CN111053914B - Mitoxantrone-containing medicine, preparation method thereof, medicine composition and application thereof - Google Patents
Mitoxantrone-containing medicine, preparation method thereof, medicine composition and application thereof Download PDFInfo
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- CN111053914B CN111053914B CN201910969428.XA CN201910969428A CN111053914B CN 111053914 B CN111053914 B CN 111053914B CN 201910969428 A CN201910969428 A CN 201910969428A CN 111053914 B CN111053914 B CN 111053914B
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Abstract
The application provides a mitoxantrone-containing medicament, a preparation method thereof, a pharmaceutical composition and application thereof. The drug comprises nucleic acid nanoparticles and mitoxantrone, and the mitoxantrone 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. The mitoxantrone-containing drug provided by the application has the advantages that the nucleic acid structure domain is modified by the target head, so that the drug has better targeting property, can stably deliver mitoxantrone and has high reliability.
Description
Technical Field
The application relates to the field of medicines, in particular to a mitoxantrone-containing medicine, a preparation method, a pharmaceutical composition and application thereof.
Background
Mitoxantrone (Mitoxanthone, CSA:65271-80-9, molecular formula: C 22 H 28 N 4 O 6 Molecular weight: 444.481) is an antitumor antibiotic with a mechanism of action similar to that of adriamycin, and is less toxic to the heart because of its absence of amino sugar structure, no generation of free radicals, and its ability to inhibit lipid peroxidation. It is mainly used for treating cancers such as breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, hepatocarcinoma, multiple myeloma, malignant mesothelioma and ovarian cancer.
Currently, antitumor antibiotics, including mitoxantrone, 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 can 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 research in cancer chemotherapy.
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, which have high in vitro transfection activity, but have the disadvantages of immunogenicity and susceptibility to mutation, and thus, the in vivo delivery brings huge safety hazards. 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.
Of the above-mentioned various targeted delivery approaches, more research is currently focused on the field of non-viral vectors, 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 mitoxantrone is one of the difficulties in solving the limited clinical application of the existing mitoxantrone drug.
Disclosure of Invention
The main purpose of the application is to provide a mitoxantrone-containing drug, a preparation method, a pharmaceutical composition and application thereof, so as to improve the delivery reliability of the mitoxantrone drug.
In order to achieve the above object, according to one aspect of the present application, there is provided a mitoxantrone-containing drug comprising a nucleic acid nanoparticle and mitoxantrone, and the mitoxantrone 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 of 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.
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 the 9 th to 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 into a structure shown in the formula (1):
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 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 '-GCUUCGCCGCCG-3', c sequence: 5'-CGGCCAUAGCCG-3', respectively; (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', respectively; (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'; (10) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3', respectively; (11) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3', respectively; (12) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3'; (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', sequence b: 5'-GGTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3' is added.
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 strand: 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 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 chain: 5' -CCCA-3', 3' end of c strand: 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 paired 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 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 extended sequence of 1 CG base pair alternating with 1 AT/AU base pair sequence.
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 mitoxantrone is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the mitoxantrone 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, interfering nucleic acid siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein, polypeptide, flavonoid, glucose, natural salicylic acid, monoclonal antibody, vitamin, phenolic lecithin and small molecule drugs except mitoxantrone.
Further, the relative molecular weight of the nucleic acid domains is denoted as N 1 The total relative molecular weight of mitoxantrone and biologically active substance is denoted as N 2 ,N 1 /N 2 ≥1:1。
Further, the bioactive substance is one or more of a target, fluorescein and miRNA, wherein the target is located on any sequence of a, b and c sequences, preferably the 5' end or the 3' end of any sequence of a, b and c, or is 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 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 any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
Further, the small molecule drug other than mitoxantrone is a drug 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 10-30 nm; further preferably 10 to 15 nm.
According to another aspect of the present application, there is also provided a method for preparing a mitoxantrone-containing medicament comprising the steps of: providing the nucleic acid nanoparticles described above; the mitoxantrone is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the mitoxantrone-containing drug.
Further, the step of mounting mitoxantrone by means of physical attachment comprises: mixing and stirring mitoxantrone, nucleic acid nanoparticles and a first solvent to obtain a premixed system; precipitating the premixed system to obtain the mitoxantrone-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 mitoxantrone-containing medicament comprises: precipitating the premixed system to obtain precipitates; washing the precipitate to remove impurities to obtain the mitoxantrone-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; a mitoxantrone-containing drug; 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 mitoxantrone-containing medicine is obtained.
Further, the step of loading mitoxantrone by covalent attachment comprises: preparing a mitoxantrone solution; reacting the mitoxantrone solution with the amino outside the G ring of the nucleic acid nanoparticles under the mediated action of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the mitoxantrone-containing medicament; preferably, the step of reacting comprises: mixing the mitoxantrone solution with the paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under the condition of keeping out of the sun to obtain a reaction system; the concentration of the preferable paraformaldehyde solution is preferably 3.7-4 wt%, the preferable 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 domain by self-assembling the single strand corresponding to the nucleic acid 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, the linker is selected from the group consisting of 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-link manner, the site of the biologically active substance precursor for the alkynyl or azido modification is selected from the group consisting of a 2 ' hydroxyl group, a carboxyl group, or an amino group, and the site of the nucleic acid domain for the alkynyl or azido modification is selected from the group consisting of a G exocyclic amino group, a 2 ' -hydroxyl group, an A amino group, or a 2 ' -hydroxyl group.
According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any one of the mitoxantrone-containing drugs described above.
According to a fourth aspect of the present application, there is also provided the use of any one of the mitoxantrone-containing medicaments described above in the preparation of a medicament for the treatment of a tumour.
Further, the tumor is any one or more of breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma and ovarian cancer.
According to a fifth aspect of the present application, there is also provided a method of preventing and/or treating a tumor, the method comprising: providing any one of the mitoxantrone-containing medicaments or pharmaceutical compositions described above; administering to a patient having a tumor an effective amount of the above mitoxantrone-containing medicament or pharmaceutical composition.
Further, the tumor is any one or more of breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma and ovarian cancer.
The mitoxantrone-containing drug provided by the application comprises nucleic acid nanoparticles and mitoxantrone, and the mitoxantrone is carried on the nucleic acid nanoparticles 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 mitoxantrone can be connected to any 5 'end and/or 3' end of the three strands as a carrier, or mitoxantrone can be stably inserted between the strands of the nucleic acid domains. According to the application, the small-molecule drug mitoxantrone is hung on the nucleic acid nanoparticles, the internal hydrophobicity, the external hydrophilicity and the stacking effect of bases of the nucleic acid nanoparticles are utilized, the coating effect on the mitoxantrone is realized, and the mitoxantrone 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, mitoxantrone can be stably delivered, and the reliability is high; meanwhile, the contact chance of mitoxantrone with non-target cells or tissues can be reduced, and the 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 results of 2% agarose gel electrophoresis detection of 7 sets 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 7 sets 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 results 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 results of 2% agarose gel electrophoresis detection of the DNA nanoparticles of groups 8 and 9 formed by self-assembly in example 4 of the present application;
FIG. 10 shows a TEM photograph of conventional sequence DNA nanoparticles D-7 formed by self-assembly in example 4 of the present application;
FIG. 11 is a graph showing a standard curve of absorbance of mitoxantrone during the detection of DNA nanoparticle loading rate in example 5 of the present application;
FIG. 12 shows the results of electrophoresis detection of DNAh-Bio-EGFRApt-Cy5-Mit nanoparticles after incubation in serum for various periods of time in example 7 of the present application;
FIG. 13 shows the inhibition of MCF-7 cell proliferation by the small molecule drug mitoxantrone;
FIG. 14 is the inhibition of the proliferation of MCF-7 cells by DNAh-Bio-EGFRApt-Cy5-Mit (targeting agent);
FIG. 15 is the inhibition of the proliferation of MCF-7 cells by DNAh-Bio-EGFRApt-Cy5 (targeting fluorescent vector);
FIG. 16 is a graph showing the inhibition of proliferation of MCF-7 cells by a blank control of DMSO;
FIG. 17 shows the result of non-denaturing PAGE gel electrophoresis detection of 7 sets of modified-segment + core short-sequence RNA self-assembly products in example 9 of the present invention;
FIG. 18 shows the dissolution curve of the RNA nanoparticle R-15 in example 9 of the present invention;
FIG. 19 shows the dissolution curve of the RNA nanoparticle R-16 in example 9 of the present invention;
FIG. 20 shows the dissolution curve of the RNA nanoparticle R-17 in example 9 of the present invention;
FIG. 21 shows the dissolution curve of the RNA nanoparticle R-18 in example 9 of the present invention;
FIG. 22 shows the dissolution curve of the RNA nanoparticle R-19 in example 9 of the present invention;
FIG. 23 shows the dissolution curve of the RNA nanoparticle R-20 in example 9 of the present invention;
FIG. 24 shows the dissolution curve of the RNA nanoparticle R-21 in example 9 of the present invention;
FIG. 25 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. 26 shows a dissolution curve of DNA nanoparticle D-8 in example 10 of the present invention;
FIG. 27 is a graph showing a dissolution curve of the DNA nanoparticle D-9 in example 10 of the present invention;
FIG. 28 shows a dissolution curve of DNA nanoparticle D-10 in example 10 of the present invention;
FIG. 29 is a graph showing a dissolution curve of DNA nanoparticle D-11 in example 10 of the present invention;
FIG. 30 is a graph showing a dissolution curve of the DNA nanoparticle D-12 in example 10 of the present invention;
FIG. 31 shows the dissolution curve of the DNA nanoparticle D-13 in example 10 of the present invention;
FIG. 32 shows a dissolution curve of the DNA nanoparticle D-14 in example 10 of the present invention;
FIG. 33 shows the result of electrophoresis detection of RNA nanoparticle R-15 in example 11 after incubation in serum for various times;
FIG. 34 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. 35 shows the result of electrophoresis detection of RNA nanoparticle R-17 in example 11 after incubation in serum for various times;
FIG. 36 shows the result of electrophoresis detection of the RNA nanoparticle R-18 in example 11 of the present invention after incubation in serum for various periods of time;
FIG. 37 shows the result of electrophoresis detection of RNA nanoparticle R-19 in example 11 after incubation in serum for various times;
FIG. 38 shows the result of electrophoresis detection of RNA nanoparticle R-20 in example 11 of the present invention after incubation in serum for various time periods;
FIG. 39 shows the result of electrophoresis detection of RNA nanoparticle R-21 in example 11 after incubation in serum for various times;
FIG. 40 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. 41 shows the result of electrophoresis detection of DNA nanoparticle D-9 in example 12 of the present invention after incubation in serum for various times;
FIG. 42 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. 43 shows the result of electrophoresis detection of DNA nanoparticle D-11 in example 12 of the present invention after incubation in serum for various times;
FIG. 44 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. 45 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. 46 shows the results of electrophoresis detection of the DNA nanoparticle D-14 of example 12 of the present invention after incubation in serum for various periods of time;
FIGS. 47a, 47b, 47c, 47D, 47e, 47f, 47g and 47h 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;
FIG. 48 shows a standard curve of daunorubicin absorbance used in the mounting ratio measurement process of example 16.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail with reference to examples.
Interpretation of terms:
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 a fluorescent substance but not containing a targeting moiety, such as Cy5-RNAh or Cy 5-DNAh.
Targeting fluorescent vector: refers to a nucleic acid nanoparticle vector containing a target and a fluorescent substance, such as Biotin-Cy5-RNAh or Biotin-Cy 5-DNAh.
Targeting drugs: refers to nucleic acid nanoparticle carrier containing target head, fluorescent substance and chemical drug, such as RNAh-Biotin-quasar670-Mit or DNAh-Biotin-quasar670-Mit, wherein Mit represents mitoxantrone.
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, the stability of RNA nanoparticles in their natural state is relatively poor, and the current improvements based on the application 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. And, 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 mitoxantrone-containing drug, which comprises nucleic acid nanoparticles and mitoxantrone, wherein the mitoxantrone 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; wherein, the sequence of a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively; 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' are provided.
The mitoxantrone-containing drug provided by the application comprises nucleic acid nanoparticles and mitoxantrone, and the mitoxantrone is carried on the nucleic acid nanoparticles. The nucleic acid nanoparticle can be used as a carrier in which mitoxantrone is linked to any of the 5 'ends and/or 3' ends of the three strands or mitoxantrone can be stably inserted between strands of the nucleic acid domain, as well as a nucleic acid domain formed by self-assembly by including the three sequences or their variants. According to the mitoxantrone-containing medicament provided by the application, the small-molecule medicament mitoxantrone is suspended on the nucleic acid nanoparticles, and the nucleic acid nanoparticles have hydrophobic property inside, hydrophilic property outside and stacking effect of basic groups, so that the mitoxantrone is coated, and the coating or covalent connection ensures that the mitoxantrone cannot be dissolved in a certain time, 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, mitoxantrone can be stably delivered, and the reliability is high; meanwhile, the contact probability of the mitoxantrone with non-target cells or tissues can be reduced, and the 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 force refers to hydrogen bonds, van der waals force, electrostatic force, hydrophobic force and the like) among a large number of atoms, ions or molecules, but a plurality of individuals are simultaneously and spontaneously 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 direction 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, ribozymes of various types, and natural RNA aptamers present in riboswitches. One advantageous feature of RNA nanotechnology is that structures comparable in size and complexity to natural RNA species can be designed. 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 a nano-particle for improving the drug loading capacity or 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 medicines obtained by mitoxantrone suspension 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 bases at certain specific positions of the sequences, so that the sequences after variation are the same as the original sequences and can be self-assembled into the nanoparticles, and the variation keeps at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of homology with the original sequences, so that the nanoparticles formed by self-assembling the sequences have the same medicine loading characteristics and similar stability, and mitoxantrone can be well suspended and delivered.
In a preferred embodiment, the above base insertions, deletions or substitutions occur in: (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) among bases 1 to 4 from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6; and/or (6) between 9 th to 12 th bases 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 mitoxantrone is loaded, in a preferred embodiment, the a sequence, the b sequence and the c sequence 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 not 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 sequences a, b and C form a nucleic acid domain having the formula (1) by self-assembly, wherein the bases at the other positions except for the non-Watson-Crick base pairs defined by N and N' 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 higher 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 nanoparticle having the structure of formula (1), the specific sequence composition of the sequence a, the sequence b and the sequence c 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), when selecting the bases paired in Watson-Crick, the bases at different positions are preferably selected according to the following principle: (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, and 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 change of microenvironment.
In a preferred embodiment, the a sequence, the b sequence and the c sequence 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', respectively; (3) a sequence (SEQ ID NO: 13): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 14): 5 '-GCUUCGCCGCCG-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', respectively; (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'; (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 into shapes, but also have the ability to carry or carry mitoxantrone drugs. Depending on the position of G-C or C-G base pairs in the above-mentioned nucleic acid nanoparticles, the amount of mitoxantrone to be carried differs.
In order to allow the nucleic acid domain to carry more mitoxantrone and bioactive substances (see below for the description of bioactive substances), in a preferred embodiment, the nucleic acid domain further comprises a first extension, wherein the first extension is a Watson-Crick paired extension, and the first extension is located at the 5 'end and/or 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 loaded substance can be obtained by adding a first extension to the 5 'end and/or the 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 extension is selected from any one of the group consisting of: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 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 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 strand: 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), c chain 3' end: 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), 5' end of c chain: 5'-GGCGGCAGG-3' (SEQ ID NO: 167); (13): a 5' end of the chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:168), the 3' end of the 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. The second extension is an extension further added on top of the first extension.
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 on itself.
The first extension and/or the second extension may be separated by unpaired base pairs.
In order to make the nucleic acid nanoparticles capable of carrying bioactive substances with larger molecular weight (see the introduction of bioactive substances below), increasing drug loading and maintaining necessary stability, in a preferred embodiment, the second extension is an extension containing both CG base pairs and AT/AU base pairs, and 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 the 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 sequences of 2-8 CG base pairs and 2-8 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 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 GGCGGC extension and TTTTTT extension in the sequence b shown by the SEQ ID NO. 50 and the positions of the GCCGCC extension and AAAAAAAA extension in the sequence c shown by the SEQ ID NO. 51 are interchanged, and the CGCCGC extension and 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 after 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) modifications 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 a 2' -F modification at the C or U base. When the modified joint is sulfydryl, the modified joint belongs to sulfo modification, the modification strength is weak, and the cost is low.
The mitoxantrone can be attached by physical and/or covalent attachment. When the mitoxantrone 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 attachment is used, mitoxantrone typically reacts with the amino group outside the G ring to form a covalent attachment. More preferably, the molar ratio between the mitoxantrone and the nucleic acid nanoparticles is 2 to 300:1, preferably 2 to 290:1, more preferably 2 to 29:1, even more preferably 10 to 50:1, and most preferably 15 to 25: 1.
In addition to the nucleic acid nanoparticles serving as delivery vehicles for mitoxantrone in the mitoxantrone-containing drugs provided herein, in a preferred embodiment, the nucleic acid nanoparticles further comprise a biologically active substance, which is linked to the nucleic acid domain, depending on the drug purpose. The bioactive substance is one or more of a 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 mitoxantrone.
In order to improve the efficiency of the nucleic acid nanoparticles in the loading of the loaded bioactive substances andthe delivery efficiency, relative molecular weight of the nucleic acid domains and the relative molecular weight of mitoxantrone and the biologically active substance are preferably matched. In a preferred embodiment, the relative molecular weight of the nucleic acid domains is denoted as N 1 The total relative molecular weight of mitoxantrone and biologically active substance is denoted as N 2 ,N 1 /N 2 ≥1:1。
The mitoxantrone-containing drugs of the present application have different performance optimizations depending on the particular type of bioactive material being loaded. For example, when the biologically active substance is biotin or folic acid, it acts to target the mitoxantrone-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 some siRNA, miRNA, protein, polypeptide, RNA antibody and small molecular drugs except mitoxantrone, the mitoxantrone-containing drugs 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 types of the biological active substances, DNA nanoparticles and RNA nanoparticles are preferably used, and can be 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 is 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 a target, fluorescein and miRNA, wherein the target is 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 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 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 any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
The target head can be covalently linked to any one of the sequences a, b and c by a linker, wherein the linker can be selected from disulfide bond, p-azido, bromopropyne or PEG. As used herein, "on any sequence" refers to any base position of any sequence of a, b, c sequences, and it is more convenient to attach to the 5 'end or 3' end, and the application is more extensive. 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 heterogeneity problem 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 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 miR-21 is relatively stronger.
When the bioactive substance capable of being carried is other small-molecule drugs except mitoxantrone, the drugs include, but are not limited to, drugs for treating liver cancer, gastric 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 disease according to the types of diseases which can be treated by different drugs; 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 mitoxantrone, the bioactive substance includes, but is not limited to, drugs containing any one or more of the following groups, depending on the molecular structure of the drug or the 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 vitamin is 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 even more 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 mitoxantrone-containing medicament described above, comprising the steps of: providing any one of the nucleic acid nanoparticles described above; the mitoxantrone is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the mitoxantrone-containing medicament.
When physically attached, mitoxantrone will typically form an insertion between the GC base pairs by physical intercalation. When covalent attachment is used, mitoxantrone typically chemically reacts with the amino group outside the G ring to form a covalent attachment. The mitoxantrone-containing drug prepared by the method has better targeting property after being modified by the target head, can stably deliver the mitoxantrone, and has high reliability.
In a preferred embodiment, the step of mounting mitoxantrone by physical attachment comprises: mixing and stirring mitoxantrone, nucleic acid nanoparticles and a first solvent to obtain a premixed system; and precipitating the premixed system to obtain the mitoxantrone-containing medicament. The dosage of the mitoxantrone 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 amount of mitoxantrone added per liter of the first solvent is preferably 0.1 to 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 mitoxantrone-containing medicament comprises: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the mitoxantrone-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 mitoxantrone-containing medicine is obtained.
In a preferred embodiment, the step of entrapping mitoxantrone by covalent attachment comprises: preparing a mitoxantrone solution; reacting the mitoxantrone solution with the amino outside the G ring of the nucleic acid nanoparticles under the mediation of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the mitoxantrone-containing medicament.
By formaldehyde-mediated form, the following reactions can occur:
preferably, the step of reacting comprises: and (3) mixing the mitoxantrone 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 in a self-assembled 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 mitoxantrone-containing drug with other functions as required for practical use, in a preferred embodiment, after obtaining the nucleic acid domain, the preparation method further comprises: the aforementioned bioactive substances are carried on the nucleic acid domain by means of physical linkage and/or covalent linkage, thereby obtaining the nucleic acid nanoparticles. 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 the biologically active substance is linked to the 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 biologically active 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 a beneficial 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 and acridine drugs are inserted, the drugs are usually inserted between GC base pairs, and the number of the 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 by the different connection modes is not limited to the range, and the drugs can be effectively released after reaching the target, so long as the efficient mounting can be realized, and no toxic effect on cells can be realized.
When the bioactive substance precursor and the nucleic acid structural 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 structural 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.
According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any one of the mitoxantrone-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 a fourth aspect of the application, there is also provided the use of any one of the mitoxantrone-containing medicaments described above in the preparation of a medicament for the treatment of a tumour. Further, the tumor is any one or more of breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma and ovarian 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 for preventing and/or treating a tumor, the method comprising: providing any one of the mitoxantrone-containing medicaments or pharmaceutical compositions described above; administering to a patient having a tumor an effective amount of the mitoxantrone-containing medicament or pharmaceutical composition described above. Further, the tumor is any one or more of breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma and ovarian 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 in breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma, or ovarian cancer. 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 is an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, e.g., to prevent or inhibit the development of breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, leukemia, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma, or ovarian cancer. A prophylactically effective amount can be determined according to the description of therapeutically effective amounts above. For any particular subject, the particular dosage can be adjusted over time according to the individual need and the professional judgment of the administering person.
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 to the drug, the water-solubility is improved. When the drugs are anthracyclines, the drugs are covalently bound to the nucleic acid domain via an-NH bond on the nucleotide guanosine (the-NH group is hundreds of times more active than other groups that may covalently bind to the drug under appropriate pH conditions), 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.
It should be noted that the nucleic acid nanoparticles formed by self-assembly of the sequences or sequence variants provided herein can also be used as basic building blocks, and can be further polymerized to form multimers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., according to the practical application.
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 the DNA nanoparticle.
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 a, b and c strands of the above-mentioned RNA nanoparticles and DNA nanoparticles were synthesized by Competition Biotechnology (Shanghai) Co., Ltd.
II, self-assembly experiment steps:
(1) mixing RNA or DNA single strands a, b and c at the same time according to the molar ratio of 1:1:1, 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 off a target strip, 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 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 larger than that of the single chain after the molecular weight is assembled, so that the position of a strip 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 electrophoresis detection result of the DNA self-assembly product is 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: 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 nanoparticles. 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 successfully self-assemble 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 nanoparticle carriers:
(1)7 sets of three polynucleotide base sequences constituting the RNA nanoparticle:
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 Venezetian Biotechnology (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;
(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 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 (6) detecting the potential.
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 is clear 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 R-1, R-4 and R-6 are dispersed but still can be seen as single bands, which indicates that the 7 groups of short sequences can be well self-assembled into RNA nanoparticle structures.
(2) Determination of potential
The determination 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;
opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
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 of 7 groups of short sequence RNA nanoparticles are as follows:
table 9:
table 10:
table 11:
table 12:
table 13:
table 14:
table 15:
from the potential detection data described above, it can be seen 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 more stable self-assembly structures.
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, by adding various functional extension fragments or connecting targeting moieties, fluorescein, etc. to these different core sequence combinations, RNA nanoparticles can be successfully assembled, and have the properties of drug loading, cell targeting, visual tracking, etc.
To further verify these properties, an extension fragment was added to example 2, see example 3. And an extension fragment is added on the basis of the DNA core sequence corresponding to the RNA core sequence of example 2, with or without target ligation, as shown in example 4.
Example 3
One, 7 groups of conventional sequence RNA nanoparticle carriers:
(1)7 sets of three polynucleotide base sequences constituting the RNA nanoparticle:
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 chainuGAcAGAuAAGGAAccuGcudTdTAs 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;
(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;
(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 strip, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at a 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.
FIG. 6 shows the electrophoresis of 4% agarose gel of 7 sets of conventional sequence RNA self-assembly products. 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 Survivin siRNA nucleic acid interference treatment fragment is modified in a 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 nucleic acid drugs and has the function of a delivery carrier of the nucleic acid drugs.
(2) Determination of 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 the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 7 groups of conventional sequence RNA nanoparticles are as follows:
table 23:
table 24:
table 25:
table 26:
table 27:
table 28:
table 29:
from the potential detection data described above, it can be seen 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 specifically example 5 and example 7).
Example 4
One, 7 groups of conventional sequence DNA nanoparticle carriers:
(1)7 sets of three polynucleotide base sequences constituting the DNA nanoparticle:
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 EGFRapt 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 above;
d-2 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRapt 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 above;
d-3 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRept 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 above;
d-4 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined section 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 set 8 DNA nanoparticles and single-stranded sequences for set 9 DNA nanoparticles were synthesized.
a chain: (SEQ ID NO:172:) 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;
c chain (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:):the front three bases of the 5' end and the rear three bases of the 3' end are respectively subjected to thio modification, and the 5' end is connected with Biotin;
b chain (SEQ ID NO: 179:):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:):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 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;
(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 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) potential measurement;
(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 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 electrophoresis picture of 4% agarose gel of 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 of the self-assembly products of the sequence DNAs from 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 7 groups of conventional sequence DNA self-assembly products are bright and clear, indicating that the 7 groups of conventional sequence DNA strands complete the self-assembly and form a stable nanoparticle structure. 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 the target heads are simultaneously connected, 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 (see specifically example 6 and example 8).
(2) Determination of potential
The determination 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 the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 3 groups of conventional sequence DNA nanoparticles are as follows:
table 37:
table 38:
table 39:
from the potential detection data described above, it can be seen that: the 3 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 stable self-assembly structures.
(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 appears, click Start starts, and the DLS measurements of the hydrodynamic size of self-assembled product D-7 result as follows:
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. a drop of sample is suspended on a 400-mesh carbon-coated copper net for 1 minute at room temperature;
2. sucking off liquid by using 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 transmission electron microscope was used for 120kv observation and photographing.
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
Mitoxantrone mounting experiments
Carrying by a chemical method:
first, experimental material and experimental method
1. Experimental materials and reagents:
(1) the DNA nucleic acid nanoparticles D-8 (hereinafter referred to as DNAh nanoparticles) formed by self-assembly of the group 8 DNA sequences in example 4 were used for mounting, and the specific information is shown in example 4.
The preparation method of the DNAh nanoparticles was similar to example 1.
(2) DEPC water: biyun Tian.
(3) PBS buffer: cellgro.
(4) 4% Paraformaldehyde
(5) Mitoxantrone.
(6) Chloroform: and (4) carrying out north transformation.
(7) Absolute ethanol: and (4) carrying out north transformation.
2. The experimental method comprises the following steps:
(1) mitoxantrone (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) while cooling in an ice-water bath, and the mixture was mixed with DNAh nanoparticles (1mg, 33.84nmoL) and reacted at 4 ℃ for 72 hours in the absence of light.
(2) 10 mu L of the reaction solution is diluted by 10 times, and HPLC analysis is carried out by taking 50 mu M mitoxantrone aqueous solution and 310 ng/mu L DNAh nano-particles as controls and injecting samples with equal volume. 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), transfer of the supernatant, washing of the solid product with ethanol (50mL) again, and evaporation of the solvent at low temperature under reduced pressure gave the blue solid product, mitoxantrone-DNAh particles.
(4) And (3) calculating the mounting rate:
1. preparing a mitoxantrone-PBS standard solution with a known concentration: 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, each 100 ul;
2. dissolving the mitoxantrone-DNAh particles in 100ul PBS;
3. placing the standard solution and the mitoxantrone-DNAh particles in a PCR plate, heating at 85 ℃ for 5min, and then cooling to room temperature;
4. measuring the absorbance of the mitoxantrone at 272nm by using a microplate reader, drawing a standard curve (see figure 11), and calculating to obtain the molar concentration of the mitoxantrone in the carried 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 molar concentration of the mitoxantrone and the mass concentration of the DNAh particles.
The specific calculation process is as follows:
C DNAh -1=32.4ug/ml,M DNAh ≈39500,100ul;C mitoxantrone -1=9.8uM,100ul;
C DNAh -2=43.8ug/ml,M DNAh ≈39500,100ul;C Mitoxantrone -2=12.32uM,100ul;
The loading rate of the mitoxantrone-DNAh obtained by taking the average value is about 11.5, and each DNAh nano-particle carrier can be loaded with about 75 mitoxantrone molecules.
In addition, on the basis of the DNAh nano-particle carrying mitoxantrone, other small molecule drugs can be further carried for the second time according to the same method as the mitoxantrone carrying method, for example, folic acid is further carried by the application to obtain the DNA nano-particles carrying two small molecule drugs of mitoxantrone and folic acid together, and the carrying rates of the two drugs can be detected by the method (numerical values are not shown).
Example 5 shows that the DNA nanoparticles with the extension segment, the targeting segment and the fluorescein all have the function of drug loading, and the small molecule drug mitoxantrone can be loaded in a covalent connection (paraformaldehyde-solvent covalent) mode and can also be loaded together with other small molecule drugs.
Example 6
Flow cytometry for detecting cell binding capacity of drug-loaded DNA nanoparticles
First, cell information (see Table 41 below)
Table 41:
secondly, the sample to be measured
Mitoxantrone targeted drugs: DNAh-Biotin-EGFRapt-Cy 5-Mit; (the DNA nanoparticles were mounted in the same manner as in example 5).
Targeting fluorescent vector: DNAh-Bio-EGFRApt-Cy5 (DNA nanoparticles provided in example 5).
Third, instrument, equipment and related reagent information (see tables 42 and 43)
Table 42:
name (R) | Brand | Goods number/model |
24-hole plate | Corning | 3526 |
Centrifugal machine | Jingli | LD5-2B |
CO2 incubator | Thermo | 3111 |
Microplate oscillator | QILINBEIER | QB-9001 |
Microscope | Olympus | IX53 |
Multifunctional enzyme mark instrument | Bio Tek | Synergy H1 |
Flow cytometer | ACEA | Novo Cyte |
Table 43:
fourthly, 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) single cell suspensions were collected by digestion and counted, adjusting cell density to 2X10 5 Planting 1 mL/hole into a 24-hole plate;
4) respectively adding the substances to be detected into corresponding cell holes, shaking and uniformly mixing the substances with the final concentration of 2 mu M;
5) incubating the cell plate in an incubator at 37 ℃ for 16 hours;
6) after incubation is finished, collecting cell suspension by trypsinization;
7) centrifuging to collect cell precipitate, and washing twice with PBS;
8) finally, 300 mu L PBS is used for resuspending the cell sediment, and the detection is carried out on a flow type machine; wherein, the detection channels of the fluorescent carrier and the mitoxantrone targeting drug are as follows: wavelength of excitation light: 488nm, emission light channel: 560 nm;
9) and (6) analyzing the data. The results of the analysis are shown in Table 44.
Table 44:
as can be seen from Table 44, the DNAh nanoparticles carrying the targeting peptide and the small molecule drug mitoxantrone had a high binding efficiency to cells, and it was clearly seen that they were efficiently bound to and internalized in human breast cancer cells MCF-7. Therefore, the mitoxantrone targeted drug DNAh-Biotin-EGFRapt-Cy5-Mit has an application prospect in treating breast cancer.
Example 7
Detection of serum stability of DNAh-Bio-EGFRApt-Cy5-Mit nanoparticle
First, experimental material, reagent and equipment
1. Experimental materials:
a sample to be tested: DNAh-Biotin-EGFRapt-Cy5-Mit, concentration 1.8 mg/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-prepared); 1 × TBE Buffer (No RNase) (self-mix); serum (FBS) (Excel); RPMI 1640 (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 6 mu L of DNAh-Biotin-EGFRatt-Cy 5-Mit nanoparticles, diluting the 6 mu L of the nanoparticles with 6 mu L of RPMI 1640 culture medium containing 10% serum, wherein the concentration of the diluted nanoparticles reaches 900 mu g/ml, respectively diluting 5 tubes, and diluting the diluted samples in a water bath at 37 ℃ for different times (0, 10min, 1h, 12h and 36 h).
(2) And (3) uniformly mixing the treated sample with 6X DNA Loading Buffer, operating on ice, and marking.
(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-Cy5-Mit nanoparticle is about 200bp, and as can be seen from FIG. 12, the DNAh-Bio-EGFRApt-Cy5-Mit nanoparticle is basically stable after being incubated at 37 ℃ for 36 h.
Example 8
Cytotoxicity of DNAh-Biotin-EGFRApt-Cy5-Mit nanoparticles in MCF-7 cells
Experimental materials and methods
1. Cell information (see Table 45)
Table 45:
2. sample to be tested (see Table 46)
Table 46:
3. consumables and equipment (see table 47):
table 47:
name(s) | Brand | Goods number/model |
96-well plate | Corning | 3599 |
Centrifugal machine | Jingli | LD5-2B |
CO 2 Culture box | Thermo | 3111 |
Microplate oscillator | QILINBEIER | QB-9001 |
Microscope | Olympus | IX53 |
Multifunctional enzyme mark instrument | Bio Tek | Synergy H1 |
4. Reagents (see table 48):
table 48:
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 2.22X 10 with growth medium 4 /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 2000;
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 mu L/hole of 10X concentration drug working solution into corresponding holes of the cell culture plate, making three multiple holes for each concentration, and obtaining the final action concentration of the drug shown in the following table 49;
table 49:
7) placing the cell culture plate in an incubator for further incubation for 96 hours;
8) CellTiter is mixedThe AQueous One Solution reagent is placed in a room temperature to melt for 90 minutes or a water bath at 37 ℃ to melt and then placed in a room temperature to balance for 30 minutes;
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 reader 490 A value;
12) and (4) processing and analyzing data.
And performing graphical processing on the data by adopting GraphPad Prism 5.0 software. To calculate IC50, the data were subjected to an "S" shaped nonlinear regression analysis to match the appropriate dose-response 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 )×100%。
Third, the experimental results (see Table 50, FIGS. 13 to 16)
TABLE 50:
as can be seen from Table 50 and FIGS. 13, 14, 15, and 16, for the MCF-7 cell line, compared with the pure DNAh targeting fluorescent carrier, both the small molecule drug mitoxantrone (Mit) and the DNAh drug-loaded particle DNAh-Bio-EGFRApt-Cy5-Mit are toxic to the MCF-7 cell.
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 51: r-15:
table 52: r-16:
table 53: r-17:
table 54: r-18:
table 55: r-19:
table 56: r-20:
table 57: r-21:
II, self-assembly testing:
(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;
(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;
(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 strip, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at a low temperature under reduced pressure;
(5) electrophoresis analysis detection and laser scanning observation.
Third, self-assembly test results
(1) Electrophoretic detection
The main reagents and instruments were as follows:
table 58:
name of reagent | Goods number | Manufacturer of the |
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 RNAse) | / | Self-matching |
Table 59:
the method comprises the following steps:
the RNA nanoparticles were diluted with ultrapure water according to the method of Table 60 below.
Table 60:
the 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 the glue running is finished, placing the dyed fabric on a horizontal shaking table for 30min, and taking pictures for 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. 17. Lanes 1 to 7 in FIG. 17 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. 17 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) Measurement of electric potential
The determination 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;
opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
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 61:
table 62:
table 63:
table 64:
table 65:
table 66:
table 67:
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;
2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
3. setting software detection parameters;
4. then clicking the determination completion setting, appearing a measurement dialog box, clicking Start, and obtaining the results of DLS measurement values of the hydrodynamic sizes of 7 groups of the extended segment variants and the core short sequence RNA as follows:
table 68:
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 the extended section deformation and 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 69:
name of reagent | Goods number | Manufacturer of the product |
AE buffer | / | Takara |
SYBR Green I dyes | / | Self-matching |
Table 70:
name(s) | 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 71:
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 length modified + core short sequence RNA nanoparticles are shown in the following, wherein the dissolution curve of R-15 is shown in FIG. 18, the dissolution curve of R-16 is shown in FIG. 19, the dissolution curve of R-17 is shown in FIG. 20, the dissolution curve of R-18 is shown in FIG. 21, the dissolution curve of R-19 is shown in FIG. 22, the dissolution curve of R-20 is shown in FIG. 23, and the dissolution curve of R-21 is shown in FIG. 24. 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 72:
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 73: d-8:
table 74: d-9:
table 75: d-10:
table 76: d-11:
table 77: d-12:
table 78: d-13:
table 79: d-14:
II, self-assembly testing:
(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;
(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 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 a TM value.
Third, self-assembly test results
(1) Electrophoretic detection
The main reagents and instruments were as follows:
table 80:
name of reagent | Goods number | Manufacturer of the |
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 |
Table 81:
the method comprises the following steps:
the DNA nanoparticles were diluted with ultrapure water according to the method of the following Table 82.
Table 82:
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 of 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are shown in FIG. 25. Lanes 1 to 7 in FIG. 25 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. 25 clearly show that the bands of the 7 sets of the products of the self-assembly of the DNA with the extended segment and the core short sequence are bright and clear, which indicates that the 7 sets of the DNA chains with the extended segment and the core short sequence complete the 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;
opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
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 extension segment deformation and core short sequence DNA nanoparticles are as follows:
table 83:
table 84:
table 85:
table 86:
table 87:
table 88:
table 89:
from the potential detection data described above, it can be seen that: the 7 groups of the extended section deformation and core short sequence DNA nano-particles have good stability, and further show that the nano-particles formed by self-assembly of the extended section deformation and the core short sequence DNA have a stable self-assembly structure.
(3) Particle size measurement
Firstly, preparing a potential sample (7 groups of extension segment 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;
secondly, opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
setting software detection parameters;
and clicking the setting after determination, generating a measurement dialog box, clicking Start, and obtaining the DLS measurement values of the hydrodynamic sizes of 7 groups of the extended segment variants and the core short sequence RNA as follows:
table 90:
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 and core short sequence DNA nano-particles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 91:
name of reagent | Goods number | Manufacturer(s) of |
AE buffer | / | Takara |
SYBR Green I dyes | / | Self-matching |
Table 92:
name (R) | 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:
② after diluting the sample with ultrapure water, mixing 5 μ g diluted sample with 2 μ L SYBR Green I dye (1: 200 dilution), the final volume is 20 μ L, the dilution concentration is as follows:
table 93:
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 DNA nanoparticles are shown in the following, the dissolution profile of D-8 is shown in FIG. 26, the dissolution profile of D-9 is shown in FIG. 27, the dissolution profile of D-10 is shown in FIG. 28, the dissolution profile of D-11 is shown in FIG. 29, the dissolution profile of D-12 is shown in FIG. 30, the dissolution profile of D-13 is shown in FIG. 31, and the dissolution profile of D-14 is shown in FIG. 32.
Table 94:
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 modified + core short sequence DNA nanoparticles shown in FIGS. 26 to 32, the TM values are all high, indicating that the sample purity is high and the self-assembled 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 95:
name of reagent | Goods number | Manufacturer of the |
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 RNAse) | / | Self-matching |
Serum (FBS) | / | Excel |
RPMI 1640 | / | GBICO |
Table 96:
the method comprises the following steps:
firstly, preparing the RNA nano-particles into the concentration shown in the following table, then diluting the prepared sample according to the method shown in the following table, diluting the sample by 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
table 97:
secondly, mixing 10 mu L of the treated sample with 2 mu L of 6 multiplied DNA Loading Buffer uniformly, operating on ice and marking;
thirdly, 8% non-denaturing PAGE gel is taken, samples with different incubation times are coated with a piece of gel, all samples processed by 12 mu L are loaded, and the procedure of 100V gel running is set 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. 33, the electrophoresis detection result of R-16 is shown in FIG. 34, the electrophoresis detection result of R-17 is shown in FIG. 35, the electrophoresis detection result of R-18 is shown in FIG. 36, the electrophoresis detection result of R-19 is shown in FIG. 37, the electrophoresis detection result of R-20 is shown in FIG. 38, and the electrophoresis detection result of R-21 is shown in FIG. 39. In fig. 33 to 39, 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 98:
name of reagent | Goods number | Manufacturer of the |
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 RNAse) | / | Self-matching |
Serum (FBS) | / | Excel |
RPMI 1640 | / | GBICO |
Table 99:
the method comprises the following steps:
preparing the DNA nano particles into the concentration shown in the table below, diluting the prepared sample by the method shown in the table below, 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 100:
mixing 5 mu L of the treated sample with 1 mu L of 6 multiplied DNA Loading Buffer, operating on ice and marking;
thirdly, 8% non-denaturing PAGE gel is taken, samples with different incubation times are coated with a piece of gel, all samples processed by 6 mu L are loaded, and the procedure of 100V gel running is set 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 D-8 is shown in FIG. 40, the electrophoresis detection result of D-9 is shown in FIG. 41, the electrophoresis detection result of D-10 is shown in FIG. 42, the electrophoresis detection result of D-11 is shown in FIG. 43, the electrophoresis detection result of D-12 is shown in FIG. 44, the electrophoresis detection result of D-13 is shown in FIG. 45, and the electrophoresis detection result of D-14 is shown in FIG. 46. In fig. 40 to 46, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and (5) 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 DNA nanoparticle sample bands at different times, indicating that the DNA nanoparticles D-8 to D-14 were relatively stable in 1640 medium of 50% FBS with no significant degradation.
Nucleic acid nanoparticle-loaded drug assay
Example 13
Doxorubicin mounting experiment:
according to the chemical method of attachment of example 5 (except for the specific limitation, the same method 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 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 the doxorubicin attachment carrier, and the doxorubicin attachment rates were 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% CO 2 And 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 adriamycin, which is respectively marked as D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin.
Third, main equipment, consumable
Table 101:
four, main reagent
Table 102:
name of reagent | Manufacturer of the product | Goods number | Remarks for note | |
DMEM (Biotin free) | Providing all the | YS3160 | ||
1%BSA-PBS | Self-matching | - |
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. after incubation, cell suspension was collected by trypsinization, centrifuged at 1000rmp for 5min, adjusted in concentration, and 2X10 cells were collected 5 -5×10 5 cells/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 given in the following table:
table 103:
2. conclusion
After incubation of HepG2 cells with D-8-adriamycin (carrier drug) and D-8 (blank carrier), 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 (vector medicine) and D-11 (blank vector), 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 medicine) and D-13 (blank vector), the binding rate is high (89.6% -98.2%).
After incubation of HepG2 cells with D-14-adriamycin (vector medicine) 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 104:
name of reagent | Manufacturer of the product | 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 (blue cloud sky) | C0038 |
Second, main consumables and instrument
Table 105:
information on cells
HepG2 (Source synergistic cell Bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), at 37 ℃ and 5% CO 2 And 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.
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 adriamycin, which is respectively marked as D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin.
The original drug substance 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 106:
number of hole | C9 | C8 | C7 | C6 | C5 | C4 | C3 | C2 | C1 |
Final concentration of drug loaded | 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 parent 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 original doxorubicin was first prepared with DMSO as 100 μ M stock solution and then diluted with complete medium (no biotin DMEM). DMSO was directly diluted with complete medium (biotin-free DMEM).
3. Adding a sample to be detected, and putting a 96-well plate into 5% CO at 37 DEG C 2 Incubate 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 with a microplate reader.
7. And (3) calculating: cell viability (%) (OD test group-OD blank). times.100%/(OD control group-OD blank), IC was calculated from GraphPad Prism 5.0 50 。
Sixth, experimental results
Table 107:
and (4) conclusion:
as can be seen from the above table and FIGS. 47a, 47b, 47c, 47D, 47e, 47f, 47g and 47h, the IC of the drug doxorubicin and the drug-loaded D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin acting on HepG2 cells 50 0.2725. 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 cells 50 Is 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) 50 Are all made of>1 μ M. It shows that compared with the pure blank vectors of D-8, D-9, D-10, D-11, D-12, D-13 and D-14, the original drug adriamycin of the small molecular drug and the drug-carrying 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 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 using a microplate reader, and a standard curve was plotted (as shown in FIG. 48).
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 mitoxantrone-containing drug is formed by hanging a small molecular drug mitoxantrone on the nucleic acid nanoparticle carrier provided by the application, so that the delivery stability of the mitoxantrone can be improved, and under the condition that the nucleic acid nanoparticle carries a target head, the mitoxantrone can be delivered to a target cell in a targeted manner to improve the bioavailability of the drug, and the toxic and side effects on non-target cells or tissues and local drug concentration are reduced due to targeted delivery, so that 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> Baiyazhida (Beijing) NanoBiotechnology Ltd
<120> mitoxantrone-containing medicine, preparation method, pharmaceutical composition and application thereof
<130> PN114941BYZD
<141> 2019-09-30
<150> 201811204164.0
<151> 2018-10-17
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<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
<210> 163
<211> 14
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(14)
<223> first extension segment
<400> 163
<210> 164
<211> 13
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(13)
<223> first extension segment
<400> 164
<210> 165
<211> 13
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(13)
<223> first extension segment
<400> 165
<210> 166
<211> 9
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> first extension segment
<400> 166
<210> 167
<211> 9
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> first extension segment
<400> 167
<210> 168
<211> 14
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(14)
<223> first extension segment
<400> 168
<210> 169
<211> 14
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(14)
<223> first extension segment
<400> 169
<210> 170
<211> 13
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(13)
<223> first extension segment
<400> 170
<210> 171
<211> 13
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(13)
<223> first extension segment
<400> 171
<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
<210> 176
<211> 9
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(9)
<223> b sequences
<400> 176
<210> 177
<211> 12
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12)
<223> c sequence
<400> 177
<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 (47)
1. A mitoxantrone-containing drug characterized in that said drug comprises a nucleic acid nanoparticle and mitoxantrone, and mitoxantrone is suspended on said 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';
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):
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' the flow of the air in the air conditioner,
b sequence: 5'-CCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCC-3';
(2) a sequence: 5'-GCAGCGUUCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCGC-3';
(3) a sequence: 5'-CGAGCGUUGC-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-GCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCG-3';
(4) a sequence: 5'-GGAGCGUUGG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5 '-CCUUCGGG-3',
c sequence: 5'-CCCCCAUAGCCC-3', respectively;
(5) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCGC-3', respectively;
(6) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
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';
(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' the flow of the air in the air conditioner,
b sequence: 5'-GCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3', respectively;
(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', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGTTCGGCG-3',
c sequence: 5'-CGCCCATAGCGC-3';
(13) a sequence: 5'-GCAGCGTTCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGTTCGGCC-3',
c sequence: 5'-GGCCCATAGCGC-3';
(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';
(15) a sequence: 5'-CGAGCGTTCC-3' the flow of the air in the air conditioner,
b sequence: 5'-GGTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3' are provided.
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 according to claim 2,
the first extension 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 chain: 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 chain: 5' -CCCA-3', 3' end of c strand: 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 one 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 medicament according to claim 4,
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 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 said 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 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 the second extension 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 drug according to any one of claims 1 to 3, wherein the mitoxantrone is entrapped on the nucleic acid nanoparticles in the form of a physical and/or covalent attachment and the molar ratio of mitoxantrone to nucleic acid nanoparticles is between 2 and 300: 1.
14. The drug of claim 13, wherein the molar ratio of mitoxantrone to nucleic acid nanoparticles is 10 to 50: 1.
15. The medicament of claim 13, wherein the molar ratio between the mitoxantrone and the nucleic acid nanoparticles is 15 to 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, the biologically active substance being 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, a natural salicylic acid, a monoclonal antibody, a vitamin, a phenolic lecithin, and a small molecule drug other than mitoxantrone.
17. The agent of claim 16, wherein the relative molecular weight of the nucleic acid domains is recorded as N 1 The total relative molecular weight of mitoxantrone and the biologically active substance is denoted as N 2 ,N 1 / N 2 ≥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 one of the a sequence, the b sequence, and the c sequence, 5 'or 3' of any one of the a sequence, the b sequence, and the c sequence, 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.
19. The drug of claim 18, wherein 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.
20. The drug of claim 16, wherein the small molecule drug other than mitoxantrone 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.
21. 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.
22. The drug according to claim 1, wherein the nucleic acid nanoparticles have a particle size of 1 to 100 nm.
23. The drug of claim 22, wherein the nucleic acid nanoparticles have a particle size of 5 to 50 nm.
24. The drug of claim 23, wherein the nucleic acid nanoparticles have a particle size of 10-30 nm.
25. The drug of claim 24, wherein the nucleic acid nanoparticles have a particle size of 10-15 nm.
26. A method for preparing a mitoxantrone-containing medicament, comprising the steps of:
providing a nucleic acid nanoparticle in the medicament of any one of claims 1 to 25;
the mitoxantrone is carried on the nucleic acid nano-particles in a physical connection and/or covalent connection mode to obtain the mitoxantrone-containing medicament.
27. The method of claim 26, wherein the step of immobilizing mitoxantrone by physical attachment comprises:
mixing and stirring the mitoxantrone, the nucleic acid nanoparticles and the first solvent to obtain a premixed system;
and precipitating the premixed system to obtain the mitoxantrone-containing medicament.
28. The method of claim 27, wherein the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
29. The method of claim 27, wherein the step of preparing,
precipitating the premixed system to obtain the mitoxantrone-containing medicament;
precipitating the premixed system to obtain a precipitate;
and washing the precipitate to remove impurities to obtain the mitoxantrone-containing medicament.
30. The method according to claim 29, 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.
31. The production method according to claim 30, wherein the precipitation is performed at a temperature of 0 to 5 ℃ to obtain the precipitate.
32. The preparation method according to claim 31, wherein the precipitate is washed with 6-12 times by volume of anhydrous ethanol to remove impurities, thereby obtaining the mitoxantrone-containing drug.
33. The method of claim 26, wherein the step of entrapping mitoxantrone by covalent attachment comprises:
preparing a mitoxantrone solution;
reacting the mitoxantrone solution with the amino outside the G ring of the nucleic acid nano-particles under the mediated action of formaldehyde to obtain a reaction system;
purifying the reaction system to obtain the mitoxantrone-containing medicament.
34. The method of claim 33,
the step of reacting comprises:
and mixing the mitoxantrone solution with a paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain the reaction system.
35. The method according to claim 34, wherein the concentration of the paraformaldehyde solution is 3.7 to 4 wt%.
36. 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.
37. The production method according to any one of claims 26 to 36, 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-assembly of single strands corresponding to the nucleic acid domain in the medicament of any one of claims 1 to 25.
38. The method of claim 37, 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 16 to 20 on the nucleic acid domain by means of physical and/or covalent attachment.
39. The method of claim 38, wherein the biologically active substance is covalently attached by solvent covalent attachment, linker covalent attachment, or click linkage.
40. The method according to 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 one or more selected from paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
41. The method of claim 39, wherein the linker is selected from the group consisting of disulfide bond, p-azido group, bromopropyne, and PEG.
42. The method of claim 39, 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.
43. The method of claim 42, 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.
44. A pharmaceutical composition comprising the mitoxantrone-containing drug of any one of claims 1 to 25.
45. Use of a mitoxantrone-containing medicament according to any of claims 1 to 25 for the preparation of a medicament for the treatment of tumors.
46. The use of claim 45, wherein the tumor is any one or more of breast cancer, malignant lymphoma, gastric cancer, intestinal cancer, bladder cancer, liver cancer, multiple myeloma, malignant mesothelioma and ovarian cancer.
47. The use of claim 45, wherein the tumor is a leukemia.
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