CN111053913B

CN111053913B - Cytarabine-containing medicine, preparation method thereof, pharmaceutical composition and application thereof

Info

Publication number: CN111053913B
Application number: CN201910969427.5A
Authority: CN
Inventors: 王力源; 王萌
Original assignee: Baiyao Zhida Beijing Nano Biotechnology Co ltd
Current assignee: Baiyao Zhida Beijing Nano Biotechnology Co ltd
Priority date: 2018-10-16
Filing date: 2019-10-12
Publication date: 2022-07-22
Anticipated expiration: 2039-10-12
Also published as: CN111053913A

Abstract

The application provides a drug containing cytarabine, a preparation method thereof, a pharmaceutical composition and application thereof. The drug comprises nucleic acid nanoparticles and cytarabine, wherein the cytarabine is carried on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence, a sequence and c sequence, the a sequence comprises a1 sequence or a sequence of a1 sequence with at least one base insertion, deletion or substitution, the b sequence comprises a sequence of b1 sequence or a sequence of b1 sequence with at least one base insertion, deletion or substitution, and the c sequence comprises a sequence of c1 sequence or a sequence of c1 sequence with at least one base insertion, deletion or substitution. The cytarabine-containing medicine provided by the application has the advantages that the nucleic acid structure domain is modified by the target head, the targeting property is better, the cytarabine can be stably delivered, and the reliability is very high.

Description

Cytarabine-containing medicine, preparation method thereof, pharmaceutical composition and application thereof

Technical Field

The application relates to the field of medicines, in particular to a cytarabine-containing medicine, a preparation method, a pharmaceutical composition and application thereof.

Background

Cytarabine was first synthesized in 1959 by Richard Walwick, Walden Roberts and Charles Dekker, university of California, Berkeley division. The U.S. food and drug administration approved cytarabine for market in 6 months of 1969; the chemical structure of this drug, originally sold under the name Cytosar-U by Upjohn, is a nucleoside in which cytosine is bound to arabinose, hence the name "cytarabine". Normally, cytosine is combined with another saccharide (deoxyribose) to form deoxycytidine, which is one of the components of DNA. However, some organisms of the phylum Polyporales use arabinose to bind cytosine to another compound (not a component of DNA) which is found in these organisms, namely cytarabine. Cytarabine is so similar to deoxycytidine that it can be incorporated into human DNA in place of the latter, however the structural difference renders the DNA incapable of replication, thereby killing the affected cells. When administered, cytarabine kills cancer cells by this mechanism of action. It is the first chemotherapeutic drug to act by altering the nucleoside itself-other earlier similar drugs (e.g. 5-fluorouracil) alter the base.

Currently, antineoplastic drugs including cytarabine must be administered at high doses of chemotherapeutic drugs in order to achieve effective therapeutic levels at the tumor site, but systemic administration of high doses may damage healthy normal cells and cause adverse effects in a range of tissues and organs. These adverse effects include immune system suppression (myelosuppression), inflammation and cleansing of the gut mucosa (mucositis), hair loss (alopecia) and organ-specific toxicity, such as cardiotoxicity and neurotoxicity. In order to avoid the adverse reactions, a tumor local administration mode is required to replace the traditional systemic administration mode so as to achieve the effects of increasing the tumor local drug concentration and reducing the systemic drug concentration. Therefore, how to achieve such local drug delivery and in vitro controlled release has become a focus of cancer chemotherapy research.

In order to reduce the side effect caused by poor targeting of the active ingredients of the medicine, the medicine delivery carrier is produced, and the function of the carrier is mainly to carry the active ingredients of the medicine and deliver the active ingredients into blood or tissue cells to treat diseases. There are a variety of approaches to achieve targeted delivery of different drugs. And is implemented by an instrument or apparatus, such as a gene gun, an electroporator, etc. The methods do not need to use a gene vector, but the transfection efficiency is generally low, the operation is complex, and the damage to tissues is large. It is also mediated by viral vectors, such as adenovirus and lentivirus, etc., and although the viral vectors have high in vitro transfection activity, the immunogenicity and the susceptibility to mutation of the viral vectors bring huge safety hazards to in vivo delivery. And non-viral vectors, especially biodegradable polymer materials are used for realizing the targeted transportation of the medicine. The advantages of non-viral vectors are mainly that under the condition of ensuring the expected transfection activity, the immunogenicity and a plurality of inflammatory reactions brought by the viral vectors can be greatly reduced.

Among the above-mentioned various targeted delivery modalities, more research is currently focused on the non-viral vector field, and is generally designed for several vectors: (a) a cationic liposome; (b) a polycationic gene vector. However, more researches are focused on the modification of polycation gene vectors and cationic liposomes, so that the polycation gene vectors and cationic liposomes are suitable for the targeted delivery of gene substances. Cationic liposomes have high transfection activity in vitro and in vivo, however, normal distribution in vivo is affected due to positive charges on the surface, and meanwhile, the cationic lipids cause immunogenicity and inflammatory reactions in animal experiments. The polycation gene vector is developed more mature at present, however, the surface of a structure is difficult to ensure by a targeting group in the structural design, a self-design contradiction between toxicity and transfection activity exists, and meanwhile, the connection of the polycation gene vector is difficult to realize nontoxic degradation in vivo.

Therefore, how to improve the delivery reliability of the existing small-molecule drug cytarabine is one of the difficulties in solving the limited clinical application of the existing cytarabine drug.

Disclosure of Invention

The main objective of the present application is to provide a cytarabine-containing drug, a preparation method thereof, a pharmaceutical composition and an application thereof, so as to improve the delivery reliability of the cytarabine-containing drug.

In order to achieve the above objects, according to one aspect of the present application, there is provided a cytarabine-containing medicament comprising a nucleic acid nanoparticle and cytarabine, and the cytarabine is suspended on the nucleic acid nanoparticle; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence a, a sequence b and a sequence c, the sequence a comprises a sequence a1 or a sequence a1 with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 with at least one base insertion, deletion or substitution; wherein, the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3'; b1 is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3'; the sequence of c1 is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' is added.

Further, when the sequence a1 is SEQ ID NO. 1, the sequence b1 is SEQ ID NO. 3, and the sequence c1 is SEQ ID NO. 5, at least one of the sequences a, b, and c comprises a sequence in which at least one base is inserted, deleted, or substituted.

Further, base insertions, deletions or substitutions occur at:

(1) 1, 2, 4 or 5 bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or

(2) Between 8 th to 10 th bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or

(3) Between the 1 st to 3 rd bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or

(4) Between 6 th to 9 th bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or

(5) Between the 1 st to 4 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; and/or

(6) Between 9 th and 12 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

Further, the sequence a, the sequence b and the sequence c self-assemble to form a structure shown in a formula (1):

wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position are each independently selected from C-G or G-C; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth 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 5 'to 3' direction is CAUA or CATA.

Further, the sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5'-CCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGCCG-3', c sequence: 5'-CGGCCAUAGCGC-3'; (3) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCG-3'; (4) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5 '-CCUUCGGG-3', c sequence: 5'-CCCCCAUAGCCC-3'; (5) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCG-3', c sequence: 5'-CGCCCAUAGCGC-3'; (6) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCC-3', c sequence: 5'-GGCCCAUAGCGC-3'; (7) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGGCG-3', c sequence: 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3'; (9) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3', respectively; (10) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3'; (11) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3'; (12) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3', respectively; (13) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3'; (14) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3'; (15) a sequence: 5'-CGAGCGTTCC-3'; b sequence: 5 '-GGTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3' are provided.

Further, the nucleic acid domain also comprises a first extension segment, wherein the first extension segment is a Watson-Crick paired extension segment, and the first extension segment is positioned at the 5 'end and/or the 3' end of any sequence in the sequences a, b and c; preferably, the first elongate section is selected from any one of the following: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3'; (2): a 3' end of the chain: 5' -GGG-3', 5' -end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of chain: 5' -CCCG-3', 3' end of c strand: 5 '-CGGG-3'; (5): a 5' end of the chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'; (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3'; (9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.

Further, the nucleic acid domain also comprises a second extension segment, the second extension segment is positioned at the 5 'end and/or the 3' end of any sequence in the sequence a, the sequence b and the sequence c, and the second extension segment is a Watson-Crick matched extension segment; preferably, the second extension is an extension of a CG base pair; more preferably, the second extension is an extension sequence of 1-10 CG base pairs.

Further, the nucleic acid domain further comprises at least one set of second stretches: a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of the chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.

Further, 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 2-50 base pairs.

Further, the second extension segment is an extension sequence formed by alternately arranging a sequence of continuous 2-8 CG base pairs and a sequence of continuous 2-8 AT/AU base pairs; alternatively, the second extension is an extension in which a sequence of 1 CG base pairs alternates with a sequence of 1 AT/AU base pairs.

Further, bases, ribose and phosphate in the sequences a, b and c have at least one modifiable site, and any modifiable site is modified through any one of the following modified linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups; preferably, the sequence a, sequence b and sequence C have a 2' -F modification at the C or U base.

Further, the cytarabine is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the cytarabine to the nucleic acid nanoparticles is 2-300: 1, preferably 10-50: 1, and more preferably 15-25: 1.

Further, the nucleic acid nanoparticle further comprises a bioactive substance, wherein the bioactive substance is connected with the nucleic acid structural domain, and the bioactive substance is one or more of a target, fluorescein, siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein, polypeptide, flavonoid, glucose, natural salicylic acid, monoclonal antibody, vitamin, phenolic lecithin and small molecule drugs except cytosine arabinoside.

Further, the relative molecular weight of the nucleic acid domains is denoted as N₁The total relative molecular weight of cytarabine and the biologically active substance is denoted as N₂，N₁/N₂≥1:1。

Further, the bioactive 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, preferably the 5' end or the 3' end of any one of the sequences a, b and c, or is inserted between GC bonds of the nucleic acid domains, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the sequence a, the 5' end and the 3' end of the sequence c; preferably, the target head 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.

Further, the small molecule drugs other than cytarabine are drugs containing any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.

Further, the protein is one or more of SOD, survivin, hTERT, EGFR and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.

Further, the particle size of the nucleic acid nanoparticles is 1-100 nm, preferably 5-50 nm; more preferably 10 to 30 nm; further preferably 10 to 15 nm.

According to another aspect of the present application, there is also provided a method for preparing a cytarabine-containing medicament, comprising the steps of: providing the nucleic acid nanoparticles described above; and (3) carrying the cytarabine on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the cytarabine-containing medicament.

Further, the step of loading cytarabine by means of physical linkage comprises: mixing cytarabine, nucleic acid nanoparticles and a first solvent, and stirring to obtain a premixed system; precipitating the premixed system to obtain the cytarabine-containing medicament; 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 precipitating the premixed system to obtain the cytarabine-containing medicament comprises the following steps: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the cytarabine-containing medicament; more preferably, the premixed system is mixed with absolute ethyl alcohol and then precipitated at the temperature lower than 10 ℃ to obtain precipitates; drugs containing cytarabine; more preferably, the precipitate is precipitated at a temperature of 0 to 5 ℃ to obtain a precipitate. More preferably, 6-12 times of volume of absolute ethyl alcohol is adopted to wash the precipitate to remove impurities, and the cytarabine-containing medicine is obtained.

Further, the step of loading cytarabine by means of covalent attachment comprises: preparing a cytarabine solution; reacting the cytarabine solution with the amino outside the G ring of the nucleic acid nano-particle under the mediation effect of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the drug containing cytarabine; preferably, the step of reacting comprises: mixing the cytarabine solution with paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain a reaction system; the concentration of the optimal paraformaldehyde solution is 3.7-4 wt%, the optimal paraformaldehyde solution is a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.

Further, the preparation method further comprises a step of preparing a nucleic acid nanoparticle, which comprises: obtaining a nucleic acid structural domain by self-assembling the single strand corresponding to the nucleic acid structural domain; preferably, after obtaining the nucleic acid domain, the method of making further comprises: the bioactive substances are carried on the nucleic acid structural domain in a physical connection and/or covalent connection mode, and then the nucleic acid nano-particles are obtained.

Further, in the process of carrying the bioactive substances in a covalent connection mode, carrying is carried out through solvent covalent connection, linker covalent connection or click link; preferably, the solvent is a third solvent used in the covalent attachment as the attachment medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, linker is selected from disulfide bond, p-azido, bromopropyne or PEG; preferably, click-linking is performed by alkynyl or azido modification of the biologically active substance precursor and the nucleic acid domain simultaneously, followed by click-linking.

Further, when the biologically active substance is linked to the nucleic acid domain in a click-linkage manner, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of 2 ' hydroxyl, carboxyl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of G exocyclic amino, 2 ' -hydroxyl, a amino or 2 ' -hydroxyl.

According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any one of the cytarabine-containing medicaments described above.

According to the fourth aspect of the application, the application of any one of the cytarabine-containing medicines in preparing the medicines for treating tumors, viral keratitis and epidemic conjunctivitis is also provided.

Further, the tumor is one or more of acute leukemia, malignant lymphoma, lung cancer, digestive tract cancer, colorectal cancer and head and neck cancer.

According to a fifth aspect of the present application, there is also provided a method for preventing and/or treating tumors, viral keratitis and epidemic conjunctivitis, the method comprising: providing any one of the above cytarabine-containing medicaments or pharmaceutical compositions; administering an effective amount of the above cytarabine-containing medicament or pharmaceutical composition to a patient with a tumor.

The cytarabine-containing medicament provided by the application comprises a nucleic acid nanoparticle and cytarabine, and the cytarabine is carried on the nucleic acid nanoparticle in a physical connection and/or covalent connection mode. In the nucleic acid nanoparticle, the three sequences or the variant sequences thereof provided by the present application can be contained, so that not only the nucleic acid domains can be formed by self-assembly, but also cytosine arabinoside can be connected to any 5 'end and/or 3' end of the three strands as a carrier, or the cytosine arabinoside can be stably inserted between the strands of the nucleic acid domains. According to the application, the small-molecule drug cytarabine is loaded on the nucleic acid nanoparticles, the 'coating effect' is realized on the cytarabine by utilizing the internal hydrophobicity, the external hydrophilicity and the stacking effect of basic groups of the nucleic acid nanoparticles, and the cytarabine cannot be dissolved in a certain time due to the coating effect or covalent connection, so that the delivery stability is improved. In addition, when the nucleic acid structure domain is modified by a target head, the targeting property is better, cytarabine can be stably delivered, and the reliability is very high; meanwhile, the probability of contacting cytarabine with non-target cells or tissues can be reduced, and toxic and side effects are reduced.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. In the drawings:

FIG. 1 shows the result of electrophoresis detection of RNA nanoparticles formed by self-assembly in example 1 of the present application;

FIG. 2 shows the result of electrophoresis detection of DNA nanoparticles formed by self-assembly in example 1 of the present application;

FIG. 3 shows the result of 2% agarose gel electrophoresis detection of the first 7 groups of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present application;

FIG. 4 shows the results of 4% agarose gel electrophoresis detection of the first 7 groups of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present application;

FIG. 5 shows the result of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;

FIG. 6 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;

FIG. 7 shows the result of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;

FIG. 8 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;

FIG. 9 shows the result of 2% agarose gel electrophoresis detection of DNA nanoparticles of group 8 and group 9 formed by self-assembly in example 4 of the present application;

FIG. 10 shows a TEM image of self-assembled DNA nanoparticles D-7 of the present application in example 4;

FIG. 11 is a graph showing a standard curve of cytarabine absorbance during the detection of the DNA nanoparticle loading rate in example 5 of the present application;

FIG. 12 shows the results of electrophoresis detection of DNAh-Bio-EGFRApt-Cy 5-cytarabine nanoparticles after incubation in serum for various periods of time in example 7 of the present application; and

FIGS. 13a to 13d show the results of detecting that DNAh-Bio-EGFRatt-Cy 5-cytarabine nanoparticles inhibit the proliferation of MCF-7 cells in example 8, wherein FIG. 13a is the proliferation inhibition of MCF-7 cells by cytarabine, a small molecule drug, FIG. 13b is the proliferation inhibition of MCF-7 cells by DNAh-Bio-EGFRatt-Cy 5-cytarabine (a targeting drug), FIG. 13c is the proliferation inhibition of MCF-7 cells by DNAh-Bio-EGFRatt-Cy 5 (a targeting fluorescent vector), and FIG. 13d is the proliferation inhibition of MCF-7 blank cells by DMSO control;

FIG. 14 shows the results of native PAGE gel electrophoresis detection of 7 sets of extended stretch modified + core short sequence RNA self-assembly products in example 9 of the present invention;

FIG. 15 shows the dissolution curve of the RNA nanoparticle R-15 in example 9 of the present invention;

FIG. 16 shows the dissolution curve of the RNA nanoparticle R-16 in example 9 of the present invention;

FIG. 17 shows the dissolution curve of the RNA nanoparticle R-17 in example 9 of the present invention;

FIG. 18 shows the dissolution curve of the RNA nanoparticle R-18 in example 9 of the present invention;

FIG. 19 shows the dissolution curve of the RNA nanoparticle R-19 in example 9 of the present invention;

FIG. 20 shows the dissolution curve of the RNA nanoparticle R-20 in example 9 of the present invention;

FIG. 21 shows the dissolution curve of the RNA nanoparticle R-21 in example 9 of the present invention;

FIG. 22 shows the result of non-denaturing PAGE gel electrophoresis detection of 7 sets of modified-segment + core short-sequence DNA self-assembly products in example 10 of the present invention;

FIG. 23 shows the dissolution curve of DNA nanoparticle D-8 in example 10 of the present invention;

FIG. 24 shows a melting curve of DNA nanoparticle D-9 in example 10 of the present invention;

FIG. 25 shows a dissolution curve of DNA nanoparticle D-10 in example 10 of the present invention;

FIG. 26 is a graph showing a dissolution curve of the DNA nanoparticle D-11 in example 10 of the present invention;

FIG. 27 is a graph showing a dissolution curve of the DNA nanoparticle D-12 in example 10 of the present invention;

FIG. 28 shows a dissolution curve of the DNA nanoparticle D-13 in example 10 of the present invention;

FIG. 29 is a graph showing the dissolution curve of the DNA nanoparticle D-14 in example 10 of the present invention;

FIG. 30 shows the result of electrophoresis detection of RNA nanoparticle R-15 in example 11 of the present invention after incubation in serum for various time periods;

FIG. 31 shows the result of electrophoresis detection of RNA nanoparticle R-16 in example 11 of the present invention after incubation in serum for various time periods;

FIG. 32 shows the result of electrophoresis detection of RNA nanoparticle R-17 in example 11 of the present invention after incubation in serum for various time periods;

FIG. 33 shows the result of electrophoresis detection of RNA nanoparticle R-18 in example 11 of the present invention after incubation in serum for various time periods;

FIG. 34 shows the result of electrophoresis detection of RNA nanoparticle R-19 in example 11 after incubation in serum for various time periods;

FIG. 35 shows the result of electrophoresis detection of RNA nanoparticle R-20 in example 11 after incubation in serum for various times;

FIG. 36 shows the result of electrophoresis detection of RNA nanoparticle R-21 in example 11 after incubation in serum for various times;

FIG. 37 shows the results of electrophoresis detection of the DNA nanoparticle D-8 of example 12 of the present invention after incubation in serum for various periods of time;

FIG. 38 shows the result of electrophoresis detection of DNA nanoparticle D-9 in example 12 after incubation in serum for various times;

FIG. 39 shows the result of electrophoresis detection of DNA nanoparticle D-10 in example 12 of the present invention after incubation in serum for various times;

FIG. 40 shows the results of electrophoresis detection of the DNA nanoparticle D-11 of example 12 of the present invention after incubation in serum for various periods of time;

FIG. 41 shows the results of electrophoresis detection of the DNA nanoparticle D-12 of example 12 of the present invention after incubation in serum for various periods of time;

FIG. 42 shows the results of electrophoresis detection of the DNA nanoparticle D-13 of example 12 of the present invention after incubation in serum for various periods of time;

FIG. 43 shows the results of electrophoresis detection of the DNA nanoparticle D-14 in example 12 of the present invention after incubation in serum for various periods of time;

FIGS. 44a, 44b, 44c, 44D, 44e, 44f, 44g and 44h show cell viability curves for DMSO and the prodrug doxorubicin, D-8 and D-8-doxorubicin, D-9 and D-9-doxorubicin, D-10 and D-10-doxorubicin, D-11 and D-11-doxorubicin, D-12 and D-12-doxorubicin, D-13 and D-13-doxorubicin and D-14-doxorubicin, respectively, in example 15 of the present invention; and

FIG. 45 shows a standard curve of daunorubicin absorbance used in the mounting rate measurement process of example 16.

Detailed Description

It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present application will be described in detail with reference to examples.

Interpretation of terms:

RNAh, DNAh or blank vector: refers to a blank nucleic acid nanoparticle vector, such as RNAh or DNAh, that does not contain any biologically active substance.

Targeting vectors: refers to a nucleic acid nanoparticle vector containing a targeting tip but not containing a fluorescent substance, such as Biotin-RNAh or Biotin-DNAh.

A fluorescent carrier: refers to a nucleic acid nanoparticle vector containing fluorescent substances but not containing a targeting head, such as Cy3-RNAh or Cy 3-DNAh.

Targeting fluorescent vector: refers to a nucleic acid nanoparticle vector containing a target and a fluorescent substance, such as RNAh-Biotin-FAM or DNAh-Biotin-FAM.

Targeting drugs: refers to a nucleic acid nanoparticle vector containing a target, fluorescent substance and chemical drug, such as RNAh-Biotin-quasar 670-cytarabine or DNAh-Biotin-quasar 670-cytarabine.

It should be noted that there is no specific format in the naming convention of each vector or bioactive substance in the present application, and the fore-and-aft position in the description does not mean that it is at the 5 'end or 3' end of RNAh or DNAh, but means that it contains the bioactive substance.

As mentioned in the background, although there are many drug carriers for improving drug delivery efficiency in the prior art, it is still difficult to solve the problem that the clinical application of drugs is limited. In order to improve the situation, the inventor of the present application has studied all available materials as drug carriers, and has conducted in-depth investigation and analysis on various carriers from the aspects of cell/tissue targeting property of the carriers, stability during transportation, activity and efficiency of entering target cells, drug release capacity after reaching target cells, toxicity to cells and the like, and found that nanostructures formed by self-assembly of emerging DNA and/or RNA molecules, for example, DNA in a self-assembly system of DNA dendrimers, have a significant effect of hindering nuclease degradation, and have very important application values in the fields of gene therapy and biomedicine.

Through analysis of nanoparticles formed by self-assembly of DNA and RNA reported in the prior art, compared with DNA nanoparticles which are relatively rigid, RNA nanoparticles have more flexibility and stronger tension due to a large number of stem-loop structures existing in molecules or among molecules, and thus have more advantages in serving as candidate drug carriers. However, RNA nanoparticles in their native state are relatively less stable, and current improvements based on the use of RNA nanocarriers have mostly been developed around improving their stability and reliability. The current research results, although providing the possibility of drug loading to some extent, focus more on the possibility and effectiveness of the loading of nucleic acid drugs, especially siRNA drugs or miRNA drugs. However, there are few reports on whether non-nucleic acids are equally effective. In addition, the existing self-assembly nanoparticles, especially those used as vectors, are self-assembled by RNA strands at present, and very few self-assembly nanoparticles adopt a form of RNA strand and DNA strand combination, but do not adopt pure DNA strands to realize self-assembly.

In order to provide a novel RNA nanoparticle carrier which is highly reliable and can be autonomously assembled, the present applicant has compared and improved existing RNA nanoparticles, developed a series of novel RNA nanoparticles, and further tried to use pure DNA strands for self-assembly in view of improvement of applicability and cost reduction, and unexpectedly found that not only self-assembly into DNA nanoparticles can be achieved by changing these DNA strands, but also the performance is as excellent as that of RNA nanoparticles. Moreover, the self-assembly of DNA nanoparticles also has the advantages of low price and easy operation. Experiments prove that the improved RNA nanoparticles and DNA nanoparticles can be used for carrying various medicaments and stably exist in serum; further experiments verify that the carrier can carry the medicine into cells, and the carrier is nontoxic to the cells. And the carrier carrying the medicine can play a role in relieving and treating corresponding diseases.

On the basis of the above research results, the applicant proposed the technical solution of the present application. The application provides a drug containing cytarabine, which comprises nucleic acid nanoparticles and cytarabine, wherein the cytarabine is carried on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence a, a sequence b and a sequence c, the sequence a comprises a sequence a1 or a sequence a1 with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 with at least one base insertion, deletion or substitution; wherein, the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3'; b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3'; c1 sequence is SEQ ID NO: 5: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' is added.

The cytarabine-containing medicament comprises a nucleic acid nanoparticle and cytarabine, and the cytarabine is loaded on the nucleic acid nanoparticle. In the nucleic acid nanoparticle, the three sequences or the variant sequences thereof can be contained, whereby not only the nucleic acid domains can be formed by self-assembly, but also cytarabine can be linked to any 5 'end and/or 3' end of the three strands as a carrier, or cytarabine can be stably inserted between the strands of the nucleic acid domains. According to the cytarabine-containing medicine provided by the application, the small-molecule medicine cytarabine is loaded on the nucleic acid nanoparticles, and the nucleic acid nanoparticles are hydrophobic inside, hydrophilic outside and stacked in base, so that the cytarabine is coated, and the coating or covalent connection ensures that the cytarabine cannot be dissolved within a certain time, so that the delivery stability is improved. In addition, when the nucleic acid structural domain is modified by a target head, the targeting property is better, cytarabine can be stably delivered, and the reliability is very high; meanwhile, the probability of contacting cytarabine with non-target cells or tissues can be reduced, and toxic and side effects are reduced.

The self-assembly refers to a technique in which basic structural units spontaneously form an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds. The self-assembly process is not a simple superposition of weak interaction forces (wherein the weak interaction forces refer to hydrogen bonds, van der waals forces, electrostatic forces, hydrophobic forces and the like) among a large number of atoms, ions or molecules, but a plurality of individuals spontaneously occur simultaneously and are connected in parallel and are combined together to form a compact and ordered whole body, and the self-assembly process is a complex synergistic action of the whole body.

The generation of self-assembly requires two conditions: self-contained power and guidance. The kinetics of self-assembly refers to the synergistic effect of weak interaction forces between molecules, which provides energy for molecular self-assembly. The guiding of self-assembly refers to the complementarity of the molecules in space, that is, the occurrence of self-assembly requires the rearrangement of the molecules to be satisfied in the size and direction of space.

The DNA nanotechnology is a mode of molecular self-assembly from bottom to top, and spontaneously forms a stable structure from a molecular conformation as a starting point based on the physical and chemical properties of nucleic acid molecules, following the strict base pairing principle of nucleic acids. A plurality of DNA fragments are connected together in a correct sequence in vitro, and a sub-assembly structure is established through a base complementary pairing principle, so that a complex multilevel structure is finally 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. The different bases can be divided into two types, including standard Watson-Crick base pair type and non-Watson-Crick base pair type, so that the RNA can form a large number of and various types of circulating structure modules, which are basic units constituting the tertiary structure of the folded RNA. RNA nanotechnology can take advantage of these naturally occurring 3D modules and their predictable interactions, where many biologically active RNA structures can have atomic-level resolution, such as ribosomes, various classes of ribozymes, and natural RNA aptamers present in riboswitches. One advantageous feature of RNA nanotechnology is that structures can be designed that are comparable in size and complexity to natural RNA species. The unique assembly properties of RNA within the native RNA complex can also be exploited.

The nucleic acid nanoparticles comprise three sequences shown by SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 or sequences after variation thereof, or three sequences shown by SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 or sequences after variation thereof, and the nucleic acid nanoparticles can be formed by self-assembly, and the specific sequence after variation can be obtained by reasonably selecting variation sites and variation types on the basis of the sequences of 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, or by prolonging suitable fragments.

The nanoparticles formed by self-assembly of SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 are RNA nanoparticles, and the nanoparticles formed by self-assembly of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6 are DNA nanoparticles. In a preferred embodiment, when the nucleic acid nanoparticle is an RNA nanoparticle, at least one of the sequences a, b, and c comprises a sequence with at least one base insertion, deletion, or substitution. The specific position and the base type of the variant sequence in the RNA nano-particle can be improved into the nano-particle for improving the drug loading capacity or the stability according to the requirement on the premise of realizing self-assembly.

In order to make the nucleic acid nanoparticles have relatively higher stability and further make the drugs obtained by cytarabine loading more stable, when base insertion, deletion or substitution is carried out on the sequences shown in SEQ ID NO 1/2, SEQ ID NO 3/4 and/or SEQ ID NO 5/6, base insertion, deletion or substitution can be carried out on the base at certain specific positions of the sequences, so that the mutated sequences can be self-assembled into the nanoparticles as the original sequences on one hand, and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of homology with the original sequences on the other hand, so that the nanoparticles formed by self-assembling the sequences have the same drug loading characteristics and similar stability, and cytarabine can be well loaded and delivered.

In a preferred embodiment, the above base insertion, deletion or substitution occurs at: (1) 1 or 2, between the 1 st, 2 nd, 4 th and 5 th bases from the 5' end of the a sequence shown in SEQ ID NO; and/or (2) between 8 th to 10 th bases from the 5' end of the sequence a shown in SEQ ID NO. 1 or 2; and/or (3) between 1 to 3 bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (4) between 6 th and 9 th bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (5) between the 1 st to 4 th bases from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6; and/or (6) between bases 9 to 12 from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6.

In the above preferred embodiment, the base positions where the mutation is limited are the non-classical Watson-Crick paired base positions or the protruding unpaired base positions in the nanostructure formed by the sequences shown 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, thereby not affecting the formation of these protruding or loop structures, and thus maintaining the flexibility and tension of the nanostructure formed by the above sequences, which helps to maintain their stability as a carrier.

In order to further improve the stability of the nucleic acid nanoparticles and further improve the stability of the drug formed after the cytarabine is loaded, in a preferred embodiment, the sequence a, the sequence b and the sequence c are self-assembled into a structure shown in formula (1):

wherein W-C represents Watson-Crick pairing, N and N ' represent non-Watson-Crick pairing, each W-C at any position is independently selected from C-G or G-C, and the two bases at the 5' end and 3' end of each of at least two of the a, b, and C sequences are non-complementary; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth 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 5 'to 3' direction is CAUA or CATA.

In the preferred embodiment, the a, b, C sequences form by self-assembly a nucleic acid domain having the formula (1), wherein the bases at the positions other than the N and N' defined non-Watson-Crick base pairs form a classical Watson-Crick pair, and the bases of the Watson-Crick pair are selected from G-C or C-G base pairs. The nucleic acid nanostructure is more stable because the force of hydrogen bonds between G-C or C-G base pairs is greater than the force of hydrogen bonds between A-U/T or U/T-A base pairs. And a bulge or loop structure formed by non-Watson-Crick pairing base brings larger tension to the nucleic acid nano-carrier, so that the adaptability of the nucleic acid nano-carrier to microenvironment change is stronger, and the stability of the nucleic acid nano-particle is higher.

In the nanoparticles having the structure of formula (1), 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 viewpoint of self-assembly of nucleic acid sequences, in order to further improve the efficiency of self-assembly of the three sequences into the nanoparticle having the structure of formula (1), it is preferable that the bases at different positions are selected according to the following principle when selecting Watson-Crick paired bases: (1) a sequence a, a sequence b and a sequence c, wherein when a single sequence is not self-complementary, a pair of sequences forms a secondary structure; (2) one end of any two sequences is complementary and matched to form a double chain, and the other end is not complementary and matched to form a Y-shaped or T-shaped structure. The principle of the base selection is to make the two ends of any chain complementary and paired with the two ends of other two chains respectively to improve the self-assembly efficiency. Of course, in addition to the Y-type or T-type structure, other variants such as tetragons other than trifurcations may be used as long as the principle that one end of any two sequences is complementarily paired to form a double strand and the other end is not complementarily paired is satisfied.

In the nanoparticle with the structure of the formula (1), in the non-Watson-Crick pairing base, the fourth N from the 5 'end in the sequence a and the first N' from the 5 'end in the sequence b can be paired with the fourth N from the 5' end in the sequence a, and can be U-U which is not matched with Watson-Crick pairing, and can also be T, A, C or G which is modified and follows the Watson-Crick pairing principle. The Watson-Crick pairing relatively improves the bonding force between chains and improves the stability, but the non-Watson-Crick pairing endows the nano particles with greater flexibility and is also beneficial to improving the stability of the nano particles in the face of microenvironment change.

In a preferred embodiment, the sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence (SEQ ID NO: 7): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 8): 5'-CCUUCGCCG-3', c sequence (SEQ ID NO: 9): 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence (SEQ ID NO: 10): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 11): 5'-CGUUCGCCG-3', c sequence (SEQ ID NO: 12): 5'-CGGCCAUAGCGC-3'; (3) a sequence (SEQ ID NO: 13): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 14): 5'-GCUUCGCCG-3', c sequence (SEQ ID NO: 15): 5'-CGGCCAUAGCCG-3', respectively; (4) a sequence (SEQ ID NO: 16): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 17): 5 '-CCUUCGGG-3', c sequence (SEQ ID NO: 18): 5'-CCCCCAUAGCCC-3'; (5) a sequence (SEQ ID NO: 19): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 20): 5'-CGUUCGGCG-3', c sequence (SEQ ID NO: 21): 5'-CGCCCAUAGCGC-3'; (6) a sequence (SEQ ID NO: 22): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 23): 5'-CGUUCGGCC-3', c sequence (SEQ ID NO: 24): 5'-GGCCCAUAGCGC-3'; (7) a sequence (SEQ ID NO: 25): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 26): 5'-GCUUCGGCG-3', c sequence (SEQ ID NO: 27): 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence (SEQ ID NO: 28): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 29): 5'-CCTTCGCCG-3', c sequence (SEQ ID NO: 30): 5'-CGGCCATAGCCC-3'; (9) a sequence (SEQ ID NO: 31): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 32): 5'-CGTTCGCCG-3', c sequence (SEQ ID NO: 33): 5'-CGGCCATAGCGC-3', respectively; (10) a sequence (SEQ ID NO: 34): 5'-CGAGCGTTGC-3', b sequence (SEQ ID NO: 35): 5'-GCTTCGCCG-3', c sequence (SEQ ID NO: 36): 5'-CGGCCATAGCCG-3'; (11) a sequence (SEQ ID NO: 37): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 38): 5'-CCTTCGGGG-3', c sequence (SEQ ID NO: 39): 5'-CCCCCATAGCCC-3', respectively; (12) a sequence (SEQ ID NO: 40): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 41): 5'-CGTTCGGCG-3', c sequence (SEQ ID NO: 42): 5'-CGCCCATAGCGC-3'; (13) a sequence (SEQ ID NO: 43): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 44): 5'-CGTTCGGCC-3', c sequence (SEQ ID NO: 45): 5'-GGCCCATAGCGC-3'; (14) a sequence (SEQ ID NO: 46): 5'-CGAGCGTTGC-3', b sequence (SEQ ID NO: 47): 5'-GCTTCGGCG-3', c sequence (SEQ ID NO: 48): 5'-CGCCCATAGCCG-3', respectively; (15) a sequence (SEQ ID NO: 175): 5'-CGAGCGTTCC-3'; b sequence (SEQ ID NO: 176): 5 '-GGTTCGCCG-3', c sequence (SEQ ID NO: 177): 5'-CGGCCATAGCCG-3' is added.

The nucleic acid nanoparticles formed by self-assembly of the fifteen groups of sequences not only have higher stability, but also have higher self-assembly efficiency.

The nucleic acid nanoparticles mentioned above can be not only self-assembled and molded, but also have the ability to carry or carry cytarabine drugs. The amount of cytarabine loaded varies depending on the position of G-C or C-G base pair in the above-mentioned nucleic acid nanoparticle.

In order to make the nucleic acid domain capable of carrying more cytarabine and bioactive substances (the introduction of the bioactive substances is described below), in a preferred embodiment, the nucleic acid domain further comprises a first extension segment, the first extension segment is a Watson-Crick paired extension segment, and the first extension segment is positioned at the 5 'end and/or the 3' end of any one of the a sequence, the b sequence and the c sequence. A certain matching relationship is required between the carrier and the carried substance, and when the molecular weight of the carrier is too small and the molecular weight of the carried substance is too large, the carrying or transporting capacity of the carrier to the carried substance is relatively reduced from the mechanical point of view. Therefore, a vector matching the size of the carried substance can be obtained by adding a first extension segment to the 5 'end and/or 3' end of any one of the a sequence, the b sequence and the c sequence based on the nucleic acid nanostructure.

The specific length of the first extension segment can be determined according to the size of the substance to be carried. In a preferred embodiment, the first elongate section is selected from any one of the following: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3'; (2): a 3' end of the chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of the chain: 5' -CCCG-3', 3' end of c chain: 5 '-CGGG-3'; (5): a 5' end of chain: 5' -CCCC-3', 3' end of c strand: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c strand: 5 '-GGG-3'. (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3'; (9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'; (10): a 5' end of chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:162), the 3' end of the c-chain: 5'-UCGCCGCUCGCCGC-3' (SEQ ID NO: 163); (11): a 3' end of the chain: 5'-GGCCGGAGGCCGG-3' (SEQ ID NO:164), 5' end of b chain: 5'-CCGGCCUCCGGCC-3' (SEQ ID NO: 165); (12) b 3' end of strand: 5' -CCAGCCGCC-3' (SEQ ID NO:166), c chain 5' end: 5'-GGCGGCAGG-3' (SEQ ID NO: 167); (13): a 5' end of chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:168), the 3' end of c-chain: 5'-TCGCCGCTCGCCGC-3' (SEQ ID NO: 169); (14): a 3' end of chain: 5'-GGCCGGAGGCCGG-3' (SEQ ID NO:170), 5' end of b chain: 5'-CCGGCCTCCGGCC-3' (SEQ ID NO: 171).

The first extension not only increases the length of any one or more of the three sequences forming the nucleic acid nanostructure, but also the first extension composed of GC bases further improves the stability of the formed nanoparticles. Moreover, the first extension segment composed of the sequence also keeps higher self-assembly activity and efficiency of the sequence a, the sequence b and the sequence c.

From the viewpoint of the size of the formed nucleic acid nanoparticles and the stability thereof when transported in vivo as a drug delivery vehicle, it is desirable to be able to transport the drug while trying not to be filtered out by the kidney until reaching the target cells. In a preferred embodiment, the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any of the a sequence, the b sequence and the c sequence, the second extension being a Watson-Crick paired extension; more preferably, the second extension is an extended sequence of CG base pairs; further preferably, the second extension is an extension sequence of 1-10 CG base pairs.

In a preferred embodiment, the above-mentioned nucleic acid domain further comprises at least one set of second stretches: a first group: a 5' end of chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'. This second extension renders the nanoparticle non-immunogenic and non-existent in the case of secondary structures to which each chain folds itself.

It is noted that the extension may be separated by unpaired base pairs.

In order to make the nucleic acid nanoparticle capable of carrying a bioactive substance with a larger molecular weight (see introduction of bioactive substances below), increasing drug loading rate and maintaining necessary stability, in a preferred embodiment, the second extension is an extension containing both CG base pairs and AT/AU base pairs, preferably the second extension is an extension of 2-50 base pairs. Here, the "/" in "AT/AU base" is in the relationship of or, specifically, the second extension is an extended sequence containing both CG base pairs and AT base pairs, or the second extension is an extended sequence containing both CG base pairs and AU base pairs.

More specifically, the sequences a, b and c after adding the above second extension may be the following sequences, respectively:

sequence a is (SEQ ID NO: 49):

b is (SEQ ID NO: 50):

sequence c is (SEQ ID NO: 51):

m in the sequence a, the sequence b and the sequence c is U or T, and when M is T, the synthesis cost of the sequences is greatly reduced.

In practical application, the specific arrangement positions of the CG base pairs and the extended sequences of AT/AU base pairs can be reasonably adjusted according to actual needs. In a more preferred embodiment, the second extension is an extension sequence formed by alternating a sequence of 2 to 8 CG base pairs and a sequence of 2 to 8 AT/AU base pairs; or the second extension is an extension sequence with 1 CG base pair sequence and 1 AT/AU base pair sequence arranged alternately.

Specifically, the positions of the CGCGCG extension and the CGCCGC extension in the sequence a shown by the SEQ ID NO. 49 and the AAAAAA extension are interchanged, the positions of the GCGGCG extension and the GGCGGC extension in the sequence b shown by the SEQ ID NO. 50 and the TTTTTT extension are interchanged, the positions of the GCCGCC extension and the AAAAAA extension in the sequence c shown by the SEQ ID NO. 51 and the CGCCGC extension and the TTTTTT extension are interchanged. The nucleic acid nanoparticles formed by self-assembly of the sequences are suitable for carrying bioactive substances with indole molecular structures (indole molecules are preferably combined with A).

Over the past years, three major challenges with RNA as a widely used building material include: 1) susceptibility to rnase degradation; 2) susceptibility to dissociation following systemic injection; 3) toxicity and adverse immune response. These three challenges have been largely overcome at present: 1) 2 '-fluoro (2' -F) or 2 '-O-methyl (2' -OMe) modification of the ribose-OH group can chemically stabilize RNA in serum; 2) certain naturally occurring linking motifs are thermodynamically stable and can keep the entire RNA nanoparticle intact at ultra-low concentrations; 3) the immunogenicity of the RNA nanoparticles is sequence and shape dependent and can be adjusted so that the RNA nanoparticles stimulate the production of inflammatory cytokines or so that the RNA nanoparticles are non-immunogenic and non-toxic when administered at 30mg/kg of repeated intravenous injections.

Therefore, in order to further reduce the susceptibility of the nucleic acid nanoparticles to rnase degradation while increasing stability during transport, in a preferred embodiment, the bases, ribose and phosphate in the a sequence, the b sequence and the c sequence have at least one modifiable site, and any modifiable site is modified by any one of the following modifying linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups; preferably, the sequence a, sequence b and sequence C have 2' -F modifications at the C or U bases. When the modified joint is sulfydryl, the modified joint belongs to sulfo modification, the modification strength is weak, and the cost is low.

The cytarabine can be carried by physical linkage and/or covalent linkage. When cytarabine is simultaneously connected with the nucleic acid domain by adopting two modes of physical intercalation and covalent connection, the physical intercalation is usually intercalated between GC base pairs, and the preferable number of intercalation sites is 1-100: the ratio of 1 was inserted. When covalent linkage is used, cytarabine usually reacts with the amino group outside the G-ring to form covalent linkage. More preferably, the molar ratio of cytarabine to nucleic acid nanoparticles is 2-300: 1, preferably 2-290: 1, more preferably 2-29: 1, further preferably 10-50: 1, and most preferably 15-25: 1.

In addition to serving as a delivery vehicle for cytarabine in the cytarabine-containing medicament provided by the present application, in a preferred embodiment, the nucleic acid nanoparticle further comprises a bioactive substance, and the bioactive substance is connected with the nucleic acid structure domain according to different medicament purposes. The bioactive substances are one or more of target, fluorescein, interfering nucleic acid siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein, polypeptide, flavonoid, glucose, natural salicylic acid, monoclonal antibody, vitamin, phenol, lecithin and small molecule drugs except cytosine arabinoside.

In order to improve the efficiency of loading and carrying the loaded bioactive substances by the nucleic acid nanoparticles, the relative molecular weight of the nucleic acid domain and the relative molecular weight of cytarabine and the bioactive substances preferably have a certain matching relationship. In a preferred embodiment, the relative molecular weight of the nucleic acid domains is denoted as N₁The total relative molecular weight of cytarabine and the biologically active substance is denoted as N₂，N₁/N₂1:1 or more; preferably, the bioactive substance is one or more of a targeting, a fluorescein, an interfering nucleic acid siRNA, a miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, a drug (generally interpreted as a small molecule drug, i.e. a chemically synthesized drug), a protein, a polypeptide, a flavonoid, glucose, natural salicylic acid, a monoclonal antibody, a vitamin, a phenolic, and lecithin.

The cytarabine containing drugs in this application have different performance optimization depending on the kind of the specific bioactive substance to be carried. For example, when the bioactive substance is biotin or folic acid, it functions to target the cytarabine-containing drug, e.g., specifically to cancer cells. When the bioactive substance is fluorescein, it acts to provide a luminescent tracer effect to the nucleic acid nanoparticles, such as may be one or more of FAM, CY3, CY5, or Quasar670, and the like. When the bioactive substances are certain siRNA, miRNA, protein, polypeptide, RNA antibody and micromolecule drugs except cytarabine, the drugs containing cytarabine can become new products with specific treatment effects, such as drugs with more excellent performance according to different biological functions. In addition, according to the different kinds of the biological active substances carried, DNA nanoparticles and RNA nanoparticles are preferably used, and can be reasonably selected according to actual needs. For example, when the bioactive substance is a drug, it is preferable that the DNA nanoparticle or the RNA nanoparticle be carried, and there is no particular requirement on the length of the single strand assembled to form the nanoparticle.

In a preferred embodiment, the bioactive substances are target heads, fluorescein and miRNA, wherein the target heads are located on any sequence of a, b and c sequences, preferably on the 5' end or the 3' end of any sequence of a, b and c, or are inserted between GC bonds of the nucleic acid structure domain, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located 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; preferably, the target head is folic acid or biotin, the fluorescein is one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.

The target head can be connected to any sequence of a sequence, b sequence and c sequence through a linker covalent connection mode, and the available linker is selected from disulfide bond, p-azido group, bromopropyne or PEG. As used herein, "on any sequence" refers to any base position of any sequence of a, b, c, and is more convenient to attach to the 5 'end or 3' end, and is more widely applicable. Folate modification can be either physical intercalation mode ligation or physical intercalation + covalent ligation.

The fluorescein can be conventional fluorescein, and is preferably one or more of FAM, CY5 and CY 3.

The miRNA can be miRNA with cancer inhibiting effect, or anti-miRNA capable of inhibiting corresponding diseases, and is reasonably selected according to medical needs in practical application. The anti-miRNA may be synthesized at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence. When anti-miRNA is synthesized at all of the above three positions, the inhibitory effect of the anti-miRNA on the corresponding miRNA is relatively stronger.

Preferably, the miR-21 is resistant to miR-21, and miR-21 is involved in the initiation and progression of various cancers and is a main oncogene for invasion and metastasis. The anti-miR-21 can effectively and simultaneously regulate a wide range of target genes, and is beneficial to solving the problem of heterogeneity of cancers. Thus, in the preferred nucleic acid nanoparticles, the target head, such as folate or biotin, can specifically target cancer cells, and after internalization in combination with the cancer cells, the anti-miR-21 is complementary to miR-21 base with very high affinity and specificity, thereby effectively reducing the expression of oncogenic miR-21. Therefore, the anti-miR-21 can 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 according to actual needs. When the anti-miR-21 is synthesized at all three positions, the inhibition effect of the anti-miR-21 on the miR-21 is relatively stronger.

When the bioactive substances capable of being carried are other small-molecule medicines except cytarabine, the medicines include, but are not limited to, medicines for treating liver cancer, stomach cancer, lung cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, melanoma, leukemia, senile dementia, ankylosing spondylitis, malignant lymphoma, bronchial cancer, rheumatoid arthritis, HBV hepatitis B, multiple myeloma, pancreatic cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, esophageal cancer, oral cancer and lupus erythematosus according to the types of diseases which can be treated by different medicines; preferably, the head and neck cancer is brain cancer, neuroblastoma or glioblastoma.

When the bioactive substance capable of being carried is a small molecule drug other than cytarabine, the bioactive substance includes, but is not limited to, drugs containing any one or more of the following groups according to the difference of the molecular structure of the drug or the difference of characteristic groups of the drug: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.

In a preferred embodiment, the protein is one or more of an antibody or aptamer to SOD (superoxide dismutase), Survivin (Survivin), hTERT (human telomerase reverse transcriptase), EGFR (epidermal growth factor receptor), PSMA (prostate specific membrane antigen); the vitamins are levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.

In a preferred embodiment, the particle size of the nucleic acid nanoparticles is 1 to 100nm, preferably 5 to 50nm, more preferably 10 to 30nm, and further preferably 10 to 15 nm. Within this range the size is suitable both for entering the cell membrane by cell surface receptor mediated phagocytosis and for being removed by renal filtration avoiding non-specific cell permeation, and therefore the favourable particle size contributes to improved pharmacokinetic, pharmacodynamic, biological and toxicological profiles.

According to a second aspect of the present application, there is also provided a method for preparing the cytarabine-containing medicament, comprising the steps of: providing any one of the nucleic acid nanoparticles described above; and (2) carrying the cytarabine on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the cytarabine-containing medicament.

When physical linkage is used, cytarabine is usually inserted between the GC base pairs by physical intercalation. When covalent linkage is used, cytarabine usually reacts with the amino group outside the G-ring to form covalent linkage. The drug containing the cytarabine prepared by the method has better targeting property after being modified by the target head, can stably deliver the cytarabine and has high reliability.

In a preferred embodiment, the step of loading cytarabine by means of physical linkage comprises: mixing and stirring cytarabine, nucleic acid nanoparticles and a first solvent to obtain a premixed system; and precipitating the premixed system to obtain the cytarabine-containing medicament. The dosage of the cytarabine and the nucleic acid nanoparticles can be adjusted according to the change of the loading amount, which can be understood by those skilled in the art and is not described in detail herein.

In order to improve the efficiency and stability of physical connection, the content of cytarabine added in each liter of the first solvent is preferably 0.1-1 g. 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 precipitating the premixed system to obtain the cytarabine-containing medicament comprises the following steps: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the cytarabine-containing medicament. More preferably, the premix system is mixed with absolute ethyl alcohol and then precipitated at a temperature of less than 10 ℃ to obtain a precipitate, and still more preferably, the precipitate is precipitated at a temperature of 0 to 5 ℃ to obtain a precipitate. More preferably, anhydrous ethanol with the volume 6-12 times that of the precipitate is adopted to wash and remove impurities, so that the cytarabine-containing medicine is obtained.

In a preferred embodiment, the step of loading cytarabine by covalent attachment comprises: preparing a cytarabine solution; reacting the cytarabine solution with the G-ring exoamino of the nucleic acid nano-particles under the mediation of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the cytarabine-containing medicament.

In a formaldehyde-mediated form, the following reactions can occur:

preferably, the step of reacting comprises: and (3) mixing the cytarabine solution with the paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain a reaction system. The paraformaldehyde solution can release formaldehyde small molecules so as to participate in the chemical reaction. In order to improve the reaction efficiency, the concentration of the paraformaldehyde solution is preferably 3.7-4 wt%, the paraformaldehyde solution is preferably a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.

In the above preparation method, the nucleic acid nanoparticles may be prepared by a self-assembly form such as: (1) mixing RNA or DNA single strands a, b and c at the same time, and dissolving in DEPC water or TMS buffer solution; (2) heating the mixed solution to 80 ℃/95 ℃ (wherein the RNA assembly temperature is 80 ℃, and the DNA assembly temperature is 95 ℃), keeping for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min; (3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃; (4) cutting a target strip, eluting in an RNA/DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at a low temperature under reduced pressure to obtain a self-assembly product, namely a nucleic acid structural domain, thereby obtaining the nucleic acid nanoparticles.

In order to provide the cytarabine-containing drug with other functions according to practical needs, in a preferred embodiment, after obtaining the nucleic acid domain, the preparation method further comprises: the bioactive substances mentioned above are loaded on the nucleic acid domain by means of physical linkage and/or covalent linkage, so as to obtain the nucleic acid nanoparticle. The means of attachment of the biologically active substance can likewise be physical and/or covalent. Forms of covalent attachment include, but are not limited to, mounting by solvent covalent attachment, linker covalent attachment, or click linkage; preferably, the solvent is a third solvent used in the covalent attachment as the attachment medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, linker is selected from disulfide bond, p-azido, bromopropyne or PEG; preferably, click-linking is performed by alkynyl or azide modification of the biologically active substance precursor and the nucleic acid domain at the same time and then by click-linking.

The above classification does not mean that a certain bioactive substance is linked to a nucleic acid domain in only one manner. Instead, some bioactive substances may be linked to the nucleic acid domain by physical intercalation, by covalent linkage, or by click linkage. However, for a particular bioactive substance, there may be only one type of attachment, or there may be multiple types of attachment, but there may be some type of attachment that has an advantageous utility.

In the above-described connection method, when different drugs are physically inserted into the nucleic acid domains, the number and binding sites of the insertion are slightly different. For example, when the anthracycline or acridine drug is inserted, it is usually inserted between GC base pairs, and the number of preferred insertion sites is 1 to 100: the ratio of 1 was inserted. When the naphthamide medicament is inserted, the naphthamide medicament is usually inserted between AA base pairs, the preferable number of insertion sites is different according to the number of the AA base pairs on a nucleic acid structural domain, and the pyridocarbazoles are inserted according to the ratio of 1-200: the ratio of 1 was inserted.

Specifically, the molar ratio of biologically active substance to nucleic acid domain can be reasonably selected for physical intercalation depending on the species of biologically active substance, the length of the a, b and c sequences forming the nucleic acid domain in the nucleic acid nanoparticle, and how many complementary base pairs of GC are present therein.

In a preferred embodiment, when the bioactive substance and the nucleic acid domain are physically intercalated and covalently linked, the molar ratio of the bioactive substance physically intercalated and linked to the drug covalently linked is 1-200: 1. the connection mode is suitable for anthracycline and acridine medicines. The proportion of the drugs connected in different connection modes is not limited to the range, and the drugs can be effectively suspended, have no toxic effect on cells and can be effectively released after reaching a target.

When the bioactive substance precursor and the nucleic acid domain are simultaneously subjected to alkynyl or azide modification and connected in a click-to-link mode, different click-to-links are selected according to different structure changes of the medicament. And the attachment position may be changed correspondingly according to the structure of the active material, which can be understood by those skilled in the art.

In a preferred embodiment, when the biologically active substance is linked to the nucleic acid domain in a click-link fashion, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of hydroxyl, carboxyl, sulfhydryl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of amino, imino or hydroxyl.

When the nucleic acid domain is bound to a drug, the nucleic acid domain is water-soluble, and many drugs have poor water-solubility, and when the nucleic acid domain is bound, the water-solubility is improved. When the drugs are anthracyclines, the drugs are covalently bound to the nucleic acid domain via an-NH bond in the guanosine nucleotide (the-NH group is hundreds of times more active at a suitable pH than other groups that may be covalently bound to the drug), thereby forming a drug-loaded nucleic acid domain. Therefore, depending on the size of the specific drug molecule and the number of GC base pairs in the a-, b-and c-sequences of the specifically designed nucleic acid domain, a binding reaction is performed with a theoretical supersaturation binding amount of 1.1 to 1.3 times, and a maximum of 35 to 45 drugs can be bound to one nucleic acid domain. When the drug has another structure, the amount of the drug to be attached is related to the occupancy of the specific drug (including but not limited to the molecular structure, form, shape and molecular weight), and therefore, the conditions for binding the active site of the drug to the-NH bond on guanosine nucleotide in the nucleic acid domain are relatively severe, and the drug can be attached in the same manner, but excessive binding is relatively difficult to occur.

In a typical embodiment, there is also provided a pharmaceutical composition comprising any one of the nucleic acid nanoparticles described above. In the drug containing the nucleic acid nanoparticles, the nucleic acid domain can be modified by a targeting head of a targeted cell to achieve good targeting, and meanwhile, the corresponding therapeutic drug and/or tracer molecule can be mounted, so that the therapeutic drug and/or tracer molecule can be stably delivered, and the reliability is high.

According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any one of the cytarabine-containing medicaments described above. Specifically, according to actual needs, a suitable combination drug or an appropriate adjuvant can be selected to form a drug combination having a combined drug effect or capable of improving certain properties (such as stability) of the drug.

According to the fourth aspect of the application, the application of any one of the cytarabine-containing medicines in preparing the medicines for treating tumors, viral keratitis and epidemic conjunctivitis is also provided. Further, the tumor is any one or more of acute leukemia, malignant lymphoma, lung cancer, digestive tract cancer, colorectal cancer and head and neck cancer. The specific application can be to improve the medicament per se on the basis of the medicament of the application to obtain a new medicament, or to prepare the medicament of the application serving as a main active ingredient into a preparation with a proper dosage form and the like.

According to a fifth aspect of the present application, there is also provided a method of preventing and/or treating tumors, viral keratitis and epidemic conjunctivitis, the method comprising: providing any one of the above cytarabine-containing medicaments or pharmaceutical compositions; administering an effective amount of the above cytarabine-containing medicament or pharmaceutical composition to a patient with a tumor. Further, the tumor is any one or more of acute leukemia, malignant lymphoma, lung cancer, digestive tract cancer, colorectal cancer and head and neck cancer.

An effective amount herein includes a prophylactically effective amount and/or a therapeutically effective amount, by which is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., reduction of symptoms. In a particular embodiment, the dosage may be adjusted to provide an optimal therapeutically responsive dose, and the therapeutically effective amount may vary depending on the following factors: the disease state, age, sex, weight of the individual and the ability of the formulation to elicit a desired response in the individual. A therapeutically effective amount is also meant to include an amount by which the beneficial effects of the treatment outweigh the toxic or detrimental effects thereof. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention or inhibition of acute leukemia, malignant lymphoma, lung cancer, cancer of the digestive tract, colorectal cancer, and head and neck cancer. A prophylactically effective amount can be determined according to the description of therapeutically effective amounts above. For any particular subject, specific dosages may be adjusted over time according to the individual need and the professional judgment of the person to whom they are administered.

It should be noted that the nucleic acid nanoparticles formed by self-assembly of the sequences or variants of the sequences provided herein can also be used as basic building blocks, and can be further polymerized to form polymers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., according to practical requirements.

The advantageous effects of the present application will be further described below with reference to specific examples.

Assembly of nucleic acid nanoparticles

Example 1

One, RNA and DNA nanoparticle vector:

(1) the three polynucleotide base sequences that make up the RNA nanoparticles are shown in table 1:

table 1:

(2) three polynucleotide base sequences of DNA nanoparticles

DNA has the same sequence as that of the RNA, except that U is replaced by T. Wherein the molecular weight of chain a is 8802.66, the molecular weight of chain b is 8280.33, and the molecular weight of chain c is 9605.2.

The strands a, b and c of the RNA nanoparticles and DNA nanoparticles were synthesized by Competition Biotechnology, Inc. (Shanghai).

II, self-assembly experiment steps:

(1) mixing and dissolving RNA or DNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;

(2) heating the mixed solution to 80 ℃/95 ℃ (wherein the RNA assembly temperature is 80 ℃ and the DNA assembly temperature is 95 ℃), keeping for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;

(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;

(4) cutting off a target band, eluting in an RNA/DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a self-assembly product;

(5) electrophoresis analysis and detection and laser scanning observation.

Third, self-assembly experimental results

(1) Results of electrophoresis

The result of electrophoresis detection of the RNA self-assembly product is shown in FIG. 1. In fig. 1, lanes 1 to 3 are, from left to right: a strand, b strand, RNA self-assembly product. As can be seen, the RNA self-assembly products are slightly dispersed, but clearly seen as a single band. And the molecular weight is the molecular weight after the assembly, and is larger than that of the single chain, so that the position of the band lags behind the a chain and the b chain, the actual situation is consistent with the theory, and the stable composite structure is formed by the self-assembly of the RNA single chains, and the RNA nano-particles are formed.

The results of the electrophoretic detection of the DNA self-assembly products are shown in FIG. 2. In FIG. 2, lanes 1 to 3 are, from left to right: a chain, b chain, DNA self-assembly product. As can be seen from the figure, the bands of the DNA self-assembly products are bright and clear, and are single bands, which proves that the DNA single strands form a stable composite structure through self-assembly, and form DNA nanoparticles.

In this example, it was verified by gel electrophoresis that: the sequences a, b and c including RNA core sequence SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 can be successfully self-assembled into RNA nano-particles. Sequences a, b and c including the DNA core sequence SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 can also be successfully self-assembled into DNA nanoparticles.

The sequences a, b and c of the RNA nanoparticles and the DNA nanoparticles include various extension sequences (including drug-loading binding sequences) that facilitate the function of loading the nucleic acid domains, and a targeting head or fluorescein linked to the nucleic acid domains, in addition to the core sequence forming the nucleic acid domains. 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 have a targeting type under the guidance of a target head, and the fluorescein can enable the nucleic acid nanoparticles to have visibility and traceability.

Example 2

One, 7 groups of short sequence RNA nano-particle carriers:

(1)7 groups of three polynucleotide base sequences composing the RNA nanoparticle are respectively shown in tables 2 to 8:

table 2: r-1

Table 3: r-2

Table 4: r-3

Table 5: r-4

Table 6: r-5

Table 7: r-6

Table 8: r-7

The single strands of the 7 groups of short-sequence RNA nanoparticle carriers are synthesized by the corporation of Venezuelan Biotechnology engineering (Shanghai).

II, self-assembly experiment steps:

(1) mixing and dissolving the RNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;

(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;

(4) cutting a target strip, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at a low temperature under reduced pressure to obtain a short-sequence RNA self-assembly product;

(5) electrophoretic analysis detection and laser scanning observation;

(6) and (4) measuring the molecular weight.

Third, self-assembly experimental results

(1) Results of electrophoresis

The 2% agarose gel electrophoresis picture of 7 groups of short sequence RNA self-assembly products is shown in FIG. 3. Lanes 1 to 7 in FIG. 3 are, from left to right: short sequences R-1, R-2, R-3, R-4, R-5, R-6 and R-7.

The 4% agarose gel electrophoresis picture of 7 groups of short sequence RNA self-assembly products is shown in FIG. 4. Lanes 1 to 7 in FIG. 4 are, from left to right: short sequences R-1, R-2, R-3, R-4, R-5, R-6 and R-7.

As can be seen from the results of FIG. 3 and FIG. 4, it can be clearly seen that the bands of R-2, R-3, R-5 and R-7 in the 7 groups of short sequence self-assembly products are bright and clear, and the bands of R-1, R-4 and R-6 are still single bands, although they are relatively dispersed, indicating that the 7 groups of short sequences can be well self-assembled into RNA nanoparticle structures.

(2) Measurement of electric potential

The measuring method comprises the following steps: preparing a potential sample (self-assembly product dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;

opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;

setting software detection parameters;

then clicking to finish setting, appearing a measurement dialog box, and clicking Start to Start;

and (3) measuring results: the potential detection results of 7 groups of short sequence RNA nanoparticles are shown in tables 9 to 15 below:

table 9:

table 10:

table 11:

table 12:

table 13:

table 14:

table 15:

from the potential detection data described above, it is found that: the 7 groups of short sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the short sequence RNAs have a stable self-assembly structure.

This example shows that: the different combinations of the core sequences a, b and c can form the RNA nano-particle with the nucleic acid structure domain through self-assembly, and the structure is stable. Based on example 1, it can be seen that various functional extension fragments or connecting targeting heads, fluorescein and the like are added on the basis of different core sequence combinations, and the RNA nanoparticles can be successfully assembled, and have the performances of drug loading, cell targeting, visual tracking and the like.

To further verify these properties, an extension fragment was added to example 2, see example 3. And adding an extension fragment on the basis of the DNA core sequence corresponding to the RNA core sequence of example 2, and simultaneously connecting the target or not connecting the target, as shown in example 4.

Example 3

One, 7 groups of conventional sequence RNA nanoparticle carriers:

(1) the base sequences of three polynucleotides constituting RNA nanoparticles of 7 groups are shown in tables 16 to 22:

table 16: r-8

Table 17: r-9

Table 18: r-10

Table 19: r-11

Table 20: r-12

Table 21: r-13

Table 22: r-14 (in the following a chain)uGAcAGAuAAGGAAccuGcudTdTAs survivin siRNA)

The 7 groups of conventional sequence RNA nanoparticle vectors are synthesized by commissioned Suzhou Jima company, wherein the sequences a, b and C in R-8 to R-14 are respectively extended RNA oligonucleotide sequences formed by adding extension segments on the basis of the sequences a, b and C in R-1 to R-7, targeting module fragments are not extended, and C/U base 2' F modification is carried out (the enzyme cutting resistance and stability are enhanced). In addition, a Survivin (Survivin) siRNA nucleic acid interference therapeutic fragment is modified in the RNA nanoparticle R-14, specifically, a sense strand of Survivin siRNA is extended at the 3 'end of the a strand (see the underline part of the a strand), and an antisense strand is extended and connected at the 5' end of the b strand (see the underline part of the b strand), so that base pair complementation is formed.

II, self-assembly experiment steps:

(1) mixing RNA single strands a, b and c at the same time according to a molar ratio of 1:1:1, and dissolving in DEPC water or TMS buffer solution;

(4) cutting off target bands, eluting in RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at low temperature under reduced pressure;

(5) electrophoretic analysis detection and laser scanning observation;

(6) and (4) measuring the potential.

Third, self-assembly experimental results

(1) Results of electrophoresis

FIG. 5 shows the electrophoresis of the 2% agarose gel of the 7 sets of conventional sequence RNA self-assembly products. Lanes 1 to 7 in FIG. 5 are, from left to right: the self-assembly products of the conventional sequence RNA are R-8, R-9, R-10, R-11, R-12, R13 and R-14.

The 4% agarose gel electrophoresis of 7 sets of conventional sequence RNA self-assembly products is shown in FIG. 6. Lanes 1 to 7 in FIG. 6 are, from left to right: the self-assembly products of the conventional sequence RNA are R-8, R-9, R-10, R-11, R-12, R13 and R-14.

As can be seen from the results of FIGS. 5 and 6, it can be clearly seen that the bands of the 7 sets of conventional sequence RNA self-assembly products are all bright and clear single bands, indicating that the 7 sets of conventional sequences can self-assemble into the nano-structure. Wherein, after a section of Survivin siRNA nucleic acid interference treatment fragment is modified in the conventional sequence RNA self-assembly product R-14, the self-assembly structure still has a stable self-assembly structure, which also indicates that the nucleic acid nano-particle can carry a nucleic acid drug and has the function of a delivery carrier of the nucleic acid drug.

(2) Measurement of electric potential

The determination method comprises the following steps: preparing a potential sample, putting the potential sample into a sample cell, opening a sample cell cover of an instrument, and putting the instrument into the instrument;

opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;

setting software detection parameters;

and (3) measuring results: the results of the potential measurements for 7 sets of conventional sequence RNA nanoparticles are shown in tables 23 to 29 below:

table 23:

table 24:

table 25:

table 26:

table 27:

table 28:

table 29:

from the potential detection data described above, it is found that: the 7 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have a stable self-assembly structure.

This example shows that: on the basis of RNA core sequences of different combinations, the addition of the extension segment can also successfully self-assemble into RNA nanoparticles with stable structure. Meanwhile, the added extension fragment enables the RNA nanoparticles to have excellent drug-loading performance (see example 5 in particular).

Example 4

One, 9 groups of conventional sequence DNA nanoparticle carriers:

(1) the three polynucleotide base sequences of the first 7 groups of DNA nanoparticles are shown in tables 30 to 36:

the EGFRatt or PSMAaptt (A9L) target is extended in part a strand:

EGFRapt(SEQ ID NO：97)：GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC；

PSMAapt(A9L，SEQ ID NO：98)：

GGGCCGAAAAAGACCTGACTTCTATACTAAGTCTACGTCCC。

table 30: d-1

Table 31: d-2

Table 32: d-3

Table 33: d-4

Table 34: d-5

Table 35: d-6

Table 36: d-7

The single strands of the 7 sets of conventional sequence DNA nanoparticles were synthesized by hong, sozhou entrusted, where:

d-1 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined section below) to the core sequence (8) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3') described previously;

d-2 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined section below) to the core sequence (9) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3') described previously;

d-3 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined section below) to the core sequence (10) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3') described previously;

d-4 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAApt target head (see underlined part below) to the core sequence (11) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3') described above;

d-5 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAApt target head (see underlined part below) to the core sequence (12) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3') described above;

d-6 is the core sequence (13) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3') added with an extension sequence not containing the targeting structure; the formed conventional sequence DNA nanoparticles;

d-7 is an extension sequence which does not contain a targeting structure and is added to the core sequence (14) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3') described above; and forming the conventional sequence DNA nano-particles.

In addition, single-stranded sequences for forming the 8 th set of DNA nanoparticles and single-stranded sequences of the 9 th set of DNA nanoparticles were synthesized.

Group 8 are conventional sequence DNA nanoparticles formed by adding an extended sequence comprising the EGFRApt target head (see bold) to the core sequence (15) described above. The specific sequence is as follows:

a chain: (SEQ ID NO:172:) 5'-CGCGCGCCCACGAGCGTTCCGGGCGCGCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC-3'; the front three bases of the 5' end and the rear three bases of the 3' end are subjected to sulfo modification respectively, the 5' end is connected with Biotin, and the bold part is an EGFRApt sequence;

b chain (SEQ ID NO: 173:): 5'-GCGCCCGGTTCGCCGCCAGCCGCCGC-3', respectively carrying out sulfo-modification on the first three bases at the 5 'end and the last three bases at the 3' end;

chain c (SEQ ID NO: 174:): 5'-GCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCG-3'; the first three bases of the 5' end and the last three bases of the 3' end are respectively subjected to sulfo modification, and the 3' end is connected with Cy5 fluorescent label.

Wherein, group 9 is the DNA nanoparticles formed after adding the extension sequence on the basis of the core sequence (15) described above. The specific sequence is as follows:

chain a (SEQ ID NO: 178:): 5'-CGCGCGCGCCCACGAGCGTTCCGGGCGCCGCCGC-3'; the front three bases of the 5' end and the rear three bases of the 3' end are subjected to thio modification respectively, and the 5' end is connected with Biotin;

b chain (SEQ ID NO: 179:): 5'-GCGGCGGCGCCCGGTTCGCCGCCAGCCGCCGCC-3', respectively; the first three bases of the 5 'end and the last three bases of the 3' end are respectively subjected to sulfo-modification;

chain c (SEQ ID NO: 180:): 5'-GGCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCGCG-3', the first three bases of the 5' end and the last three bases of the 3' end are respectively modified by sulfo, and the 5' end is connected with Cy5 fluorescent label.

II, self-assembly experiment steps:

(1) mixing and dissolving the DNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;

(2) heating the mixed solution to 95 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;

(4) cutting off a target band, eluting in a DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a conventional sequence DNA self-assembly product;

(5) electrophoretic analysis detection and laser scanning observation;

(6) detecting the potential;

(7) measuring the particle size;

(8) and (5) observing by using a transmission electron microscope.

Third, self-assembly experimental results

(1) Results of electrophoresis

The 2% agarose gel electrophoresis of the first 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 7. Lanes 1 to 7 in FIG. 7 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.

The 4% agarose gel electrophoresis of the first 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 8. Lanes 1 to 7 in FIG. 8 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.

The 2% agarose gel electrophoresis picture of the self-assembly products of the sequence DNAs of

groups

8 and 9 is shown in FIG. 9. The lanes in FIG. 9 are from right to left: the single strand of group 8 a, the DNA self-assembly products D-8 and D-9.

As can be seen from the results of FIG. 7, FIG. 8 and FIG. 9, it can be clearly seen that the bands of the self-assembly products of the 9 groups of conventional sequence DNAs are bright and clear, indicating that the self-assembly of the 9 groups of conventional sequence DNA strands is completed, and a stable nanoparticle structure is formed. The two groups of self-assembly structures D-6 and D-7 have slightly lower molecular weight because of carrying EGFRept or PSMAept target heads, the positions of the bands of the self-assembly structures are obviously more forward than those of other bands, and the actual and theoretical conditions completely conform to the conditions, thereby further proving the stability of the self-assembly structures.

This example shows that: when various functional extension fragments are added on the basis of different DNA core sequence combinations or are simultaneously connected with a target head, the DNA nano-particles can be successfully assembled, and the DNA nano-particles also have the performances of drug loading, cell targeting, visual tracking and the like.

(2) Determination of potential

setting software detection parameters;

and (3) measuring results: the potential detection results of 3 groups of conventional sequence DNA nanoparticles are shown in tables 37 to 39 below:

table 37:

table 38:

table 39:

from the potential detection data described above, it can be seen that: the 3 groups of conventional sequence DNA self-assembly products have good stability, and further show that the nanoparticles formed by the self-assembly of the conventional sequence DNA have a stable self-assembly structure.

(3) Particle size measurement

1. Preparing a potential sample (a conventional sequence DNA self-assembly product D-7) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;

2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;

3. setting software detection parameters;

4. then click on the ok setting, the measurement dialog box appears, click Start, DLS measurements of hydrodynamic size of self-assembled product D-7 result in table 40 below:

table 40:

(4) observation result of transmission electron microscope

And (3) carrying out transmission electron microscope irradiation on the conventional sequence DNA self-assembly product D-7, and comprising the following steps:

1. taking a drop of sample to suspend on a 400-mesh carbon film-coated copper net, and keeping the temperature at room temperature for 1 minute;

2. sucking the liquid by filter paper;

3. dyeing for 1 minute by using 2% uranium acetate;

4. sucking dry by filter paper, and drying at room temperature;

5. JEM-1400 was observed by 120kv using a transmission electron microscope and photographed.

The result is shown in FIG. 10, from which it is apparent that the conventional sequence DNA self-assembly product D-7 is an integral structure and can be clearly seen to have a T-shaped structure.

Example 5

Cytarabine-mounting experiment

Carrying out chemical method mounting:

first, experimental material and experimental method

1. Experimental materials and reagents:

(1) the nucleic acid nanoparticles are: DNAh-Bio-EFGRapt-Cy5, wherein three chains of DNAh are as follows:

a chain: (SEQ ID NO:172:) 5'-CGCGCGCCCACGAGCGTTCCGGGCGCGCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC-3'; the front three bases of the 5' end and the back three bases of the 3' end are respectively subjected to thio modification, the 5' end is connected with Biotin, and the bold part is an siRNA sequence of the EGFR;

chain c (SEQ ID NO: 174:): 5'-GCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCG-3', respectively; the first three bases of the 5' end and the last three bases of the 3' end are respectively subjected to sulfo modification, and the 3' end is connected with Cy5 fluorescent label.

(2) DEPC water: biyun Tian.

(3) PBS buffer: cellgro.

(4) 4% Paraformaldehyde

(5) Cytarabine (Epirarunixon).

(6) Chloroform: and (4) carrying out north transformation.

(7) Anhydrous ethanol: and (6) north transformation.

2. The experimental method comprises the following steps:

(1) cytarabine (1.354. mu. moL) was precisely weighed, dissolved in DEPC water (1.0mL) and PBS buffer (1.25mL), mixed with 4% paraformaldehyde aqueous solution (0.25mL) with cooling in an ice-water bath, and the mixture was mixed with RNA nanoparticles (33.84nmoL) and reacted at 4 ℃ for 72 hours in the absence of light.

(2) Taking 10 mu L of reaction solution to dilute by 10 times, taking 50 mu M cytarabine aqueous solution and 310 ng/mu L of RNA nano-particles as controls, and injecting sample according to the equal volume for HPLC analysis. The reaction conversion can be judged to be basically complete according to the peak area ratio of each component.

(3) The reaction mixture was extracted with chloroform (10mL x3), followed by addition of 25mL of absolute ethanol, mixing, and then sufficiently precipitating the product by keeping the mixture at 4 ℃ in the dark (4 hours). Centrifugation (4000/min) and transfer of the supernatant, re-washing of the solid product with ethanol (50mL) and evaporation of the solvent at low temperature under reduced pressure gave the product as a dark red solid.

(4) And (3) mounting rate calculation:

1. preparing a cytarabine-PBS standard solution with a known concentration: 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, each 100. mu.l;

2. dissolving the cytarabine-DNAh particles in 100. mu.l PBS;

3. placing the standard solution and the cytarabine-DNAh particles in a PCR plate, heating at 85 ℃ for 5min, and then cooling to room temperature;

4. measuring the absorbance of the cytarabine at 272nm by using a microplate reader, drawing a standard curve, and calculating the molar concentration of the cytarabine in the mounted product;

5. measuring the absorbance of the DNA at the position of 260nm by using a spectrophotometer to obtain the mass concentration of DNAh particles in each sample;

6. and calculating the mounting rate according to the measured cytosine arabinoside molar concentration and the mass concentration of the DNAh particles.

See FIG. 11 for a standard graph of DNA particles loaded with cytarabine.

C_DNAh-1＝32.4μg/ml，M_DNAh≈39500，100μl；C_Cytarabine-1＝9.8μM，100μl；

C_DNAh-2＝43.8μg/ml，M_DNAh≈39500，100μl；C_Cytarabine-2＝12.32μM，100μl；

The average value is taken to obtain that the carrying rate of the cytarabine-DNAh nucleic acid nano particles is about 89, which shows that about 89 cytarabine molecules can be carried on each nucleic acid nano particle carrier.

Example 5 shows that the DNA nanoparticles with the extension segment, the targeting segment and the fluorescein have the function of drug loading, and the small molecule drug cytarabine can be loaded in a covalent connection mode.

Example 6

Flow cytometry experiment for detecting cell binding capacity of drug-loaded DNA nanoparticles

First, cell information

MV4-11 (ATCC as source, CRL-9591 as stock), RPMI1640+ 10% FBS as culture medium, at 37 deg.C and 5% CO₂And saturation humidity.

Second, the object to be measured

Targeting drugs: DNAh-Bio-EGFRApt-Cy 5-Cy cytarabine (the product of the loading of DNA nanoparticles in example 5).

A fluorescent carrier: DNAh-Bio-EGFRApt-Cy5 (DNA nanoparticles in example 5).

Third, equipment, consumables (see table 41)

Watch 41

Reagent (see table 42)

Table 42:

and fifthly, an experimental method:

1. adjusting the cell state to logarithmic phase, changing the culture medium to a biotin-free and folic acid-free culture medium, and placing the culture medium in an incubator at 37 ℃ for overnight incubation;

2. dissolving a to-be-detected object, and preparing a to-be-detected object stock solution;

3. digesting, collecting single cell suspension, counting, and adjusting cell density to 2 × 10⁵ Planting 1 mL/hole into a 24-hole plate;

4. adding the substances to be detected into corresponding cell holes respectively, and shaking and mixing the substances to be detected with final concentrations of 0.1 mu M, 0.2 mu M and 0.4 mu M;

5. incubating the cell plate in an incubator at 37 ℃ for 2 hours;

6. after incubation is finished, collecting cell suspension by trypsinization;

7. centrifuging to collect cell precipitate, and washing twice with PBS;

8. finally, resuspending the cell precipitate with 300 μ L of PBS, and detecting on a flow machine;

9. fluorescent carrier or cytarabine detection channel: wavelength of excitation light: 488nm, emission light channel: 560 nm;

10. and (6) analyzing the data.

Sixth, the experimental results (see Table 43)

Table 43:

as can be seen from Table 43, the cytarabine targeting drug DNAh-Bio-EFGRapt-Cy 5-cytarabine was able to bind to MV4-11 cell with nearly one hundred percent binding rate; the fluorescent vector DNAh-Bio-EFGRapt-Cy5 can be combined with MV4-11 cells, and the combination rate is also nearly one hundred percent.

Example 7

Detection of stability of DNAh-Bio-EGFRApt-Cy 5-cytarabine nanoparticles in serum

First, experimental material, reagent and equipment

1. Experimental materials

DNAh-Bio-EGFRapt-Cy 5-cytarabine (same as example 6) at a concentration of 1321.8. mu.g/ml.

2. Experimental reagent

6 XDNA sample buffer (TSJ010, engine biology), 100bp DNA molecular marker (TSJ010, engine biology); 10000 x SolarGelRed nucleic acid dye (E1020, solarbio); 8% non-denaturing polyacrylamide gel (self-formulated); 1 × TBE Buffer (No RNase) (self-mix); serum (FBS) (Excel); RPMI1640 (GBICO).

Electrophoresis apparatus (PowerPac Basic, Bio-rad), vertical electrophoresis tank (Mini PROTEAN Tetra Cell, Bio-rad), decolorizing shaker (TS-3D, orbital shaker), gel imager (Tanon 3500, Tanon).

Second, Experimental methods

(1) And taking 4 mu L of DNAh-Bio-EGFRapt-Cy 5-cytarabine nano particles, diluting the obtained product with 21.8 mu L of RPMI1640 medium containing 10% serum to obtain a diluted concentration of 197.5 mu g/ml, respectively diluting the diluted product in 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h).

(2) The treated sample 20. mu.L was mixed with 4. mu.L of 6 XDNA Loading Buffer, and the mixture was labeled by ice-wash.

(3) 8% Native PAGE is taken, nanoparticle samples with different incubation times are coated with a gel, the loading amount is 20 mu L/hole/sample, and the program electrophoresis is set at 90-100V for 50 min.

(4) And after the electrophoresis is finished, dyeing, placing the mixture in a horizontal shaking table for 30min, and photographing for imaging.

Third, experimental results

The results of native PAGE gel electrophoresis are shown in FIG. 12, wherein 1 represents 0min, 2 represents 10min,3 represents 1h, 4 represents 12h, and 5 represents 36 h. The target band of the DNAh-Bio-EGFRAPT-Cy 5-cytarabine nanoparticle is about 200bp, and as can be seen from figure 12, the DNAh-Bio-EGFRAPT-Cy 5-cytarabine nanoparticle is basically stable when being incubated at 37 ℃, and slight drug release or degradation occurs after 12h and 36h of incubation.

Example 8

Cytotoxicity of DNAh-Bio-EGFRApt-Cy 5-Cytarabine nanoparticles in MV4-11 cells, respectively

First, experimental material

1. Cell information (see table 44):

table 44:

name(s)	Source	Culture medium	Culture conditions
				MV4-11	ATCC	RPMI	1640,10％FBS	37℃，5％CO₂Saturated humidity

2. Samples to be tested (see table 45):

table 45:

3. consumables and equipment (see table 46):

table 46:

name (R)	Brand	Goods number/model
			96-well plate	Corning	3599
Centrifugal machine	Jingli	LD5-2B
			CO₂Culture box	Thermo	3111
Microplate oscillator	QILINBEIER	QB-9001
			Microscope	Olympus	IX53
Multifunctional enzyme mark instrument	Bio Tek	Synergy H1

4. Reagents (see table 47):

table 47:

II, an experimental method:

1) harvesting cells in logarithmic growth phase, taking a small amount of cells, and staining and counting the cells by trypan blue to ensure that the cell activity reaches more than 98%;

2) cell density was adjusted to 1.11X 10 with growth medium⁵/mL；

3) Planting 90 mu L/well cell suspension into a 96-well plate, wherein the number of cells in each well in the plate is 10000;

4) placing the planted cell plate in an incubator at 37 ℃ for overnight incubation;

5) compound was diluted 3.16-fold in gradient from 9 concentration points;

6) taking out the cell culture plate, adding 10 μ L/hole 10X concentration drug working solution into corresponding hole of the cell culture plate, making three multiple holes for each concentration, and obtaining final action concentration of the drug shown in Table 48;

table 48:

7) placing the cell culture plate in an incubator to continue incubation for 96 hours;

8) mixing CellTiter

Melting the AQueous One Solution reagent at room temperature for 90 minutes or melting the AQueous One Solution reagent in water bath at 37 ℃, and then balancing the AQueous One Solution reagent at room temperature for 30 minutes;

9) add 20. mu.L/well CellTiter to cell culture plate

An AQueous One Solution reagent;

10) placing the cell culture plate in an incubator at 37 ℃ for further incubation for 3 hours;

11) OD of each well in the cell plate was read with microplate reader₄₉₀A value;

12) and (4) processing and analyzing data.

The data were graphically processed using GraphPad Prism 5.0 software to calculate IC50, data were subjected to "S" shaped nonlinear regression analysis to match the appropriate dose-effect curve. The survival rate was calculated as follows, and IC50 was automatically calculated in GraphPad Prism 5.0.

Cell viability (%) - (OD)_{Hole to be tested}–OD_{Blank control})/(OD_{Negative control}-OD_{Blank control})x 100％。

Third, the results of the experiment (see Table 49, FIGS. 13a to 13d)

Table 49:

as can be seen from table 49 and fig. 13a, 13b, 13c, and 13d, for the MV4-11 cell line, compared to the single DNAh targeting fluorescent vector, both the small molecule drug cytarabine and the DNAh drug-loaded particle DNAh-Bio-egfrrapt-Cy 5-cytarabine were toxic to MV4-11 cells.

Assembly of nucleic acid nanoparticles

Example 9

One, 7 groups of extended segment deformation + core short sequence RNA nano particle carriers:

(1)7 sets of three polynucleotide base sequences which form the RNA nano-particle with the extension segment deformed and the core short sequence:

table 50: r-15:

TABLE 51: r-16:

table 52: r-17:

table 53: r-18:

table 54: r-19:

table 55: r-20:

table 56: r-21:

II, self-assembly testing:

(5) electrophoresis analysis and detection and laser scanning observation.

Third, self-assembly test results

(1) Electrophoretic detection

The main reagents and instruments were as follows:

table 57:

name of reagent	Goods number	Manufacturer(s) of
			6×DNA Loading buffer	TSJ010	Organisms of Onychidae
20bp DNA Ladder	3420A	TAKARA
			10000 SolarGelRed nucleic acid dye	E1020	solarbio
8% native PAGE gel	/	Self-matching
			1 XTBE Buffer (No RNase)	/	Self-matching

Table 58:

the method comprises the following steps:

the RNA nanoparticles were diluted with ultrapure water according to the method of Table 59 below.

Table 59:

	measured concentration (μ g/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

Secondly, mixing 10 mu L (500ng) of the processed sample with 2 mu L of 6 multiplied DNA Loading Buffer, operating on ice and marking.

③ taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.

And fourthly, dyeing after glue running is finished, placing the dyed fabric on a horizontal shaking table for 30min, and photographing and imaging.

And (3) detection results:

the results of native PAGE gel for 7 sets of extended stretch-degenerate + core short sequence RNA self-assembly products are shown in FIG. 14. Lanes 1 to 7 in FIG. 14 are, from left to right: 7 groups of self-assembly products of the RNA with the extension segment deformation and the core short sequence, R-15, R-16, R-17, R-18, R-19, R-20 and R-21.

The results in fig. 14 clearly show that the bands of the 7 sets of RNA self-assembly products with the modified long segment and the short core sequence are bright and clear, which indicates that the 7 sets of RNA strands with the modified long segment and the short core sequence complete the self-assembly and form a stable nanoparticle structure.

(2) Determination of potential

setting software detection parameters;

then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;

and (3) measuring results: the potential detection results at 25 ℃ of 7 groups of extended segment deformation + core short sequence RNA nanoparticles are as follows:

table 60:

table 61:

table 62:

table 63:

table 64:

table 65:

table 66:

from the potential detection data described above, it is found that: the 7 groups of the extended segment deformation and core short sequence RNA nanoparticles have good stability, and further show that the nanoparticles formed by self-assembly of the extended segment deformation and the core short sequence RNA have a stable self-assembly structure.

(3) Particle size measurement

1. Preparing a potential sample (7 groups of extension sections are deformed and core short sequence RNA is added) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;

3. setting software detection parameters;

4. then click on the confirmed setting, a measurement dialog box appears, and Start is clicked, and the results of DLS measurement values of hydrodynamic sizes of 7 groups of extended stretch variants + core short sequence RNAs are as follows:

table 67:

	average particle diameter (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 value detection

And (3) detecting the TM values of the 7 groups of extended section deformation + core short sequence RNA nanoparticles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.

Reagents and instrumentation were as follows:

table 68:

name of reagent	Goods number	Manufacturer(s) of
			AE buffer	/	Takara
SYBR Green I dye	/	Self-matching

Table 69:

name (R)	Type number	Manufacturer of the product
			Real-Time System	CFX Connect	Bio-rad
Super clean bench	HDL	BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd.

The method comprises the following steps:

after diluting the sample with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1: 200 dilution) to give a final volume of 20. mu.L, at the following dilution concentrations:

table 70:

incubating for 30min at room temperature in a dark place;

and thirdly, detecting on a computer, wherein the program is set to be 20 ℃, the temperature is increased to 0.1-95 ℃ per second, and the reading is carried out once every 5 seconds.

And (3) detection results:

the TM values of 7 sets of extended stretch modified + core short sequence RNA nanoparticles are shown in the following, wherein the dissolution curve of R-15 is shown in FIG. 15, the dissolution curve of R-16 is shown in FIG. 16, the dissolution curve of R-17 is shown in FIG. 17, the dissolution curve of R-18 is shown in FIG. 18, the dissolution curve of R-19 is shown in FIG. 19, the dissolution curve of R-20 is shown in FIG. 20, and the dissolution curve of R-21 is shown in FIG. 21. Because of the specificity of the RNA sample, the temperature corresponding to 1/2RFUmax within the range of 20-90 ℃ is taken as the Tm value of the sample in the detection.

Table 71:

	TM value (. degree. C.)
		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℃

The TM values of 7 groups of extension segment deformation and core short sequence RNA nanoparticles are higher, which indicates that the self-assembly product has good structural stability.

Example 10

1, 7 groups of extension segment deformation + core short sequence DNA nano particle carriers:

(1)7 groups of three polynucleotide base sequences which form the extension segment deformation + core short sequence DNA nano-particles:

table 72: d-8:

table 73: d-9:

table 74: d-10:

table 75: d-11:

table 76: d-12:

table 77: d-13:

table 78: d-14:

II, self-assembly testing:

(4) cutting off a target band, eluting in a DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a DNA self-assembly product;

(5) electrophoretic analysis detection and laser scanning observation;

(6) detecting the potential;

(7) detecting the particle size;

(8) and (5) detecting the TM value.

Third, self-assembly test results

(1) Electrophoretic detection

The main reagents and instruments were as follows:

TABLE 79:

name of reagent	Goods number	Manufacturer(s) of
			6×DNA Loading buffer	TSJ010	Organisms of Onychidae
20bp DNA Ladder	3420A	TAKARA
			10000 SolarGelRed nucleic acid dye	E1020	solarbio
8% native PAGE gel	/	Self-matching
			1 × TBE Buffer (No RNA)Enzyme)	/	Self-matching

Table 80:

the method comprises the following steps:

the DNA nanoparticles were diluted with ultrapure water according to the method of the following Table 81.

Table 81:

Taking 8% non-denaturing PAGE gel, coating a piece of gel on samples with different incubation times, and completely loading 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.

And (3) detection results:

the results of native PAGE gel of 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are shown in FIG. 22. Lanes 1 to 7 in FIG. 22 are, from left to right: 7 groups of extension segment deformation + core short sequence DNA self-assembly products D-8, D-9, D-10, D-11, D-12, D-13 and D-14.

The results in fig. 22 clearly show that the bands of the 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are bright and clear, which indicates that the 7 sets of extended stretch-deformed + core short sequence DNA strands complete self-assembly and form a stable nanoparticle structure.

(2) Measurement of electric potential

The measuring method comprises the following steps: preparing a potential sample (a self-assembly product is dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;

setting software detection parameters;

and (3) measuring results: the potential detection results at 25 ℃ of 7 groups of extension segment deformation + core short sequence DNA nanoparticles are as follows:

table 82:

table 83:

table 84:

table 85:

table 86:

table 87:

table 88:

from the potential detection data described above, it is found that: the 7 groups of extension segment deformation and core short sequence DNA nanoparticles have good stability, and further show that the nanoparticles formed by the extension segment deformation and the core short sequence DNA through self-assembly have a stable self-assembly structure.

(3) Particle size measurement

Preparing a potential sample (7 groups of extension section deformation and core short sequence DNA) to be placed in a sample cell, opening a sample cell cover of an instrument, and placing the instrument;

opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;

setting software detection parameters;

and fourthly, clicking the setting after determination, generating a measurement dialog box, clicking Start, and obtaining the results of the DLS measurement values of the hydrodynamic sizes of 7 groups of the extended segment deformation and the core short sequence RNA as follows:

table 89:

	average particle diameter (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 value detection

And (3) detecting the TM values of the 7 groups of extension segment deformation + core short sequence DNA nanoparticles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.

Reagents and instrumentation were as follows:

table 90:

name of reagent	Goods number	Manufacturer of the product
			AE buffer	/	Takara
SYBR Green I dyes	/	Self-matching

Table 91:

name(s)	Model number	Manufacturer of the product
			Real-Time System	CFX Connect	Bio-rad
Super clean bench	HDL	BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd.

The method comprises the following steps:

after diluting the sample with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1: 200 dilution) to a final volume of 20. mu.L, at the following dilution concentrations:

table 92:

incubating for 30min at room temperature in a dark place;

And (3) detection results:

the TM values of 7 sets of extended length modified + core short sequence DNA nanoparticles are as follows, and the dissolution profile of D-8 is shown in FIG. 23, the dissolution profile of D-9 is shown in FIG. 24, the dissolution profile of D-10 is shown in FIG. 25, the dissolution profile of D-11 is shown in FIG. 26, the dissolution profile of D-12 is shown in FIG. 27, the dissolution profile of D-13 is shown in FIG. 28, and the dissolution profile of D-14 is shown in FIG. 29.

Table 93:

	TM value (. degree. C.)
		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

As can be seen from the dissolution curves of 7 sets of extended stretch deformation + core short sequence DNA nanoparticles shown in FIGS. 23 to 29, the TM values are all high, indicating that the sample purity is high and the self-assembly structure is stable.

Detecting stability of nucleic acid nanoparticles in serum

Example 11

And (3) characterizing the stability of the 7 groups of the extended segment deformation + core short sequence RNA nanoparticles in serum by adopting a non-denaturing PAGE method.

The main reagents and instruments were as follows:

table 94:

table 95:

the method comprises the following steps:

firstly, preparing the RNA nanoparticles into the concentrations shown in the following table, then diluting the prepared sample according to the method shown in the following table, diluting for 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);

table 96:

mixing 10 mu L of the treated sample with 2 mu L of 6 multiplied DNA Loading Buffer, operating on ice and marking;

thirdly, taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 12 mu L of processed samples, and setting a program of 100V gel running for 40 min;

fourthly, dyeing is carried out after glue running is finished, the dyeing is placed on a horizontal shaking table to be slowly oscillated for 30min, and photographing and imaging are carried out.

The electrophoresis detection result of R-15 is shown in FIG. 30, the electrophoresis detection result of R-16 is shown in FIG. 31, the electrophoresis detection result of R-17 is shown in FIG. 32, the electrophoresis detection result of R-18 is shown in FIG. 33, the electrophoresis detection result of R-19 is shown in FIG. 34, the electrophoresis detection result of R-20 is shown in FIG. 35, and the electrophoresis detection result of R-21 is shown in FIG. 36. In fig. 30 to 36, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and 0 min. From the results of the serum stability test, it can be seen that: the non-denatured gel fruits of 10min, 1h, 12h and 36h showed no significant difference in the RNA nanoparticle sample bands at different times, indicating that the RNA nanoparticles R-15 to R-21 were relatively stable in 1640 medium of 50% FBS without significant degradation.

Example 12

And (3) characterizing the stability of the 7 groups of extended segment deformation + core short sequence DNA nanoparticles in serum by adopting a non-denaturing PAGE method.

The main reagents and instruments were as follows:

table 97:

name of reagent	Goods number	Manufacturer of the product
			6×DNA Loading buffer	TSJ010	Organisms of Onychidae
20bp DNA Ladder	3420A	TAKARA
			10000 SolarGelRed nucleic acid dye	E1020	solarbio
8% non-denaturing PAGE gel	/	Self-matching
			1 × TBE Buffer (No RNAse)	/	Self-matching
Serum (FBS)	/	Excel
			RPMI 1640	/	GBICO

Table 98:

the method comprises the following steps:

preparing DNA nanoparticles into the concentrations shown in the following table, diluting the prepared sample by the method shown in the following table for 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);

table 99:

secondly, mixing 5 mu L of the treated sample with 1 mu L of 6 multiplied DNA Loading Buffer uniformly, operating on ice and marking;

thirdly, taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 6 mu L of processed samples, and setting a program of 100V gel running for 40 min;

and fourthly, dyeing after the glue running is finished, placing the dyed fabric on a horizontal shaking table to slowly oscillate for 30min, and taking pictures for imaging.

The electrophoresis detection result of D-8 is shown in FIG. 37, the electrophoresis detection result of D-9 is shown in FIG. 38, the electrophoresis detection result of D-10 is shown in FIG. 39, the electrophoresis detection result of D-11 is shown in FIG. 40, the electrophoresis detection result of D-12 is shown in FIG. 41, the electrophoresis detection result of D-13 is shown in FIG. 42, and the electrophoresis detection result of D-14 is shown in FIG. 43. In fig. 37 to 43, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and 0 min. From the results of the serum stability test, it can be seen that: the 10min, 1h, 12h and 36h non-denatured gel fruits showed no significant difference in the bands of the DNA nanoparticle samples at different times, indicating that the DNA nanoparticles D-8 to D-14 were relatively stable in 1640 medium with 50% FBS and had no significant degradation.

Nucleic acid nanoparticle-loaded drug assay

Example 13

Doxorubicin mounting experiment:

according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), RNA nanoparticles formed by self-assembly of R-15, R-16, R-17, R-18, R-19, R-20 and R-21 in the previous example 9, and DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in example 10 were used as doxorubicin-carrying carriers, and the doxorubicin-carrying rates were respectively measured as follows:

the adriamycin loading rate of the RNA nano-particle R-15 is 20.5;

the adriamycin loading rate of the RNA nano-particle R-16 is 29.4;

the adriamycin loading rate of the RNA nano-particle R-17 is 30.9;

the adriamycin loading rate of the RNA nano-particle R-18 is 34.1;

the adriamycin loading rate of the RNA nano-particle R-19 is 27.1;

the adriamycin loading rate of the RNA nano-particle R-20 is 30.2;

the adriamycin loading rate of the RNA nano-particle R-21 is 20.1;

the adriamycin loading rate of the DNA nano-particle D-8 is 28.0;

the adriamycin loading rate of the DNA nano-particle D-9 is 27.9;

the adriamycin loading rate of the DNA nano-particle D-10 is 18.9;

the adriamycin loading rate of the DNA nano-particle D-11 is 26.8;

the adriamycin loading rate of the DNA nano-particle D-12 is 27.6;

the adriamycin loading rate of the DNA nano-particle D-13 is 31.8;

the adriamycin loading rate of the DNA nanoparticle D-14 was 32.

Flow cytometry (FACS) experiment for detecting cell binding capacity of DNA nanoparticles and carrier drug

Example 14

First, cell information

HepG2 (Source synergy cell bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), culture conditions at 37 ℃ and 5% CO₂And saturation humidity.

Second, the object to be measured

Blank vector: d-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the aforementioned example 10 were self-assembled to form DNA nanoparticle carriers.

Carrier drug: according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the previous example 10 are used to carry doxorubicin, which is respectively marked as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin.

Third, main equipment and consumable

TABLE 100:

four, main reagent

Table 101:

name of reagent	Manufacturer of the product	Goods number	Remarks for note
				DMEM (Biotin free)	All-medicinal Zhida Providence	YS3160
1％BSA-PBS	Self-matching	－

And fifthly, an experimental method:

2. after incubation, cell suspension was collected by trypsinization, centrifuged at 1000rmp for 5min, adjusted in concentration, and 2X 10 cells were collected⁵-5×10⁵cells/EP tube, wash 2 times with 1 mL/tube of 1% BSA-PBS, and observe the tube bottom cells to prevent aspiration.

3. Dissolving the object to be tested, and diluting the object to be tested to the use concentration;

4. completely sucking cell supernatant, sequentially adding 100 mu L of corresponding samples into each tube, keeping out of the sun, and incubating for 2h at 37 ℃;

5. washed 2 times with 1% BSA-PBS; centrifuging at 1000rmp for 5 min;

6. finally, resuspending the cell pellet with 300. mu.L PBS and detecting it on flow machine (the blank vector used in this example was labeled by Quasar670, whereas doxorubicin in the vector drug was self-fluorescent and thus could be detected by FL4-APC and FL2-PE, respectively);

7. and (6) analyzing the data.

Sixth, experimental results

1. The results of the experiment are shown in the following table:

table 102:

2. conclusion

After incubation of HepG2 cells with D-8-adriamycin (vector medicine) and D-8 (blank vector), the binding rate is very high (93.1% -98.4%).

After incubation of HepG2 cells with D-9-adriamycin (vector drug) and D-9 (blank vector), the binding rate is very high (88.6% -98.1%).

After incubation of HepG2 cells with D-10-adriamycin (vector drug) and D-10 (blank vector), the binding rate is high (89.4% -98.3%).

After incubation of HepG2 cells with D-11-adriamycin (carrier drug) and D-11 (blank carrier), the binding rate is high (89.3% -97.8%).

After incubation of HepG2 cells with D-12-adriamycin (vector drug) and D-12 (blank vector), the binding rate is very high (94.6% -97.1%).

After incubation of HepG2 cells with D-13-adriamycin (vector drug) and D-13 (blank vector), the binding rate is high (89.6% -98.2%).

After incubation of HepG2 cells with D-14-adriamycin (vector drug) and D-14 (blank vector), the binding rate is very high (90.3% -98.3%).

Study of cytotoxicity of DNA nanoparticles and vector drugs in HepG2 cells

Example 15

The toxicity of the DNA nanoparticles and the carrier drug to HepG2 is detected by a CCK8 method.

First, main reagent

Table 103:

name of reagent	Manufacturer(s) of	Goods number
			PBS	－	－
DMSO	SIGMA	D2650
			DMEM (Biotin free)	All-medicinal Zhida Providence	YS3160
FBS	Excell Bio	FSP500
			Double antibody	gibco	15140-122
Pancreatin	gibco	25200-056
			CCK8 kit	Biyuntian (a Chinese character)	C0038

Second, main consumables and instrument

Table 104:

name(s)	Manufacturer of the product	Type number
			96-well cell culture plate	NEST	701001
Biological safety cabinet	Beijing Dong Bihaer Instrument manufacturing Co Ltd	BSC-1360ⅡA2
			Low-speed centrifuge	Zhongke Zhongjia Instrument Co., Ltd	SC-3612
CO₂Culture box	Thermo	3111
			Inverted microscope	UOP	DSZ2000X
Enzyme mark instrument	SHANGHAI OYIN EXPERIMENT EQUIPMENT Co.,Ltd.	K3

III, cell information

HepG2 (Source synergistic cell Bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), at 37 ℃ and 5% CO₂And saturation humidity.

Fourth, experimental materials

1. Sample to be tested

Blank vector: the DNA nanoparticle carriers formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the foregoing example 10 are respectively denoted as: d-8, D-9, D-10, D-11, D-12, D-13 and D-14.

A bulk drug of doxorubicin.

DMSO。

Fifth, the experimental procedure

1.HepG2 cells were harvested in the logarithmic growth phase, the Cell viability was 98.3% by trypan blue staining, and the cells were plated at 5000 cells/well in a volume of 100. mu.L/well in 8 96-well plates, 57 wells per plate, and incubated overnight at 37 ℃.

2. The samples to be tested were diluted and added as follows: removing the original culture medium, adding 100 μ L culture medium of samples to be tested with different concentrations, and repeating the wells for 3 times.

Table 105:

number of hole

C9

C8

C7

C6

C5

C4

C3

C2

C1

Final concentration of drug-carried

10μM

3.16μM

1μM

316nM

100nM

31.6nM

10nM

3.16nM

1nM

Final concentration of empty vector

1μM

316nM

100nM

31.6nM

10nM

3.16nM

1nM

0.316nM

0.1nM

Final concentration of technical doxorubicin

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

In this example, the drug-loaded and blank vehicles were each prepared as 100 μ M stock solutions in PBS and then diluted in complete medium (no biotin DMEM). The technical doxorubicin is prepared into a stock solution of 100 μ M with DMSO and then diluted with complete medium (biotin-free DMEM). DMSO was directly diluted with complete medium (biotin-free DMEM).

3. Adding a sample to be detected, and placing a 96-well plate into 5% CO at 37 DEG C₂Incubate in incubator for 72 h.

4. The kit was removed and thawed at room temperature, and 10. mu.L of CCK-8 solution was added to each well, or CCK8 solution was mixed with the medium at a ratio of 1:9 and then added to the wells at a rate of 100. mu.L/well.

5. And continuously incubating for 4 hours in the cell incubator, wherein the time depends on the type of the cells, the density of the cells and other experimental conditions.

6. Absorbance was measured at 450nm using a microplate reader.

7. And (3) calculating: cell viability (%) (OD experimental-OD blank) × 100%/(OD control-OD blank), IC calculated from GraphPad Prism 5.0₅₀。

Sixth, experimental results

Table 106:

and (4) conclusion:

as can be seen from the above table and FIGS. 44a, 44b, 44c, 44D, 44e, 44f, 44g, and 44h, the original doxorubicin and the drug-loaded D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, and the pharmaceutically acceptable salts thereof,IC of D-12-Doxorubicin, D-13-Doxorubicin, and D-14-Doxorubicin on HepG2 cells₅₀0.2725. mu.M, 0.05087. mu.M, 0.0386, 0.03955, 0.04271, 0.02294, 0.03017 and 0.03458, respectively; IC of DMSO on HepG2 cells₅₀Is composed of>0.1 percent; IC of HepG2 cells acted on by D-8 (blank vector), D-9 (blank vector), D-10 (blank vector), D-11 (blank vector), D-12 (blank vector), D-13 (blank vector) and D-14 (blank vector)₅₀Are all made of>1 μ M. The results show that compared with the pure blank vectors D-8, D-9, D-10, D-11, D-12, D-13 and D-14, the original drug adriamycin of the small molecule drug and the drug-loaded D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin are toxic to HepG2 cells of the HepG2 cell line, and the carried medicines of D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin have obvious synergistic effect compared with the original medicine of adriamycin.

Example 16

According to the chemical method of mounting in example 5 (the same method as in example 5 except for specific limitations), DNA nanoparticles formed by self-assembly of D-10 and D-14 in the previous example 10 were used as daunorubicin mounting vectors. The absorbance of daunorubicin at 492nm was measured with a microplate reader, and a standard curve was plotted (as shown in FIG. 45).

The daunorubicin loading rates were determined as follows:

the daunorubicin loading rate of the DNA nano-particles D-10 is 24.0;

the daunorubicin loading rate of the DNA nanoparticle D-14 was 25.1.

From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the present application provides a series of nucleic acid nanoparticle carriers with thermodynamic stability, chemical stability, high loading rate, and combinable multiple modules. The unique modular design of this type of vector results in a core modular structure that retains natural compatible affinities, yet has highly stable properties and diverse combinatorial features. The structure can flexibly and efficiently integrate various functional modules, including a targeting module, an imaging and probe module, a treatment module and other composite intelligent modules, so that the structure can be used for targeting delivery in vivo and realizing accurate diagnosis and treatment.

The cytarabine is a small-molecule drug and is loaded on the nucleic acid nanoparticle carrier provided by the application to form the cytarabine-containing drug, so that the delivery stability of the cytarabine can be improved, and the cytarabine can be delivered to target cells in a targeted manner under the condition that the nucleic acid nanoparticle carries a target head, so that the bioavailability of the drug is improved, and the toxic and side effects on non-target cells or tissues are reduced, the local drug concentration is reduced, and the toxic and side effects caused by high drug concentration are further reduced.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Baiyaozhima (Beijing) Nanoconstructural Co., Ltd

<120> cytarabine-containing medicine, preparation method, pharmaceutical composition and application thereof

<130> PN114942BYZD

<141> 2019-09-30

<150> 201811204344.9

<151> 2018-10-16

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<400> 60

cggccauagc gc 12

<210> 61

<211> 10

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(10)

<223> a chain

<400> 61

cgagcguugc 10

<210> 62

<211> 9

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(9)

<223> b chain

<400> 62

gcuucgccg 9

<210> 63

<211> 12

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(12)

<223> c chain

<400> 63

cggccauagc cg 12

<210> 64

<211> 10

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(10)

<223> a chain

<400> 64

ggagcguugg 10

<210> 65

<211> 9

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(9)

<223> b chain

<400> 65

ccuucgggg 9

<210> 66

<211> 12

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(12)

<223> c chain

<400> 66

cccccauagc cc 12

<210> 67

<211> 10

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(10)

<223> a chain

<400> 67

gcagcguucg 10

<210> 68

<211> 9

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(9)

<223> b chain

<400> 68

cguucggcg 9

<210> 69

<211> 12

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(12)

<223> c chain

<400> 69

cgcccauagc gc 12

<210> 70

<211> 10

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(10)

<223> a chain

<400> 70

gcagcguucg 10

<210> 71

<211> 9

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(9)

<223> b chain

<400> 71

cguucggcc 9

<210> 72

<211> 12

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(12)

<223> c chain

<400> 72

ggcccauagc gc 12

<210> 73

<211> 10

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(10)

<223> a chain

<400> 73

cgagcguugc 10

<210> 74

<211> 9

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(9)

<223> b chain

<400> 74

gcuucggcg 9

<210> 75

<211> 12

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(12)

<223> c chain

<400> 75

cgcccauagc cg 12

<210> 76

<211> 29

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 76

cgcgcgccca ggagcguugg cgggcggcg 29

<210> 77

<211> 27

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 77

cgccgcccgc cuucgccgcc agccgcc 27

<210> 78

<211> 31

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 78

ggcggcaggc ggccauagcc cugggcgcgc g 31

<210> 79

<211> 29

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 79

cgcgcgccca gcagcguucg cgggcggcg 29

<210> 80

<211> 27

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 80

cgccgcccgc guucgccgcc agccgcc 27

<210> 81

<211> 31

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 81

ggcggcaggc ggccauagcg cugggcgcgc g 31

<210> 82

<211> 29

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 82

cgcgcgccca cgagcguugc ggggcggcg 29

<210> 83

<211> 27

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 83

cgccgccccg cuucgccgcc agccgcc 27

<210> 84

<211> 31

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 84

ggcggcaggc ggccauagcc gugggcgcgc g 31

<210> 85

<211> 29

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 85

cgcgcgccca ggagcguugg cccgcggcg 29

<210> 86

<211> 27

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 86

cgccgcgggc cuucggggcc agccgcc 27

<210> 87

<211> 31

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 87

ggcggcaggc ccccauagcc cugggcgcgc g 31

<210> 88

<211> 29

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 88

cgcgcgccca gcagcguucg ccccgccgc 29

<210> 89

<211> 27

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 89

gcggcggggc guucggcggc aggcggc 27

<210> 90

<211> 31

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 90

gccgccagcc gcccauagcg cugggcgcgc g 31

<210> 91

<211> 29

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 91

cgcgcgccca gcagcguucg gggcgccgc 29

<210> 92

<211> 28

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(28)

<223> b chain

<400> 92

gcggcgcccc guucggccgg caggcggc 28

<210> 93

<211> 32

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(32)

<223> c chain

<400> 93

gccgccagcc ggcccauagc gcugggcgcg cg 32

<210> 94

<211> 40

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(40)

<223> a chain

<400> 94

cgcgcgcgag cguugcaaug acagauaagg aaccugcutt 40

<210> 95

<211> 36

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(36)

<223> b chain

<400> 95

ggcagguucc uuaucuguca aagcuucggc ggcagc 36

<210> 96

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(23)

<223> c chain

<400> 96

gcagccgccc auagccgcgc gcg 23

<210> 97

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(39)

<223> EGFRapt

<400> 97

gccttagtaa cgtgctttga tgtcgattcg acaggaggc 39

<210> 98

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(41)

<223> PSMAapt

<400> 98

gggccgaaaa agacctgact tctatactaa gtctacgtcc c 41

<210> 99

<211> 68

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(68)

<223> a chain

<400> 99

cgcgcgccca ggagcgttgg cgggcggcgg ccttagtaac gtgctttgat gtcgattcga 60

caggaggc 68

<210> 100

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 100

cgccgcccgc cttcgccgcc agccgcc 27

<210> 101

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 101

ggcggcaggc ggccatagcc ctgggcgcgc g 31

<210> 102

<211> 68

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(68)

<223> a chain

<400> 102

cgcgcgccca gcagcgttcg cgggcggcgg ccttagtaac gtgctttgat gtcgattcga 60

caggaggc 68

<210> 103

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 103

cgccgcccgc gttcgccgcc agccgcc 27

<210> 104

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 104

ggcggcaggc ggccatagcg ctgggcgcgc g 31

<210> 105

<211> 68

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(68)

<223> a chain

<400> 105

cgcgcgccca cgagcgttgc ggggcggcgg ccttagtaac gtgctttgat gtcgattcga 60

caggaggc 68

<210> 106

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 106

cgccgccccg cttcgccgcc agccgcc 27

<210> 107

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 107

ggcggcaggc ggccatagcc gtgggcgcgc g 31

<210> 108

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(71)

<223> a chain

<400> 108

cgcgcgccca ggagcgttgg cccgcggcgt gggccgaaaa agacctgact tctatactaa 60

gtctacgtcc c 71

<210> 109

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 109

cgccgcgggc cttcggggcc agccgcc 27

<210> 110

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 110

ggcggcaggc ccccatagcc ctgggcgcgc g 31

<210> 111

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(71)

<223> a chain

<400> 111

cgcgcgccca gcagcgttcg ccccgccgct gggccgaaaa agacctgact tctatactaa 60

gtctacgtcc c 71

<210> 112

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 112

gcggcggggc gttcggcggc aggcggc 27

<210> 113

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 113

gccgccagcc gcccatagcg ctgggcgcgc g 31

<210> 114

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 114

cgcgcgccca gcagcgttcg gggcgccgc 29

<210> 115

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> b chain

<400> 115

gcggcgcccc gttcggccgg caggcggc 28

<210> 116

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(32)

<223> c chain

<400> 116

gccgccagcc ggcccatagc gctgggcgcg cg 32

<210> 117

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(29)

<223> a chain

<400> 117

cgcgcgccca cgagcgttgc gggcgccgc 29

<210> 118

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(27)

<223> b chain

<400> 118

gcggcgcccg cttcggcggc aggcggc 27

<210> 119

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(31)

<223> c chain

<400> 119

gccgccagcc gcccatagcc gtgggcgcgc g 31

<210> 120

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 120

gcggcgagcg gcgaggagcg uuggggccgg aggccgg 37

<210> 121

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> b chain

<400> 121

ccggccuccg gccccuucgg ggccagccgc c 31

<210> 122

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 122

ggcggcaggc ccccauagcc cucgccgcuc gccgc 35

<210> 123

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 123

gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37

<210> 124

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 124

ccggccuccg gcccguucgc cgccagccgc c 31

<210> 125

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 125

ggcggcaggc ggccauagcg cucgccgcuc gccgc 35

<210> 126

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 126

gcggcgagcg gcgaggagcg uuggggccgg aggccgg 37

<210> 127

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 127

ccggccuccg gccccuucgc cgccagccgc c 31

<210> 128

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 128

ggcggcaggc ggccauagcc cucgccgcuc gccgc 35

<210> 129

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 129

gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37

<210> 130

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 130

ccggccuccg gcccguucgg cgccagccgc c 31

<210> 131

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 131

ggcggcaggc gcccauagcg cucgccgcuc gccgc 35

<210> 132

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 132

gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37

<210> 133

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 133

ccggccuccg gcccguucgg ccccagccgc c 31

<210> 134

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 134

ggcggcaggg gcccauagcg cucgccgcuc gccgc 35

<210> 135

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 135

gcggcgagcg gcgacgagcg uugcggccgg aggccgg 37

<210> 136

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 136

ccggccuccg gccgcuucgc cgccagccgc c 31

<210> 137

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 137

ggcggcaggc ggccauagcc gucgccgcuc gccgc 35

<210> 138

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 138

gcggcgagcg gcgacgagcg uugcggccgg aggccgg 37

<210> 139

<211> 31

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 139

ccggccuccg gccgcuucgg cgccagccgc c 31

<210> 140

<211> 35

<212> RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 140

ggcggcaggc gcccauagcc gucgccgcuc gccgc 35

<210> 141

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 141

gcggcgagcg gcgaggagcg ttggggccgg aggccgg 37

<210> 142

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 142

ccggcctccg gccccttcgg ggccagccgc c 31

<210> 143

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 143

ggcggcaggc ccccatagcc ctcgccgctc gccgc 35

<210> 144

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 144

gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37

<210> 145

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 145

ccggcctccg gcccgttcgc cgccagccgc c 31

<210> 146

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 146

ggcggcaggc ggccatagcg ctcgccgctc gccgc 35

<210> 147

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 147

gcggcgagcg gcgaggagcg ttggggccgg aggccgg 37

<210> 148

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 148

ccggcctccg gccccttcgc cgccagccgc c 31

<210> 149

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 149

ggcggcaggc ggccatagcc ctcgccgctc gccgc 35

<210> 150

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 150

gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37

<210> 151

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 151

ccggcctccg gcccgttcgg cgccagccgc c 31

<210> 152

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 152

ggcggcaggc gcccatagcg ctcgccgctc gccgc 35

<210> 153

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 153

gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37

<210> 154

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 154

ccggcctccg gcccgttcgg ccccagccgc c 31

<210> 155

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 155

ggcggcaggg gcccatagcg ctcgccgctc gccgc 35

<210> 156

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 156

gcggcgagcg gcgacgagcg ttgcggccgg aggccgg 37

<210> 157

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 157

ccggcctccg gccgcttcgc cgccagccgc c 31

<210> 158

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 158

ggcggcaggc ggccatagcc gtcgccgctc gccgc 35

<210> 159

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(37)

<223> a chain

<400> 159

gcggcgagcg gcgacgagcg ttgcggccgg aggccgg 37

<210> 160

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(31)

<223> b chain

<400> 160

ccggcctccg gccgcttcgg cgccagccgc c 31

<210> 161

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(35)

<223> c chain

<400> 161

ggcggcaggc gcccatagcc gtcgccgctc gccgc 35

<210> 162

<211> 14

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(14)

<223> first extension segment

<400> 162

gcggcgagcg gcga 14

<210> 163

<211> 14

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(14)

<223> first extension segment

<400> 163

ucgccgcucg ccgc 14

<210> 164

<211> 13

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(13)

<223> first extension segment

<400> 164

ggccggaggc cgg 13

<210> 165

<211> 13

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(13)

<223> first extension segment

<400> 165

ccggccuccg gcc 13

<210> 166

<211> 9

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(9)

<223> first extension segment

<400> 166

ccagccgcc 9

<210> 167

<211> 9

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(9)

<223> first extension segment

<400> 167

ggcggcagg 9

<210> 168

<211> 14

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(14)

<223> first extension segment

<400> 168

gcggcgagcg gcga 14

<210> 169

<211> 14

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(14)

<223> first extension segment

<400> 169

tcgccgctcg ccgc 14

<210> 170

<211> 13

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(13)

<223> first extension segment

<400> 170

ggccggaggc cgg 13

<210> 171

<211> 13

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(13)

<223> first extension segment

<400> 171

ccggcctccg gcc 13

<210> 172

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(65)

<223> a chain

<400> 172

cgcgcgccca cgagcgttcc gggcgcgcct tagtaacgtg ctttgatgtc gattcgacag 60

gaggc 65

<210> 173

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(26)

<223> b chain

<400> 173

gcgcccggtt cgccgccagc cgccgc 26

<210> 174

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(33)

<223> c chain

<400> 174

gcggcggcag gcggccatag ccgtgggcgc gcg 33

<210> 175

<211> 10

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(10)

<223> a sequence

<400> 175

cgagcgttcc 10

<210> 176

<211> 9

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(9)

<223> b sequences

<400> 176

ggttcgccg 9

<210> 177

<211> 12

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(12)

<223> c sequence

<400> 177

cggccatagc cg 12

<210> 178

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(34)

<223> a chain

<400> 178

cgcgcgcgcc cacgagcgtt ccgggcgccg ccgc 34

<210> 179

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(33)

<223> b chain

<400> 179

gcggcggcgc ccggttcgcc gccagccgcc gcc 33

<210> 180

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(36)

<223> c chain

<400> 180

ggcggcggca ggcggccata gccgtgggcg cgcgcg 36

Claims

1. A drug containing cytarabine, wherein the drug comprises a nucleic acid nanoparticle and cytarabine, and the cytarabine is carried on the nucleic acid nanoparticle;

the nucleic acid nanoparticle comprises a nucleic acid domain comprising a sequence a comprising a variation of the sequence a1, a sequence b comprising a variation of the sequence b1, and a sequence c comprising a variation of the sequence c 1;

wherein the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively;

the b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3';

the c1 sequence is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3', respectively;

the sequence a, the sequence b and the sequence c self-assemble to form a structure shown in formula (1):

… … … formula (1) is shown,

wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position is independently selected from C-G or G-C;

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 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 from the 5 'end to the 3' end is CAUA or CATA;

the sequence a, the sequence b and the sequence c are any one of the following groups:

(1) a sequence: 5'-GGAGCGUUGG-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-CCUUCGCCG-3',

c sequence: 5'-CGGCCAUAGCCC-3';

(2) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,

b sequence: 5'-CGUUCGCCG-3',

c sequence: 5'-CGGCCAUAGCGC-3';

(3) a sequence: 5'-CGAGCGUUGC-3' the flow of the air in the air conditioner,

b sequence: 5'-GCUUCGCCG-3',

c sequence: 5'-CGGCCAUAGCCG-3';

(4) a sequence: 5'-GGAGCGUUGG-3' the flow of the air in the air conditioner,

b sequence: 5 '-CCUUCGGG-3',

c sequence: 5'-CCCCCAUAGCCC-3';

(5) a sequence: 5'-GCAGCGUUCG-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-CGUUCGGCG-3',

c sequence: 5'-CGCCCAUAGCGC-3', respectively;

(6) a sequence: 5'-GCAGCGUUCG-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-CGUUCGGCC-3',

c sequence: 5'-GGCCCAUAGCGC-3';

(7) a sequence: 5'-CGAGCGUUGC-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-GCUUCGGCG-3',

c sequence: 5'-CGCCCAUAGCCG-3', respectively;

(8) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,

b sequence: 5'-CCTTCGCCG-3',

c sequence: 5'-CGGCCATAGCCC-3';

(9) a sequence: 5'-GCAGCGTTCG-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-CGTTCGCCG-3',

c sequence: 5'-CGGCCATAGCGC-3', respectively;

(10) a sequence: 5'-CGAGCGTTGC-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-GCTTCGCCG-3',

c sequence: 5'-CGGCCATAGCCG-3';

(11) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,

b sequence: 5'-CCTTCGGGG-3',

c sequence: 5'-CCCCCATAGCCC-3', respectively;

(12) a sequence: 5'-GCAGCGTTCG-3' the flow of the air in the air conditioner,

b sequence: 5'-CGTTCGGCG-3',

c sequence: 5'-CGCCCATAGCGC-3';

(13) a sequence: 5'-GCAGCGTTCG-3' the flow of the air in the air conditioner,

b sequence: 5'-CGTTCGGCC-3',

c sequence: 5'-GGCCCATAGCGC-3', respectively;

(14) a sequence: 5'-CGAGCGTTGC-3', and the adhesive tape is used for adhering the film to a substrate,

b sequence: 5'-GCTTCGGCG-3',

c sequence: 5'-CGCCCATAGCCG-3', respectively;

(15) a sequence: 5'-CGAGCGTTCC-3';

b sequence: 5 '-GGTTCGCCG-3',

c sequence: 5'-CGGCCATAGCCG-3' is added.

2. The agent of claim 1, further comprising a first extension in the nucleic acid domain, wherein the first extension is a Watson-Crick paired extension located 5 'and/or 3' to any of the a, b, and c sequences.

3. The medicament of claim 2, wherein the first extension is selected from at least any one of the group consisting of:

(1): a 5' end of chain: 5' -CCCA-3', 3' end of c strand: 5 '-UGGG-3';

(2): a 3' end of chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3';

(3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3';

(4): a 5' end of the chain: 5' -CCCG-3', 3' end of c strand: 5 '-CGGG-3';

(5): a 5' end of the chain: 5' -CCCC-3', 3' end of c strand: 5 '-GGGG-3';

(6): b 3' end of strand: 5' -CCC-3', 5' -end of c strand: 5 '-GGG-3';

(7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3';

(8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3';

(9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.

4. The agent of any one of claims 1 to 3, wherein the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any of the a, b, and c sequences, wherein the second extension is a Watson-Crick paired extension.

5. The agent of claim 4, wherein said second extension is an extension of CG base pairs.

6. The drug of claim 5, wherein the second extension is an extension of 1 to 10 CG base pairs.

7. The agent of claim 4, wherein said nucleic acid domain further comprises at least one second set of extensions of:

a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3';

second group: a 3' end of chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3';

third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.

8. The agent of claim 4, wherein the second extension is an extended sequence comprising both CG base pairs and AT/AU base pairs.

9. The agent of claim 8, wherein the second extension is an extended sequence of 2 to 50 base pairs.

10. The drug of claim 8, wherein the second extension is an extension sequence in which a sequence of 2 to 8 CG base pairs in succession alternates with a sequence of 2 to 8 AT/AU base pairs in succession; or alternatively

The second extension segment is an extension sequence formed by alternating sequences of 1 CG base pair and 1 AT/AU base pair.

11. The agent according to any one of claims 1 to 3, wherein the bases, ribose and phosphate in the a sequence, the b sequence and the c sequence have at least one modifiable site, and any of the modifiable sites is modified by any one of the following modifying linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups.

12. The agent of claim 11, wherein the sequence a, the sequence b and the sequence C have 2' -F modifications at the C or U bases.

13. The medicament of any one of claims 1 to 3, wherein the cytarabine is loaded on the nucleic acid nanoparticles in a form of physical and/or covalent attachment and the molar ratio between the cytarabine and the nucleic acid nanoparticles is 2-300: 1.

14. The medicament of claim 13, wherein the molar ratio of cytarabine to the nucleic acid nanoparticles is 10-50: 1.

15. The medicament of claim 14, wherein the molar ratio of cytarabine to the nucleic acid nanoparticles is 15-25: 1.

16. The drug of any one of claims 1 to 3, wherein the nucleic acid nanoparticle further comprises a biologically active substance attached to the nucleic acid domain, wherein the biologically active substance is one or more of a target, a fluorescein, an interfering nucleic acid (siRNA), a miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, a protein, a polypeptide, a flavonoid, glucose, natural salicylic acid, a monoclonal antibody, a vitamin, a phenol, lecithin, and a small molecule drug other than cytarabine.

17. The agent of claim 16, wherein the relative molecular weight of the nucleic acid domains is recorded as N₁The total relative molecular weight of cytarabine and the biologically active substance is denoted as N₂，N₁/ N₂≥1:1。

18. The drug of claim 16, wherein the biologically active substance is one or more of the target, the fluorescein, and the miRNA,

wherein the target head is located on any sequence of the a sequence, the b sequence and the c sequence.

19. The agent of claim 18, wherein the sequence a, the sequence b, the sequence c, or either of the sequences is 5 'or 3' to the nucleic acid domain, or is inserted between GC bonds of the nucleic acid domains,

the miRNA is an anti-miRNA, the fluorescein is modified at the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence.

20. The drug of claim 18, wherein the target is folate or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.

21. The drug of claim 16, wherein the small molecule drug other than cytarabine is a drug comprising any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.

22. The medicament of claim 16, wherein the protein is one or more of SOD, survivin, hTERT, EGFR, and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.

23. The drug according to claim 1, wherein the nucleic acid nanoparticles have a particle size of 1 to 100 nm.

24. The drug of claim 23, wherein the nucleic acid nanoparticles have a particle size of 5 to 50 nm.

25. The drug of claim 24, wherein the nucleic acid nanoparticles have a particle size of 10-30 nm.

26. The drug of claim 25, wherein the nucleic acid nanoparticles have a particle size of 10-15 nm.

27. A preparation method of a cytarabine-containing medicament is characterized by comprising the following steps of:

providing a nucleic acid nanoparticle in a medicament according to any one of claims 1 to 26;

and (2) carrying the cytarabine on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the cytarabine-containing medicament.

28. The method of claim 27, wherein the step of loading cytarabine by physical linkage comprises:

mixing and stirring the cytarabine, the nucleic acid nanoparticles and the first solvent to obtain a premixed system;

and precipitating the premixed system to obtain the cytarabine-containing medicament.

29. The method of claim 28, wherein the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.

30. The method for preparing a cytarabine containing drug according to claim 28, wherein the step of precipitating the pre-mixed system to obtain the cytarabine containing drug comprises;

precipitating the premixed system to obtain precipitates;

and washing the precipitate to remove impurities to obtain the cytarabine-containing medicament.

31. The method according to claim 30, wherein the precipitation is performed at a temperature of less than 10 ℃ after the premix system is mixed with absolute ethanol to obtain the precipitate.

32. The method according to claim 31, wherein the precipitation is performed at a temperature of 0 to 5 ℃ to obtain the precipitate.

33. The preparation method according to claim 31, wherein the precipitate is washed with 6 to 12 times by volume of absolute ethanol to remove impurities, thereby obtaining the cytarabine-containing medicament.

34. The method of claim 27, wherein the step of loading cytarabine by covalent linkage comprises:

preparing a cytarabine solution;

reacting the cytarabine arabine solution with the amino outside the G ring of the nucleic acid nano-particle under the mediation effect of formaldehyde to obtain a reaction system;

purifying the reaction system to obtain the cytarabine-containing medicament.

35. The method of claim 34, wherein the step of reacting comprises:

and mixing the cytarabine solution, a paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain the reaction system.

36. The method according to claim 35, wherein the concentration of the paraformaldehyde solution is 3.7 to 4 wt%.

37. The method according to claim 35, wherein the paraformaldehyde solution is a mixture of paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.

38. The production method according to any one of claims 27 to 37, characterized in that the production method further comprises a step of producing the nucleic acid nanoparticle, which comprises: the nucleic acid domain is obtained by self-assembling single strands corresponding to the nucleic acid domain in the nucleic acid nanoparticle in the medicament according to any one of claims 1 to 16.

39. The method of claim 38, wherein after obtaining the nucleic acid domain, the method further comprises: the nucleic acid nanoparticle is obtained by mounting the bioactive substance in the drug according to any one of claims 27 to 37 on the nucleic acid domain by means of physical and/or covalent attachment.

40. The method of claim 38, wherein the biologically active substance is covalently attached by solvent covalent attachment, linker covalent attachment, or click linkage.

41. The method of claim 39, wherein a third solvent used in the covalent linkage of the solvents is used as a linking medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.

42. The method of claim 40, wherein the linker is selected from the group consisting of disulfide bond, p-azido, bromopropyne, and PEG.

43. The method of claim 40, wherein the click-through linkage is performed by modifying the biologically active substance precursor and the nucleic acid domain with an alkynyl or azide at the same time and then by click-through linkage.

44. The method of claim 40, wherein the biologically active substance is linked to the nucleic acid domain by a click-link, wherein the site of the alkyne or azide modification of the biologically active substance precursor is selected from the group consisting of a 2 ' hydroxyl, a carboxyl, and an amino group, and wherein the site of the alkyne or azide modification of the nucleic acid domain is selected from the group consisting of a G exocyclic amino, a 2 ' -hydroxyl, an A amino, and a 2 ' -hydroxyl.

45. A pharmaceutical composition comprising the cytarabine-containing medicament of any one of claims 1 to 26.

46. Use of a cytarabine-containing medicament of any one of claims 1 to 26 in the manufacture of a medicament for the treatment of a tumor, viral keratitis and epidemic conjunctivitis.

47. The use according to claim 46, wherein the tumour is any one or more of malignant lymphoma, lung cancer, cancer of the digestive tract, colorectal cancer and cancer of the head and neck.

48. The use of claim 46, wherein the tumor is acute leukemia.