CN111053919B - Medicine containing idarubicin, preparation method thereof, pharmaceutical composition and application thereof - Google Patents
Medicine containing idarubicin, preparation method thereof, pharmaceutical composition and application thereof Download PDFInfo
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- CN111053919B CN111053919B CN201910969108.4A CN201910969108A CN111053919B CN 111053919 B CN111053919 B CN 111053919B CN 201910969108 A CN201910969108 A CN 201910969108A CN 111053919 B CN111053919 B CN 111053919B
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Abstract
The application provides a medicine containing idarubicin, a preparation method, a pharmaceutical composition and application thereof. The medicine comprises nucleic acid nanoparticles and idarubicin, and the idarubicin is hung on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence, a sequence and c sequence, the a sequence comprises a1 sequence or a sequence of a1 sequence with at least one base insertion, deletion or substitution, the b sequence comprises a sequence of b1 sequence or a sequence of b1 sequence with at least one base insertion, deletion or substitution, and the c sequence comprises a sequence of c1 sequence or a sequence of c1 sequence with at least one base insertion, deletion or substitution. The idarubicin-containing medicament provided by the application has the advantages that the nucleic acid structure domain is modified by the target head, so that the better targeting property is realized, the idarubicin can be stably delivered, and the reliability is very high.
Description
Technical Field
The application relates to the field of medicines, in particular to a medicine containing idarubicin, a preparation method, a pharmaceutical composition and application thereof.
Background
Idarubicin (Idarubicin, molecular formula: C)26H27NO9Molecular weight: 497.49) is an anthracycline cell cycle nonspecific anticancer drug, and has effects of inhibiting DNA synthesis, interfering RNA polymerase, and inhibiting topoisomerase II. The titer intensity is stronger than that of both daunorubicin and adriamycin. It is mainly used for treating acute non-lymphocytic leukemia, advanced breast cancer, myelodysplastic syndrome and non-Hodgkin's lymphoma.
Currently, antitumor antibiotics, including idarubicin, 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 needs to be used for replacing 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 gene vectors, but the transfection efficiency is generally low, the operation is complex, and the damage to tissues is large. It is also mediated by viral vectors, such as adenovirus and lentivirus, etc., and although the viral vectors have high in vitro transfection activity, the immunogenicity and the susceptibility to mutation of the viral vectors bring huge safety hazards to in vivo delivery. And non-viral vectors, especially biodegradable polymer materials are used for realizing the targeted transportation of the medicine. The non-viral vector has the advantages that under the condition of ensuring the expected transfection activity, the immunogenicity and a plurality of inflammatory reactions brought by the viral vector can be greatly reduced.
Among the above-mentioned various targeted delivery modalities, more research is currently focused on the non-viral vector field, and is generally designed for several vectors: (a) a cationic liposome; (b) a polycationic gene vector. However, much research is focused on modification of polycationic gene vectors and cationic liposomes to make them suitable for 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 medicament idarubicin is one of the difficulties in solving the limited clinical application of the existing idarubicin medicament.
Disclosure of Invention
The main purpose of the present application is to provide an idarubicin-containing drug, a preparation method thereof, a pharmaceutical composition and an application thereof, so as to improve the delivery reliability of the idarubicin drug.
In order to achieve the above objects, according to one aspect of the present application, there is provided an idarubicin-containing pharmaceutical comprising a nucleic acid nanoparticle and idarubicin, and the idarubicin 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', respectively; b1 is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3'; the sequence of c1 is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' is added.
Further, when the sequence a1 is SEQ ID NO. 1, the sequence b1 is SEQ ID NO. 3, and the sequence c1 is SEQ ID NO. 5, at least one of the sequences a, b, and c comprises a sequence in which at least one base is inserted, deleted, or substituted.
Further, base insertions, deletions or substitutions occur at:
(1) 1, 2, 4 or 5 bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(2) Between 8 th to 10 th bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(3) Between the 1 st to 3 rd bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(4) Between 6 th to 9 th bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(5) Between the 1 st to 4 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; and/or
(6) Between 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 to form a structure shown in a formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position are each independently selected from C-G or G-C; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; among the c sequences, 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', respectively; (3) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCG-3'; (4) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5 '-CCUUCGGG-3', c sequence: 5'-CCCCCAUAGCCC-3', respectively; (5) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCG-3', c sequence: 5'-CGCCCAUAGCGC-3', respectively; (6) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCC-3', c sequence: 5'-GGCCCAUAGCGC-3'; (7) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGGCG-3', c sequence: 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3', respectively; (9) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3', respectively; (10) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3', 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', respectively; (13) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3', respectively; (14) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3', respectively; (15) a sequence: 5'-CGAGCGTTCC-3', respectively; b sequence: 5 '-GGTTCGCCG-3', c sequence: 5' -CGGCCATAGCCG.
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 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 chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'; (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of the chain: 5' -CCCA-3', 3' end of c 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'.
Furthermore, the second extension is an extension sequence containing both CG base pairs and AT/AU base pairs, and preferably the second extension is an extension sequence of 2-50 base pairs.
Further, the second extension segment is an extension sequence formed by alternately arranging a sequence of continuous 2-8 CG base pairs and a sequence of continuous 2-8 AT/AU base pairs; alternatively, the second extension is an extension in which a sequence of 1 CG base pairs alternates with a sequence of 1 AT/AU base pairs.
Further, bases, ribose and phosphate in the sequence a, the sequence b and the sequence c have at least one modifiable site, and any modifiable site is modified by any one of the following modification 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.
Furthermore, the idarubicin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the idarubicin 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 idarubicin.
Further, the relative molecular weight of the nucleic acid domains is denoted as N1The total relative molecular weight of idarubicin and biologically active substance is denoted as N2,N1/N2≥1:1。
Further, the bioactive substance is one or more of a target, fluorescein and miRNA, wherein the target is located on any one of the sequences a, b and c, preferably the 5' end or the 3' end of any one of the sequences a, b and c, or is inserted between GC bonds of the nucleic acid domains, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the sequence a, the 5' end and the 3' end of the sequence c; preferably, the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
Further, the small molecule drugs other than idarubicin are drugs containing any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
Further, the protein is one or more of SOD, survivin, hTERT, EGFR and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
Further, the particle size of the nucleic acid nanoparticles is 1-100 nm, preferably 5-50 nm; more preferably 10 to 30 nm; further preferably 10 to 15 nm.
According to another aspect of the present application, there is also provided a method for preparing an idarubicin-containing medicament, comprising the steps of: providing the nucleic acid nanoparticle described above; the idarubicin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the idarubicin-containing medicine.
Further, the step of mounting the idarubicin by means of physical connection comprises: mixing and stirring idarubicin, nucleic acid nanoparticles and a first solvent to obtain a premixed system; precipitating the premixed system to obtain the medicine containing idarubicin; 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 premix system to obtain the idarubicin-containing pharmaceutical composition comprises: precipitating the premixed system to obtain precipitates; washing the precipitate to remove impurities to obtain the medicine containing idarubicin; more preferably, the premixed system is mixed with absolute ethyl alcohol and then precipitated at the temperature lower than 10 ℃ to obtain precipitates; idarubicin-containing drugs; 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 of that of the precipitate is adopted to wash and remove impurities, so that the idarubicin-containing medicine is obtained.
Further, the step of loading idarubicin by covalent attachment comprises: preparing an idarubicin solution; enabling the idarubicin solution to react 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 idarubicin-containing medicament; preferably, the step of reacting comprises: mixing the idarubicin solution, the paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain a reaction system; the concentration of the optimal paraformaldehyde solution is 3.7-4 wt%, the optimal paraformaldehyde solution is a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
Further, the preparation method further comprises a step of preparing a nucleic acid nanoparticle, which comprises: obtaining a nucleic acid structural domain by self-assembling the single strand corresponding to the nucleic acid structural domain; preferably, after obtaining the nucleic acid domain, the method of making further comprises: the bioactive substances are carried on the nucleic acid structural domain in a physical connection and/or covalent connection mode, and then the nucleic acid nano-particles are obtained.
Further, in the process of carrying the bioactive substances in a covalent connection mode, carrying is carried out through solvent covalent connection, linker covalent connection or click link; preferably, the solvent is a third solvent used in the covalent attachment as the attachment medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, 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-linkage manner, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of 2 ' hydroxyl, carboxyl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of G exocyclic amino, 2 ' -hydroxyl, a amino or 2 ' -hydroxyl.
According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any of the idarubicin-containing drugs described above.
According to a fourth aspect of the present application, there is also provided the use of any of the idarubicin-containing medicaments described above in the manufacture of a medicament for the treatment of a tumour or myelodysplastic syndrome.
Further, the tumor is any one or more of acute non-lymphocytic leukemia, advanced breast cancer, and non-hodgkin lymphoma.
According to a fifth aspect of the present application, there is also provided a method of preventing and/or treating a tumor or myelodysplastic syndrome, the method comprising: providing any of the above idarubicin-containing medicaments or pharmaceutical compositions; administering to the patient an effective amount of the above described idarubicin-containing medicament or pharmaceutical composition.
Further, the tumor is any one or more of acute non-lymphocytic leukemia, advanced breast cancer, and non-hodgkin's lymphoma.
The medicine containing idarubicin comprises nucleic acid nanoparticles and idarubicin, and the idarubicin is mounted 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 application can be contained, so that not only the nucleic acid structure domain can be formed by self assembly, but also the idarubicin can be connected to any 5 'end and/or 3' end of the three strands as a carrier, or the idarubicin can be stably inserted between the strands of the nucleic acid structure domain. According to the application, the micromolecular drug idarubicin is hung on the nucleic acid nanoparticles, the internal hydrophobicity, the external hydrophilicity and the stacking effect of basic groups of the nucleic acid nanoparticles are utilized, the 'coating effect' is achieved on the idarubicin, the idarubicin cannot be dissolved within a certain time due to the coating effect or covalent connection, and the delivery stability is improved. In addition, when the nucleic acid structure domain is modified by a target head, the targeting property is better, idarubicin can be stably delivered, and the reliability is high; meanwhile, the contact chance of the idarubicin 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, are included to provide a further understanding of the application, and the description of the exemplary embodiments and illustrations of the application are intended 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 results of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;
FIG. 6 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;
FIG. 7 shows the result of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;
FIG. 8 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;
FIG. 9 shows the result of 2% agarose gel electrophoresis detection of DNA nanoparticles of group 8 and group 9 formed by self-assembly in example 4 of the present application;
FIG. 10 shows a TEM image of self-assembled DNA nanoparticles D-7 of the present application in example 4;
FIG. 11 shows a standard curve of the absorbance of idarubicin during the DNA nanoparticle loading rate measurement in example 5 of the present application;
FIG. 12 shows the results of electrophoresis detection of DNAh-Biotin-EGFRatt-Cy 5-idarubicin nanoparticles after incubation in serum for various periods of time in example 6 of the present application;
FIGS. 13a to 13d show the results of detecting that DNAh-Bio-EGFRatpt-Cy 5-idarubicin nanoparticles inhibit the proliferation of MCF-7 cells in example 8, wherein FIG. 13a is the proliferation inhibition of MCF-7 cells by the small molecule drug idarubicin, FIG. 13b is the proliferation inhibition of MCF-7 cells by DNAh-Bio-EGFRatpt-Cy 5-idarubicin (targeting agent), FIG. 13c is the proliferation inhibition of MCF-7 cells by DNAh-Bio-EGFRatpt-Cy 5 (targeting fluorescent vector), and FIG. 13d is the proliferation inhibition of MCF-7 cells by the blank control of DMSO;
FIGS. 14a to 14d show the results of detecting that DNAh-Bio-EGFRaptt-Cy 5-idarubicin nanoparticles inhibit the proliferation of MV4-11 cells in example 8, wherein FIG. 14a is the proliferation inhibition of MV4-11 cells by the small molecule drug idarubicin, FIG. 14b is the proliferation inhibition of MV4-11 cells by DNAh-Bio-EGFRaptt-Cy 5-idarubicin (targeting drug), FIG. 14c is the proliferation inhibition of MV4-11 cells by DNAh-Bio-EGFRaptt-Cy 5 (targeting fluorescent vector), and FIG. 14d is the proliferation inhibition of MV4-11 cells by the blank control of DMSO;
FIG. 15 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. 16 shows the dissolution curve of the RNA nanoparticle R-15 in example 9 of the present invention;
FIG. 17 shows the dissolution curve of the RNA nanoparticle R-16 in example 9 of the present invention;
FIG. 18 shows the dissolution curve of the RNA nanoparticle R-17 in example 9 of the present invention;
FIG. 19 shows the dissolution curve of the RNA nanoparticle R-18 in example 9 of the present invention;
FIG. 20 shows the dissolution curve of RNA nanoparticle R-19 in example 9 of the present invention;
FIG. 21 shows the dissolution curve of the RNA nanoparticle R-20 in example 9 of the present invention;
FIG. 22 shows the dissolution curve of the RNA nanoparticle R-21 in example 9 of the present invention;
FIG. 23 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. 24 shows a melting curve of DNA nanoparticle D-8 in example 10 of the present invention;
FIG. 25 shows a melting curve of DNA nanoparticle D-9 in example 10 of the present invention;
FIG. 26 shows a dissolution curve of DNA nanoparticle D-10 in example 10 of the present invention;
FIG. 27 is a graph showing a dissolution curve of the DNA nanoparticle D-11 in example 10 of the present invention;
FIG. 28 shows a dissolution curve of the DNA nanoparticle D-12 in example 10 of the present invention;
FIG. 29 is a graph showing the dissolution curve of the DNA nanoparticle D-13 in example 10 of the present invention;
FIG. 30 shows a dissolution curve of DNA nanoparticle D-14 in example 10 of the present invention;
FIG. 31 shows the result of electrophoresis detection of RNA nanoparticle R-15 in example 11 after incubation in serum for various times;
FIG. 32 shows the result of electrophoresis detection of RNA nanoparticle R-16 in example 11 after incubation in serum for various times;
FIG. 33 shows the result of electrophoresis detection of RNA nanoparticle R-17 in example 11 of the present invention after incubation in serum for various time periods;
FIG. 34 shows the result of electrophoresis detection of RNA nanoparticle R-18 in example 11 after incubation in serum for various times;
FIG. 35 shows the result of electrophoresis detection of RNA nanoparticle R-19 in example 11 after incubation in serum for various times;
FIG. 36 shows the result of electrophoresis detection of RNA nanoparticle R-20 in example 11 after incubation in serum for various times;
FIG. 37 shows the result of electrophoresis detection of RNA nanoparticle R-21 in example 11 after incubation in serum for various times;
FIG. 38 shows the result of electrophoresis detection of DNA nanoparticle D-8 in example 12 after incubation in serum for various times;
FIG. 39 shows the result of electrophoresis detection of DNA nanoparticle D-9 in example 12 after incubation in serum for various times;
FIG. 40 shows the results of electrophoresis detection of the DNA nanoparticle D-10 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-11 in example 12 of the present invention after incubation in serum for various times;
FIG. 42 shows the result of electrophoresis detection of the DNA nanoparticle D-12 in example 12 of the present invention after incubation in serum for various times;
FIG. 43 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. 44 shows the results of electrophoresis detection of the DNA nanoparticle D-14 in example 12 of the present invention after incubation in serum for various periods of time;
FIGS. 45a, 45b, 45c, 45D, 45e, 45f, 45g and 45h 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. 46 shows a standard curve of daunorubicin absorbance used in the mounting rate measurement process of example 16.
Detailed Description
It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present application will be described in detail with reference to examples.
Interpretation of terms:
blank vector: refers to a blank nucleic acid nanoparticle vector, such as RNAh or DNAh, that does not contain any biologically active substance.
Targeting vectors: refers to a nucleic acid nanoparticle vector containing a targeting tip but not containing a fluorescent substance, such as Biotin-RNAh or Biotin-DNAh.
A fluorescent carrier: refers to a nucleic acid nanoparticle vector containing fluorescent substances but not containing a targeting head, such as 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 a nucleic acid nanoparticle vector containing a target, a fluorescent substance and a chemical drug, such as RNAh-Biotin-Cy 5-idarubicin or DNAh-Biotin-Cy 5-idarubicin.
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 art, although there are many drug carriers for improving drug delivery efficiency in the prior art, it is difficult to solve the problem that the clinical application of drugs is limited. In order to improve the situation, the inventor of the present application has studied all available materials as drug carriers, and has conducted in-depth investigation and analysis on various carriers from the aspects of cell/tissue targeting property of the carriers, stability during transportation, activity and efficiency of entering target cells, drug release capacity after reaching target cells, toxicity to cells and the like, and found that nanostructures formed by self-assembly of emerging DNA and/or RNA molecules, for example, DNA in a self-assembly system of DNA dendrimers, have a significant effect of hindering nuclease degradation, and have very important application values in the fields of gene therapy and biomedicine.
Through analysis of nanoparticles formed by self-assembly of DNA and RNA reported in the prior art, compared with DNA nanoparticles which are relatively rigid, RNA nanoparticles have more flexibility and stronger tension due to a large number of stem-loop structures existing in molecules or among molecules, and thus have more advantages in serving as candidate drug carriers. However, RNA nanoparticles in their native state are relatively less stable, and current improvements based on the use of RNA nanocarriers have mostly been developed around improving their stability and reliability. The current research results, although providing the possibility of drug loading to some extent, focus more on the possibility and effectiveness of the loading of nucleic acid drugs, especially siRNA drugs or miRNA drugs. However, there are few reports on whether non-nucleic acids are equally effective. In addition, the existing self-assembly nanoparticles, especially those used as vectors, are self-assembled by RNA strands at present, and very few self-assembly nanoparticles adopt a form of RNA strand and DNA strand combination, but do not adopt pure DNA strands to realize self-assembly.
In order to provide a novel RNA nanoparticle carrier which is highly reliable and can be self-assembled, the applicant has compared and improved existing RNA nanoparticles, developed a series of novel RNA nanoparticles, and further tried to perform self-assembly using pure DNA strands from the viewpoint of improving applicability and reducing cost. And the self-assembly of the 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 idarubicin and can stably exist in serum; further experiments verify that the idarubicin can be carried into cells, and the carrier alone is non-toxic to the cells. And the carrier carrying the idarubicin 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 medicine containing idarubicin, which comprises nucleic acid nanoparticles and idarubicin, wherein the idarubicin 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 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'; the sequence of c1 is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' is added.
The idarubicin-containing medicine provided by the application comprises nucleic acid nanoparticles and idarubicin, and the idarubicin is mounted on the nucleic acid nanoparticles. The nucleic acid nanoparticles can be used as a carrier, in which idarubicin is linked to any of the 5 'ends and/or 3' ends of the three strands, or can be stably inserted between strands of the nucleic acid domains, as well as a nucleic acid domain formed by self-assembly by including the three sequences or their variant sequences. According to the idarubicin-containing drug provided by the application, the small-molecule drug idarubicin is loaded on the nucleic acid nanoparticles, and due to the fact that the nucleic acid nanoparticles are hydrophobic inside and hydrophilic outside and the base has a stacking effect, the idarubicin is coated, and the coating or covalent connection enables the idarubicin not to be dissolved within 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, idarubicin can be stably delivered, and the reliability is high; meanwhile, the contact chance of the idarubicin 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. In the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under non-covalent bond-based interactions. 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 guiding of self-assembly refers to the complementarity of the molecules in space, that is, the occurrence of self-assembly requires the rearrangement of the molecules to be satisfied in the size and direction of space.
The DNA nanotechnology is a mode of molecular self-assembly from bottom to top, spontaneously forming a stable structure based on the physical and chemical properties of nucleic acid molecules, with molecular architecture as the starting point, following strict principles of nucleic acid base pairing. 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 formed finally. Unlike DNA, RNA can be structured beyond the limitations 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 Watton-Crick base pair type and non-Watton-Crick base pair type, so that the RNA can form a large number of and various types of circulating structure modules, and the modules are basic units forming 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.
In the nucleic acid nanoparticles, the nucleic acid nanoparticles comprise three sequences shown by the sequences SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 or sequences after variation thereof, or three sequences shown by the sequences 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.
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 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 medicament obtained by idarubicin suspension more stable, when base insertion, deletion or substitution is carried out on the sequence shown in SEQ ID NO:1/2, SEQ ID NO:3/4 and/or SEQ ID NO:5/6, base insertion, deletion or substitution can be carried out on the base at certain specific positions of the sequence, on one hand, the sequence after variation is the same as the original sequence and can be self-assembled into the nanoparticles, and on the other hand, the variation keeps at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of homology with the original sequence, so that the nanoparticles formed by self-assembling the sequence have the same medicament loading property and similar stability, and can well suspend and deliver the idarubicin.
In a preferred embodiment, the above base insertion, deletion or substitution occurs at: (1) 1 or 2 between the 1 st, 2 nd, 4 th and 5 th bases from the 5' end of the a sequence shown in SEQ ID NO; and/or (2) among bases 8 to 10 from the 5' end of the sequence a shown in SEQ ID NO 1 or 2; and/or (3) among 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 to 9 th bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (5) between the 1 st to 4 th bases from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6; and/or (6) between 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 idarubicin is loaded, in a preferred embodiment, the sequence a, the sequence b and the sequence c are self-assembled into a structure shown in formula (1):
wherein W-C represents Watson-Crick pairing, N and N ' represent non-Watson-Crick pairing, each W-C at any position is independently selected from C-G or G-C, and the two bases at the 5' end and 3' end of each of at least two of the a, b, and C sequences are non-complementary; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; in the c sequence, the NNNN sequence in the 5 'to 3' direction is CAUA or CATA.
In the preferred embodiment, the a, b, C sequences form by self-assembly a nucleic acid domain having the formula (1), wherein the bases at the positions other than the N and N' defined non-Watson-Crick base pairs form a classical Watson-Crick pair, and the bases of the Watson-Crick pair are selected from G-C or C-G base pairs. The nucleic acid nanostructure is more stable because the force of hydrogen bonds between G-C or C-G base pairs is greater than the force of hydrogen bonds between A-U/T or U/T-A base pairs. And a bulge or loop structure formed by non-Watson-Crick pairing base brings larger tension to the nucleic acid nano-carrier, so that the adaptability of the nucleic acid nano-carrier to microenvironment change is stronger, and the stability of the nucleic acid nano-particle is higher.
In the 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), it is preferable that the bases at different positions are selected according to the following principle when selecting Watson-Crick paired bases: (1) a sequence a, a sequence b and a sequence c, wherein when a single sequence is not self-complementary, a pair of sequences forms a secondary structure; (2) one end of any two sequences is complementary and matched to form a double chain, and the other end is not complementary and matched to form a Y-shaped or T-shaped structure. The principle of the base selection is to make the two ends of any chain complementary and paired with the two ends of other two chains respectively to improve the self-assembly efficiency. Of course, in addition to the Y-type or T-type structure, other variants such as quadrilateral, etc. may be used as long as the principle that one end of any two sequences is complementary and paired to form a double strand and the other end is not complementary and 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 a sequence and the first N ' from the 5' end in the b sequence can be non-Watson-Crick pairing U-U, or modified T, A, C or G following 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 sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence (SEQ ID NO: 7): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 8): 5'-CCUUCGCCG-3', c sequence (SEQ ID NO: 9): 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence (SEQ ID NO: 10): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 11): 5 '-CGUUCGCCGC-3', c sequence (SEQ ID NO: 12): 5'-CGGCCAUAGCGC-3'; (3) a sequence (SEQ ID NO: 13): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 14): 5'-GCUUCGCCG-3', c sequence (SEQ ID NO: 15): 5'-CGGCCAUAGCCG-3'; (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', respectively; (5) a sequence (SEQ ID NO: 19): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 20): 5'-CGUUCGGCG-3', c sequence (SEQ ID NO: 21): 5'-CGCCCAUAGCGC-3'; (6) a sequence (SEQ ID NO: 22): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 23): 5'-CGUUCGGCC-3', c sequence (SEQ ID NO: 24): 5'-GGCCCAUAGCGC-3'; (7) a sequence (SEQ ID NO: 25): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 26): 5'-GCUUCGGCG-3', c sequence (SEQ ID NO: 27): 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence (SEQ ID NO: 28): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 29): 5'-CCTTCGCCG-3', c sequence (SEQ ID NO: 30): 5'-CGGCCATAGCCC-3'; (9) a sequence (SEQ ID NO: 31): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 32): 5'-CGTTCGCCG-3', c sequence (SEQ ID NO: 33): 5'-CGGCCATAGCGC-3', respectively; (10) a sequence (SEQ ID NO: 34): 5'-CGAGCGTTGC-3', b sequence (SEQ ID NO: 35): 5'-GCTTCGCCG-3', c sequence (SEQ ID NO: 36): 5'-CGGCCATAGCCG-3', respectively; (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'; (15) a sequence (SEQ ID NO: 175): 5'-CGAGCGTTCC-3', respectively; b sequence (SEQ ID NO: 176): 5 '-GGTTCGCCG-3', c sequence (SEQ ID NO: 177): 5' -CGGCCATAGCCG.
The nucleic acid nanoparticles formed by self-assembly of the fifteen groups of sequences have higher stability and 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 idarubicin drugs. The amount of idarubicin loaded varies according to the position of G-C or C-G base pairs in the nucleic acid nanoparticles.
In order to make the nucleic acid domain capable of carrying more idarubicin and bioactive substances (the description of the bioactive substances is given below), in a preferred embodiment, the nucleic acid domain further comprises a first extension, the first extension is a Watson-Crick paired extension, and the first extension is located at the 5 'end and/or the 3' end of any one of the a sequence, the b sequence and the c sequence. A certain matching relationship is required between the carrier and the carried substance, and when the molecular weight of the carrier is too small and the molecular weight of the carried substance is too large, the carrying or transporting capacity of the carrier to the carried substance is relatively reduced from the mechanical point of view. Therefore, a vector matching the size of the carried substance can be obtained by adding a first extension segment to the 5 'end and/or 3' end of any one of the a sequence, the b sequence and the c sequence based on the nucleic acid nanostructure.
The specific length of the first extension segment can be determined according to the size of the substance to be carried. In a preferred embodiment, the first 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 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 the chain: 5' -CCCG-3', 3' end of c chain: 5 '-CGGG-3'; (5): a 5' end of chain: 5' -CCCC-3', 3' end of c 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'; (10): a 5' end of the chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:162), the 3' end of the c-chain: 5'-UCGCCGCUCGCCGC-3' (SEQ ID NO: 163); (11): a 3' end of the chain: 5'-GGCCGGAGGCCGG-3' (SEQ ID NO:164), 5' end of b chain: 5'-CCGGCCUCCGGCC-3' (SEQ ID NO: 165); (12) b 3' end of strand: 5' -CCAGCCGCC-3' (SEQ ID NO:166), 5' end of c chain: 5'-GGCGGCAGG-3' (SEQ ID NO: 167); (13): a 5' end of chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:168), the 3' end of c-chain: 5'-TCGCCGCTCGCCGC-3' (SEQ ID NO: 169); (14): a 3' end of the 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 sequences also enables the a sequence, the b sequence and the c sequence to maintain higher self-assembly activity and efficiency.
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-, b-and c-sequences, 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 the basis of the first extension.
In a preferred embodiment, the nucleic acid domain further comprises at least one second extension selected from the group consisting of: a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'. This second extension renders the nanoparticle non-immunogenic and non-existent in the case of secondary structures to which each chain folds 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):
c is (SEQ ID NO: 51):
m in the sequence a, the sequence b and the sequence c is U or T, and when M is T, the synthesis cost of the sequences is greatly reduced.
In practical application, the specific arrangement positions of the CG base pairs and the extended sequences of AT/AU base pairs can be reasonably adjusted according to actual needs. In a more preferred embodiment, the second extension is an extension sequence formed by alternating 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 the CGCCGC extension in the sequence a shown by the SEQ ID NO. 49 and the AAAAAA extension are interchanged, the positions of the GCGGCG extension and the GGCGGC extension in the sequence b shown by the SEQ ID NO. 50 and the TTTTTT extension are interchanged, the positions of the GCCGCC extension and the AAAAAA extension in the sequence c shown by the SEQ ID NO. 51 and the CGCCGC extension and the TTTTTT extension are interchanged. The nucleic acid nanoparticles formed by self-assembly of the sequences are suitable for carrying bioactive substances with indole molecular structures (indole molecules are preferably combined with A).
Over the past years, three major challenges with RNA as a widely used building material include: 1) susceptibility to rnase degradation; 2) susceptibility to dissociation following systemic injection; 3) toxicity and adverse immune response. Currently, these three challenges have been largely overcome: 1) 2 '-fluoro (2' -F) or 2 '-O-methyl (2' -OMe) modification of the ribose-OH group can chemically stabilize RNA in serum; 2) certain naturally occurring linking motifs are thermodynamically stable and can keep the entire RNA nanoparticle intact at ultra-low concentrations; 3) the immunogenicity of the RNA nanoparticles is sequence and shape dependent and can be adjusted to allow the RNA nanoparticles to stimulate the production of inflammatory cytokines or to render the RNA nanoparticles non-immunogenic and non-toxic for repeated intravenous administration of 30 mg/kg.
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 idarubicin can be mounted by physical and/or covalent attachment. When idarubicin is simultaneously linked to a nucleic acid domain by both physical intercalation and covalent linkage, the physical intercalation is usually between GC base pairs, and the preferred number of intercalation sites is 1-100: 1, and inserting. When covalent attachment is used, idarubicin typically reacts chemically with the exocyclic amino group of the G ring to form a covalent attachment. More preferably, the molar ratio of idarubicin to nucleic acid nanoparticles is 2-300: 1, preferably 2-290: 1, more preferably 2-29: 1, even more preferably 10-50: 1, and most preferably 15-25: 1.
In addition to the idarubicin-containing drug, the nucleic acid nanoparticle is used as a delivery vehicle for idarubicin, and in a preferred embodiment, the nucleic acid nanoparticle further comprises a bioactive substance, and the bioactive substance is linked to the nucleic acid domain according to different purposes of the drug. The bioactive substance is one or more of a target, fluorescein, siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein, polypeptide, flavonoid, glucose, natural salicylic acid, monoclonal antibody, vitamin, phenol, lecithin and small molecule drugs except idarubicin.
In order to improve the efficiency of the nucleic acid nanoparticles in loading and delivery of the biologically active substance to be loaded, the relative molecular weights of the nucleic acid domains preferably match the relative molecular weights of idarubicin and the biologically active substance. In a preferred embodiment, the relative molecular weight of the nucleic acid domains is denoted as N1The total relative molecular weight of idarubicin and biologically active substance is denoted as N2,N1/N2≥1:1。
The idarubicin-containing drugs of the present application have different performance optimizations depending on the type of bioactive substance specifically loaded. For example, when the bioactive substance is biotin or folic acid, it acts to target the idarubicin-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 micromolecule medicines except idarubicin, the medicine containing the idarubicin can become a new product with specific treatment effect according to different biological functions, such as a medicine with more excellent performance. 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 be carried, and there is no particular requirement on the length of the single strand assembled to form the nanoparticle.
In a preferred embodiment, the bioactive substances are target heads, fluorescein and miRNA, wherein the target heads are located on any sequence of a, b and c sequences, preferably on the 5' end or the 3' end of any sequence of a, b and c, or are inserted between GC bonds of the nucleic acid structure domain, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more positions of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence; preferably, the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
The target head can be connected to any sequence of a sequence, b sequence and c sequence through a linker covalent connection mode, and the available linker is selected from disulfide bond, p-azido group, bromopropyne or PEG. As used herein, "on any sequence" refers to any base position of any sequence of a, b, c 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 may be any one or more of conventional fluorescein, preferably FAM, CY5 and CY 3.
The miRNA can be miRNA with cancer inhibition effect, and can also be anti-miRNA capable of inhibiting corresponding diseases, and reasonable selection is carried out 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 cancer cells, the anti-miR-21 is complementary to miR-21 base with very high affinity and specificity, thereby effectively reducing expression of oncogenic miR-21. Therefore, the anti-miR-21 can be synthesized at any one or more positions of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence according to actual needs. When the anti-miR-21 is synthesized at all three positions, the inhibition effect of the anti-miR-21 on the miR-21 is relatively stronger.
When the biological active substance capable of being carried is other small molecule drugs except idarubicin, the drugs include, but are not limited to, drugs for treating liver cancer, stomach cancer, lung cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, melanoma, leukemia, senile dementia, ankylosing spondylitis, malignant lymphoma, bronchial cancer, rheumatoid arthritis, HBV hepatitis B, multiple myeloma, pancreatic cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, esophageal cancer, oral cancer, lupus erythematosus and other diseases which can be treated by different drugs; preferably, the head and neck cancer is brain cancer, neuroblastoma or glioblastoma.
When the biologically active substance capable of being carried is a small molecule drug other than idarubicin, the biologically active 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 further preferably 10 to 15 nm. Within this range the size is suitable both for entering the cell membrane by cell surface receptor mediated phagocytosis and for being removed by renal filtration avoiding non-specific cell permeation, and therefore the favourable particle size contributes to improved pharmacokinetic, pharmacodynamic, biological and toxicological profiles.
According to a second aspect of the present application, there is also provided a method for preparing the idarubicin-containing medicament, comprising the steps of: providing any one of the nucleic acid nanoparticles described above; the idarubicin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the idarubicin-containing medicament.
When physically linked, idarubicin will typically form insertions between GC base pairs in a physical insertion. When covalent attachment is used, idarubicin typically reacts with the amino group outside the G ring to form a covalent attachment. The medicine containing the idarubicin prepared by the method has better targeting property after being modified by the target head, can stably deliver the idarubicin and has high reliability.
In a preferred embodiment, the step of mounting the idarubicin by way of a physical connection comprises: mixing and stirring idarubicin, nucleic acid nanoparticles and a first solvent to obtain a premixed system; precipitating the premixed system to obtain the idarubicin-containing medicament. The specific amounts of idarubicin and nucleic acid nanoparticles can be adjusted according to the variation of the loading amount, which can be understood by those skilled in the art and will not be described herein again.
In order to improve the efficiency and stability of physical connection, the amount of idarubicin added per liter of first solvent is preferably 0.1-1 g. Preferably, the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid. Preferably, the step of precipitating the premix system to obtain the idarubicin-containing pharmaceutical composition comprises: precipitating the premixed system to obtain precipitates; washing the precipitate to remove impurities, and obtaining the idarubicin-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 of that of the precipitate is adopted to wash and remove impurities, so that the idarubicin-containing medicine is obtained.
In a preferred embodiment, the step of loading idarubicin by covalent attachment comprises: preparing an idarubicin solution; enabling the idarubicin solution to react with the amino outside the G ring of the nucleic acid nanoparticles under the mediation effect of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the idarubicin-containing medicament.
By formaldehyde-mediated form, the following reactions can occur:
preferably, the step of reacting comprises: mixing the idarubicin solution, 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 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 other functions to the idarubicin-containing drug according to practical application requirements, 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, 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.
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: 1, and inserting. When the naphthamide drug is inserted, the naphthamide drug 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 the nucleic acid structural domain, and the pyridocarbazoles are inserted according to the difference of the number of the AA base pairs in the range of 1-200: the ratio of 1 was inserted.
Specifically, depending on the species of the bioactive substance, the length of the a, b, and c sequences forming the nucleic acid domains in the nucleic acid nanoparticles, and the number of GC-complementary base pairs therein, the molar ratio of the bioactive substance to the nucleic acid domains can be rationally selected for physical intercalation.
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 covalently 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 domain are simultaneously subjected to alkynyl or azide modification and connected in a click-to-link mode, different click-to-links are selected according to different structure changes of the medicament. And the attachment position may be changed correspondingly according to the structure of the active material, which can be understood by those skilled in the art.
In a preferred embodiment, when the biologically active substance is linked to the nucleic acid domain in a click-link fashion, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of hydroxyl, carboxyl, sulfhydryl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of amino, imino or hydroxyl.
When the nucleic acid domain is bound to a drug, the nucleic acid domain is water-soluble, and many drugs have poor water-solubility, and when the nucleic acid domain is bound 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, according to the size of a specific drug molecule and the number of GC base pairs on the sequence a, the sequence b and the sequence c of a specifically designed nucleic acid structural domain, when in combination, the combination reaction is carried out according to the supersaturation combination amount which is 1.1-1.3 times of the theoretical amount, and 35-45 drugs can be combined on one nucleic acid structural domain at most. When the drug has other structure, the loading amount is related to the occupancy of the specific drug (including but not limited to molecular structure, form, shape and molecular weight), so that the binding condition of the active site of the drug and the-NH bond on the nucleotide guanosine of the nucleic acid domain is relatively severe, and the drug can be loaded but is relatively difficult to be excessively bound.
According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any of the idarubicin-containing medicaments described above. Specifically, according to actual needs, a suitable combination drug or 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 present application, there is also provided the use of any of the above idarubicin-containing medicaments for the preparation of a medicament for the treatment of a tumor or myelodysplastic syndrome. Further, the tumor is any one or more of acute non-lymphocytic leukemia, advanced breast cancer, and non-hodgkin lymphoma. Specific application can be to improve the medicament per se on the basis of the medicament to obtain a new medicament, or to prepare the medicament as a main active ingredient into a preparation with a proper dosage form and the like.
According to a fifth aspect of the present application, there is also provided a method of preventing and/or treating a tumor or myelodysplastic syndrome, the method comprising: providing any of the above idarubicin-containing medicaments or pharmaceutical compositions; administering to the patient an effective amount of the above described idarubicin-containing medicament or pharmaceutical composition. Further, the tumor is any one or more of acute non-lymphocytic leukemia, advanced breast cancer, and non-hodgkin lymphoma.
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., acute non-lymphocytic leukemia, advanced breast cancer remission. In a particular embodiment, the dosage may be adjusted to provide the optimum therapeutically responsive dosage, 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., prevention or inhibition of acute non-lymphocytic leukemia, advanced breast 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.
It should be noted that the nucleic acid nanoparticles formed by self-assembly of the sequences or variants of the sequences provided herein can also be used as basic building blocks, and can be further polymerized to form polymers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., according to practical requirements.
The advantageous effects of the present application will be further described with reference to specific examples.
Assembly of nucleic acid nanoparticles
Example 1
One, RNA and DNA nanoparticle vector:
(1) the three polynucleotide base sequences that make up the RNA nanoparticles are shown in table 1:
table 1:
(2) three polynucleotide base sequences of DNA nanoparticles
The DNA has the same sequence as that of the RNA described above except that T is substituted for U. Wherein the molecular weight of the a chain is 8802.66, the molecular weight of the b chain is 8280.33, and the molecular weight of the c chain 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) and (4) electrophoretic analysis and detection.
Third, self-assembly experimental results
Results of electrophoresis
The result of electrophoresis detection of the RNA self-assembly product is shown in FIG. 1. In fig. 1, lanes 1 to 3 are, from left to right: a strand, b strand, RNA self-assembly product. As can be seen, the RNA self-assembly products are slightly dispersed, but clearly seen as a single band. And the molecular weight is the molecular weight after the assembly, and is larger than that of the single chain, so that the position of the band lags behind the a chain and the b chain, the actual situation is consistent with the theory, and the stable composite structure is formed by the self-assembly of the RNA single chains, and the RNA nano-particles are formed.
The 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 be successfully self-assembled into DNA nanoparticles.
The sequences a, b and c of the RNA nanoparticles and the DNA nanoparticles include various extension sequences (including drug-loading binding sequences) that facilitate the function of loading the nucleic acid domains, and a targeting head or fluorescein linked to the nucleic acid domains, in addition to the core sequence forming the nucleic acid domains. It can be seen that the presence of substances other than these core sequences does not affect the formation of nucleic acid domains and the successful self-assembly of nucleic acid nanoparticles. The self-assembled nucleic acid nanoparticles can have a targeting type under the guidance of a target head, and the fluorescein can enable the nucleic acid nanoparticles to have visibility and traceability.
Example 2
One, 7 groups of short sequence RNA nano-particle carriers:
(1)7 groups of three polynucleotide base sequences composing the RNA nanoparticle are respectively shown in tables 2 to 8:
table 2: r-1
Table 3: r-2
Table 4: r-3
Table 5: r-4
Table 6: r-5
Table 7: r-6
Table 8: r-7
The single strands of the 7 groups of short-sequence RNA nanoparticle carriers are synthesized by the corporation of Venezetian Biotechnology (Shanghai).
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 a target band, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at 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 can be clearly seen that the bands of R-2, R-3, R-5 and R-7 in the 7 groups of short sequence self-assembly products are bright and clear, and the bands of R-1, R-4 and R-6 are still single bands, although they are relatively dispersed, indicating that the 7 groups of short sequences can be well self-assembled into RNA nanoparticle structures.
(2) Measurement of electric potential
The 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 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 short sequence RNA nanoparticles are shown in tables 9 to 15 below:
table 9:
table 10:
table 11:
table 12:
table 13:
table 14:
table 15:
from the potential detection data described above, it is found that: the 7 groups of short sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the short sequence RNAs have 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 structural domain through self-assembly, and the structure is stable. Based on example 1, it can be seen that various functional extension fragments or connecting targeting heads, fluorescein and the like are added on the basis of different core sequence combinations, and the RNA nanoparticles can be successfully assembled, and have the performances of drug loading, cell targeting, visual tracking and the like.
To further verify these properties, an extension fragment was added to example 2, see example 3. And 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 groups of three polynucleotide base sequences constituting the RNA nanoparticles are respectively shown in tables 16 to 22:
table 16: r-8
Table 17: r-9
Table 18: r-10
Table 19: r-11
Table 20: r-12
Table 21: r-13
Table 22: r-14 (in the following a chain)uGAcAGAuAAGGAAccuGcudTdTAs survivin siRNA)
The 7 groups of conventional sequence RNA nanoparticle vectors are synthesized by commissioned Suzhou Jima company, wherein the sequences a, b and C in R-8 to R-14 are respectively extended RNA oligonucleotide sequences formed by adding extension segments on the basis of the sequences a, b and C in R-1 to R-7, targeting module fragments are not extended, and C/U base 2' F modification is carried out (the enzyme cutting resistance and stability are enhanced). In addition, a Survivin (Survivin) siRNA nucleic acid interference therapeutic fragment is modified in the RNA nanoparticle R-14, specifically, a sense strand of Survivin siRNA is extended at the 3 'end of the a strand (see the underlined part of the a strand), and an antisense strand is extended and connected at the 5' end of the b strand (see the underlined 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 target bands, eluting in RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at low temperature under reduced pressure;
(5) electrophoretic analysis detection and laser scanning observation;
(6) and (4) measuring the potential.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis of 7 sets of conventional sequence RNA self-assembly products is shown in FIG. 5. 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 FIG. 5 and FIG. 6, it can be clearly seen that the bands of the 7 sets of conventional sequence RNA self-assembly products are all bright and clear single bands, indicating that the 7 sets of conventional sequences can self-assemble into the nano-structure. Wherein, after a section of Survivin siRNA nucleic acid interference treatment fragment is modified in the conventional sequence RNA self-assembly product R-14, the self-assembly structure still has a stable self-assembly structure, which also indicates that the nucleic acid nano-particle can carry a nucleic acid drug and has the function of a delivery carrier of the nucleic acid drug.
(2) Determination of potential
The determination method comprises the following steps: preparing a potential sample, putting the potential sample into a sample cell, opening a sample cell cover of an instrument, and putting the instrument into the instrument;
opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the results of the potential measurements for 7 sets of conventional sequence RNA nanoparticles are shown in tables 23 to 29 below:
table 23:
table 24:
table 25:
table 26:
table 27:
table 28:
table 29:
from the potential detection data described above, it is found that: the 7 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have a stable self-assembly structure.
This example shows that: on the basis of RNA core sequences of different combinations, the RNA nano-particles with stable structures can be successfully self-assembled by adding the extension segments. Meanwhile, the added extension segment enables the RNA nanoparticles to have excellent drug-carrying performance (see example 5 in particular).
Example 4
One, 9 groups of conventional sequence DNA nanoparticle carriers:
(1) the three polynucleotide base sequences of the first 7 groups of DNA nanoparticles are shown in tables 30 to 36:
part a of the table has extended the EGFRApt or PSMAApt (A9L) head:
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-chain of the 7 groups of conventional DNA nanoparticle sequences were synthesized by hong, Suzhou, entrusted with:
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 EGFRatt target head (see underlined section below) to the core sequence (9) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3') described previously;
d-3 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the 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 part below) to the core sequence (11) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3') described above;
d-5 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined section below) to the core sequence (12) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3') described previously;
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 nanoparticles.
In addition, single-stranded sequences for forming the 8 th set of DNA nanoparticles and single-stranded sequences of the 9 th set of DNA nanoparticles were synthesized.
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:): 5'-CGCGCGCGCCCACGAGCGTTCCGGGCGCCGCCGC-3'; 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:): 5'-GCGGCGGCGCCCGGTTCGCCGCCAGCCGCCGCC-3', respectively; the first three bases of the 5 'end and the last three bases of the 3' end are respectively subjected to sulfo-modification;
chain c (SEQ ID NO: 180:): 5'-GGCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCGCG-3', the first three bases of the 5' end and the last three bases of the 3' end are respectively modified by sulfo, and the 5' end is connected with Cy5 fluorescent label.
II, self-assembly experiment steps:
(1) mixing and dissolving the DNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 95 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(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 and detection;
(6) measuring the potential;
(7) measuring the particle size;
(8) and (5) observing by using a transmission electron microscope.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis of the first 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 7. Lanes 1 to 7 in FIG. 7 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.
The 4% agarose gel electrophoresis of the first 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 8. Lanes 1 to 7 in FIG. 8 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.
The 2% agarose gel electrophoresis 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 strands of group 8, a, and DNA self-assembly products D-8 and D-9.
As can be seen from the results of FIG. 7, FIG. 8 and FIG. 9, it can be clearly seen that the bands of the self-assembly products of the 9 groups of conventional sequence DNAs are bright and clear, indicating that the self-assembly of the 9 groups of conventional sequence DNA strands is completed, and a stable nanoparticle structure is formed. Wherein, the two groups of self-assembly structures D-6 and D-7 carry EGFRatt or PSMAaptt target heads, the molecular weight is slightly lower, the position of the strip is obviously more ahead than that of other strips, the actual condition and the theoretical condition completely conform to each other, and the stability of the self-assembly structures is further proved.
This example shows that: when various functional extension fragments are added on the basis of different DNA core sequence combinations or are simultaneously connected with a target head, the DNA nano-particles can be successfully assembled, and the DNA nano-particles also have the performances of drug loading, cell targeting, visual tracking and the like.
(2) Determination of potential
The determination method comprises the following steps: preparing a potential sample, putting the potential sample into a sample cell, opening a sample cell cover of an instrument, and putting the instrument into the instrument;
opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking to finish setting, appearing a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 3 groups of conventional sequence DNA nanoparticles are shown in tables 37 to 39 below:
table 37:
table 38:
table 39:
from the potential detection data described above, it is found 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 a stable self-assembly structure.
(3) Particle size measurement
1. Preparing a potential sample (a conventional sequence DNA self-assembly product D-7) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
3. setting software detection parameters;
4. then click on the ok setting, the measurement dialog box appears, click Start, DLS measurements of hydrodynamic size of self-assembled product D-7 result in table 40 below:
table 40:
(4) observation results of transmission electron microscope
And (3) carrying out transmission electron microscope irradiation on the conventional sequence DNA self-assembly product D-7, and comprising the following steps:
1. taking a drop of sample to suspend on a 400-mesh carbon film-coated copper net, and keeping the temperature at room temperature for 1 minute;
2. sucking 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 was observed by 120kv using a transmission electron microscope and photographed.
The result is shown in FIG. 10, from which it is apparent that the product D-7 of the conventional sequence DNA self-assembly is an integral structure and can be clearly seen to have a T-shaped structure.
Example 5
Idarubicin mounting experiment
Carrying out chemical method mounting:
first, experimental material and experimental method
1. Experimental materials and reagents:
(1) the DNA nucleic acid nanoparticles D-8 formed by self-assembly of the 8 th group of DNA sequences in example 4 are used for carrying out the mounting, and the specific information is shown in example 4.
(2) DEPC water: biyun Tian.
(3) PBS buffer: cellgro.
(4) 4% Paraformaldehyde
(5) Idarubicin.
(6) Chloroform: and (6) north transformation.
(7) Absolute ethanol: and (6) north transformation.
2. The experimental method comprises the following steps:
(1) idarubicin (1.354 μmoL) was precisely weighed, dissolved in DEPC water (1.0mL) and PBS buffer (1.25mL), mixed with 4% paraformaldehyde aqueous solution (0.25mL) with cooling in an ice-water bath, and the mixture was mixed with D-8 nucleic acid nanoparticles (33.84nmoL) and reacted at 4 ℃ for 72 hours in the absence of light.
(2) Taking 10 mu L of reaction solution to dilute by 10 times, taking 50 mu M of idarubicin water solution and 310 ng/mu L of D-8 nucleic acid nanoparticles as controls, and carrying out HPLC analysis according to the equal volume injection. 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). Centrifuging (4000/min), transferring the supernatant, washing the solid product with ethanol (50mL) again, and evaporating the solvent at low temperature under reduced pressure to obtain solid product idarubicin-DNAh particles.
(4) Determination of idarubicin-DNAh mounting rate
1. Preparing an idarubicin-methanol standard solution with a known concentration: 2uM, 4uM, 6uM, 8uM, 10uM, each 100 ul;
2. dissolving the idarubicin-DNAh particles in 100ul PBS;
3. placing the standard solution and the idarubicin-DNAh particles in a PCR plate, heating at 85 ℃ for 5min, and then cooling to room temperature;
4. measuring the absorbance of idarubicin at 485nm by using a microplate reader, drawing a standard curve (see figure 11), and calculating to obtain the molar concentration of idarubicin in the mounted product;
5. measuring the absorbance of the DNA at the position of 260nm by using a spectrophotometer to obtain the mass concentration of DNAh particles in each sample;
6. and calculating the mounting rate according to the measured molar concentration of the idarubicin and the mass concentration of the DNAh particles.
The calculation process is specifically as follows:
CDNAh-1=23.32ug/ml,MDNAh≈39500,100ul;Cidarubicin (Idarubicin)-1=18.37uM,100ul;
CDNAh-2=45.07ug/ml,MDNAh≈39500,100ul;CIdarubicin (Idarubicin)-2=32.52uM,100ul;
The average value is taken to obtain that the loading rate of the idarubicin-DNAh particles is about 30, which indicates that about 30 idarubicin can be loaded on each DNA rice particle carrier.
In addition, on the basis that idarubicin is carried by the DNA nanoparticles, other small molecule drugs can be further carried by a method similar to the method for carrying idarubicin, for example, folic acid is further carried by the present application to obtain DNA nanoparticles carrying two small molecule drugs of idarubicin and folic acid together, and the carrying rates of the two drugs can be obtained by detecting according to the above method (the values are not shown).
Example 5 shows that the DNA nanoparticles with the extension segment, the targeting segment and the fluorescein have the function of drug loading, and the small molecule drug idarubicin 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
Idarubicin targeted drug: DNAh-Biotin-EGFRapt-Cy 5-idarubicin; (the product of DNA nanoparticle loading in example 5).
Targeting fluorescent vector: DNAh-Bio-EGFRapt-Cy 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) single cell suspensions were collected by digestion and counted, adjusting cell density to 2X105mL, planting 1 mL/well into 24-well plate;
3) dissolving a to-be-detected object, and preparing a to-be-detected object stock solution;
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) the cell plate was incubated in an incubator at 37 ℃ for 16 hours;
6) after incubation, collecting cell suspension by trypsinization;
7) centrifuging to collect cell precipitate, and washing twice with PBS;
8) finally, resuspending the cell precipitate with 300 μ L of PBS, and detecting on a flow machine;
9) the data were analyzed and the results are shown in Table 44.
Table 44:
from table 44, it can be seen that the binding rate of the DNA nucleic acid nanoparticles carrying the targeting head and the small molecule drug idarubicin to cells is very high, and it is evident that the DNA nucleic acid nanoparticles can be internalized in combination with the corresponding tumor cell line cells. Furthermore, as can be seen from the above Table 46, DNAh-Bio-EFGRapt-Cy 5-idarubicin was able to bind internalization not only with the human breast cancer cell line MCF-7 cells with high efficiency, but also with the acute monocytic leukemia cell MV 4-11. Therefore, the idarubicin targeting drug DNAh-Bio-EFGRapt-Cy 5-idarubicin has an application prospect of treating breast cancer and leukemia.
Example 7
Detection of DNAh-Bio-EGFRApt-Cy 5-Idarubicin nanoparticle serum stability
First, experimental material, reagent and equipment
1. Experimental materials:
a sample to be tested: DNAh-Bio-EGFRapt-Cy 5-idarubicin at a concentration of 1.32 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-formulated); 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) Taking 4 mu L of DNAh drug-loaded nanoparticles, diluting with 21.7 mu L of RPMI 1640 culture medium containing 10% serum until the concentration reaches 197.5 mu g/ml, respectively diluting with 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h).
(2) And (3) uniformly mixing 20 mu l of the processed sample with 6 XDNADLoading Buffer, and performing operation on ice to mark the mixture.
(3) And (3) taking 8% Native PAGE, coating a piece of gel on the nanoparticle samples with different incubation times, loading 20 mu L of gel per hole per sample, and setting a program to perform electrophoresis at 90-100V for 50 min.
(4) And after the electrophoresis is finished, dyeing, placing the mixture on a horizontal shaking table for 30min, and photographing and imaging.
Third, experimental results
The results of native PAGE gel electrophoresis are shown in FIG. 12. Wherein, 1 represents 0min, 2 represents 10min,3 represents 1h, 4 represents 12h, and 5 represents 36 h. The target band of the DNAh-Bio-EGFRAPT-Cy 5-idarubicin nanoparticle is about 200bp, and as can be seen from FIG. 12, the DNAh-Bio-EGFRAPT-Cy 5-idarubicin nanoparticle is basically stable when incubated at 37 ℃ for 36 h.
Example 8
Cytotoxicity of DNAh-Biotin-EGFRApt-Cy 5-idarubicin nanoparticles in MCF-7 and MV4-11 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 (R) | Brand | Goods number/model |
96-well plate | Corning | 3599 |
Centrifugal machine | Jingli | LD5-2B |
CO2Culture box | Thermo | 3111 |
Microplate oscillator | QILINBEIER | QB-9001 |
Microscope | Olympus | IX53 |
Multifunctional enzyme mark instrument | Bio Tek | Synergy H1 |
4. Reagents (see table 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 medium4/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 μ L/hole 10X concentration drug working solution into corresponding hole of the cell culture plate, making three multiple holes for each concentration, and obtaining the final action concentration of the drug shown in 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 reader490A value;
12) and (4) processing and analyzing data.
And performing graphical processing on the data by adopting GraphPad Prism5.0 software. To calculate IC50And performing S-shaped nonlinear regression analysis on the data to match the adaptive dose-response curve. The survival rate is calculated as follows, IC50It can be automatically calculated in GraphPad prism 5.0.
Cell viability (%) - (OD)Hole to be tested–ODBlank control)/(ODNegative control-ODBlank control)x 100%。
Third, the experimental results (see Table 50, FIGS. 13a to 13d and FIGS. 14a to 14d)
Table 50:
as can be seen from Table 50 and FIGS. 13a, 13b, 13c, and 13d, for the MCF-7 cell line, compared to the pure DNAh targeting fluorescent vector, the small molecule drug idarubicin and the DNAh drug-loaded particle DNAh-Bio-EGFRApt-Cy 5-idarubicin are toxic to the MCF-7 cell. Similarly, it can be seen from table 50 and fig. 14a, 14b, 14c, and 14d that, for MV4-11 cell line, the small molecule drug idarubicin and the DNAh drug-loaded particle DNAh-Bio-EGFRapt-Cy 5-idarubicin are toxic to MV4-11 cells, compared to the pure DNAh targeting fluorescent vector.
Assembly of nucleic acid nanoparticles
Example 9
1, 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 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 off target bands, eluting in RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at 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% non-denaturing PAGE gel | / | Self-matching |
1 XTBE 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:
measured concentration (μ g/mL) | |
R-15 | 165.937 |
R-16 | 131.706 |
R-17 | 144.649 |
R-18 | 164.743 |
R-19 | 126.377 |
R-20 | 172.686 |
R-21 | 169.455 |
Secondly, mixing 10 mu L (500ng) of the processed sample with 2 mu L of 6 multiplied DNA Loading Buffer, operating on ice and marking.
③ taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.
And fourthly, dyeing after glue running is finished, placing the dyed fabric on a horizontal shaking table for 30min, and photographing and imaging.
And (3) detection results:
the results of native PAGE gel for 7 sets of extended stretch-degenerate + core short sequence RNA self-assembly products are shown in FIG. 15. Lanes 1 to 7 in FIG. 15 are, from left to right: 7 groups of extension segment deformation + core short sequence RNA self-assembly products R-15, R-16, R-17, R-18, R-19, R-20 and R-21.
The results in fig. 15 clearly show that the bands of the 7 sets of RNA self-assembly products with the modified long segment and the short core sequence are bright and clear, which indicates that the 7 sets of RNA strands with the modified long segment and the short core sequence complete the self-assembly and form a stable nanoparticle structure.
(2) Determination of potential
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 at 25 ℃ of 7 groups of extension segment deformation and 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 can be seen 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 the extended segment deformation and the core short sequence RNA through self-assembly 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 click on the confirmed setting, a measurement dialog box appears, and Start is clicked, and the results of DLS measurement values of hydrodynamic sizes of 7 groups of extended stretch variants + core short sequence RNAs are as follows:
table 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 extended section deformation + core short sequence RNA nanoparticles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 69:
name of reagent | Goods number | Manufacturer of the product |
AE buffer | / | Takara |
SYBR Green I dyes | / | Self-matching |
Table 70:
name (R) | Type number | Manufacturer of the product |
Real-Time System | CFX Connect | Bio-rad |
Super clean bench | HDL | BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd. |
The method comprises the following steps:
after diluting the sample with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1:200 dilution) to 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. 16, the dissolution curve of R-16 is shown in FIG. 17, the dissolution curve of R-17 is shown in FIG. 18, the dissolution curve of R-18 is shown in FIG. 19, the dissolution curve of R-19 is shown in FIG. 20, the dissolution curve of R-20 is shown in FIG. 21, and the dissolution curve of R-21 is shown in FIG. 22. 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
The first and the 7 groups of the extended 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(s) of |
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:
② mixing 10 microliter (500ng) of the treated sample with 2 microliter of 6 multiplied by DNA Loading Buffer, operating on ice and marking.
Taking 8% non-denaturing PAGE gel, coating a piece of gel on samples with different incubation times, and completely loading 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.
And 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 of 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are shown in FIG. 23. Lanes 1 to 7 in FIG. 23 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.
It can be clearly seen from the results of fig. 23 that the bands of the 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are bright and clear, which indicates that the 7 sets of extended stretch-deformed + core short sequence DNA strands complete self-assembly and form a stable nanoparticle structure.
(2) Determination of 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 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 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 extension segment deformation and core short sequence DNA nanoparticles have good stability, and further show that the nanoparticles formed by the extension segment deformation and the core short sequence DNA through self-assembly have a stable self-assembly structure.
(3) Particle size measurement
Preparing a potential sample (7 groups of extension section deformation and core short sequence DNA) to be placed in a sample cell, opening a sample cell cover of an instrument, and placing the instrument;
opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
thirdly, 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:
(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(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:
② after samples were diluted with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1:200 dilution), the final volume was 20. mu.L, the dilution concentration was as follows:
table 93:
② incubating for 30min at room temperature in dark place;
and thirdly, detecting on a computer, setting a program to start at 20 ℃, raising the temperature to between 0.1 and 95 ℃ per second, and reading once every 5 seconds.
And (3) detection results:
the TM values of 7 sets of extended length modified + core short sequence DNA nanoparticles are shown in the following, and the dissolution profile of D-8 is shown in FIG. 24, the dissolution profile of D-9 is shown in FIG. 25, the dissolution profile of D-10 is shown in FIG. 26, the dissolution profile of D-11 is shown in FIG. 27, the dissolution profile of D-12 is shown in FIG. 28, the dissolution profile of D-13 is shown in FIG. 29, and the dissolution profile of D-14 is shown in FIG. 30.
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 the 7 sets of extended length modified + core short sequence DNA nanoparticles shown in FIGS. 24 to 30, the TM values are all higher, indicating that the sample purity is higher and the self-assembly structure is stable.
Detecting stability of nucleic acid nanoparticles in serum
Example 11
And (3) characterizing the stability of the 7 groups of the extended segment deformation + core short sequence RNA nanoparticles in serum by adopting a non-denaturing PAGE method.
The main reagents and instruments were as follows:
table 95:
table 96:
the method comprises the following steps:
firstly, preparing the RNA nanoparticles into the concentrations shown in the following table, then diluting the prepared sample according to the method shown in the following table, diluting for 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
table 97:
mixing 10 mu L of the treated sample with 2 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 12 mu L are loaded, and the procedure of 100V gel running is set for 40 min;
and fourthly, dyeing after the glue running is finished, placing the dyed fabric on a horizontal shaking table to slowly oscillate for 30min, and taking pictures for imaging.
The electrophoresis detection result of R-15 is shown in FIG. 31, the electrophoresis detection result of R-16 is shown in FIG. 32, the electrophoresis detection result of R-17 is shown in FIG. 33, the electrophoresis detection result of R-18 is shown in FIG. 34, the electrophoresis detection result of R-19 is shown in FIG. 35, the electrophoresis detection result of R-20 is shown in FIG. 36, and the electrophoresis detection result of R-21 is shown in FIG. 37. In fig. 31 to 37, 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 show that there is no obvious difference in the RNA nanoparticle sample bands at different times, which indicates that the RNA nanoparticles R-15 to R-21 are relatively stable in 1640 medium of 50% FBS without obvious degradation.
Example 12
The stability of 7 groups of extended length modified + core short sequence DNA nanoparticles in serum was characterized by non-denaturing PAGE.
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 XTBE Buffer (No RNase) | / | Self-matching |
Serum (FBS) | / | Excel |
RPMI 1640 | / | GBICO |
TABLE 99:
the method comprises the following steps:
preparing the DNA nanoparticles into the concentration shown in the following table, diluting the prepared sample by the method shown in the following table for 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
TABLE 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, taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 6 mu L of processed samples, and setting a program of 100V gel running for 40 min;
and fourthly, dyeing after the glue running is finished, placing the dyed fabric on a horizontal shaking table to slowly oscillate for 30min, and taking pictures for imaging.
The electrophoresis detection result of D-8 is shown in FIG. 38, the electrophoresis detection result of D-9 is shown in FIG. 39, the electrophoresis detection result of D-10 is shown in FIG. 40, the electrophoresis detection result of D-11 is shown in FIG. 41, the electrophoresis detection result of D-12 is shown in FIG. 42, the electrophoresis detection result of D-13 is shown in FIG. 43, and the electrophoresis detection result of D-14 is shown in FIG. 44. In fig. 38 to 44, 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 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 synergistic cell Bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), at 37 ℃ and 5% CO2And saturation humidity.
Second, the object to be measured
Blank vector: d-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the aforementioned example 10 were self-assembled to form DNA nanoparticle carriers.
Carrier drug: according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the previous example 10 are used to carry 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:
manufacturer of the product | Model number | |
Biological safety cabinet | Beijing Dong Bihaer Instrument manufacturing Co Ltd | BSC-1360IIA2 |
Low-speed centrifuge | Zhongke Zhongjia Instrument Co., Ltd | SC-3612 |
CO2Culture box | Thermo | 3111 |
Inverted microscope | UOP | DSZ2000X |
Flow cytometer | BD | BD FACSCaliburTM |
Four, main reagent
Table 102:
name of reagent | Manufacturer of the product | Goods number | |
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 collected5-5×105cells/EP tube, wash 2 times with 1 mL/tube of 1% BSA-PBS, and observe the tube bottom cells to prevent aspiration.
3. Dissolving the object to be tested, and diluting the object to be tested to the using 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 medicine) 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 medicine) and D-10 (blank vector), the binding rate is high (89.4% -98.3%).
After incubation of HepG2 cells with D-11-adriamycin (carrier drug) and D-11 (blank carrier), the binding rate is high (89.3% -97.8%).
After incubation of HepG2 cells with D-12-adriamycin (carrier drug) and D-12 (blank carrier), 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 (a Chinese character) | C0038 |
Second, main consumables and instrument
Table 105:
III, cell information
HepG2 (Source synergistic cell Bank) in DMEM + 10% FBS + 1% double antibodygibco, 15140-122) at 37 ℃ in the presence of 5% CO2And saturation humidity.
Fourth, experimental materials
1. Sample to be tested
Blank vector: the DNA nanoparticle carriers formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the foregoing example 10 were 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 doxorubicin, which is respectively marked as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin.
The original drug substance doxorubicin.
DMSO。
Fifth, the experimental procedure
1.HepG2 cells in the logarithmic growth phase were collected, counted using trypan blue staining for Cell viability of 98.3%, 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-carried | 10μM | 3.16μM | 1μM | 316nM | 100nM | 31.6nM | 10nM | 3.16nM | 1nM |
Final concentration of empty vector | 1μM | 316nM | 100nM | 31.6nM | 10nM | 3.16nM | 1nM | 0.316nM | 0.1nM |
Final concentration of 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, each of the drug-loaded and blank vehicles was first prepared as a 100 μ M stock solution in PBS and then diluted in complete medium (biotin-free 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 C2Incubate in incubator for 72 h.
4. The kit was removed and thawed at room temperature, and 10. mu.L of CCK-8 solution was added to each well, or CCK8 solution was mixed with the medium at a ratio of 1:9 and then added to the wells at a rate of 100. mu.L/well.
5. And continuously incubating for 4 hours in the cell incubator, wherein the time depends on the type of the cells, the density of the cells and other experimental conditions.
6. Absorbance was measured at 450nm using a microplate reader.
7. And (3) calculating: cell viability (%) (OD test group-OD blank). times.100%/(OD control group-OD blank), IC was calculated from GraphPad Prism5.050。
Sixth, experimental results
Table 107:
and (4) conclusion:
as can be seen from the above table and FIGS. 45a, 45b, 45c, 45D, 45e, 45f, 45g, and 45h, 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 cells500.2725. mu.M, 0.05087. mu.M, 0.0386, 0.03955, 0.04271, 0.02294, 0.03017 and 0.03458, respectively; IC of DMSO on HepG2 cells50Is composed of>0.1 percent; IC of HepG2 cells acted on by D-8 (blank vector), D-9 (blank vector), D-10 (blank vector), D-11 (blank vector), D-12 (blank vector), D-13 (blank vector) and D-14 (blank vector)50Are all made of>1 μ M. 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 the mounting 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-10 and D-14 in the previous example 10 were used as the daunorubicin mounting carrier. The absorbance of daunorubicin at 492nm was measured using a microplate reader, and a standard curve was plotted (as shown in FIG. 46).
The daunorubicin carrying rates are respectively measured 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-described 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.
By loading the small-molecule drug idarubicin on the nucleic acid nanoparticle carrier provided by the application to form the drug containing idarubicin, the delivery stability of the idarubicin can be improved, and under the condition that the nucleic acid nanoparticle carries a target head, the idarubicin is delivered to target cells in a targeted manner on one hand, so that the bioavailability of the drug is improved, and on the other hand, toxic and side effects on non-target cells or tissues are reduced, the local drug concentration is reduced, and the toxic and side effects caused by high drug concentration are further reduced.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Baiyaozhima (Beijing) Nanoconstructural Co., Ltd
<120> idarubicin-containing medicine, preparation method thereof, pharmaceutical composition and application thereof
<130> PN114940BYZD
<141> 2019-09-30
<150> 201811204156.6
<151> 2018-10-16
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<400> 65
<210> 66
<211> 12
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12)
<223> c chain
<400> 66
<210> 67
<211> 10
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(10)
<223> a chain
<400> 67
<210> 68
<211> 9
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(9)
<223> b chain
<400> 68
<210> 69
<211> 12
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12)
<223> c chain
<400> 69
<210> 70
<211> 10
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(10)
<223> a chain
<400> 70
<210> 71
<211> 9
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(9)
<223> b chain
<400> 71
<210> 72
<211> 12
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12)
<223> c chain
<400> 72
<210> 73
<211> 10
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(10)
<223> a chain
<400> 73
<210> 74
<211> 9
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(9)
<223> b chain
<400> 74
<210> 75
<211> 12
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12)
<223> c chain
<400> 75
<210> 76
<211> 29
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 76
cgcgcgccca ggagcguugg cgggcggcg 29
<210> 77
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 77
cgccgcccgc cuucgccgcc agccgcc 27
<210> 78
<211> 31
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 78
ggcggcaggc ggccauagcc cugggcgcgc g 31
<210> 79
<211> 29
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 79
cgcgcgccca gcagcguucg cgggcggcg 29
<210> 80
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 80
cgccgcccgc guucgccgcc agccgcc 27
<210> 81
<211> 31
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 81
ggcggcaggc ggccauagcg cugggcgcgc g 31
<210> 82
<211> 29
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 82
cgcgcgccca cgagcguugc ggggcggcg 29
<210> 83
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 83
cgccgccccg cuucgccgcc agccgcc 27
<210> 84
<211> 31
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 84
ggcggcaggc ggccauagcc gugggcgcgc g 31
<210> 85
<211> 29
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 85
cgcgcgccca ggagcguugg cccgcggcg 29
<210> 86
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 86
cgccgcgggc cuucggggcc agccgcc 27
<210> 87
<211> 31
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 87
ggcggcaggc ccccauagcc cugggcgcgc g 31
<210> 88
<211> 29
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 88
cgcgcgccca gcagcguucg ccccgccgc 29
<210> 89
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 89
gcggcggggc guucggcggc aggcggc 27
<210> 90
<211> 31
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 90
gccgccagcc gcccauagcg cugggcgcgc g 31
<210> 91
<211> 29
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 91
cgcgcgccca gcagcguucg gggcgccgc 29
<210> 92
<211> 28
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(28)
<223> b chain
<400> 92
gcggcgcccc guucggccgg caggcggc 28
<210> 93
<211> 32
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(32)
<223> c chain
<400> 93
gccgccagcc ggcccauagc gcugggcgcg cg 32
<210> 94
<211> 40
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(40)
<223> a chain
<400> 94
cgcgcgcgag cguugcaaug acagauaagg aaccugcutt 40
<210> 95
<211> 36
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(36)
<223> b chain
<400> 95
ggcagguucc uuaucuguca aagcuucggc ggcagc 36
<210> 96
<211> 23
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(23)
<223> c chain
<400> 96
gcagccgccc auagccgcgc gcg 23
<210> 97
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(39)
<223> EGFRapt
<400> 97
gccttagtaa cgtgctttga tgtcgattcg acaggaggc 39
<210> 98
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(41)
<223> PSMAapt
<400> 98
gggccgaaaa agacctgact tctatactaa gtctacgtcc c 41
<210> 99
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(68)
<223> a chain
<400> 99
cgcgcgccca ggagcgttgg cgggcggcgg ccttagtaac gtgctttgat gtcgattcga 60
caggaggc 68
<210> 100
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 100
cgccgcccgc cttcgccgcc agccgcc 27
<210> 101
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 101
ggcggcaggc ggccatagcc ctgggcgcgc g 31
<210> 102
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(68)
<223> a chain
<400> 102
cgcgcgccca gcagcgttcg cgggcggcgg ccttagtaac gtgctttgat gtcgattcga 60
caggaggc 68
<210> 103
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 103
cgccgcccgc gttcgccgcc agccgcc 27
<210> 104
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 104
ggcggcaggc ggccatagcg ctgggcgcgc g 31
<210> 105
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(68)
<223> a chain
<400> 105
cgcgcgccca cgagcgttgc ggggcggcgg ccttagtaac gtgctttgat gtcgattcga 60
caggaggc 68
<210> 106
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 106
cgccgccccg cttcgccgcc agccgcc 27
<210> 107
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 107
ggcggcaggc ggccatagcc gtgggcgcgc g 31
<210> 108
<211> 71
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(71)
<223> a chain
<400> 108
cgcgcgccca ggagcgttgg cccgcggcgt gggccgaaaa agacctgact tctatactaa 60
gtctacgtcc c 71
<210> 109
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 109
cgccgcgggc cttcggggcc agccgcc 27
<210> 110
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 110
ggcggcaggc ccccatagcc ctgggcgcgc g 31
<210> 111
<211> 71
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(71)
<223> a chain
<400> 111
cgcgcgccca gcagcgttcg ccccgccgct gggccgaaaa agacctgact tctatactaa 60
gtctacgtcc c 71
<210> 112
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 112
gcggcggggc gttcggcggc aggcggc 27
<210> 113
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 113
gccgccagcc gcccatagcg ctgggcgcgc g 31
<210> 114
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 114
cgcgcgccca gcagcgttcg gggcgccgc 29
<210> 115
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> b chain
<400> 115
gcggcgcccc gttcggccgg caggcggc 28
<210> 116
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(32)
<223> c chain
<400> 116
gccgccagcc ggcccatagc gctgggcgcg cg 32
<210> 117
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(29)
<223> a chain
<400> 117
cgcgcgccca cgagcgttgc gggcgccgc 29
<210> 118
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(27)
<223> b chain
<400> 118
gcggcgcccg cttcggcggc aggcggc 27
<210> 119
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(31)
<223> c chain
<400> 119
gccgccagcc gcccatagcc gtgggcgcgc g 31
<210> 120
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 120
gcggcgagcg gcgaggagcg uuggggccgg aggccgg 37
<210> 121
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> b chain
<400> 121
ccggccuccg gccccuucgg ggccagccgc c 31
<210> 122
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 122
ggcggcaggc ccccauagcc cucgccgcuc gccgc 35
<210> 123
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 123
gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37
<210> 124
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 124
ccggccuccg gcccguucgc cgccagccgc c 31
<210> 125
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 125
ggcggcaggc ggccauagcg cucgccgcuc gccgc 35
<210> 126
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 126
gcggcgagcg gcgaggagcg uuggggccgg aggccgg 37
<210> 127
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 127
ccggccuccg gccccuucgc cgccagccgc c 31
<210> 128
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 128
ggcggcaggc ggccauagcc cucgccgcuc gccgc 35
<210> 129
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 129
gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37
<210> 130
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 130
ccggccuccg gcccguucgg cgccagccgc c 31
<210> 131
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 131
ggcggcaggc gcccauagcg cucgccgcuc gccgc 35
<210> 132
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 132
gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37
<210> 133
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 133
ccggccuccg gcccguucgg ccccagccgc c 31
<210> 134
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 134
ggcggcaggg gcccauagcg cucgccgcuc gccgc 35
<210> 135
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 135
gcggcgagcg gcgacgagcg uugcggccgg aggccgg 37
<210> 136
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 136
ccggccuccg gccgcuucgc cgccagccgc c 31
<210> 137
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 137
ggcggcaggc ggccauagcc gucgccgcuc gccgc 35
<210> 138
<211> 37
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 138
gcggcgagcg gcgacgagcg uugcggccgg aggccgg 37
<210> 139
<211> 31
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 139
ccggccuccg gccgcuucgg cgccagccgc c 31
<210> 140
<211> 35
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 140
ggcggcaggc gcccauagcc gucgccgcuc gccgc 35
<210> 141
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 141
gcggcgagcg gcgaggagcg ttggggccgg aggccgg 37
<210> 142
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 142
ccggcctccg gccccttcgg ggccagccgc c 31
<210> 143
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 143
ggcggcaggc ccccatagcc ctcgccgctc gccgc 35
<210> 144
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 144
gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37
<210> 145
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 145
ccggcctccg gcccgttcgc cgccagccgc c 31
<210> 146
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 146
ggcggcaggc ggccatagcg ctcgccgctc gccgc 35
<210> 147
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 147
gcggcgagcg gcgaggagcg ttggggccgg aggccgg 37
<210> 148
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 148
ccggcctccg gccccttcgc cgccagccgc c 31
<210> 149
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 149
ggcggcaggc ggccatagcc ctcgccgctc gccgc 35
<210> 150
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 150
gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37
<210> 151
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 151
ccggcctccg gcccgttcgg cgccagccgc c 31
<210> 152
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 152
ggcggcaggc gcccatagcg ctcgccgctc gccgc 35
<210> 153
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 153
gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37
<210> 154
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 154
ccggcctccg gcccgttcgg ccccagccgc c 31
<210> 155
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 155
ggcggcaggg gcccatagcg ctcgccgctc gccgc 35
<210> 156
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 156
gcggcgagcg gcgacgagcg ttgcggccgg aggccgg 37
<210> 157
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 157
ccggcctccg gccgcttcgc cgccagccgc c 31
<210> 158
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 158
ggcggcaggc ggccatagcc gtcgccgctc gccgc 35
<210> 159
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(37)
<223> a chain
<400> 159
gcggcgagcg gcgacgagcg ttgcggccgg aggccgg 37
<210> 160
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(31)
<223> b chain
<400> 160
ccggcctccg gccgcttcgg cgccagccgc c 31
<210> 161
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(35)
<223> c chain
<400> 161
ggcggcaggc gcccatagcc gtcgccgctc gccgc 35
<210> 162
<211> 14
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(14)
<223> first extension segment
<400> 162
<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 (46)
1. An idarubicin-containing medicament comprising a nucleic acid nanoparticle and idarubicin, wherein idarubicin is suspended on the nucleic acid nanoparticle;
the nucleic acid nanoparticle comprises a nucleic acid domain comprising a sequence a comprising a variant sequence of a1 sequence, a sequence b comprising a variant sequence of b1 sequence, and a sequence c comprising a variant sequence of c1 sequence;
wherein the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively;
the b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3';
the c1 sequence is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3';
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 are each independently selected from C-G or G-C;
in the a sequence, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G;
in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C;
in the c sequence, the NNNN sequence from the 5 'end to the 3' end is CAUA or CATA;
the sequence a, the sequence b and the sequence c are any one of the following groups:
(1) a sequence: 5'-GGAGCGUUGG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCC-3', respectively;
(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', respectively;
(3) a sequence: 5'-CGAGCGUUGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCG-3';
(4) a sequence: 5'-GGAGCGUUGG-3' the flow of the air in the air conditioner,
b sequence: 5 '-CCUUCGGG-3',
c sequence: 5'-CCCCCAUAGCCC-3', 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' the flow of the air in the air conditioner,
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';
(10) a sequence: 5'-CGAGCGTTGC-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-GCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3', respectively;
(11) a sequence: 5'-GGAGCGTTGG-3', and the adhesive tape is used for adhering the film to a substrate,
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' the flow of the air in the air conditioner,
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', respectively;
(15) a sequence: 5'-CGAGCGTTCC-3', respectively;
b sequence: 5 '-GGTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3' is added.
2. The agent of claim 1, wherein the nucleic acid domain further comprises a first extension that 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 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 chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3';
(6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3';
(7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3';
(8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3';
(9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.
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 3, wherein said second extension is an extension of CG base pairs.
6. The drug of claim 5, wherein the second extension is an extension of 1 to 10 CG base pairs.
7. The agent of claim 4, wherein said nucleic acid domain further comprises at least one second extension selected from the group consisting of:
a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' end of c chain: 5 '-CGCGCG-3';
second group: a 3' end of the chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3';
third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.
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 medicament according to claim 8,
the second extension segment is an extension sequence formed by alternately arranging a continuous sequence with 2-8 CG base pairs and a continuous sequence with 2-8 AT/AU base pairs; 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 medicament according to claim 11,
the sequence a, the sequence b and the sequence C have 2' -F modification on the C or U base.
13. The medicament according to any one of claims 1 to 3, wherein the idarubicin is suspended on the nucleic acid nanoparticles in a physical and/or covalent linkage, and the molar ratio between the idarubicin and the nucleic acid nanoparticles is 2-300: 1.
14. The medicament according to claim 13, wherein the molar ratio between idarubicin and the nucleic acid nanoparticles is 10-50: 1.
15. The medicament according to claim 13, wherein the molar ratio between idarubicin and the nucleic acid nanoparticles is 15-25: 1.
16. The drug of any one of claims 1 to 3, wherein the nucleic acid nanoparticle further comprises a biologically active substance attached to the nucleic acid domain, wherein the biologically active substance is one or more of a target, a fluorescein, an interfering nucleic acid siRNA, a miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, a protein, a polypeptide, a flavonoid, glucose, natural salicylic acid, a monoclonal antibody, a vitamin, a phenolic lecithin, and a small molecule drug other than idarubicin.
17. The drug of claim 16, wherein the bioactive agent is one or more of the target, the fluorescein and the miRNA, wherein the target is located at the 5' end or the 3' end of any one of the a, b, c sequences, or is inserted between the GC bonds of the nucleic acid domain, 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.
18. The drug of claim 17, 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.
19. The drug of claim 16, wherein the small molecule drug other than idarubicin 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.
20. 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.
21. The agent of claim 16, wherein the relative molecular weight of the nucleic acid domains is recorded as N1The total relative molecular weight of idarubicin and the biologically active substance is denoted as N2,N1/ N2≥1:1。
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 preparation method of a medicine containing idarubicin is characterized by comprising the following steps:
providing a nucleic acid nanoparticle in a medicament according to any one of claims 1 to 25;
the idarubicin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode to obtain the idarubicin-containing medicine.
27. The method of claim 26, wherein the step of mounting idarubicin by means of a physical connection comprises:
mixing and stirring the idarubicin, the nucleic acid nanoparticles and the first solvent to obtain a premixed system;
and precipitating the premixed system to obtain the idarubicin-containing medicament.
28. The method according to claim 27, wherein the first solvent is one or more selected from the group consisting of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
29. The method of claim 27, wherein the step of precipitating the premix system to obtain the idarubicin-containing pharmaceutical comprises;
precipitating the premixed system to obtain a precipitate;
and washing the precipitate to remove impurities to obtain the idarubicin-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 29, wherein the precipitate is washed with 6-12 times by volume of absolute ethanol to remove impurities, so as to obtain the idarubicin-containing medicament.
33. The method of claim 26, wherein the step of loading idarubicin by covalent attachment comprises:
preparing an idarubicin solution;
enabling the idarubicin solution to react with the amino outside the G ring of the nucleic acid nanoparticles under the mediation effect of formaldehyde to obtain a reaction system;
purifying the reaction system to obtain the idarubicin-containing medicament.
34. The method of claim 33, wherein the step of preparing,
the step of reacting comprises:
and mixing the idarubicin solution, the 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 corresponding single strands of the nucleic acid domain in the medicament of any one of claims 1 to 25.
38. The method of claim 37, wherein the step of preparing,
after obtaining the nucleic acid domain, the method of making further comprises: the nucleic acid nanoparticle is obtained by mounting a bioactive substance in the drug according to any one of claims 16 to 21 on the nucleic acid domain by means of physical and/or covalent attachment.
39. The method according to claim 38, wherein the biologically active substance is immobilized by covalent bonding via solvent covalent bonding, linker covalent bonding or click bonding.
40. The method of claim 39,
the solvent is a third solvent used in covalent linking, 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,
the bioactive substance is connected with the nucleic acid structural domain in a click chain mode, the site of alkynyl or azide modification of the bioactive substance precursor is selected from 2 ' hydroxyl, carboxyl or amino, and the site of alkynyl or azide modification of the nucleic acid structural domain is selected from G exocyclic amino, 2 ' -hydroxyl, A amino or 2 ' -hydroxyl.
44. A pharmaceutical composition comprising the idarubicin-containing pharmaceutical of any one of claims 1 to 25.
45. Use of an idarubicin-containing medicament according to any one of claims 1 to 25 in the manufacture of a medicament for the treatment of a tumour or myelodysplastic syndrome.
46. The use of claim 45, wherein the tumor is any one or more of acute non-lymphocytic leukemia, advanced breast cancer, and non-Hodgkin's lymphoma.
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