WO2020011248A1 - 核酸纳米颗粒、包含其的药物组合物、含阿霉素的药物及其制备方法 - Google Patents

核酸纳米颗粒、包含其的药物组合物、含阿霉素的药物及其制备方法 Download PDF

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WO2020011248A1
WO2020011248A1 PCT/CN2019/095766 CN2019095766W WO2020011248A1 WO 2020011248 A1 WO2020011248 A1 WO 2020011248A1 CN 2019095766 W CN2019095766 W CN 2019095766W WO 2020011248 A1 WO2020011248 A1 WO 2020011248A1
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sequence
nucleic acid
doxorubicin
seq
nanoparticles
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PCT/CN2019/095766
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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 AU2019302604A priority Critical patent/AU2019302604B2/en
Priority to EP19833361.9A priority patent/EP3821911A4/en
Priority to KR1020217004271A priority patent/KR102656600B1/ko
Priority to US17/259,201 priority patent/US20220409742A1/en
Priority to JP2021524092A priority patent/JP7267416B2/ja
Publication of WO2020011248A1 publication Critical patent/WO2020011248A1/zh

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    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5107—Excipients; Inactive ingredients
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to the field of delivery carriers, and in particular, to a nucleic acid nanoparticle, a pharmaceutical composition containing the same, a doxorubicin-containing drug, and a preparation method thereof.
  • Cancer has become the most important disease that endangers human health.
  • Drug treatment including cell-targeted drug delivery, gene therapy, RNAi, etc.
  • many anticancer drugs have the disadvantages of poor solubility in water or poor stability.
  • doxorubicin is difficult to be used by organisms because of its poor solubility. Solving its water solubility problem is the key to the clinical application of this type of pharmaceutical preparations.
  • the effects of tumor treatment and diagnostic drugs are mostly non-selective, and usually have large toxic and side effects on normal tissues and organs at therapeutic doses.
  • siRNAs are rapidly degraded by serum factors and cannot reach their target site.
  • platinum drugs are widely used in ovarian cancer, small cell lung cancer, testicular cancer, head and neck squamous cell carcinoma, cervical cancer, non-small cell lung cancer, bladder cancer, pleural mesothelioma, melanoma and uterus Chemotherapy for Endometrial Cancer.
  • the platinum drugs are poorly targeted, and the general drug dose cannot achieve the therapeutic effect. Excessive doses will lead to a large amount of drugs acting on normal cells and tissues, causing human bone marrow suppression, gastrointestinal reactions, renal toxicity, and neurotoxicity after chemotherapy. And other adverse reactions, thus limiting the use of platinum chemotherapy drugs.
  • a delivery vector delivers a chemotherapeutic drug into a cancer cell, so that the active ingredient of the drug interacts with the DNA in the cancer cell, and has an inhibitory effect on the tumor.
  • platinum-based chemotherapy drug delivery vehicles include liposomes, micelles, nanocapsules, polymer-platinum conjugates, and carbon nanotubes.
  • 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 structure 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 existing research on non-viral vectors has focused more on nucleic acid drugs, and the delivery effect of non-nucleic acid drugs has not been reported with any value.
  • the main purpose of the present invention is to provide a nucleic acid nano particle, a pharmaceutical composition containing the same, a doxorubicin-containing drug, and a preparation method thereof, so as to provide a reliable drug carrier to solve the current problem of limited clinical application of drugs.
  • a nucleic acid nanoparticle which has a nucleic acid domain, the nucleic acid domain comprises an a sequence, a b sequence, and a c sequence, the a sequence comprises an a1 sequence or at least one of the a1 sequences A sequence of base insertion, deletion, or replacement.
  • the b sequence includes the sequence of b1 or at least one base insertion, deletion, or replacement
  • the c sequence includes the c1 sequence or at least one base insertion, deletion, or replacement of c1 sequence.
  • a1 sequence is SEQ ID NO: 1: 5'-CCAGCGUUCC-3 'or SEQ ID ID NO: 2: 5'-CCAGCGTTCC-3';
  • the b1 sequence is SEQ ID NO: 3: 5'-GGUUCGCCG-3 'Or SEQ ID NO: 4: 5'-GGTTCGCCG-3';
  • c1 sequence is SEQ ID NO: 5: 5'-CGGCCAUAGCGG-3 'or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3'.
  • the a1 sequence is SEQ ID NO: 1
  • the b1 sequence is SEQ ID NO: 3
  • the c1 sequence is SEQ ID NO: 5
  • at least one of the a sequence, the b sequence, and the c sequence includes at least one base insertion , Missing or replaced sequences.
  • WC represents Watson-Crick pairing
  • WC at any position 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 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:
  • 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 of the sequences a, b, and c. / Or 3 'end; preferably, the first extension is selected from any one of the following: (1): the 5' end of the a chain: 5'-CCCA-3 ', and the 3' end of the 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
  • 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 sequences a, b and c, 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 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'; 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 extension is an extension sequence containing both CG base pairs and AT / AU base pairs, and preferably the second extension is an extension sequence of 2 to 50 base pairs.
  • the second extended segment is an extended sequence in which a sequence of 2 to 8 CG base pairs is consecutively alternated with a continuous 2 to 8 AT / AU base pair sequence; or the second extended segment is in a sequence of 1 CG base.
  • the paired sequence is an extended sequence that is alternately arranged with one AT / AU base pair sequence.
  • the 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 a 2'-F modification.
  • the nucleic acid nanoparticle further includes a biologically active substance, and the biologically active substance is connected to a nucleic acid domain.
  • the ratio of the relative molecular weight of the nucleic acid domain to the total relative molecular weight of the biologically active substance is ⁇ 1: 1; preferably, the biologically active substance is a target, fluorescein, interference nucleic acid siRNA, miRNA, ribozyme, riboswitch, suitable One or more of a body, an RNA antibody, a drug, a protein, a peptide, a flavonoid, glucose, a natural salicylic acid, a monoclonal antibody, a vitamin, a phenol, and a lecithin.
  • the biologically active substance is a target, fluorescein, interference nucleic acid siRNA, miRNA, ribozyme, riboswitch, suitable One or more of a body, an RNA antibody, a drug, a protein, a peptide, a flavonoid, glucose, a natural salicylic acid, a monoclonal antibody, a vitamin, a phenol, and
  • 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, and preferably the 5 'end or the 3' end of any of the sequences of a, b, and c.
  • the miRNA is anti-miRNA
  • the fluorescein is modified at the 5 'end or 3' end of the anti-miRNA
  • the miRNA is located at the 3 'end of the a sequence, the 5' end of the c sequence, and 3 Any one or more positions in the 'end; preferably, the target is folic acid or biotin, fluorescein is any one or more of FAM, CY5, and CY3, and the anti-miRNA is anti-miR-21.
  • the medicine is for treating 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 Drugs for hepatitis B, multiple myeloma, pancreatic cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal cancer, esophageal cancer, oral cancer, lupus erythematosus; preferably, head and neck cancer is brain cancer, neuroblastoma or glioblastoma Cell tumor.
  • the 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 antibodies or aptamers of SOD, survivin, hTERT, EGFR, and PSMA; vitamins are L-C and / or esterified C; phenols are tea polyphenols and / or grapes Polyphenols.
  • the biologically active substance is connected to the nucleic acid domain by any of the following methods: Method 1: Physical insertion; Method 2: Covalent connection.
  • the biologically active substance and the nucleic acid domain are connected in a physical intercalation manner, the biologically active substance and the nucleic acid domain are physically intercalated at a molar ratio of 1 to 200: 1.
  • the molar ratio of the biologically active substance connected with the physical insertion method and the covalently connected drug is 1 to 200: 1.
  • the biologically active substance connected in the covalent connection manner is covalently connected by a solvent, covalently linked by a linker, or clicked on a link;
  • the solvent is selected from paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, or glacial acetic acid.
  • the linker is selected from the group consisting of disulfide bonds, p-phenylazide, bromopropyne, or PEG; preferably, clicking the link is to simultaneously perform alkynyl or azide modification on the biologically active substance precursor and the nucleic acid domain, and then By clicking on the link.
  • the site of the biologically active substance precursor that is alkynyl or azide modified is selected from the 2 ′ hydroxyl, carboxyl, or amino group
  • the nucleic acid domain is alkynyl or
  • the azide-modified site is selected from the group consisting of an G-ring amino group, a 2'-hydroxyl group, an A amino group, or a 2'-hydroxyl group.
  • 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.
  • composition including the above-mentioned nucleic acid nanoparticles.
  • a doxorubicin-containing drug is also provided.
  • the doxorubicin-containing drug includes doxorubicin and the aforementioned (non-mounted biologically active substance) nucleic acid nanoparticles.
  • the doxorubicin is mounted on the nucleic acid nanoparticle through a physical connection and / or a covalent connection, and the molar ratio between the doxorubicin and the nucleic acid nanoparticle is 2 to 300: 1, and 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, an interfering nucleic acid siRNA, miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, and a protein.
  • the relative molecular weight of the nucleic acid domain is referred to as N 1
  • the total relative molecular weight of adriamycin and the biologically active substance is referred to 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.
  • small-molecule drugs other than doxorubicin are drugs containing any one or more of the following groups: amino group, hydroxyl group, carboxyl group, thiol group, benzene ring group, and acetamino group group.
  • the protein is one or more of SOD, survivin, hTERT, EGFR, and PSMA;
  • the vitamin is L-C and / or esterified C;
  • the phenols are tea polyphenols and / or grape polyphenols.
  • a method for preparing a doxorubicin-containing drug which includes the following steps: providing the right nucleic acid nanoparticles of any one of the foregoing (without mounting a biologically active substance); Doxorubicin is mounted on the nucleic acid nanoparticles by means of physical linking and / or covalent linking to obtain a doxorubicin-containing drug.
  • the step of mounting doxorubicin by means of physical connection includes: mixing and stirring doxorubicin, nucleic acid nanoparticles, and the first solvent to obtain a premixed system; removing free substances in the premixed system to obtain doxorubicin-containing Drugs of mycin; preferably, the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, the step of removing free materials in the premixed system includes: The premixed system is mixed with absolute ethanol, and the doxorubicin-containing drug is precipitated at a temperature lower than 10 ° C; more preferably, the doxorubicin-containing drug is precipitated at a temperature of 0-5 ° C.
  • the step of mounting doxorubicin by means of covalent linking includes: configuring a doxorubicin solution; and reacting the doxorubicin solution with formaldehyde-mediated interaction with the G-ring amino group of the nucleic acid nanoparticle to obtain a reaction System; purifying the reaction system to obtain a doxorubicin-containing drug; preferably, the step of the reaction includes: mixing the doxorubicin solution with a paraformaldehyde solution and nucleic acid nanoparticles, and performing the reaction under the condition of avoiding light to obtain a reaction system; Among them, the concentration of the paraformaldehyde solution is preferably 3.7 to 4% by weight.
  • the paraformaldehyde solution is preferably a solution of paraformaldehyde and a second solvent.
  • the second solvent is DCM, DCC, DMAP, Py, DMSO, PBS, and One or more of glacial acetic acid.
  • the above preparation method further includes a step of preparing nucleic acid nanoparticles, which comprises: self-assembling a single strand corresponding to a nucleic acid domain in any of the aforementioned (non-mounted biologically active substance) nucleic acid nanoparticles to obtain A nucleic acid domain; preferably, after the nucleic acid domain is obtained, the preparation method further comprises: mounting the biologically active substance on the nucleic acid domain through physical and / or covalent attachment, thereby obtaining a nucleic acid nanoparticle, wherein,
  • the drugs in the biologically active substance are small molecule drugs other than doxorubicin.
  • the solvent is covalently connected, the linker is covalently connected, or the link is clicked to mount; preferably, the third solvent used in the solvent covalent connection is used as the connection.
  • the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, 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 by clicking on the link.
  • the site of the biologically active substance precursor that is alkynyl or azide modified is selected from the 2 ′ hydroxyl, carboxyl, or amino group
  • the nucleic acid domain is alkynyl or
  • the azide-modified site is selected from the group consisting of an G-ring amino group, a 2'-hydroxyl group, an A amino group, or a 2'-hydroxyl group.
  • the nucleic acid nanoparticle provided by the present invention can not only self-assemble to form a nucleic acid domain by including the three sequences described above or a variant sequence thereof, but also can be used as a carrier to connect an siRNA drug at any 5 'end and / or 3' end of the three strands or In the process of forming the above-mentioned nanoparticles, the miRNA drug reduces the degradation effect of the nuclease on the mounted nucleic acid drug due to the existence of the nucleic acid domain, and improves the reliability and stability of drug delivery.
  • FIG. 1 shows the results of electrophoretic detection of RNA nanoparticles formed by self-assembly in Example 1 of the present invention
  • Example 2 shows the results of electrophoretic detection of DNA nanoparticles formed by self-assembly in Example 1 of the present invention
  • Example 3 shows the results of 2% agarose gel electrophoresis detection of 7 groups of short-sequence RNA nanoparticles formed by self-assembly in Example 2 of the present invention
  • FIG. 7 shows the results of 2% agarose gel electrophoresis detection of 7 groups of conventional sequence DNA nanoparticles formed by self-assembly in Example 4 of the present invention
  • FIG. 8 shows the results of 4% agarose gel electrophoresis detection of 7 groups of conventional sequence DNA nanoparticles formed by self-assembly in Example 4 of the present invention
  • FIG. 10 shows the results of electrophoresis detection of the adriamycin-mounted product in Example 5 of the present invention
  • FIG. 11 shows a standard curve of the doxorubicin absorbance used in the detection process of the mounting rate in Example 5 of the present invention
  • Example 13 shows the results of binding and internalization of different nanoparticles with HepG2 cells in Example 7 of the present invention
  • Example 14 shows the results of electrophoresis detection of RNA nanoparticles in Example 9 of the present invention after incubation in serum for different times under the Coomassie Blue program;
  • FIG. 15 shows the results of electrophoretic detection of RNA nanoparticles in Example 9 of the present invention after being incubated in serum for different times under the Stain Free Gel program;
  • FIG. 16 shows detection results of HepG2 cell proliferation of different nanoparticles in Example 10 of the present invention
  • FIG. 17 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 11 of the present invention
  • FIG. 18 shows a dissolution curve of RNA nanoparticles R-15 in Example 11 of the present invention
  • FIG. 19 shows a dissolution curve of RNA nanoparticles R-16 in Example 11 of the present invention
  • FIG. 20 shows a dissolution curve of RNA nanoparticles R-17 in Example 11 of the present invention
  • FIG. 21 shows a dissolution curve of RNA nanoparticles R-18 in Example 11 of the present invention
  • FIG. 22 shows a dissolution curve of RNA nanoparticles R-19 in Example 11 of the present invention
  • FIG. 23 shows a dissolution curve of the RNA nanoparticle R-20 in Example 11 of the present invention
  • FIG. 24 shows a dissolution curve of RNA nanoparticles R-21 in Example 11 of the present invention
  • FIG. 25 shows the results of non-denaturing PAGE gel electrophoresis detection of 7 groups of extended segments + core short sequence DNA self-assembly products in Example 12 of the present invention
  • FIG. 26 shows a dissolution curve of the DNA nanoparticle D-8 in Example 12 of the present invention
  • FIG. 27 shows a dissolution curve of the DNA nanoparticle D-9 in Example 12 of the present invention
  • FIG. 28 shows a dissolution curve of the DNA nanoparticle D-10 in Example 12 of the present invention
  • FIG. 28 shows the dissolution profile of DNA nanoparticles D-11 in Example 12 of the present invention
  • FIG. 30 shows a dissolution curve of the DNA nanoparticle D-12 in Example 12 of the present invention
  • FIG. 31 shows a dissolution curve of the DNA nanoparticle D-13 in Example 12 of the present invention
  • FIG. 32 shows the dissolution profile of DNA nanoparticles D-14 in Example 12 of the present invention
  • FIG. 33 shows the results of electrophoresis detection of RNA nanoparticles R-15 in Example 13 of the present invention after different incubation in serum;
  • FIG. 34 shows the results of electrophoresis detection of RNA nanoparticles R-16 in Example 13 of the present invention after different incubation in serum;
  • FIG. 35 shows the results of electrophoresis detection of RNA nanoparticles R-17 in Example 13 of the present invention after different incubations in serum;
  • FIG. 36 shows the results of electrophoresis detection of RNA nanoparticles R-18 in serum of Example 13 after different incubation times in serum;
  • FIG. 37 shows the results of electrophoresis detection of RNA nanoparticles R-19 in Example 13 of the present invention after different incubation in serum;
  • FIG. 38 shows the results of electrophoresis detection of RNA nanoparticles R-20 in Example 13 of the present invention after different incubations in serum;
  • FIG. 39 shows the results of electrophoresis detection of RNA nanoparticles R-21 in Example 13 of the present invention after different incubation in serum;
  • FIG. 40 shows the results of electrophoresis detection of DNA nanoparticles D-8 in Example 14 of the present invention after being incubated in serum for different times;
  • FIG. 42 shows the results of electrophoresis detection of DNA nanoparticle D-10 in Example 14 of the present invention after being incubated in serum for different times;
  • FIG. 43 shows the results of electrophoresis detection of DNA nanoparticles D-11 in Example 14 of the present invention after being incubated in serum for different times;
  • FIG. 44 shows the results of electrophoresis detection of DNA nanoparticle D-12 in Example 14 of the present invention after different incubation in serum;
  • FIG. 45 shows the results of electrophoresis detection of DNA nanoparticle D-13 in Example 14 of the present invention after being incubated in serum for different times;
  • Figure 47a, Figure 47b, Figure 47c, Figure 47d, Figure 47e, Figure 47f, Figure 47g, and Figure 47h show the DMSO and the original drug doxorubicin, D-8 and D-8-A in Example 17 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. 48 shows a standard curve of daunorubicin absorbance used in the mounting rate detection process of Example 18.
  • RNA nanoparticles have a larger number of stem-loop structures within or between molecules, which has a more stable structure. 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 some 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.
  • both the RNA nanoparticles and DNA nanoparticles improved by the inventors can mount various drugs and can stably exist in serum; further experiments have verified that they can carry drugs into cells, and separate carrier pairs The cells are non-toxic. Carriers carrying drugs can alleviate and treat corresponding diseases.
  • a nucleic acid nanoparticle is provided, the nucleic acid nanoparticle has a nucleic acid domain, the nucleic acid domain comprises an a sequence, a b sequence, and a c sequence, the a sequence comprises the a1 sequence or the a1 sequence occurs at least A base insertion, deletion, or replacement sequence.
  • the b sequence contains the b1 sequence or the sequence where at least one base insertion, deletion, or replacement occurs, and the c sequence contains the c1 sequence or the c1 sequence has at least one base insertion, deletion, or replacement.
  • sequence of a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3 'or SEQ ID NO: 2: 5'-CCAGCGTTCC-3';
  • sequence of b1 is SEQ ID NO: 3: 5'-GGUUCGCCG- 3 'or SEQ ID NO: 4: 5'-GGTTCGCCG-3';
  • c1 sequence is SEQ ID NO: 5: 5'-CGGCCAUAGCGG-3 'or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3'.
  • 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 an siRNA drug or miRNA drug to any 5 'end and / or 3' end of the three strands.
  • the existence of the nucleic acid domain reduces the degradation effect of the nuclease on the mounted nucleic acid drug, and improves the reliability and stability of drug delivery.
  • the aforementioned 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 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 nucleic acid molecules, 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 subassembly structure is established, and finally a complex multilevel 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.
  • An advantage 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 by the sequences SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 or a variant thereof, or the sequences SEQ ID NO: 2 and SEQ:
  • the three sequences shown in IDNO: 4 and SEQ IDNO: 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 sequence can be obtained by reasonable selection of the mutation site and its mutation type, or by extending the appropriate fragment.
  • the nanoparticles formed by SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 self-assembly are RNA nanoparticles, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 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 drug loading capacity or improve the stability under the premise that self-assembly can be realized.
  • the sequence shown in the above SEQ ID ID NO: 1/2, SEQ ID ID NO: 3/4 and / or SEQ ID ID NO: 5/6 When inserting, deleting, or replacing a base, 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, and on the other hand, the mutation remains the same as the original sequence.
  • the above-mentioned base insertion, deletion or substitution occurs at: (1) 1, 2, 4, and 5 starting from the 5 ′ end of the a sequence shown in 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 NO : 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 starting 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, b-sequence, and c-sequence self-assemble into a structure represented by formula (1):
  • 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 respectively 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 addition to the 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 three sequences described above into the structure of the formula (1), when selecting bases paired by Watson-Crick, it is best to follow the base selection at different positions
  • 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 maximize the self-assembly efficiency by maximally efficiently making the two ends of any one strand complementary to the ends of the other two strands, respectively.
  • 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 strand, 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 paired UU, or it can be an improved T, A, C, or G that follows 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 ID NO: 11): 5'-CGUUCGCCG-3 ', c sequence (SEQ ID ID NO: 12): 5'-CGGCCAUAGCGC-3'; (3) a sequence (SEQ ID ID NO: 13): 5'-CGAGCGUUGC-3 ', b sequence (SEQ ID NO: 14): 5'-GCUUCGCCG-3', c sequence (SEQ ID NO: 15): 5'-CGGCCAUAGCCG-3 ';
  • the nucleic acid nanoparticles formed by the self-assembly of the fourteen sets 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.
  • the amount of the drug to be mounted also varies.
  • nucleic acid drugs the 5 'end and / or the 3' end of any of the a, b, and c sequences can be mounted by extension.
  • 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 and / or 3 of any of the a, b, and c sequences. 'end. A certain matching relationship is required between the carrier and the substance to be mounted.
  • the carrier When the molecular weight of the carrier is too small and the molecular weight of the substance to be mounted is too large, from a mechanical point of view, the carrier's ability to carry or transport the substance is relatively reduced. Therefore, by adding the first extension segment to the 5 'end and / or 3' end of any of the a sequence, b sequence, and c sequence based on the aforementioned nucleic acid nanostructure, it is possible to obtain a size that matches the size of the mounted substance. Carrier.
  • 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): the 5 'end of the a chain: 5'-CCCA-3', and the 3 'end of the 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
  • the first extension not only increases the length of any one or more of the three sequences that form 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 segment, the second extension segment 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
  • 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 further added to the first extension.
  • the above-mentioned nucleic acid domain further includes at least one second extended segment as follows: the first group: 5 ′ end of a chain: 5′-CGCGCG-3 ′, 3 ′ end of 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 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 segment contains both CG base pairs and AT
  • the extension sequence of the / AU base pair is preferably an extension sequence of 2 to 50 base pairs in the second extension segment.
  • the "/" in the "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 An extended sequence in which a sequence of one CG base pair and an sequence of one AT / AU base pair 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 the above SEQ ID ID NO: 51 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 chemical drugs with an indole molecular structure (the indole drug 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 substance capable of being mounted on the nucleic acid nanoparticle provided as a carrier in the present application may be any substance having a biologically active effect. Therefore, in a preferred embodiment, the above-mentioned nucleic acid nanoparticles further include a biologically active substance, and the biologically active substance is connected to the nucleic acid domain.
  • the ratio of the relative molecular weight of the nucleic acid domain to the total relative molecular weight of the biologically active substance is ⁇ 1: 1; preferably, the biologically active substance is a target, fluorescein, interfering nucleic acid siRNA, miRNA, nuclear Enzymes, riboswitches, aptamers, RNA antibodies, drugs (usually interpreted as small molecule drugs, chemically synthesized drugs), proteins, peptides, flavonoids, glucose, natural salicylic acid, monoclonal antibodies, vitamins, phenols, and lecithin One or more of them.
  • the biologically active substance is a target, fluorescein, interfering nucleic acid siRNA, miRNA, nuclear Enzymes, riboswitches, aptamers, RNA antibodies, drugs (usually interpreted as small molecule drugs, chemically synthesized drugs), proteins, peptides, flavonoids, glucose, natural salicylic acid, monoclonal antibodies, vitamins, phenols, and lecithin
  • the bioactive substance is biotin or folic acid
  • its role is to make the nucleic acid nanoparticles 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 certain siRNA, miRNA, drug (usually interpreted as a small molecule drug), protein, peptide or RNA antibody
  • the nucleic acid nanoparticle may become a new one with a specific therapeutic effect according to different biological functions. Products, such as better-performing drugs.
  • 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 of the sequences a, b, and c, preferably 5 ′ of any of the sequences of a, b, and c.
  • the miRNA is anti-miRNA
  • fluorescein is modified at the 5' end or 3 'end of the anti-miRNA
  • the 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 aforementioned anti-miRNA is anti- miR-21.
  • 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 in any sequence of the a, b, and c sequences, and it is more convenient to connect to the 5 'end or the 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 can 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, in the above-mentioned preferred nucleic acid nanoparticles, the target, such as folic acid or biotin, can specifically target cancer cells, and 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.
  • the biologically active substance that can be mounted above is a drug
  • the drug includes but is not limited to the treatment of liver cancer, gastric 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 ,
  • a drug for lupus erythematosus disease preferably, the head and neck cancer is brain cancer, neuroblastoma or glioblastoma.
  • the drug includes, but is not limited to, drugs containing any one or more of the following groups: amino Groups, hydroxyl groups, carboxyl groups, mercapto groups, benzene ring groups, and acetamino groups.
  • the aforementioned proteins are SOD (Superoxide Dismutase), Survivin, hTERT (Human Telomerase Reverse Transcriptase), EGFR (epidermal growth factor Receptor), and PSMA (Prostate Specific Sex membrane antigen) antibodies or aptamers;
  • the above vitamins are L-C and / or esterified C;
  • the above phenols are tea polyphenols and / or grape polyphenols.
  • a suitable connection method can be selected for connection with the above-mentioned nucleic acid nanocarrier.
  • the biologically active substance is connected to the nucleic acid domain by any of the following methods: Method 1: Physical intercalation; Method 2: Covalent connection.
  • the above classification does not mean that there is only one connection method between a certain biologically active substance and a nucleic acid nanocarrier. Instead, some biologically active substances can be connected to the nucleic acid nanocarrier by means of physical intercalation, or they can be connected to the nucleic acid nanocarrier by means of physical intercalation and covalent connection. At the same time, it may also be connected by clicking on the link . But 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 preferred number of 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.
  • anthracyclines and acridines are usually intercalated between GC base pairs during intercalation.
  • the preferred intercalation The number of dots is interpolated at a ratio of 1 to 100: 1 according to the number of GC base pairs on the nucleic acid domain.
  • naphthamide drugs are intercalated, they are usually interposed between AA base pairs.
  • the preferred number of 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 biologically active substance and the nucleic acid domain can be reasonably selected.
  • the molar ratio is physically interpolated.
  • the biologically active substance and the nucleic acid domain are connected in a physical intercalation manner, the biologically active substance and the nucleic acid domain are physically intercalated at a molar ratio of 1 to 200: 1.
  • This connection method is suitable for anthracyclines and acridines. Physical intercalation within this ratio range can meet both the mounting needs and the efficacy requirements.
  • the molar ratio of the biologically active substance connected with the physical insertion and the covalently connected drug is 1 ⁇ 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 bioactive substances connected in a covalent connection manner are covalently connected through a solvent, covalently linkeder or clicked on a linker; preferably, the solvent is selected from paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS or glacial acetic acid; preferably, the linker is selected from disulfide bonds, p-phenylazide, bromopropyne or PEG.
  • clicking the link is to perform alkynyl or azide modification on the biologically active substance precursor and the nucleic acid domain simultaneously, and then click the link.
  • the alkynyl or azide modification site of the biologically active substance precursor is selected from the group consisting of a hydroxyl group, a carboxyl group, a thiol group, or an amino group.
  • the site for the alkynyl or azide modification of the domain is selected from amino, imino or hydroxyl.
  • the nucleic acid domain when the nucleic acid domain is combined with a drug, the nucleic acid domain is water-soluble, and most drugs are poorly water-soluble. 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 will be 1.1 to 1.3 times the theoretical supersaturation.
  • the binding reaction can be carried out in a large amount, and a maximum of 35 to 45 drugs can be bound to one nucleic acid domain.
  • the loading amount is related to the occupancy of the specific drug (including but not limited to molecular structure, morphology, shape, and molecular weight). Therefore, the active site of the drug and the nucleotides of the nucleic acid domain.
  • the binding conditions of the -NH bond on guanosine are relatively severe, and they can also be mounted but it is more difficult to have excessive binding.
  • 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, the favorable particle size helps to improve the pharmacokinetics Kinetics, pharmacodynamics, biological distribution, and toxicology.
  • a pharmaceutical composition is also provided, and the pharmaceutical composition includes any one of the nucleic acid nanoparticles described above.
  • the nucleic acid domain can be modified by the target of the target cell to have good targeting, and at the same time, the corresponding therapeutic drug and / or tracer can be mounted. Sex molecules, enabling stable delivery of therapeutic drugs and / or tracer molecules with high reliability.
  • a doxorubicin-containing drug in a third typical embodiment, includes doxorubicin and any of the aforementioned (non-mounted biologically active substance) nucleic acid nanoparticles;
  • the doxorubicin-containing drug is any one of the aforementioned nucleic acid nanoparticles (on which a biologically active substance is mounted), wherein the biologically active substance is at least a drug, and the drug includes doxorubicin.
  • the doxorubicin-containing drugs provided above include nucleic acid nanoparticles and doxorubicin, and doxorubicin is 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 adriamycin at any 5 'end and / or 3' end of the three strands, Doxorubicin can be stably inserted between the strands of the nucleic acid domain.
  • the doxorubicin-containing drug provided by the present invention has a target region modified by a nucleic acid domain, can have better targeting, can stably deliver doxorubicin, and has high reliability.
  • doxorubicin when the biologically active substance is a drug and the drug is doxorubicin, doxorubicin can be mounted in the form of physical connection and / or covalent connection.
  • the physical insertion is usually inserted between the GC base pairs.
  • the preferred number of insertion sites is based on the nucleic acid domain.
  • the number of GC base pairs is interpolated at a ratio of 1 to 100: 1.
  • covalent linking is used, doxorubicin usually chemically reacts with the amino group outside the G ring to form a covalent link. More preferably, the molar ratio between doxorubicin and the nucleic acid nanoparticles is 2 to 300: 1, preferably 10 to 50: 1, and more preferably 15 to 25: 1.
  • the nucleic acid nanoparticles are used as a delivery vehicle for doxorubicin.
  • the nucleic acid nanoparticles further include A biologically active substance, which is connected 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 other than doxorubicin.
  • the relative molecular weight of the nucleic acid domain and the relative molecular weights of doxorubicin and the biologically active 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 doxorubicin and the biologically active substance is recorded as N 2
  • the doxorubicin-containing drug in the present invention is optimized in different performances.
  • the bioactive substance is biotin or folic acid
  • its role is to make the doxorubicin-containing 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 certain siRNA, miRNA, protein, peptide, RNA antibody, and small molecule drugs other than doxorubicin
  • the doxorubicin-containing drug may be made to have a specific treatment according to different biological functions. Effective new products, such as better-performing drugs.
  • the biologically active substance is a target, fluorescein, and miRNA, wherein the target is located on any of the sequences a, b, and c, preferably 5 ′ of any of the sequences of a, b, and c.
  • the miRNA is anti-miRNA
  • fluorescein is modified at the 5' end or 3 'end of the anti-miRNA
  • the miRNA is located at the 3' end of the a sequence
  • the c sequence At 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 Anti-miR-21.
  • 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 in any sequence of the a, b, and c sequences, and it is more convenient to connect to the 5 'end or the 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 can 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, in the above-mentioned preferred nucleic acid nanoparticles, the target, such as folic acid or biotin, can specifically target cancer cells, and 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.
  • the drugs include, but are not limited to, liver cancer, gastric 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, Drugs for nasopharyngeal cancer, esophageal cancer, oral cancer, and lupus erythematosus; preferably, the head and neck cancer is brain cancer, neuroblastoma, or glioblastoma.
  • bioactive substances that can be mounted are small-molecule drugs other than doxorubicin, depending on the molecular structure of the drug or the different characteristic groups, it includes, but is not limited to, any one of the following or A variety of groups of drugs: amino groups, hydroxyl groups, carboxyl groups, thiol groups, benzene ring groups, and acetamino groups.
  • the aforementioned proteins are SOD (Superoxide Dismutase), Survivin, hTERT (Human Telomerase Reverse Transcriptase), EGFR (epidermal growth factor Receptor), and PSMA (Prostate Specific Sex membrane antigen) antibodies or aptamers;
  • the above vitamins are L-C and / or esterified C;
  • the above 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, the favorable particle size helps to improve the pharmacokinetics Kinetics, pharmacodynamics, biological distribution, and toxicology.
  • a method for preparing the above-mentioned doxorubicin-containing medicine which includes the following steps: providing the above-mentioned (non-bioactive substance-containing) nucleic acid nanoparticles; Doxorubicin is mounted on nucleic acid nanoparticles by means of linking and / or covalent linking to obtain a doxorubicin-containing drug.
  • doxorubicin When the physical connection method is used, doxorubicin is usually inserted between GC base pairs by physical insertion. When covalent linking is used, doxorubicin usually chemically reacts with the amino group outside the G ring to form a covalent link. Doxorubicin-containing drugs prepared by the method described above can have better targeting after modified target, can stably deliver doxorubicin, and have high reliability.
  • the step of mounting doxorubicin by means of physical connection includes: mixing and stirring doxorubicin, nucleic acid nanoparticles, and the first solvent to obtain a premixed system; removing the premixed system from the premixed system; Free substance to get a drug containing adriamycin.
  • the specific amount of doxorubicin 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 adriamycin added to the first solvent is preferably 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 removing the free substance in the premixed system includes: mixing the premixed system with anhydrous ethanol, and precipitating a drug containing adriamycin at a temperature lower than 10 ° C; more preferably at a temperature of 0 to 5 ° C Under the conditions, doxorubicin-containing drugs were precipitated.
  • the step of mounting doxorubicin by means of covalent attachment includes: configuring a doxorubicin solution; and adducting the doxorubicin solution outside the G ring of the nucleic acid nanoparticle under the mediation of formaldehyde.
  • the amino group is reacted to obtain a reaction system; the reaction system is purified to obtain a doxorubicin-containing drug.
  • the reaction step includes: mixing the doxorubicin solution with the paraformaldehyde solution and the 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, One or more of DMSO, PBS and glacial acetic acid.
  • the nucleic acid nanoparticles can be prepared by self-assembly, for example: (1) simultaneously mixing 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 100V in TBM buffer at 4 ° C; (4) Cut the target band and elute in 37 ° C in RNA / DNA elution buffer After that, it was precipitated with ethanol 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 and / or covalently connecting the biologically active substance described above.
  • the method of linking is mounted on the nucleic acid domain to obtain the 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 connection. At the same time, it may also be connected by clicking on the link. . But 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 preferred number of 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 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 connected with the physical insertion and the covalently connected drug is 1 ⁇ 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, thiol or amino, and the nucleic acid
  • the site for the alkynyl or azide modification of the domain is selected from amino, imino or hydroxyl.
  • nucleic acid nanoparticles formed by self-assembly of the sequences or sequence deformations can also be used as basic structural units, and can be further polymerized to form multimers, such as dimers and trimers according to the needs of practical applications. , 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 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 position of the band lags behind the a and b strands.
  • the actual situation is consistent with the theory, which proves that the above RNA single strands form a stable composite structure through self-assembly To form RNA nanoparticles.
  • RNA core sequences SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 can successfully self-assemble into RNA nanoparticles 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 core sequence that forms a nucleic acid domain, as well as various extended sequences (including drug-mount binding sequences) that promote the mounting function of 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 7 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, R-7.
  • Measurement method Prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell. Open the sample cell cover of the instrument and place it in the instrument.
  • 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 target heads, 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 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.
  • the extended RNA oligonucleotide sequence formed by adding an extension to the a, b, and c sequences of the sequence, 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 therapeutic 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 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 images of 7 groups of conventional sequence RNA self-assembly products are 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 modified a Survivin siRNA nucleic acid interference therapy fragment and still has a stable self-assembling structure.
  • the nucleic acid nanoparticles in the present invention can mount nucleic acid drugs and have nucleic acid drug delivery. Carrier function.
  • Measurement method Prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell. Open the sample cell cover of the instrument and place it in the instrument.
  • extension fragments can also successfully self-assemble into structurally stable RNA nanoparticles.
  • the added extension fragments enable the RNA nanoparticles to have superior drug-mounting properties (for details, see Examples 5 and 7).
  • 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 ') ,
  • the conventional sequence DNA nanoparticle formed after adding the 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 ') , The conventional sequence DNA nanoparticle formed after adding the 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 ') , The conventional sequence DNA nanoparticle formed after adding the 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 of DNA nanoparticles formed by adding an extended sequence containing a PSMAapt target (see underlined section);
  • D-5 is based on the core sequence (12) described above (a sequence: 5'-GCAGCGTTCG-3 ', b sequence: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3 ') , The conventional sequence of DNA nanoparticles formed by adding an extended sequence containing a 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; the 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.
  • the target band is cut and eluted in a DNA elution buffer at 37 ° C, followed by ethanol precipitation overnight, and then 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.
  • DNA nanoparticles can also be successfully assembled, and they also have drug-loading, cell-targeting, and See traceability and other properties (for details, see Example 6 and Example 8).
  • Measurement method Prepare a potential sample (the self-assembled product is dissolved in ultrapure water) and place it in the sample cell. Open the sample cell cover of the instrument and place it in the instrument.
  • the conventional sequence DNA self-assembly product D-7 is irradiated with a transmission electron microscope, and the steps are as follows:
  • Nucleic acid nanoparticles RNA nanoparticles from Example 1.
  • Dox Doxorubicin
  • reaction solution was extracted with chloroform (10 mL ⁇ 3), and then 10 times the volume of absolute ethanol was added, and after mixing, the product was analyzed at 4 ° C. in the dark to allow the product to fully analyze (4 hours). After centrifugation, the supernatant was transferred, and the solid product was washed with ethanol again. The solvent was evaporated under reduced pressure at low temperature to obtain the mounted product as a dark red solid.
  • RNAh -2 1.21ug / ul, M RNAh ⁇ 30,000, 100ul;
  • C Doxorubicin -1 9.200uM, 100ul;
  • RNA particles Dissolve the RNA particles in 200ul DEPC water and add to the adriamycin-PBS mixed solution, mix well and adjust the pH to about 7.5;
  • Example 5 shows that the RNA nanoparticles (in Example 1) with the extension, target and fluorescein have the function of loading drugs, and can be physically inserted and covalently linked (paraformaldehyde-solvent co- Price) way to achieve drug mounting.
  • Example 5 According to the chemical method of mounting method in Example 5 (except for the special limitation, the method is the same as that in Example 5), using the DNA nanoparticles in Example 1 and R-1, R-2, and R in Example 2 respectively. -3, RNA nanoparticles formed by R-4, R-5, R-6, and R-7 self-assembly, and DNA nanoparticles formed by D-2, D-6, and D-7 in Example 4
  • the carrier of doxorubicin, the measured mount rates of doxorubicin are as follows:
  • the doxorubicin mount ratio of the DNA nanoparticles in Example 1 was 300 (in this method, doxorubicin was 1.2 mg, DEPC water was 0.5 mg, PBS buffer solution was 8.5 ml, and 4% paraformaldehyde aqueous solution was 1 ml. DNA nanoparticles are 2.5nmol, DNA nanoparticles are dissolved in 20 ⁇ l of water).
  • Doxorubicin mount rate of RNA nanoparticle R-1 is 3.5;
  • Doxorubicin mount rate of RNA nanoparticle R-2 is 2.4;
  • Doxorubicin mount ratio of RNA nanoparticles R-3 is 4.8;
  • Doxorubicin mount rate of RNA nanoparticle R-4 is 3.5;
  • Doxorubicin mount rate of RNA nanoparticles R-5 is 12.5;
  • Doxorubicin mount rate of RNA nanoparticles R-6 is 2.8;
  • DNA nanoparticle D-2 has a doxorubicin mount rate of 14;
  • Doxorubicin mount rate of DNA nanoparticle D-6 is 11;
  • DNA nanoparticle D-7 has a doxorubicin mount factor of 10.
  • RNAh 28083 PBS 2 RNAh-Biotin-quasar670 29552.6 PBS 3 RNAh-Biotin-quasar670-Dox 41232.6 DMSO
  • RNAh in the table refers to the control nanoparticles without biotin modification in the self-assembled RNA nanoparticles in Example 1
  • RNAh-Biotine-quasar670 refers to the RNA nanoparticles formed by self-assembly in Example 1. Nanoparticles formed after the quasar670 fluorescein is modified at the 5 ′ end.
  • RNAh-Biotine-quasar670-Dox refers to the nanoparticles formed after further mounting the doxorubicin drug (chemically mounted 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 Stetemcell, 07901-500mL
  • DMSO Sigma, D5879-1L
  • Inverted Microscope (Olympus IX71, TH4-200); Flow Cytometer (Life Science, Attune NxT).
  • HepG2 cells were cultured in RPMI1640 + 10% FBS + 1% PS medium at 37 ° C and 5% CO 2 .
  • A corresponds to the HepG2 cell control group
  • B corresponds to the 200nM RNAh control nanoparticles
  • C corresponds to the 200nM RNAh-Biotin-quasar670 nanoparticles
  • D corresponds to the 200nM RNAh-Biotin-quasar670-Dox nanoparticles
  • E Corresponding to 400nM RNAh control nanoparticles
  • F corresponds to 400nM RNAh-Biotin-quasar670 nanoparticles
  • G corresponds to 400nM RNAh-Biotin-quasar670-Dox nanoparticles.
  • RNAh-Biotin-quasar670 and RNAh-Biotin-quasar670-Dox nanoparticles have a strong ability to bind HepG2 cells (P ⁇ 0.0001).
  • Figure 13 shows the results of microscopic detection of nanoparticle binding and internalization with HepG2 cells.
  • the results of cell binding and internalization experiments showed that both RNAh-Biotin-quasar670 and RNAh-Biotin-quasar670-Dox nanoparticles can bind to HepG2 cells and internalize (Among them, adriamycin-loaded nanoparticles RNAh-Biotin-quasar670- After co-incubation of Dox and HepG2 cells, the cells were obviously stained red, and the color became darker as the concentration and time of RNAh-Biotin-quasar670-Dox nanoparticles increased.
  • RNAh-Bio-quasar670 also has the ability to bind and internalize HepG2 cells, but because it does not contain Dox, it cannot be stained red).
  • DOX-D-1-EGFR refers to the nanoparticles formed by self-assembly of DNA nanoparticles D-1 in the aforementioned Example 4 after doxorubicin is mounted (the mounting steps are the same as in Example 5, and the same below) ( D-1 itself is mounted with EGFR, which is expressed here as DOX-D-1-EGFR to clarify the target type and doxorubicin mount, the same below);
  • DOX-D-2-EGFR refers to the aforementioned implementation
  • the self-assembled DNA nanoparticle D-2 is a nanoparticle formed after mounting doxorubicin;
  • DOX-D-5-PSMA refers to the self-assembled DNA nanoparticle D-5 mounted in the previous embodiment. Nanoparticles formed after mycin.
  • FBS fetal bovine serum (GBICO, Cat # 10099141).
  • the cells are recovered to the corresponding medium, and cultured in a 5% CO 2 cell incubator at 37 ° C.
  • the sample should be protected from light before loading on the flow cytometer.
  • nucleic acid nanoparticles including RNA nanoparticles and remaining DNA nanoparticles
  • DOX-D-1-DNAh-EGFR, DOX-D-2-EGFR or DOX-D-5-PSMA has the same target EGFRapt or PSMAapt, and therefore both have binding efficiency equivalent to corresponding cells.
  • Test sample RNA nanoparticles prepared in Example 1 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. 14 shows the electrophoresis results of 8% non-denaturing gel (Coomassie Blue program)
  • FIG. 15 shows the electrophoresis results of 8% non-denaturing gel (Stain Free Gel program).
  • the results of serum stability test of RNA nanoparticles showed that the results of non-denaturing gels at 10min, 1h, 12h, and 36h ( Figure 14 and Figure 15) showed no significant difference in the bands of RNA nanoparticles at different times, indicating that % FBS in 1640 medium is relatively stable without significant degradation.
  • the samples to be tested are the three samples in Example 7.
  • 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
  • Dox H3SUN Pharm, H33021980-10mg
  • CCTG CellTiter-Glo Luminescent Cell Viability Assay Kit
  • Inverted Microscope (Olympus IX71, TH4-200); 96-well Plate Reader (Molecular Devices, Flexstation 3).
  • HepG2 cells were cultured in RPMI1640 + 10% FBS + 1% PS medium at 37 ° C and 5% CO 2 .
  • HepG2 cells were trypsinized, 100 ⁇ L of 5000 cells per well was seeded in a 96-well plate, and cultured overnight at 37 ° C. and 5% CO 2 .
  • RNAh, RNAh-Biotine, RNAh-Dox, and Dox were respectively added to each of the plated cells in 100 ⁇ L, and each sample was repeated 4 times.
  • a corresponds to the cell proliferation result of PBS
  • b corresponds to the cell proliferation result of DMSO
  • c corresponds to the cell proliferation result of Dox (doxorubicin)
  • d corresponds to the cell proliferation result of RNAh
  • e corresponds to the cell proliferation result of RNAh-Biotin-quasar670
  • f corresponds to the cell proliferation result of RNAh-Biotin-quasar670-Dox.
  • 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. Open the sample cell cover of the instrument and place it in the instrument.
  • the dissolution curve method was used to detect the TM values 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 18, the dissolution profile of R-16 is shown in Figure 19, and the dissolution profile of R-17 is shown in Figure 20.
  • the dissolution profile of R-18 is shown in Figure 21, the dissolution profile of R-19 is shown in Figure 22, the dissolution profile of R-20 is shown in Figure 23, and the dissolution profile of R-21 is shown in Figure 24. Due to the specificity of the RNA sample, the temperature corresponding to 1/2 of the 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 had good structural stability.
  • 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. Open the sample cell cover of the instrument and place it in the instrument.
  • 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 as follows.
  • the dissolution profile of D-8 is shown in Figure 26
  • the dissolution profile of D-9 is shown in Figure 27, and the dissolution profile of D-10 is shown in Figure 28.
  • the dissolution profile of D-11 is shown in Figure 29
  • the dissolution profile of D-12 is shown in Figure 30
  • the dissolution profile of D-13 is shown in Figure 31
  • the dissolution profile of D-14 is shown in Figure 32.
  • the non-denaturing PAGE method was used to characterize the stability of 7 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 samples according to the method in the table, dilute 5 tubes, and dilute the samples 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 Fig. 33
  • the electrophoretic test results of R-16 are shown in Fig. 34
  • the electrophoretic test results of R-17 are shown in Fig. 35
  • the electrophoretic test results of R-18 are shown in Fig. 36
  • the electrophoretic test results of R-19 The results are shown in Figure 37
  • the electrophoretic results of R-20 are shown in Figure 38
  • the electrophoretic results of R-21 are shown in Figure 39.
  • 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 segment deformation + core short sequence DNA nanoparticles in serum.
  • the main reagents and instruments are as follows:
  • the electrophoretic test result of D-8 is shown in Figure 40
  • the electrophoretic test result of D-9 is shown in Figure 41
  • the electrophoretic test result of D-10 is shown in Figure 42
  • the electrophoretic test result of D-11 is shown in Figure 43
  • the electrophoretic test of D-12 is shown
  • the results are shown in Figure 44
  • the results of D-13 are shown in Figure 45
  • the results of D-14 are shown in Figure 46.
  • the lanes from left to right are M: marker; 1: 36h; 2: 12h; 3: 1h; 4: 10min; 5: 0min.
  • Example 12 According to the mounting method of the chemical method of Example 5 (except for the special limitation, the method is the same as that of Example 5), and R-15, R-16, R-17, R-18, and R-19 in the aforementioned Example 11 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 formed in Example 12
  • the DNA nanoparticles were used as a carrier for doxorubicin, and the measured mount rates of doxorubicin were as follows:
  • Doxorubicin mount rate of RNA nanoparticles R-15 is 20.5;
  • RNA nanoparticles R-16 The 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 12 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.
  • Reagent name Manufacturer Article number Remark DMEM biotin-free Provided by Baiyao Zhida YS3160 Zh 1% BSA-PBS Self-provisioning - Zh
  • 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 Example 12 described as: 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 12 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 method for preparing the sample in the C8 well is: complete medium 324 ⁇ L, and then aspirate from the C9
  • the mounting 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).
  • the original drug doxorubicin was first prepared 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 12 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 48).
  • the measured mounting rates of daunorubicin are as follows:
  • the mount rate of daunorubicin of DNA nanoparticle D-10 is 24.0;
  • the mount rate of daunorubicin for DNA nanoparticles D-14 was 25.1.
  • the present application provides a series of nucleic acid nanoparticle carriers with thermodynamic stability, chemical stability, high loading rate, and multivalent combination modules. .
  • the unique modular design of this type of carrier results in a core module structure that not only maintains a natural compatible affinity, but also has a high degree of stability and a variety of combined features.
  • This structure can flexibly and efficiently integrate various functional modules, including targeting modules, imaging and probe modules, treatment modules, and other composite intelligent modules, so that it can be used for targeted delivery in vivo to achieve precise diagnosis and treatment.

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Abstract

一种核酸纳米颗粒、包含其的药物组合物、含阿霉素的药物及其制备方法。该核酸纳米颗粒具有核酸结构域，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***、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***、缺失或替换的序列，c序列包含d序列或者d序列发生至少一个碱基***、缺失或替换的序列。该核酸纳米颗粒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。作为载体时不仅能够挂载和递送核酸药物，还可适用于化学药物等其他生物活性物质的挂载和递送。

Description

核酸纳米颗粒、包含其的药物组合物、含阿霉素的药物及其制备方法 技术领域
本发明涉及递送载体领域，具体而言，涉及一种核酸纳米颗粒、包含其的药物组合物、含阿霉素的药物及其制备方法。
背景技术
癌症已成为危害人类健康的最主要疾病，药物治疗，包括细胞靶向给药、基因治疗、RNAi等均是攻克癌症的前沿研究领域。然而许多抗癌药物存在难溶于水或稳定性差等缺陷。如阿霉素等都因溶解度差而难以被生物体很好地利用，解决其水溶性问题是这类药物制剂临床应用的关键。此外，肿瘤治疗和诊断药物的作用大多是非选择性的，通常在治疗剂量下对正常组织器官的毒副作用大。与许多蛋白类物质的递送一样，核酸类物质在胃肠道中的降解和较低的生物利用度是siRNA的口服递送的主要障碍。即使对于静脉内递送，常规siRNA被血清因子快速降解，从而不能到达其靶位置。
药物活性成分往往由于无法有效到达病灶部位或过量作用于正常细胞和组织，而无法实现治疗目的或产生毒副作用。例如比较典型的有铂类药物，其广泛用于卵巢癌、小细胞肺癌、睾丸癌、头颈部鳞癌、子***、非小细胞肺癌、膀胱癌、胸膜间皮瘤、黑色素瘤及子宫内膜癌的化疗治疗。但是铂类药物的靶向性差，一般药物剂量无法达到治疗效果，过多剂量又会导致药物大量作用于正常细胞和组织，造成化疗后人体骨髓抑制作、胃肠道反应、肾毒性、神经毒性等不良反应，因而限制了铂类化疗药物的使用。
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。例如，在癌症的化疗治疗中，递送载体将化疗药物输送至癌细胞内，使药物活性成分与癌细胞内DNA相互作用，对肿瘤产生抑制作用。目前常用的铂类化疗药物递送载体包括脂质体、胶束、纳米囊、聚合物-铂偶联物和碳纳米管等。
此外，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。然而，由上述可知，现有的非病毒载体的研究更多地着眼于核酸药物，而针对非核酸类的药物的递送效果尚无有价值的报道。
因此，如何提供一种可靠的药物载体是解决目前药物临床应用受限的关键。
发明内容
本发明的主要目的在于提供一种核酸纳米颗粒、包含其的药物组合物、含阿霉素的药物及其制备方法，以提供一种可靠的药物载体来解决目前药物临床应用受限的问题。
为了实现上述目的，根据本发明的一个方面，提供了一种核酸纳米颗粒，其具有核酸结构域，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***、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***、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***、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***、缺失或替换的序列。
进一步地，碱基***、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 PCTCN2019095766-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，核酸结构域中，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，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。
进一步地，核酸结构域的相对分子量与生物活性物质的总相对分子量之比≥1:1；优选地，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、药物、蛋白、多肽、类黄酮、葡萄糖、天然水杨酸、单抗、维生素、酚类以及卵磷脂中的一种或多种。
进一步地，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。
进一步地，药物为治疗肝癌、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、***癌、鼻咽癌、食道癌、口腔癌、红斑狼疮的药物；优选地，头颈癌为脑癌、神经母细胞瘤或胶质母细胞瘤。
进一步地，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、EGFR及PSMA的抗体或适配体中的一种或多种；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，生物活性物质通过如下任一方式与核酸结构域相连：方式一：物理嵌插；方式二：共价连接。
进一步地，生物活性物质与核酸结构域以物理嵌插方式相连时，生物活性物质与核酸结构域按照1～200：1的摩尔比进行物理嵌插。
进一步地，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。
进一步地，共价连接方式连接的生物活性物质通过溶剂共价连接、linker共价连接或点击链接；优选地，溶剂选自多聚甲醛、DCM、DCC、DMAP、Py、DMSO、PBS或冰醋酸；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。
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。
根据本发明的另一方面，还提供了一种药物组合物，该药物组合物包括上述的核酸纳米颗粒。
根据本发明的第三个方面，还提供了一种含阿霉素的药物，该含阿霉素的药物包括阿霉素及前述的(不挂载生物活性物质的)核酸纳米颗粒。
进一步地，阿霉素通过物理连接和/或共价连接的形式挂载在核酸纳米颗粒上，且阿霉素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、酚类卵磷脂以及除阿霉素以外的小分子药物中的一种或多种。
进一步地，将核酸结构域的相对分子量记为N 1，将阿霉素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，除阿霉素以外的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。
根据本发明的第四个方面，还提供了一种含阿霉素的药物的制备方法，该制备方法包括以下步骤：提供权前述任一种(不挂载生物活性物质的)核酸纳米颗粒；通过物理连接和/或共价连接的方式将阿霉素挂载在核酸纳米颗粒上，得到含阿霉素的药物。
进一步地，通过物理连接的方式挂载阿霉素的步骤包括：将阿霉素、核酸纳米颗粒及第一溶剂混合并搅拌，得到预混体系；去除预混体系中的游离物质，得到含阿霉素的药物；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，去除预混体系中的游离物质的步骤包括：将预混体系与无水乙醇混合，在低于10℃的温度条件下析出含阿霉素的药物；更优选在0～5℃温度条件下析出含阿霉素的药物。
进一步地，通过共价连接的方式挂载阿霉素的步骤包括：配置阿霉素溶液；使阿霉素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，得到含阿霉素的药物；优选地，反应的步骤包括：将阿霉素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，上述制备方法还包括制备核酸纳米颗粒的步骤，其包括：通过将前述任一种(不挂载生物活性物质的)核酸纳米颗粒中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，其中，生物活性物质中的药物为除阿霉素之外的小分子药物。
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。
本发明提供的核酸纳米颗粒，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siRNA药物或miRNA药物，在形成上述纳米颗粒的过程中，由于核酸结构域的存在，减少了核酸酶对所挂载的核酸药物的降解作用，提高了药物的递送的可靠性和稳定性。
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发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明实施例1中自组装形成的RNA纳米颗粒的电泳检测结果；
图2示出了本发明实施例1中自组装形成的DNA纳米颗粒的电泳检测结果；
图3示出了本发明实施例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示出了本发明实施例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示出了本发明实施例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示出了本发明实施例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示出了本发明实施例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示出了本发明实施例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示出了本发明实施例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示出了本发明实施例5中的阿霉素挂载产物的电泳检测结果；
图11示出了本发明实施例5中挂载率检测过程中采用的阿霉素吸光度的标准曲线；
图12示出了本发明实施例7中不同纳米颗粒的FACS荧光信号强度检测结果；
图13示出了本发明实施例7中不同纳米颗粒与HepG2细胞结合和内化结果；
图14示出了本发明实施例9中RNA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15示出了本发明实施例9中RNA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16示出了本发明实施例10中不同纳米颗粒的HepG2细胞增殖情况检测结果；
图17示出了本发明实施例11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18示出了本发明实施例11中RNA纳米颗粒R-15的溶解曲线；
图19示出了本发明实施例11中RNA纳米颗粒R-16的溶解曲线；
图20示出了本发明实施例11中RNA纳米颗粒R-17的溶解曲线；
图21示出了本发明实施例11中RNA纳米颗粒R-18的溶解曲线；
图22示出了本发明实施例11中RNA纳米颗粒R-19的溶解曲线；
图23示出了本发明实施例11中RNA纳米颗粒R-20的溶解曲线；
图24示出了本发明实施例11中RNA纳米颗粒R-21的溶解曲线；
图25示出了本发明实施例12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26示出了本发明实施例12中DNA纳米颗粒D-8的溶解曲线；
图27示出了本发明实施例12中DNA纳米颗粒D-9的溶解曲线；
图28示出了本发明实施例12中DNA纳米颗粒D-10的溶解曲线；
图28示出了本发明实施例12中DNA纳米颗粒D-11的溶解曲线；
图30示出了本发明实施例12中DNA纳米颗粒D-12的溶解曲线；
图31示出了本发明实施例12中DNA纳米颗粒D-13的溶解曲线；
图32示出了本发明实施例12中DNA纳米颗粒D-14的溶解曲线；
图33示出了本发明实施例13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34示出了本发明实施例13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35示出了本发明实施例13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36示出了本发明实施例13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37示出了本发明实施例13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38示出了本发明实施例13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39示出了本发明实施例13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40示出了本发明实施例14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41示出了本发明实施例14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42示出了本发明实施例14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43示出了本发明实施例14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44示出了本发明实施例14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45示出了本发明实施例14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46示出了本发明实施例14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47a、图47b、图47c、图47d、图47e、图47f、图47g、图47h分别示出了本发明实施例17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48示出了实施例18的挂载率检测过程中采用的柔红霉素吸光度的标准曲线。
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发明。
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主装的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各种药物，并能在血清中稳定存在；进一步的实验验证，其能携带药物进入细胞，且单独的载体对细胞无毒性。而携带药物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。在一种典型的实施方式中，提供了一种核酸纳米颗粒，该核酸纳米颗粒具有核酸结构域，该核酸结构域包含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***、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***、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***、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’。
上述核酸纳米颗粒，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siRNA药物或miRNA药物。在形成上述纳米颗粒的过程中，由于核酸结构域的存在，减少了核酸酶对所挂载的核酸药物的降解作用，提高了药物的递送的可靠性和稳定性。
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***、缺失或替换的序列。该RNA纳米颗粒中变异序列的具***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。
为了使所制备的核酸纳米颗粒具有相对更高的稳定性，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***、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，不仅能够挂载和递送核酸药物，同样适用于化学药物等其他生物活性物质的挂载和递送，因而使其具有相对普适性。
在一种优选的实施例中，上述碱基***、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，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 PCTCN2019095766-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以及所欲挂载的药物的种类或性质的区别，所挂载的药物的量也有所差异。同样，对于核酸类药物，可以在a序列、b序列和c序列中任一序列的5'端和/或3'端通过延伸挂载。
根据所挂载的药物的分子量的大小，或者在挂载时的结构上位阻的差异，为了使上述核酸药物能够挂载分子量相对较大的生物活性物质，在一种优选的实施例中，上述核酸结构域中，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、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 PCTCN2019095766-appb-000003
b序列为(SEQ ID NO:50)：
Figure PCTCN2019095766-appb-000004
c序列为(SEQ ID NO:51)：
Figure PCTCN2019095766-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化学药物的挂载之用(吲哚类药物分子优选与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。
本申请所提供的上述核酸纳米颗粒作为载体之用所能挂载的物质可以是任何具有生物活性作用的物质。因而，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。
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生物活性物质的总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，核酸结构域的相对分子量与生物活性物质的总相对分子量之比≥1:1；优选地，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、药物(通常解释为小分子药物，即化学合成药物)、蛋白、多肽、类黄酮、葡萄糖、天然水杨酸、单抗、维生素、酚类以及卵磷脂中的一种或多种。
根据具体挂载的生物活性物质的种类的不同，其对本申请的核酸纳米颗粒的性能优化作用并不相同。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核酸纳米颗粒 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、药物(通常解释为小分子药物)、蛋白、多肽或RNA抗体时，根据不同生物学功能的不同，可能使得该核酸纳米颗粒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，前述抗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药物时，根据不同药物所能治疗的疾病类型，药物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、***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，药物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(epidermal growth factor receptor)、PSMA(***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。
根据所挂载的生物活性物质的不同，可以选择合适的连接方式与上述核酸纳米载体进行连接。在一种优选的实施例中，上述生物活性物质通过如下任意一种方式与核酸结构域相连：方式一：物理嵌插；方式二：共价连接。
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纳米载体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纳米载体连接，也可以通过物理嵌插与共价连接的方式与核酸纳米载体连接，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。
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，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相连时，生物活性物质与核酸结构域按照1～200：1的摩尔比进行物理嵌插。该连接方式适用于蒽环类、吖啶类药物。在该比例范围内进行物理嵌插既能满足挂载需求，又能满足药效需求。
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。
在一种优选的实施例中，共价连接方式连接的生物活性物质通过溶剂共价连接、linker共价连接或点击链接；优选地，溶剂选自多聚甲醛、DCM、DCC、DMAP、Py、DMSO、PBS或冰醋酸；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。
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。
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。
在第二种典型的实施方式中，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颗粒。含有本申请所提供规定上述核酸纳米颗粒的药物中，核酸结构域可经靶向目的细胞的靶头修饰而具有良好的靶向性，同时还可以挂载相应的治疗性药物和/或示踪性分子，从而能够稳定地递送治疗性药物和/或示踪性分子，可靠性很高。
在第三种典型的实施方式中，提供了一种含阿霉素的药物，该含阿霉素的药物包括阿霉素及前述任一种(不挂载生物活性物质的)核酸纳米颗粒；或者，该含阿霉素的药物为前述任一种(挂载生物活性物质的)核酸纳米颗粒，其中，生物活性物质至少为药物，该药物包括阿霉素。
上述提供的含阿霉素的药物中包括核酸纳米颗粒和阿霉素，且阿霉素挂载在核酸纳米颗粒上。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'末端连接阿霉素，或者能够使阿霉素稳定地嵌插在核酸结构域的链间。本发明提供的含阿霉素的药物，其核酸结构域经过靶头修饰后，可具有较好的靶向性，能够稳定地递送阿霉素，可靠性很高。
如前述，当生物活性物质为药物，且药物为阿霉素时，阿霉素可以通过物理连接和/或共价连接的形式进行挂载。当阿霉素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，阿霉素通常会与G环外氨基发生化学反应形成共价连接。更优选地，阿霉素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。
本申请所提供的含阿霉素的药物中，核酸纳米颗粒是作为阿霉素的递送载体，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、卵磷脂以及除阿霉素以外的小分子药物中的一种或多种。
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阿霉素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，将阿霉素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，本发明中含阿霉素的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含阿霉素的药物具有靶向性，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、除阿霉素以外的小分子药物时，根据不同生物学功能的不同，可能使得该含阿霉素的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。
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，所述抗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阿霉素以外的其他小分子药物时，根据不同药物所能治疗的疾病类型，药物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、***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阿霉素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(epidermal growth factor receptor)、PSMA(***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。
根据本发明的第三种典型的实施方式中，还提供了一种上述含阿霉素的药物的制备方法，其包括以下步骤：提供上述(不挂生物活性物质的)核酸纳米颗粒；通过物理连接和/或共价连接的方式将阿霉素挂载在核酸纳米颗粒上，得到含阿霉素的药物。
当采用物理连接方式时，阿霉素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，阿霉素通常会与G环外氨基发生化学反应形成共价连接。利用上述 方法制备的含阿霉素的药物，其经过靶头修饰后，可具有较好的靶向性，能够稳定地递送阿霉素，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阿霉素的步骤包括：将阿霉素、核酸纳米颗粒及第一溶剂混合并搅拌，得到预混体系；去除预混体系中的游离物质，得到含阿霉素的药物。具体的阿霉素、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阿霉素量为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，去除预混体系中的游离物质的步骤包括：将预混体系与无水乙醇混合，在低于10℃的温度条件下析出含阿霉素的药物；更优选在0～5℃温度条件下析出含阿霉素的药物。
在一种优选的实施例中，通过共价连接的方式挂载阿霉素的步骤包括：配置阿霉素溶液；使阿霉素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，得到含阿霉素的药物。
通过甲醛介导的形式，可以发生如下反应：
Figure PCTCN2019095766-appb-000006
优选地，反应的步骤包括：将阿霉素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。
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。
为了使上述含阿霉素的药物具有其他功能，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所述核酸结构域上，进而得到所述核酸纳米颗粒。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，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，具体见表1：
表1：
Figure PCTCN2019095766-appb-000007
Figure PCTCN2019095766-appb-000008
(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：
表2：R-1：
Figure PCTCN2019095766-appb-000009
表3：R-2：
Figure PCTCN2019095766-appb-000010
表4：R-3：
Figure PCTCN2019095766-appb-000011
Figure PCTCN2019095766-appb-000012
表5：R-4：
Figure PCTCN2019095766-appb-000013
表6：R-5：
Figure PCTCN2019095766-appb-000014
表7：R-6：
Figure PCTCN2019095766-appb-000015
Figure PCTCN2019095766-appb-000016
表8：R-7：
Figure PCTCN2019095766-appb-000017
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
(1)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：
Figure PCTCN2019095766-appb-000018
表10：
Figure PCTCN2019095766-appb-000019
表11：
Figure PCTCN2019095766-appb-000020
表12：
Figure PCTCN2019095766-appb-000021
表13：
Figure PCTCN2019095766-appb-000022
表13：
Figure PCTCN2019095766-appb-000023
表14：
Figure PCTCN2019095766-appb-000024
Figure PCTCN2019095766-appb-000025
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：
表15：R-8：
Figure PCTCN2019095766-appb-000026
表16：R-9：
Figure PCTCN2019095766-appb-000027
Figure PCTCN2019095766-appb-000028
表17：R-10：
Figure PCTCN2019095766-appb-000029
表18：R-11：
Figure PCTCN2019095766-appb-000030
表19：R-12：
Figure PCTCN2019095766-appb-000031
Figure PCTCN2019095766-appb-000032
表20：R-13：
Figure PCTCN2019095766-appb-000033
表21：R-14：(下述a链中的 uGAcAGAuAAGGAAccuGcudTdT为survivin siRNA)
Figure PCTCN2019095766-appb-000034
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，也说明了本发明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22：
Figure PCTCN2019095766-appb-000035
Figure PCTCN2019095766-appb-000036
表23：
Figure PCTCN2019095766-appb-000037
表24：
Figure PCTCN2019095766-appb-000038
表25：
Figure PCTCN2019095766-appb-000039
表26：
Figure PCTCN2019095766-appb-000040
表27：
Figure PCTCN2019095766-appb-000041
表28：
Figure PCTCN2019095766-appb-000042
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和实施例7)。
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：
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)：
GGGCCGAAAAAGACCTGACTTCTATACTAAGTCTACGTCCC。
表29：D-1：
Figure PCTCN2019095766-appb-000043
Figure PCTCN2019095766-appb-000044
表30：D-2：
Figure PCTCN2019095766-appb-000045
表31：D-3：
Figure PCTCN2019095766-appb-000046
Figure PCTCN2019095766-appb-000047
表32：D-4：
Figure PCTCN2019095766-appb-000048
表33：D-5：
Figure PCTCN2019095766-appb-000049
Figure PCTCN2019095766-appb-000050
表34：D-6：
Figure PCTCN2019095766-appb-000051
表35：D-7：
Figure PCTCN2019095766-appb-000052
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)电位检测；
(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(具体见实施例6和实施例8)。
(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36：
Figure PCTCN2019095766-appb-000053
表37：
Figure PCTCN2019095766-appb-000054
Figure PCTCN2019095766-appb-000055
表38：
Figure PCTCN2019095766-appb-000056
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39：
Figure PCTCN2019095766-appb-000057
(4)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。
阿霉素(Dox)挂载实验
实施例5
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)阿霉素(Dox)。
(6)氯仿：北化。
(7)无水乙醇：北化。
2.实验方法：
(1)精密称取阿霉素(5.0mg，8.6μmol，40eq.)溶于DEPC水(1.8mL)及PBS缓冲液(2.1mL)，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(215nmol)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的阿霉素水溶液及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10倍体积的无水乙醇，混匀后避光置于4℃使产物充分析出(4小时)。离心，转移上清，固体产物再次以乙醇洗，于低温下减压挥干溶剂得挂载产物为暗红色固体。
(4)取少量产物溶于DEPC水，上样于8％PAGE胶中，在TBM缓冲液中4℃条件下100V电泳1小时，电泳结果见图10。图10中，从左至右，泳道1至5分别是1)20bp DNA ladder、2-4)RNA纳米颗粒空白颗粒和5)阿霉素挂载产物。从图10可以看到阿霉素挂载产物条带位于RNA纳米颗粒空白颗粒后面一点。
(5)挂载率计算：
1.配置已知浓度的阿霉素-PBS标准液：2uM、4uM、6uM、8uM、10uM，各100ul；
2.将阿霉素挂载产物溶解在100ul PBS中；
3.将标准液与阿霉素挂载产物置于PCR板中，于85℃加热5min，随后冷却至室温；
4.利用酶标仪测量492nm处阿霉素的吸光度，绘制标准曲线(如图11所示)，计算得出挂载产物中阿霉素的摩尔浓度；
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阿霉素挂载产物的质量浓度；
6 根据测量得到的阿霉素摩尔浓度及阿霉素挂载产物的质量浓度，计算挂载率。
计算具体过程如下：
C RNAh-1＝9.5ug/ul，M RNAh≈30000，100ul；C 阿霉素-1＝8.033uM，100ul；
C RNAh-2＝1.21ug/ul，M RNAh≈30000，100ul；C 阿霉素-1＝9.200uM，100ul；
Figure PCTCN2019095766-appb-000058
取N-1和N-2的平均值得到RNAh-阿霉素的挂载率约为24，及表示每一个核酸纳米颗粒载体上能够挂载约24个阿霉素分子。
物理法挂载：
1)阿霉素与RNA纳米颗粒的质量比为1:1；
2)称取0.1mg阿霉素原药溶于50ul DMSO，随后加入300ul PBS混匀；
3)将RNA颗粒溶于200ul DEPC水中，并加入到阿霉素-PBS混合液中，混匀，调pH约为7.5；
4)将全部溶液置于55℃水浴锅中，反应3h；
5)反应结束后，直接加入10倍体积的无水乙醇，4℃析出4h；
6)以10倍的无水乙醇洗涤4遍，并转移至1.5mL EP管中。随后，进行挂载率的测定，测定方法同上，测定结果为阿霉素挂载率为15.5。
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，而且可以通过物理嵌插和共价连接(多聚甲醛—溶剂共价)的方式来实现药物挂载。
实施例6
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1中的DNA纳米颗粒、实施例2中的R-1、R-2、R-3、R-4、R-5、R-6、R-7自组装形成的RNA纳米颗粒、实施例4中D-2、D-6和D-7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：
实施例1中的DNA纳米颗粒的阿霉素挂载率为300(该方法中，阿霉素为1.2mg，DEPC水为0.5mg，PBS缓冲液为8.5ml，4％多聚甲醛水溶液为1ml，DNA纳米颗粒为2.5nmol，DNA纳米颗粒溶于20μl水中)。
RNA纳米颗粒R-1的阿霉素挂载率为3.5；
RNA纳米颗粒R-2的阿霉素挂载率为2.4；
RNA纳米颗粒R-3的阿霉素挂载率为4.8；
RNA纳米颗粒R-4的阿霉素挂载率为3.5；
RNA纳米颗粒R-5的阿霉素挂载率为12.5；
RNA纳米颗粒R-6的阿霉素挂载率为2.8；
DNA纳米颗粒D-2的阿霉素挂载率为14；
DNA纳米颗粒D-6的阿霉素挂载率为11；
DNA纳米颗粒D-7的阿霉素挂载率为10。
流式细胞仪(FACS)实验检测RNA纳米颗粒的细胞结合能力
实施例7
一、实验材料和实验方法：
1.待测样品见表40：
表40：
纳米颗粒 MW 溶解试剂
1 RNAh 28083 PBS
2 RNAh-Biotin-quasar670 29552.6 PBS
3 RNAh-Biotin-quasar670-Dox 41232.6 DMSO
注：表中RNAh指的是实施例1中自组装形成的RNA纳米颗粒中不进行Biotin修饰的对照纳米颗粒，RNAh-Biotine-quasar670指的是在实施例1中自组装形成的RNA纳米颗粒的5’端修饰quasar670荧光素后形成的纳米颗粒，RNAh-Biotine-quasar670-Dox指的是进一步挂载阿霉素药物后(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)。
3.所用到的实验仪器如下：
倒置显微镜(Inverted Microscope)(Olympus IX71,TH4-200)；流式细胞仪(Flow Cytometer)(Life Science,Attune NxT)。
4.实验方法:
(1)用RPMI1640+10％FBS+1％PS培养基，于37℃和5％CO 2中培养HepG2细胞。
(2)胰酶消化HepG2细胞，并用PBS洗一遍。
(3)分别将2x10 5个细胞与RNAh、RNAh-Biotin-quasar670、RNAh-Biotin-quasar670-Dox纳米颗粒一起于37℃和5％CO 2中孵育1h，每个样品分别有200nM和400nM两个浓度，每个浓度下每个样品有3次重复。
(4)细胞用PBS洗涤后，再用PBS缓冲液重悬，并用FACS仪器检测。
(5)收集收据并统计分析。
二、实验结果
实验结果见表41、图12和图13。
表41：荧光阳性HepG2细胞(％)Mean±SEM(n＝3)
Figure PCTCN2019095766-appb-000059
Figure PCTCN2019095766-appb-000060
图12中，A对应HepG2细胞对照组，B对应200nM浓度的RNAh对照纳米颗粒，C对应200nM浓度的RNAh-Biotin-quasar670纳米颗粒，D对应200nM浓度的RNAh-Biotin-quasar670-Dox纳米颗粒，E对应400nM浓度的RNAh对照纳米颗粒，F对应400nM浓度的RNAh-Biotin-quasar670纳米颗粒，G对应400nM浓度的RNAh-Biotin-quasar670-Dox纳米颗粒。
从表41和图12中可以看出，单纯的不修饰靶头的RNA纳米颗粒不具备细胞靶向性，当挂载生物素之后能够与HepG2细胞结合。此外，图12的FACS结果显示，RNAh-Biotin-quasar670和RNAh-Biotin-quasar670-Dox纳米颗粒与HepG2细胞结合能力较强(P<0.0001)。
图13显示的是显微镜检测纳米颗粒与HepG2细胞结合和内化结果。细胞结合和内化实验结果显示，RNAh-Biotin-quasar670和RNAh-Biotin-quasar670-Dox纳米颗粒均能够与HepG2细胞结合并内化(其中，挂载阿霉素的纳米颗粒RNAh-Biotin-quasar670-Dox与HepG2细胞共孵育后，细胞明显被染红，并且随着RNAh-Biotin-quasar670-Dox纳米颗粒浓度和时间的增加而颜色加深，可以看出，载药RNA纳米颗粒与HepG2细胞结合和内化能力较强。RNAh-Bio-quasar670同样具有与HepG2细胞结合和内化的能力，只因不含Dox，因而不能被染成红色)。
实施例8
一、实验材料和试验方法：
1.待测样品见表42：
表42：
Figure PCTCN2019095766-appb-000061
注：DOX-D-1-EGFR指的是前述实施例4中自组装形成的DNA纳米颗粒D-1挂载阿霉素(挂载步骤同实施例5，下同)后形成的纳米颗粒(D-1中本身挂载有EGFR，此处表述为 DOX-D-1-EGFR是为了明晰靶头类型和阿霉素挂载，下同)；DOX-D-2-EGFR指的是前述实施例中自组装形成的DNA纳米颗粒D-2挂载阿霉素后形成的纳米颗粒；DOX-D-5-PSMA指的是前述实施例中自组装形成的DNA纳米颗粒D-5挂载阿霉素后形成的纳米颗粒。
2.细胞信息见表43：
表43：
Figure PCTCN2019095766-appb-000062
3.所用到的实验试剂及其来源如下：
RPMI-1640培养基(YY0167-500Ml)；
MEM(YS4150-500mL)；
MEM NEAA(100×)(GBICO,Cat#1872982)；
FBS胎牛血清(GBICO,Cat#10099141)。
4.所用到的实验仪器如下：
流式细胞仪Guava EasyCyte 8HT(Millipore)；
SpectraMax多标记微孔板检测仪，MD，2104-0010A。
5.实验方法：
5.1细胞培养
a)细胞复苏至对应的培养基中，于37℃、5％CO 2细胞培养箱中培养。
b)细胞在T75细胞培养瓶中达到对数生长期时(约80％汇合率)，更换原培养基为不含叶酸和生物素的培养基。
5.2结合实验
a)第一天收集细胞并计数，以2×10 5cell/well的密度铺至24孔板中。
b)第二天，使用PBS稀释样品。使用PBS将所有样品稀释至100μM，配制1μM溶液，在酶标仪上检测荧光基团(阿霉素：Ex＝480nm、Em＝580nm；)是否正常发光。
c)用PBS洗涤细胞2次。
d)加入溶于培养基的纳米颗粒，37℃的CO 2培养箱中孵育细胞16h。纳米颗粒浓度为2μM，样品顺序如下表44。
表44：
Figure PCTCN2019095766-appb-000063
e)用PBS洗涤细胞2次。
f)收集胰蛋白酶消化的细胞并用PBS洗涤细胞2次。
g)PBS洗涤过的细胞重悬于400uLPBS,转移到一个5mL的流式细胞管中。
h)流式细胞仪上样之前样品需要避光。
i)流式细胞仪检测。阿霉素在Ex＝480nm、Em＝580nm(黄色通道)检测细胞荧光强度。
j)用FlowJo软件分析FACS数据。
k)根据空白细胞组本底荧光强度设门，分析各DNA纳米颗粒与细胞结合比例。
二、实验结果
实验结果见表45、46和47
表45：样品酶标仪荧光检测结果
编号 样品 阿霉素，Ex＝480nm、Em＝580nm
  PBS 4.37
1 DOX-D-1-EGFR 280.178
2 DOX-D-2-EGFR 260.175
3 DOX-D-5-PSMA 295.964
表46：流式检测样品与细胞结合率
Figure PCTCN2019095766-appb-000064
Figure PCTCN2019095766-appb-000065
表47：流式检测MFI
Figure PCTCN2019095766-appb-000066
由上述表46、47的数据结果可知：DOX-D-1-DNAh-EGFR与U87MG细胞的结合能力很强，当给药浓度为2uM，给药时间为16h时结合效率为100％。DOX-D-2-EGFR与MDA-MB-231细胞的结合能力很强，当给药浓度为2uM，给药时间为16h时结合效率为100％。DOX-D-3-EGFR与HCC-78细胞的结合能力很强，当给药浓度为2uM，给药时间为16h时结合效率为100％。
关于其他核酸纳米颗粒(包括RNA纳米颗粒及剩余DNA纳米颗粒)，由于均携带或可以通过增加延长段的方式使其携带与DOX-D-1-DNAh-EGFR、DOX-D-2-EGFR或DOX-D-5-PSMA相同的靶头EGFRapt或PSMAapt，因而也都具有与相应细胞相当的结合效率。此外，均携带与DOX-D-1-DNAh-EGFR、DOX-D-2-EGFR或DOX-D-5-PSMA相同的药物挂载序列(GC挂载位点序列)，因而也都具有相当的药物挂载功能。
检测核酸纳米颗粒在血清中的稳定性
实施例9
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1中制备的RNA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 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***(Imaging System)(Bio-Rad,ChemiDoc MP)。
4.实验方法：
(1)吸取350μl PBS加到RNA纳米颗粒样品中，充分混匀。
(2)将2μM的RNA纳米颗粒置于含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 TM 8％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48：RNA定量结果及上样体积
Figure PCTCN2019095766-appb-000067
电泳检测结果见图14和图15。其中，图14示出了8％非变性胶的电泳结果(Coomassie Blue程序)，图15示出了8％非变性胶的电泳结果(Stain Free Gel程序)。RNA纳米颗粒的血清稳定性试验结果显示：10min、1h、12h和36h的非变性胶结果显示(图14和图15)，不同时 间RNA纳米颗粒样品条带无明显差别，表明RNA纳米颗粒在10％FBS的1640培养基中比较稳定，无明显降解。
研究RNA纳米颗粒在HepG2细胞中的细胞毒性
实施例10
一、实验材料和实验方法
1.待测样品为实施例7中的三个样品。
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)；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HepG2细胞。
(2)胰酶消化HepG2细胞，以每孔5000个细胞100μL接种在96孔板中，于37℃和5％CO 2中培养过夜。
(3)第二天去除细胞上清，使用培养基稀释待测样品，分别加入200nM的RNAh、RNAh-Biotine、RNAh-Dox和Dox，各100μL至铺板细胞中，每个样品4次重复。
(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49：HepG2细胞增殖(％)Mean±SEM(n＝4)
Figure PCTCN2019095766-appb-000068
实验结果见表49和图16，图16中，a对应PBS的细胞增殖结果，b对应DMSO的细胞增殖结果，c对应Dox(阿霉素)的细胞增殖结果，d对应RNAh的细胞增殖结果，e对应RNAh-Biotin-quasar670的细胞增殖结果，f对应RNAh-Biotin-quasar670-Dox的细胞增殖结果。
从表49和图16中可以看出，CTG结果显示，200nM载药纳米颗粒RNAh-Biotine-Dox对于HepG2细胞有明显细胞毒性(P<0.0001)，而200nM RNAh-Biotine对于HepG2细胞无细胞毒性。
核酸纳米颗粒的组装
实施例11
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50：R-15：
Figure PCTCN2019095766-appb-000069
表51：R-16：
Figure PCTCN2019095766-appb-000070
Figure PCTCN2019095766-appb-000071
表52：R-17：
Figure PCTCN2019095766-appb-000072
表53：R-18：
Figure PCTCN2019095766-appb-000073
Figure PCTCN2019095766-appb-000074
表54：R-19：
Figure PCTCN2019095766-appb-000075
表55：R-20：
Figure PCTCN2019095766-appb-000076
Figure PCTCN2019095766-appb-000077
表56：R-21：
Figure PCTCN2019095766-appb-000078
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；
(6)电位测定；
(7)粒径测量；
(8)Tm值测定。
三、自组装试验结果
(1)电泳检测
主要试剂和仪器如下：
表57：
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58：
Figure PCTCN2019095766-appb-000079
步骤：
①将RNA纳米颗粒按下表59方法采用超纯水进行稀释。
表59：
  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17。图17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17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60：
Figure PCTCN2019095766-appb-000080
表61：
Figure PCTCN2019095766-appb-000081
Figure PCTCN2019095766-appb-000082
表62：
Figure PCTCN2019095766-appb-000083
表63：
Figure PCTCN2019095766-appb-000084
表64：
Figure PCTCN2019095766-appb-000085
表65：
Figure PCTCN2019095766-appb-000086
表66：
Figure PCTCN2019095766-appb-000087
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67：
  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68：
试剂名称 货号 厂家
AE buffer / Takara
SYBR Green I染料 / 自配
表69：
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70：
Figure PCTCN2019095766-appb-000088
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18，R-16的溶解曲线图见图19，R-17的溶解曲线图见图20，R-18的溶解曲线图见图21，R-19的溶解曲线图见图22，R-20的溶解曲线图见图23，R-21的溶解曲线图见图24。因RNA样本特殊性，本次检测以20～90℃范围内1/2 RFUmax值所对应的温度为样本Tm值。
表71：
  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12
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72：D-8：
Figure PCTCN2019095766-appb-000089
表73：D-9：
Figure PCTCN2019095766-appb-000090
Figure PCTCN2019095766-appb-000091
表74：D-10：
Figure PCTCN2019095766-appb-000092
表75：D-11：
Figure PCTCN2019095766-appb-000093
Figure PCTCN2019095766-appb-000094
表76：D-12：
Figure PCTCN2019095766-appb-000095
表77：D-13：
Figure PCTCN2019095766-appb-000096
表78：D-14：
Figure PCTCN2019095766-appb-000097
Figure PCTCN2019095766-appb-000098
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79：
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80：
Figure PCTCN2019095766-appb-000099
步骤：
①将DNA纳米颗粒按下表81方法采用超纯水进行稀释。
表81：
Figure PCTCN2019095766-appb-000100
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25。图25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37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82：
Figure PCTCN2019095766-appb-000101
表83：
Figure PCTCN2019095766-appb-000102
表84：
Figure PCTCN2019095766-appb-000103
表85：
Figure PCTCN2019095766-appb-000104
Figure PCTCN2019095766-appb-000105
表86：
Figure PCTCN2019095766-appb-000106
表87：
Figure PCTCN2019095766-appb-000107
表88：
Figure PCTCN2019095766-appb-000108
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89：
  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90：
试剂名称 货号 厂家
AE buffer / Takara
SYBR Green I染料 / 自配
表91：
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92：
Figure PCTCN2019095766-appb-000109
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26，D-9的溶解曲线图见图27，D-10的溶解曲线图见图28，D-11的溶解曲线图见图29，D-12的溶解曲线图见图30，D-13的溶解曲线图见图31，D-14的溶解曲线图见图32。
表93：
  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26至32可知，7组延长段变形+核心短序列DNA纳米颗粒的溶解曲线的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13
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94：
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95：
Figure PCTCN2019095766-appb-000110
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96：
Figure PCTCN2019095766-appb-000111
Figure PCTCN2019095766-appb-000112
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33，R-16的电泳检测结果见图34，R-17的电泳检测结果见图35，R-18的电泳检测结果见图36，R-19的电泳检测结果见图37，R-20的电泳检测结果见图38，R-21的电泳检测结果见图39。图33至39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14
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97：
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98：
Figure PCTCN2019095766-appb-000113
Figure PCTCN2019095766-appb-000114
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99：
Figure PCTCN2019095766-appb-000115
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40，D-9的电泳检测结果见图41，D-10的电泳检测结果见图42，D-11的电泳检测结果见图43，D-12的电泳检测结果见图44，D-13的电泳检测结果见图45，D-14的电泳检测结果见图46。图40至46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15
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11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16
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100：
  生产厂家 型号
生物安全柜 北京东联哈尔仪器制造公司 BSC-1360 Ⅱ A2
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101：
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Quasar670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102：
Figure PCTCN2019095766-appb-000116
Figure PCTCN2019095766-appb-000117
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17
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103：
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104：
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-1360 Ⅱ A2
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，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105：
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
C8孔中样品的配制方法是：完全培养基324μL，然后从C9孔中吸取
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106：
Figure PCTCN2019095766-appb-000118
Figure PCTCN2019095766-appb-000119
结论：
从上表和图47a、图47b、图47c、图47d、图47e、图47f、图47g、图47h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。
柔红霉素挂载实验
实施例18
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12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48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。
从以上的描述中，可以看出，本发明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、高负载率以及多价组合模块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。
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 (37)

  1. 一种核酸纳米颗粒，其特征在于，所述核酸纳米颗粒具有核酸结构域，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***、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***、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***、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所述的核酸纳米颗粒，其特征在于，所述a1序列为SEQ ID NO:1，所述所述b1序列为SEQ ID NO:3，所述c1序列为SEQ ID NO:5时，所述a序列、b序列、所述c序列中的至少一个序列包含至少一个碱基***、缺失或替换的序列。
  3. 根据权利要求1或2所述的核酸纳米颗粒，其特征在于，所述碱基***、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。
  4. 根据权利要求1或2所述的核酸纳米颗粒，其特征在于，所述a序列、b序列和c序列自组装成式(1)所示结构：
    Figure PCTCN2019095766-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。
  5. 根据权利要求4所述的核酸纳米颗粒，其特征在于，所述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'。
  6. 根据权利要求4所述的核酸纳米颗粒，其特征在于，所述核酸结构域中，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、
    b序列和c序列中任一序列的5'端和/或3'端；
    优选地，所述第一延长段至少选自如下任意一组：
    (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'。
  7. 根据权利要求1至6中任一项所述的核酸纳米颗粒，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、b序列和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。
  8. 根据权利要求7所述的核酸纳米颗粒，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’。
  9. 根据权利要求7所述的核酸纳米颗粒，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。
  10. 根据权利要求9所述的核酸纳米颗粒，其特征在于，
    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。
  11. 根据权利要求1至10中任一项所述的核酸纳米颗粒，其特征在于，所述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、b序列和c序列中的C或U碱基上具有2’-F修饰。
  12. 根据权利要求1至11中任一项所述的核酸纳米颗粒，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。
  13. 根据权利要求12所述的核酸纳米颗粒，其特征在于，所述核酸结构域的相对分子量与所述生物活性物质的总相对分子量之比≥1:1；
    优选地，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、药物、蛋白、多肽、类黄酮、葡萄糖、天然水杨酸、单抗、维生素、酚类以及卵磷脂中的一种或多种。
  14. 根据权利要求13所述的核酸纳米颗粒，其特征在于，所述生物活性物质为所述靶头、所述荧光素以及所述miRNA，
    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；
    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。
  15. 根据权利要求13所述的核酸纳米颗粒，其特征在于，所述药物为治疗肝癌、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、***癌、鼻咽癌、食道癌、口腔癌、红斑狼疮的药物；优选地，所述头颈癌为脑癌、神经母细胞瘤或胶质母细胞瘤。
  16. 根据权利要求13所述的核酸纳米颗粒，其特征在于，所述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。
  17. 根据权利要求13所述的核酸纳米颗粒，其特征在于，所述蛋白为SOD、生存素、hTERT、EGFR及PSMA的抗体或适配体中的一种或多种；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。
  18. 根据权利要求12所述的核酸纳米颗粒，其特征在于，所述生物活性物质通过如下任一方式与所述核酸结构域相连：
    方式一：物理嵌插；
    方式二：共价连接。
  19. 根据权利要求18所述的核酸纳米颗粒，其特征在于，所述生物活性物质与所述核酸结构域以物理嵌插方式相连时，所述生物活性物质与所述核酸结构域按照1～200：1的摩尔比进行物理嵌插。
  20. 根据权利要求19所述的核酸纳米颗粒，其特征在于，所述生物活性物质与所述核酸结构域以物理嵌插方式与共价连接方式相连时，所述物理嵌插方式连接的生物活性物质与所述共价连接方式连接的药物的摩尔比为1～200：1。
  21. 根据权利要求18所述的核酸纳米颗粒，其特征在于，所述共价连接方式连接的生物活性物质通过溶剂共价连接、linker共价连接或点击链接；
    优选地，所述溶剂选自多聚甲醛、DCM、DCC、DMAP、Py、DMSO、PBS或冰醋酸；
    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。
  22. 根据权利要求21所述的核酸纳米颗粒，其特征在于，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时，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。
  23. 根据权利要求1所述的核酸纳米颗粒，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。
  24. 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23中任一项所述的核酸纳米颗粒。
  25. 一种含阿霉素的药物，其特征在于，所述含阿霉素的药物包括阿霉素及权利要求1至12中任一项或权利要求23所述的核酸纳米颗粒。
  26. 根据权利要求25所述的含阿霉素的药物，其特征在于，阿霉素通过物理连接和/或共价连接的形式挂载在所述核酸纳米颗粒上，且阿霉素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。
  27. 根据权利要求25所述的含阿霉素的药物，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、酚类卵磷脂以及除阿霉素以外的小分子药物中的一种或多种。
  28. 根据权利要求27所述的含阿霉素的药物，其特征在于，将所述核酸结构域的相对分子量记为N 1，将阿霉素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。
  29. 根据权利要求27所述的含阿霉素的药物，其特征在于，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；
    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。
  30. 根据权利要求27所述的含阿霉素的药物，其特征在于，所述除阿霉素以外的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。
  31. 根据权利要求27所述的含阿霉素的药物，其特征在于，所述蛋白为SOD、生存素、hTERT及EGFR、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。
  32. 一种含阿霉素的药物的制备方法，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颗粒；
    通过物理连接和/或共价连接的方式将阿霉素挂载在所述核酸纳米颗粒上，得到所述含阿霉素的药物。
  33. 根据权利要求32所述的制备方法，其特征在于，通过物理连接的方式挂载阿霉素的步骤包括：
    将阿霉素、所述核酸纳米颗粒及第一溶剂混合并搅拌，得到预混体系；
    去除所述预混体系中的游离物质，得到所述含阿霉素的药物；
    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，去除所述预混体系中的游离物质的步骤包括：将所述预混体系与无水乙醇混合，在低于10℃的温度条件下析出所述含阿霉素的药物；更优选在0～5℃温度条件下析出所述含阿霉素的药物。
  34. 根据权利要求32所述的制备方法，其特征在于，通过共价连接的方式挂载阿霉素的步骤包括：
    配置阿霉素溶液；
    使所述阿霉素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，得到所述含阿霉素的药物；
    优选地，所述反应的步骤包括：
    将所述阿霉素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优选为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。
  35. 根据权利要求32至35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颗粒中的所述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13至17中任一项所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所述核酸结构域上，进而得到所述核酸纳米颗粒，其中，所述生物活性物质中的药物为除阿霉素之外的小分子药物。
  36. 根据权利要求34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。
  37. 根据权利要求35所述的制备方法，其特征在于，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时，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。
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