CN110960534B - Medicine containing pentafluorouracil, preparation method thereof, pharmaceutical composition and application thereof - Google Patents
Medicine containing pentafluorouracil, preparation method thereof, pharmaceutical composition and application thereof Download PDFInfo
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- CN110960534B CN110960534B CN201910944613.3A CN201910944613A CN110960534B CN 110960534 B CN110960534 B CN 110960534B CN 201910944613 A CN201910944613 A CN 201910944613A CN 110960534 B CN110960534 B CN 110960534B
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Abstract
The application provides a drug containing pentafluorouracil, a preparation method thereof, a pharmaceutical composition and application. The medicine comprises nucleic acid nanoparticles and pentafluorouracil, and the pentafluorouracil is carried on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence, a sequence and c sequence, the a sequence comprises a1 sequence or a sequence of a1 sequence with at least one base insertion, deletion or substitution, the b sequence comprises a sequence of b1 sequence or a sequence of b1 sequence with at least one base insertion, deletion or substitution, and the c sequence comprises a sequence of c1 sequence or a sequence of c1 sequence with at least one base insertion, deletion or substitution. The medicine containing the pentafluorouracil has the advantages that the nucleic acid structural domain is modified by the target head, the targeting property is good, the pentafluorouracil can be stably delivered, and the reliability is high.
Description
Technical Field
The application relates to the field of medicines, in particular to a medicine containing pentafluorouracil, a preparation method, a pharmaceutical composition and an application thereof.
Background
At present, cancer has become one of the major diseases threatening human health and quality of life, and the search for effective drugs for treating cancer and therapeutic strategies thereof has become a problem to be solved urgently in the whole medical field all over the world.
The pentafluorouracil is a commonly used medicine for treating liver cancer, colon cancer, rectal cancer, stomach cancer, breast cancer, ovarian cancer, chorioepithelioma, malignant hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer and the like, can inhibit DNA synthesis by inhibiting thymidylate synthetase, has a certain inhibiting effect on RNA synthesis, and plays an important role in the treatment of the medical oncology.
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. This can be accomplished with instruments or apparatus, such as gene guns, electroporators, etc. The methods do not need to use a gene vector, but the transfection efficiency is generally low, the operation is complex, and the damage to tissues is large. It is also mediated by viral vectors, such as adenovirus and lentivirus, etc., and although the viral vectors have high in vitro transfection activity, the immunogenicity and the susceptibility to mutation of the viral vectors bring huge safety hazards to in vivo delivery. And non-viral vectors, especially biodegradable polymer materials are used for realizing the targeted transportation of the medicine. The 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 micromolecular drug of the pentafluorouracil is one of the difficulties in solving the limited clinical application of the current pentafluorouracil drugs.
Disclosure of Invention
The application mainly aims to provide a drug containing the pentafluorouracil, a preparation method, a pharmaceutical composition and application thereof, so as to improve the delivery reliability of the pentafluorouracil drug.
In order to achieve the above objects, according to one aspect of the present application, there is provided a pentafluorouracil-containing drug, including a nucleic acid nanoparticle and pentafluorouracil, and the pentafluorouracil is carried 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 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.
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 sequence a, the sequence b, and the sequence c comprises a sequence in which at least one base is inserted, deleted, or substituted.
Further, base insertions, deletions or substitutions occur at:
(1) 1, 2, 4 or 5 bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(2) Between 8 th to 10 th bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(3) Between the 1 st to 3 rd bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(4) Between 6 th to 9 th bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(5) Between the 1 st to 4 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; and/or
(6) Between 9 th and 12 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.
Further, the sequence a, the sequence b and the sequence c self-assemble to form a structure shown in a formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position is independently selected from C-G or G-C; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; 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'; (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', respectively; (4) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5 '-CCUUCGGG-3', c sequence: 5'-CCCCCAUAGCCC-3'; (5) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCG-3', c sequence: 5'-CGCCCAUAGCGC-3'; (6) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCC-3', c sequence: 5'-GGCCCAUAGCGC-3', respectively; (7) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGGCG-3', c sequence: 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3', respectively; (9) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3'; (10) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3', respectively; (11) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3', respectively; (12) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3'; (13) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3', respectively; (14) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3' is added.
Further, the nucleic acid domain also comprises a first extension segment, wherein the first extension segment is a Watson-Crick paired extension segment, and the first extension segment is positioned at the 5 'end and/or the 3' end of any sequence in the sequences a, b and c; preferably, the first elongate section is selected from any one of the following: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c 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 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 strand: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'; (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3'; (9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.
Further, the nucleic acid domain also comprises a second extension segment, the second extension segment is positioned at the 5 'end and/or the 3' end of any sequence in the sequence a, the sequence b and the sequence c, and the second extension segment is a Watson-Crick 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 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 sequences a, b and c have at least one modifiable site, and any modifiable site is modified through any one of the following modified linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups; preferably, the sequence a, sequence b and sequence C have a 2' -F modification at the C or U base.
Further, the pentafluorouracil is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the pentafluorouracil 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 for pentafluorouracil.
Further, the relative molecular weight of the nucleic acid domains is denoted as N 1 The total relative molecular weight of the pentafluorouracil and the bioactive substance is denoted as N 2 ，N 1 /N 2 ≥1:1。
Further, the bioactive substance is one or more of a target, fluorescein and miRNA, wherein the target is located on any sequence of a, b and c sequences, preferably the 5' end or the 3' end of any sequence of a, b and c, or is inserted between GC bonds of the nucleic acid structure domain, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence; preferably, the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
Further, the small molecule drugs except for the pentafluorouracil 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 10-30 nm; further preferably 10 to 15 nm.
According to another aspect of the application, a preparation method of a medicament containing the pentafluorouracil is also provided, and the preparation method comprises the following steps: providing the nucleic acid nanoparticles described above; the drug containing the pentafluorouracil is obtained by carrying the pentafluorouracil on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode.
Further, the step of mounting the pentafluorouracil in a physical connection manner comprises: mixing and stirring pentafluorouracil, nucleic acid nanoparticles and a first solvent to obtain a premixed system; precipitating the premixed system to obtain a medicament containing the pentafluorouracil; preferably, the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid; preferably, the step of precipitating the premixed system to obtain the drug containing the pentafluorouracil comprises the following steps: precipitating the premixed system to obtain precipitates; washing the precipitate to remove impurities to obtain a medicament containing the pentafluorouracil; more preferably, the premixed system is mixed with absolute ethyl alcohol and then precipitated at the temperature lower than 10 ℃ to obtain precipitates; drugs containing pentafluorouracil; more preferably, the precipitate is precipitated at a temperature of 0 to 5 ℃ to obtain a precipitate. More preferably, absolute ethyl alcohol with the volume of 6-12 times is adopted to wash the precipitate to remove impurities, and the medicine containing the pentafluorouracil is obtained.
Further, the step of mounting the pentafluorouracil by means of covalent attachment comprises: preparing a pentafluorouracil solution; reacting the pentafluorouracil solution with the amino outside the G ring of the nucleic acid nano-particle under the mediation effect of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the drug containing the pentafluorouracil; preferably, the step of reacting comprises: mixing a pentafluorouracil solution, a paraformaldehyde solution and 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 one of the above-mentioned pentafluorouracil-containing drugs.
According to the fourth aspect of the application, the application of any one of the medicines containing the pentafluorouracil in preparing medicines for treating liver cancer, colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, chorioepithelial cancer, malignant hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer is also provided.
According to a fifth aspect of the present application, there is also provided a method of preventing and/or treating liver cancer, colon cancer, rectal cancer, stomach cancer, breast cancer, ovarian cancer, chorioepithelial cancer, hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer, the method comprising: providing a medicament or pharmaceutical composition comprising pentafluorouracil according to any of the preceding claims; the medicine or the medicine composition containing the pentafluorouracil is administered to patients with liver cancer, colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, chorioepithelioma, malignant hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer in effective dose.
The application provides a drug containing the pentafluorouracil, which comprises nucleic acid nanoparticles and the pentafluorouracil, wherein the pentafluorouracil is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode. In the nucleic acid nanoparticle, by containing the three sequences or the variant sequences thereof provided by the application, not only the nucleic acid domain can be formed by self-assembly, but also the five-fluorouracil can be connected to any 5 'end and/or 3' end of the three strands as a carrier, or the five-fluorouracil can be stably inserted between the strands of the nucleic acid domain. This application is through hanging micromolecule medicine pentafluorouracil on nucleic acid nanoparticle, utilizes the inside hydrophobicity of nucleic acid nanoparticle, outside hydrophilicity and the heap effect of basic group, has played "coating effect" to pentafluorouracil, and coating effect or covalent coupling make pentafluorouracil can not dissolved in certain time moreover, have improved the stability of delivering. In addition, when the nucleic acid structural domain is modified by a target head, the target can have better targeting property, can stably deliver the pentafluorouracil, and has high reliability; meanwhile, the contact probability of the pentafluorouracil with non-target cells or tissues can be reduced, and the toxic and side effects are reduced.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. In the drawings:
FIG. 1 shows the result of electrophoresis detection of RNA nanoparticles formed by self-assembly in example 1 of the present invention;
FIG. 2 shows the result of electrophoresis detection of DNA nanoparticles formed by self-assembly in example 1 of the present invention;
FIG. 3 shows the result of 2% agarose gel electrophoresis detection of 7 sets of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present invention;
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 invention;
FIG. 5 shows the result of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present invention;
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 invention;
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 invention;
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 invention;
FIG. 9 shows a TEM image of self-assembled conventional sequenced DNA nanoparticles D-7 in example 4 of the present invention;
FIG. 10 shows a standard curve of absorbance of pentafluorouracil during the detection of the loading rate in example 5 of the present application;
FIG. 11 shows the microscopic observations of the binding and internalization of the RNAh-Biotin-quasar670 nanoparticles and of the RNAh-Biotin-quasar670-flu nanoparticles with HepG2 cells in example 6 of the present application;
FIG. 12 shows the results of electrophoresis of RNAh-Biotin-quasar670-flu nanoparticles in example 7 of the present application after incubation in serum for various times under the Coomassie Blue program;
FIG. 13 shows the results of electrophoresis of RNAh-Biotin-quasar670-flu nanoparticles in example 7 of the present application after incubation in serum for various times under the Stain Free Gel program; and
FIG. 14 shows the results of detection of inhibition of HepG2 by small molecule Pentafluorouracil and RNAh-Biotin-quasar670-flu nanoparticles in example 8 of the present application;
FIG. 15 shows the results of detecting the inhibition of HepG2 cell proliferation by the fluorescent targeting vector RNAh-Biotin-FAM in example 8 of the present application;
FIG. 16 shows the result of native PAGE gel electrophoresis detection of 7 sets of extended stretch modified + core short sequence RNA self-assembly products in example 9 of the present invention;
FIG. 17 shows the dissolution curve of the RNA nanoparticle R-15 in example 9 of the present invention;
FIG. 18 shows the dissolution curve of the RNA nanoparticle R-16 in example 9 of the present invention;
FIG. 19 shows the dissolution curve of the RNA nanoparticle R-17 in example 9 of the present invention;
FIG. 20 shows the dissolution curve of the RNA nanoparticle R-18 in example 9 of the present invention;
FIG. 21 shows the dissolution curve of the RNA nanoparticle R-19 in example 9 of the present invention;
FIG. 22 shows the dissolution curve of the RNA nanoparticle R-20 in example 9 of the present invention;
FIG. 23 shows the dissolution curve of the RNA nanoparticle R-21 in example 9 of the present invention;
FIG. 24 shows the results of native PAGE gel electrophoresis detection of 7 sets of extended stretch-deformed + core short-sequence DNA self-assembly products in example 10 of the present invention;
FIG. 25 shows a dissolution curve of DNA nanoparticle D-8 in example 10 of the present invention;
FIG. 26 is a graph showing a dissolution curve of the DNA nanoparticle D-9 in example 10 of the present invention;
FIG. 27 is a graph showing a dissolution curve of DNA nanoparticle D-10 in example 10 of the present invention;
FIG. 28 is a graph showing a dissolution curve of the DNA nanoparticle D-11 in example 10 of the present invention;
FIG. 29 is a graph showing a dissolution curve of the DNA nanoparticle D-12 in example 10 of the present invention;
FIG. 30 shows a dissolution curve of DNA nanoparticle D-13 in example 10 of the present invention;
FIG. 31 is a graph showing the dissolution curve of the DNA nanoparticle D-14 in example 10 of the present invention;
FIG. 32 shows the result of electrophoresis detection of RNA nanoparticle R-15 in example 11 after incubation in serum for various times;
FIG. 33 shows the result of electrophoresis detection of RNA nanoparticle R-16 in example 11 of the present invention after incubation in serum for various time periods;
FIG. 34 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. 35 shows the result of electrophoresis detection of RNA nanoparticle R-18 in example 11 of the present invention after incubation in serum for various time periods;
FIG. 36 shows the result of electrophoresis detection of RNA nanoparticle R-19 in example 11 after incubation in serum for various time periods;
FIG. 37 shows the result of electrophoresis detection of RNA nanoparticle R-20 in example 11 after incubation in serum for various times;
FIG. 38 shows the result of electrophoresis detection of RNA nanoparticle R-21 in example 11 of the present invention after incubation in serum for various time periods;
FIG. 39 shows the result of electrophoresis detection of DNA nanoparticle D-8 in example 12 after incubation in serum for various times;
FIG. 40 shows the results of electrophoresis detection of DNA nanoparticle D-9 in example 12 after incubation in serum for various periods of time;
FIG. 41 shows the result of electrophoresis detection of DNA nanoparticle D-10 in example 12 of the present invention after incubation in serum for various times;
FIG. 42 shows the results of electrophoresis detection of the DNA nanoparticle D-11 of example 12 of the present invention after incubation in serum for various periods of time;
FIG. 43 shows the result of electrophoresis detection of DNA nanoparticle D-12 in example 12 of the present invention after incubation in serum for various times;
FIG. 44 shows the result of electrophoresis detection of DNA nanoparticle D-13 in example 12 after incubation in serum for various times;
FIG. 45 shows the result of electrophoresis detection of DNA nanoparticle D-14 in example 12 after incubation in serum for various times;
FIGS. 46a, 46b, 46c, 46D, 46e, 46f, 46g and 46h 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. 47 shows a standard curve of daunorubicin absorbance used in the mounting ratio measurement process of example 16.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail with reference to examples.
Interpretation of terms:
RNAh or empty 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 carrier containing a target, fluorescent substances and chemicals, such as pentafluorouracil-Biotin-Cy 5-RNAh.
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, the stability of RNA nanoparticles in their natural state is relatively poor, and the current improvements based on the application of RNA nanocarriers have mostly been developed around improving their stability and reliability. The current research results, although providing the possibility of drug loading to some extent, focus more on the possibility and effectiveness of the loading of nucleic acid drugs, especially siRNA drugs or miRNA drugs. However, there are few reports on whether non-nucleic acids are equally effective. In addition, the existing self-assembly nanoparticles, especially the self-assembly nanoparticles used as carriers, are self-assembled by using RNA strands at present, and rarely self-assembled by using a combination of RNA strands and DNA strands, but the self-assembly is not realized by using pure DNA strands.
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. Moreover, the self-assembly of DNA nanoparticles also has the advantages of low price and easy operation. Experiments prove that the improved RNA nanoparticles and DNA nanoparticles can be used for carrying various medicaments and stably exist in serum; further experiments verify that the carrier can carry the medicine into cells, and the carrier is nontoxic to the cells. And the carrier carrying the medicine can play a role in relieving and treating the corresponding diseases.
On the basis of the above research results, the applicant proposed the technical solution of the present application. The application provides a drug containing pentafluorouracil, the drug comprises nucleic acid nanoparticles and pentafluorouracil, and the pentafluorouracil is carried on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence a, a sequence b and a sequence c, the sequence a comprises a sequence a1 or a sequence a1 with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 with at least one base insertion, deletion or substitution; wherein, the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3'; b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3'; c1 sequence is SEQ ID NO: 5: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' are provided.
The drug containing the pentafluorouracil provided by the application comprises nucleic acid nanoparticles and the pentafluorouracil, and the pentafluorouracil is carried on the nucleic acid nanoparticles. The nucleic acid nanoparticle can be used as a vector to connect the pentafluorouracil to any of the 5 'end and/or 3' end of the three strands, or can allow the pentafluorouracil to be stably inserted between strands of the nucleic acid domain, as well as to form a nucleic acid domain by self-assembly by including the three sequences or their variant sequences. The application provides a medicine containing pentafluorouracil, through hanging micromolecular medicine pentafluorouracil on nucleic acid nanoparticle, because of nucleic acid nanoparticle's inside has hydrophobicity, the outside has hydrophilicity and base has the heap effect, has played "coating effect" to pentafluorouracil in other words, and coating or covalent coupling make pentafluorouracil can not dissolved in certain time, have improved the stability of delivering. In addition, when the nucleic acid structural domain is modified by a target head, the target can have better targeting property, can stably deliver the pentafluorouracil, and has high reliability; meanwhile, the contact probability of the pentafluorouracil with non-target cells or tissues can be reduced, and the toxic and side effects are reduced.
The self-assembly refers to a technique in which basic structural units spontaneously form an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds. The self-assembly process is not a simple superposition of weak interaction forces (wherein the weak interaction force refers to hydrogen bonds, van der waals force, electrostatic force, hydrophobic force and the like) among a large number of atoms, ions or molecules, but a plurality of individuals are simultaneously and spontaneously connected in parallel and are combined together to form a compact and ordered whole body, and the self-assembly process is a complex synergistic action of the whole body.
The generation of self-assembly requires two conditions: self-contained power and guidance. The kinetics of self-assembly refers to the synergistic effect of weak interaction forces between molecules, which provide energy for molecular self-assembly. The direction of self-assembly refers to the complementarity of the molecules in space, that is, the occurrence of self-assembly requires the rearrangement of the molecules to be satisfied in the size and direction of space.
The DNA nanotechnology is a mode of molecular self-assembly from bottom to top, and spontaneously forms a stable structure from a molecular conformation as a starting point based on the physical and chemical properties of nucleic acid molecules, following the strict base pairing principle of nucleic acids. A plurality of DNA fragments are connected together in a correct sequence in vitro, and a sub-assembly structure is established through a base complementary pairing principle, so that a complex multilevel structure is formed finally. Unlike DNA, the structure of RNA can exceed the limits of the double helix. RNA can form a series of different base pairs with at least two hydrogen bonds between the base pairs. The different bases can be divided into two types, including standard 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, various classes of ribozymes, 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 a sequence with at least one base insertion, deletion, or substitution. The specific position and the base type of the variant sequence in the RNA nano-particle can be improved into a nano-particle for improving the drug loading capacity or stability according to the requirement on the premise of realizing self-assembly.
In order to enable the nucleic acid nanoparticles to have relatively higher stability and further enable the drugs obtained by the pentafluorouracil loading to be more stable, when base insertion, deletion or substitution is carried out on the sequences shown in SEQ ID NO 1/2, SEQ ID NO 3/4 and/or SEQ ID NO 5/6, base insertion, deletion or substitution can be carried out on bases at certain specific positions of the sequences, on one hand, the sequences after variation are the same as the original sequences and can be self-assembled into nanoparticles, and on the other hand, the variations retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of homology with the original sequences, so that the nanoparticles formed by the self-assembly of the sequences have the same drug loading characteristics and similar stability, and the pentafluorouracil can be well loaded and delivered.
In a preferred embodiment, the above base insertion, deletion or substitution occurs at: (1) 1 or 2, between the 1 st, 2 nd, 4 th and 5 th bases from the 5' end of the a sequence shown in SEQ ID NO; and/or (2) between 8 th to 10 th bases from the 5' end of the sequence a shown in SEQ ID NO. 1 or 2; and/or (3) between 1 to 3 bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (4) between 6 th and 9 th bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (5) among bases 1 to 4 from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6; and/or (6) between bases 9 to 12 from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6.
In the preferred embodiment, the base positions with variations are defined as the non-classical Watson-Crick paired base positions or the bulge 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 bulges or loop structures, thereby maintaining the flexibility and tension of the nanostructure formed by the sequences and helping to maintain the stability of the nanostructure 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 pentafluorouracil is carried, 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; among the c sequences, 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 higher tension to the nucleic acid nano-carrier, so that the adaptability of the nucleic acid nano-carrier to microenvironment change is stronger, and the stability of the nucleic acid nano-particle is higher.
In the nanoparticle having the structure of formula (1), the specific sequence composition of the sequence a, the sequence b and the sequence c is not particularly limited as long as the structure can be formed. From the viewpoint of self-assembly of nucleic acid sequences, in order to further improve the efficiency of self-assembly of the three sequences into the nanoparticle having the structure of formula (1), 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 one sequence is independent, self-complementary pairing is not performed to form a secondary structure; (2) one end of any two sequences is complementary and matched to form a double chain, and the other end is not complementary and matched to form a Y-shaped or T-shaped structure. The principle of the base selection is to make the two ends of any chain complementary and paired with the two ends of other two chains respectively to improve the self-assembly efficiency. Of course, in addition to the Y-type or T-type structure, other variants such as tetragons other than trifurcations may be used as long as the principle that one end of any two sequences is complementarily paired to form a double strand and the other end is not complementarily paired is satisfied.
In the nanoparticle with the structure of the formula (1), in the non-Watson-Crick pairing base, the fourth N from the 5 'end in the sequence a and the first N' from the 5 'end in the sequence b can be paired with the fourth N from the 5' end in the sequence a, and can be U-U which is not matched with Watson-Crick pairing, and can also be T, A, C or G which is modified and follows the Watson-Crick pairing principle. The Watson-Crick pairing relatively improves the bonding force between chains and improves the stability, and the non-Watson-Crick pairing endows the nano-particles with greater flexibility and is also beneficial to improving the stability of the nano-particles in the face of change of microenvironment.
In a preferred embodiment, the a sequence, the b sequence and the c sequence are any one of the following groups: (1) a sequence (SEQ ID NO: 7): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 8): 5'-CCUUCGCCG-3', c sequence (SEQ ID NO: 9): 5'-CGGCCAUAGCCC-3'; (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', respectively; (3) a sequence (SEQ ID NO: 13): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 14): 5 '-GCUUCGCCGCCG-3', c sequence (SEQ ID NO: 15): 5'-CGGCCAUAGCCG-3'; (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', respectively; (6) a sequence (SEQ ID NO: 22): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 23): 5'-CGUUCGGCC-3', c sequence (SEQ ID NO: 24): 5'-GGCCCAUAGCGC-3'; (7) a sequence (SEQ ID NO: 25): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 26): 5'-GCUUCGGCG-3', c sequence (SEQ ID NO: 27): 5'-CGCCCAUAGCCG-3'; (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'; (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', respectively; (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' is added.
The nucleic acid nanoparticles formed by self-assembly of the fourteen groups of sequences not only have higher stability, but also have higher self-assembly efficiency.
The nucleic acid nanoparticles can be assembled and formed by self, and have the capability of carrying or carrying a pentafluorouracil medicament. The amount of the carried pentafluorouracil differs depending on the position of G-C or C-G base pair in the above nucleic acid nanoparticles.
In order to make the nucleic acid domain capable of carrying more pentafluorouracil 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 segment, the first extension segment is a Watson-Crick paired extension segment, and the first extension segment 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 elongate section is selected from any one of the following: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3'; (2): a 3' end of chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of the chain: 5' -CCCG-3', 3' end of c strand: 5 '-CGGG-3'; (5): a 5' end of the chain: 5' -CCCC-3', 3' end of c strand: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'. (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3'; (9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'; (10): a 5' end of 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 the chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:168), the 3' end of the c-chain: 5'-TCGCCGCTCGCCGC-3' (SEQ ID NO: 169); (14): a 3' end of chain: 5'-GGCCGGAGGCCGG-3' (SEQ ID NO:170), 5' end of b chain: 5'-CCGGCCTCCGGCC-3' (SEQ ID NO: 171).
The first extension not only increases the length of any one or more of the three sequences forming the nucleic acid nanostructure, but also the first extension composed of GC bases further improves the stability of the formed nanoparticles. Moreover, the first extension segment composed of the sequence also keeps higher self-assembly activity and efficiency of the sequence a, the sequence b and the sequence c.
From the viewpoint of the size of the formed nucleic acid nanoparticles and the stability thereof when transported in vivo as a drug delivery vehicle, it is desirable to be able to transport the drug while trying not to be filtered out by the kidney until reaching the target cells. In a preferred embodiment, the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any of the a sequence, the b sequence and the c sequence, the second extension being a Watson-Crick paired extension; more preferably, the second extension is an extended sequence of CG base pairs; further preferably, the second extension is an extension sequence of 1-10 CG base pairs. The second extension is an extension further added on top of the first extension.
In a preferred embodiment, the above-mentioned nucleic acid domain further comprises at least one set of second stretches: a first group: a 5' end of chain: 5' -CGCGCG-3 ', 3' end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'. This second extension renders the nanoparticle non-immunogenic and non-existent in the case of secondary structures to which each chain folds on itself.
The first extension and/or the second extension may be separated by unpaired base pairs.
In order to make the nucleic acid nanoparticle capable of carrying a bioactive substance with a larger molecular weight (see introduction of bioactive substances below), increasing drug loading rate and maintaining necessary stability, in a preferred embodiment, the second extension is an extension containing both CG base pairs and AT/AU base pairs, preferably the second extension is an extension of 2-50 base pairs. Here, "/" in "AT/AU base" is in the relationship of or, specifically, the second extension is an extended sequence containing both CG base pairs and AT base pairs, or the second extension is an extended sequence containing both CG base pairs and AU base pairs.
More specifically, the sequences a, b and c after adding the above second extension may be the following sequences, respectively:
sequence a is (SEQ ID NO: 49):
b is (SEQ ID NO: 50):
sequence c is (SEQ ID NO: 51):
m in the sequence a, the sequence b and the sequence c is U or T, and when M is T, the synthesis cost of the sequences is greatly reduced.
In practical application, the specific arrangement positions of the CG base pairs and the extended sequences of the AT/AU base pairs can be reasonably adjusted according to actual needs. In a more preferred embodiment, the second extension is an extension sequence formed by alternating a sequence of 2 to 8 CG base pairs and a sequence of 2 to 8 AT/AU base pairs; or the second extension is an extension sequence with 1 CG base pair sequence and 1 AT/AU base pair sequence arranged alternately.
Specifically, the positions of the CGCGCG extension and CGCCGC extension in the sequence a shown by the SEQ ID NO. 49 and the AAAAAA extension are interchanged, the positions of the GCGGCG extension and GGCGGC extension and TTTTTT extension in the sequence b shown by the SEQ ID NO. 50 and the positions of the GCCGCC extension and AAAAAAAA extension in the sequence c shown by the SEQ ID NO. 51 are interchanged, and the CGCCGC extension and TTTTTT extension are interchanged. The nucleic acid nanoparticles formed by self-assembly of the sequences are suitable for carrying bioactive substances with indole molecular structures (indole molecules are preferably combined with A).
Three major challenges that have existed as building materials for widespread use in RNA over the past years include: 1) susceptibility to rnase degradation; 2) susceptibility to dissociation after 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) modifications of the ribose-OH group can chemically stabilize RNA in serum; 2) certain naturally occurring linking motifs are thermodynamically stable and can keep the entire RNA nanoparticle intact at ultra-low concentrations; 3) the immunogenicity of the RNA nanoparticles is sequence and shape dependent and can be adjusted 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 above-mentioned pentafluorouracil may be carried by physical linkage and/or covalent linkage. When the pentafluorouracil is simultaneously connected with the nucleic acid domain by adopting two ways of physical intercalation and covalent connection, the physical intercalation is usually intercalated between GC base pairs, and the preferred number of intercalation sites is 1-100: 1, and inserting. When covalent linkage is adopted for linkage, the pentafluorouracil generally reacts with the amino group outside the G ring to form covalent linkage. More preferably, the molar ratio of the pentafluorouracil to the nucleic acid nanoparticles is 2-300: 1, preferably 2-290: 1, more preferably 2-29: 1, further preferably 10-50: 1, and most preferably 15-25: 1.
In addition to the nucleic acid nanoparticles serving as delivery vehicles for pentafluorouracil in the drugs containing pentafluorouracil provided herein, in a preferred embodiment, the nucleic acid nanoparticles further include a bioactive substance, and the bioactive substance is linked to the nucleic acid domain according to different purposes of the drugs. The bioactive substances are one or more of targets, fluorescein, siRNA (interfering nucleic acid), miRNA, ribozymes, riboswitches, aptamers, RNA antibodies, proteins, polypeptides, flavonoids, glucose, natural salicylic acid, monoclonal antibodies, vitamins, phenols, lecithin and small-molecule drugs except for pentafluorouracil.
In order to improve the efficiency of the nucleic acid nanoparticles in loading and carrying the loaded bioactive substances, the relative molecular weights of the nucleic acid domains and the relative molecular weights of the pentafluorouracil and the bioactive substances preferably have a certain matching relationship. In a preferred embodiment, the relative molecular weight of the nucleic acid domains is denoted as N 1 The total relative molecular weight of the pentafluorouracil and the bioactive substance is denoted as N 2 ，N 1 /N 2 ≥1:1。
The drugs containing the pentafluorouracil in the present application have different performance optimization according to the kinds of the specific carried bioactive substances. For example, when the bioactive substance is biotin or folic acid, it acts to target the drug containing pentafluorouracil, e.g., specifically to cancer cells. When the bioactive substance is fluorescein, the bioactive substance plays a role in enabling the nucleic acid nanoparticles to have a luminescent tracing effect. When the bioactive substances are certain siRNA, miRNA, protein, polypeptide, RNA antibody and micromolecule drugs except for the pentafluorouracil, the drugs containing the pentafluorouracil can become new products with specific treatment effects, such as drugs with more excellent performance, according to different biological functions. In addition, according to the different kinds of the biological active substances carried, DNA nanoparticles and RNA nanoparticles are preferably used, and can be reasonably selected according to actual needs. For example, when the bioactive substance is a drug, it is preferable that the DNA nanoparticle or the RNA nanoparticle is carried, and there is no particular requirement on the length of the single strand assembled to form the nanoparticle.
In a preferred embodiment, the bioactive substances are a target, fluorescein and miRNA, wherein the target is located on any sequence of a, b and c sequences, preferably on the 5' end or the 3' end of any sequence of a, b and c, or is inserted between GC bonds of the nucleic acid domains, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence; preferably, the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
The target head can be 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 of ligation or physical intercalation + covalent ligation.
The fluorescein can be conventional fluorescein, and is preferably one or more of FAM, CY5 and CY 3.
The miRNA can be miRNA with cancer 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 problem of heterogeneity of cancers. Thus, in the preferred nucleic acid nanoparticles, the target head, such as folate or biotin, can specifically target cancer cells, and after internalization in combination with 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 bioactive substances capable of being carried are other small-molecule drugs except for pentafluorouracil, 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 and lupus erythematosus according to the types of diseases which can be treated by different drugs; preferably, the head and neck cancer is brain cancer, neuroblastoma or glioblastoma.
When the bioactive substance capable of being carried is a small molecule drug other than pentafluorouracil, the drug may include, but is not limited to, drugs containing any one or more of the following groups according to differences in molecular structures of the drugs or differences in characteristic groups of the drugs: 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 antibodies or aptamers to SOD (superoxide dismutase), Survivin (Survivin), hTERT (human telomerase reverse transcriptase) and EGFR (epidermal growth factor receptor), PSMA (prostate specific membrane antigen); the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
In a preferred embodiment, the particle size of the nucleic acid nanoparticles is 1 to 100nm, preferably 5 to 50nm, more preferably 10 to 30nm, and even more preferably 10 to 15 nm. Within this range the size is suitable both for entering the cell membrane by cell surface receptor mediated phagocytosis and for being removed by renal filtration avoiding non-specific cell permeation, and therefore the favourable particle size contributes to improved pharmacokinetic, pharmacodynamic, biological and toxicological profiles.
According to a second aspect of the application, a preparation method of the medicine containing the pentafluorouracil is also provided, and the preparation method comprises the following steps: providing any one of the nucleic acid nanoparticles described above; the pentafluorouracil is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, so that the medicine containing the pentafluorouracil is obtained.
When physical attachment is used, the pentafluorouracil will typically intercalate between the GC base pairs by physical intercalation. When covalent attachment is used, the pentafluorouracil generally reacts with the exo-amino group of the G ring to form a covalent attachment. The medicine containing the pentafluorouracil prepared by the method has better targeting property after being modified by a target head, can stably deliver the pentafluorouracil, and has high reliability.
In a preferred embodiment, the step of mounting the pentafluorouracil by means of a physical attachment comprises: mixing and stirring the pentafluorouracil, the nucleic acid nanoparticles and the first solvent to obtain a premixed system; precipitating the premixed system to obtain the medicament containing the pentafluorouracil. The specific dosage of the pentafluorouracil and the nucleic acid nanoparticles can be adjusted according to the change of the loading capacity, which can be understood by those skilled in the art, and is not described herein again.
In order to improve the efficiency and stability of physical connection, the amount of the pentafluorouracil added per liter of the first solvent is preferably 0.1-1 g. Preferably, the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid. Preferably, the step of precipitating the premixed system to obtain the drug containing the pentafluorouracil comprises the following steps: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the medicament containing the pentafluorouracil. 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, absolute ethyl alcohol with the volume of 6-12 times is adopted to wash the precipitate to remove impurities, and the medicine containing the pentafluorouracil is obtained.
In a preferred embodiment, the step of mounting the pentafluorouracil by covalent attachment comprises: preparing a pentafluorouracil solution; reacting the pentafluorouracil solution with the amino outside the G ring of the nucleic acid nano-particle under the mediation effect of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the medicament containing the pentafluorouracil.
By formaldehyde-mediated form, the following reactions can occur:
preferably, the step of reacting comprises: mixing the pentafluorouracil solution, the paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain a reaction system. The paraformaldehyde solution can release formaldehyde small molecules so as to participate in the chemical reaction. In order to improve the reaction efficiency, the concentration of the paraformaldehyde solution is preferably 3.7-4 wt%, the paraformaldehyde solution is preferably a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
In the above preparation method, the nucleic acid nanoparticles may be prepared in a self-assembled form such as: (1) mixing RNA or DNA single strands a, b and c at the same time, and dissolving in DEPC water or TMS buffer solution; (2) heating the mixed solution to 80 ℃/95 ℃ (wherein the RNA assembly temperature is 80 ℃ and the DNA assembly temperature is 95 ℃), keeping for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min; (3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃; (4) cutting off a target band, eluting in 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, namely a nucleic acid structural domain, thereby obtaining the nucleic acid nanoparticles.
In order to provide the above-mentioned drug containing pentafluorouracil with other functions according to the practical application, in a preferred embodiment, after obtaining the nucleic acid domain, the preparation method further comprises: the bioactive substances mentioned above are loaded on the nucleic acid domain by means of physical linkage and/or covalent linkage, so as to obtain the nucleic acid nanoparticle. The biologically active substance may also be attached by physical and/or covalent attachment. 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 azide modification of the biologically active substance precursor and the nucleic acid domain at the same time and then by click-linking.
The above classification does not mean that a certain bioactive substance is linked to a nucleic acid domain in only one manner. Instead, some bioactive substances may be linked to the nucleic acid domain by physical intercalation, by covalent linkage, or by click linkage. However, for a particular 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 or acridine drug is inserted, it is usually inserted between GC base pairs, and the number of preferred insertion sites is 1 to 100: the ratio of 1 was inserted. When the naphthamide medicament is inserted, the naphthamide medicament is usually inserted between AA base pairs, the preferable number of insertion sites is different according to the number of the AA base pairs on a nucleic acid structural domain, and the pyridocarbazoles are inserted according to the ratio of 1-200: the ratio of 1 was inserted.
Specifically, 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 linked to the drug covalently linked is 1-200: 1. the connection mode is suitable for anthracycline and acridine medicines. The proportion of the drugs connected in different connection modes is not limited to the range, and the drugs can be effectively suspended, have no toxic effect on cells and can be effectively released after reaching a target.
When the bioactive substance precursor and the nucleic acid structural domain are simultaneously subjected to alkynyl or azide modification and connected in a click-to-link mode, different click-to-links are selected according to different structural changes of the medicament. And the attachment position may be changed correspondingly according to the structure of the active material, which can be understood by those skilled in the art.
In a preferred embodiment, when the biologically active substance is linked to the nucleic acid domain in a click-link fashion, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of hydroxyl, carboxyl, sulfhydryl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of amino, imino or hydroxyl.
When the nucleic acid domain is bound to a drug, the nucleic acid domain is water-soluble, and many drugs have poor water-solubility, and when the nucleic acid domain is bound, the water-solubility is improved. When the drugs are anthracyclines, the drugs are covalently bound to the nucleic acid domain via an-NH bond on the nucleotide guanosine (the-NH group is hundreds of times more active than other groups that may covalently bind to the drug under appropriate pH conditions), thereby forming a drug-loaded nucleic acid domain. Therefore, depending on the size of the specific drug molecule and the number of GC base pairs in the a-, b-and c-sequences of the specifically designed nucleic acid domain, a binding reaction is performed with a theoretical supersaturation binding amount of 1.1 to 1.3 times, and a maximum of 35 to 45 drugs can be bound to one nucleic acid domain. When the drug has 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 one of the above-mentioned pentafluorouracil-containing medicaments. 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 the fourth aspect of the application, the application of any one of the medicines containing the pentafluorouracil in preparing medicines for treating liver cancer, colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, chorioepithelial cancer, malignant hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer is also provided. 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 liver cancer, colon cancer, rectal cancer, stomach cancer, breast cancer, ovarian cancer, chorioepithelial cancer, hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer, the method comprising: providing a medicament or pharmaceutical composition comprising pentafluorouracil; the medicine or the pharmaceutical composition containing the pentafluorouracil is administered to patients with liver cancer, colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, chorioepithelial cancer, malignant hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer in effective amount. An effective amount herein includes a prophylactically effective amount and/or a therapeutically effective amount, by which is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., reduction in liver, colon, rectal, gastric, breast, ovarian, chorioepithelial, hydatidiform mole, head and neck squamous cell, skin, lung, cervical, pancreatic or bladder cancer. 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 effect of the treatment exceeds its toxic or detrimental effects. A prophylactically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention or inhibition of the occurrence of liver cancer, colon cancer, rectal cancer, stomach cancer, breast cancer, ovarian cancer, chorioepithelial cancer, hydatidiform mole, head and neck squamous carcinoma, skin cancer, lung cancer, cervical cancer, pancreatic cancer or bladder cancer. A prophylactically effective amount can be determined according to the description of a therapeutically effective amount 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 below with reference to specific examples.
Assembly of nucleic acid nanoparticles
Example 1
One, RNA and DNA nanoparticle vector:
(1) the three polynucleotide base sequences that make up the RNA nanoparticles are shown in table 1: :
(2) three polynucleotide base sequences of the DNA nanoparticle.
DNA has the same sequence as that of the RNA, except that U is replaced by T. Wherein the molecular weight of 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) electrophoresis analysis and detection and laser scanning observation.
Third, self-assembly experimental results
(1) Results of electrophoresis
The result of electrophoresis detection of the RNA self-assembly product is shown in FIG. 1. In fig. 1, lanes 1 to 3 are, from left to right: a strand, b strand, RNA self-assembly product. As can be seen, the RNA self-assembly products are slightly dispersed, but clearly seen as a single band. And the molecular weight is the molecular weight after the assembly, and is larger than that of the single chain, so that the position of the band lags behind the a chain and the b chain, the actual situation is consistent with the theory, and the stable composite structure is formed by the self-assembly of the RNA single chains, and the RNA nano-particles are formed.
The results of the electrophoretic detection of the DNA self-assembly products are shown in FIG. 2. In FIG. 2, lanes 1 to 3 are, from left to right: a chain, b chain, DNA self-assembly product. As can be seen from the figure, the bands of the DNA self-assembly products are bright and clear, and are single bands, which proves that the DNA single strands form a stable composite structure through self-assembly, and form DNA nanoparticles.
In this example, it was verified by gel electrophoresis that: the sequences a, b and c including RNA core sequence SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 can be successfully self-assembled into RNA nano-particles. Sequences a, b, and c, including the DNA core sequence SEQ ID NO 2, SEQ ID NO 4, and SEQ ID NO 6, can also successfully self-assemble into DNA nanoparticles.
The sequences a, b and c of the RNA nanoparticles and the DNA nanoparticles include various extension sequences (including drug-loading binding sequences) that facilitate the function of loading the nucleic acid domains, and a targeting head or fluorescein linked to the nucleic acid domains, in addition to the core sequence forming the nucleic acid domains. It can be seen that the presence of substances other than these core sequences does not affect the formation of nucleic acid domains and the successful self-assembly of nucleic acid nanoparticles. The self-assembled nucleic acid nanoparticles can have a targeting type under the guidance of a target head, and the fluorescein can enable the nucleic acid nanoparticles to have visibility and traceability.
Example 2
One, 7 groups of short sequence RNA nano-particle carriers:
(1)7 sets of three polynucleotide base sequences constituting the RNA nanoparticle:
table 2: r-1:
table 3: r-2:
table 4: r-3:
table 5: r-4:
table 6: r-5:
table 7: r-6:
table 8: r-7:
the single strands of the 7 groups of short-sequence RNA nanoparticle carriers are synthesized by the corporation of Venezetian Biotechnology (Shanghai).
II, self-assembly experiment steps:
(1) mixing and dissolving the RNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting a target 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 of the 7 sets 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) 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 the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking to finish setting, appearing a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 7 groups of short sequence RNA nanoparticles are as follows:
table 9:
table 10:
table 11:
table 12:
table 13:
table 14:
table 15:
from the potential detection data described above, it can be seen that: the 7 groups of short sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the short sequence RNAs have a stable self-assembly structure.
This example shows that: the different combinations of the core sequences a, b and c can form the RNA nano-particle with the nucleic acid structural domain through self-assembly, and the structure is stable. Based on example 1, it can be seen that, by adding various functional extension fragments or connecting targeting moieties, fluorescein, etc. to these different core sequence combinations, RNA nanoparticles can be successfully assembled, and have the properties of drug loading, cell targeting, visual tracking, etc.
To further verify these properties, an extension fragment was added to example 2, see example 3. And an extension fragment is added on the basis of the DNA core sequence corresponding to the RNA core sequence of example 2, with or without target ligation, as shown in example 4.
Example 3
One, 7 groups of conventional sequence RNA nanoparticle carriers:
(1)7 sets of three polynucleotide base sequences constituting the RNA nanoparticle:
table 16: r-8:
table 17: r-9:
table 18: r-10:
table 19: r-11:
table 20: r-12:
table 21: r-13:
table 22: r-14: (in the following a chainuGAcAGAuAAGGAAccuGcudTdTAs survivin siRNA)
The 7 groups of conventional sequence RNA nanoparticle vectors are synthesized by commissioned Suzhou Jima company, wherein the sequences a, b and C in R-8 to R-14 are respectively extended RNA oligonucleotide sequences formed by adding extension segments on the basis of the sequences a, b and C in R-1 to R-7, targeting module fragments are not extended, and C/U base 2' F modification is carried out (the enzyme cutting resistance and stability are enhanced). In addition, a Survivin (Survivin) siRNA nucleic acid interference therapeutic fragment is modified in the RNA nanoparticle R-14, specifically, a sense strand of Survivin siRNA is extended at the 3 'end of the a strand (see the underline part of the a strand), and an antisense strand is extended and connected at the 5' end of the b strand (see the underline part of the b strand), so that base pair complementation is formed.
II, self-assembly experiment steps:
(1) mixing RNA single strands a, b and c at the same time according to a molar ratio of 1:1:1, and dissolving in DEPC water or TMS buffer solution;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off a target strip, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at a low temperature under reduced pressure;
(5) electrophoretic analysis detection and laser scanning observation;
(6) and (4) measuring the potential.
Third, self-assembly experimental results
(1) Results of electrophoresis
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 (self-assembly product dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start; and (3) measuring results: the potential detection results of 7 groups of conventional sequence RNA nanoparticles are as follows:
table 23:
table 24:
table 25:
table 26:
table 27:
table 28:
table 29:
from the potential detection data described above, it is found that: the 7 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have a stable self-assembly structure.
This example shows that: on the basis of RNA core sequences of different combinations, the addition of the extension segment can also successfully self-assemble into RNA nanoparticles with stable structure. Meanwhile, the added extension fragment enables the RNA nanoparticles to have excellent drug-carrying performance (see example 5 and example 7 in particular).
Example 4
One, 7 groups of conventional sequence DNA nanoparticle carriers:
(1)7 sets of three polynucleotide base sequences constituting the DNA nanoparticle:
the EGFRatt or PSMAaptt (A9L) target is extended in part a strand:
EGFRapt(SEQ ID NO：97)：GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC；PSMAapt(A9L，SEQ ID NO：98)：
GGGCCGAAAAAGACCTGACTTCTATACTAAGTCTACGTCCC。
table 30: d-1:
table 31: d-2:
table 32: d-3:
table 33: d-4:
table 34: d-5:
table 35: d-6:
table 36: d-7:
the single chains of the 7 groups of conventional sequence DNA nanoparticles were synthesized by Suzhou Hongxin entrustment, in which:
d-1 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined section below) to the core sequence (8) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3') described previously;
d-2 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRapt target head (see underlined section below) to the core sequence (9) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3') described above;
d-3 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRept target head (see underlined section below) to the core sequence (10) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3') described above;
d-4 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined section below) to the core sequence (11) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3') described above;
d-5 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined 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.
II, self-assembly experiment steps:
(1) mixing and dissolving the DNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 95 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off a target band, eluting in a DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a conventional sequence DNA self-assembly product;
(5) electrophoretic analysis detection and laser scanning observation;
(6) potential measurement;
(7) measuring the particle size;
(8) and (5) observing by using a transmission electron microscope.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis pattern of the 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 7. Lanes 1 to 7 in FIG. 7 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.
The electrophoresis picture of 4% agarose gel of 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 8. Lanes 1 to 7 in FIG. 8 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.
As can be seen from the results of FIG. 7 and FIG. 8, it is clear that the bands of the 7 sets of conventional sequence DNA self-assembly products are all bright and clear, indicating that the 7 sets of conventional sequence DNA strands complete self-assembly and form stable nanoparticle structures. 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, the DNA nano-particles can be successfully assembled, and the DNA nano-particles also have the performances of drug loading, cell targeting, visual tracking and the like (see example 6 and example 8).
(2) Measurement of electric potential
The measuring method comprises the following steps: preparing a potential sample (a self-assembly product is dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking 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 as follows:
table 37:
table 38:
table 39:
from the potential detection data described above, it can be seen that: the 3 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have stable self-assembly structures.
(3) Particle size measurement
1. Preparing a potential sample (a conventional sequence DNA self-assembly product D-7) and putting the potential sample into a sample cell, opening a sample cell cover of an instrument, and putting the instrument into the instrument;
2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
3. setting software detection parameters;
4. then click on the ok setting, the measurement dialog appears, click Start starts, and the DLS measurements of the hydrodynamic size of self-assembled product D-7 result as follows:
table 40:
(4) observation result of transmission electron microscope
And (3) carrying out transmission electron microscope irradiation on the conventional sequence DNA self-assembly product D-7, and comprising the following steps:
1. a drop of sample is suspended on a 400-mesh carbon-coated copper net for 1 minute at room temperature;
2. sucking the liquid by filter paper;
3. dyeing for 1 minute by using 2% uranium acetate;
4. sucking dry by filter paper, and drying at room temperature;
5. JEM-1400 transmission electron microscope was used for 120kv observation and photographing.
The result is shown in FIG. 9, from which it is apparent that the above-mentioned conventional sequence DNA self-assembly product D-7 is an integral structure, and it can be clearly seen that it has a T-type structure.
Example 5
Pentafluorouracil (5-fluoroouracil) mounting experiment
Carrying by a chemical method:
first, experimental material and experimental method
1. Experimental materials and reagents:
(1) nucleic acid nanoparticles: RNA nanoparticles from example 1.
(2) DEPC water: biyun Tian.
(3) PBS buffer: cellgro.
(4) 4% Paraformaldehyde
(5) Pentafluorouracil (5-fluorouracil).
(6) Chloroform: and (4) carrying out north transformation.
(7) Anhydrous ethanol: and (4) carrying out north transformation.
2. The experimental method comprises the following steps:
(1) pentafluorouracil (0.823. mu. moL) was precisely weighed, dissolved in DEPC water (1.0mL) and PBS buffer (1.25mL), mixed with 4% paraformaldehyde aqueous solution (0.4mL) while cooling in an ice-water bath, and the mixture was mixed with RNA nanoparticles (1mg, 33.84nmoL) in total and reacted at 4 ℃ for 72 hours in the absence of light.
(2) And taking 10 mu L of reaction liquid to dilute by 10 times, and taking 50 mu M aqueous solution of pentafluorouracil and 310 ng/mu L RNA nanoparticles as controls, and carrying out HPLC analysis according to 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 under reduced pressure at low temperature to obtain the carried product.
(4) And (3) mounting rate calculation:
1. preparing different known-concentration pentafluorouracil-PBS standard solutions, wherein the standard solutions are respectively taken from the point values of 0-25 mu M and are 100 ul;
2. dissolving the pentafluorouracil-RNAh particles in 100ul PBS;
3. placing the standard solution and the pentafluorouracil-RNAh particles in a PCR plate, heating at 85 ℃ for 5min, and then cooling to room temperature;
4. measuring the absorbance of the pentafluorouracil at 265nm by using a microplate reader, drawing a standard curve (shown in figure 10), and calculating to obtain the molar concentration of the pentafluorouracil in the mounted product;
5. measuring the absorbance of RNA at 260nm by using a spectrophotometer to obtain the mass concentration of the pentafluorouracil-RNAh particles contained in each sample;
6. according to the measured molar concentration of the pentafluorouracil and the mass concentration of the RNAh particles, the mounting rate is calculated, the RNAh-pentafluorouracil mounting rate is about 0.31, and the result shows that about 0.31 pentafluorouracil molecules can be mounted on each nucleic acid nanoparticle carrier.
RNAh-pentafluorouracil particles with the mounting rates of 10, 20, 28, 50 and the like can also be obtained by changing the relative dosage of the pentafluorouracil and the RNA nanoparticles, and the description is omitted here.
In addition, on the basis that the RNA nanoparticles are used for carrying the pentafluorouracil, other small molecule drugs can be further carried for the second time according to the same method as that for carrying the pentafluorouracil, for example, folic acid is further carried in the present application, so that the RNA nanoparticles carrying two small molecule drugs of the pentafluorouracil and the folic acid together are obtained, and the carrying rates of the two drugs can be detected by referring to the above method (the values are not shown).
Example 5 shows that the RNA nanoparticles (in example 1) with the extension fragment, the targeting head and the fluorescein have the function of drug loading, and the small molecule drug of the pentafluorouracil can be loaded in a covalent connection mode (paraformaldehyde-solvent covalent), and can also be loaded together with other small molecule drugs.
Example 6
Flow cytometry and confocal microscope experiment detection of cell binding capacity of drug-loaded RNA nanoparticles
First, experimental materials and experimental methods:
1. the samples to be tested are shown in Table 41:
table 41:
note: in the table, RNAh-Biotin-quasar670, which is a control, refers to nanoparticles prepared by performing Biotin modification at the 5 '-ends of the a-and b-strands and performing fluorescein modification at the 3' -end of the c-strand according to the self-assembly method in example 1, and RNAh-Biotin-quasar670-flu refers to nanoparticles formed after further loading of pentafluorouracil (loading according to the chemical method in example 5).
2. The experimental reagents used and their sources were as follows:
MEM medium (YS3160-500 mL); MEM NEAA (Gibco,11140-050 + 100 mL); fetal Bovine Serum (FBS) (ExCell Bio, FNA500-500 mL); Penicillin/Streptomycin (Penicilin/Streptomyces, PS) (Gibco,15140-122-100 mL); PBS buffer (Gibco, C20012500BT-500 mL); Trypsin-EDTA (Stemcell,07901-500 mL); DMSO (Sigma, D5879-1L); prolong Gold antibody mounting anti-quencher (Thermo, P36941-2 mL); DAPI (Yeasen,36308ES11-4 mL).
3. The experimental equipment used was as follows:
inverted Microscope (Inverted Microscope) (Olympus BX53, U-RFL-T); BD Falcon (Corning, 354118); cytospin (TXD 3); flow Cytometer (Flow Cytometer) (Life Science, Atttune NxT).
4. The experimental method comprises the following steps:
confocal microscope experiments:
(1) HepG2 cells were cultured in EMEM + 10% FBS + 1% PS at 37 ℃ and 5% CO 2 Culturing under the condition.
(2) The cells were trypsinized and washed once with PBS at 1X10 per well 5 Individual cells were added to the cell culture slide.
(3) After the cells adhere to the wall, the slides are rinsed with medium.
(3) Cells were incubated with 200nM and 400nM of RNAh-Biotin-quasar670 and RNAh-Biotin-quasar670-flu nanoparticles at 37 ℃ and 5% CO 2 And (4) incubating for 2h and 4 h.
(4) Adherent cells were washed with PBS and stained with anti-quencher (Prolong Gold antibody Mount) overnight at room temperature.
(5) The cells were stained with DAPI at room temperature for 5min and mounted.
(6) And taking a picture under a microscope and storing.
Flow cytometry detection:
(1) with RPMI1640+ 10% FBS + 1% PS medium at 37 ℃ and 5% CO 2 HepG2 cells were cultured.
(2) HepG2 cells were trypsinized and washed once with PBS.
(3) 2x10 5 The individual cells were incubated with RNAh-Biotin-quasar670-flu nanoparticles at 37 ℃ and 5% CO 2 And incubated for 1h at two concentrations of 0.2. mu.M and 0.4. mu.M, respectively, with 3 replicates per sample at each concentration.
(4) After washing the cells with PBS, they were resuspended in PBS buffer and detected with FACS machine.
(5) Receipts were collected and statistically analyzed.
Second, experimental results
The results of the confocal microscope observation are shown in FIG. 11. As can be seen from FIG. 11, the results of the cell binding and internalization experiments indicate that both the RNAh-Biotin-quasar670 and the RNAh-Biotin-quasar670-flu nanoparticles are capable of binding to and internalizing into cells because they carry the targeting head, Biotin (Biotin).
The flow cytometer measurement results are shown in table 42:
table 42:
as can be seen from Table 42, the RNAh-Biotin-quasar670-flu nanoparticles have a strong binding ability to HepG2 cells.
Example 7
Detection of stability of Pentafluorouracil-containing drugs on nucleic acid nanoparticles in serum
First, experimental material and experimental method
1. A sample to be tested: RNAh-Biotin-quasar670-flu nanoparticles prepared in example 5.
2. The experimental reagent:
RPMI-1640 medium (YS3160-500 mL); fetal Bovine Serum (FBS) (ExCell Bio, FNA500-500 mL); Penicillin/Streptomycin (Penicilin/Streptomyces, PS) (Gibco,15140-122-100 mL); PBS buffer (Gibco, C20012500BT-500 mL); novex TM Tris-Glycine Native Sample Buffer(2X)(Invitrogen,LC2673-20 mL)；Novex TM 8% Tris-Glycine Mini Gels (Invitrogen, XP00080BOX-1.0 mm); Tris-Glycine Native Running buffer (10 ×) (Life science, LC2672-500 mL); g250 staining solution (Beyotime, P0017-250 mL).
3. An experimental instrument:
spectrophotometer (Spectrophotometer) (Thermo, ND 2000C); mini Gel Tank (Invitrogen, PS 0301); imaging System (Imaging System) (Bio-Rad, ChemiDoc MP).
4. The experimental method comprises the following steps:
(1) mu.L of 10. mu.M sample was incubated in 90. mu.L of 10% serum in RPMI1640 medium.
(2) Samples were taken after incubation at 37 ℃ for 10min, 1h, 12h, 36h, respectively.
(3) After quantification with NanoDrop, 200ng of RNAh-Biotin-quasar670-flu nanoparticles were added to the same volume of Tris-Glycine SDS Sample Buffer (2X) and mixed well.
(4) Get a block Novex TM 8% Tris-Glycine Mini gel, load in order, set program 200V, 30min, startAnd (4) electrophoresis.
(5) And (5) after the electrophoresis is finished, carrying out G250 staining, placing on a horizontal shaking table for 30min-1h, and photographing for imaging.
Second, experimental results
Table 43: quantification results and sample loading volume.
The results of the electrophoretic measurements are shown in FIGS. 12 and 13. In this figure 12 shows the results of electrophoresis on 8% non-denatured Gel (Coomassie Blue program) and figure 13 shows the results of electrophoresis on 8% non-denatured Gel (Stain Free Gel program). The result of the serum stability test shows that: at 0min, 10min, 1h, 12h and 36h, under different time lengths, the RNAh-Biotin-quasar670-flu nanoparticle sample bands have no obvious difference, which indicates that the RNAh-Biotin-quasar670-flu nanoparticles are relatively stable in a 1640 culture medium of 10% FBS and have no obvious degradation.
Example 8
Study of the cytotoxicity of RNAh-Biotin-quasar670-flu nanoparticles in HepG2 cells
First, experimental material and experimental method
1. The sample to be detected is micromolecular pentafluorouracil and RNAh-Biotin-quasar670-flu nano particles.
2. Experimental reagent:
cell Titer-Glo luminescennt Cell Viability Assay (Promega, G7572-100 mL); EMEM medium (Gibco); PBS buffer (Gibco); fetal Bovine Serum (FBS) (Excel Bio, C20012500BT-500 mL); Penicillin/Streptomycin (Penicillin/Streptomycin liquid, Invitrogen); 96-well white plate (Costar).
3. An experimental instrument:
inverted Microscope (Inverted Microscope) (Olympus IX71, TH 4-200); 96-well Plate Reader (96-well Plate Reader) (Molecular Devices, Flexstation 3); perkin Elmer Envision 2104Multilabel Reader (No. 01-094-.
4. The experimental method comprises the following steps:
(1) cell culture and plating
Cells were supplemented with 10% FBS and 1% PS, respectively, in the corresponding basal medium at 37 ℃ and 5% CO 2 Culturing under the condition. The cell density used in the experiment was above 80%. Cells were harvested, centrifuged at 1000rpm for 4 minutes, the medium resuspended, cell concentration adjusted, and added to 96-well plates in a volume of 50 μ L of 3000 cells, 3 wells per group.
(2) Gradient drug concentration formulation and administration
After 24 hours, the 2X compound solution was transferred to each well, 50 ul/well, according to the following experimental design. The final concentrations obtained were: 5uM,1.667uM,0.556uM,0.185uM,0.062uM,0.021uM,0.0069uM,0.0023 uM; and (3) cell administration, which is divided into micromolecular pentafluorouracil and RNAh-Biotin-quasar670-flu nanoparticles.
(3) Culturing after cell administration
The medicated cells were incubated at 37 deg.C and 5% CO 2 Cultured under the conditions for 72 hours.
(4) Detection kit for treating cells
The plate was brought to room temperature in advance and left to stand for 30 minutes. Add 100. mu.l to each well of the well plate
The reagents were mixed on a shaker for 2 minutes to facilitate cell lysis. Values were read and recorded using a Perkin Elmer Envision 2104Multilabel Reader instrument.
(5) Acquiring and processing experimental data
The obtained experimental data were analyzed using excel software and curve analysis was fitted using GraphPad Prism 5 software, the results of which are shown in fig. 14.
II, experimental results:
table 44: cell inhibition at 5. mu.M (%)
| Cell lines | Time of treatment | Micromolecular pentafluorouracil chemicals | RNAh-Biotin-quasar670-flu |
| HepG2 | 72h | 39.02％ | 52.98％ |
The results of the experiments are shown in Table 44 and FIG. 14, and it can be seen from Table 44 and FIG. 14 that 5. mu.M RNA nanoparticle RNAh-Biotin-quasar670-flu carrying pentafluorouracil is significantly cytotoxic to HepG2 cells and is unexpected: compared with the proliferation inhibition effect of micromolecular pentafluorouracil medicaments on cells, the proliferation inhibition effect of 5 mu M of RNAh-Biotin-quasar670-flu on HepG2 cells is more obvious, and the inhibition rate of the cells after treatment of the micromolecular pentafluorouracil medicaments is improved by at least 25 percent (improved to 52.98 percent) on the basis that the inhibition rate of the cells after treatment of the micromolecular pentafluorouracil medicaments is 39.02 percent.
To further confirm that RNA nanoparticles not carrying pentafluorouracil were not significantly cytotoxic to HepG2 cells, the inventors further designed toxicity experiments (drug administration gradients: 100. mu.M, 31.6. mu.M, 10. mu.M, 3.16. mu.M, 1. mu.M, 316nM, 100nM, 31.6nM, 10nM, 0 (10% PBS) in experiments) of this targeted fluorescent vector to HepG2 cells, and the results are shown in Table 45 and FIG. 15. IC from Table 45 50 As can be seen from the values and FIG. 26, the targeted fluorescent vector without pentafluorouracil itself has no significant toxicity to the experimental cells.
Table 45:
assembly of nucleic acid nanoparticles
Example 9
One, 7 groups of extended segment deformation + core short sequence RNA nano particle carriers:
(1)7 groups of three polynucleotide base sequences which form the RNA nano-particle with the extension segment deformation and the core short sequence:
table 46: r-15:
table 47: r-16:
table 48: r-17:
table 49: r-18:
table 50: r-19:
table 51: r-20:
table 52: 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 a target strip, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at a low temperature under reduced pressure;
(5) electrophoresis analysis detection and laser scanning observation.
Third, self-assembly test results
(1) Electrophoretic detection
The main reagents and instruments were as follows:
table 53:
| name of reagent | Goods number | Manufacturer of the |
| 6×DNA Loading buffer | TSJ010 | Organisms of Onychidae |
| 20bp DNA Ladder | 3420A | TAKARA |
| 10000 SolarGelRed nucleic acid dye | E1020 | solarbio |
| 8% non-denaturing PAGE gel | / | Self-matching |
| 1 × TBE Buffer (No RNAse) | / | Self-matching |
Table 54:
the method comprises the following steps:
the RNA nanoparticles were diluted with ultrapure water according to the method of Table 55 below.
Table 55:
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 for 7 sets of extended stretch-degenerate + core short sequence RNA self-assembly products are shown in FIG. 16. Lanes 1 to 7 in FIG. 16 are, from left to right: 7 groups of self-assembly products of the RNA with the extension segment deformation and the core short sequence, R-15, R-16, R-17, R-18, R-19, R-20 and R-21.
The results in fig. 16 clearly show that the bands of the 7 sets of RNA self-assembly products with the modified long segment and the short core sequence are bright and clear, which indicates that the 7 sets of RNA strands with the modified long segment and the short core sequence complete the self-assembly and form a stable nanoparticle structure.
(2) Measurement of electric potential
The measuring method comprises the following steps: preparing a potential sample (self-assembly product dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening the software, clicking the menu measurei @ 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 at 25 ℃ of 7 groups of extension segment deformation and core short sequence RNA nanoparticles are as follows:
table 56:
table 57:
table 58:
table 59:
table 60:
table 61:
table 62:
from the potential detection data described above, it is found that: the 7 groups of the extended segment deformation and core short sequence RNA nanoparticles have good stability, and further show that the nanoparticles formed by self-assembly of the extended segment deformation and the core short sequence RNA have a stable self-assembly structure.
(3) Particle size measurement
1. Preparing a potential sample (7 groups of extension sections and core short sequence RNA) 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 63:
(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 64:
| name of reagent | Goods number | Manufacturer(s) of |
| AE buffer | / | Takara |
| SYBR Green I dye | / | Self-matching |
Table 65:
| name(s) | Type number | Manufacturer of the product |
| Real-Time System | CFX Connect | Bio-rad |
| Super clean bench | HDL | BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd. |
The method comprises the following steps:
after diluting the sample with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1:200 dilution) to give a final volume of 20. mu.L, at the following dilution concentrations:
table 66:
② incubating for 30min at room temperature in dark place;
and thirdly, detecting on a computer, wherein the program is set to be 20 ℃, the temperature is increased to 0.1-95 ℃ per second, and the reading is carried out once every 5 seconds.
And (3) detection results:
the TM values of 7 sets of extended stretch modified + core short sequence RNA nanoparticles are shown in the following, wherein the dissolution curve of R-15 is shown in FIG. 17, the dissolution curve of R-16 is shown in FIG. 18, the dissolution curve of R-17 is shown in FIG. 19, the dissolution curve of R-18 is shown in FIG. 20, the dissolution curve of R-19 is shown in FIG. 21, the dissolution curve of R-20 is shown in FIG. 22, and the dissolution curve of R-21 is shown in FIG. 23. 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 67:
| TM value (. degree. C.) | |
| R-15 | 57.5℃ |
| R-16 | 53.8℃ |
| R-17 | 55.2℃ |
| R-18 | 54.5℃ |
| R-19 | 54.0℃ |
| R-20 | 59.5℃ |
| R-21 | 65.0℃ |
The TM values of 7 groups of extension segment deformation and core short sequence RNA nanoparticles are higher, which indicates that the self-assembly product has good structural stability.
Example 10
1, 7 groups of extension segment deformation + core short sequence DNA nano particle carriers:
(1)7 groups of three polynucleotide base sequences which form the extension segment deformation + core short sequence DNA nano-particles:
table 68: d-8:
table 69: d-9:
table 70: d-10:
table 71: d-11:
table 72: d-12:
table 73: d-13:
table 74: 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 the TM value.
Third, self-assembly test results
(1) Electrophoretic detection
The main reagents and instruments were as follows:
table 75:
| 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 76:
the method comprises the following steps:
the DNA nanoparticles were diluted with ultrapure water according to the method of the following Table 77.
Table 77:
② mixing 10 microliter (500ng) of the treated sample with 2 microliter of 6 multiplied by DNA Loading Buffer, operating on ice and marking.
③ taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.
And fourthly, dyeing after the glue running is finished, placing the dyed fabric on a horizontal shaking table for 30min, and taking pictures for imaging.
And (3) detection results:
the results of native PAGE gel of 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are shown in FIG. 24. Lanes 1 to 7 in FIG. 24 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. 24 that the bands of the 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are bright and clear, which indicates that the 7 sets of extended stretch-deformed + core short sequence DNA strands complete self-assembly and form a stable nanoparticle structure.
(2) Measurement of electric potential
The 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 to finish setting, appearing 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 + core short sequence DNA nanoparticles are as follows:
table 78:
TABLE 79:
table 80:
table 81:
table 82:
table 83:
table 84:
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;
setting software detection parameters;
and fourthly, clicking the setting after determination, generating a measurement dialog box, clicking Start, and obtaining the results of the DLS measurement values of the hydrodynamic sizes of 7 groups of the extended segment deformation and the core short sequence RNA as follows:
table 85:
| average particle diameter (nm) | |
| D-8 | 7.460 |
| D-9 | 7.920 |
| D-10 | 7.220 |
| D-11 | 7.472 |
| D-12 | 6.968 |
| D-13 | 7.012 |
| D-14 | 6.896 |
(4) TM value detection
And (3) detecting the TM values of the 7 groups of extension segment deformation + core short sequence DNA nanoparticles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 86:
| name of reagent | Goods number | Manufacturer(s) of |
| AE buffer | / | Takara |
| SYBR Green I dyes | / | Self-matching |
Table 87:
| name (R) | Type number | Manufacturer of the product |
| Real-Time System | CFX Connect | Bio-rad |
| Super clean bench | HDL | Beijing Union haar manufacturing Co., Ltd |
The method comprises the following steps:
② after diluting the sample with ultrapure water, mixing 5 μ g diluted sample with 2 μ L SYBR Green I dye (1:200 dilution), the final volume is 20 μ L, the dilution concentration is as follows:
table 88:
incubating for 30min at room temperature in a 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. 25, the dissolution profile of D-9 is shown in FIG. 26, the dissolution profile of D-10 is shown in FIG. 27, the dissolution profile of D-11 is shown in FIG. 28, the dissolution profile of D-12 is shown in FIG. 29, the dissolution profile of D-13 is shown in FIG. 30, and the dissolution profile of D-14 is shown in FIG. 31.
Table 89:
as can be seen from the dissolution curves of the 7 sets of extended length modified + core short sequence DNA nanoparticles shown in FIGS. 25 to 31, the TM values are all high, indicating that the sample purity is high and the self-assembly structure is stable.
Detecting stability of nucleic acid nanoparticles in serum
Example 11
The stability of 7 groups of extended segment deformation + core short sequence RNA nanoparticles in serum is characterized by adopting a non-denaturing PAGE method.
The main reagents and instruments were as follows:
table 90:
| 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 |
| Serum (FBS) | / | Excel |
| RPMI 1640 | / | GBICO |
Table 91:
the method comprises the following steps:
firstly, RNA nano-particles are prepared into the concentration shown in the following table, then the prepared sample is diluted according to the method shown in the following table,
diluting 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
table 92:
secondly, mixing 10 mu L of the treated sample with 2 mu L of 6 multiplied DNA Loading Buffer uniformly, operating on ice and marking;
thirdly, taking 8% non-denaturing PAGE gel, applying a piece of gel on samples with different incubation times, completely applying 12 mu L of processed samples, and setting a program of 100V gel running for 40 min;
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. 32, the electrophoresis detection result of R-16 is shown in FIG. 33, the electrophoresis detection result of R-17 is shown in FIG. 34, the electrophoresis detection result of R-18 is shown in FIG. 35, the electrophoresis detection result of R-19 is shown in FIG. 36, the electrophoresis detection result of R-20 is shown in FIG. 37, and the electrophoresis detection result of R-21 is shown in FIG. 38. In fig. 32 to 38, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and (5) 0 min. From the results of the serum stability test, it can be seen that: the non-denatured gel fruits of 10min, 1h, 12h and 36h showed no significant difference in the RNA nanoparticle sample bands at different times, indicating that the RNA nanoparticles R-15 to R-21 were relatively stable in 1640 medium of 50% FBS without significant degradation.
Example 12
And (3) characterizing the stability of the 7 groups of extended segment deformation + core short sequence DNA nanoparticles in serum by adopting a non-denaturing PAGE method.
The main reagents and instruments were as follows:
table 93:
table 94:
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 95:
mixing 5 mu L of the treated sample with 1 mu L of 6 multiplied DNA Loading Buffer, operating on ice and marking;
thirdly, 8% non-denaturing PAGE gel is taken, samples with different incubation times are coated with a piece of gel, all samples processed by 6 mu L are loaded, and the procedure of 100V gel running is set for 40 min;
fourthly, dyeing is carried out after glue running is finished, the dyeing is placed on a horizontal shaking table to be slowly oscillated for 30min, and photographing and imaging are carried out.
The electrophoresis detection result of D-8 is shown in FIG. 39, the electrophoresis detection result of D-9 is shown in FIG. 40, the electrophoresis detection result of D-10 is shown in FIG. 41, the electrophoresis detection result of D-11 is shown in FIG. 42, the electrophoresis detection result of D-12 is shown in FIG. 43, the electrophoresis detection result of D-13 is shown in FIG. 44, and the electrophoresis detection result of D-14 is shown in FIG. 45. In fig. 39 to 45, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and (5) 0 min. From the results of the serum stability test, it can be seen that: the non-denatured gel fruits of 10min, 1h, 12h and 36h showed no significant difference in the DNA nanoparticle sample bands at different times, indicating that the DNA nanoparticles D-8 to D-14 were relatively stable in 1640 medium of 50% FBS with no significant degradation.
Nucleic acid nanoparticle-loaded drug assay
Example 13
Doxorubicin mounting experiment:
according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), RNA nanoparticles formed by self-assembly of R-15, R-16, R-17, R-18, R-19, R-20 and R-21 in the previous example 9, and DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in example 10 were used as doxorubicin-carrying carriers, and the doxorubicin-carrying rates were respectively measured as follows:
the adriamycin loading rate of the RNA nano-particle R-15 is 20.5;
the adriamycin loading rate of the RNA nano-particle R-16 is 29.4;
the adriamycin loading rate of the RNA nano-particle R-17 is 30.9;
the adriamycin loading rate of the RNA nano-particle R-18 is 34.1;
the adriamycin loading rate of the RNA nano-particle R-19 is 27.1;
the adriamycin loading rate of the RNA nano-particle R-20 is 30.2;
the adriamycin loading rate of the RNA nano-particle R-21 is 20.1;
the adriamycin loading rate of the DNA nano-particle D-8 is 28.0;
the adriamycin loading rate of the DNA nano-particle D-9 is 27.9;
the adriamycin loading rate of the DNA nano-particle D-10 is 18.9;
the adriamycin loading rate of the DNA nano-particle D-11 is 26.8;
the adriamycin loading rate of the DNA nano-particle D-12 is 27.6;
the adriamycin loading rate of the DNA nano-particle D-13 is 31.8;
the adriamycin loading rate of the DNA nanoparticle D-14 was 32.
Flow cytometry (FACS) experiment for detecting cell binding capacity of DNA nanoparticles and carrier drug
Example 14
First, cell information
HepG2 (Source synergy cell bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), culture conditions at 37 ℃ and 5% CO 2 And saturation humidity.
Second, the object to be measured
Blank vector: d-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the aforementioned example 10 were self-assembled to form DNA nanoparticle carriers.
Carrier drug: according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the previous example 10 are used to carry doxorubicin, which is respectively marked as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin.
Third, main equipment and consumable
Table 96:
| manufacturer of the product | Type number | |
| Biological safety cabinet | Beijing Dong Bihaer Instrument manufacturing Co Ltd | BSC-1360ⅡA2 |
| Low-speed centrifugal machine | Zhongke Zhongjia Instrument Co Ltd | SC-3612 |
| CO 2 Culture box | Thermo | 3111 |
| Inverted microscope | UOP | DSZ2000X |
| Flow cytometer | BD | BD FACSCalibur TM |
Four, main reagent
Table 97:
| name of reagentBalance with scale | Manufacturer of the product | Goods number | Remarks for note | |
| DMEM (Biotin free) | Providing all the | YS3160 | ||
| 1％BSA-PBS | Self-matching | － |
And fifthly, an experimental method:
1. adjusting the cell state to logarithmic phase, changing the culture medium to a biotin-free and folic acid-free culture medium, and placing the culture medium in an incubator at 37 ℃ for overnight incubation;
2. after incubation, cell suspension was collected by trypsinization, centrifuged at 1000rmp for 5min, adjusted to concentration, and 2X10 cells were collected 5 -5×10 5 cells/EP tube, wash 2 times with 1 mL/tube of 1% BSA-PBS, and observe the tube bottom cells to prevent aspiration.
3. Dissolving the object to be tested, and diluting the object to be tested to the use concentration;
4. completely sucking cell supernatant, sequentially adding 100 mu L of corresponding samples into each tube, keeping out of the sun, and incubating for 2h at 37 ℃;
5. washed 2 times with 1% BSA-PBS; centrifuging at 1000rmp for 5 min;
6. finally resuspending the cell pellet with 300. mu.L PBS and detecting it on flow machine (blank vector used in this example was labeled by Quasar670, whereas doxorubicin in the vector drug is self-fluorescent and therefore can be detected by FL4-APC and FL2-PE, respectively);
7. and (6) analyzing the data.
Sixth, experimental results
1. The results of the experiment are shown in the following table:
table 98:
2. conclusion
After incubation of HepG2 cells with D-8-adriamycin (vector medicine) and D-8 (blank vector), the binding rate is very high (93.1% -98.4%).
After incubation of HepG2 cells with D-9-adriamycin (vector 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 (vector medicine) and D-11 (blank vector), the binding rate is high (89.3% -97.8%).
After incubation of HepG2 cells with D-12-adriamycin (vector drug) and D-12 (blank vector), the binding rate is very high (94.6% -97.1%).
After incubation of HepG2 cells with D-13-adriamycin (vector medicine) and D-13 (blank vector), the binding rate is high (89.6% -98.2%).
After incubation of HepG2 cells with D-14-adriamycin (vector medicine) and D-14 (blank vector), the binding rate is very high (90.3% -98.3%).
Study of cytotoxicity of DNA nanoparticles and vector drugs in HepG2 cells
Example 15
The toxicity of the DNA nanoparticles and the carrier drug to HepG2 is detected by a CCK8 method.
First, main reagent
Table 99:
| name of reagent | Manufacturer(s) of | Goods number |
| PBS | － | － |
| DMSO | SIGMA | D2650 |
| DMEM (Biotin free) | All-medicinal Zhida Providence | YS3160 |
| FBS | Excell Bio | FSP500 |
| Double antibody | gibco | 15140-122 |
| Pancreatin | gibco | 25200-056 |
| CCK8 kit | Biyuntian (a Chinese character) | C0038 |
Second, main consumables and instrument
TABLE 100:
| name (R) | Manufacturer of the product | Type number |
| 96-well cell culture plate | NEST | 701001 |
| Biological safety cabinet | Beijing Dong gang haar Instrument manufacturing Co Ltd | BSC-1360ⅡA2 |
| Low-speed centrifugal machine | Zhongke Zhongjia Instrument Co Ltd | SC-3612 |
| CO 2 Culture box | Thermo | 3111 |
| Inverted microscope | UOP | DSZ2000X |
| Enzyme-linked immunosorbent assay (ELISA) instrument | Shanghai Ouyao experimental equipment comprisesLimited company | K3 |
Information on cells
HepG2 (Source synergy cell bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), culture conditions at 37 ℃ and 5% CO 2 And saturation humidity.
Fourth, experimental materials
1. Sample to be tested
Blank vector: the DNA nanoparticle carriers formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the foregoing example 10 are respectively denoted as: d-8, D-9, D-10, D-11, D-12, D-13 and D-14.
Carrier drug: according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the previous example 10 are used to carry 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.
A bulk drug of 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 original culture medium, adding 100 μ L culture medium of samples to be tested with different concentrations, and repeating each group for 3 multiple wells.
Table 101:
| number of holes | 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, the drug-loaded and blank vehicles were each prepared as 100 μ M stock solutions in PBS and then diluted in complete medium (no biotin DMEM). The original doxorubicin was first prepared with DMSO as 100 μ M stock solution and then diluted with complete medium (no biotin DMEM). DMSO was directly diluted with complete medium (biotin-free DMEM).
3. Adding a sample to be detected, and putting a 96-well plate into 5% CO at 37 DEG C 2 Incubate in incubator for 72 h.
4. The kit was removed and thawed at room temperature, and 10. mu.L of CCK-8 solution was added to each well, or CCK8 solution was mixed with the medium at a ratio of 1:9 and then added to the wells at a rate of 100. mu.L/well.
5. And continuously incubating for 4 hours in the cell incubator, wherein the time depends on the type of the cells, the density of the cells and other experimental conditions.
6. Absorbance was measured at 450nm using a microplate reader.
7. And (3) calculating: cell viability (%) (OD experimental-OD blank) × 100%/(OD control-OD blank), IC calculated from GraphPad Prism 5.0 50 。
Sixth, experimental results
Table 102:
and (4) conclusion:
as can be seen from the above table and FIGS. 46a, 46b, 46c, 46D, 46e, 46f, 46g and 46h, the IC of the drug doxorubicin and the drug-loaded D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin acting on HepG2 cells 50 0.2725. mu.M, 0.05087. mu.M, 0.0386, 0.03955, 0.04271, 0.02294, 0.03017 and 0.03458, respectively; IC of DMSO on HepG2 cells 50 Is composed of>0.1 percent; IC of HepG2 cells acted on by D-8 (blank vector), D-9 (blank vector), D-10 (blank vector), D-11 (blank vector), D-12 (blank vector), D-13 (blank vector) and D-14 (blank vector) 50 Are all made of>1 μ M. The results show that compared with the simple empty vectors 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, and the drug-carrying D-8-adriamycin, D-9-adriamycin and the drug-carrying D-14-adriamycin are toxic to HepG2 cellsThe adriamycin, the D-10-adriamycin, the D-11-adriamycin, the D-12-adriamycin, the D-13-adriamycin and the D-14-adriamycin have obvious synergistic effect relative to the original adriamycin.
Example 16
According to the chemical method of mounting in example 5 (the same method as in example 5 except for specific limitations), DNA nanoparticles formed by self-assembly of D-10 and D-14 in the previous example 10 were used as daunorubicin mounting vectors. The absorbance of daunorubicin at 492nm was measured using a microplate reader, and a standard curve was plotted (as shown in FIG. 47).
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 that can be combined with multiple modules. The carrier is subjected to unique modular design, so that a core module structure which not only maintains natural compatible affinity, but also has high stable property and various combination characteristics is obtained. The structure can flexibly and efficiently integrate various functional modules, including a targeting module, an imaging and probe module, a treatment module and other composite intelligent modules, so that the structure can be used for targeting delivery in vivo and realizing accurate diagnosis and treatment.
The micromolecular drug pentafluorouracil is hung on the nucleic acid nanoparticle carrier provided by the application to form the drug containing pentafluorouracil, so that the delivery stability of the pentafluorouracil can be improved, and the pentafluorouracil can be delivered to target cells in a targeted manner under the condition that the nucleic acid nanoparticles carry target heads, so that the bioavailability of the drug is improved, and 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> Baiyazhida (Beijing) NanoBiotechnology Ltd
<120> medicament containing pentafluorouracil, preparation method, pharmaceutical composition and application thereof
<130> PN114934BYZD
<141> 2019-09-30
<150> 201811161985.0
<151> 2018-09-30
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<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
Claims (45)
1. A drug containing pentafluorouracil, wherein the drug comprises a nucleic acid nanoparticle and pentafluorouracil, and the pentafluorouracil is carried on the nucleic acid nanoparticle;
the nucleic acid nanoparticle comprises a nucleic acid domain comprising a sequence a comprising a variation of the sequence a1, a sequence b comprising a variation of the sequence b1, and a sequence c comprising a variation of the sequence c 1;
wherein the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively;
the b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3';
the c1 sequence is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3';
the sequence a, the sequence b and the sequence c self-assemble to form a structure shown in formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position is independently selected from C-G or G-C;
in the a sequence, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G;
in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C;
in the c sequence, the NNNN sequence along the direction from the 5 'end to the 3' end is CAUA or CATA;
the sequence a, the sequence b and the sequence c are any one of the following groups:
(1) a sequence: 5'-GGAGCGUUGG-3' the flow of the air in the air conditioner,
b sequence: 5'-CCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCC-3';
(2) a sequence: 5'-GCAGCGUUCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCGC-3', respectively;
(3) a sequence: 5'-CGAGCGUUGC-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-GCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCG-3';
(4) a sequence: 5'-GGAGCGUUGG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5 '-CCUUCGGG-3',
c sequence: 5'-CCCCCAUAGCCC-3', respectively;
(5) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCGC-3';
(6) a sequence: 5'-GCAGCGUUCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGUUCGGCC-3',
c sequence: 5'-GGCCCAUAGCGC-3';
(7) a sequence: 5'-CGAGCGUUGC-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-GCUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCCG-3', respectively;
(8) a sequence: 5'-GGAGCGTTGG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCC-3';
(9) a sequence: 5'-GCAGCGTTCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGTTCGCCG-3',
c sequence: 5'-CGGCCATAGCGC-3';
(10) a sequence: 5'-CGAGCGTTGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3', respectively;
(11) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,
b sequence: 5'-CCTTCGGGG-3',
c sequence: 5'-CCCCCATAGCCC-3', respectively;
(12) a sequence: 5'-GCAGCGTTCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGTTCGGCG-3',
c sequence: 5'-CGCCCATAGCGC-3', respectively;
(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' are provided.
2. The agent of claim 1, further comprising a first extension in the nucleic acid domain, wherein the first extension is a Watson-Crick paired extension located 5 'and/or 3' to any of the a, b, and c sequences.
3. The medicament according to claim 2,
the first extension is selected from any one of the following:
(1): a 5' end of 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 chain: 5 '-CGGG-3';
(5): a 5' end of the chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3';
(6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3';
(7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3';
(8): a 5' end of 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'.
4. The agent of any one of claims 1 to 3, wherein the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any one of the a, b, and c sequences, wherein the second extension is a Watson-Crick paired extension.
5. The agent of claim 4, wherein said second extension is an extension of CG base pairs.
6. The drug of claim 5, wherein the second extension is an extension of 1-10 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 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 drug of claim 8, wherein the second extension is an extended sequence of 2 to 50 base pairs.
10. The medicament according to claim 9,
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 segment is an extension sequence formed by alternating a sequence of 1 CG base pair and a sequence of 1 AT/AU base pair.
11. The agent according to any one of claims 1 to 3, wherein the bases, ribose and phosphate in the a sequence, the b sequence and the c sequence have at least one modifiable site, and any of the modifiable sites is modified by any one of the following modifying linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups.
12. The agent of claim 11, wherein the sequence a, the sequence b and the sequence C have 2' -F modifications at the C or U bases.
13. The medicament according to claim 11,
the pentafluorouracil is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the pentafluorouracil to the nucleic acid nanoparticles is 2-300: 1.
14. The drug according to claim 13, wherein the molar ratio between the pentafluorouracil and the nucleic acid nanoparticles is 10-50: 1.
15. The drug according to claim 14, wherein the molar ratio between the pentafluorouracil and the nucleic acid nanoparticles is 15-25: 1.
16. The medicament according to claim 11,
the nucleic acid nanoparticle further comprises a bioactive substance, wherein the bioactive substance is connected with the nucleic acid structural domain, and the bioactive substance is one or more of a target, fluorescein, siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein, polypeptide, flavonoid, glucose, natural salicylic acid, monoclonal antibody, vitamin, phenol, lecithin and a small molecule drug except for pentafluorouracil.
17. The medicament according to claim 16,
the relative molecular weight of the nucleic acid domains is noted as N 1 The total relative molecular weight of the pentafluorouracil to the biologically active substance is recordedN 2 ，N 1 / N 2 ≥1:1。
18. The medicament according to claim 16,
the bioactive substance is one or more of the target head, the fluorescein and the miRNA, wherein the target head is positioned at the 5' end or the 3' end of any one of the a sequence, the b sequence and the c sequence or is inserted between GC bonds of the nucleic acid structure domain, the miRNA is anti-miRNA, the fluorescein is modified at the 5' end or the 3' end of the anti-miRNA, and the miRNA is positioned at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence.
19. The medicament according to claim 18,
the target head is folic acid or biotin, the fluorescein is one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
20. The drug of claim 16, wherein the small molecule drug other than pentafluorouracil is a drug comprising any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
21. The medicament of claim 16, wherein the protein is one or more of SOD, survivin, hTERT, EGFR, and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
22. The drug according to claim 1, wherein the nucleic acid nanoparticles have a particle size of 1 to 100 nm.
23. The drug of claim 22, wherein the nucleic acid nanoparticles have a particle size of 5 to 50 nm.
24. The drug of claim 23, wherein the nucleic acid nanoparticles have a particle size of 10-30 nm.
25. The drug of claim 24, wherein the nucleic acid nanoparticles have a particle size of 10-15 nm.
26. A preparation method of a medicament containing pentafluorouracil 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 pentafluorouracil is carried on the nucleic acid nano-particles in a physical connection and/or covalent connection mode, so that the medicine containing the pentafluorouracil is obtained.
27. The method of claim 26, wherein the step of attaching the pentafluorouracil by physical attachment comprises:
mixing and stirring the pentafluorouracil, the nucleic acid nanoparticles and a first solvent to obtain a premixed system;
and precipitating the premixed system to obtain the medicament containing the pentafluorouracil.
28. The method of claim 27, wherein the first solvent is selected from one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
29. The method of claim 27, wherein,
precipitating the premixed system to obtain the medicament containing the pentafluorouracil, wherein the step of obtaining the medicament containing the pentafluorouracil comprises the following steps of:
precipitating the premixed system to obtain a precipitate;
washing the precipitate to remove impurities to obtain the medicament containing the pentafluorouracil.
30. The method of claim 29, wherein,
and mixing the premixed system with absolute ethyl alcohol, and then carrying out precipitation at the temperature lower than 10 ℃ 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 method of claim 29,
and washing the precipitate by adopting absolute ethyl alcohol with the volume of 6-12 times to remove impurities, thereby obtaining the medicament containing the pentafluorouracil.
33. The method of claim 26, wherein the step of loading the pentafluorouracil by covalent bonding comprises:
preparing a pentafluorouracil solution;
reacting the pentafluorouracil solution with the amino outside the G ring of the nucleic acid nano-particle under the mediation effect of formaldehyde to obtain a reaction system;
purifying the reaction system to obtain the medicament containing the pentafluorouracil.
34. The method of claim 33, wherein the step of reacting comprises:
and mixing the pentafluorouracil solution, a paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain the reaction system.
35. The method according to claim 34, wherein the concentration of the paraformaldehyde solution is 3.7 to 4 wt%.
36. The method according to claim 35, wherein the paraformaldehyde solution is a mixture of paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
37. The production method according to any one of claims 26 to 36, characterized in that the production method further comprises a step of producing the nucleic acid nanoparticle, which comprises: the nucleic acid domain is obtained by self-assembly of single strands corresponding to the nucleic acid domain in the medicament of any one of claims 1 to 25.
38. The method of claim 37,
after obtaining the nucleic acid domain, the method of making further comprises: the nucleic acid nanoparticle is obtained by mounting the bioactive substance in the drug according to any one of claims 16 to 21 on the nucleic acid domain by means of physical linkage and/or covalent linkage.
39. The method of claim 38, wherein the step of preparing,
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 linkage.
40. The method of claim 39, wherein a third solvent used in the covalent linkage of the solvents is used as a linking medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
41. The method of claim 39, wherein the linker is selected from the group consisting of disulfide bond, p-azido, bromopropyne, and PEG.
42. The method of claim 39, wherein the click-through linkage is performed by modifying the nucleic acid domain and the bioactive substance precursor with an alkynyl or azide modification at the same time and then by click-through linkage.
43. The method of claim 42, wherein the biologically active substance is linked to the nucleic acid domain by a click-link, wherein the site of the alkyne or azide modification of the biologically active substance precursor is selected from the group consisting of a 2 ' hydroxyl, a carboxyl, and an amino group, and wherein the site of the alkyne or azide modification of the nucleic acid domain is selected from the group consisting of a G exocyclic amino, a 2 ' -hydroxyl, an A amino, and a 2 ' -hydroxyl.
44. A pharmaceutical composition comprising a pentafluorouracil-containing medicament according to any of claims 1 to 25.
45. Use of a pentafluorouracil-containing medicament according to any one of claims 1 to 25 in the manufacture of a medicament for the treatment of liver, colon, rectal, stomach, breast, ovarian, chorioepithelial carcinoma, hydatidiform mole, head and neck squamous carcinoma, skin carcinoma, lung carcinoma, cervical carcinoma, pancreatic carcinoma or bladder carcinoma.
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