequence shown in SEQ ID NO: 5 or 6.
  • the defined base positions for mutation are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ
  • the flexibility and tension of the formed nanostructures help maintain their stability as a carrier.
  • the a sequence, the b sequence, and the c sequence are self-assembled into the formula (1) structure:
  • WC represents Watson-Crick pairing
  • WC at any position is independently selected from CG or GC
  • at least two sequences of a sequence, b sequence and c sequence are each The two bases at the 5 'end and the 3' end are not complementary; in the a sequence, the first N from the 5 'end is A, the second N is G, the third N is U or T, and the fourth Each N is any one of U, T, A, C, or G; in the b sequence, the first N ′ from the 5 ′ end is any one of U, T, A, C, or G; the second N ′ is U or T, and the third N ′ is C; in the c sequence, the NNNN sequence in the direction from the 5 ′ end to the 3 ′ end is CAUA or CATA.
  • the a, b, and c sequences are self-assembled to form a nucleic acid domain having the formula (1), in which, except for non-Watson-Crick paired bases defined by N and N ′, The bases form a classic Watson-Crick pairing, and the bases of the above Watson-Crick pairings all select GC or CG base pairs. Because the force of hydrogen bonding between G-C or C-G base pairs is greater than the force of hydrogen bonding between A-U / T or U / T-A base pairs, the nucleic acid nanostructure is more stable.
  • the raised or loop structure formed by non-Watson-Crick base pairs brings greater tension to the nucleic acid nanocarrier, making it more adaptable to changes in the microenvironment, and thus the stability of the nucleic acid nanoparticle is higher. .
  • the specific sequence composition of the a sequence, the b sequence, and the c sequence is not particularly limited as long as the structure can be formed. From the perspective of self-assembly of nucleic acid sequences, in order to further improve the efficiency of self-assembly of the above three sequences into the structure of the above formula (1), when selecting bases paired by Watson-Crick, the selection of bases at different positions is best to follow The following principles: (1) a sequence, b sequence and c sequence, a single sequence does not self-complementary pairing to form a secondary structure; (2) a sequence, b sequence and c sequence, one pair of complementary complementary ends between any two sequences A double strand is formed, and the other ends are not complementary paired to form a Y-type or T-type structure.
  • the above-mentioned principle of base selection is to make the two ends of any one strand complementary to the two ends of the other two strands, respectively, to maximize the self-assembly efficiency.
  • a quadrilateral other than a trigeminal can also be used to deform other forms, as long as the complementary pairing between one end of any two sequences forms a double-stranded pair, and the other end is not complementary pairing.
  • the fourth N from the 5 ′ end in the a sequence and the first N from the 5 ′ end in the b sequence are matched with it.
  • ' It can be a non-Watson-Crick pairing UU, or it can be an improved T, A, C or G following the Watson-Crick pairing principle.
  • Watson-Crick pairing relatively improves the binding force between chains and improves stability, while non-Watson-Crick pairing gives the nanoparticles greater flexibility and flexibility. It also helps to improve nanoparticles when facing changes in the microenvironment. The stability.
  • the a sequence, the b sequence, and the c sequence are any of the following: (1) a sequence (SEQ ID NO: 7): 5'-GGAGCGUUGG-3 ', b sequence (SEQ ID ID NO : 8): 5'-CCUUCGCCG-3 ', c sequence (SEQ ID NO: 9): 5'-CGGCCAUAGCCC-3'; (2) a sequence (SEQ ID NO: 10): 5'-GCAGCGUUCG-3 ' , B sequence (SEQ ID NO: 11): 5'-CGUUCGCCG-3 ', c sequence (SEQ ID NO: 12): 5'-CGGCCAUAGCGC-3'; (3) a sequence (SEQ ID ID ID: 13): 5'-CGAGCGUUGC-3 ', b sequence (SEQ ID NO: 14): 5'-GCUUCGCCG-3', c sequence (SEQ ID NO: 15): 5'-CGGCCAUAGCCG-3 '; (4) a sequence (SEQ
  • the nucleic acid nanoparticles formed by the self-assembly of the fourteen groups of sequences described above not only have higher stability, but also have higher self-assembly efficiency.
  • nucleic acid nanoparticles are not only capable of self-assembly, but also have the ability to carry or mount drugs. Depending on the positions of the G-C or C-G base pairs in the nucleic acid nanoparticles described above, the amount of the drug mounted also varies.
  • the above-mentioned nucleic acid domain further includes a first extension segment.
  • An extension is a Watson-Crick paired extension, and the first extension is located at the 5 ′ end and / or the 3 ′ end of any of the a sequence, the b sequence, and the c sequence.
  • a certain matching relationship is required between the carrier and the substance to be mounted.
  • the specific length of the first extension section can be determined according to the size of the substance to be mounted.
  • the first extension is selected from any one of the following groups: (1): 5 'end of a chain: 5'-CCCA-3', 3 'end of c chain: 5'-UGGG-3 '; (2): 3' end of a chain: 5'-GGG-3 ', 5' end of b chain: 5'-CCC-3 '; (3): 3' end of b chain: 5'-CCA-3 ', 5' end of c chain: 5'-UGG-3 '; (4): 5' end of a chain: 5'-CCCG-3 ', 3' end of c chain: 5'-CGGG-3 '; (5) ): 5 'end of a chain: 5'-CCCC-3', 3 'end of c chain: 5'-GGGG-3'; (6): 3 'end of b chain: 5'-CCC-3', c chain 5 'end: 5 'end
  • the above first extension not only increases the length of any one or more of the three sequences forming the nucleic acid nanostructure, but also the first extension of the GC base composition further improves the stability of the formed nanoparticles.
  • the first extension composed of the above sequences also maintained high self-assembly activity and efficiency of the a, b, and c sequences.
  • the nucleic acid domain further includes a second extension, and the second extension is located at the 5 ′ end and / or the 3 ′ end of any one of the a sequence, the b sequence, and the c sequence, and the second extension
  • the segment is an extended segment of Watson-Crick pairing; more preferably, the second extended segment is an extended sequence of CG base pairs; further preferably, the second extended segment is an extended sequence of 1 to 10 CG base pairs.
  • the second extension is an extension added to the first extension.
  • the above-mentioned nucleic acid domain further includes at least one second extended segment as follows: the first group: the 5 'end of the a chain: 5'-CGCGCG-3', and the 3 'end of the c chain: 5' -CGCGCG-3 '; second group: 3' end of a chain: 5'-CGCCGC-3 ', 5' end of b chain: 5'-GCGGCG-3 '; third group: 3' end of b chain: 5 ' -GGCGGC-3 ', 5' end of the c chain: 5'-GCCGCC-3 '.
  • This second extension makes the nanoparticles non-immunogenic and there is no secondary structure in which each chain folds and binds to itself.
  • first extended segment and / or the second extended segment may be separated by unpaired base pairs.
  • the second extension section In order to include an extended sequence of both CG base pairs and AT / AU base pairs, the second extended segment is preferably an extended sequence of 2 to 50 base pairs.
  • the "/" in "AT / AU base” is an OR relationship.
  • the second extension is an extension sequence containing both CG base pairs and AT base pairs, or the second extension contains both Extended sequences of CG base pairs and AU base pairs.
  • sequences after adding the second extension may be the following sequences, respectively:
  • the b sequence is (SEQ ID NO: 50):
  • the c sequence is (SEQ ID NO: 51):
  • M in the a, b, and c sequences is U or T.
  • M is T, the synthesis cost of the above sequence is greatly reduced.
  • the second extended segment is an extended sequence in which consecutive 2-8 CG base pair sequences are alternately arranged with consecutive 2-8 AT / AU base pair sequences; or the second extended segment It is an extended sequence in which one CG base pair sequence and one AT / AU base pair sequence are alternately arranged.
  • the GCGGCG in the b sequence shown in the above SEQ ID NO: 50 is extended
  • the positions of the GGCGGC extension and the TTTTTT extension are interchanged.
  • the GCCGCC extension in the c sequence shown in SEQ ID NO: 51 above is interchanged with the AAAAAA extension
  • the CGCCGC extension is interchanged with the TTTTTT extension.
  • the nucleic acid nanoparticles formed by the self-assembly of the above sequence are suitable for the mounting of biologically active substances with an indole molecular structure (indole molecules are preferably combined with A).
  • RNA as a widely used construction material include: 1) sensitivity to RNase degradation; 2) sensitivity to dissociation after systemic injection; and 3) toxicity and adverse immune response.
  • these three major challenges have been largely overcome: 1) 2'-fluoro (2'-F) or 2'-O-methyl (2'-OMe) modification of the ribose-OH group can be Makes RNA chemically stable in serum; 2) Some naturally occurring linking motifs are thermodynamically stable and can keep the entire RNA nanoparticles intact at ultra-low concentrations; 3)
  • the immunogenicity of RNA nanoparticles is sequence and shape Dependent and can be adjusted to make RNA nanoparticles stimulate inflammatory cytokine production or make RNA nanoparticles non-immunogenic and non-toxic when administered repeatedly at 30 mg / kg intravenously.
  • the bases, riboses and The phosphate ester has at least one modifiable site, and any modifiable site is modified by any of the following modified linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, thiol, and aldehyde group; preferably, The C or U base in the a sequence, the b sequence, and the c sequence has a 2'-F modification.
  • the modified linker is a thiol group, it is a thio modification, and the modification strength is weak and the cost is low.
  • the aforementioned drugs can be mounted in the form of physical connection and / or covalent connection.
  • physical intercalation is usually intervening between GC base pairs.
  • the number of preferred intervening sites is based on the GC base on the nucleic acid domain.
  • the number of base pairs is different, and the interpolation is performed according to a ratio of 1 to 100: 1.
  • covalent linking the above-mentioned drugs usually chemically react with the amino group outside the G ring to form a covalent link. More preferably, the molar ratio between the drug and the nucleic acid nanoparticles is 2 to 300: 1, preferably 2 to 290: 1, more preferably 2 to 29: 1, even more preferably 10 to 50: 1, and most preferably 15 ⁇ 25: 1.
  • nucleic acid nanocarrier drugs are used as drug delivery carriers.
  • nucleic acid nanoparticles are used as drug delivery carriers.
  • the above-mentioned nucleic acid nanoparticles also include a biologically active substance.
  • the biologically active substance is linked to a nucleic acid domain.
  • Bioactive substances are targets, fluorescein, interfering nucleic acid siRNA, miRNA, ribozymes, riboswitches, aptamers, RNA antibodies, proteins, peptides, flavonoids, glucose, natural salicylic acid, monoclonal antibodies, vitamins, phenols, One or more of lecithin and small molecule drugs, wherein the small molecule drugs do not include taclaline, epirubicin, methotrexate, pirarubicin, daunorubicin, pentafluorouracil, 10- Hydroxycamptothecin, aspirin and gemcitabine.
  • the relative molecular weight of the nucleic acid domain and the relative molecular weight of the drug and the bioactive substance preferably have a certain matching relationship.
  • the relative molecular weight of the nucleic acid domain is recorded as N 1
  • the total relative molecular weight of the drug and the biologically active substance is recorded as N 2
  • the nucleic acid nanocarrier drug in this application is optimized in different performances.
  • the bioactive substance is biotin or folic acid
  • its role is to make the nucleic acid nanocarrier drug targeted, for example, to specifically target cancer cells.
  • the biologically active substance is fluorescein
  • its role is to make the nucleic acid nanoparticles have a luminescent tracer effect.
  • the biologically active substance is some siRNA, miRNA, protein, peptide, RNA antibody, and small molecule drug
  • the nucleic acid nanocarrier drug may become a new product with a specific therapeutic effect according to different biological functions. Excellent drug.
  • DNA nanoparticles and RNA nanoparticles are preferably used, and can be reasonably selected according to actual needs.
  • the biologically active substance is a drug
  • the biologically active substance is a target, fluorescein, and miRNA, wherein the target is located on any one of the sequences a, b, and c, preferably 5 ′ of any one of the sequences a, b, and c.
  • miRNA is anti-miRNA
  • fluorescein is modified at the 5' end or 3 'end of the anti-miRNA
  • miRNA is located at the 3' end of the a sequence
  • the c sequence Any one or more of the 5 'end and the 3' end; preferably, the target is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is an anti-miR -twenty one.
  • the target can be linked to any of the a, b, and c sequences by a covalent linker.
  • the available linker is selected from disulfide bonds, p-phenylazide, bromopropyne, or PEG.
  • "on any sequence” refers to a base at any position of any sequence of the a, b, and c sequences, and it is more convenient to connect to the 5 'end or 3' end and it is more widely used.
  • Folic acid modification can be a physical intercalation mode connection or a physical intercalation + covalent connection.
  • the above-mentioned fluorescein may be a commonly used fluorescein, and is preferably any one or more of FAM, CY5, and CY3.
  • the above miRNA may be a miRNA having a tumor suppressing effect, or an anti-miRNA capable of suppressing a corresponding disorder, and is reasonably selected according to medical needs in practical applications.
  • the anti-miRNA may be synthesized at any one or more of the 3 'end of the a sequence, the 5' end of the c sequence, and the 3 'end. When anti-miRNAs are synthesized at the above three positions, the inhibitory effect of anti-miRNAs on the corresponding miRNAs is relatively stronger.
  • Anti-miR-21 is preferred, miR-21 is involved in the initiation and progression of various cancers, and is the main oncogene for invasion and metastasis. Anti-miR-21 can effectively regulate a wide range of target genes simultaneously, which is conducive to solving the problem of cancer heterogeneity. Therefore, among the above-mentioned preferred nucleic acid nanoparticles, the target, such as folic acid or biotin, can specifically target cancer cells. After binding and internalization with cancer cells, anti-miR-21 has a very high affinity and specificity with miR- 21 bases are complementary, which effectively reduces the expression of oncogenic miR-21.
  • the anti-miR-21 may be synthesized at any one or more positions of the 3 'end of the a sequence, the 5' end and the 3 'end of the c sequence.
  • anti-miR-21 was synthesized at all three positions, the inhibitory effect of anti-miR-21 on miR-21 was relatively stronger.
  • nucleic acid nanocarrier drugs are based on the types of diseases that can be treated by different drugs, including but not limited to the treatment of liver cancer, stomach cancer, lung cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, melanoma, leukemia, dementia , Ankylosing spondylitis, malignant lymphoma, bronchial cancer, rheumatoid arthritis, HBV hepatitis B, multiple myeloma, pancreatic cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal cancer, esophageal cancer, oral cancer, lupus erythematosus disease
  • the head and neck cancer is
  • the above-mentioned bioactive substances that can be mounted are small-molecule drugs other than the above-mentioned taclarine, etc., depending on the molecular structure of the drug or the characteristic group it has, it includes but is not limited to containing any of the following: Drugs of one or more groups: amino group, hydroxyl group, carboxyl group, thiol group, benzene ring group, and acetamino group.
  • the aforementioned proteins are SOD (superoxide dismutase), survivin, hTERT (human telomerase reverse transcriptase), and EGFR (epidermal growth factor receptor, epidermal growth factor receptor). ), One or more of antibodies or aptamers of PSMA (Prostate Specific Membrane Antigen); the above-mentioned vitamins are L-C and / or esterified C; the above-mentioned phenols are tea polyphenols and / or grape polyphenols.
  • the particle size of the nucleic acid nanoparticles is 1 to 100 nm, preferably 5 to 50 nm, more preferably 10 to 30 nm, and even more preferably 10 to 15 nm. In this range, the size is suitable, which can enter the cell membrane through cell surface receptor-mediated cell phagocytosis, and avoid non-specific cell penetration and filtration and removal by the kidney. Therefore, favorable particle size helps to improve pharmacogenetics Kinetics, pharmacodynamics, biological distribution, and toxicology.
  • a method for preparing the above-mentioned nucleic acid nanocarrier drug which includes the following steps: providing any one of the above-mentioned nucleic acid nanoparticles; and suspending the drug by means of physical connection and / or covalent connection. Loaded on nucleic acid nanoparticles to obtain nucleic acid nanocarrier drugs.
  • the drug When the physical connection method is used, the drug is usually inserted between the GC base pairs by physical insertion. When covalent linking is used, the drug usually reacts with the amino group outside the G ring to form a covalent link.
  • the nucleic acid nanocarrier drug prepared by the above method can have better targeting after being modified by the target, can stably deliver the drug, and has high reliability.
  • the step of mounting the drug by means of physical connection includes: mixing and stirring the drug, the nucleic acid nanoparticles, and the first solvent to obtain a premixed system; and precipitating the premixed system to obtain nucleic acids.
  • Nanocarrier drugs The amount of specific drugs and nucleic acid nanoparticles can be adjusted according to the change in the amount of mounting, which can be understood by those skilled in the art, and will not be repeated here.
  • the amount of the drug added per liter of the first solvent is 0.1 to 1 g.
  • the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid.
  • the step of precipitating the premixed system to obtain a nucleic acid nanocarrier drug includes: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain a nucleic acid nanocarrier drug.
  • the premixed system is mixed with anhydrous ethanol to perform precipitation under a temperature condition of less than 10 ° C to obtain a precipitate, and further preferably to precipitate under a temperature condition of 0 to 5 ° C to obtain a precipitate. More preferably, the precipitate is washed with 6 to 12 times the volume of absolute ethanol to remove impurities to obtain a nucleic acid nanocarrier drug.
  • the step of mounting the drug by covalent linking includes: configuring a drug solution; allowing the drug solution to react with the G-ring amino group of the nucleic acid nanoparticle under the mediation of formaldehyde to obtain a reaction System; purification of the reaction system to obtain nucleic acid nanocarrier drugs.
  • the above-mentioned reaction step includes: mixing a drug solution with a paraformaldehyde solution and nucleic acid nanoparticles, and performing the reaction under the condition of avoiding light to obtain a reaction system.
  • the paraformaldehyde solution can release small molecules of formaldehyde, thereby participating in the above-mentioned chemical reaction.
  • the concentration of the paraformaldehyde solution is preferably 3.7 to 4% by weight.
  • the paraformaldehyde solution is preferably a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is DCM, DCC, DMAP, Py, and DMSO. Or more of PBS, PBS and glacial acetic acid.
  • the nucleic acid nanoparticles can be prepared by self-assembly, such as: (1) simultaneous mixing of RNA or DNA single strands a, b, and c in DEPC water or TMS buffer; (2) heating and mixing Solution to 80 ° C / 95 ° C (where the RNA assembly temperature is 80 ° C and the DNA assembly temperature is 95 ° C), and after keeping for 5 minutes, slowly cool down to room temperature at a rate of 2 ° C / min; (3) load the product to 8% ( m / v) Purification of the complex on a non-denaturing PAGE gel and electrophoresis at 100 V in TBM buffer at 4 ° C; (4) Cut the target band and elute in 37 ° C in RNA / DNA elution buffer After that, ethanol was precipitated overnight, and dried under reduced pressure and low temperature to obtain a self-assembling product, and a nucleic acid domain was obtained, and then nucleic acid nanoparticles were obtained.
  • the preparation method further includes: physically connecting the biologically active substance mentioned above and And / or covalently attached to the nucleic acid domain to obtain nucleic acid nanoparticles.
  • the mounting method of the biologically active substance may also be a physical connection and / or a covalent connection.
  • the form of covalent connection includes, but is not limited to, mounting by solvent covalent connection, linker covalent connection, or clicking on a link; preferably, the third solvent used in the solvent covalent connection is used as the connection medium, and the third solvent is selected from multiple One or more of polyoxymethylene, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, the linker is selected from disulfide bonds, p-phenylazide, bromopropyne, or PEG; preferably, Clicking the link is to modify the precursor of the biologically active substance and the nucleic acid domain at the same time by alkynyl or azide, and then click the link.
  • the third solvent used in the solvent covalent connection is used as the connection medium, and the third solvent is selected from multiple One or more of polyoxymethylene, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, the linker is selected from disulfide bonds
  • the above classification does not mean that there is only one way to connect a certain biologically active substance to a nucleic acid domain. Instead, some biologically active substances can be connected to the nucleic acid domain by means of physical intercalation, or can be connected to the nucleic acid domain by means of physical intercalation and covalent attachment. . However, for a specific biologically active substance, there may be only one connection method, or there may be multiple connection methods, but one of the connection efficiency may have practical value of advantage.
  • the binding sites and number of intercalation are also slightly different.
  • anthracyclines and acridines are intercalated, they are usually intercalated between GC base pairs.
  • the number of preferred intervening sites depends on the number of GC base pairs on the nucleic acid domain. Interpolation was performed at a ratio of 100: 1.
  • naphthamide drugs are intercalated, they are usually interposed between AA base pairs.
  • the number of preferred intercalation sites depends on the number of AA base pairs on the nucleic acid domain.
  • Pyridocarbazoles are based on AA bases. The number of pairs is interpolated according to a ratio of 1 to 200: 1.
  • the length of the a, b, and c sequences that form the nucleic acid domain in the nucleic acid nanoparticle and the number of GC complementary base pairs therein, the biologically active substance and the nucleic acid domain can be reasonably selected
  • the molar ratio is physically interpolated.
  • the molar ratio of the biologically active substance and the covalently connected drug is 1 to 200: 1.
  • This connection method is suitable for anthracycline and acridine drugs.
  • the proportion of the drugs connected by the different connection methods is not limited to the above range, as long as it can meet the efficient mounting, has no toxic effect on the cells, and achieve effective drug release after reaching the target.
  • the alkynyl or azide modification site of the biologically active substance precursor is selected from the group consisting of hydroxyl, carboxyl, sulfhydryl, or amino.
  • the site for the alkynyl or azide modification of the domain is selected from amino, imino or hydroxyl.
  • nucleic acid domain when the above-mentioned nucleic acid domain is combined with a drug, the nucleic acid domain is water-soluble, and most drugs have poor water solubility. After binding to the nucleic acid domain, the water solubility is improved.
  • these drugs are anthracyclines, these drugs pass the -NH bond on the nucleotide guanosine (at a suitable pH value, the -NH group is more active than other groups that may covalently bind to the drug Hundred times higher activity) and covalently bind to the nucleic acid domain, thereby forming a drug-loaded nucleic acid domain.
  • the binding reaction is carried out in a quantity, and a maximum of 35 to 45 drugs can be bound to one nucleic acid domain.
  • the amount of loading is related to the occupancy of the specific drug (including but not limited to molecular structure, morphology, shape, and molecular weight).
  • a pharmaceutical composition is also provided, and the pharmaceutical composition includes any one of the foregoing nucleic acid nanocarrier drugs.
  • an appropriate combination of drugs or excipients can be selected to form a drug combination that has a combined drug effect or can improve certain aspects of the drug's performance (such as stability).
  • nucleic acid nanocarrier drugs in the preparation of a medicament for treating Alzheimer's disease, tumor, autoimmune disease or heart disease.
  • Specific applications can be based on the drug of the present application to improve the drug itself to obtain a new drug, or use the drug of the present application as the main active ingredient to prepare it into a suitable formulation.
  • the diseases that can be treated are also different.
  • Drugs in nucleic acid nanocarrier drugs include taclaline which can be used to prepare drugs for the preparation of Alzheimer's disease.
  • the drug includes epirubicin
  • the above nucleic acid nanocarrier drugs can be used to prepare drugs for treating tumors.
  • the tumors can be acute leukemia, malignant lymphoma, breast cancer, bronchial lung cancer, ovarian cancer, nephroblastoma, soft tissue sarcoma, protozoa Any one or more of primary hepatocellular carcinoma, metastatic liver cancer, and medullary thyroid cancer.
  • the above-mentioned nucleic acid nanocarrier drugs can be used to prepare drugs for preventing and / or treating tumors or autoimmune diseases.
  • the tumors targeted are acute leukemia, breast cancer, chorionic epithelial cancer, malignant grape Any one or more of fetal, head and neck tumors, bone tumors, leukemia meningeal spinal infiltration, lung cancer, reproductive system tumors, and liver cancer.
  • the autoimmune diseases are refractory psoriasis, systemic lupus erythematosus, mandatory spondylitis, and Any one or more of dermatomyositis.
  • the aforementioned nucleic acid nanocarrier drug can be used to prepare a drug for treating tumors.
  • the tumor is any one or more of breast cancer, head and neck cancer, bladder cancer, ureter cancer, renal pelvis cancer, ovarian cancer, and cervical cancer.
  • the aforementioned nucleic acid nanocarrier drugs can also be used to prepare drugs for treating tumors.
  • the tumor is acute lymphocytic leukemia or myeloid leukemia.
  • the aforementioned nucleic acid nanocarrier drugs can also be used to prepare drugs for treating tumors.
  • the drugs can be used for treating liver cancer, colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, and chorionic epithelial cancer. , Malignant hydatidiform mole, squamous cell carcinoma of the head and neck, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer.
  • the above-mentioned nucleic acid nanocarrier drugs can also be used to prepare drugs for treating liver cancer, stomach cancer, head and neck cancer or leukemia.
  • the above nucleic acid nanocarrier drugs can be used to prepare antipyretic and analgesic, prevent heart disease and cerebral thrombosis, anti-inflammatory and rheumatism, treat arthritis, reduce skin and mucosal lymph node syndrome in patients with Kawasaki disease, fight cancer and prevent digestive tract Tumor drugs.
  • the aforementioned nucleic acid nanocarrier drugs can also be used to prepare drugs for treating tumors.
  • the drugs can be used for treating pancreatic cancer, non-small cell lung cancer, ovarian cancer, breast cancer, bladder cancer, cervical cancer, Drugs for liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumor, lymphoma, mesothelioma or head and neck cancer.
  • a method for preventing and / or treating Alzheimer's disease, tumor, autoimmune disease, or heart disease includes: providing any one of the nucleic acid nanocarrier drugs or drug combinations described above.
  • the effective amount herein includes a prophylactically effective amount and / or a therapeutically effective amount.
  • a therapeutically effective amount refers to an amount effective to achieve a desired therapeutic result, such as a reduction in dementia, within a necessary dose and time period.
  • the dose can be adjusted to provide the optimal therapeutic response dose, and the therapeutically effective amount can vary depending on the individual's disease state, age, gender, weight, and formulation to cause the desired response in the individual Ability.
  • a therapeutically effective amount also includes an amount in which the beneficial effects of the treatment exceed its toxic or deleterious effects.
  • a prophylactically effective amount refers to an amount effective to achieve a desired preventive result, such as preventing or inhibiting the occurrence of Alzheimer's disease, within the necessary dose and time period.
  • a prophylactically effective amount can be determined from the above description of a therapeutically effective amount. For any particular subject, a particular dose can be adjusted over time according to the needs of the individual and the professional judgment of the person administering it.
  • nucleic acid nanoparticles formed by self-assembly of the sequences or sequence deformations provided in this application can also be used as basic structural units, and can be further polymerized to form multimers, such as dimers and trimers, according to actual application needs. , Tetramer, pentamer, hexamer or heptamer, etc.
  • RNA and DNA nanoparticle carriers I. RNA and DNA nanoparticle carriers:
  • DNA uses the same sequence as the RNA above, with only T replacing U. Among them, the molecular weight of the a chain is 8802.66, the molecular weight of the b chain is 8802.33, and the molecular weight of the c chain is 9605.2.
  • RNA nanoparticles and DNA nanoparticles described above were all entrusted to biosynthetic engineering (Shanghai) Co., Ltd. for synthesis.
  • RNA or DNA single strands a, b, and c are simultaneously mixed and dissolved in DEPC water or TMS buffer at a molar ratio of 1: 1: 1;
  • the target band is cut and eluted in an RNA / DNA elution buffer at 37 ° C, followed by ethanol precipitation overnight, and dried under reduced pressure and low temperature to obtain a self-assembled product;
  • RNA self-assembly products The results of electrophoretic detection of RNA self-assembly products are shown in Figure 1.
  • lanes 1 to 3 are in order from left to right: a-chain, b-chain, and RNA self-assembly products.
  • the RNA self-assembly product diffuses slightly, but it can be clearly seen that it is a single band.
  • the molecular weight is the assembled molecular weight, which is larger than the single-stranded molecular weight, the band position lags behind the a and b strands.
  • the actual situation is consistent with the theory, which proves that the above-mentioned RNA single strands form a stable complex structure through self-assembly.
  • RNA core sequences SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 can successfully self-assemble into RNA nanometers. Particles.
  • the a, b, and c sequences including the DNA core sequences SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 can also successfully self-assemble into DNA nanoparticles.
  • RNA nanoparticles and DNA nanoparticles have a variety of extended sequences (including drug-mounting binding sequences) that facilitate the mounting function of the nucleic acid domain in addition to the core sequence that forms the nucleic acid domain, and A target or luciferin linked to a nucleic acid domain. It can be seen that the presence of substances other than these core sequences does not affect the formation of nucleic acid domains and the successful self-assembly of nucleic acid nanoparticles.
  • the self-assembled nucleic acid nanoparticles can be targeted under the guidance of the target, and fluorescein can make the nucleic acid nanoparticles visible and traceable.
  • the single strands of the above seven groups of short-sequence RNA nanoparticle carriers were all entrusted to biosynthetic engineering (Shanghai) Co., Ltd. for synthesis.
  • RNA single strands a, b, and c are mixed and dissolved in DEPC water or TMS buffer at a molar ratio of 1: 1: 1;
  • FIG. 4 The 4% agarose gel electrophoresis images of 7 groups of short-sequence RNA self-assembly products are shown in FIG. 4. From left to right, lanes 1 to 7 in FIG. 4 are: short sequences R-1, R-2, R-3, R-4, R-5, R-6, and R-7.
  • Measurement method prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell.
  • Example 2 shows that different combinations of a, b, and c core sequences can form RNA nanoparticles with a nucleic acid domain through self-assembly, and the structure is stable. Based on Example 1, it can be seen that adding various functional extension fragments or connecting targets, fluorescein, etc. on the basis of these different core sequence combinations can also successfully assemble into RNA nanoparticles, and has mounted drugs and cell targets. Directivity and visual traceability.
  • Example 4 In order to further verify these performances, an extended segment is added on the basis of Example 2, as described in Example 3. Based on the DNA core sequence corresponding to the RNA core sequence of Example 2, an extension fragment is added, and the target is connected or not connected. For details, see Example 4.
  • RNA nanoparticle carriers The single strands of the above-mentioned 7 groups of conventional sequence RNA nanoparticle carriers were all entrusted to Suzhou Gima for synthesis, in which the a, b, and c sequences in R-8 to R-14 were in R-1 to R-7, respectively.
  • a sequence of extended RNA oligos formed on the basis of the a, b, and c sequences, without the extension of the targeting module fragment, and C / U base 2'F modification (enhanced anti-digestion Sex and stability).
  • RNA nanoparticle R-14 a survivin siRNA nucleic acid interference therapy fragment was modified in the above-mentioned RNA nanoparticle R-14, specifically, the positive strand of Survivin siRNA was extended at the 3 'end of the a chain (see the underlined part of the a chain), and at b The 5 'end of the strand is connected to the antisense strand (see underlined part of the b strand) to form base pair complementarity.
  • RNA single strands a, b, and c are mixed and dissolved in DEPC water or TMS buffer at a molar ratio of 1: 1: 1;
  • FIG. 5 The 2% agarose gel electrophoresis images of 7 groups of conventional sequence RNA self-assembly products are shown in FIG. 5. From left to right, lanes 1 to 7 in FIG. 5 are: conventional sequence RNA self-assembly products R-8, R-9, R-10, R-11, R-12, R13, R-14.
  • FIG. 6 The 4% agarose gel electrophoresis of 7 groups of conventional sequence RNA self-assembly products is shown in FIG. 6. From left to right, lanes 1 to 7 in FIG. 6 are: conventional sequence RNA self-assembly products R-8, R-9, R-10, R-11, R-12, R13, R-14.
  • the bands of the 7 groups of conventional sequence RNA self-assembly products are bright and clear single bands, indicating that the 7 groups of conventional sequences can self-assemble into nanostructures.
  • the conventional sequence RNA self-assembly product R-14 has a modified Survivin siRNA nucleic acid interference therapy fragment and still has a stable self-assembly structure. It also shows that the nucleic acid nanoparticles can be used for nucleic acid drugs and have nucleic acid drug delivery in this application. Carrier function.
  • Measurement method prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell.
  • extension fragments can also successfully self-assemble into structurally stable RNA nanoparticles.
  • the added extension fragments make the RNA nanoparticles have superior drug-loading properties (see Example 5 for details).
  • the EGFRapt target or PSMAapt (A9L) target is extended in some a chains in the table:
  • EGFRapt (SEQ ID NO: 97): GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC;
  • PSMAapt (A9L, SEQ ID NO: 98):
  • D-1 is based on the core sequence (8) (a sequence: 5'-GGAGCGTTGG-3 ', b sequence: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3 ') , Conventional sequence DNA nanoparticles formed after adding an extended sequence containing the EGFRapt target (see underlined section);
  • D-2 is based on the core sequence (9) (a sequence: 5'-GCAGCGTTCG-3 ', b sequence: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3 ') , Conventional sequence DNA nanoparticles formed after adding an extended sequence containing the EGFRapt target (see underlined section);
  • D-3 is based on the core sequence (10) (a sequence: 5'-CGAGCGTTGC-3 ', b sequence: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3 ') , Conventional sequence DNA nanoparticles formed after adding an extended sequence containing the EGFRapt target (see underlined section);
  • D-4 is based on the core sequence (11) (a sequence: 5'-GGAGCGTTGG-3 ', b sequence: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3 ') , The conventional sequence DNA nanoparticle formed after adding the extended sequence containing the PSMAapt target (see underlined section);
  • D-5 is based on the core sequence (12) (a sequence: 5'-GCAGCGTTCG-3 ', b sequence: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3 ').
  • the conventional sequence DNA nanoparticle formed after adding the extended sequence containing the PSMAapt target see underlined section;
  • D-6 is based on the core sequence (13) described above (a sequence: 5'-GCAGCGTTCG-3 ', b sequence: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3 ') After adding an extended sequence that does not include the target structure; a conventional sequence of DNA nanoparticles is formed;
  • D-7 is based on the core sequence (14) described above (a sequence: 5'-CGAGCGTTGC-3 ', b sequence: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3 ') After adding an extended sequence that does not include the target structure; a conventional sequence of DNA nanoparticles is formed.
  • DNA single strands a, b, and c are simultaneously mixed and dissolved in DEPC water or TMS buffer at a molar ratio of 1: 1: 1;
  • the target band is cut and eluted in a DNA elution buffer at 37 ° C, followed by ethanol precipitation overnight, and dried under reduced pressure at low temperature to obtain a conventional sequence DNA self-assembly product;
  • FIG. 7 The 2% agarose gel electrophoresis images of 7 groups of conventional sequence DNA self-assembly products are shown in FIG. 7. From left to right, lanes 1 to 7 in FIG. 7 are: conventional sequence DNA self-assembly products D-1, D-2, D-3, D-4, D-5, D-6, and D-7.
  • FIG. 8 The 4% agarose gel electrophoresis images of 7 groups of conventional sequence DNA self-assembly products are shown in FIG. 8. From left to right, lanes 1 to 7 in FIG. 8 are: conventional sequence DNA self-assembly products D-1, D-2, D-3, D-4, D-5, D-6, D-7.
  • the bands of the 7 groups of conventional sequence DNA self-assembly products are bright and clear, indicating that the 7 groups of conventional sequence DNA strands have completed self-assembly and formed stable nanoparticles. structure.
  • the self-assembled structures of D-6 and D-7 are slightly lower in molecular weight because they carry EGFRapt or PSMAapt targets, and their band positions are significantly higher than other bands. The actual and theoretical conditions are in full agreement, further proving the self-assembled structure The stability.
  • DNA nanoparticles can also be successfully assembled, and they also have drug-loading, cell-targeting, and See traceability and other performance.
  • Measurement method prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell.
  • Nucleic acid nanoparticles RNA nanoparticles from Example 1.
  • reaction solution was extracted with chloroform (10 mL ⁇ 3), and then 25 mL of anhydrous ethanol was added, and after mixing, the product was fully analyzed in the dark at 4 ° C. (4 hours). Centrifuge (4000 / min), transfer the supernatant, wash the solid product with ethanol (50 mL) again, and dry the solvent under reduced pressure at low temperature to obtain the mounted product.
  • RNA nanoparticles on the basis of mounting tacroline on RNA nanoparticles, other small molecule drugs can be mounted for the second time in the same way as taclaline.
  • folic acid is further mounted in this application to obtain RNA nanoparticles with two small-molecule drugs, tacrine and folic acid, are mounted together.
  • the loading rate of the two drugs can be detected by referring to the above method (values are not shown).
  • Example 5 shows that the RNA nanoparticles (in Example 1) with extended fragments, targets and fluorescein have the function of loading drugs, and the small molecule drug tacrine can be covalently linked (paraformaldehyde-solvent Covalent) to achieve mounting, and can also be co-mounted with other small molecule drugs.
  • RNAh-Biotine-quasar670 in the table is used as a control, which refers to the biotin modification on the 5 ′ end of the a and b chains prepared according to the self-assembly method in Example 1, and quasar670 on the 3 ′ end of the c chain.
  • Nanoparticles formed by fluorescein modification, and RNAh-Biotin-quasar670-tacrine refers to nanoparticles formed after further mounting taclarine (mounted chemically according to Example 5).
  • RPMI-1640 medium Gibco, C11875500BT-500mL
  • DMEM Gibco, C11995500BT-500mL
  • Fetal Bovine Serum FBS
  • FBS Fetal Bovine Serum
  • PBS buffer Gibco, C20012500BT-500mL
  • Trypsin-EDTA Stemcell, 07901-500mL
  • DMSO Sigma, D5879-1L
  • Prolong Gold Antifade Mountant Inhibitor Thermo, P36941-2 mL
  • DAPI Yaman, 36308ES11-4 mL
  • Inverted Microscope (Olympus BX53, U-RFL-T); BD Falcon (Corning, 354118); Cytospin (TXD3).
  • SH-SY5Y cells (neuroblastoma cell line) were cultured in RPMI1640 + 10% FBS + 1% PS medium at 37 ° C and 5% CO 2 .
  • SH-SY5Y cells were trypsinized, washed with PBS, and added to a cell culture slide at 1 ⁇ 10 5 cells per well.
  • RNAh-Biotin-quasar670 and RNAh-Biotin-quasar670-tacrine nanoparticles were incubated with 200nM and 400nM RNAh-Biotin-quasar670 and RNAh-Biotin-quasar670-tacrine nanoparticles at 37 ° C and 5% CO 2 for 1 h and 4 h.
  • Test sample RNAh-Biotin-quasar670-tacrine nanoparticles prepared in Example 5 dissolved in PBS solution.
  • RPMI-1640 medium Gibco, C11875500BT-500mL
  • Fetal bovine serum FBS
  • Penicillin / Streptomycin PS
  • PBS buffer Gibco, C20012500BT-500mL
  • Novex TM Tris-Glycine Native Sample Buffer (2X) Invitrogen, LC2673-20mL
  • Novex TM 8% Tris-Glycine Mini Gels Invitrogen, XP00080BOX-1.0mm
  • Tris-Glycine Native Running buffer (10x) Life science, LC2672-500mL
  • G250 staining solution Beyotime, P0017-250mL
  • Spectrophotometer Thermo, ND2000C
  • Mini Gel Tank Invitrogen, PS0301
  • Imaging System Bio-Rad, ChemiDoc MP
  • FIG. 12 shows the electrophoresis results of the 8% non-denaturing gel (Coomassie Blue program)
  • FIG. 13 shows the electrophoresis results of the 8% non-denaturing gel (Stain Free Gel program).
  • the results of the serum stability test showed that at 0min, 10min, 1h, 12h, and 36h, the RNAh-Biotin-quasar670-tacrine nanoparticle sample bands had no significant difference under different time lengths, indicating that the RNAh-Biotin-quasar670-tacrine nanoparticle was at % FBS in 1640 medium is relatively stable without significant degradation.
  • the samples to be tested are DMSO control, small molecule drug tacrine and RNAh-Biotin-quasar670-tacrine nanoparticles.
  • RPMI-1640 medium Gibco, C11875500BT-500mL
  • DMEM Gibco, C11995500BT-500mL
  • Fetal Bovine Serum FBS
  • FBS Fetal Bovine Serum
  • PBS buffer Gibco, C20012500BT-500mL
  • Trypsin-EDTA Stemcell, 07901-500mL
  • DMSO Sigma, D5879-1L
  • Dox HSUN Pharm, H33021980 -10mg
  • CTG CellTiter-Glo Luminescent Cell Viability Assay Kit
  • Inverted Microscope (Olympus IX71, TH4-200); 96-well Plate Reader (Molecular Devices, Flexstation 3).
  • SH-SY5Y cells were cultured in RPMI1640 + 10% FBS + 1% PS medium at 37 ° C and 5% CO 2 .
  • RNA nanoparticles that do not carry tacrolime have no obvious cytotoxicity to SH-SY5Y cells
  • the inventors further designed RNAh-Biotin-Cy5, a targeted fluorescent carrier, to test the toxicity of SH-SY5Y cells, and used another
  • the small molecule drug Cisplatin was used as a control (the highest drug concentration in the experiment was 5 ⁇ M).
  • Table 44 and FIG. 15 It can be seen from the IC 50 values in Table 44 and in Figure 15 that the targeted fluorescent carrier that does not carry tacrolimin itself has no significant toxicity to the experimental cells.
  • RNA single strands a, b, and c are mixed and dissolved in DEPC water or TMS buffer at a molar ratio of 1: 1: 1;
  • the main reagents and instruments are as follows:
  • Measurement method prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell.
  • the dissolution curve method was used to detect the TM value of the 7 groups of extended deformation + core short-sequence RNA nanoparticles. The samples were consistent with the potential samples.
  • the reagents and instruments are as follows:
  • the program is set to start at 20 ° C, and the temperature is raised from 0.1 ° C to 95 ° C per second, and the reading is taken every 5s.
  • the TM values of the 7 groups of extended deformation + core short-sequence RNA nanoparticles are as follows.
  • the dissolution profile of R-15 is shown in Figure 17, the dissolution profile of R-16 is shown in Figure 18, and the dissolution profile of R-17 is shown in Figure 19.
  • the dissolution profile of R-18 is shown in Figure 20, the dissolution profile of R-19 is shown in Figure 21, the dissolution profile of R-20 is shown in Figure 22, and the dissolution profile of R-21 is shown in Figure 23. Due to the particularity of the RNA sample, the temperature corresponding to the 1/2 RFUmax value in the range of 20 to 90 ° C was used as the sample Tm value.
  • TM values of the 7 groups of extended deformation + core short-sequence RNA nanoparticles were higher, indicating that the self-assembled products have good structural stability.
  • DNA single strands a, b, and c are simultaneously mixed and dissolved in DEPC water or TMS buffer at a molar ratio of 1: 1: 1;
  • the target band is cut and eluted in a DNA elution buffer at 37 ° C, followed by ethanol precipitation overnight, and dried under reduced pressure at low temperature to obtain a DNA self-assembly product;
  • the main reagents and instruments are as follows:
  • Measurement method prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell.
  • the dissolution curve method was used to detect the TM value of 7 groups of extended deformation + core short-sequence DNA nanoparticles. The samples were consistent with the potential samples.
  • the reagents and instruments are as follows:
  • the program is set to start at 20 ° C, and the temperature is raised from 0.1 ° C to 95 ° C per second, and the reading is taken every 5s.
  • the TM values of the 7 groups of extended deformation + core short-sequence DNA nanoparticles are shown below.
  • the dissolution profile of D-8 is shown in Figure 25
  • the dissolution profile of D-9 is shown in Figure 26
  • the dissolution profile of D-10 is shown in Figure 27.
  • the dissolution profile of D-11 is shown in Figure 28
  • the dissolution profile of D-12 is shown in Figure 29
  • the dissolution profile of D-13 is shown in Figure 30, and the dissolution profile of D-14 is shown in Figure 31.
  • the non-denaturing PAGE method was used to characterize the stability of the seven groups of extended deformation + core short-sequence RNA nanoparticles in serum.
  • the main reagents and instruments are as follows:
  • RNA nanoparticles Formulate the RNA nanoparticles to the following concentrations, and then dilute the prepared sample according to the method in the table, dilute 5 tubes, and dilute the sample at 37 ° C in a water bath for different times (0, 10min, 1h, 12h, 36h);
  • the electrophoretic test results of R-15 are shown in Figure 32
  • the electrophoretic test results of R-16 are shown in Figure 33
  • the electrophoretic test results of R-17 are shown in Figure 34
  • the electrophoretic test results of R-18 are shown in Figure 35
  • the electrophoretic tests of R-19 are shown
  • the results are shown in Figure 36
  • the electrophoretic results of R-20 are shown in Figure 37
  • the electrophoretic results of R-21 are shown in Figure 38. From Figure 32 to Figure 38, the lanes from left to right are M: marker; 1: 36h; 2: 12h; 3: 1h; 4: 10min; 5: 0min.
  • the non-denaturing PAGE method was used to characterize the stability of 7 groups of extended deformation + core short sequence DNA nanoparticles in serum.
  • the main reagents and instruments are as follows:
  • the electrophoretic test results of D-8 are shown in Figure 39
  • the electrophoretic test results of D-9 are shown in Figure 40
  • the electrophoretic test results of D-10 are shown in Figure 41
  • the electrophoretic test results of D-11 are shown in Figure 42
  • the electrophoretic tests of D-12 are shown
  • the results are shown in Figure 43
  • the results of D-13 are shown in Figure 44
  • the results of D-14 are shown in Figure 45.
  • the lanes from left to right are M: marker; 1: 36h; 2: 12h; 3: 1h; 4: 10min; 5: 0min.
  • Example 5 According to the chemical method mounting method of Example 5 (except for the special limitation, the method is the same as that of Example 5), and the R-15, R-16, R-17, R-18, and R-19 in the foregoing Example 9 are used respectively.
  • R-20 and R-21 self-assembled RNA nanoparticles, D-8, D-9, D-10, D-11, D-12, D-13 and D-14 self-assembled in Example 10
  • the DNA nanoparticles were used as a carrier for adriamycin, and the rates of adriamycin were determined as follows:
  • Doxorubicin mount rate of RNA nanoparticles R-15 is 20.5;
  • Doxorubicin mount rate of RNA nanoparticles R-16 is 29.4;
  • Doxorubicin mount rate of RNA nanoparticles R-17 is 30.9;
  • Doxorubicin mount rate of RNA nanoparticles R-18 is 34.1;
  • Doxorubicin mount rate of RNA nanoparticles R-19 is 27.1;
  • Doxorubicin mount rate of RNA nanoparticle R-20 is 30.2;
  • Doxorubicin mount rate of RNA nanoparticles R-21 is 20.1;
  • Doxorubicin mount rate of DNA nanoparticle D-8 is 28.0;
  • Doxorubicin mount rate of DNA nanoparticle D-9 is 27.9;
  • Doxorubicin mount rate of DNA nanoparticle D-10 is 18.9;
  • Doxorubicin mount rate of DNA Nanoparticle D-11 is 26.8;
  • Doxorubicin mount rate of DNA nanoparticle D-12 is 27.6;
  • DNA nanoparticle D-13 has a doxorubicin mount rate of 31.8;
  • DNA nanoparticle D-14 has a doxorubicin mount factor of 32.
  • HepG2 (derived from Concord Cell Bank), the medium was DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), and the culture conditions were 37 ° C, 5% CO 2 , and saturated humidity.
  • Blank carrier DNA nanoparticle carrier formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13, and D-14 in the aforementioned Example 12.
  • Carrier drug According to the chemical method mounting method of Example 5 (except for the special limitation, the method is the same as that of Example 5), using D-8, D-9, D-10, D-11, D in Example 10 Doxorubicin is mounted on DNA nanoparticles formed by self-assembly of -12, D-13, and D-14, and are recorded as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-Doxorubicin, D-12-Doxorubicin, D-13-Doxorubicin, and D-14-Doxorubicin.
  • CCK8 method was used to detect the toxicity of DNA nanoparticles and carrier drugs to HepG2.
  • HepG2 (derived from Concord Cell Bank), the medium was DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), and the culture conditions were 37 ° C, 5% CO 2 , and saturated humidity.
  • Blank carrier DNA nanoparticle carriers formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13, and D-14 in the foregoing Example 10, respectively, denoted as: D- 8. D-9, D-10, D-11, D-12, D-13 and D-14.
  • Carrier drug According to the chemical method mounting method of Example 5 (except for the special limitation, the method is the same as that of Example 5), using D-8, D-9, D-10, D-11, D in Example 10 Doxorubicin is mounted on DNA nanoparticles formed by self-assembly of -12, D-13, and D-14, and are recorded as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-Doxorubicin, D-12-Doxorubicin, D-13-Doxorubicin, and D-14-Doxorubicin.
  • test sample Dilute the test sample and add it according to the following table: remove the original medium, add 100 ⁇ L of medium with different concentrations of the test sample, 3 replicates per group.
  • the mounted drug and the blank carrier are respectively prepared into a 100 ⁇ M stock solution with PBS, and then diluted with a complete medium (biotin-free DMEM).
  • biotin-free DMEM a complete medium
  • the original drug doxorubicin was first made into a 100 ⁇ M stock solution with DMSO, and then diluted with complete medium (biotin-free DMEM).
  • DMSO was directly diluted in complete medium (biotin-free DMEM).
  • the small molecule drug doxorubicin And mounted drugs D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin And D-14-doxorubicin are toxic to HepG2 cells, and the drugs D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin Compared with the original drug doxorubicin, D-12, doxorubicin, D-13-doxorubicin and D-14-doxorubicin have significant synergistic effects.
  • Example 5 According to the chemical mounting method of Example 5 (except for the special limitation, the method is the same as that of Example 5), the DNA nanoparticles formed by self-assembly of D-10 and D-14 in Example 10 were used as daunorubicin ⁇ carrier. Measure the absorbance of daunorubicin at 492nm with a microplate reader and draw a standard curve (as shown in Figure 47).
  • the mount rate of daunorubicin of DNA nanoparticle D-10 is 24.0;
  • the mount rate of daunorubicin of DNA nanoparticle D-14 was 25.1.
  • RNA nanoparticles in 1 are similar, except that the fluorescent label on the c chain is Cy5) as a carrier, and they are mounted separately: epirubicin, methotrexate, pirarubicin, daunorubicin, and pentafluorouracil. , 10-hydroxycamptothecin, aspirin and gemcitabine.
  • the measured mounting rates are as follows:
  • the mounting rate of epirubicin-RNAh nucleic acid nanoparticles is about 9.3, which means that about 9.3 epirubicin molecules can be mounted on each nucleic acid nanoparticle carrier.
  • each nucleic acid nanoparticle carrier can mount about 11.5 pirarubicin molecules.
  • RNAh-pentafluorouracil The calculated loading rate of RNAh-pentafluorouracil is about 0.31, and it indicates that each nucleic acid nanoparticle carrier can mount about 0.31 pentafluorouracil molecules.
  • RNAh-pentafluorouracil particles with mounting rates of 10, 20, 28, 50, etc. can also be obtained, which will not be repeated here.
  • RNAh-10-hydroxycamptothecin particles with mounting rates of 10, 20, 28, 50, 70, 80, 100, 200, etc. can also be obtained. I will not repeat them here.
  • RNAh -2 26.8 ⁇ g / ml, M RNAh ⁇ 30,000, 100 ⁇ l;
  • C Aspirin -2 18.4 ⁇ M, 100 ⁇ l;
  • the mounting method and the calculation method of the mounting rate are the same as the above RNA nucleic acid nanoparticles.
  • the specific nucleic acid nanoparticles used are: DNAh-Bio-EFGRapt-Cy5, where the three strands of DNAh are:
  • c chain (SEQ ID NO: 174 :): 5'-GCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCG-3 '; the first three bases at the 5' end and the last three bases at the 3 'end are thio-modified, respectively, and a Cy5 fluorescent tag is attached to the 3' end.
  • the mounting rate of epirubicin-DNAh nanoparticles is about 27.6, which means that about 27.6 epirubicin can be mounted on each DNA rice particle carrier.
  • RNA nanoparticles and DNA nanoparticles mounting epirubicin and other drugs mentioned above it is possible to further mount other small-molecule drugs in the same way as mounting epirubicin, such as In this application, folic acid is further mounted to obtain RNA nanoparticles and DNA nanoparticles that jointly mount two small molecule drugs, epirubicin and folic acid.
  • the mounting rate of the two drugs can be detected by referring to the above method ( Value not shown).
  • RNA nanoparticles in Example 1 and DNA nanoparticles with extensions, targets and fluorescein have the function of loading drugs, and the small molecule drug epirubicin can be covalently linked (Paraformaldehyde-solvent covalent) to achieve mounting, and can also be mounted with other small molecule drugs.
  • RNAh-Bio-670 in the table is used as a control, which refers to the biotin modification on the 5 'end of the a and b chains prepared according to the self-assembly method in Example 1, and quasar670 on the 3' end of the c chain.
  • Nanoparticles formed by fluorescein modification, while RNAh-Bio-670-EPB and the like refer to nanoparticles formed by further mounting drugs such as epirubicin (mounted in accordance with the chemical method in Example 5).
  • RPMI-1640 Medium Gibco, C11875500BT-500mL
  • Fetal Bovine Serum Fetal Bovine Serum
  • PS Penicillin / Streptomycin
  • PBS buffer Gibco, C20012500BT-500mL
  • Trypsin-EDTA Stemcell, 07901-500mL
  • DMSO Sigma, D5879-1L
  • Prolong Gold Antifade Mountant Thermo, P36941-2mL
  • DAPI Yaman, 36308ES11-4 mL.
  • Inverted Microscope (Olympus BX53, U-RFL-T); BD Falcon (Corning, 354118); Cytospin (TXD3).
  • the cells used for each drug are respectively placed in RPMI1640 + 10% FBS + 1% PS medium, and cultured at 37 ° C and 5% CO 2 ;
  • Epirubicin HL60 cells, acute leukemia cell line
  • MOLT4 cells human acute lymphoblastic leukemia line
  • Pirarubicin MCF-7 cells (human breast cancer cell line)
  • Pentafluorouracil HepG2 cells
  • RNAh-Bio-670 and RNAh-Bio-670-EPB nanoparticles were incubated with 200nM and 400nM RNAh-Bio-670 and RNAh-Bio-670-EPB nanoparticles at 37 ° C and 5% CO 2 for 1 to 2 hours and 4 hours.
  • RNAh-Bio-670 and RNAh-Bio-670-EPB nanoparticles both carry a target, Biotin, and are therefore capable of binding to and internalizing cells. It can be seen that the drug RNAh-Bio-670-EPB nanoparticles containing epirubicin have strong ability to bind and internalize HL60 cells.
  • RNAh-Bio-670 and RNAh-Bio-670-MTX nanoparticles can bind to and internalize cells because they carry a target, Biotin. This result indicates that the methotrexate-containing drug RNAh-Bio-670-MTX nanoparticles have strong ability to bind and internalize MOLT4 cells.
  • RNAh-Biotin-quasar670 and RNAh-Biotin-quasar670-flu nanoparticles are both able to interact with Biotin because they both carry the target Cell binding and internalization.
  • MCF-7 source ATCC, article number HTB-22
  • NCI-H1975 source ATCC, article number CRL-5908
  • medium was MEM + 10% FBS
  • culture conditions were 37 ° C, 5% CO 2 , and saturated humidity.
  • Targeted drug DNAh-Bio-EGFRapt-Cy5-EPB (mounted in accordance with the mounting method of DNA nanoparticles in Example 5).
  • Targeted fluorescent vector DNAh-Bio-EGFRapt-Cy5.
  • test substance to the corresponding cell wells, the final concentration is 2 ⁇ M, and shake and mix;
  • Fluorescence carrier or epirubicin detection channel excitation light wavelength: 488nm, emission light channel: 560nm;
  • the epirubicin-targeting drug DNAh-Bio-EFGRapt-Cy5-EPB can bind to MCF-7 cells and NCI-H1975 cells with a binding rate of nearly 100%; and the fluorescent vector DNAh- Bio-Cy5 can also bind to MCF-7 cells and NCI-H1975 cells with a binding rate of 100%.
  • RNAh-Biotin-Cy5-DNR The preparation method of RNAh-Biotin-Cy5 is the same as that of RNAh-Biotin-quasar670, except that the fluorescent substance is replaced by quasar670 to Cy5.
  • RNAh-Biotin-Cy5-DNR is a nanoparticle formed after RNAh-Biotin-Cy5 is further mounted with DNR (mounted in accordance with the method in Example 5).
  • the conditions for fluorescence detection are as follows:
  • Excitation light is 640 nm
  • emission light is 675 nm
  • detection height is 7 mm
  • measurement value / data point 10
  • detection speed: normal extension: 100 ms.
  • the binding rate of RNAh-Biotin-Cy5-DNR nanoparticles and MCF-7 cells can be as high as 84% or more when the treatment time and concentration are appropriate.
  • the RNA Drug-loaded particles have strong ability to bind and internalize MCF-7 cells.
  • Pirrubicin-targeted drug DNAh-Biotin-EGFRapt-Cy5-THP; (mounted in accordance with the mounting method of DNA nanoparticles in Example 5)
  • Targeted fluorescent vector DNAh-Bio-EGFRapt-Cy5.
  • the detection channel of the fluorescent carrier and pirarubicin targeting drug excitation light wavelength: 488 nm, emission light channel: 560 nm;
  • DNA nucleic acid nanoparticles carrying the target and the small molecule drug pirarubicin have a high binding rate to cells, and it can be clearly seen that they can be internalized by binding to the corresponding tumor cell line cells.
  • DNAh-Bio-EFGRapt-Cy5-THP can not only efficiently bind and internalize with the human gastric cancer cell line SGC-7901 cells, but also with the human ovarian cancer cell line SKOV3 cells. It can be seen that the DNAh-Bio-EFGRapt-Cy5-THP targeted drug, pirarubicin, has both application prospects for treating gastric cancer and ovarian cancer.
  • RNAh-Biotin-Cy5-THP The preparation method of RNAh-Biotin-Cy5 is the same as that of RNAh-Biotin-quasar670, except that the fluorescent substance is replaced by quasar670 to Cy5.
  • RNAh-Biotin-Cy5-THP is a nanoparticle formed after RNAh-Biotin-Cy5 is further mounted with THP (mounted according to the method in Example 5).
  • the conditions for fluorescence detection are as follows:
  • Excitation light is 640 nm
  • emission light is 675 nm
  • detection height is 7 mm
  • measurement value / data point 10
  • detection speed: normal extension: 100 ms.
  • the binding rate of RNAh-Biotin-Cy5-THP nanoparticles to MCF-7 cells is as high as 96% or more. Compared to a blank control containing only culture medium, the binding of RNA-loaded particles to MCF-7 cells And strong internalization.
  • RNAh-Biotin-Cy5-Gemcitabine The preparation method of RNAh-Biotin-Cy5 is the same as that of RNAh-Biotin-quasar670, except that the fluorescent substance is replaced by quasar670 to Cy5.
  • RNAh-Biotin-Cy5- gemcitabine is a nanoparticle formed after RNAh-Biotin-Cy5 is further mounted with gemcitabine (mounted in accordance with the method in Example 5).
  • the conditions for fluorescence detection are as follows:
  • Excitation light is 640 nm
  • emission light is 675 nm
  • detection height is 7 mm
  • measurement value / data point 10
  • detection speed: normal extension: 100 ms.
  • the binding rate of RNAh-Biotin-Cy5-Gemcistat nanoparticles and BxPC-3 cells can be as high as 98% or more when the treatment time and concentration are appropriate, compared to a blank control containing only media.
  • the ability of RNA-loaded particles to bind and internalize BxPC-3 cells is strong.
  • Test sample RNAh-Bio-670-EPB nanoparticles dissolved in PBS solution;
  • RPMI-1640 medium Gibco, C11875500BT-500mL
  • Fetal bovine serum FBS
  • Penicillin / Streptomycin PS
  • PBS buffer Gibco, C20012500BT-500mL
  • Novex TM Tris-Glycine Native Sample Buffer (2X) Invitrogen, LC2673-20mL
  • Novex TM 8% Tris-Glycine Mini Gels Invitrogen, XP00080BOX-1.0mm
  • Tris-Glycine Native Running buffer (10x) Life science, LC2672-500mL
  • G250 staining solution Beyotime, P0017-250mL
  • Spectrophotometer Thermo, ND2000C
  • Mini Gel Tank Invitrogen, PS0301
  • Imaging System Bio-Rad, ChemiDoc MP
  • FIG. 64 shows the electrophoresis results of the 8% non-denaturing gel (Coomassie Blue program)
  • FIG. 65 shows the electrophoresis results of the 8% non-denaturing gel (Stain Free Gel program).
  • the results of the serum stability test showed that at 0min, 10min, 1h, 12h, and 36h, the RNAh-Bio-670-EPB nanoparticle sample bands had no significant difference under different time lengths, indicating that they were compared in 10% FBS 1640 medium. Stable without significant degradation.

Abstract

提供了一种核酸纳米载体药物、其制备方法、药物组合物及应用。该药物包括核酸纳米颗粒和药物，药物挂载在核酸纳米颗粒上，药物包括他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨中的一种或多种；核酸纳米颗粒包括核酸结构域，核酸结构域包含a序列、b序列和c序列，a序列包含a1序列或a1序列发生至少一个碱基插入、缺失或替换的序列，b序列包含b1序列或b1序列发生至少一个碱基插入、缺失或替换的序列，c序列包含c1序列或c1序列发生至少一个碱基插入、缺失或替换的序列。该核酸纳米载体药物因核酸结构域经过靶头修饰后具有较好的靶向性而形成稳定递送的靶向药。

Description

核酸纳米载体药物、其制备方法、药物组合物及其应用 技术领域
本申请涉及医药领域，具体而言，涉及一种核酸纳米载体药物、其制备方法、药物组合物及应用。
背景技术
为减轻药物活性成分靶向性差所产生的副作用，药物递送载体应运而生，其作用主要是承载药物活性成分，将活性成分输送至血液或组织细胞内以治疗疾病。目前已经有多种多样的方法来实现不同药物的靶向运输。有用仪器或器械实现的，比如基因枪、电穿孔仪等。这些方法无需使用基因载体，但是转染效率普遍很低、操作复杂，对组织的损伤也比较大。也有用病毒载体介导的，如腺病毒、慢病毒等，病毒载体虽然有较高的体外转染活性，然而，其免疫原性与易导致突变的缺点为体内输送带来了巨大的安全隐患。还有非病毒载体，尤其是生物可降解的高分子材料来实现药物的靶向运输。非病毒载体的优势主要在于，在保证预期的转染活性的条件下，可以大大降低病毒载体所带来的免疫原性与诸多炎症反应。
上述多种靶向运输方式中，目前更多的研究集中在非病毒载体领域，且一般为以下几种载体设计：(a)阳离子脂质体；(b)聚阳离子基因载体。而目前研究更多的主要集中于聚阳离子基因载体与阳离子脂质体的修饰，使之适用于基因物质的靶向输送。阳离子脂质体具有较高的体内外转染活性，然而，由于表面的正电荷影响其体内的正常分布，同时，阳离子脂质会在动物试验中引起免疫原性与炎症反应。聚阳离子基因载体目前发展已经较为成熟，然而在结构设计中难以保证靶向基团在结构的表面，而且存在一个毒性与转染活性的自身设计矛盾，同时,其连接难以在体内实现无毒化降解。
因此，如何改进现有小分子药物的递送可靠性是解决目前的药物在临床应用受限的难点之一。
发明内容
本申请的主要目的在于提供一种核酸纳米载体核酸纳米载体药物、其制备方法、药物组合物及应用，以提高药物的递送可靠性。
为了实现上述目的，根据本申请的一个方面，提供了一种核酸纳米载体药物，其包括核酸纳米颗粒和挂载在核酸纳米颗粒上的药物，药物包括他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨中的任意一种或多种；核酸纳米颗粒包括核酸结构域，核酸结构域包含a序列、b序列和c序列，a序列包含a1序列或者a1序列发生至少一个碱基插入、缺失或替换的序列，b序列包含b1序列或者b1序列发生至少一个碱基插入、缺失或替换的序列，c序列包含c1序列或者c1序列发生至少一个碱 基插入、缺失或替换的序列；其中，a1序列为SEQ ID NO:1：5’-CCAGCGUUCC-3’或者SEQ ID NO:2：5’-CCAGCGTTCC-3’；b1序列为SEQ ID NO:3：5’-GGUUCGCCG-3’或者SEQ ID NO:4：5’-GGTTCGCCG-3’；c1序列为SEQ ID NO:5：5’-CGGCCAUAGCGG-3’或者SEQ ID NO:6：5’-CGGCCATAGCGG-3’。
进一步地，a1序列为SEQ ID NO:1，b1序列为SEQ ID NO:3，c1序列为SEQ ID NO:5时，a序列、b序列、c序列中的至少一个序列包含至少一个碱基插入、缺失或替换的序列。
进一步地，碱基插入、缺失或替换发生在：
(1)SEQ ID NO:1或SEQ ID NO:2所示的序列的5’端起始的第1、2、4或5位碱基上；和/或
(2)SEQ ID NO:1或SEQ ID NO:2所示的序列的5’端起始的第8～10位碱基之间；和/或
(3)SEQ ID NO:3或SEQ ID NO:4所示的序列的5’端起始的第1～3位碱基之间；和/或
(4)SEQ ID NO:3或SEQ ID NO:4所示的序列的5’端起始的第6～9位碱基之间；和/或
(5)SEQ ID NO:5或SEQ ID NO:6所示的序列的5’端起始的第1～4位碱基之间；和/或
(6)SEQ ID NO:5或SEQ ID NO:6所示的序列的5’端起始的第9～12位碱基之间。
进一步地，a序列、b序列和c序列自组装成式(1)所示结构：
Figure PCTCN2019109563-appb-000001
其中，W-C表示Watson-Crick配对，N和N’表示非Watson-Crick配对，任一位置的W-C各自独立地选自C-G或G-C；在a序列中，从5’端起的第一个N为A，第二个N为G，第三个N为U或T，第四个N为U、T、A、C或G中的任意一个；在b序列中，从5’端起的第一个N’为U、T、A、C或G中的任意一个；第二个N’为U或T，第三个N’为C；在c序列中，沿5’端至3’端方向上的NNNN序列为CAUA或CATA。
进一步地，a序列、b序列和c序列为如下任意一组：(1)a序列：5'-GGAGCGUUGG-3'，b序列：5'-CCUUCGCCG-3'，c序列：5'-CGGCCAUAGCCC-3'；(2)a序列：5'-GCAGCGUUCG-3'，b序列：5'-CGUUCGCCG-3'，c序列：5'-CGGCCAUAGCGC-3'；(3)a序列：5'-CGAGCGUUGC -3'，b序列：5'-GCUUCGCCG-3'，c序列：5'-CGGCCAUAGCCG-3'；(4)a序列：5'-GGAGCGUUGG-3'，b序列：5'-CCUUCGGGG-3'，c序列：5'-CCCCCAUAGCCC-3'；(5)a序列：5'-GCAGCGUUCG-3'，b序列：5'-CGUUCGGCG-3'，c序列：5'-CGCCCAUAGCGC-3'；(6)a序列：5'-GCAGCGUUCG-3'，b序列：5'-CGUUCGGCC-3'，c序列：5'-GGCCCAUAGCGC-3'；(7)a序列：5'-CGAGCGUUGC-3'，b序列：5'-GCUUCGGCG-3'，c序列：5'-CGCCCAUAGCCG-3'；(8)a序列：5'-GGAGCGTTGG-3'，b序列：5'-CCTTCGCCG-3'，c序列：5'-CGGCCATAGCCC-3'；(9)a序列：5'-GCAGCGTTCG-3'，b序列：5'-CGTTCGCCG-3'，c序列：5'-CGGCCATAGCGC-3'；(10)a序列：5'-CGAGCGTTGC-3'，b序列：5'-GCTTCGCCG-3'，c序列：5'-CGGCCATAGCCG-3'；(11)a序列：5'-GGAGCGTTGG-3'，b序列：5'-CCTTCGGGG-3'，c序列：5'-CCCCCATAGCCC-3'；(12)a序列：5'-GCAGCGTTCG-3'，b序列：5'-CGTTCGGCG-3'，c序列：5'-CGCCCATAGCGC-3'；(13)a序列：5'-GCAGCGTTCG-3'，b序列：5'-CGTTCGGCC-3'，c序列：5'-GGCCCATAGCGC-3'；(14)a序列：5'-CGAGCGTTGC-3'，b序列：5'-GCTTCGGCG-3'，c序列：5'-CGCCCATAGCCG-3'。
进一步地，核酸结构域还包括第一延长段，第一延长段为Watson-Crick配对的延长段，第一延长段位于a序列、b序列和c序列中任一序列的5'端和/或3'端；优选地，第一延长段选自如下任意一组：(1)：a链5'端：5'-CCCA-3'，c链3'端：5'-UGGG-3'；(2)：a链3'端：5'-GGG-3'，b链5'端：5'-CCC-3'；(3)：b链3'端：5'-CCA-3'，c链5'端：5'-UGG-3'；(4)：a链5'端：5'-CCCG-3'，c链3'端：5'-CGGG-3'；(5)：a链5'端：5'-CCCC-3'，c链3'端：5'-GGGG-3'；(6)：b链3'端：5'-CCC-3'，c链5'端：5'-GGG-3'；(7)：b链3'端：5'-CCG-3'，c链5'端：5'-CGG-3'；(8)：a链5'端：5'-CCCA-3'，c链3'端：5'-TGGG-3'；(9)：b链3'端：5'-CCA-3'，c链5'端：5'-TGG-3'。
进一步地，核酸结构域还包括第二延长段，第二延长段位于a序列、b序列和c序列中任一序列的5’端和/或3’端，第二延长段为Watson-Crick配对的延长段；优选地，第二延长段为CG碱基对的延长序列；更优选，第二延长段为1～10个CG碱基对的延长序列。
进一步地，核酸结构域还包括如下至少一组第二延长段：第一组：a链5’端：5’-CGCGCG-3’，c链3’端：5’-CGCGCG-3’；第二组：a链3’端：5’-CGCCGC-3’，b链5’端：5’-GCGGCG-3’；第三组：b链3’端：5’-GGCGGC-3’，c链5’端：5’-GCCGCC-3’。
进一步地，第二延长段为同时含有CG碱基对和AT/AU碱基对的延长序列，优选第二延长段为2～50个碱基对的延长序列。
进一步地，第二延长段为连续2～8个CG碱基对的序列与连续2～8个AT/AU碱基对序列交替设置的延长序列；或者，第二延长段为1个CG碱基对的序列与1个AT/AU碱基对序列交替设置的延长序列。
进一步地，a序列、b序列和c序列中碱基、核糖和磷酸酯具有至少一个可修饰位点，任一可修饰位点通过以下任意一种修饰接头进行修饰：-F、甲基、氨基、二硫化物、羰基、羧基、巯基及醛基；优选地，a序列、b序列和c序列中的C或U碱基上具有2’-F修饰。
进一步地，药物通过物理连接和/或共价连接的形式挂载在核酸纳米颗粒上，且他克林与核酸纳米颗粒之间的摩尔比为2～300:1，优选为10～50:1，更优选为15～25:1。
进一步地，核酸纳米颗粒还包括生物活性物质，生物活性物质与核酸结构域相连，生物活性物质为靶头、荧光素、干扰核酸siRNA、miRNA、核酶、核糖开关、适体、RNA抗体、蛋白、多肽、类黄酮、葡萄糖、天然水杨酸、单抗、维生素、酚类卵磷脂以及小分子药物中的一种或多种，小分子药物为除他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨之外的小分子药物。
进一步地，将核酸结构域的相对分子量记为N 1，将药物与生物活性物质的总相对分子量记为N 2，N 1/N 2≥1:1。
进一步地，生物活性物质为靶头、荧光素以及miRNA中的一种或多种，其中，靶头位于a、b、c序列中任一序列上，优选a、b、c任一序列的5’端或3’端，或嵌插于核酸结构域的GC键之间，miRNA为抗miRNA，荧光素修饰于抗miRNA的5’端或3’端，miRNA位于a序列的3’端、c序列的5’端和3’端中的任意一个或多个位置；优选地，靶头为叶酸或生物素，荧光素为FAM、CY5及CY3中的任意一种或多种，抗miRNA为抗miR-21。
进一步地，小分子药物为含有如下任意一种或多种基团的药物：氨基基团、羟基基团、羧基基团、巯基基团、苯环基团以及乙酰氨基基团。
进一步地，蛋白为SOD、生存素、hTERT及EGFR、PSMA中的一种或多种；维生素为左旋C和/或酯化C；酚类为茶多酚和/或葡萄多酚。
进一步地，核酸纳米颗粒的粒径为1～100nm，优选为5～50nm；更优选10～30nm；进一步优选10～15nm。
根据本申请的另一方面，还提供了一种核酸纳米载体药物的制备方法，其包括以下步骤：提供上述的核酸纳米颗粒；通过物理连接和/或共价连接的方式将药物挂载在核酸纳米颗粒上，得到核酸纳米载体药物。
进一步地，通过物理连接的方式挂载药物的步骤包括：将药物、核酸纳米颗粒及第一溶剂混合并搅拌，得到预混体系；对预混体系进行沉淀析出，得到核酸纳米载体药物；优选地，第一溶剂选自DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种；优选地，对预混体系进行沉淀析出，得到核酸纳米载体药物的步骤包括：对预混体系进行沉淀析出，得到析出物；对析出物进行洗涤去除杂质,得到核酸纳米载体药物；更优选地，将预混体系与无水乙醇混合后在低于10℃的温度条件下沉淀析出，得到析出物；核酸纳米载体药物；更优选在0～5℃温度条件下沉淀析出，得到析出物。更优选地，采用6～12倍体积的无水乙醇对析出物进行洗涤去除杂质，得到核酸纳米载体药物。
进一步地，通过共价连接的方式挂载药物的步骤包括：配置药物溶液；使药物溶液在甲醛的介导作用下与核酸纳米颗粒的G环外氨基进行反应，得到反应体系；提纯反应体系，得到核酸纳米载体药物；优选地，反应的步骤包括：将药物溶液与多聚甲醛溶液、核酸纳米颗 粒混合，在避光条件下进行反应，得到反应体系；其中优选多聚甲醛溶液的浓度优选为3.7～4wt％，优选多聚甲醛溶液为多聚甲醛和第二溶剂混合形成的溶液，且第二溶剂为DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种。
进一步地，制备方法还包括制备核酸纳米颗粒的步骤，其包括：通过将上述的核酸结构域对应的单链进行自组装，得到核酸结构域；优选地，在得到核酸结构域之后，制备方法还包括：将上述的生物活性物质通过物理连接和/或共价连接的方式挂载在核酸结构域上，进而得到核酸纳米颗粒。
进一步地，通过共价连接的方式挂载生物活性物质的过程中，通过溶剂共价连接、linker共价连接或点击链接进行挂载；优选地，溶剂共价连接中采用的第三溶剂作为连接介质，且第三溶剂选自多聚甲醛、DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种；优选地，linker选自二硫键、对苯叠氮基、溴丙炔或PEG；优选地，点击链接是在对生物活性物质前体和核酸结构域同时进行炔基或叠氮修饰，然后通过点击链接。
进一步地，生物活性物质与核酸结构域以点击链接的方式相连时，生物活性物质前体进行炔基或叠氮修饰的位点选自2’羟基、羧基或氨基，核酸结构域进行炔基或叠氮修饰的位点选自G环外氨基、2’-羟基、A氨基或2’-羟基。
根据本申请的第三个方面，还提供了一种药物组合物，该药物组合物包括上述任一种核酸纳米载体药物。
根据本申请的第四个方面，还提供了上述任一种核酸纳米载体药物在制备用于治疗老年痴呆症、肿瘤、自身免疫疾病或心脏病相关的药物中的应用。
进一步地，肿瘤为如下任意一种或多种：胰腺癌、卵巢癌、乳腺癌、膀胱癌、宫颈癌、肝癌、胆道癌、鼻咽癌、睾丸肿瘤、淋巴瘤、间皮瘤、头颈部癌、胃癌、白血病、结肠癌、直肠癌、绒毛膜上皮癌、恶性葡萄胎、皮肤癌、肺癌、输尿管癌、肾盂癌、绒毛膜上皮癌、骨肿瘤、白血病脑膜脊髓浸润、肾母细胞瘤、软组织肉瘤及甲状腺髓样癌；自身免疫疾病为顽固性牛皮藓、系统性红斑狼疮、强制性脊柱炎或皮肌炎。
进一步地，白血病为急性白血病，优选急性白血病为急性淋巴细胞白血病或粒细胞白血病。
进一步地，肺癌包括支气管肺癌或非小细胞肺癌。
进一步地，肝癌包括原发性肝细胞癌或转移性肝癌。
根据本申请的第五个方面，还提供了一种预防和/或治疗老年痴呆症、肿瘤、自身免疫疾病或心脏病的方法，该方法包括：提供上述任一种核酸纳米载体药物或药物组合物；给患者相应有效量的上述核酸纳米载体药物或药物组合物。
本申请提供的核酸纳米载体药物中包括核酸纳米颗粒和药物，且药物通过物理连接和/或共价连接的形式挂载在核酸纳米颗粒上。该核酸纳米颗粒中，通过包含本申请所提供的三条 序列或其变异序列，不仅能够自组装形成核酸结构域，而且可以作为载体在三条链的任意5'端和/或3'末端连接该药物，或者能够使该药物稳定地嵌插在核酸结构域的链间。本申请通过将药物挂载于核酸纳米颗粒上，利用核酸纳米颗粒的内部疏水性、外部的亲水性以及碱基的堆砌效应，对药物起到了“包被作用”，而且包被作用或共价连接使药物在一定的时间内不会被溶解，提高了递送的稳定性。此外，当核酸结构域经过靶头修饰后，可具有较好的靶向性，能够稳定地递送药物，可靠性很高；同时能减少药物与非目标细胞或组织的接触的机会，降低毒副作用。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1示出了本申请实施例1中自组装形成的RNA纳米颗粒的电泳检测结果；
图2示出了本申请实施例1中自组装形成的DNA纳米颗粒的电泳检测结果；
图3本申请实施例2中自组装形成的7组短序列RNA纳米颗粒的2％琼脂糖凝胶电泳检测结果；
图4示出了本申请实施例2中自组装形成的7组短序列RNA纳米颗粒的4％琼脂糖凝胶电泳检测结果；
图5示出了本申请实施例3中自组装形成的7组常规序列RNA纳米颗粒的2％琼脂糖凝胶电泳检测结果；
图6示出了本申请实施例3中自组装形成的7组常规序列RNA纳米颗粒的4％琼脂糖凝胶电泳检测结果；
图7示出了本申请实施例4中自组装形成的7组常规序列DNA纳米颗粒的2％琼脂糖凝胶电泳检测结果；
图8示出了本申请实施例4中自组装形成的7组常规序列DNA纳米颗粒的4％琼脂糖凝胶电泳检测结果；
图9示出了本申请实施例4中自组装形成的常规序列DNA纳米颗粒D-7的透射电镜照片；
图10示出了本申请实施例5中挂载率检测过程中他克林吸光度的标准曲线；
图11示出了本申请实施例6中RNAh-Biotin-quasar670纳米颗粒及RNAh-Biotin-quasar670-tacrine纳米颗粒与SH-SY5Y细胞的结合和内化的显微镜观察结果；
图12示出了本申请实施例7中RNAh-Biotin-quasar670-tacrine纳米颗粒在Coomassie Blue程序下，在血清中孵育不同时间后的电泳检测结果；
图13示出了本申请实施例7中RNAh-Biotin-quasar670-tacrine纳米颗粒在Stain Free Gel程序下，在血清中孵育不同时间后的电泳检测结果；
图14示出了本申请实施例8中小分子药物他克林及RNAh-Biotin-quasar670-tacrine纳米颗粒抑制SH-SY5Y细胞增殖情况检测结果；
图15示出了本申请实施例8中荧光靶向载体Bio-Cy5-RNAh抑制SH-SY5Y细胞增殖情况的检测结果；
图16示出了本发明实施例9中7组延长段变形+核心短序列RNA自组装产物的非变性PAGE胶电泳检测结果；
图17示出了本发明实施例9中RNA纳米颗粒R-15的溶解曲线；
图18示出了本发明实施例9中RNA纳米颗粒R-16的溶解曲线；
图19示出了本发明实施例9中RNA纳米颗粒R-17的溶解曲线；
图20示出了本发明实施例9中RNA纳米颗粒R-18的溶解曲线；
图21示出了本发明实施例9中RNA纳米颗粒R-19的溶解曲线；
图22示出了本发明实施例9中RNA纳米颗粒R-20的溶解曲线；
图23示出了本发明实施例9中RNA纳米颗粒R-21的溶解曲线；
图24示出了本发明实施例10中7组延长段变形+核心短序列DNA自组装产物的非变性PAGE胶电泳检测结果；
图25示出了本发明实施例10中DNA纳米颗粒D-8的溶解曲线；
图26示出了本发明实施例10中DNA纳米颗粒D-9的溶解曲线；
图27示出了本发明实施例10中DNA纳米颗粒D-10的溶解曲线；
图28示出了本发明实施例10中DNA纳米颗粒D-11的溶解曲线；
图29示出了本发明实施例10中DNA纳米颗粒D-12的溶解曲线；
图30示出了本发明实施例10中DNA纳米颗粒D-13的溶解曲线；
图31示出了本发明实施例10中DNA纳米颗粒D-14的溶解曲线；
图32示出了本发明实施例11中RNA纳米颗粒R-15在血清中孵育不同时间后的电泳检测结果；
图33示出了本发明实施例11中RNA纳米颗粒R-16在血清中孵育不同时间后的电泳检测结果；
图34示出了本发明实施例11中RNA纳米颗粒R-17在血清中孵育不同时间后的电泳检测结果；
图35示出了本发明实施例11中RNA纳米颗粒R-18在血清中孵育不同时间后的电泳检测结果；
图36示出了本发明实施例11中RNA纳米颗粒R-19在血清中孵育不同时间后的电泳检测结果；
图37示出了本发明实施例11中RNA纳米颗粒R-20在血清中孵育不同时间后的电泳检测结果；
图38示出了本发明实施例11中RNA纳米颗粒R-21在血清中孵育不同时间后的电泳检测结果；
图39示出了本发明实施例12中DNA纳米颗粒D-8在血清中孵育不同时间后的电泳检测结果；
图40示出了本发明实施例12中DNA纳米颗粒D-9在血清中孵育不同时间后的电泳检测结果；
图41示出了本发明实施例12中DNA纳米颗粒D-10在血清中孵育不同时间后的电泳检测结果；
图42示出了本发明实施例12中DNA纳米颗粒D-11在血清中孵育不同时间后的电泳检测结果；
图43示出了本发明实施例12中DNA纳米颗粒D-12在血清中孵育不同时间后的电泳检测结果；
图44示出了本发明实施例12中DNA纳米颗粒D-13在血清中孵育不同时间后的电泳检测结果；
图45示出了本发明实施例12中DNA纳米颗粒D-14在血清中孵育不同时间后的电泳检测结果；
图46a、图46b、图46c、图46d、图46e、图46f、图46g、图46h分别示出了本发明实施例15中DMSO和原药阿霉素、D-8和D-8-阿霉素、D-9和D-9-阿霉素、D-10和D-10-阿霉素、D-11和D-11-阿霉素、D-12和D-12-阿霉素、D-13和D-13-阿霉素、D-14和D-14-阿霉素所对应的细胞存活率曲线；
图47示出了实施例16的挂载率检测过程中采用的柔红霉素吸光度的标准曲线；
图48a和图48b、图49、图50a和图50b、图51、图52、图53、图54a和图54b、图55依次示出了本申请实施例17中挂载率检测过程中表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨吸光度的标准曲线；
图56至图63分别示出了挂载有表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨的核酸纳米颗粒与细胞的结合内化能力；
图64至图81分别示出了挂载有表柔比星(图64至图66)、甲氨蝶呤(图67和图68)、吡柔比星(图69、70和图71)、柔红霉素(图72和图73)、五氟尿嘧啶(图74和图75)、10-羟基喜树碱(图76和图77)、阿司匹林(图78和图79)及吉西他滨(图80和图81)的核酸纳米颗粒在血清中的稳定性；
图82至图101分别示出了挂载有表柔比星(图82、图83、图84a至84d及图85a至85d)、甲氨蝶呤(图86和87)、吡柔比星(图88、89、及91a至91d)、柔红霉素(图92和93)、五氟尿嘧啶(图94和95)、10-羟基喜树碱(图96和97)、阿司匹林(图98和99)及吉西他滨(图100和101)的核酸纳米颗粒对细胞的毒性。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本申请。
术语解释：
空白载体：指不含任何生物活性物质的空白核酸纳米颗粒载体，如RNAh或DNAh。
靶向载体：指含有靶头但不含有荧光物质的核酸纳米颗粒载体，如Biotin-RNAh或Biotin-DNAh。
荧光载体：指含有荧光物质但不含有靶头的核酸纳米颗粒载体，如Cy5-RNAh或Cy5-DNAh。
靶向荧光载体：指含有靶头和荧光物质的核酸纳米颗粒载体，如Biotin-Cy5-RNAh或Biotin-Cy5-DNAh。
靶向药：指含有靶头、荧光物质和化药的核酸纳米颗粒载体，如他克林-Biotin-Cy5-RNAh或他克林-Biotin-Cy5-DNAh。
需要说明的是，本申请中的各载体或生物活性物质的命名规则并无特殊格式，其在表述中的前后位置并不代表其在RNAh或DNAh的5’端或3’端，仅表示含有该生物活性物质。
如背景技术所提到的，现有技术中尽管已有多种提高药物递送效率的药物载体，但仍难以解决药物在临床上应用受限的问题。为了改善这一状况，本申请的发明人对现有所有可用作药物载体的材料进行了研究，并从载体的细胞/组织靶向性、运输过程中的稳定性、进入靶细胞的活性和效率、到达靶细胞后的药物释放能力以及对细胞的毒性等方面对各种载体进行了深入考察和分析，发现采用新兴的DNA和/或RNA分子自组装形成的纳米结构，比如，DNA树枝状大分子的自组装体系中DNA对核酸酶的降解有显著的阻碍作用，在基因治疗和生物医学领域有非常重要的应用价值。
通过对现有报道的DNA和RNA自组装形成的纳米颗粒进行分析发现，相对于比较刚性的DNA纳米颗粒而言，RNA纳米颗粒由于分子内或分子间存在大量的茎-环结构，其具有更大柔性和更强的张力，因而在作为候选药物载体方面更具优势。然而，自然状态的RNA纳米颗粒稳定性相对较差，而目前基于RNA纳米载体应用方面的改进，大多都是围绕提高其稳定性和可靠性而展开的。目前的研究结果尽管在一定程度上提供了挂载药物的可能性，但更侧重于对核酸药物，尤其是siRNA药物或miRNA药物等挂载的可能性和有效性进行研究。而对于非核酸类的药物是否同样有效，目前报道很少。此外，现有的自组装纳米颗粒，尤其是作为载体应用的自组装纳米颗粒，目前都是采用RNA链进行自组装成的，极少数采用了RNA链和DNA链组合的形式进行自组装的，但却并没有采用纯粹的DNA链来实现自组装的。
为了提供一种新的可靠性好且能够自组装的RNA纳米颗粒载体，申请人对现有的RNA纳米颗粒进行了比较和改进，开发出了一系列新的RNA纳米颗粒，而且，从提高适用性及降低成本角度考虑，进一步尝试了采用纯粹的DNA链来进行自组装，意外发现改为这些DNA单链不仅能够实现自组装成DNA纳米颗粒，而且性能与RNA纳米颗粒同样优异。且，DNA纳米颗粒的自组装还具有价格廉价和易操作的优势。并经过实验验证，发明人所改进的RNA纳米颗粒和DNA纳米颗粒均能够挂载药物，并能在血清中稳定存在；进一步的实验验证，其能携带这些药物进入细胞，且单独的核酸纳米颗粒载体对细胞无毒性。而携带药物后的载体能够对相应疾病起到缓解和治疗作用。
在上述研究结果的基础上，申请人提出了本申请的技术方案。本申请提供了一种核酸纳米载体药物，该药物包括核酸纳米颗粒和药物，药物挂载在核酸纳米颗粒上，且药物包括他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨中的一种或多种；该核酸纳米颗粒包括核酸结构域，核酸结构域包含a序列、b序列和c序列，a序列包含a1序列或者a1序列发生至少一个碱基插入、缺失或替换的序列，b序列包含b1序列或者b1序列发生至少一个碱基插入、缺失或替换的序列，c序列包含c1序列或者c1序列发生至少一个碱基插入、缺失或替换的序列；其中，a1序列为SEQ ID NO:1：5’-CCAGCGUUCC-3’或者SEQ ID NO:2：5’-CCAGCGTTCC-3’；b1序列为SEQ ID NO:3：5’-GGUUCGCCG-3’或者SEQ ID NO:4：5’-GGTTCGCCG-3’；c1序列为SEQ ID NO:5：5’-CGGCCAUAGCGG-3’或者SEQ ID NO:6：5’-CGGCCATAGCGG-3’。
本申请提供的核酸纳米载体药物中包括核酸纳米颗粒和药物，且上述任一种或多种药物挂载在该核酸纳米颗粒上。该核酸纳米颗粒中，通过包含上述三条序列或其变异序列，不仅能够自组装形成核酸结构域，而且可以作为载体，在三条链的任意5'端和/或3'末端连接药物，或者能够使药物稳定地嵌插在核酸结构域的链间。本申请提供的核酸纳米载体药物，通过将上述药物挂载于核酸纳米颗粒上，因核酸纳米颗粒的内部具有疏水性、外部具有亲水性以及碱基具有堆砌效应，相当于对药物起到了“包被作用”，而包被或共价连接使药物在一定的时间内不会被溶解，提高了递送的稳定性。此外，当核酸结构域经过靶头修饰后，可具有较好的靶向性，能够稳定地递送药物，可靠性很高；同时能减少相应药物与非目标细胞或组织的接触的机会，降低毒副作用。
上述自组装是指基本结构单元自发形成有序结构的一种技术。在自组装的过程中，基本结构单元在基于非共价键的相互作用下自发地组织或聚集为一个稳定、具有一定规则几何外观的结构。自组装过程并不是大量原子、离子或分子之间弱相互作用力(其中“弱相互作用力”指氢键、范德华力、静电力、疏水作用力等)的简单叠加，而是若干个体之间同时自发的发生并联并集合在一起形成一个紧密而又有序的整体，是一种整体的复杂的协同作用。
自组装的产生需要两方面的条件：自主装的动力和导向作用。自组装的动力指分子间的弱相互作用力的协同作用，它为分子自组装提供能量。自组装的导向作用指的是分子在空间的互补性，也就是说自组装发生需要在空间的尺寸和方向上满足分子重排的要求。
DNA纳米技术是一种自下而上的分子自组装模式，由分子构造为起点基于核酸分子的物理和化学性质自发地形成稳定结构，遵循严格的核酸碱基配对原则。多个DNA片段在体外以正确顺序连接在一起，通过碱基互补配对原则，建立亚组装结构，最终形成复杂的多级结构。与DNA不同，RNA的结构可以超出双螺旋的限制。RNA可以形成一系列不同的碱基对，碱基对之间至少形成两个氢键。不同的碱基可以分为两种个类型，包括标准的Waston-Crick碱基对型和非Waston-Crick碱基对型，可以使得RNA形成大量和多种类型的循环结构模块，这些模块就是构成折叠RNA三级结构的基本单元。RNA纳米技术可以利用这些天然存在的3D模块及其可以预知的相互作用，其中，很多具有生物学活性的RNA结构都可以具有原子级别的分辨率，比如核糖体、各类核酶以及存在于核糖开关内的天然RNA适配体。RNA纳米技术的一个优越特性在于，可以设计出在大小和复杂性上都能够与天然RNA物质相媲美的结构。还可以对天然RNA复合体内的RNA的独特组装性质加以利用。
本申请的上述核酸纳米颗粒中，包含序列SEQ ID NO:1、SEQ ID NO:3和SEQ ID NO:5所示的三条序列或其变异后的序列，或者包含序列SEQ ID NO:2、SEQ ID NO:4和SEQ ID NO:6所示的三条序列或其变异后的序列，均以能够通过自组装形成核酸纳米颗粒为准，具体变异后的序列可以在SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6序列基础上合理选择变异位点及其变异类型得到，或者通过延长合适片段得到。
SEQ ID NO:1、SEQ ID NO:3和SEQ ID NO:5自组装形成的纳米颗粒为RNA纳米颗粒，SEQ ID NO:2、SEQ ID NO:4和SEQ ID NO:6自组装形成的纳米颗粒为DNA纳米颗粒。在一种优选的实施例中，上述核酸纳米颗粒为RNA纳米颗粒时，且a序列、b序列、c序列中的至少一个序列包含至少一个碱基插入、缺失或替换的序列。该RNA纳米颗粒中变异序列的具体位置和碱基类型可以在能够实现自组装的前提下，根据需要改进为提高药物挂载量或提高稳定性的纳米颗粒。
为了使核酸纳米颗粒具有相对更高的稳定性，进而使经过药物挂载得到的药物更稳定，在对上述SEQ ID NO:1/2、SEQ ID NO:3/4和/或SEQ ID NO:5/6所示的序列进行碱基插入、缺失或替换时，可以在上述序列的某些特定位置的碱基上进行，一方面使得变异后的序列与原序列一样，能够自组装成纳米颗粒，另一方面变异保留与原序列至少50％、55％、60％、65％、 70％、75％、80％、85％、90％或95％的同源性，使得其与上述序列自组装形成的纳米颗粒具有同样的载药特性和类似的稳定性，能够很好地挂载和递送药物。
在一种优选的实施例中，上述碱基插入、缺失或替换发生在：(1)SEQ ID NO:1或2所示的a序列的5’端起始的第1、2、4和5位碱基之间；和/或(2)SEQ ID NO:1或2所示的a序列的5’端起始的第8～10位碱基之间；和/或(3)SEQ ID NO:3或4所示的b序列的5’端起始的第1～3位碱基之间；和/或(4)SEQ ID NO:3或4所示的b序列的5’端起始的第6～9位碱基之间；和/或(5)SEQ ID NO:5或6所示的c序列的5’端起始的第1～4位碱基之间；和/或(6)SEQ ID NO:5或6所示的c序列的5’端起始的第9～12位碱基之间。
上述优选的实施例中，所限定的发生变异的碱基位置，是在SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的序列形成的纳米结构中的非经典Watson-Crick配对碱基位置或凸出的未配对碱基位置，因而不影响这些凸出或loop结构的形成，从而保持了上述序列形成的纳米结构的柔性和张力，有助于维持其作为载体的稳定性。
为了进一步提高上述核酸纳米颗粒的稳定性，进而提高药物挂载后形成的药物的稳定性，在一种优选的实施例中，a序列、b序列和c序列自组装成式(1)所示结构：
Figure PCTCN2019109563-appb-000002
其中，W-C表示Watson-Crick配对，N和N’表示非Watson-Crick配对，任一位置的W-C各自独立地选自C-G或G-C，且a序列、b序列和c序列中至少两条序列各自的5’端和3’端的两个碱基不互补；在a序列中，从5’端起的第一个N为A，第二个N为G，第三个N为U或T，第四个N为U、T、A、C或G中的任意一个；在b序列中，从5’端起的第一个N’为U、T、A、C或G中的任意一个；第二个N’为U或T，第三个N’为C；在c序列中，沿5’端至3’端方向上的NNNN序列为CAUA或CATA。
上述优选的实施例中，a、b、c序列通过自组装形成具有式(1)所示的核酸结构域，其中，除了N和N’限定的非Watson-Crick配对碱基外，其余位置的碱基均形成的经典的Watson-Crick配对，并且上述Watson-Crick配对的碱基均选择G-C或C-G碱基对。由于G-C或C-G碱基对间的氢键的作用力大于A-U/T或U/T-A碱基对间的氢键的作用力，因而使得该 核酸纳米结构更稳定。而非Watson-Crick配对碱基所形成的凸起或loop结构，为核酸纳米载体带来更大的张力，使得其对微环境变化的适应性更强，因而该核酸纳米颗粒的稳定性更高。
上述式(1)结构的纳米颗粒中，a序列、b序列和c序列的具体序列组成并无特殊限定，只要能够形成上述结构即可。从核酸序列自组装的角度考虑，为了进一步提高上述三条序列自组装成上述式(1)结构的纳米颗粒的效率，在选择Watson-Crick配对的碱基时，不同位置的碱基选择最好遵循如下原则：(1)a序列、b序列和c序列，单独一条序列时并不自我互补配对形成二级结构；(2)a序列、b序列和c序列，任意两条序列之间一端互补配对形成双链，另一端不互补配对，形成Y型或T型结构。上述碱基选择的原则是最大效率地使任意一条链的两端分别与其他两条链的两端分别互补配对，从而提高自组装效率。当然，除了Y型或T型结构，也可以是三叉以外的四边形等替他变形形式，只要满足任意两条序列之间一端互补配对形成双链，另一端不互补配对的原则即可。
上述式(1)结构的纳米颗粒中，非Watson-Crick配对碱基中，a序列中从5’端起的第四个N及b序列中与其配对的从5’端起的第一个N’，可以是非Watson-Crick配对的U-U，也可以是改进后的遵循Watson-Crick配对原则的T、A、C或G。Watson-Crick配对相对提高链间的结合力，提高稳定性，而非Watson-Crick配对赋予了纳米颗粒更大的柔性和灵活性，在面对微环境变化的时候，同样有助于提高纳米颗粒的稳定性。
在一种优选的实施例中，a序列、b序列和c序列为如下任意一组：(1)a序列(SEQ ID NO：7)：5'-GGAGCGUUGG-3'，b序列(SEQ ID NO：8)：5'-CCUUCGCCG-3'，c序列(SEQ ID NO：9)：5'-CGGCCAUAGCCC-3'；(2)a序列(SEQ ID NO：10)：5'-GCAGCGUUCG-3'，b序列(SEQ ID NO：11)：5'-CGUUCGCCG-3'，c序列(SEQ ID NO：12)：5'-CGGCCAUAGCGC-3'；(3)a序列(SEQ ID NO：13)：5'-CGAGCGUUGC-3'，b序列(SEQ ID NO：14)：5'-GCUUCGCCG-3'，c序列(SEQ ID NO：15)：5'-CGGCCAUAGCCG-3'；(4)a序列(SEQ ID NO：16)：5'-GGAGCGUUGG-3'，b序列(SEQ ID NO：17)：5'-CCUUCGGGG-3'，c序列(SEQ ID NO：18)：5'-CCCCCAUAGCCC-3'；(5)a序列(SEQ ID NO：19)：5'-GCAGCGUUCG-3'，b序列(SEQ ID NO：20)：5'-CGUUCGGCG-3'，c序列(SEQ ID NO：21)：5'-CGCCCAUAGCGC-3'；(6)a序列(SEQ ID NO：22)：5'-GCAGCGUUCG-3'，b序列(SEQ ID NO：23)：5'-CGUUCGGCC-3'，c序列(SEQ ID NO：24)：5'-GGCCCAUAGCGC-3'；(7)a序列(SEQ ID NO：25)：5'-CGAGCGUUGC-3'，b序列(SEQ ID NO：26)：5'-GCUUCGGCG-3'，c序列(SEQ ID NO：27)：5'-CGCCCAUAGCCG-3'；(8)a序列(SEQ ID NO：28)：5'-GGAGCGTTGG-3'，b序列(SEQ ID NO：29)：5'-CCTTCGCCG-3'，c序列(SEQ ID NO：30)：5'-CGGCCATAGCCC-3'；(9)a序列(SEQ ID NO：31)：5'-GCAGCGTTCG-3'，b序列(SEQ ID NO：32)：5'-CGTTCGCCG-3'，c序列(SEQ ID NO：33)：5'-CGGCCATAGCGC-3'；(10)a序列(SEQ ID NO：34)：5'-CGAGCGTTGC-3'，b序列(SEQ ID NO：35)：5'-GCTTCGCCG-3'，c序列(SEQ ID NO：36)：5'-CGGCCATAGCCG-3'；(11)a序列(SEQ ID NO：37)：5'-GGAGCGTTGG-3'，b 序列(SEQ ID NO：38)：5'-CCTTCGGGG-3'，c序列(SEQ ID NO：39)：5'-CCCCCATAGCCC-3'；(12)a序列(SEQ ID NO：40)：5'-GCAGCGTTCG-3'，b序列(SEQ ID NO：41)：5'-CGTTCGGCG-3'，c序列(SEQ ID NO：42)：5'-CGCCCATAGCGC-3'；(13)a序列(SEQ ID NO：43)：5'-GCAGCGTTCG-3'，b序列(SEQ ID NO：44)：5'-CGTTCGGCC-3'，c序列(SEQ ID NO：45)：5'-GGCCCATAGCGC-3'；(14)a序列(SEQ ID NO：46)：5'-CGAGCGTTGC-3'，b序列(SEQ ID NO：47)：5'-GCTTCGGCG-3'，c序列(SEQ ID NO：48)：5'-CGCCCATAGCCG-3'。
上述十四组序列所自组装形成的核酸纳米颗粒，不仅具有更高的稳定性，而且自组装效率更高。
上述所提到的核酸纳米颗粒不仅能够自我组装成型，而且也具备携带或挂载药物药物的能力。根据上述核酸纳米颗粒中G-C或C-G碱基对的位置的不同，所挂载的药物的量也有所差异。
为了使上述核酸结构域能够挂载更多的药物以及其他的生物活性物质(生物活性物质的介绍见下文)，在一种优选的实施例中，上述核酸结构域还包括第一延长段，第一延长段为Watson-Crick配对的延长段，第一延长段位于a序列、b序列和c序列中任一序列的5'端和/或3'端。载体与所挂载的物质之间需要一定的匹配关系，当载体的分子量过小而所挂载的物质分子量过大时，从力学角度考虑，载体对挂载物质的携带或运输能力相对降低。因而，通过在前述核酸纳米结构基础上，通过在a序列、b序列和c序列中任一序列的5'端和/或3'端增加第一延长段，能够获得与挂载物质大小相匹配的载体。
上述第一延长段的具体长度，可以根据所欲挂载的物质的大小而定。在一种优选的实施例中，第一延长段选自如下任意一组：(1)：a链5'端：5'-CCCA-3'，c链3'端：5'-UGGG-3'；(2)：a链3'端：5'-GGG-3'，b链5'端：5'-CCC-3'；(3)：b链3'端：5'-CCA-3'，c链5'端：5'-UGG-3'；(4)：a链5'端：5'-CCCG-3'，c链3'端：5'-CGGG-3'；(5)：a链5'端：5'-CCCC-3'，c链3'端：5'-GGGG-3'；(6)：b链3'端：5'-CCC-3'，c链5'端：5'-GGG-3'。(7)：b链3'端：5'-CCG-3'，c链5'端：5'-CGG-3'；(8)：a链5'端：5'-CCCA-3'，c链3'端：5'-TGGG-3'；(9)：b链3'端：5'-CCA-3'，c链5'端：5'-TGG-3'；(10)：a链5'端：5'-GCGGCGAGCGGCGA-3'(SEQ ID NO:162)，c链3'端：5'-UCGCCGCUCGCCGC-3'(SEQ ID NO:163)；(11)：a链3'端：5'-GGCCGGAGGCCGG-3'(SEQ ID NO:164)，b链5'端：5'-CCGGCCUCCGGCC-3'(SEQ ID NO:165)；(12)b链3'端：5'-CCAGCCGCC-3'(SEQ ID NO:166)，c链5'端：5'-GGCGGCAGG-3'(SEQ ID NO:167)；(13)：a链5'端：5'-GCGGCGAGCGGCGA-3'(SEQ ID NO:168)，c链3'端：5'-TCGCCGCTCGCCGC-3'(SEQ ID NO:169)；(14)：a链3'端：5'-GGCCGGAGGCCGG-3'(SEQ ID NO:170)，b链5'端：5'-CCGGCCTCCGGCC-3'(SEQ ID NO:171)。
上述第一延长段不仅增加了形成核酸纳米结构的三条序列中任意一条或多条的长度，而且，GC碱基组成的第一延长段进一步提高了所形成的纳米颗粒的稳定性。而且，上述序列组成的第一延长段同样使a序列、b序列和c序列保持了较高的自组装活性和效率。
从所形成的核酸纳米颗粒的大小及其作为药物递送载体在体内运输时的稳定性考虑，需要能够在运输药物的同时，尽量在达到靶细胞之前不被肾脏过滤出去。在一种优选的实施例中，核酸结构域还包括第二延长段，第二延长段位于a序列、b序列和c序列中任一序列的5’端和/或3’端，第二延长段为Watson-Crick配对的延长段；更优选地，第二延长段为CG碱基对的延长序列；进一步优选地，第二延长段为1～10个CG碱基对的延长序列。第二延长段是在第一延长段的基础上进一步添加的延长段。
在一种优选的实施例中，上述核酸结构域还包括如下至少一组第二延长段：第一组：a链5’端：5’-CGCGCG-3’，c链3’端：5’-CGCGCG-3’；第二组：a链3’端：5’-CGCCGC-3’，b链5’端：5’-GCGGCG-3’；第三组：b链3’端：5’-GGCGGC-3’，c链5’端：5’-GCCGCC-3’。这种第二延长段，使得纳米颗粒不存在免疫原性，而且不存在每条链自身折叠结合的二级结构的情况。
需说明的是，上述第一延长段和/或第二延长段中也可以间隔有非配对的碱基对。
为了使上述核酸纳米颗粒能够挂载更大分子量的生物活性物质(生物活性物质的介绍见下文)、增加载药量以及维持必要的稳定性，在一种优选的实施例中，第二延长段为同时含有CG碱基对和AT/AU碱基对的延长序列，优选第二延长段为2～50个碱基对的延长序列。此处“AT/AU碱基”中的“/”是或的关系，具体地，第二延长段为同时含有CG碱基对和AT碱基对的延长序列，或者第二延长段为同时含有CG碱基对和AU碱基对的延长序列。
更具体地，添加上述第二延长段之后的a、b和c序列可以分别是如下序列：
a序列为(SEQ ID NO:49)：
Figure PCTCN2019109563-appb-000003
b序列为(SEQ ID NO:50)：
Figure PCTCN2019109563-appb-000004
c序列为(SEQ ID NO:51)：
Figure PCTCN2019109563-appb-000005
上述a序列、b序列和c序列中的M为U或T，当M为T时，上述序列的合成成本大大降低。
在实际应用中，可以根据实际需要合理调整上述CG碱基对以及AT/AU碱基对的延长序列的具体设置位置。在一种更优选的实施例中，第二延长段为连续2～8个CG碱基对的序列与连续2～8个AT/AU碱基对序列交替设置的延长序列；或者第二延长段为1个CG碱基对的序列与1个AT/AU碱基对序列交替设置的延长序列。
具体地，如将上述SEQ ID NO:49所示的a序列中的CGCGCG延长段和CGCCGC延长段与AAAAAA延长段的位置互换，将上述SEQ ID NO:50所示的b序列中的GCGGCG延长段和GGCGGC延长段与TTTTTT延长段的位置互换，将上述SEQ ID NO:51所示的c序列中的GCCGCC延长段与AAAAAA延长段互换，同时将CGCCGC延长段与TTTTTT延长段互换。上述序列自组装形成的核酸纳米颗粒适用于吲哚类分子结构的生物活性物质的挂载之用(吲哚类分子优选与A结合)。
过去多年里，RNA作为广泛应用的构建材料所存在的三大挑战包括：1)RNA酶降解的敏感性；2)全身注射后对解离的敏感性；3)毒性和不良免疫应答。目前，这三大挑战已经在很大程度上得到了克服：1)核糖-OH基团的2’-氟(2’-F)或者2’-O-甲基(2’-OMe)修饰可以使RNA在血清中化学稳定；2)某些天然存在的连接基序是热力学稳定的，并且可以保持整个RNA纳米颗粒在超低浓度下完整；3)RNA纳米颗粒的免疫原性是序列和形状依赖性的，并且可以调节，以使RNA纳米颗粒刺激炎性细胞因子的产生，或使得RNA纳米颗粒在30mg/kg的重复静脉注射施用时具有非免疫原性和无毒性。
因此，为了进一步降低上述核酸纳米颗粒对RNA酶降解的敏感性，同时提高在运输过程中的稳定性，在一种优选的实施例中，a序列、b序列和c序列中碱基、核糖和磷酸酯具有至少一个可修饰位点，任一可修饰位点通过以下任意一种修饰接头进行修饰：-F、甲基、氨基、二硫化物、羰基、羧基、巯基及醛基；优选地，a序列、b序列和c序列中的C或U碱基上具有2’-F修饰。当修饰接头为巯基时，属于硫代修饰，修饰强度较弱，成本低。
上述药物可以通过物理连接和/或共价连接的形式进行挂载。当药物采用物理嵌插与共价连接两种方式同时与核酸结构域进行连接时，物理嵌插通常是嵌插在GC碱基对之间，优选的嵌插位点数目根据核酸结构域上GC碱基对的数目的不同，按照1～100：1的比例进行嵌插。而采用共价连接方式进行连接时，上述药物通常会与G环外氨基发生化学反应形成共价连接。更优选地，药物与核酸纳米颗粒之间的摩尔比为2～300:1，优选为2～290:1，更优选为2～29:1，进一步优选为10～50:1，最优选为15～25:1。
本申请所提供的核酸纳米载体药物中，核酸纳米颗粒是作为药物的递送载体，除此以外，根据不同的药物目的，在一种优选的实施例中，上述核酸纳米颗粒还包括生物活性物质，生物活性物质与核酸结构域相连。生物活性物质为靶头、荧光素、干扰核酸siRNA、miRNA、核酶、核糖开关、适体、RNA抗体、蛋白、多肽、类黄酮、葡萄糖、天然水杨酸、单抗、维生素、酚类、卵磷脂以及小分子药物中的一种或多种，其中，小分子药物不包括他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨。
为了提高核酸纳米颗粒对所挂载的生物活性物质的挂载效率和运载效率，核酸结构域的相对分子量与药物及生物活性物质的相对分子量最好存在一定的匹配关系。在一种优选的实施例中，将核酸结构域的相对分子量记为N 1，将药物与生物活性物质的总相对分子量记为N 2，N 1/N 2≥1:1。
根据具体挂载的生物活性物质的种类的不同，本申请中核酸纳米载体药物具有不同性能方面的优化。比如，当生物活性物质为生物素或叶酸时，其所起到的作用是使该核酸纳米载体药物具有靶向性，如，特异靶向癌细胞。当生物活性物质为荧光素时，其所起到的作用是使核酸纳米颗粒具有发光示踪效果。而生物活性物质为某些siRNA、miRNA、蛋白、多肽、RNA抗体、小分子药物时，根据不同生物学功能的不同，可能使得该核酸纳米载体药物成为具有特定治疗效果的新产品，比如性能更优异的药物。此外，根据具体挂载的生物活性物质的种类的不同，其具体优选使用的是DNA纳米颗粒和RNA纳米颗粒，可以根据实际需要进行合理选择。比如，当生物活性物质为药物时，优选DNA纳米颗粒或RNA纳米颗粒进行挂载，且对组装形成纳米颗粒的单链长度无特殊要求。
在一种优选的实施例中，生物活性物质为靶头、荧光素以及miRNA，其中，靶头位于a、b、c序列中任一序列上，优选a、b、c任一序列的5’端或3’端，或嵌插于核酸结构域的GC键之间，miRNA为抗miRNA，荧光素修饰于抗miRNA的5’端或3’端，miRNA位于a序列的3’端、c序列的5’端和3’端中的任意一个或多个位置；优选地，靶头为叶酸或生物素，荧光素为FAM、CY5及CY3中的任意一种或多种，抗miRNA为抗miR-21。
上述靶头可以通过linker共价连接的方式连接于a、b、c序列中的任一序列上，可用的linker选自二硫键、对苯叠氮基、溴丙炔或PEG。此处所说的“任一序列上”是a、b、c序列任一序列的任一位置的碱基上，而连在5’端或3’端更方便，应用更广泛。叶酸修饰可以是物理嵌插模式连接或者是物理嵌插+共价连接。
上述荧光素可以现有常用的荧光素，优选为FAM、CY5及CY3中的任意一种或多种。
上述miRNA可以是具有抑癌效果的miRNA，也可以是能够抑制对应病症的抗miRNA，实际应用中根据医疗需要合理选择。上述抗miRNA可以合成于上述a序列的3’端、c序列的5’端和3’端中的任意一个或多个位置。当在上述三个位置上均合成有抗miRNA时，抗miRNA对相应miRNA的抑制作用相对更强。
优选为抗miR-21，miR-21参与多种癌症的起始和进展，是侵袭和转移的主要致癌基因。抗miR-21能够有效地同时调节广泛的靶基因，有利于解决癌症的异质性问题。因而，上述优选的核酸纳米颗粒中，靶头，比如叶酸或生物素，能够特异地靶向癌细胞，与癌细胞结合内化后，抗miR-21以非常高的亲和力和特异性与miR-21碱基互补，从而有效降低致癌miR-21的表达。因此，根据实际需要，上述抗miR-21可以合成于上述a序列的3’端、c序列的5’端和3’端中的任意一个或多个位置。当在上述三个位置上均合成有抗miR-21时，抗miR-21对miR-21的抑制作用相对更强。
上述所能够挂载的生物活性物质为除了他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨以外的其他小分子药物时，核酸纳米载体药物根据不同药物所能治疗的疾病类型，包括但不仅限于治疗肝癌、胃癌、肺癌、乳腺癌、头颈癌、子宫癌、卵巢癌、黑色素瘤、白血病、老年痴呆、强直性脊柱炎、恶性淋巴瘤、支气管癌、类风湿关节炎、HBV乙肝、多发性骨髓瘤、胰腺癌、非小细胞肺癌、前列腺 癌、鼻咽癌，食道癌，口腔癌，红斑狼疮疾病的药物；优选地，头颈癌为脑癌、神经母细胞瘤或胶质母细胞瘤。
上述所能够挂载的生物活性物质为除了上述他克林等以外的小分子药物时，根据药物的分子结构的不同或者所具有的特征性基团的不同，其包括但不仅限于含有如下任意一种或多种基团的药物：氨基基团、羟基基团、羧基基团、巯基基团、苯环基团以及乙酰氨基基团。
在一种优选的实施例中，上述蛋白为SOD(超氧化物歧化酶)、生存素(Survivin)、hTERT(人端粒酶逆转录酶)及EGFR(表皮生长因子受体，epidermal growth factor receptor)、PSMA(前列腺特异性膜抗原)的抗体或适配体中的一种或多种；上述维生素为左旋C和/或酯化C；上述酚类为茶多酚和/或葡萄多酚。
在一种优选的实施例中，核酸纳米颗粒的粒径为1～100nm，优选为5～50nm，更优选为10～30nm，进一步优选为10～15nm。在该范围内大小合适，既能通过细胞表面受体介导的细胞吞噬现象而进入细胞膜，又避免非特异性的细胞渗透而被肾脏过滤除去，因而，有利的粒径尺寸有助于改进药代动力学、药效学、生物学分布和毒理学的分布。
根据本申请的第二方面，还提供了一种上述核酸纳米载体药物的制备方法，其包括以下步骤：提供上述任一种核酸纳米颗粒；通过物理连接和/或共价连接的方式将药物挂载在核酸纳米颗粒上，得到核酸纳米载体药物。
当采用物理连接方式时，药物通常会以物理嵌插形成嵌插在GC碱基对之间。而采用共价连接方式进行连接时，药物通常会与G环外氨基发生化学反应形成共价连接。利用上述方法制备的核酸纳米载体药物，其经过靶头修饰后，可具有较好的靶向性，能够稳定地递送药物，可靠性很高。
在一种优选的实施例中，通过物理连接的方式挂载药物的步骤包括：将药物、核酸纳米颗粒及第一溶剂混合并搅拌，得到预混体系；对预混体系进行沉淀析出，得到核酸纳米载体药物。具体的药物、核酸纳米颗粒的用量可以根据挂载量的变化进行调整，这是本领域技术人员都能够理解的，在此不再赘述。
为了提高物理连接的效率和稳定性，优选每升第一溶剂中添加的药物量为0.1～1g。优选地，第一溶剂选自DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种。优选地，对预混体系进行沉淀析出，得到核酸纳米载体药物的步骤包括：对预混体系进行沉淀析出，得到析出物；对析出物进行洗涤去除杂质，得到核酸纳米载体药物。更优选地，将预混体系与无水乙醇混合后在低于10℃的温度条件下进行沉淀析出，得到析出物，进一步优选在0～5℃温度条件下沉淀析出，得到析出物。更优选地，采用6～12倍体积的无水乙醇对析出物进行洗涤去除杂质，得到核酸纳米载体药物。
在一种优选的实施例中，通过共价连接的方式挂载药物的步骤包括：配置药物溶液；使药物溶液在甲醛的介导作用下与核酸纳米颗粒的G环外氨基进行反应，得到反应体系；提纯反应体系，得到核酸纳米载体药物。
通过甲醛介导的形式，可以发生如下反应：
Figure PCTCN2019109563-appb-000006
优选地，上述反应的步骤包括：将药物溶液与多聚甲醛溶液、核酸纳米颗粒混合，在避光条件下进行反应，得到反应体系。多聚甲醛溶液能够释放甲醛小分子，从而参与上述化学反应。为了提高反应效率，优选多聚甲醛溶液的浓度为3.7～4wt％，优选多聚甲醛溶液为多聚甲醛和第二溶剂混合形成的溶液，且第二溶剂为DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种。
上述制备方法中，核酸纳米颗粒可以通过自组装的形式进行制备，比如：(1)将RNA或DNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；(2)加热混合溶液至80℃/95℃(其中RNA组装温度为80℃，DNA组装温度为95℃)，保持5min后以2℃/min的速率缓慢降温到室温；(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；(4)切下目的条带并在RNA/DNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干，得到自组装产物，即可得到核酸结构域，进而得到核酸纳米颗粒。
根据实际应用需要，为了使上述核酸纳米载体药物具有其他功能，在一种优选的实施例中，在得到核酸结构域之后，制备方法还包括：将前文所提到的生物活性物质通过物理连接和/或共价连接的方式挂载在所述核酸结构域上，进而得到核酸纳米颗粒。生物活性物质的挂载方式同样可以是物理连接和/或共价连接。共价连接的形式包括但不限于通过溶剂共价连接、linker共价连接或点击链接进行挂载；优选地，溶剂共价连接中采用的第三溶剂作为连接介质，且第三溶剂选自多聚甲醛、DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种；优选地，linker选自二硫键、对苯叠氮基、溴丙炔或PEG；优选地，点击链接是在对生物活性物质前体和核酸结构域同时进行炔基或叠氮修饰，然后通过点击链接。
需要说明的是，上述分类并不意味着某种生物活性物质与核酸结构域的连接方式仅有一种。而是，有的生物活性物质，既可以通过物理嵌插的方式与核酸结构域连接，也可以通过物理嵌插与共价连接的方式与核酸结构域连接，同时还可能利用点击链接的方式实现连接。但对某种特定的生物活性物质而言，可能仅有其中一种连接方式，也可能有多种连接方式，但可能其中某种连接效率具有优势的实用价值。
上述连接方式中，不同药物在与核酸结构域通过物理嵌插的方式进行连接时，嵌插的结合位点及数目也略有不同。比如，蒽环类、吖啶类药物在嵌插时，通常嵌插在GC碱基对之间，优选的嵌插位点数目根据核酸结构域上GC碱基对的数目的不同，按照1～100：1的比例进行嵌插。而萘酰胺药物在嵌插时，通常嵌插在AA碱基对之间，优选的嵌插位点数目根据核酸结构域上AA碱基对的数目的不同，吡啶并咔唑类根据AA碱基对的数目的不同按照1～200：1的比例进行嵌插。
具体地，根据生物活性物质种类的不同、核酸纳米颗粒中形成核酸结构域的a、b和c序列的长度以及其中GC互补碱基对的数目的多少，可以合理选择生物活性物质与核酸结构域的摩尔比进行物理嵌插。
在一种优选的实施例中，生物活性物质与核酸结构域以物理嵌插方式与共价连接方式相连时，物理嵌插方式连接的生物活性物质与共价连接方式连接的药物的摩尔比为1～200：1。该连接方式适用于蒽环类、吖啶类的药物。上述不同连接方式连接的药物比例并不局限于上述范围，只要能够满足高效挂载，对细胞无毒性作用，且在达到靶标后实现药物的有效释放即可。
当采用对生物活性物质前体和核酸结构域同时进行炔基或叠氮修饰，通过点击链接的方式连接时，随药物不同结构的变化选择不同的点击连接。且随着活性物质结构的不同，连接位置也有可能会发生相应改变，这是本领域技术人员能够理解的。
在一种优选的实施例中，生物活性物质与核酸结构域以点击链接的方式相连时，生物活性物质前体进行炔基或叠氮修饰的位点选自羟基、羧基、巯基或氨基，核酸结构域进行炔基或叠氮修饰的位点选自氨基、亚氨基或羟基。
需要说明的是，上述核酸结构域与药物结合时，核酸结构域为水溶性的，多数药物的水溶性较差，将其与核酸结构域结合后，水溶性提高。当上述药物为蒽环类时，这些药物通过核苷酸鸟苷上的-NH键(在合适的pH值条件下，该-NH基团的活性比其他可能与药物发生共价结合的基团的活性高上百倍)与核酸结构域发生共价结合，从而形成挂载药物的核酸结构域。因而，根据具体药物分子的大小及具体所设计的核酸结构域上的a序列，b序列和c序列上的GC碱基对的数量，在结合时，按照理论上1.1～1.3倍的过饱和结合量进行结合反应，一个核酸结构域上最多可结合35～45个药物。当上述药物为其他结构时，挂载量与具体药物的占位情况有关(包括但不仅限于分子结构、形态、形状及分子量大小)，因此，药物的活性位点与核酸结构域的核苷酸鸟苷上的-NH键的结合条件相对严苛，同样能挂载但比较难以出现过量结合的情况。根据本申请的第三个方面，还提供了一种药物组合物，该药物组合物包括上述任一种核酸纳米载体药物。具体可以根据实际需要，选择合适的联用药或辅料来形成具有联合药效或能使药物某方面性能(比如稳定性)得以提升的药物组合。
根据本申请的第四个方面，还提供了上述任一种核酸纳米载体药物在制备用于治疗老年痴呆老年痴呆症、肿瘤、自身免疫疾病或心脏病的药物中的应用。具体应用可以在本申请的 药物基础上对药物本身进行改进而获得新的药物，或者将本申请的药物作为主要活性成分将其制备成适合剂型的制剂等。
具体地，根据核酸纳米载体药物中的药物不同，所能够治疗的疾病也有所差异。核酸纳米载体药物中的药物包括他克林时能够用于制备制备老年痴呆症的药物。药物包括表柔比星时，上述核酸纳米载体药物能够用于制备治疗肿瘤的药物，肿瘤可以为急性白血病、恶性淋巴瘤、乳腺癌、支气管肺癌、卵巢癌、肾母细胞瘤、软组织肉瘤、原发性肝细胞癌、转移性肝癌以及甲状腺髓样癌中的任意一种或多种。
药物包括甲氨蝶呤时，上述核酸纳米载体药物能够制备用于预防和/或治疗肿瘤或自身免疫疾病的药物，优选其所针对的肿瘤为急性白血病、乳腺癌、绒毛膜上皮癌、恶性葡萄胎、头颈部肿瘤、骨肿瘤、白血病脑膜脊髓浸润、肺癌、生殖系统肿瘤、肝癌中的任意一种或多种，自身免疫疾病为顽固性牛皮藓、系统性红斑狼疮、强制性脊柱炎及皮肌炎中的任意一种或多种。
药物包括吡柔比星时，上述核酸纳米载体药物能够制备用于治疗肿瘤的药物。优选肿瘤为乳腺癌、头颈癌、膀胱癌、输尿管癌、肾盂癌、卵巢癌及宫颈癌中的任意一种或多种。
药物包括柔红霉素时，上述核酸纳米载体药物同样能够制备用于治疗肿瘤的药物。优选肿瘤为急性淋巴细胞白血病或粒细胞白血病。
药物包括五氟尿嘧啶时，上述核酸纳米载体药物同样能够制备用于治疗治疗肿瘤的药物，优选地，能够制备用于治疗肝癌、结肠癌、直肠癌、胃癌、乳腺癌、卵巢癌、绒毛膜上皮癌、恶性葡萄胎、头颈部鳞癌、皮肤癌、肺癌、宫颈癌、胰腺癌或膀胱癌的药物。
药物为10-羟基喜树碱时，上述核酸纳米载体药物同样能够制备用于治疗肝癌、胃癌、头颈部癌或白血病的药物。
药物为阿司匹林时，上述核酸纳米载体药物能够用于制备解热镇痛、预防心脏病及脑血栓、消炎抗风湿、治疗关节炎、减轻川崎病患者皮肤粘膜淋巴结综合症、抵抗癌症及预防消化道肿瘤的药物。
药物包括吉西他滨时，上述核酸纳米载体药物同样能够制备用于治疗治疗肿瘤的药物，优选地，能够制备用于治疗胰腺癌、非小细胞肺癌、卵巢癌、乳腺癌、膀胱癌、子宫颈癌、肝癌、胆道癌、鼻咽癌、睾丸肿瘤、淋巴瘤、间皮瘤或头颈部癌的药物。
根据本申请的第五个方面，还提供了一种预防和/或治疗老年痴呆症、肿瘤、自身免疫疾病或心脏病的方法，该方法包括：提供上述任一种核酸纳米载体药物或药物组合物；给予老年痴呆症患者、肿瘤患者、自身免疫疾病患者或心脏病患者有效量的上述核酸纳米载体药物或药物组合物。此处的有效量包括预防有效量和/或治疗有效量，治疗有效量指在必要的剂量和时间期限内，有效实现所希望的治疗结果，如老年痴呆症减轻的量。在一个具体的实施方案中，可以通过调节剂量以提供最佳治疗应答剂量，治疗有效量可以根据如下因素而变：个体的疾病状态、年龄、性别、体重以及制剂在个体中引起所希望的应答的能力。治疗有效量 的涵义还包括治疗的有益效果超过其毒性或者有害效果的量。预防有效量指在必要的剂量和时间期限内，有效实现所希望的预防结果，如预防或抑制老年痴呆症发生的量。可以根据上述对治疗有效量的描述确定预防有效量。对于任何具体受试者，可以根据个体需要和施用人的职业判断随时间调节特定剂量。
需要说明的是，本申请所提供的序列或序列的变形通过自组装形成的核酸纳米颗粒也可以作为基本结构单元，根据实际应用需要可以进一步聚合形成多聚体，比如二聚体、三聚体、四聚体、五聚体、六聚体或七聚体等。
下面将结合具体的实施例来进一步说明本申请的有益效果。
核酸纳米颗粒的组装
实施例1
一、RNA和DNA纳米颗粒载体：
(1)组成RNA纳米颗粒的三条多核苷酸碱基序列见表1：
表1：
Figure PCTCN2019109563-appb-000007
(2)DNA纳米颗粒的三条多核苷酸碱基序列
DNA采用与上述RNA同样的序列，仅是T替代U。其中，a链的分子量为8802.66，b链的分子量为8280.33，c链的分子量为9605.2。
上述RNA纳米颗粒和DNA纳米颗粒的a、b和c链，均是委托生工生物工程(上海)股份有限公司合成。
二、自组装实验步骤：
(1)按1:1:1的摩尔比将RNA或DNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；
(2)加热混合溶液至80℃/95℃(其中RNA组装温度为80℃，DNA组装温度为95℃)，保持5min后以2℃/min的速率缓慢降温到室温；
(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；
(4)切下目的条带并在RNA/DNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干，得到自组装产物；
(5)电泳分析检测与激光扫描观察。
三、自组装实验结果
电泳检测结果
RNA自组装产物的电泳检测结果见图1。图1中，泳道1至3从左到右依次为：a链、b链、RNA自组装产物。由图中可知，RNA自组装产物随稍有弥散，但明显可以看出是单一条带。且由于分子量为组装后的分子量，较单链分子量大，因此条带位置落后于a链和b链，实际情况与理论相符，证明了上述RNA单链之间经自组装形成了稳定的复合结构，形成了RNA纳米颗粒。
DNA自组装产物的电泳检测结果见图2。图2中，泳道1至3从左到右依次为：a链、b链、DNA自组装产物。由图中可知，DNA自组装产物的条带明亮清晰，为单一条带，证明了上述DNA单链之间经自组装形成了稳定的复合结构，形成了DNA纳米颗粒。
该实施例中，通过凝胶电泳验证了：包括RNA核心序列SEQ ID NO:1,SEQ ID NO:3和SEQ ID NO:5在内的a、b、c序列，能够成功自组装成RNA纳米颗粒。包括DNA核心序列SEQ ID NO:2,SEQ ID NO:4和SEQ ID NO:6在内的a、b、c序列，也能够成功自组装成DNA纳米颗粒。
上述RNA纳米颗粒和DNA纳米颗粒的a、b、c序列中除了具有形成核酸结构域的核心序列外，还具有各种促进核酸结构域挂载功能的延长序列(包括药物挂载结合序列)以及与核酸结构域连接的靶头或荧光素。可见，这些核心序列以外的物质存在并不影响核酸结构域的形成和核酸纳米颗粒的成功自组装。而所自组装而成的核酸纳米颗粒在靶头的引导下，能够具有靶向型，荧光素能使该核酸纳米颗粒具有可视性和可追踪性。
实施例2
一、7组短序列RNA纳米颗粒载体：
(1)7组组成RNA纳米颗粒的三条多核苷酸碱基序列见表2至表8：
表2：R-1
Figure PCTCN2019109563-appb-000008
表3：R-2
Figure PCTCN2019109563-appb-000009
表4：R-3
Figure PCTCN2019109563-appb-000010
表5：R-4
Figure PCTCN2019109563-appb-000011
表6：R-5
Figure PCTCN2019109563-appb-000012
表7：R-6
Figure PCTCN2019109563-appb-000013
表8：R-7
Figure PCTCN2019109563-appb-000014
上述7组短序列RNA纳米颗粒载体的单链均是委托生工生物工程(上海)股份有限公司进行合成。
二、自组装实验步骤：
(1)按1:1:1的摩尔比将RNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；
(2)加热混合溶液至80℃，保持5min后以2℃/min的速率缓慢降温到室温；
(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；
(4)切下目的条带并在RNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干，得到短序列RNA自组装产物；
(5)电泳分析检测与激光扫描观察；
(6)电位检测。
三、自组装实验结果
电泳检测结果
7组短序列RNA自组装产物的2％琼脂糖凝胶电泳图见图3。图3中泳道1至7从左到右依次为：短序列R-1、R-2、R-3、R-4、R-5、R-6、R-7。
7组短序列RNA自组装产物的4％琼脂糖凝胶电泳图见图4。图4中泳道1至7从左到右依次为：短序列R-1、R-2、R-3、R-4、R-5、R-6、R-7。
由图3和图4结果可以看出，可以清楚地看出7组短序列自组装产物中R-2、R-3、R-5、R-7的条带明亮清晰，R-1、R-4、R-6虽然较为弥散，但仍然可以看出为单一条带，表明7组短序列均能较好地自组装成RNA纳米颗粒结构。
(2)电位测定
测定方法：准备好电位样品(自组装产物溶于超纯水中)放入样品池中，打开仪器的样品池盖，放入仪器；
打开软件，点击菜单MeasUre€ManUal，出现手动测量参数设置对话框；
设置软件检测参数；
然后点击确定完毕设置，出现测量对话框，点击Start开始；
测定结果：7组短序列RNA纳米颗粒的电位检测结果如下表9至表15：
表9：
Figure PCTCN2019109563-appb-000015
表10：
Figure PCTCN2019109563-appb-000016
表11：
Figure PCTCN2019109563-appb-000017
表12：
Figure PCTCN2019109563-appb-000018
Figure PCTCN2019109563-appb-000019
表13：
Figure PCTCN2019109563-appb-000020
表14：
Figure PCTCN2019109563-appb-000021
表15：
Figure PCTCN2019109563-appb-000022
由上述电位检测数据可知：7组短序列RNA自组装产物均具有良好的稳定性，进一步表明各短序列RNA自组装而成的纳米颗粒具有较稳定的自组装结构。
该实施例表明：不同的a、b、c核心序列组合能够通过自组装形成具有核酸结构域的RNA纳米颗粒，且结构稳定。在实施例1的基础上可知，在这些不同的核心序列组合基础上增加各种功能延长片段或者连接靶头、荧光素等，同样能成功组装成RNA纳米颗粒，并具有挂载药物、细胞靶向性及可视可追踪等性能。
为了进一步验证这些性能，在实施例2基础上增加延长片段，具体见实施例3。并在与实施例2的RNA核心序列相对应的DNA核心序列基础上，增加延长片段，同时连接靶头或不连接靶头，具体见实施例4。
实施例3
一、7组常规序列RNA纳米颗粒载体：
(1)7组组成RNA纳米颗粒的三条多核苷酸碱基序列见表16至表22：
表16：R-8
Figure PCTCN2019109563-appb-000023
表17：R-9
Figure PCTCN2019109563-appb-000024
表18：R-10
Figure PCTCN2019109563-appb-000025
Figure PCTCN2019109563-appb-000026
表19：R-11
Figure PCTCN2019109563-appb-000027
表20：R-12
Figure PCTCN2019109563-appb-000028
表21：R-13
Figure PCTCN2019109563-appb-000029
Figure PCTCN2019109563-appb-000030
表22：R-14(下述a链中的 uGAcAGAuAAGGAAccuGcudTdT为survivin siRNA)
Figure PCTCN2019109563-appb-000031
上述7组常规序列RNA纳米颗粒载体的单链均是委托苏州吉玛公司进行合成，其中R-8至R-14中的a序列、b序列、c序列分别是在R-1至R-7的a序列、b序列、c序列基础上增加延长段后形成的延展RNA寡核苷酸序列，没有延展靶向模块片段，并进行了C/U碱基2’F修饰(增强了抗酶切性和稳定性)。另外，上述RNA纳米颗粒R-14中修饰了一段生存素(Survivin)的siRNA核酸干扰治疗片段，具体是在a链3’端延展了Survivin siRNA的正义链(见a链下划线部分)，在b链的5’端延展连接了反义链(见b链下划线部分)，形成碱基对互补。
二、自组装实验步骤：
(1)按1:1:1的摩尔比将RNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；
(2)加热混合溶液至80℃，保持5min后以2℃/min的速率缓慢降温到室温；
(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；
(4)切下目的条带并在RNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干；
(5)电泳分析检测与激光扫描观察；
(6)电位测定。
三、自组装实验结果
(1)电泳检测结果
7组常规序列RNA自组装产物的2％琼脂糖凝胶电泳图见图5。图5中泳道1至7从左到右依次为：常规序列RNA自组装产物R-8、R-9、R-10、R-11、R-12、R13、R-14。
7组常规序列RNA自组装产物的4％琼脂糖凝胶电泳图见图6。图6中泳道1至7从左到右依次为：常规序列RNA自组装产物R-8、R-9、R-10、R-11、R-12、R13、R-14。
由图5和图6结果可以看出，可以清楚地看出7组常规序列RNA自组装产物的条带均为明亮清晰的单一条带，表明7组常规序列均能自组装成纳米结构。其中常规序列RNA自组装产物R-14中修饰了一段Survivin siRNA核酸干扰治疗片段后，仍旧具有稳定的自组装结构，也说明了本申请中核酸纳米颗粒能够挂载核酸药，具有核酸药的递送载体功能。
(2)电位测定
测定方法：准备好电位样品(自组装产物溶于超纯水中)放入样品池中，打开仪器的样品池盖，放入仪器；
打开软件，点击菜单MeasUre€ManUal，出现手动测量参数设置对话框；
设置软件检测参数；
然后点击确定完毕设置，出现测量对话框，点击Start开始；
测定结果：7组常规序列RNA纳米颗粒的电位检测结果如下表23至表29：
表23：
Figure PCTCN2019109563-appb-000032
表24：
Figure PCTCN2019109563-appb-000033
Figure PCTCN2019109563-appb-000034
表25：
Figure PCTCN2019109563-appb-000035
表26：
Figure PCTCN2019109563-appb-000036
表27：
Figure PCTCN2019109563-appb-000037
表28：
Figure PCTCN2019109563-appb-000038
表29：
Figure PCTCN2019109563-appb-000039
由上述电位检测数据可知：7组常规序列RNA自组装产物均具有良好的稳定性，进一步表明各常规序列RNA自组装而成的纳米颗粒具有较稳定的自组装结构。
该实施例表明：在不同组合的RNA核心序列基础上，添加延长片段同样能够成功自组装成结构稳定的RNA纳米颗粒。同时，添加的延长片段使得RNA纳米颗粒具有优越的药物挂载性能(具体见实施例5)。
实施例4
一、7组常规序列DNA纳米颗粒载体：
(1)7组组成DNA纳米颗粒的三条多核苷酸碱基序列见下表30至表36：
表中部分a链中延展了EGFRapt靶头或PSMAapt(A9L)靶头：
EGFRapt(SEQ ID NO：97)：GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC；
PSMAapt(A9L，SEQ ID NO：98)：
Figure PCTCN2019109563-appb-000040
表30：D-1
Figure PCTCN2019109563-appb-000041
Figure PCTCN2019109563-appb-000042
表31：D-2
Figure PCTCN2019109563-appb-000043
表32：D-3
Figure PCTCN2019109563-appb-000044
表33：D-4
Figure PCTCN2019109563-appb-000045
表34：D-5
Figure PCTCN2019109563-appb-000046
表35：D-6
Figure PCTCN2019109563-appb-000047
Figure PCTCN2019109563-appb-000048
表36：D-7
Figure PCTCN2019109563-appb-000049
上述7组常规序列DNA纳米颗粒的单链均是委托苏州泓迅进行合成，其中：
D-1是在前文所述核心序列(8)(a序列：5'-GGAGCGTTGG-3'，b序列：5'-CCTTCGCCG-3'，c序列：5'-CGGCCATAGCCC-3')的基础上，增加包含EGFRapt靶头(见下划线部分)的延展序列后形成的常规序列DNA纳米颗粒；
D-2是在前文所述核心序列(9)(a序列：5'-GCAGCGTTCG-3'，b序列：5'-CGTTCGCCG-3'，c序列：5'-CGGCCATAGCGC-3')的基础上，增加包含EGFRapt靶头(见下划线部分)的延展序列后形成的常规序列DNA纳米颗粒；
D-3是在前文所述核心序列(10)(a序列：5'-CGAGCGTTGC-3'，b序列：5'-GCTTCGCCG-3'，c序列：5'-CGGCCATAGCCG-3')的基础上，增加包含EGFRapt靶头(见下划线部分)的延展序列后形成的常规序列DNA纳米颗粒；
D-4是在前文所述核心序列(11)(a序列：5'-GGAGCGTTGG-3'，b序列：5'-CCTTCGGGG-3'，c序列：5'-CCCCCATAGCCC-3')的基础上，增加包含PSMAapt靶头(见下划线部分)的延展序列后形成的常规序列DNA纳米颗粒；
D-5是在前文所述核心序列(12)(a序列：5'-GCAGCGTTCG-3'，b序列：5'-CGTTCGGCG-3'，c序列：5'-CGCCCATAGCGC-3')的基础上，增加包含PSMAapt靶头(见下划线部分)的延展序列后形成的常规序列DNA纳米颗粒；
D-6是在前文所述核心序列(13)(a序列：5'-GCAGCGTTCG-3'，b序列：5'-CGTTCGGCC-3'，c序列：5'-GGCCCATAGCGC-3')的基础上，增加了不包含靶头结构的延展序列后；形成的常规序列DNA纳米颗粒；
D-7是在前文所述核心序列(14)(a序列：5'-CGAGCGTTGC-3'，b序列：5'-GCTTCGGCG-3'，c序列：5'-CGCCCATAGCCG-3')的基础上，增加了不包含靶头结构的延展序列后；形成的常规序列DNA纳米颗粒。
二、自组装实验步骤：
(1)按1:1:1的摩尔比将DNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；
(2)加热混合溶液至95℃，保持5min后以2℃/min的速率缓慢降温到室温；
(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；
(4)切下目的条带并在DNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干，得到常规序列DNA自组装产物；
(5)电泳分析检测与激光扫描观察；
(6)电位测定；
(7)粒径测量；
(8)透射电镜观察。
三、自组装实验结果
(1)电泳检测结果
7组常规序列DNA自组装产物的2％琼脂糖凝胶电泳图见图7。图7中泳道1至7从左到右依次为：常规序列DNA自组装产物D-1、D-2、D-3、D-4、D-5、D-6、D-7。
7组常规序列DNA自组装产物的4％琼脂糖凝胶电泳图见图8。图8中泳道1至7从左到右依次为：常规序列DNA自组装产物D-1、D-2、D-3、D-4、D-5、D-6、D-7。
由图7和图8结果可以看出，可以清楚地看出7组常规序列DNA自组装产物的条带均明亮清晰，表明7组常规序列DNA链均完成了自组装，形成了稳定的纳米颗粒结构。其中D-6、D-7两组自组装结构因为携带EGFRapt或PSMAapt靶头，分子量略低，其条带位置明显比其他条带靠前，实际与理论情况完全符合，进一步证明了自组装结构的稳定性。
该实施例表明：在这些不同的DNA核心序列组合基础上增加各种功能延长片段或者同时连接靶头时，同样能成功组装成DNA纳米颗粒，并同样具有挂载药物、细胞靶向性及可视可追踪等性能。
(2)电位测定
测定方法：准备好电位样品(自组装产物溶于超纯水中)放入样品池中，打开仪器的样品池盖，放入仪器；
打开软件，点击菜单MeasUre€ManUal，出现手动测量参数设置对话框；
设置软件检测参数；
然后点击确定完毕设置，出现测量对话框，点击Start开始；
测定结果：3组常规序列DNA纳米颗粒的电位检测结果如下表37至表39：
表37：
Figure PCTCN2019109563-appb-000050
表38：
Figure PCTCN2019109563-appb-000051
表39：
Figure PCTCN2019109563-appb-000052
Figure PCTCN2019109563-appb-000053
由上述电位检测数据可知：3组常规序列RNA自组装产物均具有良好的稳定性，进一步表明各常规序列RNA自组装而成的纳米颗粒具有较稳定的自组装结构。
(3)粒径测量
1.准备好电位样品(常规序列DNA自组装产物D-7)放入样品池中，打开仪器的样品池盖，放入仪器；
2.打开软件，点击菜单，出现手动测量参数设置对话框；
3.设置软件检测参数；
4.然后点击确定完毕设置，出现测量对话框，点击Start开始，自组装产物D-7的流体动力学尺寸的DLS测量值结果如下表40：
表40：
Figure PCTCN2019109563-appb-000054
(5)透射电镜观察结果
对上述常规序列DNA自组装产物D-7进行透射电镜照射，步骤如下：
1、取一滴样本悬浮在400目覆碳膜铜网上，室温1分钟；
2、滤纸吸去液体；
3、2％醋酸铀染色1分钟；
4、滤纸吸干，室温干燥；
5、JEM-1400透射电子显微镜120kv观察、拍照。
结果如图9所示，从图中明显看出上述常规序列DNA自组装产物D-7为一个整体结构，且能够清晰地看出其具有T型结构。
实施例5
他克林(Tacrine)挂载实验
(一)化学法挂载：
一、实验材料和实验方法
1.实验材料及试剂：
(1)核酸纳米颗粒：来自实施例1中的RNA纳米颗粒。
(2)DEPC水：碧云天。
(3)PBS缓冲液：cellgro。
(4)4％多聚甲醛
(5)他克林(Tacrine)。
(6)氯仿：北化。
(7)无水乙醇：北化。
2.实验方法：
(1)精密称取他克林(0.32mg，1.354μmoL)溶于DEPC水(1.0mL)及PBS缓冲液(1.25mL)，冰水浴冷却下加入4％多聚甲醛水溶液(0.4mL)混匀，将此混合液全部与RNA纳米颗粒(1mg，33.84nmoL)混匀，并在避光条件下于4℃反应72小时。
(2)取反应液10μL稀释10倍，以50μM的他克林水溶液及310ng/μL的RNA纳米颗粒为对照，按等体积进样进行HPLC分析。根据各组分峰面积比可判断反应转化基本完全。
(3)将反应液以氯仿萃取(10mLx3)，随后加入25mL的无水乙醇，混匀后避光置于4℃使产物充分析出(4小时)。离心(4000/min)，转移上清，固体产物再次以乙醇(50mL)洗，于低温下减压挥干溶剂得挂载产物。
(4)挂载率计算：
1.配置已知浓度的他克林-HCl标准液(HCl浓度为0.1M)：2μM、4μM、6μM、8μM、10μM，各100ul；
2.将他克林-RNAh颗粒溶解在100ul PBS中；
3.将标准液与他克林-RNAh颗粒置于PCR板中，于85℃加热5min，随后冷却至室温；
4.利用酶标仪测量240nm处他克林的吸光度，绘制标准曲线(如图10所示)，计算得出挂载产物中他克林的摩尔浓度；
5.利用分光光度计测量260nm处RNA的吸光度，得到每个样品中含有他克林-RNAh颗粒的质量浓度；
6.根据测量得到的他克林摩尔浓度及RNAh颗粒的质量浓度，计算挂载率。
计算具体过程如下：
C RNAh-1＝76.0ug/ul，M RNAh≈30000，100ul；C 他克林-1＝42.24μM，100ul；
C RNAh-2＝52.0ug/ul，M RNAh≈30000，100ul；C 他克林-1＝24.0μM，100ul；
Figure PCTCN2019109563-appb-000055
取N-1和N-2的平均值得到RNAh-他克林的挂载率约为15，及表示每一个核酸纳米颗粒载体上能够挂载约15个他克林分子。
此外，在RNA纳米颗粒挂载他克林的基础上，可进一步按照与挂载他克林一样的方法进行第二次挂载其他小分子药物，比如，本申请还进一步挂载了叶酸，得到共同挂载了他克林和叶酸两种小分子药物的RNA纳米颗粒，两种药物的挂载率可以参照上述方法进行检测得到(数值未显示)。
实施例5表明，带有延长片段、靶头和荧光素的RNA纳米颗粒(实施例1中的)具有挂载药物的功能，小分子药物他克林可以通过共价连接(多聚甲醛—溶剂共价)的方式来实现挂载，而且还可以与其他小分子药物实现共同挂载。
实施例6
共聚焦显微镜实验检测载药RNA纳米颗粒的细胞结合能力
一、实验材料和实验方法：
1.待测样品见表41：
表41：
Figure PCTCN2019109563-appb-000056
注：表中RNAh-Biotine-quasar670作为对照，其指的是按照实施例1中自组装方法制备的在a链和b链的5’端进行Biotin修饰，而在c链的3’端进行quasar670荧光素修饰而形成的纳米颗粒，而RNAh-Biotin-quasar670-tacrine指的是进一步挂载他克林后(按照实施例5中化学法挂载)形成的纳米颗粒。
2.所用到的实验试剂及其来源如下：
RPMI-1640培养基(Gibco,C11875500BT-500mL)；DMEM(Gibco,C11995500BT-500mL)；胎牛血清(Fetal bovine serum，FBS)(ExCell Bio,FNA500-500mL)；盘尼西林/链霉素(Penicillin/Streptomycin，PS)(Gibco,15140-122-100mL)；PBS缓冲液(Gibco,C20012500BT-500mL)；Trypsin-EDTA(Stemcell,07901-500mL)；DMSO(Sigma,D5879-1L)；Prolong Gold Antifade Mountant防猝灭剂(Thermo,P36941-2mL)；DAPI(Yeasen,36308ES11-4mL)。
3.所用到的实验仪器如下：
倒置显微镜(Inverted Microscope)(Olympus BX53,U-RFL-T)；BD Falcon(Corning,354118)；细胞离心涂片机(Cytospin)(TXD3)。
4.实验方法:
(1)SH-SY5Y细胞(神经母细胞瘤细胞系)在RPMI1640+10％FBS+1％PS培养基中，于37℃和5％CO 2条件下培养。
(2)胰酶消化SH-SY5Y细胞，并用PBS洗一遍，以每孔1x10 5个细胞加到细胞培养载玻片中。
(3)细胞贴壁后用培养基冲洗载玻片。
(4)将细胞与200nM和400nM的RNAh-Biotin-quasar670和RNAh-Biotin-quasar670-tacrine纳米颗粒一起于37℃和5％CO 2中孵育1h和4h。
(5)贴壁细胞用PBS洗涤后，防猝灭剂(Prolong Gold Antifade Mountant)处理，室温过夜。
(6)再用DAPI染色室温5min，然后封片。
(7)显微镜下拍照，保存。
二、实验结果
实验结果见图11。从图11中可以看出，细胞结合和内化实验结果表明，RNAh-Biotin-quasar670和RNAh-Biotin-quasar670-tacrine纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。此结果表明，含他克林的药物RNAh-Biotin-quasar670-tacrine纳米颗粒与SH-SY5Y细胞结合和内化能力较强。
实施例7
检测挂载于核酸纳米颗粒上的含他克林的药物在血清中的稳定性
一、实验材料和实验方法
1.待测样品：溶解在PBS溶液中的实施例5中制备的RNAh-Biotin-quasar670-tacrine纳米颗粒。
2.实验试剂：
RPMI-1640培养基(Gibco,C11875500BT-500mL)；胎牛血清(Fetal bovine serum，FBS)(ExCell Bio,FNA500-500mL)；盘尼西林/链霉素(Penicillin/Streptomycin，PS)(Gibco,15140-122-100mL)；PBS缓冲液(Gibco,C20012500BT-500mL)；Novex TMTris-Glycine Native Sample Buffer(2X)(Invitrogen,LC2673-20mL)；Novex TM8％Tris-Glycine Mini Gels(Invitrogen,XP00080BOX-1.0mm)；Tris-Glycine Native Running buffer(10x)(Life science,LC2672-500mL)；G250染色液(Beyotime,P0017-250mL)。
3.实验仪器：
分光光度计(Spectrophotometer)(Thermo,ND2000C)；Mini Gel Tank(Invitrogen,PS0301)；成像系统(Imaging System)(Bio-Rad,ChemiDoc MP)。
4.实验方法：
(1)将RNAh-Biotin-quasar670-tacrine纳米颗粒配成100μM，充分混匀。
(2)取其中1μL置于99μL含10％血清的RPMI 1640培养基中孵育。
(3)在37℃孵育10min、1h、12h、36h后分别取样。
(4)采用NanoDrop定量后，取200ng的RNA纳米颗粒，加入相同体积的Tris-Glycine SDS样品缓冲液(2X)，充分混匀。
(5)取一块Novex TM8％Tris-Glycine Mini gel，按照顺序上样，设置程序200V，30min，开始电泳。
(6)电泳结束，进行G250染色，置于水平摇床30min-1h，拍照成像。
二、实验结果
表42：定量结果及上样体积。
Figure PCTCN2019109563-appb-000057
电泳检测结果见图12和图13。其中，图12示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图13示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Biotin-quasar670-tacrine纳米颗粒样品条带无明显差别，表明RNAh-Biotin-quasar670-tacrine纳米颗粒在10％FBS的1640培养基中比较稳定，无明显降解。
实施例8
研究RNAh-Biotin-quasar670-tacrine纳米颗粒在SH-SY5Y细胞中的细胞毒性
一、实验材料和实验方法
1.待测样品为DMSO对照、小分子药物他克林及RNAh-Biotin-quasar670-tacrine纳米颗粒。
2.实验试剂:
RPMI-1640培养基(Gibco,C11875500BT-500mL)；DMEM(Gibco,C11995500BT-500mL)；胎牛血清(Fetal bovine serum，FBS)(ExCell Bio,FNA500-500mL)；盘尼西林/链霉素(Penicillin/Streptomycin，PS)(Gibco,15140-122-100mL)；PBS缓冲液(Gibco,C20012500BT-500mL)；Trypsin-EDTA(Stemcell,07901-500mL)；DMSO(Sigma,D5879-1L)；Dox(HISUN Pharm,H33021980-10mg)；CellTiter-Glo Luminescent Cell Viability Assay kit(CTG)(Promega,G7572-100mL)。
3.实验仪器:
倒置显微镜(Inverted Microscope)(Olympus IX71,TH4-200)；96孔板阅读仪(96-well Plate Reader)(Molecular Devices,Flexstation 3)。
4.实验方法:
(1)用RPMI1640+10％FBS+1％PS培养基，于37℃和5％CO 2中培养SH-SY5Y细胞。
(2)收集细胞，800rpm 5分钟离心，培养基重悬，调整细胞浓度，以5000个细胞90μL的体积加到96孔板中。
(3)第二天用培养基稀释待测样品，每个样品分别加入200nM，每个样品有4个复孔进行重复。
(4)培养72h后，每孔加入CTG试剂100μL，振荡2min，室温静置10min，全程避光。
(5)最后使用Soft Max Pro5软件读数。
二、实验结果:
表43：细胞增殖率(％)
细胞系 处理时间 他克林(Tacrine) RNAh-Biotin-quasar670-tacrine
SH-SY5Y 72h 39.75 13.04
实验结果见表43和图14，从表43和图14中可以看出，200nM的携带他克林的RNA纳米颗粒RNAh-Biotin-quasar670-tacrine对于SH-SY5Y细胞有明显细胞毒性(P<0.0001)，而且让人难以预料的是：与小分子药物他克林对细胞的增殖抑制作用相比，200nM的RNAh-Biotin-quasar670-tacrine对SH-SY5Y细胞的增殖抑制更显著，其在小分子药物他克林处理后细胞的增殖率为39.75％的基础上，使细胞的增殖率又降低了至少2/3(降低至13.04％)。
为了进一步确认不携带他克林的RNA纳米颗粒对SH-SY5Y细胞无明显细胞毒性，发明人进一步设计了RNAh-Biotin-Cy5这一靶向荧光载体对SH-SY5Y细胞的毒性实验，并用另一小分子化药顺铂(Cisplatin)作为对照(实验中药物的最高给药浓度为5μM)，其结果如表44和图15所示。从表44的IC 50值和图15中可以看出，不携带他克林的靶向荧光载体本身对实验细胞并无明显毒性。
表44：
  Bio-Cy5-RNAh 顺铂(Cisplatin)
IC50(μM) >5 0.51
核酸纳米颗粒的组装
实施例9
一、7组延长段变形+核心短序列RNA纳米颗粒载体：
(1)7组组成延长段变形+核心短序列RNA纳米颗粒的三条多核苷酸碱基序列：
表45：R-15：
Figure PCTCN2019109563-appb-000058
Figure PCTCN2019109563-appb-000059
表46：R-16：
Figure PCTCN2019109563-appb-000060
表47：R-17：
Figure PCTCN2019109563-appb-000061
Figure PCTCN2019109563-appb-000062
表48：R-18：
Figure PCTCN2019109563-appb-000063
表49：R-19：
Figure PCTCN2019109563-appb-000064
表50：R-20：
Figure PCTCN2019109563-appb-000065
表51：R-21：
Figure PCTCN2019109563-appb-000066
二、自组装试验步骤：
(1)按1:1:1的摩尔比将RNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；
(2)加热混合溶液至80℃，保持5min后以2℃/min的速率缓慢降温到室温；
(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；
(4)切下目的条带并在RNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干；
(5)电泳分析检测与激光扫描观察。
三、自组装试验结果
(1)电泳检测
主要试剂和仪器如下：
表52：
试剂名称 货号 厂家
6×DNA Loading buffer TSJ010 擎科生物
20bp DNA Ladder 3420A TAKARA
10000*SolarGelRed核酸染料 E1020 solarbio
8％非变性PAGE凝胶 / 自配
1×TBE Buffer(无RNA酶) / 自配
表53：
Figure PCTCN2019109563-appb-000067
步骤：
①将RNA纳米颗粒按下表54方法采用超纯水进行稀释。
表54：
编号 实测浓度(μg/mL)
R-15 165.937
R-16 131.706
R-17 144.649
R-18 164.743
R-19 126.377
R-20 172.686
R-21 169.455
②取处理后的样品10μL(500ng)与2μL 6×DNA Loading Buffer混匀，冰上操作，做好标记。
③取8％非变性PAGE凝胶，将不同孵育时间的样品上一块胶，将12μL处理的样品全部上样，设置程序100V跑胶40min。
④跑胶结束，进行染色，置于水平摇床30min，拍照成像。
检测结果：
7组延长段变形+核心短序列RNA自组装产物的非变性PAGE胶跑胶结果见图16。图16中泳道1至7从左到右依次为：7组延长段变形+核心短序列RNA自组装产物R-15、R-16、R-17、R-18、R-19、R-20、R-21。
由图16结果可以清楚地看出7组延长段变形+核心短序列RNA自组装产物的条带均明亮清晰，表明7组延长段变形+核心短序列RNA链均完成了自组装，形成了稳定的纳米颗粒结构。
(2)电位测定
测定方法：准备好电位样品(自组装产物溶于超纯水中)放入样品池中，打开仪器的样品池盖，放入仪器；
打开软件，点击菜单MeasUre€ManUal，出现手动测量参数设置对话框；
设置软件检测参数；
然后点击确定完毕设置，出现测量对话框，点击Start开始；
测定结果：7组延长段变形+核心短序列RNA纳米颗粒的25℃电位检测结果如下：
表55：
Figure PCTCN2019109563-appb-000068
表56：
Figure PCTCN2019109563-appb-000069
表57：
Figure PCTCN2019109563-appb-000070
表58：
Figure PCTCN2019109563-appb-000071
表59：
Figure PCTCN2019109563-appb-000072
表60：
Figure PCTCN2019109563-appb-000073
Figure PCTCN2019109563-appb-000074
表61：
Figure PCTCN2019109563-appb-000075
由上述电位检测数据可知：7组延长段变形+核心短序列RNA纳米颗粒均具有良好的稳定性，进一步表明各延长段变形+核心短序列RNA自组装而成的纳米颗粒具有较稳定的自组装结构。
(3)粒径测量
1.准备好电位样品(7组延长段变形+核心短序列RNA)放入样品池中，打开仪器的样品池盖，放入仪器；
2.打开软件，点击菜单，出现手动测量参数设置对话框；
3.设置软件检测参数；
4.然后点击确定完毕设置，出现测量对话框，点击Start开始，7组延长段变形+核心短序列RNA的流体动力学尺寸的DLS测量值结果分别如下：
表62：
编号 平均粒径(nm)
R-15 6.808
R-16 6.978
R-17 7.592
R-18 7.520
R-19 6.936
R-20 7.110
R-21 6.720
(4)TM值检测
采用溶解曲线法，对7组延长段变形+核心短序列RNA纳米颗粒的TM值进行检测，样品与电位样品一致。
试剂和仪器如下：
表63：
试剂名称 货号 厂家
AE buffer / Takara
SYBR Green I染料 / 自配
表64：
名称 型号 生产厂家
Real-Time System CFX Connect Bio-rad
超净工作台 HDL 北京东联哈尔仪器制造有限公司
步骤：
①样品采用超纯水进行稀释后，将5μg稀释所得样品与2μL SYBR Green I染料(1∶200稀释)进行混合，终体积20μL，稀释浓度如下：
表65：
Figure PCTCN2019109563-appb-000076
②室温避光孵育30min；
③上机检测，程序设置为20℃开始，每秒升温0.1℃至95℃，每5s读数一次。
检测结果：
7组延长段变形+核心短序列RNA纳米颗粒的TM值如下，R-15的溶解曲线图见图17，R-16的溶解曲线图见图18，R-17的溶解曲线图见图19，R-18的溶解曲线图见图20，R-19的 溶解曲线图见图21，R-20的溶解曲线图见图22，R-21的溶解曲线图见图23。因RNA样本特殊性，本次检测以20～90℃范围内1/2RFUmax值所对应的温度为样本Tm值。
表66：
  TM值(℃)
R-15 57.5℃
R-16 53.8℃
R-17 55.2℃
R-18 54.5℃
R-19 54.0℃
R-20 59.5℃
R-21 65.0℃
7组延长段变形+核心短序列RNA纳米颗粒的TM值均较高，表明自组装产物具有良好的结构稳定性。
实施例10
一、7组延长段变形+核心短序列DNA纳米颗粒载体：
(1)7组组成延长段变形+核心短序列DNA纳米颗粒的三条多核苷酸碱基序列：
表67：D-8：
Figure PCTCN2019109563-appb-000077
表68：D-9：
Figure PCTCN2019109563-appb-000078
表69：D-10：
Figure PCTCN2019109563-appb-000079
表70：D-11：
Figure PCTCN2019109563-appb-000080
Figure PCTCN2019109563-appb-000081
表71：D-12：
Figure PCTCN2019109563-appb-000082
表72：D-13：
Figure PCTCN2019109563-appb-000083
表73：D-14：
Figure PCTCN2019109563-appb-000084
二、自组装试验步骤：
(1)按1:1:1的摩尔比将DNA单链a，b，c同时混合溶于DEPC水或TMS缓冲液中；
(2)加热混合溶液至95℃，保持5min后以2℃/min的速率缓慢降温到室温；
(3)将产物上样到8％(m/v)非变性PAGE凝胶上并在TBM缓冲液中4℃条件下，以100V电泳纯化复合体；
(4)切下目的条带并在DNA洗脱缓冲液中37℃洗脱，之后乙醇沉淀过夜，减压低温挥干，得到DNA自组装产物；
(5)电泳分析检测与激光扫描观察；
(6)电位检测；
(7)粒径检测；
(8)TM值检测。
三、自组装试验结果
(1)电泳检测
主要试剂和仪器如下：
表74：
试剂名称 货号 厂家
6×DNA Loading buffer TSJ010 擎科生物
20bp DNA Ladder 3420A TAKARA
10000*SolarGelRed核酸染料 E1020 solarbio
8％非变性PAGE凝胶 / 自配
1×TBE Buffer(无RNA酶) / 自配
表75：
Figure PCTCN2019109563-appb-000085
步骤：
①将DNA纳米颗粒按下表76方法采用超纯水进行稀释。
表76：
Figure PCTCN2019109563-appb-000086
②取处理后的样品10μL(500ng)与2μL 6×DNA Loading Buffer混匀，冰上操作，做好标记。
③取8％非变性PAGE凝胶，将不同孵育时间的样品上一块胶，将12μL处理的样品全部上样，设置程序100V跑胶40min。
④跑胶结束，进行染色，置于水平摇床30min，拍照成像。
检测结果：
7组延长段变形+核心短序列DNA自组装产物的非变性PAGE胶跑胶结果见图24。图24中泳道1至7从左到右依次为：7组延长段变形+核心短序列DNA自组装产物D-8、D-9、D-10、D-11、D-12、D-13、D-14。
由图24结果可以清楚地看出7组延长段变形+核心短序列DNA自组装产物的条带均明亮清晰，表明7组延长段变形+核心短序列DNA链均完成了自组装，形成了稳定的纳米颗粒结构。
(2)电位测定
测定方法：准备好电位样品(自组装产物溶于超纯水中)放入样品池中，打开仪器的样品池盖，放入仪器；
打开软件，点击菜单MeasUre€ManUal，出现手动测量参数设置对话框；
设置软件检测参数；
然后点击确定完毕设置，出现测量对话框，点击Start开始；
测定结果：7组延长段变形+核心短序列DNA纳米颗粒的25℃电位检测结果如下：
表77：
Figure PCTCN2019109563-appb-000087
表78：
Figure PCTCN2019109563-appb-000088
表79：
Figure PCTCN2019109563-appb-000089
Figure PCTCN2019109563-appb-000090
表80：
Figure PCTCN2019109563-appb-000091
表81：
Figure PCTCN2019109563-appb-000092
表82：
Figure PCTCN2019109563-appb-000093
表83：
Figure PCTCN2019109563-appb-000094
由上述电位检测数据可知：7组延长段变形+核心短序列DNA纳米颗粒均具有良好的稳定性，进一步表明各延长段变形+核心短序列DNA自组装而成的纳米颗粒具有较稳定的自组装结构。
(3)粒径测量
①准备好电位样品(7组延长段变形+核心短序列DNA)放入样品池中，打开仪器的样品池盖，放入仪器；
②打开软件，点击菜单，出现手动测量参数设置对话框；
③设置软件检测参数；
④然后点击确定完毕设置，出现测量对话框，点击Start开始，7组延长段变形+核心短序列RNA的流体动力学尺寸的DLS测量值结果分别如下：
表84：
编号 平均粒径(nm)
D-8 7.460
D-9 7.920
D-10 7.220
D-11 7.472
D-12 6.968
D-13 7.012
D-14 6.896
(4)TM值检测
采用溶解曲线法，对7组延长段变形+核心短序列DNA纳米颗粒的TM值进行检测，样品与电位样品一致。
试剂和仪器如下：
表85：
试剂名称 货号 厂家
AE buffer / Takara
SYBR Green I染料 / 自配
表86：
名称 型号 生产厂家
Real-Time System CFX Connect Bio-rad
超净工作台 HDL 北京东联哈尔仪器制造有限公司
步骤：
②样品采用超纯水进行稀释后，将5μg稀释所得样品与2μL SYBR Green I染料(1∶200稀释)进行混合，终体积20μL，稀释浓度如下：
表87：
Figure PCTCN2019109563-appb-000095
②室温避光孵育30min；
③上机检测，程序设置为20℃开始，每秒升温0.1℃至95℃，每5s读数一次。
检测结果：
7组延长段变形+核心短序列DNA纳米颗粒的TM值如下，D-8的溶解曲线图见图25，D-9的溶解曲线图见图26，D-10的溶解曲线图见图27，D-11的溶解曲线图见图28，D-12的溶解曲线图见图29，D-13的溶解曲线图见图30，D-14的溶解曲线图见图31。
表88：
编号 TM值(℃)
D-8 48.5
D-9 52.5
D-10 54.5～55.0
D-11 48.7
D-12 51.5
D-13 51.0
D-14 49.2
由图25至图31所示的7组延长段变形+核心短序列DNA纳米颗粒的溶解曲线可以看出，TM值均较高，表明样本纯度较高且自组装结构稳定。
检测核酸纳米颗粒在血清中的稳定性
实施例11
采用非变性PAGE法对7组延长段变形+核心短序列RNA纳米颗粒在血清中的稳定性进行表征。
主要试剂和仪器如下：
表89：
试剂名称 货号 厂家
6×DNA Loading buffer TSJ010 擎科生物
20bp DNA Ladder 3420A TAKARA
10000*SolarGelRed核酸染料 E1020 solarbio
8％非变性PAGE凝胶 / 自配
1×TBE Buffer(无RNA酶) / 自配
血清(FBS) / Excel
RPMI 1640 / GBICO
表90：
Figure PCTCN2019109563-appb-000096
步骤：
①将RNA纳米颗粒配制为下表浓度，然后将配制后的样品按下表中的方法进行稀释，稀释5管，稀释后样品37℃水浴不同时间(0、10min、1h、12h、36h)；
表91：
Figure PCTCN2019109563-appb-000097
Figure PCTCN2019109563-appb-000098
②取处理后的样品10μL与2μL 6×DNA Loading Buffer混匀，冰上操作，做好标记；
③取8％非变性PAGE凝胶，将不同孵育时间的样品上一块胶，将12μL处理的样品全部上样，设置程序100V跑胶40min；
④跑胶结束，进行染色，置于水平摇床缓慢振荡30min，拍照成像。
R-15的电泳检测结果见图32，R-16的电泳检测结果见图33，R-17的电泳检测结果见图34，R-18的电泳检测结果见图35，R-19的电泳检测结果见图36，R-20的电泳检测结果见图37，R-21的电泳检测结果见图38。图32至图38中，从左到右的泳道分别为M：marker；1：36h；2：12h；3：1h；4：10min；5：0min。从血清稳定性试验结果可知：10min、1h、12h和36h的非变性胶结果显示，不同时间RNA纳米颗粒样品条带无明显差别，表明RNA纳米颗粒R-15至R-21在50％FBS的1640培养基中比较稳定，无明显降解。
实施例12
采用非变性PAGE法对7组延长段变形+核心短序列DNA纳米颗粒在血清中的稳定性进行表征。
主要试剂和仪器如下：
表92：
试剂名称 货号 厂家
6×DNA Loading buffer TSJ010 擎科生物
20bp DNA Ladder 3420A TAKARA
10000*SolarGelRed核酸染料 E1020 solarbio
8％非变性PAGE凝胶 / 自配
1×TBE Buffer(无RNA酶) / 自配
血清(FBS) / Excel
RPMI 1640 / GBICO
表93：
Figure PCTCN2019109563-appb-000099
步骤：
②将DNA纳米颗粒配制为下表浓度，然后将配制后的样品按下表中的方法进行稀释，稀释5管，稀释后样品37℃水浴不同时间(0、10min、1h、12h、36h)；
表94：
Figure PCTCN2019109563-appb-000100
②取处理后的样品5μL与1μL 6×DNA Loading Buffer混匀，冰上操作，做好标记；
③取8％非变性PAGE凝胶，将不同孵育时间的样品上一块胶，将6μL处理的样品全部上样，设置程序100V跑胶40min；
④跑胶结束，进行染色，置于水平摇床缓慢振荡30min，拍照成像。
D-8的电泳检测结果见图39，D-9的电泳检测结果见图40，D-10的电泳检测结果见图41，D-11的电泳检测结果见图42，D-12的电泳检测结果见图43，D-13的电泳检测结果见图44，D-14的电泳检测结果见图45。图39至图45中，从左到右的泳道分别为M：marker；1：36h；2：12h；3：1h；4：10min；5：0min。从血清稳定性试验结果可知：10min、1h、12h和36h的非变性胶结果显示，不同时间DNA纳米颗粒样品条带无明显差别，表明DNA纳米颗粒D-8至D-14在50％FBS的1640培养基中比较稳定，无明显降解。
核酸纳米颗粒挂载药物试验
实施例13
阿霉素挂载实验：
根据实施例5的化学法挂载方法(除特殊限定外，方法同实施例5相同)，分别采用前述实施例9中R-15、R-16、R-17、R-18、R-19、R-20和R-21自组装形成的RNA纳米颗粒、实施例10中D-8、D-9、D-10、D-11、D-12、D-13和D-14自组装形成的DNA纳米颗粒作为阿霉素挂载载体，测得阿霉素挂载率分别如下：
RNA纳米颗粒R-15的阿霉素挂载率为20.5；
RNA纳米颗粒R-16的阿霉素挂载率为29.4；
RNA纳米颗粒R-17的阿霉素挂载率为30.9；
RNA纳米颗粒R-18的阿霉素挂载率为34.1；
RNA纳米颗粒R-19的阿霉素挂载率为27.1；
RNA纳米颗粒R-20的阿霉素挂载率为30.2；
RNA纳米颗粒R-21的阿霉素挂载率为20.1；
DNA纳米颗粒D-8的阿霉素挂载率为28.0；
DNA纳米颗粒D-9的阿霉素挂载率为27.9；
DNA纳米颗粒D-10的阿霉素挂载率为18.9；
DNA纳米颗粒D-11的阿霉素挂载率为26.8；
DNA纳米颗粒D-12的阿霉素挂载率为27.6；
DNA纳米颗粒D-13的阿霉素挂载率为31.8；
DNA纳米颗粒D-14的阿霉素挂载率为32。
流式细胞仪(FACS)实验检测DNA纳米颗粒及载体药的细胞结合能力
实施例14
一、细胞信息
HepG2(来源协和细胞库)，培养基为DMEM+10％FBS+1％双抗(gibco，15140-122)，培养条件为37℃，5％CO 2，饱和湿度。
二、待测物
空白载体：前述实施例12中D-8、D-9、D-10、D-11、D-12、D-13和D-14自组装形成的DNA纳米颗粒载体。
载体药：根据实施例5的化学法挂载方法(除特殊限定外，方法同实施例5相同)，采用前述实施例10中D-8、D-9、D-10、D-11、D-12、D-13和D-14自组装形成的DNA纳米颗粒挂载阿霉素，分别记为D-8-阿霉素、D-9-阿霉素、D-10-阿霉素、D-11-阿霉素、D-12-阿霉素、D-13-阿霉素和D-14-阿霉素。
三、主要设备、耗材
表95：
  生产厂家 型号
生物安全柜 北京东联哈尔仪器制造公司 BSC-1360ⅡA2
低速离心机 中科中佳仪器有限公司 SC-3612
CO 2培养箱 Thermo 3111
倒置显微镜 UOP DSZ2000X
流式细胞仪 BD BD FACSCalibur TM
四、主要试剂
表96：
试剂名称 生产厂家 货号 备注
DMEM(无生物素) 百药智达提供 YS3160  
1％BSA-PBS 自配 －  
五、实验方法：
1.调整细胞状态到对数生长期，更换培养基为无生物素无叶酸的培养基，置于37℃培养箱中孵育过夜；
2.孵育结束后，胰酶消化收集细胞悬液，1000rmp离心5min，调整浓度后，取2×10 5-5×10 5细胞/EP管，用1mL/管1％BSA-PBS洗2次，观察管底细胞，以防被吸走。
3.溶解待测物，稀释待测物到使用浓度；
4.将细胞上清液吸净，每管按顺序加入100μL相应样品，避光，37℃孵育2h；
5.用1％BSA-PBS洗2次；1000rmp离心5min；
6.最后用300μL PBS重悬细胞沉淀，流式上机检测(本实施例所用的空白载体是由Quasar 670标记的，而载体药中的阿霉素自带荧光，因此可以分别通过FL4-APC和FL2-PE进行检测)；
7.数据分析。
六、实验结果
1.实验结果见下表：
表97：
Figure PCTCN2019109563-appb-000101
Figure PCTCN2019109563-appb-000102
2.结论
1.HepG2细胞与D-8-阿霉素(载体药)及D-8(空白载体)孵育后，均有很高(93.1％～98.4％)的结合率。
2.HepG2细胞与D-9-阿霉素(载体药)及D-9(空白载体)孵育后，均有很高(88.6％～98.1％)的结合率。
3.HepG2细胞与D-10-阿霉素(载体药)及D-10(空白载体)孵育后，均有很高(89.4％～98.3％)的结合率。
4.HepG2细胞与D-11-阿霉素(载体药)及D-11(空白载体)孵育后，均有很高(89.3％～97.8％)的结合率。
5.HepG2细胞与D-12-阿霉素(载体药)及D-12(空白载体)孵育后，均有很高(94.6％～97.1％)的结合率。
6.HepG2细胞与D-13-阿霉素(载体药)及D-13(空白载体)孵育后，均有很高(89.6％～98.2％)的结合率。
7.HepG2细胞与D-14-阿霉素(载体药)及D-14(空白载体)孵育后，均有很高(90.3％～98.3％)的结合率。
研究DNA纳米颗粒及载体药在HepG2细胞中的细胞毒性
实施例15
采用CCK8法检测DNA纳米颗粒及载体药对HepG2的毒性。
一、主要试剂
表98：
试剂名称 厂家 货号
PBS － －
DMSO SIGMA D2650
DMEM(无生物素) 百药智达提供 YS3160
FBS Excell Bio FSP500
双抗 gibco 15140-122
胰酶 gibco 25200-056
CCK8试剂盒 碧云天 C0038
二、主要耗材和仪器
表99：
名称 生产厂家 型号
96孔细胞培养板 NEST 701001
生物安全柜 北京东联哈尔仪器制造公司 BSC-1360ⅡA2
低速离心机 中科中佳仪器有限公司 SC-3612
CO 2培养箱 Thermo 3111
倒置显微镜 UOP DSZ2000X
酶标仪 上海欧颖实验设备有限公司 K3
三、细胞信息
HepG2(来源协和细胞库)，培养基为DMEM+10％FBS+1％双抗(gibco，15140-122)，培养条件为37℃，5％CO 2，饱和湿度。
四、实验材料
1.待测样品
空白载体：前述实施例10中D-8、D-9、D-10、D-11、D-12、D-13和D-14自组装形成的DNA纳米颗粒载体，分别记作：D-8、D-9、D-10、D-11、D-12、D-13和D-14。
载体药：根据实施例5的化学法挂载方法(除特殊限定外，方法同实施例5相同)，采用前述实施例10中D-8、D-9、D-10、D-11、D-12、D-13和D-14自组装形成的DNA纳米颗粒挂载阿霉素，分别记为D-8-阿霉素、D-9-阿霉素、D-10-阿霉素、D-11-阿霉素、D-12-阿霉素、D-13-阿霉素和D-14-阿霉素。
原药阿霉素。
DMSO。
五、实验步骤
1.取对数生长期的HepG2细胞，使用台盼蓝染色计数细胞活率为98.3％，以5000个Cell/孔进行铺板，体积为100μL/孔，铺8个96孔板，每板57个孔，37℃孵育过夜。
2.按照下表稀释待测样品并加入：去除原有培养基，加入100μL不同浓度待测样品的培养基，每组3个复孔。
表100：
孔号 C9 C8 C7 C6 C5 C4 C3 C2 C1
挂载药终浓度 10μM 3.16μM 1μM 316nM 100nM 31.6nM 10nM 3.16nM 1nM
空载体终浓度 1μM 316nM 100nM 31.6nM 10nM 3.16nM 1nM 0.316nM 0.1nM
原药阿霉素终浓度 10μM 3.16μM 1μM 316nM 100nM 31.6nM 10nM 3.16nM 1nM
DMSO(％) 0.1 0.0316 0.01 0.00316 0.001 0.00036 0.0001 0.000036 0.00001
在本实施例中，挂载药和空白载体分别先用PBS配制成100μM的原液，再用完全培养基(无生物素DMEM)进行稀释。原药阿霉素先用DMSO配制成100μM的原液，再用完全培养基(无生物素DMEM)进行稀释。DMSO直接用完全培养基(无生物素DMEM)进行稀释。
3.加待测样品后将96孔板放入37℃5％CO 2培养箱中孵育72h。
4.将试剂盒取出室温融化，每孔加入10μL CCK-8溶液，也可将CCK8溶液与培养基以1:9混合，然后以100μL/孔的量加入孔中。
5.在细胞培养箱内继续孵育4h，时间的长短根据细胞的类型和细胞的密度等实验情况而定。
6.用酶标仪在450nm处测定吸光度。
7.计算：细胞活力(％)＝(OD实验组-OD空白组)×100％/(OD对照组-OD空白组)，由GraphPad Prism 5.0计算得到IC 50。
六、实验结果
表101：
Figure PCTCN2019109563-appb-000103
Figure PCTCN2019109563-appb-000104
结论：
从上表和图46a、图46b、图46c、图46d、图46e、图46f、图46g、图46h中可以看出，原药阿霉素及挂载药D-8-阿霉素、D-9-阿霉素、D-10-阿霉素、D-11-阿霉素、D-12-阿霉素、D-13-阿霉素和D-14-阿霉素作用于HepG2细胞的IC 50分别为0.2725μM、0.05087μM、0.0386、0.03955、0.04271、0.02294、0.03017和0.03458；DMSO作用于HepG2细胞的IC 50为>0.1％；D-8(空白载体)、D-9(空白载体)、D-10(空白载体)、D-11(空白载体)、D-12(空白载体)、D-13(空白载体)和D-14(空白载体)作用于HepG2细胞的IC 50均>1μM。说明针对HepG2细胞系而言，相比单纯的空白载体D-8、D-9、D-10、D-11、D-12、D-13和D-14，小分子药物原药阿霉素及挂载药D-8-阿霉素、D-9-阿霉素、D-10-阿霉素、D-11-阿霉素、D-12-阿霉素、D-13-阿霉素和D-14-阿霉素都对HepG2细胞有毒性，且挂载药D-8-阿霉素、D-9-阿霉素、D-10-阿霉素、D-11-阿霉素、D-12-阿霉素、D-13-阿霉素和D-14-阿霉素相对于原药阿霉素有明显的增效作用。
实施例16
根据实施例5的化学法挂载方法(除特殊限定外，方法同实施例5相同)，采用前述实施例10中D-10和D-14自组装形成的DNA纳米颗粒作为柔红霉素挂载载体。利用酶标仪测量492nm处柔红霉素的吸光度，绘制标准曲线(如图47所示)。
测得柔红霉素挂载率分别如下：
DNA纳米颗粒D-10的柔红霉素挂载率为24.0；
DNA纳米颗粒D-14的柔红霉素挂载率为25.1。
实施例17
表柔比星挂载实验
(一)RNA纳米颗粒的挂载
根据实施例5的化学法挂载方法(除特殊限定外，方法同实施例5相同，相同摩尔数的表柔比星作为挂载的用量)，采用核酸纳米颗粒(分子量为29550，与实施例1中的RNA纳米颗粒类似，不同之处在于c链上的荧光标记为Cy5)作为载体，分别挂载：表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨。
其中，绘制标准曲线时，分别测量上述药物在酶标仪上的吸光度。表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨分别检测如下波长下的吸光度：492nm、303nm、492nm、492nm、265nm、384nm、225nm及268nm。对应得到的标准曲线分别见图48a、图49、图50a、图51、图52、图53、图54a及图55。
测得各挂载率分别如下：
表柔比星：
C RNAh-1＝21.0ug/ml，M RNAh≈30000，100ul；C 表柔比星-1＝7.158uM，100ul；
C RNAh-2＝33.5ug/ml，M RNAh≈30000，100ul；C 表柔比星-2＝9.263uM，100ul；
Figure PCTCN2019109563-appb-000105
取其平均值得到表柔比星-RNAh核酸纳米颗粒的挂载率约为9.3，表示每一个核酸纳米颗粒载体上能够挂载约9.3个表柔比星分子。
甲氨蝶呤：
C RNAh-1＝45.0ug/ul，M RNAh≈30000，100ul；C 甲氨蝶呤-1＝16.9uM，100ul；
C RNAh-2＝36.0ug/ul，M RNAh≈30000，100ul；C 甲氨蝶呤-2＝10.85uM，100ul；
Figure PCTCN2019109563-appb-000106
取其平均值得到甲氨蝶呤-RNAh的挂载率约为10，及表示每一个核酸纳米颗粒载体上能够挂载约10个甲氨蝶呤分子。
吡柔比星：
C RNAh-1＝23.2ug/ml，M RNAh≈30000，100ul；C 吡柔比星-1＝8.500uM，100ul；
C RNAh-2＝48.1ug/ml，M RNAh≈30000，100ul；C 吡柔比星-2＝19.24uM，100ul；
Figure PCTCN2019109563-appb-000107
取其平均值得到吡柔比星-RNAh的挂载率约为11.5，每一个核酸纳米颗粒载体上能够挂载约11.5吡柔比星分子。
柔红霉素：
C RNAh-1＝58.8ug/ml，M RNAh≈30000，100ul；C 柔红霉素-1＝11.76uM，100ul；
C RNAh-2＝39.8ug/ml，M RNAh≈30000，100ul；C 柔红霉素-2＝7.506uM，100ul；
Figure PCTCN2019109563-appb-000108
取其平均值得到柔红霉素-RNAh的挂载率约为6，表示每一个核酸纳米颗粒载体上能够挂载约6个柔红霉素分子。
五氟尿嘧啶：
计算得到RNAh-五氟尿嘧啶的挂载率约为0.31，及表示每一个核酸纳米颗粒载体上能够挂载约0.31个五氟尿嘧啶分子。
通过改变五氟尿嘧啶和RNA纳米颗粒的相对用量，也可以获得挂载率为10、20、28、50等的RNAh-五氟尿嘧啶颗粒，在此不再赘述。
10-羟基喜树碱：
C RNAh-1＝73.3ug/ml，M RNAh≈30000，100ul；C 10羟基喜树碱-1＝28.88uM，100ul；
C RNAh-2＝65.8ug/ml，M RNAh≈30000，100ul；C 10羟基喜树碱-2＝15.2uM，100ul；
Figure PCTCN2019109563-appb-000109
取其平均值得到10-羟基喜树碱-RNAh的挂载率约为9，即表示每一个核酸纳米颗粒载体上能够挂载约16个10-羟基喜树碱分子。
通过改变10-羟基喜树碱和RNA纳米颗粒的相对用量，也可以获得挂载率为10、20、28、50、70、80、100、200等的RNAh-10-羟基喜树碱颗粒，在此不再赘述。
阿司匹林：
C RNAh-1＝68.4μg/ml，M RNAh≈30000，100μl；C 阿司匹林-1＝52.5μM，100μl；
C RNAh-2＝26.8μg/ml，M RNAh≈30000，100μl；C 阿司匹林-2＝18.4μM，100μl；
Figure PCTCN2019109563-appb-000110
取N-1和N-2的平均值得到阿司匹林-RNAh的挂载率约为22，及表示每一个核酸纳米颗粒载体上能够挂载约22个阿司匹林分子。
吉西他滨：
C RNAh-1＝26.9ug/ml，M RNAh≈30000，100ul；C 吉西他滨-1＝18.23uM，100ul；
C RNAh-2＝29.8ug/ul，M RNAh≈30000，100ul；C 吉西他滨-2＝21.65uM，100ul；
Figure PCTCN2019109563-appb-000111
取N-1和N-2的平均值得到吉西他滨-RNAh的挂载率约为21，及表示每一个核酸纳米颗粒载体上能够挂载约21个吉西他滨分子。
(二)DNA核酸纳米颗粒的挂载实验
挂载方法及挂载率的计算方式同上述RNA核酸纳米颗粒，具体所使用的核酸纳米颗粒为：DNAh-Bio-EFGRapt-Cy5，其中，DNAh的三条链分别为：
a链：(SEQ ID NO:172:)5’-CGCGCGCCCACGAGCGTTCCGGGCGCGCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC-3’；5’端前三个及3’的后三个碱基分别进行硫代修饰，5’端连接Biotin(生物素)，黑体加粗部分为EGFRapt序列；
b链(SEQ ID NO:173:)：5’-GCGCCCGGTTCGCCGCCAGCCGCCGC-3’，5’端前三个及3’的后三个碱基分别进行硫代修饰；
c链(SEQ ID NO:174:)：5’-GCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCG-3’；5’端前三个及3’的后三个碱基分别进行硫代修饰，3’端连接Cy5荧光标记。
上述DNA核酸纳米颗粒挂载表柔比星的标准曲线见图48b，具体计算过程如下：
C DNAh-1＝22.19ug/ml，M DNAh≈39500，100ul；C 表柔比星-1＝17.06uM，100ul；
C DNAh-2＝32.57ug/ml，M DNAh≈39500，100ul；C 表柔比星-2＝20.40uM，100ul；
Figure PCTCN2019109563-appb-000112
取其平均值得到表柔比星-DNAh纳米颗粒的挂载率约为27.6，表示每一个DNA米颗粒载体上能够挂载约27.6个表柔比星。
上述DNA核酸纳米颗粒挂载吡柔比星的标准曲线见图50b，具体计算过程如下：
C DNAh-1＝18.64ug/ml，M DNAh≈39500，100ul；C 吡柔比星-1＝11.7uM，100ul；
C DNAh-2＝41.23ug/ml，M DNAh≈39500，100ul；C 吡柔比星-2＝19.73uM，100ul；
Figure PCTCN2019109563-appb-000113
取其平均值得到吡柔比星-DNAh纳米颗粒的挂载率约为21.9，表示每一个DNA米颗粒载体上能够挂载约21.9个吡柔比星。
上述DNA核酸纳米颗粒挂载阿司匹林的标准曲线见图54b，具体计算过程如下：
C DNAh-1＝10.97μg/ml，M DNAh≈39500，100μl；C 阿司匹林-1＝3.7μM，100μl；
C DNAh-2＝21.56μg/ml，M DNAh≈39500，100μl；C 阿司匹林-2＝7.67μM，100μl；
Figure PCTCN2019109563-appb-000114
取其平均值得到阿司匹林-DNAh纳米颗粒的挂载率约为14，表示每一个DNA米颗粒载体上能够挂载约14个阿司匹林。
此外，在上述RNA纳米颗粒及DNA纳米颗粒挂载表柔比星等上述各药物的基础上，可进一步按照与挂载表柔比星一样的方法进行第二次挂载其他小分子药物，比如，本申请还进一步挂载了叶酸，得到共同挂载了表柔比星和叶酸两种小分子药物的RNA纳米颗粒及DNA纳米颗粒，两种药物的挂载率可以参照上述方法进行检测得到(数值未显示)。
该实施例表明，带有延长片段、靶头和荧光素的RNA纳米颗粒(实施例1中的)和DNA纳米颗粒均具有挂载药物的功能，小分子药物表柔比星可以通过共价连接(多聚甲醛—溶剂共价)的方式来实现挂载，而且还可以与其他小分子药物实现共同挂载。
实施例18
共聚焦显微镜实验检测挂载药物的RNA纳米颗粒与细胞的结合能力
一、实验材料和实验方法：
1.待测样品见表102：
表102：
Figure PCTCN2019109563-appb-000115
Figure PCTCN2019109563-appb-000116
注：表中RNAh-Bio-670作为对照，其指的是按照实施例1中自组装方法制备的在a链和b链的5’端进行Biotin修饰，而在c链的3’端进行quasar670荧光素修饰而形成的纳米颗粒，而RNAh-Bio-670-EPB等指的是进一步挂载表柔比星等药物后(按照实施例5中化学法挂载)形成的纳米颗粒。
2.所用到的实验试剂及其来源如下：
RPMI-1640培养基(Gibco,C11875500BT-500mL)；胎牛血清(Fetal bovine serum，FBS)(ExCell Bio,FNA500-500mL)；盘尼西林/链霉素(Penicillin/Streptomycin，PS)(Gibco,15140-122-100mL)；PBS缓冲液(Gibco,C20012500BT-500mL)；Trypsin-EDTA(Stemcell,07901-500mL)；DMSO(Sigma,D5879-1L)；Prolong Gold Antifade Mountant防猝灭剂(Thermo,P36941-2mL)；DAPI(Yeasen,36308ES11-4mL)。
3.所用到的实验仪器如下：
倒置显微镜(Inverted Microscope)(Olympus BX53,U-RFL-T)；BD Falcon(Corning,354118)；细胞离心涂片机(Cytospin)(TXD3)。
4.实验方法:
(1)将各药物所采用的细胞分别置于RPMI1640+10％FBS+1％PS培养基中，于37℃和5％CO 2条件下培养；
表柔比星：HL60细胞，急性白血病细胞系；
甲氨蝶呤：MOLT4细胞(人急性淋巴母细胞白血病系)
吡柔比星：MCF-7细胞(人乳腺癌细胞系)
柔红霉素：MCF-7细胞
五氟尿嘧啶：HepG2细胞；
10-羟基喜树碱：SGC7901；
阿司匹林：SH-SY5Y细胞；
吉西他滨：BxPC-3细胞。
(2)收集细胞，用PBS洗一遍，以每孔1x10 5个细胞加到48孔板中。
(3)将细胞与200nM和400nM的RNAh-Bio-670和RNAh-Bio-670-EPB纳米颗粒一起于37℃和5％CO 2中孵育1～2h和4h。
(4)细胞用PBS洗涤后，通过离心图片法将细胞加到载玻片上，防猝灭剂(Prolong Gold Antifade Mountant)处理，室温过夜。
(5)再用DAPI染色室温5min，然后封片。
(6)用激光扫描共聚焦显微镜的DAPI和FAM通道评估细胞结合和内化，显微镜下拍照，保存。
二、实验结果
实验结果见图56至图63。从图56中可以看出，RNAh-Bio-670和RNAh-Bio-670-EPB纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。可见，含表柔比星的药物RNAh-Bio-670-EPB纳米颗粒与HL60细胞结合和内化能力较强。
从图57中可以看出，RNAh-Bio-670和RNAh-Bio-670-MTX纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。此结果表明，含甲氨蝶呤的药物RNAh-Bio-670-MTX纳米颗粒与MOLT4细胞结合和内化能力较强。
从图58中可以看出，细胞结合和内化实验结果表明，RNAh-Bio-670和RNAh-Bio-670-THP纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。此结果表明，含吡柔比星的药物RNAh-Bio-670-THP纳米颗粒与MCF-7细胞结合和内化能力较强。
从图59中可以看出，细胞结合和内化实验结果表明，RNAh-Bio-670和RNAh-Bio-670-DNR纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。此结果表明，含柔红霉素的药物RNAh-Bio-670-DNR纳米颗粒与MCF-7细胞结合和内化能力较强。
从图60中可以看出，细胞结合和内化实验结果表明，RNAh-Biotin-quasar670和RNAh-Biotin-quasar670-flu纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。
从图61中可以看出，细胞结合和内化实验结果表明，RNAh-Biotin-quasar670和RNAh-Biotin-quasar670-hdcp纳米颗粒因均携带靶头---生物素(Biotin)，因而都能够与细胞结合与内化。
从图62和图63的细胞结合和内化实验结果可以看出，阿司匹林-RNA纳米颗粒与SH-SY5Y细胞结合内化较强。吉西他滨RNA纳米颗粒与BxPC-3细胞结合内化较强。
实施例19
(一)流式细胞实验检测DNAh-Bio-EGFRapt-Cy5-EPB纳米颗粒与细胞的结合能力
一、细胞信息
MCF-7(来源ATCC，货号HTB-22)，NCI-H1975(来源ATCC，货号CRL-5908)；培养基为MEM+10％FBS，培养条件为37℃，5％CO 2，饱和湿度。
二、待测物
靶向药物：DNAh-Bio-EGFRapt-Cy5-EPB(按照实施例5中的DNA纳米颗粒的挂载方式进行挂载)。
靶向荧光载体：DNAh-Bio-EGFRapt-Cy5。
三、设备、耗材
表103
Figure PCTCN2019109563-appb-000117
四、试剂
表104：
Figure PCTCN2019109563-appb-000118
五、实验方法：
1.调整细胞状态到对数生长期，更换培养基为无生物素无叶酸的培养基，置于37℃培养箱中孵育过夜；
2.溶解待测物，配置待测物储液；
3.消化收集单细胞悬液并计数，调整细胞密度到2 X10 5/mL，种植1mL/孔到24孔板中；
4.分别将待测物加入相应的细胞孔中，终浓度2μM，震荡混匀；
5.将细胞板置于37℃培养箱中孵育2小时；
6.孵育结束后，胰酶消化收集细胞悬液；
7.离心收集细胞沉淀，并用PBS清洗两次；
8.最后用300μL PBS重悬细胞沉淀，流式上机检测；
9.荧光载体或表柔比星检测通道：激发光波长：488nm，发射光通道：560nm；
10.数据分析。
六、实验结果
表105：
Figure PCTCN2019109563-appb-000119
从上表可以看出，表柔比星靶向药DNAh-Bio-EFGRapt-Cy5-EPB能够与MCF-7细胞和NCI-H1975细胞结合，且结合率都近乎百分之百；而靶向荧光载体DNAh-Bio-Cy5同样能够与MCF-7细胞结合和NCI-H1975细胞，结合率也是百分之百。
(二)流式细胞仪检测RNAh-Biotin-Cy5-DNR纳米颗粒的细胞结合能力
一、待测样品
靶向药：RNAh-Biotin-Cy5-DNR，其中，RNAh-Biotin-Cy5的制备方法同RNAh-Biotin-quasar670，不同之处仅在于荧光物质由quasar670替换为Cy5。 RNAh-Biotin-Cy5-DNR是RNAh-Biotin-Cy5进一步挂载DNR后形成的纳米颗粒(按照实施例5中的方法挂载)。
二、实验细胞及培养条件(MCF-7细胞，具体同前述，此处不再赘述)
三、荧光检测
荧光检测的条件如下:
激发光640nm，发射光675nm，检测高度为7mm，测量值/数据点＝10，检测速度：正常，延长：100ms。
四、检测结果
表106：
Figure PCTCN2019109563-appb-000120
从上表可以看出，RNAh-Biotin-Cy5-DNR纳米颗粒与MCF-7细胞在处理时间和浓度合适的情况下，结合率可高达84％以上，相比仅含培养基的空白对照，RNA载药颗粒与MCF-7细胞的结合和内化的能力较强。
(三)流式细胞仪检测吡柔比星载药DNA纳米颗粒的细胞结合能力
一、细胞信息
表107：
Figure PCTCN2019109563-appb-000121
二、待测样品
吡柔比星靶向药物：DNAh-Biotin-EGFRapt-Cy5-THP；(按照实施例5中的DNA纳米颗粒的挂载方式进行挂载)。
靶向荧光载体：DNAh-Bio-EGFRapt-Cy5。
三、仪器、设备及相关试剂信息(同前述)
四、实验方法：
1)调整细胞状态到对数生长期，更换培养基为无生物素无叶酸的培养基，置于37℃培养箱中孵育过夜；
2)溶解待测物，配置待测物储液；
3)消化收集单细胞悬液并计数，调整细胞密度到2X10 5/mL，种植1mL/孔到24孔板中；
4)分别将待测物加入相应的细胞孔中，终浓度2μM，震荡混匀；
5)将细胞板置于37℃培养箱中孵育2小时；
6)孵育结束后，胰酶消化收集细胞悬液；
7)离心收集细胞沉淀，并用PBS清洗两次；
8)最后用300μL PBS重悬细胞沉淀，流式上机检测；其中，荧光载体及吡柔比星靶向药的检测通道：激发光波长：488nm，发射光通道：560nm；
9)数据分析。分析结果见下表。
表108：
Figure PCTCN2019109563-appb-000122
从上表可以看出，携带靶头和小分子药物吡柔比星的DNA核酸纳米颗粒与细胞的结合率很高，明显可以看出能够与相应的肿瘤细胞系细胞结合内化。而且，从上表还可以看出，DNAh-Bio-EFGRapt-Cy5-THP不仅与人胃癌细胞系SGC-7901细胞能够高效结合内化，而且能够与人卵巢癌细胞系SKOV3细胞进行结合内化。可见，吡柔比星靶向药DNAh-Bio-EFGRapt-Cy5-THP既具有治疗胃癌的应用前景，又具有治疗卵巢癌的应用前景。
(四)流式细胞仪检测RNAh-Biotin-Cy5-THP纳米颗粒的细胞结合能力
一、待测样品
靶向药：RNAh-Biotin-Cy5-THP，其中，RNAh-Biotin-Cy5的制备方法同RNAh-Biotin-quasar670，不同之处仅在于荧光物质由quasar670替换为Cy5。RNAh-Biotin-Cy5-THP是RNAh-Biotin-Cy5进一步挂载THP后形成的纳米颗粒(按照实施例5中的方法挂载)。
二、实验细胞及培养条件(MCF-7细胞，具体同前述实施例6共聚焦显微镜实验，此处不再赘述)
三、荧光检测
荧光检测的条件如下:
激发光640nm，发射光675nm，检测高度为7mm，测量值/数据点＝10，检测速度：正常，延长：100ms。
四、检测结果
表109：
Figure PCTCN2019109563-appb-000123
从上表可以看出，RNAh-Biotin-Cy5-THP纳米颗粒与MCF-7细胞的结合率高达96％以上，相比仅含培养基的空白对照，RNA载药颗粒与MCF-7细胞的结合和内化的能力较强。
(五)流式细胞仪检测RNAh-Biotin-Cy5-吉西他纳米颗粒与细胞的结合能力
一、待测样品
靶向药：RNAh-Biotin-Cy5-吉西他滨，其中，RNAh-Biotin-Cy5的制备方法同RNAh-Biotin-quasar670，不同之处仅在于荧光物质由quasar670替换为Cy5。RNAh-Biotin-Cy5-吉西他滨是RNAh-Biotin-Cy5进一步挂载吉西他滨后形成的纳米颗粒(按照实施例5中的方法挂载)。
二、实验细胞及培养条件(BxPC-3细胞，同前述)
三、荧光检测
荧光检测的条件如下:
激发光640nm，发射光675nm，检测高度为7mm，测量值/数据点＝10，检测速度：正常，延长：100ms。
四、检测结果
表110：
Figure PCTCN2019109563-appb-000124
Figure PCTCN2019109563-appb-000125
从上表可以看出，RNAh-Biotin-Cy5-吉西他纳米颗粒与BxPC-3细胞在处理时间和浓度合适的情况下，结合率可高达98％以上，相比仅含培养基的空白对照，RNA载药颗粒与BxPC-3细胞的结合和内化的能力较强。
实施例20
检测纳米颗粒在血清中的稳定性
(一)RNAh-Bio-670-EPB纳米颗粒在血清中的稳定性
一、实验材料和实验方法
1.待测样品：溶解在PBS溶液中的RNAh-Bio-670-EPB纳米颗粒；
2.实验试剂：
RPMI-1640培养基(Gibco,C11875500BT-500mL)；胎牛血清(Fetal bovine serum，FBS)(ExCell Bio,FNA500-500mL)；盘尼西林/链霉素(Penicillin/Streptomycin，PS)(Gibco,15140-122-100mL)；PBS缓冲液(Gibco,C20012500BT-500mL)；Novex TMTris-Glycine Native Sample Buffer(2X)(Invitrogen,LC2673-20mL)；Novex TM8％Tris-Glycine Mini Gels(Invitrogen,XP00080BOX-1.0mm)；Tris-Glycine Native Running buffer(10x)(Life science,LC2672-500mL)；G250染色液(Beyotime,P0017-250mL)。
3.实验仪器：
分光光度计(Spectrophotometer)(Thermo,ND2000C)；Mini Gel Tank(Invitrogen,PS0301)；成像系统(Imaging System)(Bio-Rad,ChemiDoc MP)。
4.实验方法：
(1)取10μM的RNAh-Bio-670-EPB纳米颗粒10μL置于90μL含10％血清的RPMI 1640培养基中孵育。
(2)在37℃孵育10min、1h、12h、36h后分别取样。
(3)采用NanoDrop定量后，取200ng的RNA纳米颗粒，加入相同体积的Tris-Glycine SDS样品缓冲液(2X)，充分混匀。
(4)取一块Novex TM8％Tris-Glycine Mini gel，按照顺序上样，设置程序200V，30min，开始电泳。
(5)电泳结束，进行G250染色，置于水平摇床30min，拍照成像。
二、实验结果
表111：定量结果及上样体积
Figure PCTCN2019109563-appb-000126
电泳检测结果见图64和图65。其中，图64示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图65示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Bio-670-EPB纳米颗粒样品条带无明显差别，表明其在10％FBS的1640培养基中比较稳定，无明显降解。
(二)DNAh-Bio-EGFRapt-Cy5-EPB纳米颗粒在血清中的稳定性
一、实验材料、试剂及设备
1.实验材料
DNAh-Bio-EGFRapt-Cy5-EPB纳米颗粒。
2.实验试剂
6×DNA上样缓冲液(TSJ010，擎科生物)，100bp DNA分子标记(TSJ010，擎科生物)；10000*SolarGelRed核酸染料(E1020，solarbio)；8％非变性聚丙烯酰胺凝胶(自配)；1×TBE Buffer(无RNA酶)(自配)；血清(FBS)(Excel)；RPMI 1640(GBICO)。
电泳仪(PowerPac Basic，Bio-rad)，垂直电泳槽(Mini PROTEAN Tetra Cell，Bio-rad)，脱色摇床(TS-3D，orbital shaker)，凝胶成像仪(Tanon 3500,Tanon)。
二、实验方法
(1)将DNAh-Bio-EGFRapt-Cy5-EPB纳米颗粒取6μL，用含10％血清的RPMI 1640培养基6μL进行稀释，稀释后浓度达900μg/ml，分别稀释5管，稀释后样品37℃水浴不同时间(0、10min、1h、12h、36h)。
(2)取处理后的样品与6×DNA Loading Buffer混匀，冰上操作，做好标记。
(3)取8％Native PAGE，将不同孵育时间的纳米颗粒样品上一块胶，上样量20μL/孔/样，设置程序90-100V电泳50min。
(4)电泳结束，进行染色，置于水平摇床30min，拍照成像。
三、实验结果
非变性PAGE胶电泳结果见图66，其中，1代表0min，2代表10min,3代表1h，4代表12h，5代表36h。DNAh-Bio-EGFRapt-Cy5-EPB纳米颗粒的目的条带在200bp左右，从图66上可以看出，表柔比星-DNAh纳米颗粒在37℃孵育至36h基本稳定。
(三)检测挂载于核酸纳米颗粒上的含甲氨蝶呤的药物在血清中的稳定性
一、除待测样品为：RNAh-Bio-670-MTX纳米颗粒外，其余同(一)。
二、实验结果
表112：定量结果及上样体积
Figure PCTCN2019109563-appb-000127
电泳检测结果见图67和图68。其中，图67示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图68示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Bio-670-MTX纳米颗粒样品条带无明显差别，表明其在10％FBS的1640培养基中比较稳定，无明显降解。
(四)检测靶向药RNAh-Bio-670-THP纳米颗粒在血清中的稳定性
一、除待测样品为：RNAh-Bio-670-THP纳米颗粒外，其余同(一)。
二、实验结果
表113：定量结果及上样体积
Figure PCTCN2019109563-appb-000128
电泳检测结果见图69和图70。其中，图69示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图70示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Bio-670-THP纳米颗粒样品条带无明显差别，表明其在10％FBS的1640培养基中比较稳定，无明显降解。
(五)检测DNAh-Bio-EGFRapt-Cy5-THP纳米颗粒血清稳定性
一、除待测样品为：DNAh-Bio-EGFRapt-Cy5-THP，浓度为1.8mg/ml外，其余同(一)。
二、实验结果
非变性PAGE胶电泳结果见图71。其中，1代表0min，2代表10min,3代表1h，4代表12h，5代表36h。DNAh-Bio-EGFRapt-Cy5-THP纳米颗粒的目的条带在200bp左右，从图71上可以看出，DNAh-Bio-EGFRapt-Cy5-THP纳米颗粒在37℃孵育至36h基本稳定。
(六)检测挂载于核酸纳米颗粒上的含柔红霉素的药物在血清中的稳定性
一、除待测样品为：RNAh-Bio-670-DNR纳米颗粒外，其余同(一)。
二、实验结果
表114：定量结果及上样体积
Figure PCTCN2019109563-appb-000129
电泳检测结果见图72和图73。其中，图72示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图73示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Bio-670-DNR纳米颗粒样品条带无明显差别，表明其在10％FBS的1640培养基中比较稳定，无明显降解。
(七)检测挂载于核酸纳米颗粒上的含五氟尿嘧啶的药物在血清中的稳定性
一、除待测样品为：RNAh-Biotin-quasar670-flu纳米颗粒外，其余同(一)。
二、实验结果
表115：定量结果及上样体积。
Figure PCTCN2019109563-appb-000130
Figure PCTCN2019109563-appb-000131
电泳检测结果见图74和图75。其中，图74示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图75示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Biotin-quasar670-flu纳米颗粒样品条带无明显差别，表明RNAh-Biotin-quasar670-flu纳米颗粒在10％FBS的1640培养基中比较稳定，无明显降解。
(八)检测挂载于核酸纳米颗粒上的含10-羟基喜树碱的药物在血清中的稳定性
一、实验材料和实验方法
1.待测样品：实施例5中制备的RNAh-Biotin-quasar670-hdcp纳米颗粒。
2.实验试剂：同(一)
3.实验仪器：同(一)
4.实验方法：同(一)
二、实验结果
表116：定量结果及上样体积。
Figure PCTCN2019109563-appb-000132
电泳检测结果见图76和图77。其中，图76示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图77示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Biotin-quasar670-hdcp纳米颗粒样品条带无明显差别，表明RNAh-Biotin-quasar670-hdcp纳米颗粒在10％FBS的1640培养基中比较稳定，无明显降解。
(九)检测挂载于核酸纳米颗粒上的含阿司匹林的药物在血清中的稳定性
一、除待测样品为：RNAh-Biotin-quasar670-阿司匹林纳米颗粒外，其余同(一)
二、实验结果
表117：定量结果及上样体积。
Figure PCTCN2019109563-appb-000133
电泳检测结果见图78和图79。其中，图78示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图79示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Biotin-quasar670-阿司匹林纳米颗粒样品条带无明显差别，表明RNAh-Biotin-quasar670-阿司匹林纳米颗粒在10％FBS的1640培养基中比较稳定，无明显降解。
(十)检测挂载于核酸纳米颗粒上的含吉西他滨的药物在血清中的稳定性
一、除待测样品为：RNAh-Bio-670-吉西他滨纳米颗粒外，其余同(一)。
二、实验结果
表118：定量结果及上样体积
Figure PCTCN2019109563-appb-000134
电泳检测结果见图80和图81。其中，图80示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图81示出了8％非变性胶的电泳结果(Stain Free Gel程序)。血清稳定性试验结果显示：0min、10min、1h、12h和36h，不同时间长度下，RNAh-Bio-670-吉西他滨纳米颗粒样品条带无明显差别，表明其在10％FBS的1640培养基中比较稳定，无明显降解。
实施例21研究载药纳米颗粒对细胞的毒性
(一)研究RNAh-Bio-670-EPB纳米颗粒在HL60细胞中的细胞毒性
一、实验材料和实验方法
1.实验材料：
待测样品：小分子药物EPB及RNAh-Bio-670-EPB纳米颗粒；
药物浓度配制：
准备现配试剂到对应容积容器中，加PBS定量到10uM.
用培养基制备连续稀释溶剂，从10μM依次到3.33μM,1.11μM,0.370μM,0.124μM,0.041μM,0.014μM,0.0046μM,0.0015μM。
将50μl溶液转移到每个孔以得到分别为5uM,1.667uM,0.556uM,0.185uM,0.062uM,0.021uM,0.0069uM,0.0023uM的终浓度。
2.实验试剂:
(Promega)；RPMI-1640培养基(Gibco,C11875500BT-500mL)；胎牛血清(Fetal bovine serum，FBS)(ExCell Bio,FNA500-500mL)；盘尼西林/链霉素(Penicillin/Streptomycin，PS)(Gibco,15140-122-100mL)；PBS缓冲液(Gibco,C20012500BT-500mL)；Trypsin-EDTA(Stemcell,07901-500mL)；DMSO(Sigma,D5879-1L)；CellTiter-Glo Luminescent Cell Viability Assay kit(CTG)(Promega,G7572-100mL)。
3.实验仪器:
倒置显微镜(Inverted Microscope)(Olympus IX71,No.112A-1)；96孔板阅读仪(96-well Plate Reader)(Molecular Devices,Flexstation 3)；Perkin Elmer Envision 2104 Multilabel Reader(No.01-094-0002)。
4.实验方法:
1)细胞培养及铺板
细胞在对应的基础培养基中分别添加10％FBS及1％PS中，于37℃和5％CO 2条件下培养。用于实验的细胞密度在80％以上。收集细胞，1000rpm 4分钟离心，培养基重悬，调整细胞浓度，以3000个细胞50μL的体积加到96孔板中，每组3个复孔。
2)梯度药物浓度配制及给药
在24小时后，将化合物溶液转移到每个孔中，50μL/孔。最后得到终浓度分别为:5uM,1.667uM,0.556uM,0.185uM,0.062uM,0.021uM,0.0069uM,0.0023uM的溶液；
溶剂对照＝DMSO
培养基(未处理)对照:仅有细胞没有化合物处理
空白对照:没有细胞，用于仪器校零
3)细胞给药后培养
将上述加药后细胞于37℃和5％CO 2条件下培养72小时。
4)检测试剂盒处理细胞
将孔板提前拿至室温静置30分钟。向孔板的每个孔中加100μL
Figure PCTCN2019109563-appb-000135
试剂，在摇床上2分钟混匀促进细胞裂解。使用Perkin Elmer Envision 2104Multilabel Reader仪器读值并记录。
5)获取实验数据并处理
将获取的实验数据使用excel软件分析处理，使用GraphPad Prism 5软件拟合曲线分析。
二、实验结果:
表119：IC 50值
Figure PCTCN2019109563-appb-000136
实验结果见表119和图82，从表119和图82中可以看出，表柔比星(EPB)与RNAh-Bio-670-EPB纳米颗粒对于HL60细胞增殖均有明显抑制作用，而且让人难以预料的是：给药浓度为5μM时，两种药物对细胞的抑制率分别99.25％，99.93％，而当对细胞增殖的抑制率为50％时，IC 50分别是0.06977μM和0.3015μM。可见，RNAh-Bio-670-EPB纳米颗粒对细胞增殖的抑制活性更强，其IC 50的药物浓度几乎是小分子药物EPB浓度的1/5，因此可显著减少药物用量，减少毒副作用。
进一步地，为了确认靶向荧光载体本身对HL60细胞无明显毒性，本申请进一步设计了RNAh-Bio-FAM靶向荧光载体对HL60细胞的毒性实验，以小分子化药顺铂(Cisplatin)对HL60细胞的毒性作为对照，具体结果见图83(其中，最高给药浓度为10μM，此时RNAh-Bio-FAM靶向荧光载体对HL60细胞的抑制率为8.75％，而对照顺铂对细胞的抑制率为99.96％)。从图83可以看出，荧光载体本身对HL60细胞无明显毒性。
(二)DNAh-Bio-EGFRapt-Cy5-EPB纳米颗粒分别在MCF-7和NCI-H1975细胞中的细胞毒性
一、实验材料
1.细胞信息：
表120：
Figure PCTCN2019109563-appb-000137
2.待测样品
表121：
Figure PCTCN2019109563-appb-000138
Figure PCTCN2019109563-appb-000139
3.耗材与设备
表123：
名称 品牌 货号/型号
96孔板 Corning 3599
离心机 京立 LD5-2B
CO 2培养箱 Thermo 3111
微孔板振荡器 QILINBEIER QB-9001
显微镜 Olympus IX53
多功能酶标仪 Bio Tek Synergy H1
4.试剂
表124：
Figure PCTCN2019109563-appb-000140
二、实验方法：
1)收获对数生长期的细胞，取少量用台盼蓝染色计数，保证细胞活力达到98％以上；
2)用生长培养基调整细胞密度到2.22×10 4/mL；
3)种植90μL/孔细胞悬液到96孔板中，孔板中每孔细胞数量为2000；
4)将种植好的细胞板置于37℃培养箱中孵育过夜；
5)对化合物做9个浓度点的3.16倍梯度稀释；
6)取出细胞培养板，将10μL/孔的10X浓度药物工作液加入到细胞培养板的相应孔中，每个浓度做三个复孔，药物最终作用浓度见下表；
表125：
Figure PCTCN2019109563-appb-000141
7)将细胞培养板置于培养箱中继续孵育96小时；
8)将CellTiter
Figure PCTCN2019109563-appb-000142
AQueous One Solution试剂置于室温融化90分钟或37℃水浴融化后置于室温平衡30分钟；
9)向细胞培养板中加入20μL/孔的CellTiter
Figure PCTCN2019109563-appb-000143
AQueous One Solution试剂；
10)将细胞培养板置于37℃培养箱中继续孵育3小时；
11)用酶标仪读取细胞板中各孔的OD 490值；
12)数据处理与分析。
采用GraphPad Prism 5.0软件对数据进行图形化处理。为计算IC 50，对数据进行“S”形非线性回归分析，匹配相适应的剂量-效应曲线。细胞存活率的计算公式如下，IC 50可在GraphPad Prism 5.0中自动计算得出。
细胞存活率(％)＝(OD 待测孔–OD 空白对照)/(OD 阴性对照-OD 空白对照)x 100％。
三、实验结果(见表126、图84a至84d及图85a至85d)
表126：
Figure PCTCN2019109563-appb-000144
从表126及图84a，84b，84c，84d可以看出，针对MCF-7细胞系而言，相比单纯的DNAh靶向荧光载体，小分子药物EPB及DNAh载药颗粒DNAh-Bio-EGFRapt-Cy5-EPB都对MCF-7细胞有毒性，且DNAh载药颗粒DNAh-Bio-EGFRapt-Cy5-EPB的IC 50药物浓度是小分子药物 EPB的IC 50药物浓度的一半。类似地，从表126及图85a，85b，85c，85d可以看出，针对NCI-H1975细胞系而言，相比单纯的DNAh靶向荧光载体，小分子药物EPB及DNAh载药颗粒DNAh-Bio-EGFRapt-Cy5-EPB都对NCI-H1975细胞有毒性，且DNAh载药颗粒DNAh-Bio-EGFRapt-Cy5-EPB的IC 50药物浓度是小分子药物EPB的IC 50药物浓度的一半。
从上述毒性实验可以看出，本申请的载药纳米颗粒比小分子药物具有更强的细胞增殖抑制作用，在达到相同药效时可减少药物用量，同时减少毒副作用。
(三)研究RNAh-Bio-670-MTX纳米颗粒在MOLT4细胞中的细胞毒性
一、实验材料和实验方法
1.实验材料：
待测样品：小分子药物MTX及RNAh-Bio-670-MTX纳米颗粒。
2.实验方法:
1)MOLT4细胞在RPMI1640+10％FBS+1％PS培养基中，于37℃和5％CO 2条件下培养。
2)收集细胞，800rpm 5分钟离心，培养基重悬，调整细胞浓度，以5000个细胞90μL的体积加到96孔板中。
3)第二天用培养基稀释待测样品，每孔每个样品200nM，4个复孔。
4)培养72h后，每孔加入CTG试剂100μL，振荡2min，室温静置10min，全程避光。
5)最后使用SoftMax Pro5软件读数。
二、实验结果:
表127：细胞活率(％)
细胞系 处理时间 甲氨蝶呤(MTX) RNAh-Bio-670-MTX
MOLT-4 72h 2.88 4.72
实验结果见表127和图86，从表127和图86中可以看出，在体外细胞MOLT4中，甲氨蝶呤(MTX)与RNAh-Bio-670-MTX纳米颗粒对于MOLT4细胞增殖均有明显抑制作用，且效果无明显差异。
进一步通过荧光靶向载体Bio-Cy5-RNAh与小分子化药顺铂(Cisplatin)对MOLT4细胞的毒性实验证明，荧光靶向载体Bio-Cy5-RNAh对MOLT4细胞增殖无明显的抑制作用(具体见图87)(只有在最大给药浓度5μm时，Bio-Cy5-RNAh对MOLT4细胞增殖抑制率达47.38％，而此时对照顺铂对细胞的增殖抑制率为99.94％)。
(四)RNAh-Bio-670-THP纳米颗粒在MCF-7细胞中的细胞毒性
一、实验材料和实验方法
1.待测样品：小分子药物THP及RNAh-Bio-670-THP纳米颗粒；
2.实验方法:
1)除为MCF-7细胞外，其余同(三)
二、实验结果:
表128：细胞存活率(％)
细胞系 处理时间 吡柔比星(THP) RNAh-Bio-670-THP
MCF-7 72h 13.85 12.93
实验结果见表128和图88，从表128和图88中可以看出，RNAh-Bio-670-THP纳米颗粒对MCF-7细胞增殖有显著的抑制作用，且比小分子药物吡柔比星(THP)的抑制效果还略强。
进一步地，为了确认载体本身对MCF-7细胞无明显毒性，本申请进一步设计了RNAh-Bio-FAM靶向荧光载体对MCF-7细胞的毒性实验，以10％PBS作为阴性对照，培养基作为空白对照，具体结果见图89。从图89可以看出，靶向荧光载体本身对MCF-7细胞无明显毒性。
(五)DNAh-Biotin-EGFRapt-Cy5-THP纳米颗粒在SGC-7901及SKOV3细胞中的细胞毒性
一、实验材料和方法
1.细胞信息
表129：
Figure PCTCN2019109563-appb-000145
2.待测样品
表130：
Figure PCTCN2019109563-appb-000146
二、实验设备与方法同(二)
三、实验结果
表131：
Figure PCTCN2019109563-appb-000147
从表131及图90a，90b，90c，90d可以看出，针对SKOV3细胞系而言，相比单纯的DNAh靶向荧光载体，小分子药物THP及DNAh载药颗粒DNAh-Bio-EGFRapt-Cy5-THP都对SKOV3细胞有毒性。类似地，从表131及图91a，91b，91c，91d可以看出，针对SGC-7901细胞系而言，相比单纯的DNAh靶向荧光载体，小分子药物THP及DNAh载药颗粒DNAh-Bio-EGFRapt-Cy5-THP都对SGC-7901细胞有毒性。
(六)研究RNAh-Bio-670-DNR纳米颗粒在MCF-7细胞中的细胞毒性
一、待测样品：小分子药物DNR及RNAh-Bio-670-DNR纳米颗粒；
二、实验结果:
表132：细胞存活率(％)
细胞系 处理时间 柔红霉素(DNR) RNAh-Bio-670-DNR
MCF-7 72h 17.33 15.36
实验结果见表132和图92，从表132和图92中可以看出，RNAh-Bio-670-DNR纳米颗粒对MCF-7细胞增殖有明显抑制作用，而且比小分子药物柔红霉素(DNR)对细胞增殖的抑制作用还略强。
进一步地，为了确认载体本身对MCF-7细胞无明显毒性，本申请进一步设计了RNAh-Bio-FAM靶向荧光载体对MCF-7细胞的毒性实验，以10％PBS作为阴性对照，培养基作为空白对照，具体结果见图93。从图93可以看出，靶向荧光载体本身对MCF-7细胞无明显毒性。
(七)研究RNAh-Biotin-quasar670-flu纳米颗粒在HepG2细胞中的细胞毒性
一、待测样品：小分子五氟尿嘧啶化药和RNAh-Biotin-quasar670-flu纳米颗粒。
二、实验结果:
表133：给药浓度为5μM时的细胞抑制率(％)
细胞系 处理时间 小分子五氟尿嘧啶化药 RNAh-Biotin-quasar670-flu
HepG2 72h 39.02％ 52.98％
实验结果见表133和图94，从表133和图94中可以看出，5μM的携带五氟尿嘧啶的RNA纳米颗粒RNAh-Biotin-quasar670-flu对于HepG2细胞有明显细胞毒性，而且让人难以预料的是：与小分子五氟尿嘧啶药物对细胞的增殖抑制作用相比，5μM的RNAh-Biotin-quasar670-flu对HepG2细胞的增殖抑制更显著，其在小分子五氟尿嘧啶药物处理后细胞的抑制率为39.02％的基础上，对细胞的抑制率又提高了至少25％(提高至52.98％)。
为了进一步确认不携带五氟尿嘧啶的RNA纳米颗粒对HepG2细胞无明显细胞毒性，发明人进一步设计了RNAh-Biotin-FAM(FAM为荧光标记)这一靶向荧光载体对HepG2细胞的毒性实验(实验中药物的给药梯度如下：100μM、31.6μM、10μM、3.16μM、1μM、316nM、100nM、31.6nM、10nM、0(10％PBS))，其结果如表134和图95所示。从表134的IC 50值和图95中可以看出，不携带五氟尿嘧啶的靶向荧光载体本身对实验细胞并无明显毒性。
表134：
Figure PCTCN2019109563-appb-000148
(八)研究RNAh-Biotin-quasar670-hdcp纳米颗粒在SGC7901细胞中的细胞毒性
一、待测样品：小分子10-羟基喜树碱化药和RNAh-Biotin-quasar670-hdcp纳米颗粒。
二、实验结果:
表135：给药浓度为5μM时的细胞抑制率(％)
Figure PCTCN2019109563-appb-000149
实验结果见表135和图96，从表135和图96中可以看出，5μM的携带10-羟基喜树碱的RNA纳米颗粒RNAh-Biotin-quasar670-hdcp对于SGC7901细胞有明显细胞毒性，而且让人难以预料的是：与小分子10-羟基喜树碱药物对细胞的增殖抑制作用相比，5μM的RNAh-Biotin-quasar670-hdcp对SGC7901细胞的增殖抑制更显著，其在小分子10-羟基喜树碱药物处理后细胞的抑制率为75.43％的基础上，对细胞的抑制率又提高了至少25％(提高至94.52％)。
为了进一步确认不携带10-羟基喜树碱的RNA纳米颗粒对SGC7901细胞无明显细胞毒性，发明人进一步设计了RNAh-Biotin-FAM(FAM为荧光标记)这一靶向荧光载体对SGC7901细胞的毒性实验(实验中药物的给药梯度如下：100μM、31.6μM、10μM、3.16μM、1μM、316nM、100nM、31.6nM、10nM、0(10％PBS))，其结果如表136和图97所示。从表136的IC 50值和图97中可以看出，不携带10-羟基喜树碱的靶向荧光载体本身对实验细胞并无明显毒性。
表136：
Figure PCTCN2019109563-appb-000150
(九)研究RNAh-Biotin-quasar670-阿司匹林纳米颗粒在SH-SY5Y细胞中的细胞毒性
实验目的：研究化合物对目的肿瘤细胞系增殖的影响。
实验设计：通过将化合物进行8个浓度梯度稀释，依次加入目的肿瘤细胞系中共同孵育72h，使用CTG试剂盒检测化合物对细胞增殖的影响。
一、待测样品：RNAh-Biotin-quasar670-阿司匹林及阿司匹林
二、实验结果:
IC 50值见表137。
表137：
细胞系 处理时间 RNAh-Biotin-quasar670-阿司匹林IC 50(μM) 阿司匹林IC 50(μM)
SH-SY5Y 72h 0.2744 1430
具体实验结果见图98和表137。从图98和表137中可以看出，RNA载体挂载阿司匹林化药组(RNAh-Biotin-quasar670-阿司匹林)对SH-SY5Y细胞增殖有明显抑制效果；阿司匹林化药组对SH-SY5Y细胞无明显抑制作用；给药浓度为5μM时，细胞抑制率分别98.77％，17.72％；IC 50分别是0.2744μM和1430μM。
进一步地，为了确认靶向荧光载体本身对SH-SY5Y细胞无明显毒性，本申请进一步设计了RNAh-Bio-Cy5靶向荧光载体对SH-SY5Y细胞的毒性实验，以小分子化药顺铂(Cisplatin)对SH-SY5Y细胞的毒性作为对照，具体结果见图99(其中，最高给药浓度为10μM，此时RNAh-Bio-Cy5(也写作Bio-Cy5-RNAh)靶向荧光载体对SH-SY5Y细胞的抑制率为29.34％，而对照顺铂对细胞的抑制率为99.81％)。从图99可以看出，荧光载体本身对SH-SY5Y细胞无明显毒性。
(十)研究RNAh-Bio-670-吉西他滨纳米颗粒在BXPC3细胞中的细胞毒性
一、待测样品：小分子药物吉西他滨及RNAh-Bio-670-吉西他滨纳米颗粒；
二、实验结果:
IC 50值见表138。
表138：
细胞系 处理时间 RNAh-Bio-670-吉西他滨IC 50(μM) 吉西他滨IC 50(μM)
BXPC3 72h 0.5916 0.03418
实验结果见表138和图100，从表138和图100中可以看出，RNA载体挂载吉西他滨组化药(RNAh-Bio-670-吉西他滨)和吉西他滨化药组对BxPC3细胞增殖均有明显抑制作用；给药浓度为5μM时，细胞抑制率分别99.68％，82.96％；IC50分别是0.5916μM和0.0341μM。
进一步地，为了确认载体本身对BXPC3细胞无明显毒性，本申请进一步设计了RNAh-Bio-FAM靶向荧光载体对BXPC3细胞的毒性实验，以10％PBS作为阴性对照，培养基作为空白对照，具体结果见图101。从图101可以看出，靶向荧光载体本身对BXPC3细胞无明显毒性。
从以上的描述中，可以看出，本申请上述的实施例实现了如下技术效果：本申请提供了一系列具有热力学稳定性、化学稳定性、高负载率以及可多种模块组合的核酸纳米颗粒载体。对该类载体进行独特的模块化设计的，得到既保持天然相容的亲和力，又具有高度稳定性质和多样组合特征的核心模块结构。该结构可以灵活高效的集成各种功能性模块，包括靶向模块、成像和探针模块、治疗模块和其它复合智能模块，从而能够用于体内靶向投送，实现精准诊疗。
通过将小分子药物他克林等挂载于本申请所提供的核酸纳米颗粒载体上形成核酸纳米载体药物，不仅能够提高药物的递送稳定性，而且能够在核酸纳米颗粒携带靶头的情况下，一方面将药物靶向递送到目标细胞，提高药物的生物利用度，另一方面因靶向递送既降低了对非目标细胞或组织的毒副作用，又降低了局部用药浓度，进一步减少了因用药浓度高而带来的毒副作用。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (20)

  1. 一种核酸纳米载体药物，其特征在于，所述核酸纳米载体药物包括核酸纳米颗粒和挂载在所述核酸纳米颗粒上的药物，所述药物包括他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨中的一种或多种；
    所述核酸纳米颗粒包括核酸结构域，所述核酸结构域包含a序列、b序列和c序列，所述a序列包含a1序列或者所述a1序列发生至少一个碱基插入、缺失或替换的序列，所述b序列包含b1序列或者所述b1序列发生至少一个碱基插入、缺失或替换的序列，所述c序列包含c1序列或者所述c1序列发生至少一个碱基插入、缺失或替换的序列；
    其中，所述a1序列为SEQ ID NO:1：5’-CCAGCGUUCC-3’或者SEQ ID NO:2：5’-CCAGCGTTCC-3’；
    所述b1序列为SEQ ID NO:3：5’-GGUUCGCCG-3’或者SEQ ID NO:4：5’-GGTTCGCCG-3’；
    所述c1序列为SEQ ID NO:5：5’-CGGCCAUAGCGG-3’或者SEQ ID NO:6：5’-CGGCCATAGCGG-3’。
  2. 根据权利要求1所述的核酸纳米载体药物，其特征在于，所述a1序列为SEQ ID NO:1，所述b1序列为SEQ ID NO:3，所述c1序列为SEQ ID NO:5时，所述a序列、所述b序列、所述c序列中的至少一个序列包含至少一个碱基插入、缺失或替换的序列。
  3. 根据权利要求1或2所述的核酸纳米载体药物，其特征在于，所述碱基插入、缺失或替换发生在：
    (1)SEQ ID NO:1或SEQ ID NO:2所示的序列的5’端起始的第1、2、4或5位碱基上；和/或
    (2)SEQ ID NO:1或SEQ ID NO:2所示的序列的5’端起始的第8～10位碱基之间；和/或
    (3)SEQ ID NO:3或SEQ ID NO:4所示的序列的5’端起始的第1～3位碱基之间；和/或
    (4)SEQ ID NO:3或SEQ ID NO:4所示的序列的5’端起始的第6～9位碱基之间；和/或
    (5)SEQ ID NO:5或SEQ ID NO:6所示的序列的5’端起始的第1～4位碱基之间；和/或
    (6)SEQ ID NO:5或SEQ ID NO:6所示的序列的5’端起始的第9～12位碱基之间；
    优选地，所述a序列、所述b序列和所述c序列自组装成式(1)所示结构：
    Figure PCTCN2019109563-appb-100001
    其中，W-C表示Watson-Crick配对，N和N’表示非Watson-Crick配对，任一位置的W-C各自独立地选自C-G或G-C；
    在所述a序列中，从5’端起的第一个N为A，第二个N为G，第三个N为U或T，第四个N为U、T、A、C或G中的任意一个；
    在所述b序列中，从5’端起的第一个N’为U、T、A、C或G中的任意一个；第二个N’为U或T，第三个N’为C；
    在所述c序列中，沿5’端至3’端方向上的NNNN序列为CAUA或CATA；
    更优选地，所述a序列、所述b序列和所述c序列为如下任意一组：
    (1)a序列：5'-GGAGCGUUGG-3'，
    b序列：5'-CCUUCGCCG-3'，
    c序列：5'-CGGCCAUAGCCC-3'；
    (2)a序列：5'-GCAGCGUUCG-3'，
    b序列：5'-CGUUCGCCG-3'，
    c序列：5'-CGGCCAUAGCGC-3'；
    (3)a序列：5'-CGAGCGUUGC-3'，
    b序列：5'-GCUUCGCCG-3'，
    c序列：5'-CGGCCAUAGCCG-3'；
    (4)a序列：5'-GGAGCGUUGG-3'，
    b序列：5'-CCUUCGGGG-3'，
    c序列：5'-CCCCCAUAGCCC-3'；
    (5)a序列：5'-GCAGCGUUCG-3'，
    b序列：5'-CGUUCGGCG-3'，
    c序列：5'-CGCCCAUAGCGC-3'；
    (6)a序列：5'-GCAGCGUUCG-3'，
    b序列：5'-CGUUCGGCC-3'，
    c序列：5'-GGCCCAUAGCGC-3'；
    (7)a序列：5'-CGAGCGUUGC-3'，
    b序列：5'-GCUUCGGCG-3'，
    c序列：5'-CGCCCAUAGCCG-3'；
    (8)a序列：5'-GGAGCGTTGG-3'，
    b序列：5'-CCTTCGCCG-3'，
    c序列：5'-CGGCCATAGCCC-3'；
    (9)a序列：5'-GCAGCGTTCG-3'，
    b序列：5'-CGTTCGCCG-3'，
    c序列：5'-CGGCCATAGCGC-3'；
    (10)a序列：5'-CGAGCGTTGC-3'，
    b序列：5'-GCTTCGCCG-3'，
    c序列：5'-CGGCCATAGCCG-3'；
    (11)a序列：5'-GGAGCGTTGG-3'，
    b序列：5'-CCTTCGGGG-3'，
    c序列：5'-CCCCCATAGCCC-3'；
    (12)a序列：5'-GCAGCGTTCG-3'，
    b序列：5'-CGTTCGGCG-3'，
    c序列：5'-CGCCCATAGCGC-3'；
    (13)a序列：5'-GCAGCGTTCG-3'，
    b序列：5'-CGTTCGGCC-3'，
    c序列：5'-GGCCCATAGCGC-3'；
    (14)a序列：5'-CGAGCGTTGC-3'，
    b序列：5'-GCTTCGGCG-3'，
    c序列：5'-CGCCCATAGCCG-3'。
  4. 根据权利要求3所述的核酸纳米载体药物，其特征在于，所述核酸结构域还包括第一延长段，所述第一延长段为Watson-Crick配对的延长段，所述第一延长段位于所述a序列、所述b序列和所述c序列中任一序列的5'端和/或3'端；
    优选地，所述第一延长段选自如下任意一组：
    (1)：a链5'端：5'-CCCA-3'，c链3'端：5'-UGGG-3'；
    (2)：a链3'端：5'-GGG-3'，b链5'端：5'-CCC-3'；
    (3)：b链3'端：5'-CCA-3'，c链5'端：5'-UGG-3'；
    (4)：a链5'端：5'-CCCG-3'，c链3'端：5'-CGGG-3'；
    (5)：a链5'端：5'-CCCC-3'，c链3'端：5'-GGGG-3'；
    (6)：b链3'端：5'-CCC-3'，c链5'端：5'-GGG-3'；
    (7)：b链3'端：5'-CCG-3'，c链5'端：5'-CGG-3'；
    (8)：a链5'端：5'-CCCA-3'，c链3'端：5'-TGGG-3'；
    (9)：b链3'端：5'-CCA-3'，c链5'端：5'-TGG-3'。
  5. 根据权利要求1至4中任一项所述的核酸纳米载体药物，其特征在于，所述核酸结构域还包括第二延长段，所述第二延长段位于所述a序列、所述b序列和所述c序列中任一序列的5’端和/或3’端，所述第二延长段为Watson-Crick配对的延长段；
    优选地，所述第二延长段为CG碱基对的延长序列；
    更优选，所述第二延长段为1～10个CG碱基对的延长序列。
  6. 根据权利要求5所述的核酸纳米载体药物，其特征在于，所述核酸结构域还包括如下至少一组第二延长段：
    第一组：a链5’端：5’-CGCGCG-3’，c链3’端：5’-CGCGCG-3’；
    第二组：a链3’端：5’-CGCCGC-3’，b链5’端：5’-GCGGCG-3’；
    第三组：b链3’端：5’-GGCGGC-3’，c链5’端：5’-GCCGCC-3’。
  7. 根据权利要求5所述的核酸纳米载体药物，其特征在于，所述第二延长段为同时含有CG碱基对和AT/AU碱基对的延长序列，优选所述第二延长段为2～50个碱基对的延长序列；更优选，所述第二延长段为连续2～8个CG碱基对的序列与连续2～8个AT/AU碱基对序列交替设置的延长序列；或者所述第二延长段为1个CG碱基对的序列与1个AT/AU碱基对序列交替设置的延长序列。
  8. 根据权利要求1至7中任一项所述的核酸纳米载体药物，其特征在于，所述a序列、所述b序列和所述c序列中碱基、核糖和磷酸酯具有至少一个可修饰位点，任一所述可修饰位点通过以下任意一种修饰接头进行修饰：-F、甲基、氨基、二硫化物、羰基、羧基、巯基及醛基；
    优选地，所述a序列、所述b序列和所述c序列中的C或U碱基上具有2’-F修饰。
  9. 根据权利要求1至8中任一项所述的核酸纳米载体药物，其特征在于，所述药物通过物理连接和/或共价连接的形式挂载在所述核酸纳米颗粒上，且所述药物与所述核酸纳米颗粒之间的摩尔比为2～300:1，优选为10～50:1，更优选为15～25:1。
  10. 根据权利要求1至9中任一项所述的核酸纳米载体药物，其特征在于，所述核酸纳米颗粒还包括生物活性物质，所述生物活性物质与所述核酸结构域相连，所述生物活性物质为靶头、荧光素、干扰核酸siRNA、miRNA、核酶、核糖开关、适体、RNA抗体、蛋白、多肽、类黄酮、葡萄糖、天然水杨酸、单抗、维生素、酚类卵磷脂以及小分子药物中的一种或多种，所述小分子药物不包括他克林、表柔比星、甲氨蝶呤、吡柔比星、柔红霉素、五氟尿嘧啶、10-羟基喜树碱、阿司匹林及吉西他滨；
    优选地，所述生物活性物质为所述靶头、所述荧光素以及所述miRNA中的一种或多种，其中，所述靶头位于所述a、b、c序列中任一序列上，优选a、b、c任一序列的5’端或3’端，或嵌插于所述核酸结构域的GC键之间，所述miRNA为抗miRNA，所述荧光素修饰于所述抗miRNA的5’端或3’端，所述miRNA位于所述a序列的3’端、c序列的5’端和3’端中的任意一个或多个位置，更优选地，所述靶头为叶酸或生物素，所述荧光素为FAM、CY5及CY3中的任意一种或多种，所述抗miRNA为抗miR-21；
    优选地，所述小分子药物为含有如下任意一种或多种基团的药物：氨基基团、羟基基团、羧基基团、巯基基团、苯环基团以及乙酰氨基基团；
    优选地，所述蛋白为SOD、生存素、hTERT、EGFR及PSMA中的一种或多种；所述维生素为左旋C和/或酯化C；所述酚类为茶多酚和/或葡萄多酚。
  11. 根据权利要求10所述的核酸纳米载体药物，其特征在于，将所述核酸结构域的相对分子量记为N 1，将所述药物与所述生物活性物质的总相对分子量记为N 2，N 1/N 2≥1:1。
  12. 根据权利要求1所述的核酸纳米载体药物，其特征在于，所述核酸纳米颗粒的粒径为1～100nm，优选为5～50nm；更优选10～30nm；进一步优选10～15nm。
  13. 一种核酸纳米载体药物的制备方法，其特征在于，所述制备方法包括以下步骤：
    提供权利要求1至12中任一项所述的核酸纳米载体药物中的核酸纳米颗粒；
    通过物理连接和/或共价连接的方式将所述药物挂载在所述核酸纳米颗粒上，得到所述核酸纳米载体药物。
  14. 根据权利要求13所述的制备方法，其特征在于，通过物理连接的方式挂载所述药物的步骤包括：
    将所述药物、所述核酸纳米颗粒及第一溶剂混合并搅拌，得到预混体系；
    对所述预混体系进行沉淀析出，得到所述核酸纳米载体药物；
    优选地，所述第一溶剂选自DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种；
    优选地，对所述预混体系进行沉淀析出，得到所述核酸纳米载体药物的步骤包括；
    对所述预混体系进行沉淀析出，得到析出物；
    对所述析出物进行洗涤去除杂质,得到所述核酸纳米载体药物；
    更优选地，将所述预混体系与无水乙醇混合后在低于10℃的温度条件下进行所述沉淀析出，得到所述析出物；进一步优选在0～5℃温度条件下进行所述沉淀析出，得到所述析出物；
    更优选地，采用6～12倍体积的无水乙醇对所述析出物进行洗涤去除杂质，得到所述核酸纳米载体药物。
  15. 根据权利要求14所述的制备方法，其特征在于，通过共价连接的方式挂载所述药物的步骤包括：
    配置药物溶液；
    使所述药物溶液在甲醛的介导作用下与所述核酸纳米颗粒的G环外氨基进行反应，得到反应体系；
    提纯所述反应体系，得到所述核酸纳米载体药物；
    优选地，所述反应的步骤包括：
    将所述药物溶液与多聚甲醛溶液、所述核酸纳米颗粒混合，在避光条件下进行反应，得到所述反应体系；其中优选所述多聚甲醛溶液的浓度为3.7～4wt％，优选所述多聚甲醛溶液为多聚甲醛和第二溶剂混合形成的溶液，且所述第二溶剂为DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种。
  16. 根据权利要求13至15中任一项所述的制备方法，其特征在于，所述制备方法还包括制备 所述核酸纳米颗粒的步骤，其包括：通过将权利要求1至12中任一项所述的核酸纳米载体药物中的核酸结构域对应的单链进行自组装，得到所述核酸结构域；
    优选地，在得到所述核酸结构域之后，所述制备方法还包括：将权利要求10或11所述的药物中的所述生物活性物质通过物理连接和/或共价连接的方式挂载在所述核酸结构域上，进而得到所述核酸纳米颗粒。
  17. 根据权利要求16所述的制备方法，其特征在于，通过共价连接的方式挂载所述生物活性物质的过程中，通过溶剂共价连接、linker共价连接或点击链接进行挂载；
    优选地，所述溶剂共价连接中采用的第三溶剂作为连接介质，且所述第三溶剂选自多聚甲醛、DCM、DCC、DMAP、Py、DMSO、PBS及冰醋酸中的一种或多种；
    优选地，所述linker选自二硫键、对苯叠氮基、溴丙炔或PEG；
    优选地，所述点击链接是在对生物活性物质前体和所述核酸结构域同时进行炔基或叠氮修饰，然后通过点击链接；
    更优选地，所述生物活性物质与所述核酸结构域以点击链接的方式相连，所述生物活性物质前体进行炔基或叠氮修饰的位点选自2’羟基、羧基或氨基，所述核酸结构域进行炔基或叠氮修饰的位点选自G环外氨基、2’-羟基、A氨基或2’-羟基。
  18. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1至12中任一项所述的核酸纳米载体药物。
  19. 权利要求1至12中任一项所述的核酸纳米载体药物在制备用于治疗老年痴呆症、肿瘤、自身免疫疾病或心脏病的药物中的应用。
  20. 根据权利要求19所述的应用，其特征在于，所述肿瘤为如下任意一种或多种：胰腺癌、卵巢癌、乳腺癌、膀胱癌、宫颈癌、肝癌、胆道癌、鼻咽癌、睾丸肿瘤、淋巴瘤、间皮瘤、头颈部癌、胃癌、白血病、结肠癌、直肠癌、绒毛膜上皮癌、恶性葡萄胎、皮肤癌、肺癌、输尿管癌、肾盂癌、绒毛膜上皮癌、骨肿瘤、白血病脑膜脊髓浸润、肾母细胞瘤、软组织肉瘤及甲状腺髓样癌；
    所述自身免疫疾病为顽固性牛皮藓、系统性红斑狼疮、强制性脊柱炎或皮肌炎；
    优选地，所述白血病为所述急性白血病，更优选所述急性白血病为急性淋巴细胞白血病或粒细胞白血病；
    优选地，所述肺癌包括支气管肺癌或非小细胞肺癌；
    优选地，所述肝癌包括原发性肝细胞癌或转移性肝癌。
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