CN110960684A - Medicine containing 10-hydroxycamptothecin, preparation method thereof, pharmaceutical composition and application thereof - Google Patents
Medicine containing 10-hydroxycamptothecin, preparation method thereof, pharmaceutical composition and application thereof Download PDFInfo
- Publication number
- CN110960684A CN110960684A CN201910945833.8A CN201910945833A CN110960684A CN 110960684 A CN110960684 A CN 110960684A CN 201910945833 A CN201910945833 A CN 201910945833A CN 110960684 A CN110960684 A CN 110960684A
- Authority
- CN
- China
- Prior art keywords
- sequence
- nucleic acid
- hydroxycamptothecin
- chain
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003814 drug Substances 0.000 title claims abstract description 176
- HAWSQZCWOQZXHI-FQEVSTJZSA-N 10-Hydroxycamptothecin Chemical compound C1=C(O)C=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 HAWSQZCWOQZXHI-FQEVSTJZSA-N 0.000 title claims abstract description 162
- HAWSQZCWOQZXHI-UHFFFAOYSA-N CPT-OH Natural products C1=C(O)C=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 HAWSQZCWOQZXHI-UHFFFAOYSA-N 0.000 title claims abstract description 159
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 title claims abstract description 159
- 229940079593 drug Drugs 0.000 title claims abstract description 133
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 239000002105 nanoparticle Substances 0.000 claims abstract description 287
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 189
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 184
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 184
- 238000003780 insertion Methods 0.000 claims abstract description 23
- 230000037431 insertion Effects 0.000 claims abstract description 23
- 238000012217 deletion Methods 0.000 claims abstract description 18
- 230000037430 deletion Effects 0.000 claims abstract description 18
- 238000006467 substitution reaction Methods 0.000 claims abstract description 18
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 279
- 239000000126 substance Substances 0.000 claims description 60
- 238000000034 method Methods 0.000 claims description 56
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 50
- 238000011068 loading method Methods 0.000 claims description 47
- 230000000975 bioactive effect Effects 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 239000002679 microRNA Substances 0.000 claims description 30
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 28
- 239000002904 solvent Substances 0.000 claims description 27
- 239000002953 phosphate buffered saline Substances 0.000 claims description 26
- 230000004048 modification Effects 0.000 claims description 23
- 238000012986 modification Methods 0.000 claims description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 19
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 18
- 229940088623 biologically active substance Drugs 0.000 claims description 18
- 229920002866 paraformaldehyde Polymers 0.000 claims description 18
- 108091070501 miRNA Proteins 0.000 claims description 17
- 239000002244 precipitate Substances 0.000 claims description 17
- 230000001376 precipitating effect Effects 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 239000011616 biotin Substances 0.000 claims description 15
- 229960002685 biotin Drugs 0.000 claims description 15
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 14
- 235000020958 biotin Nutrition 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 14
- 229940126586 small molecule drug Drugs 0.000 claims description 14
- 201000010536 head and neck cancer Diseases 0.000 claims description 12
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 12
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 11
- 206010017758 gastric cancer Diseases 0.000 claims description 11
- 208000032839 leukemia Diseases 0.000 claims description 11
- 201000007270 liver cancer Diseases 0.000 claims description 11
- 208000014018 liver neoplasm Diseases 0.000 claims description 11
- 201000011549 stomach cancer Diseases 0.000 claims description 11
- 125000000304 alkynyl group Chemical group 0.000 claims description 10
- 150000001540 azides Chemical class 0.000 claims description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 10
- JVVRCYWZTJLJSG-UHFFFAOYSA-N 4-dimethylaminophenol Chemical compound CN(C)C1=CC=C(O)C=C1 JVVRCYWZTJLJSG-UHFFFAOYSA-N 0.000 claims description 9
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 9
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-dimethylaminopyridine Substances CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 9
- 108020004459 Small interfering RNA Proteins 0.000 claims description 9
- 102000000763 Survivin Human genes 0.000 claims description 9
- 108010002687 Survivin Proteins 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- 235000019152 folic acid Nutrition 0.000 claims description 8
- 239000011724 folic acid Substances 0.000 claims description 8
- 235000013824 polyphenols Nutrition 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000002243 precursor Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 6
- 229960000304 folic acid Drugs 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 6
- 229940088594 vitamin Drugs 0.000 claims description 6
- 229930003231 vitamin Natural products 0.000 claims description 6
- 235000013343 vitamin Nutrition 0.000 claims description 6
- 239000011782 vitamin Substances 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 5
- XXFUZSHTIOFGNV-UHFFFAOYSA-N 1-bromoprop-1-yne Chemical compound CC#CBr XXFUZSHTIOFGNV-UHFFFAOYSA-N 0.000 claims description 4
- 108091023037 Aptamer Proteins 0.000 claims description 4
- 102000053642 Catalytic RNA Human genes 0.000 claims description 4
- 108090000994 Catalytic RNA Proteins 0.000 claims description 4
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims description 4
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims description 4
- 108020004422 Riboswitch Proteins 0.000 claims description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 108091092562 ribozyme Proteins 0.000 claims description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 4
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 3
- 244000269722 Thea sinensis Species 0.000 claims description 3
- 229910052770 Uranium Inorganic materials 0.000 claims description 3
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 3
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 3
- 240000006365 Vitis vinifera Species 0.000 claims description 3
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 3
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 claims description 3
- 125000003172 aldehyde group Chemical group 0.000 claims description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 3
- 229930003935 flavonoid Natural products 0.000 claims description 3
- 150000002215 flavonoids Chemical class 0.000 claims description 3
- 235000017173 flavonoids Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 230000002452 interceptive effect Effects 0.000 claims description 3
- 229940067606 lecithin Drugs 0.000 claims description 3
- 239000000787 lecithin Substances 0.000 claims description 3
- 235000010445 lecithin Nutrition 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 150000002989 phenols Chemical class 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- WHALSQRTWNBBCV-UHFFFAOYSA-N s-aminosulfanylthiohydroxylamine Chemical compound NSSN WHALSQRTWNBBCV-UHFFFAOYSA-N 0.000 claims description 3
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 238000001556 precipitation Methods 0.000 claims 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 claims 1
- 230000008685 targeting Effects 0.000 abstract description 25
- 230000008901 benefit Effects 0.000 abstract description 5
- 108020004414 DNA Proteins 0.000 description 232
- 238000001338 self-assembly Methods 0.000 description 124
- 210000004027 cell Anatomy 0.000 description 102
- 238000001514 detection method Methods 0.000 description 70
- 239000000047 product Substances 0.000 description 69
- 239000000523 sample Substances 0.000 description 61
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 50
- 238000001962 electrophoresis Methods 0.000 description 49
- 239000013598 vector Substances 0.000 description 41
- 229940009456 adriamycin Drugs 0.000 description 37
- 229960004679 doxorubicin Drugs 0.000 description 37
- 238000004090 dissolution Methods 0.000 description 31
- 238000011534 incubation Methods 0.000 description 31
- 239000000499 gel Substances 0.000 description 29
- 210000002966 serum Anatomy 0.000 description 28
- 238000002474 experimental method Methods 0.000 description 27
- 238000005259 measurement Methods 0.000 description 26
- 230000000694 effects Effects 0.000 description 22
- 239000003153 chemical reaction reagent Substances 0.000 description 21
- 230000002829 reductive effect Effects 0.000 description 18
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 17
- 239000012091 fetal bovine serum Substances 0.000 description 17
- 239000000872 buffer Substances 0.000 description 16
- 239000000969 carrier Substances 0.000 description 14
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 239000012634 fragment Substances 0.000 description 11
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 10
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 10
- 239000012980 RPMI-1640 medium Substances 0.000 description 9
- 238000000246 agarose gel electrophoresis Methods 0.000 description 9
- 230000000295 complement effect Effects 0.000 description 9
- 238000001816 cooling Methods 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 229910021642 ultra pure water Inorganic materials 0.000 description 9
- 239000012498 ultrapure water Substances 0.000 description 9
- 235000019441 ethanol Nutrition 0.000 description 8
- 238000010438 heat treatment Methods 0.000 description 8
- 239000012160 loading buffer Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000002539 nanocarrier Substances 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 231100000331 toxic Toxicity 0.000 description 8
- 230000002588 toxic effect Effects 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 230000015556 catabolic process Effects 0.000 description 7
- 238000005520 cutting process Methods 0.000 description 7
- 238000006731 degradation reaction Methods 0.000 description 7
- 239000012149 elution buffer Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000009830 intercalation Methods 0.000 description 7
- 230000002687 intercalation Effects 0.000 description 7
- 239000011259 mixed solution Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000002086 nanomaterial Substances 0.000 description 7
- 108091033319 polynucleotide Proteins 0.000 description 7
- 102000040430 polynucleotide Human genes 0.000 description 7
- 239000002157 polynucleotide Substances 0.000 description 7
- 239000013603 viral vector Substances 0.000 description 7
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 229960000975 daunorubicin Drugs 0.000 description 6
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 108091062762 miR-21 stem-loop Proteins 0.000 description 6
- 108091041631 miR-21-1 stem-loop Proteins 0.000 description 6
- 108091044442 miR-21-2 stem-loop Proteins 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 125000002091 cationic group Chemical group 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 238000004043 dyeing Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 4
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 4
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 4
- 239000000980 acid dye Substances 0.000 description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000012470 diluted sample Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- -1 formaldehyde small molecules Chemical class 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 3
- 108060006698 EGF receptor Proteins 0.000 description 3
- 102000001301 EGF receptor Human genes 0.000 description 3
- 101000655352 Homo sapiens Telomerase reverse transcriptase Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 102000019197 Superoxide Dismutase Human genes 0.000 description 3
- 108010012715 Superoxide dismutase Proteins 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 229940045799 anthracyclines and related substance Drugs 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 2
- 238000007702 DNA assembly Methods 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 108091060592 XDNA Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- SPTYHKZRPFATHJ-HYZXJONISA-N dT6 Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)CO)[C@@H](O)C1 SPTYHKZRPFATHJ-HYZXJONISA-N 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 229940014144 folate Drugs 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005232 molecular self-assembly Methods 0.000 description 2
- RMHJJUOPOWPRBP-UHFFFAOYSA-N naphthalene-1-carboxamide Chemical compound C1=CC=C2C(C(=O)N)=CC=CC2=C1 RMHJJUOPOWPRBP-UHFFFAOYSA-N 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- MALZYBOXBVXCSN-UHFFFAOYSA-N 11h-pyrido[3,2-a]carbazole Chemical class C1=CC2=NC=CC=C2C2=C1C1=CC=CC=C1N2 MALZYBOXBVXCSN-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000004566 building material Substances 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012054 celltiter-glo Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000013878 renal filtration Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 231100000583 toxicological profile Toxicity 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nanotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Optics & Photonics (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application provides a medicine containing 10-hydroxycamptothecin, a preparation method, a pharmaceutical composition and application thereof. The medicine comprises nucleic acid nanoparticles and 10-hydroxycamptothecin, and the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence a, a sequence b and a sequence c, the sequence a comprises a sequence a1 or a sequence a1 with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 with at least one base insertion, deletion or substitution. The drug containing 10-hydroxycamptothecin provided by the application has the advantages that the nucleic acid structure domain of the drug is modified by the target head, the targeting property is good, the 10-hydroxycamptothecin 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 10-hydroxycamptothecin, a preparation method, a pharmaceutical composition and 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 10-hydroxycamptothecin is a commonly used medicine for treating liver cancer, gastric cancer, head and neck cancer or leukemia and the like, and plays an important role in the medical treatment of tumors.
In order to reduce the side effect caused by poor targeting of the active ingredients of the medicine, the medicine delivery carrier is produced, and the function of the carrier is mainly to carry the active ingredients of the medicine and deliver the active ingredients into blood or tissue cells to treat diseases. There are a variety of approaches to achieve targeted delivery of different drugs. And is implemented by an instrument or apparatus, such as a gene gun, an electroporator, etc. The methods do not need to use a gene vector, but the transfection efficiency is generally low, the operation is complex, and the damage to tissues is large. It is also mediated by viral vectors, such as adenovirus and lentivirus, etc., and although the viral vectors have high in vitro transfection activity, the immunogenicity and the susceptibility to mutation of the viral vectors bring huge safety hazards to in vivo delivery. And non-viral vectors, especially biodegradable high molecular 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.
Of the above-mentioned various targeted delivery approaches, more research is currently focused on the field of non-viral vectors, and is generally designed for several vectors: (a) a cationic liposome; (b) a polycationic gene vector. However, more researches are focused on the modification of polycation gene vectors and cationic liposomes, so that the polycation gene vectors and cationic liposomes are suitable for the targeted delivery of gene substances. Cationic liposomes have high transfection activity in vitro and in vivo, however, normal distribution in vivo is affected due to positive charges on the surface, and meanwhile, the cationic lipids cause immunogenicity and inflammatory reactions in animal experiments. The polycation gene vector is developed more mature at present, however, the surface of a structure is difficult to ensure by a targeting group in the structural design, a self-design contradiction between toxicity and transfection activity exists, and meanwhile, the connection of the polycation gene vector is difficult to realize nontoxic degradation in vivo.
Therefore, how to improve the delivery reliability of the existing small-molecule drug 10-hydroxycamptothecin is one of the difficulties in solving the limited clinical application of the existing 10-hydroxycamptothecin drug.
Disclosure of Invention
The main objective of the present application is to provide a drug containing 10-hydroxycamptothecin, a preparation method, a pharmaceutical composition and an application thereof, so as to improve the delivery reliability of the 10-hydroxycamptothecin drug.
In order to achieve the above objects, according to one aspect of the present application, there is provided a 10-hydroxycamptothecin-containing drug, which includes a nucleic acid nanoparticle and 10-hydroxycamptothecin, and the 10-hydroxycamptothecin is suspended on the nucleic acid nanoparticle; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence a, a sequence b and a sequence c, the sequence a comprises a sequence a1 or a sequence a1 with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 with at least one base insertion, deletion or substitution; wherein, the sequence of a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively; b1 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.
Further, when the sequence a1 is SEQ ID NO. 1, the sequence b1 is SEQ ID NO. 3, and the sequence c1 is SEQ ID NO. 5, at least one of the sequences a, b, and c comprises a sequence in which at least one base is inserted, deleted, or substituted.
Further, base insertions, deletions or substitutions occur at:
(1) 1, 2, 4 or 5 bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(2) Between 8 th to 10 th bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(3) Between the 1 st to 3 rd bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(4) Between 6 th to 9 th bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(5) Between the 1 st to 4 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; and/or
(6) Between the 9 th to 12 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.
Further, the sequence a, the sequence b and the sequence c self-assemble into a structure shown in the formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position is independently selected from C-G or G-C; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; among the c sequences, the NNNN sequence in the 5 'to 3' direction is CAUA or CATA.
Further, the sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5'-CCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGCCG-3', c sequence: 5'-CGGCCAUAGCGC-3', respectively; (3) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5 '-GCUUCGCCGCCG-3', c sequence: 5'-CGGCCAUAGCCG-3', respectively; (4) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5 '-CCUUCGGG-3', c sequence: 5'-CCCCCAUAGCCC-3', respectively; (5) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCG-3', c sequence: 5'-CGCCCAUAGCGC-3', respectively; (6) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCC-3', c sequence: 5'-GGCCCAUAGCGC-3', 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', respectively; (10) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3', respectively; (11) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3', respectively; (12) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3', respectively; (13) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3', respectively; (14) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3' are provided.
Further, the nucleic acid domain also comprises a first extension segment, wherein the first extension segment is a Watson-Crick paired extension segment, and the first extension segment is positioned at the 5 'end and/or the 3' end of any sequence in the sequences a, b and c; preferably, the first elongate section is selected from any one of the following: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3'; (2): a 3' end of the chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of 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 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 the chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of the chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.
Further, the second extension is an extension sequence containing both CG base pairs and AT/AU base pairs, and preferably the second extension is an extension sequence of 2-50 base pairs.
Further, the second extension segment is an extension sequence formed by alternately arranging a sequence of continuous 2-8 CG base pairs and a sequence of continuous 2-8 AT/AU base pairs; alternatively, the second extension is an extended sequence of 1 CG base pair alternating with 1 AT/AU base pair sequence.
Further, bases, ribose and phosphate in the sequences a, b and c have at least one modifiable site, and any modifiable site is modified through any one of the following modified linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups; preferably, the sequence a, sequence b and sequence C have a 2' -F modification at the C or U base.
Further, the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the 10-hydroxycamptothecin 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 10-hydroxycamptothecin.
Further, the relative molecular weight of the nucleic acid domains is denoted as N1The total relative molecular weight of 10-hydroxycamptothecin and bioactive substance is recorded as N2，N1/N2≥1:1。
Further, the bioactive substance is one or more of a target, fluorescein and miRNA, wherein the target is located on any 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 other than 10-hydroxycamptothecin are drugs containing any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
Further, the protein is one or more of SOD, survivin, hTERT, EGFR and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
Further, the particle size of the nucleic acid nanoparticles is 1-100 nm, preferably 5-50 nm; more preferably 10 to 30 nm; further preferably 10 to 15 nm.
According to another aspect of the present application, there is also provided a method for preparing a 10-hydroxycamptothecin-containing drug, which comprises the steps of: providing the nucleic acid nanoparticles described above; the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the medicine containing the 10-hydroxycamptothecin is obtained.
Further, the step of carrying the 10-hydroxycamptothecin by means of physical connection comprises: mixing and stirring 10-hydroxycamptothecin, the nucleic acid nanoparticles and the first solvent to obtain a premixed system; precipitating the premixed system to obtain a medicine containing 10-hydroxycamptothecin; 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 10-hydroxycamptothecin comprises: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the medicine containing 10-hydroxycamptothecin; more preferably, the premixed system is mixed with absolute ethyl alcohol and then precipitated at the temperature lower than 10 ℃ to obtain precipitates; a drug containing 10-hydroxycamptothecin; more preferably, the precipitate is precipitated at a temperature of 0 to 5 ℃ to obtain a precipitate. More preferably, 6-12 times of volume of absolute ethyl alcohol is adopted to wash the precipitate to remove impurities, and the medicine containing 10-hydroxycamptothecin is obtained.
Further, the step of carrying the 10-hydroxycamptothecin by means of covalent linkage comprises: preparing 10-hydroxycamptothecin solution; reacting the 10-hydroxycamptothecin solution with the amino outside the G ring of the nucleic acid nano-particles under the mediated action of formaldehyde to obtain a reaction system; purifying the reaction system to obtain a drug containing 10-hydroxycamptothecin; preferably, the step of reacting comprises: mixing the 10-hydroxycamptothecine solution with a paraformaldehyde solution and nucleic acid nanoparticles, and reacting under a dark condition to obtain a reaction system; the concentration of the preferable paraformaldehyde solution is preferably 3.7-4 wt%, the preferable paraformaldehyde solution is a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
Further, the preparation method further comprises a step of preparing a nucleic acid nanoparticle, which comprises: obtaining a nucleic acid 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 azide modification of the biologically active substance precursor and the nucleic acid domain at the same time and then 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 10-hydroxycamptothecin-containing drugs described above.
According to the fourth aspect of the application, the application of any one of the medicines containing 10-hydroxycamptothecin in preparing medicines for treating liver cancer, gastric cancer, head and neck cancer or leukemia is also provided.
According to a fifth aspect of the present application, there is also provided a method for preventing and/or treating liver cancer, gastric cancer, head and neck cancer, or leukemia, the method comprising: providing any one of the above 10-hydroxycamptothecin-containing drugs or pharmaceutical compositions; administering an effective amount of the above drug or pharmaceutical composition containing 10-hydroxycamptothecin to patients with liver cancer, gastric cancer, head and neck cancer or leukemia.
The drug containing 10-hydroxycamptothecin provided by the application comprises nucleic acid nanoparticles and 10-hydroxycamptothecin, and the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode. In the nucleic acid nanoparticles, the three sequences or variant sequences thereof provided by the application can be contained, so that not only can the nucleic acid domains be formed by self assembly, but also 10-hydroxycamptothecin can be connected to any 5 'end and/or 3' end of the three chains as a carrier, or the 10-hydroxycamptothecin can be stably inserted between the chains of the nucleic acid domains. According to the application, the small-molecule drug 10-hydroxycamptothecin is hung on the nucleic acid nanoparticles, the internal hydrophobicity, the external hydrophilicity and the stacking effect of basic groups of the nucleic acid nanoparticles are utilized, the 10-hydroxycamptothecin is coated, the 10-hydroxycamptothecin cannot be dissolved within a certain time due to the coating effect or covalent connection, and the delivery stability is improved. In addition, when the nucleic acid structural domain is modified by a target head, the target head has better targeting property, can stably deliver 10-hydroxycamptothecin, and has high reliability; meanwhile, the probability of contact of the 10-hydroxycamptothecin with non-target cells or tissues can be reduced, and toxic and side effects are reduced.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. In the drawings:
FIG. 1 shows the result of electrophoresis detection of RNA nanoparticles formed by self-assembly in example 1 of the present application;
FIG. 2 shows the result of electrophoresis detection of DNA nanoparticles formed by self-assembly in example 1 of the present application;
FIG. 3 shows the results of 2% agarose gel electrophoresis detection of 7 sets of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present application;
FIG. 4 shows the results of 4% agarose gel electrophoresis detection of 7 sets of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present application;
FIG. 5 shows the results of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;
FIG. 6 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;
FIG. 7 shows the results of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;
FIG. 8 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;
FIG. 9 shows a TEM image of self-assembled DNA nanoparticles D-7 of the present application in example 4;
FIG. 10 shows a standard curve of absorbance of 10-hydroxycamptothecin in the process of the loading rate measurement in example 5 of the present application;
FIG. 11 shows the microscopic observations of the binding and internalization of the RNAh-Biotin-quasar670 nanoparticle and RNAh-Biotin-quasar670-hdcp nanoparticle with SGC7901 cells in example 6 of the present application;
FIG. 12 shows the results of electrophoresis of the RNAh-Biotin-quasar670-hdcp nanoparticles of example 7 of the present application after incubation in serum for various periods of time under the Coomassie Blue program;
FIG. 13 shows the results of electrophoresis of the RNAh-Biotin-quasar670-hdcp nanoparticles of example 7 of the present application after incubation in serum for various periods of time under the Stainfree Gel program; and
FIG. 14 shows the results of detecting the inhibition of SGC7901 cell proliferation by small molecule 10-hydroxycamptothecin and RNAh-Biotin-quasar670-hdcp nanoparticles in example 8 of the present application;
FIG. 15 shows the results of detecting the inhibition of SGC7901 cell proliferation by the fluorescent targeting vector RNAh-Biotin-FAM in example 8 of the present application;
FIG. 16 shows the result of non-denaturing PAGE gel electrophoresis detection of 7 sets of modified-segment + core short-sequence RNA self-assembly products in example 9 of the present invention;
FIG. 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 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 result of non-denaturing PAGE gel electrophoresis detection of 7 sets of modified-segment + core short-sequence DNA self-assembly products in example 10 of the present invention;
FIG. 25 shows a dissolution curve of DNA nanoparticle D-8 in example 10 of the present invention;
FIG. 26 shows a dissolution curve of the DNA nanoparticle D-9 in example 10 of the present invention;
FIG. 27 shows the dissolution curve of DNA nanoparticle D-10 in example 10 of the present invention;
FIG. 28 shows a dissolution curve of the DNA nanoparticle D-11 in example 10 of the present invention;
FIG. 29 is a graph showing the dissolution profile 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 shows 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 after incubation in serum for various times;
FIG. 34 shows the result of electrophoresis detection of RNA nanoparticle R-17 in example 11 after incubation in serum for different time periods;
FIG. 35 shows the result of electrophoresis detection of RNA nanoparticle R-18 in example 11 after incubation in serum for various times;
FIG. 36 shows the result of electrophoresis detection of RNA nanoparticle R-19 in example 11 after incubation in serum for various times;
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 after incubation in serum for various times;
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 result of electrophoresis detection of DNA nanoparticle D-11 in example 12 of the present invention after incubation in serum for various times;
FIG. 43 shows the result of electrophoresis detection of DNA nanoparticle D-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 blank vector: refers to a blank nucleic acid nanoparticle vector, such as RNAh or DNAh, that does not contain any biologically active substance.
Targeting vectors: refers to a nucleic acid nanoparticle vector containing a targeting tip but not containing a fluorescent substance, such as Biotin-RNAh or Biotin-DNAh.
A fluorescent carrier: refers to a nucleic acid nanoparticle vector containing a fluorescent substance but not containing a targeting moiety, such as Cy5-RNAh or Cy 5-DNAh.
Targeting fluorescent carrier: refers to a nucleic acid nanoparticle vector containing a target and a fluorescent substance, such as Biotin-Cy5-RNAh or Biotin-Cy 5-DNAh.
Targeting drugs: refers to nucleic acid nanoparticle carrier containing target, fluorescent substance and chemical, such as 10-hydroxycamptothecin-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, although there are many drug carriers for improving drug delivery efficiency in the prior art, it is still difficult to solve the problem that the clinical application of drugs is limited. In order to improve the situation, the inventor of the present application has studied all available materials as drug carriers, and has conducted in-depth investigation and analysis on various carriers from the aspects of cell/tissue targeting property of the carriers, stability during transportation, activity and efficiency of entering target cells, drug release capacity after reaching target cells, toxicity to cells and the like, and found that nanostructures formed by self-assembly of emerging DNA and/or RNA molecules, for example, DNA in a self-assembly system of DNA dendrimers, have a significant effect of hindering nuclease degradation, and have very important application values in the fields of gene therapy and biomedicine.
Through analysis of nanoparticles formed by self-assembly of DNA and RNA reported in the prior art, compared with DNA nanoparticles which are relatively rigid, RNA nanoparticles have more flexibility and stronger tension due to a large number of stem-loop structures existing in molecules or between 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 it can carry drugs into cells, and the carrier alone is not toxic to cells. And the carrier carrying the medicine can play a role in relieving and treating corresponding diseases.
On the basis of the above research results, the applicant proposed the technical solution of the present application. The application provides a drug containing 10-hydroxycamptothecin, which comprises nucleic acid nanoparticles and 10-hydroxycamptothecin, wherein the 10-hydroxycamptothecin 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 a1 sequence or a sequence a1 sequence with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 sequence or a sequence b1 sequence with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 sequence or a sequence c1 sequence with at least one base insertion, deletion or substitution; wherein, the sequence of a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively; b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3'; c1 sequence is SEQ ID NO: 5: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' are provided.
The drug containing 10-hydroxycamptothecin provided by the application comprises nucleic acid nanoparticles and 10-hydroxycamptothecin, and the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles. The nucleic acid nanoparticles can be used as a carrier to connect 10-hydroxycamptothecin to any 5 'end and/or 3' end of the three strands, or to stably intercalate 10-hydroxycamptothecin between strands of the nucleic acid domains, as well as to form a nucleic acid domain by self-assembly by including the three sequences or their variant sequences. According to the drug containing 10-hydroxycamptothecin, the small-molecule drug 10-hydroxycamptothecin is hung on the nucleic acid nanoparticles, and the nucleic acid nanoparticles have hydrophobicity inside, hydrophilicity outside and stacking effect of basic groups, so that the 10-hydroxycamptothecin is coated, and the coating or covalent connection ensures that the 10-hydroxycamptothecin cannot be dissolved in a certain time, so that the delivery stability is improved. In addition, when the nucleic acid structural domain is modified by a target head, the target head has better targeting property, can stably deliver 10-hydroxycamptothecin, and has high reliability; meanwhile, the probability of contact of the 10-hydroxycamptothecin with non-target cells or tissues can be reduced, and toxic and side effects are reduced.
The self-assembly refers to a technique in which basic structural units spontaneously form an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds. The self-assembly process is not a simple superposition of weak interaction forces (wherein the weak interaction 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, spontaneously forming a stable structure based on the physical and chemical properties of nucleic acid molecules, with molecular architecture as the starting point, following strict principles of nucleic acid base pairing. A plurality of DNA fragments are connected together in a correct sequence in vitro, and a sub-assembly structure is established through a base complementary pairing principle, so that a complex multilevel structure is finally formed. Unlike DNA, RNA can be structured beyond the limitations of the double helix. RNA can form a series of different base pairs with at least two hydrogen bonds between the base pairs. The different bases can be divided into two types, including standard Watton-Crick base pair type and non-Watton-Crick base pair type, so that the RNA can form a large number of and various types of circulating structure modules, and the modules are basic units forming the tertiary structure of the folded RNA. RNA nanotechnology can take advantage of these naturally occurring 3D modules and their predictable interactions, where many biologically active RNA structures can have atomic-level resolution, such as ribosomes, ribozymes of various types, and natural RNA aptamers present in riboswitches. One advantageous feature of RNA nanotechnology is that structures comparable in size and complexity to natural RNA species can be designed. The unique assembly properties of RNA within the native RNA complex can also be exploited.
In the nucleic acid nanoparticles, the nucleic acid nanoparticles comprise three sequences shown by SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 or sequences after variation thereof, or three sequences shown by SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6 or sequences after variation thereof, and the nucleic acid nanoparticles can be formed by self-assembly, and the specific sequence after variation can be obtained by reasonably selecting variation sites and variation types thereof on the basis of the sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or by extending suitable fragments.
The nanoparticles formed by self-assembly of SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 are RNA nanoparticles, and the nanoparticles formed by self-assembly of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 are DNA nanoparticles. In a preferred embodiment, when the nucleic acid nanoparticle is an RNA nanoparticle, at least one of the sequences a, b, and c comprises a sequence with at least one base insertion, deletion, or substitution. The specific position and the base type of the variant sequence in the RNA nano-particle can be improved into the nano-particle for improving the drug loading capacity or the stability according to the requirement on the premise of realizing self-assembly.
In order to ensure that the nucleic acid nano-particles have relatively higher stability and further ensure that the medicine obtained by carrying 10-hydroxycamptothecin is more stable, in the case of the above-described SEQ ID NO:1/2, SEQ ID NO:3/4 and/or SEQ ID NO:5/6 in the case of base insertion, deletion or substitution, can be carried out on the bases of the above sequence at specific positions, on the one hand, so that the mutated sequence can self-assemble into nanoparticles as the original sequence, and on the other hand, the mutation retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% homology with the original sequence, the nano-particles formed by self-assembly of the sequences have the same drug-loading characteristics and similar stability, and can well carry and deliver 10-hydroxycamptothecin.
In a preferred embodiment, the above base insertions, deletions or substitutions occur in: (1) 1 or 2 between the 1 st, 2 nd, 4 th and 5 th bases from the 5' end of the a sequence shown in SEQ ID NO; and/or (2) between 8 th to 10 th bases from the 5' end of the sequence a shown in SEQ ID NO. 1 or 2; and/or (3) between 1 to 3 bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (4) between 6 th and 9 th bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (5) between the 1 st to 4 th bases from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6; and/or (6) between 9 th to 12 th bases from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6.
In the preferred embodiment, the base positions where the mutation is limited are the non-classical Watson-Crick paired base positions or the protruding unpaired base positions in the nanostructure formed by the sequences shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, thereby not affecting the formation of these protruding or loop structures, thereby maintaining the flexibility and tension of the nanostructure formed by the above sequences and helping to maintain the stability as a carrier.
In order to further improve the stability of the nucleic acid nanoparticles and further improve the stability of the drug formed after the 10-hydroxycamptothecin 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 not complementary; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; 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 nanoparticles having the structure of formula (1), the specific sequence composition of the a sequence, the b sequence and the c sequence is not particularly limited as long as the structure can be formed. From the viewpoint of self-assembly of nucleic acid sequences, in order to further improve the efficiency of self-assembly of the three sequences into the nanoparticle having the structure of formula (1), when selecting the bases paired in Watson-Crick, the bases at different positions are preferably selected according to the following principle: (1) a sequence a, a sequence b and a sequence c, wherein when a single sequence is not self-complementary, a pair of sequences forms a secondary structure; (2) one end of any two sequences is complementary and matched to form a double chain, and the other end is not complementary and matched to form a Y-shaped or T-shaped structure. The principle of the base selection is to make the two ends of any one strand complementary and paired with the two ends of the other two strands respectively to improve the self-assembly efficiency. Of course, in addition to the Y-type or T-type structure, other variants such as quadrilateral, etc. may be used as long as the principle that one end of any two sequences is complementary and paired to form a double strand and the other end is not complementary and paired is satisfied.
In the nanoparticle with the structure of the formula (1), in the non-Watson-Crick pairing base, the fourth N from the 5 'end in the sequence a and the first N' from the 5 'end in the sequence b can be paired with the fourth N from the 5' end in the sequence a, and can be U-U which is not matched with Watson-Crick pairing, and can also be T, A, C or G which is modified and follows the Watson-Crick pairing principle. The Watson-Crick pairing relatively improves the bonding force between chains and improves the stability, but the non-Watson-Crick pairing endows the nano particles with greater flexibility and is also beneficial to improving the stability of the nano particles in the face of microenvironment change.
In a preferred embodiment, the sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence (SEQ ID NO: 7): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 8): 5'-CCUUCGCCG-3', c sequence (SEQ ID NO: 9): 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence (SEQ ID NO: 10): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 11): 5 '-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', respectively; (4) a sequence (SEQ ID NO: 16): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 17): 5 '-CCUUCGGG-3', c sequence (SEQ ID NO: 18): 5'-CCCCCAUAGCCC-3', 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', respectively; (7) a sequence (SEQ ID NO: 25): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 26): 5'-GCUUCGGCG-3', c sequence (SEQ ID NO: 27): 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence (SEQ ID NO: 28): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 29): 5'-CCTTCGCCG-3', c sequence (SEQ ID NO: 30): 5'-CGGCCATAGCCC-3', respectively; (9) a sequence (SEQ ID NO: 31): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 32): 5'-CGTTCGCCG-3', c sequence (SEQ ID NO: 33): 5'-CGGCCATAGCGC-3', respectively; (10) a sequence (SEQ ID NO: 34): 5'-CGAGCGTTGC-3', b sequence (SEQ ID NO: 35): 5'-GCTTCGCCG-3', c sequence (SEQ ID NO: 36): 5'-CGGCCATAGCCG-3', respectively; (11) a sequence (SEQ ID NO: 37): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 38): 5'-CCTTCGGGG-3', c sequence (SEQ ID NO: 39): 5'-CCCCCATAGCCC-3', respectively; (12) a sequence (SEQ ID NO: 40): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 41): 5'-CGTTCGGCG-3', c sequence (SEQ ID NO: 42): 5'-CGCCCATAGCGC-3', respectively; (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' are provided.
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 mentioned above can be not only self-assembled and molded, but also have the ability to carry or carry 10-hydroxycamptothecin drugs. The amount of the 10-hydroxycamptothecin carried differs depending on the position of G-C or C-G base pairs in the nucleic acid nanoparticles.
In order to make the nucleic acid domain capable of carrying more 10-hydroxycamptothecin and bioactive substances (the bioactive substances are described below), in a preferred embodiment, the nucleic acid domain further comprises a first extension, the first extension is a Watson-Crick paired extension, and the first extension is located at the 5 'end and/or the 3' end of any one of the a sequence, the b sequence and the c sequence. A certain matching relationship is required between the carrier and the carried substance, and when the molecular weight of the carrier is too small and the molecular weight of the carried substance is too large, the carrying or transporting capacity of the carrier to the carried substance is relatively reduced from the mechanical point of view. Therefore, a vector matching the size of the carried substance can be obtained by adding a first extension segment to the 5 'end and/or 3' end of any one of the a sequence, the b sequence and the c sequence based on the nucleic acid nanostructure.
The specific length of the first extension segment can be determined according to the size of the substance to be carried. In a preferred embodiment, the first extension is selected from any one of the group consisting of: (1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3'; (2): a 3' end of the chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of the chain: 5' -CCCG-3', 3' end of c chain: 5 '-CGGG-3'; (5): a 5' end of the chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'. (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3'; (9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'; (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 the chain: 5'-GGCCGGAGGCCGG-3' (SEQ ID NO:170), 5' end of b chain: 5'-CCGGCCTCCGGCC-3' (SEQ ID NO: 171).
The first extension not only increases the length of any one or more of the three sequences forming the nucleic acid nanostructure, but also the first extension composed of GC bases further improves the stability of the formed nanoparticles. Moreover, the first extension segment composed of the 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 the basis of the first extension.
In a preferred embodiment, the nucleic acid domain further comprises at least one second extension selected from the group consisting of: a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of the chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'. This second extension renders the nanoparticle non-immunogenic and non-existent in the case of secondary structures to which each chain folds itself.
The first extension and/or the second extension may be separated by unpaired base pairs.
In order to make the nucleic acid nanoparticles capable of carrying bioactive substances with larger molecular weight (see the introduction of bioactive substances below), increasing drug loading and maintaining necessary stability, in a preferred embodiment, the second extension is an extension containing both CG base pairs and AT/AU base pairs, and preferably the second extension is an extension of 2-50 base pairs. Here, the "/" in "AT/AU base" is in the relationship of or, specifically, the second extension is an extended sequence containing both CG base pairs and AT base pairs, or the second extension is an extended sequence containing both CG base pairs and AU base pairs.
More specifically, the sequences a, b and c after adding the above second extension may be the following sequences, respectively:
sequence a is (SEQ ID NO: 49):
b is (SEQ ID NO: 50):
sequence c is (SEQ ID NO: 51):
m in the sequence a, the sequence b and the sequence c is U or T, and when M is T, the synthesis cost of the sequences is greatly reduced.
In practical application, the specific arrangement positions of the CG base pairs and the extended sequences of the AT/AU base pairs can be reasonably adjusted according to actual needs. In a more preferred embodiment, the second extension is an extension sequence formed by alternating sequences of 2-8 CG base pairs and 2-8 AT/AU base pairs; or the second extension is an extension sequence with 1 CG base pair sequence and 1 AT/AU base pair sequence arranged alternately.
Specifically, the positions of the CGCGCG extension and the CGCCGC extension in the sequence a shown by the SEQ ID NO. 49 and the AAAAAA extension are interchanged, the positions of the GCGGCG extension and the GGCGGC extension in the sequence b shown by the SEQ ID NO. 50 and the TTTTTT extension are interchanged, the positions of the GCCGCC extension and the AAAAAA extension in the sequence c shown by the SEQ ID NO. 51 and the CGCCGC extension and the TTTTTT extension are interchanged. The nucleic acid nanoparticles formed by self-assembly of the sequences are suitable for carrying bioactive substances with indole molecular structures (indole molecules are preferably combined with A).
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 10-hydroxycamptothecin can be carried by physical connection and/or covalent connection. When the 10-hydroxycamptothecin is simultaneously connected with the nucleic acid domain by adopting two modes of physical intercalation and covalent connection, the physical intercalation is usually intercalated between GC base pairs, and the preferable number of intercalation sites is 1-100: the ratio of 1 was inserted. When covalent linkage is used, 10-hydroxycamptothecin will usually react with the amino group outside the G ring to form covalent linkage. More preferably, the molar ratio of the 10-hydroxycamptothecin to the nucleic acid nanoparticles is 2-300: 1, preferably 2-290: 1, more preferably 2-29: 1, even more preferably 10-50: 1, and most preferably 15-25: 1.
In addition to the nucleic acid nanoparticles serving as delivery vehicles for 10-hydroxycamptothecin in the 10-hydroxycamptothecin-containing drugs provided herein, in a preferred embodiment, the nucleic acid nanoparticles further comprise a bioactive substance, and the bioactive substance is linked to the nucleic acid domain, according to different drug purposes. The bioactive substances are one or more of target, fluorescein, interfering nucleic acid siRNA, miRNA, ribozyme, riboswitch, aptamer, RNA antibody, protein, polypeptide, flavonoid, glucose, natural salicylic acid, monoclonal antibody, vitamin, phenol, lecithin and small molecule drugs except 10-hydroxycamptothecin.
In order to improve the efficiency of loading and carrying the loaded bioactive substances by the nucleic acid nanoparticles, the relative molecular weight of the nucleic acid domain and the relative molecular weight of the 10-hydroxycamptothecin and the bioactive substances preferably have a certain matching relationship. In a preferred embodiment, the relative molecular weight of the nucleic acid domains is denoted as N1The total relative molecular weight of 10-hydroxycamptothecin and bioactive substance is recorded as N2，N1/N2≥1:1。
The 10-hydroxycamptothecin-containing drugs of the present application are optimized in terms of different properties depending on the kind of the specific bioactive substance to be carried. For example, when the bioactive substance is biotin or folic acid, it acts to target the 10-hydroxycamptothecin-containing drug, 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 10-hydroxycamptothecin, the drugs containing 10-hydroxycamptothecin 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 target heads, fluorescein and miRNA, wherein the target heads are located on any sequence of a, b and c sequences, preferably on the 5' end or the 3' end of any sequence of a, b and c, or are inserted between GC bonds of the nucleic acid structure domain, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more positions of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence; preferably, the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
The target head can be connected to any sequence of a sequence, b sequence and c sequence through a linker covalent connection mode, and the available linker is selected from disulfide bond, p-azido group, bromopropyne or PEG. As used herein, "on any sequence" refers to any base position of any sequence of a, b, c sequences, and it is more convenient to attach to the 5 'end or 3' end, and the application is more extensive. Folate modification can be either physical intercalation mode ligation or physical intercalation + covalent ligation.
The fluorescein may be any one or more of conventional fluorescein, preferably FAM, CY5 and CY 3.
The miRNA can be miRNA with cancer inhibiting effect, or anti-miRNA capable of inhibiting corresponding diseases, and is reasonably selected according to medical needs in practical application. The anti-miRNA may be synthesized at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence. When anti-miRNA is synthesized at all of the above three positions, the inhibitory effect of the anti-miRNA on the corresponding miRNA is relatively stronger.
Preferably, the miR-21 is resistant to miR-21, and miR-21 is involved in the initiation and progression of various cancers and is a main oncogene for invasion and metastasis. The anti-miR-21 can effectively and simultaneously regulate a wide range of target genes, and is beneficial to solving the problem of heterogeneity of cancers. Thus, in the preferred nucleic acid nanoparticles, the target head, such as folate or biotin, can specifically target cancer cells, and after internalization in combination with 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 substance can be other small molecule drugs except 10-hydroxycamptothecin, the drugs include, but are not limited to, drugs for treating liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, melanoma, leukemia, senile dementia, ankylosing spondylitis, malignant lymphoma, bronchial cancer, rheumatoid arthritis, HBV hepatitis B, multiple myeloma, pancreatic cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, esophageal cancer, oral cancer, and lupus erythematosus 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 biologically active substance capable of being carried is a small molecule drug other than 10-hydroxycamptothecin, the biologically active substance includes, but is not limited to, drugs containing any one or more of the following groups, depending on the molecular structure of the drug or the characteristic groups of the drug: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
In a preferred embodiment, the protein is one or more of an antibody or aptamer to SOD (superoxide dismutase), Survivin (Survivin), hTERT (human telomerase reverse transcriptase), EGFR (epidermal growth factor receptor), PSMA (prostate specific membrane antigen); the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
In a preferred embodiment, the particle size of the nucleic acid nanoparticles is 1 to 100nm, preferably 5 to 50nm, more preferably 10 to 30nm, and even more preferably 10 to 15 nm. Within this range the size is suitable both to enter the cell membrane by cell surface receptor mediated phagocytosis and to avoid non-specific cell penetration and removal by renal filtration, so that the favourable particle size contributes to improved pharmacokinetic, pharmacodynamic, biological and toxicological profiles.
According to a second aspect of the present application, there is also provided a method for preparing the above drug containing 10-hydroxycamptothecin, which comprises the steps of: providing any one of the nucleic acid nanoparticles described above; the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the medicine containing the 10-hydroxycamptothecin is obtained.
When physical attachment is used, 10-hydroxycamptothecin will typically form insertions between GC base pairs by physical insertions. When covalent linkage is used, 10-hydroxycamptothecin will usually react with the amino group outside the G ring to form covalent linkage. The medicine containing 10-hydroxycamptothecin prepared by the method has better targeting property after being modified by the target head, can stably deliver the 10-hydroxycamptothecin, and has high reliability.
In a preferred embodiment, the step of loading the 10-hydroxycamptothecin by means of physical attachment comprises: mixing and stirring 10-hydroxycamptothecin, the nucleic acid nanoparticles and the first solvent to obtain a premixed system; precipitating the premixed system to obtain the medicine containing 10-hydroxycamptothecin. The dosage of the specific 10-hydroxycamptothecin and the nucleic acid nanoparticles can be adjusted according to the change of the loading amount, which can be understood by those skilled in the art and will not be described in detail herein.
In order to improve the efficiency and stability of physical connection, the amount of 10-hydroxycamptothecin 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 10-hydroxycamptothecin comprises: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the medicine containing 10-hydroxycamptothecin. 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, 6-12 times of volume of absolute ethyl alcohol is adopted to wash the precipitate to remove impurities, and the medicine containing 10-hydroxycamptothecin is obtained.
In a preferred embodiment, the step of loading the 10-hydroxycamptothecin by covalent attachment comprises: preparing 10-hydroxycamptothecin solution; reacting the 10-hydroxycamptothecin solution with the amino outside the G ring of the nucleic acid nano-particles under the mediated action of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the medicine containing 10-hydroxycamptothecin.
In a formaldehyde-mediated form, the following reactions can occur:
preferably, the step of reacting comprises: mixing the 10-hydroxycamptothecine solution with a paraformaldehyde solution and nucleic acid nanoparticles, and reacting under the condition of keeping out of the sun to obtain a reaction system. The paraformaldehyde solution can release formaldehyde small molecules so as to participate in the chemical reaction. In order to improve the reaction efficiency, the concentration of the paraformaldehyde solution is preferably 3.7-4 wt%, the paraformaldehyde solution is preferably a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
In the above preparation method, the nucleic acid nanoparticles may be prepared by a self-assembly form such as: (1) mixing RNA or DNA single strands a, b and c at the same time, and dissolving in DEPC water or TMS buffer solution; (2) heating the mixed solution to 80 ℃/95 ℃ (wherein the RNA assembly temperature is 80 ℃, and the DNA assembly temperature is 95 ℃), keeping for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min; (3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃; (4) cutting 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 10-hydroxycamptothecin-containing drugs with other functions, according to the practical application requirements, in a preferred embodiment, after obtaining the nucleic acid domain, the preparation method further comprises: the aforementioned bioactive substances are carried on the nucleic acid domain by means of physical linkage and/or covalent linkage, thereby obtaining the nucleic acid nanoparticles. The 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 bioactive substance, there may be only one type of attachment, or there may be multiple types of attachment, but there may be some type of attachment that has an advantageous utility.
In the above connection method, when different drugs are physically inserted into the nucleic acid domains, the number and binding sites of the insertion are slightly different. For example, when the anthracycline and acridine drugs are inserted, the drugs are usually inserted between GC base pairs, and the number of the preferred insertion sites is 1 to 100: the ratio of 1 was inserted. When the naphthamide drug is inserted, the naphthamide drug is usually inserted between AA base pairs, the preferable number of insertion sites is different according to the number of the AA base pairs on the nucleic acid structural domain, and the pyridocarbazoles are inserted according to the difference of the number of the AA base pairs in the range of 1-200: the ratio of 1 was inserted.
Specifically, the molar ratio of biologically active substance to nucleic acid domain can be reasonably selected for physical intercalation depending on the species of biologically active substance, the length of the a, b and c sequences forming the nucleic acid domain in the nucleic acid nanoparticle, and how many complementary base pairs of GC are present therein.
In a preferred embodiment, when the bioactive substance and the nucleic acid domain are physically intercalated and covalently linked, the molar ratio of the bioactive substance physically intercalated and linked to the drug covalently linked is 1-200: 1. the connection mode is suitable for anthracycline and acridine medicines. The proportion of the drugs connected in different connection modes is not limited to the range, and the drugs can be effectively suspended, have no toxic effect on cells and can be effectively released after reaching a target.
When the bioactive substance precursor and the nucleic acid domain are simultaneously subjected to alkynyl or azide modification and connected in a click-to-link mode, different click-to-links are selected according to different structure changes of the medicament. And the attachment position may be changed correspondingly according to the structure of the active material, which can be understood by those skilled in the art.
In a preferred embodiment, when the biologically active substance is linked to the nucleic acid domain in a click-link fashion, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of hydroxyl, carboxyl, sulfhydryl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of amino, imino or hydroxyl.
According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any one of the 10-hydroxycamptothecin-containing drugs described above. Specifically, according to actual needs, a suitable combination drug or adjuvant can be selected to form a drug combination having a combined drug effect or capable of improving certain properties (such as stability) of the drug.
According to the fourth aspect of the application, the application of any one of the medicines containing 10-hydroxycamptothecin in preparing medicines for treating liver cancer, gastric cancer, head and neck cancer or leukemia is also provided. The specific application can be to improve the medicament per se on the basis of the medicament of the application to obtain a new medicament, or to prepare the medicament of the application serving as a main active ingredient into a preparation with a proper dosage form and the like.
According to a fifth aspect of the present application, there is also provided a method for preventing and/or treating liver cancer, gastric cancer, head and neck cancer, or leukemia, the method comprising: providing any one of the above 10-hydroxycamptothecin-containing drugs or pharmaceutical compositions; administering an effective amount of the above drug or pharmaceutical composition containing 10-hydroxycamptothecin to patients with liver cancer, gastric cancer, head and neck cancer or leukemia. An effective amount herein includes a prophylactically effective amount and/or a therapeutically effective amount, by which is meant an amount effective to achieve the desired therapeutic result, such as reduction of liver cancer, stomach cancer, head and neck cancer, or leukemia, at the necessary dosage and for a period of time. In a particular embodiment, the dosage may be adjusted to provide an optimal therapeutically responsive dose, and the therapeutically effective amount may vary depending on the following factors: the disease state, age, sex, weight of the individual and the ability of the formulation to elicit a desired response in the individual. A therapeutically effective amount is also meant to include an amount by which the beneficial effect of the treatment exceeds its toxic or detrimental effects. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention or inhibition of the occurrence of liver cancer, gastric cancer, head and neck cancer, or leukemia. A prophylactically effective amount can be determined according to the description of therapeutically effective amounts above. For any particular subject, specific dosages may be adjusted over time according to the individual need and the professional judgment of the person to whom they are administered.
When the nucleic acid domain is bound to a drug, the nucleic acid domain is water-soluble, and many drugs have poor water-solubility, and when the nucleic acid domain is bound to the drug, the water-solubility is improved. When the drugs are anthracyclines, the drugs are covalently bound to the nucleic acid domain via an-NH bond on the nucleotide guanosine (the-NH group is hundreds of times more active than other groups that may covalently bind to the drug under appropriate pH conditions), thereby forming a drug-loaded nucleic acid domain. Therefore, according to the size of a specific drug molecule and the number of GC base pairs on the sequence a, the sequence b and the sequence c of a specifically designed nucleic acid structural domain, when in combination, the combination reaction is carried out according to the supersaturation combination amount which is 1.1-1.3 times of the theoretical amount, and 35-45 drugs can be combined on one nucleic acid structural domain at most. When the drug has other structure, the loading amount is related to the occupancy of the specific drug (including but not limited to molecular structure, form, shape and molecular weight), so that the binding condition of the active site of the drug and the-NH bond on the nucleotide guanosine of the nucleic acid domain is relatively severe, and the drug can be loaded but is relatively difficult to be excessively bound.
It should be noted that the nucleic acid nanoparticles formed by self-assembly of the sequences or sequence variants provided herein can also be used as basic building blocks, and can be further polymerized to form multimers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., according to the practical application.
The advantageous effects of the present application will be further described with reference to specific examples.
Assembly of nucleic acid nanoparticles
Example 1
One, RNA and DNA nanoparticle vector:
(1) the three polynucleotide base sequences that make up the RNA nanoparticles are shown in table 1:
table 1:
(2) three polynucleotide base sequences of the DNA nanoparticle.
The DNA has the same sequence as that of the RNA described above except that T is substituted for U. Wherein the molecular weight of the a chain is 8802.66, the molecular weight of the b chain is 8280.33, and the molecular weight of the c chain is 9605.2.
The a, b and c strands of the above-mentioned RNA nanoparticles and DNA nanoparticles were synthesized by Competition Biotechnology (Shanghai) Co., Ltd.
II, self-assembly experiment steps:
(1) mixing RNA or DNA single strands a, b and c at the same time according to the molar ratio of 1:1:1, and dissolving in DEPC water or TMS buffer solution;
(2) heating the mixed solution to 80 ℃/95 ℃ (wherein the RNA assembly temperature is 80 ℃, and the DNA assembly temperature is 95 ℃), keeping for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off a target band, eluting in an RNA/DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a self-assembly product;
(5) electrophoresis analysis detection and laser scanning observation.
Third, self-assembly experimental results
(1) Results of electrophoresis
The results of the electrophoretic detection of the RNA self-assembly products are 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: sequences a, b and c including RNA core sequence SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 can be successfully self-assembled into RNA nanoparticles. Sequences a, b and c including DNA core sequence SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 can also be successfully self-assembled into DNA nanoparticles.
The sequences a, b and c of the RNA nanoparticles and the DNA nanoparticles include various extension sequences (including drug-loading binding sequences) that facilitate the function of loading the nucleic acid domains, and a targeting head or fluorescein linked to the nucleic acid domains, in addition to the core sequence forming the nucleic acid domains. It can be seen that the presence of substances other than these core sequences does not affect the formation of nucleic acid domains and the successful self-assembly of nucleic acid nanoparticles. The self-assembled nucleic acid nanoparticles can have a targeting type under the guidance of a target head, and the fluorescein can enable the nucleic acid nanoparticles to have visibility and traceability.
Example 2
One, 7 groups of short sequence RNA nano-particle carriers:
(1)7 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 RNA single strands a, b and c at the same time according to a molar ratio of 1:1:1, and dissolving in DEPC water or TMS buffer solution;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting a target band, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a short-sequence RNA self-assembly product;
(5) electrophoretic analysis detection and laser scanning observation;
(6) and (6) detecting the potential.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis of the 7 sets 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 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 MeasUre € ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 7 groups of short sequence RNA nanoparticles are as follows:
table 9:
table 10:
table 11:
table 12:
table 13:
table 14:
table 15:
from the potential detection data described above, it can be seen that: the 7 groups of short sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the short sequence RNAs have 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 various functional extension fragments or connecting targeting heads, fluorescein and the like are added on the basis of different core sequence combinations, and the RNA nanoparticles can be successfully assembled, and have the performances of drug loading, cell targeting, visual tracking and the like.
To further verify these properties, an extension fragment was added to example 2, see example 3. And adding an extension fragment on the basis of the DNA core sequence corresponding to the RNA core sequence of example 2, and simultaneously connecting the target or not connecting the target, as shown in example 4.
Example 3
One, 7 groups of conventional sequence RNA nanoparticle carriers:
(1)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 single strands of the 7 groups of conventional sequence RNA nanoparticle carriers are synthesized by consignment of Jima of Suzhou, 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 of R-1 to R-7, targeting module fragments are not extended, and C/U base 2' F modification (the enzyme cleavage resistance and stability are enhanced) is carried out. 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 target bands, eluting in RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at low temperature under reduced pressure;
(5) electrophoretic analysis detection and laser scanning observation;
(6) and (4) measuring the potential.
Third, self-assembly experimental results
(1) Results of electrophoresis
FIG. 5 shows the electrophoresis of the 2% agarose gel of the 7 sets of conventional sequence RNA self-assembly products. Lanes 1 to 7 in FIG. 5 are, from left to right: the self-assembly products of the conventional sequence RNA are R-8, R-9, R-10, R-11, R-12, R13 and R-14.
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 MeasUre € ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 7 groups of conventional sequence RNA nanoparticles are as follows:
table 23:
table 24:
table 25:
table 26:
table 27:
table 28:
table 29:
from the potential detection data described above, it can be seen that: the 7 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have a stable self-assembly structure.
This example shows that: on the basis of RNA core sequences of different combinations, the addition of the extension segment can also successfully self-assemble into RNA nanoparticles with stable structure. Meanwhile, the added extension fragment enables the RNA nanoparticles to have excellent drug-carrying performance (see example 5 and example 7 in particular).
Example 4
1, 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 part below) to the core sequence (8) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3') described above;
d-2 is a regular sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined part 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 EGFRatt target head (see underlined section below) to the core sequence (10) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3') described above;
d-4 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined part below) to the core sequence (11) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3') described above;
d-5 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAApt target head (see underlined part below) to the core sequence (12) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3') described above;
d-6 is the core sequence (13) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3') added with an extension sequence not containing the targeting structure; the formed conventional sequence DNA nanoparticles;
d-7 is an extension sequence which does not contain a targeting structure and is added to the core sequence (14) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3') described above; and forming the conventional sequence DNA nano-particles.
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) measuring the potential;
(7) measuring the particle size;
(8) and (5) observing by using a transmission electron microscope.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis of the 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 pattern of 4% agarose gel of the 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 can be clearly seen that the bands of the 7 groups of conventional sequence DNA self-assembly products are all bright and clear, indicating that the 7 groups of conventional sequence DNA strands complete the self-assembly and form a stable nanoparticle structure. 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) 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 MeasUre € ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 3 groups of conventional sequence DNA nanoparticles are as follows:
table 37:
table 38:
table 39:
from the potential detection data described above, it can be seen that: the 3 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have a stable self-assembly structure.
(3) Particle size measurement
1. Preparing a potential sample (a conventional sequence DNA self-assembly product D-7) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
3. setting software detection parameters;
4. then click on the ok setting, the measurement dialog 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 results of transmission electron microscope
And (3) carrying out transmission electron microscope irradiation on the conventional sequence DNA self-assembly product D-7, and comprising the following steps:
1. taking a drop of sample to suspend on a 400-mesh carbon film-coated copper net, and keeping the temperature at room temperature for 1 minute;
2. sucking off liquid by using filter paper;
3. dyeing for 1 minute by using 2% uranium acetate;
4. sucking dry by filter paper, and drying at room temperature;
5. JEM-1400 was observed by 120kv using a transmission electron microscope and photographed.
As a result, as shown in FIG. 9, it is apparent that the product D-7 of the conventional DNA sequence is a whole structure and can be clearly seen to have a T-type structure.
Example 5
Experiment for carrying 10-hydroxycamptothecin
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) 10-hydroxycamptothecin.
(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) 10-hydroxycamptothecin (1.355. mu. moL) is precisely weighed, dissolved in DEPC water (1.0mL) and PBS buffer (1.25mL), added with 4% paraformaldehyde aqueous solution (0.4mL) to be mixed uniformly under the cooling of ice-water bath, and the mixture is completely mixed with RNA nanoparticles (1mg, 33.84nmoL) to react for 72 hours at 4 ℃ in a dark condition.
(2) 10 mu L of the reaction solution is diluted by 10 times, and HPLC analysis is carried out by taking 50 mu M10-hydroxycamptothecin solution and 310 ng/mu L RNA nanoparticles as controls and injecting samples in equal volume. The reaction conversion can be judged to be basically complete according to the peak area ratio of each component.
(3) The reaction mixture was extracted with chloroform (10mL x3), followed by addition of 25mL of absolute ethanol, mixing, and then sufficiently precipitating the product by keeping the mixture at 4 ℃ in the dark (4 hours). Centrifuging (4000/min), transferring supernatant, washing solid product with ethanol (50mL) again, and evaporating solvent under reduced pressure at low temperature to obtain the carried product.
(4) And (3) calculating the mounting rate:
1. preparing a standard solution of 10-hydroxycamptothecin-50% methanol with known concentration: 2uM, 4uM, 6uM, 8uM, 10uM, each 100 ul;
2. dissolving 10-hydroxycamptothecin-RNAh granules in 100ul PBS;
3. placing the standard solution and 10-hydroxycamptothecin-RNAh particles in a PCR plate, heating at 85 deg.C for 5min, and cooling to room temperature;
4. measuring the absorbance of 10-hydroxycamptothecin at 384nm by using a microplate reader, drawing a standard curve (shown in figure 10), and calculating the molar concentration of 10-hydroxycamptothecin in the mounted product;
5. measuring the absorbance of RNA at 260nm by using a spectrophotometer to obtain the mass concentration of the RNAh particles in each sample;
6. and calculating the mounting rate according to the measured molar concentration of the 10-hydroxycamptothecin and the mass concentration of the RNAh particles.
CRNAh-1＝73.3ug/ml，MRNAh≈30000，100ul；C10 hydroxycamptothecin-1＝28.88uM，100ul；
CRNAh-2＝65.8ug/ml，MRNAh≈30000，100ul；C10 hydroxycamptothecin-2＝15.2uM，100ul；
The average value is taken to obtain the loading rate of 10-hydroxycamptothecin-RNAh to be about 9, namely, the loading rate represents that each nucleic acid nanoparticle carrier can load about 16 10-hydroxycamptothecin molecules.
By changing the relative dosage of 10-hydroxycamptothecin and RNA nano-particles, RNAh-10-hydroxycamptothecin particles with the carrying rate of 10, 20, 28, 50, 70, 80, 100, 200 and the like can also be obtained, and the details are not repeated.
In addition, on the basis of the 10-hydroxycamptothecin carried by the RNA nanoparticles, other small molecule drugs can be carried for the second time by the same method as the method for carrying the 10-hydroxycamptothecin, for example, folic acid is further carried by the application to obtain the RNA nanoparticles carrying the two small molecule drugs, namely the 10-hydroxycamptothecin and the folic acid, and the carrying rates of the two drugs can be detected by the method (the value is not shown).
Example 5 shows that the RNA nanoparticles (example 1) with the extension fragment, the targeting head and the fluorescein have the function of drug loading, and the small molecule drug 10-hydroxycamptothecin can be loaded by means of covalent connection (paraformaldehyde-solvent covalent), and can also be loaded together with other small molecule drugs. .
Example 6
Confocal microscope experiment and flow cytometry 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 is used as a control, and 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-hdcp refers to nanoparticles formed after further loading of 10-hydroxycamptothecin (loading by the chemical method in example 5).
2. The experimental reagents used and their sources were as follows:
RPMI-1640 medium (Gibco, C11875500BT-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); Trypsin-EDTA (Stemcell, 07901-; DMSO (Sigma, D5879-1L); prolong Gold antibody mount 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:
| cell lines | Culture Medium |
| SGC7901 | RPMI1640+10％FBS |
Confocal microscopy experiments:
(1) SGC7901 cells in RPMI1640+ 10% FBS + 1% PS medium at 37 ℃ and 5% CO2Culturing under the condition.
(2) The cells were trypsinized and washed once with PBS at 1X10 per well5Individual cells were loaded onto cell culture slides.
(3) After the cells were attached to the wall, the slides were rinsed with medium.
(4) Cells were incubated with 200nM and 400nM of RNAh-Biotin and chemotherapeutics RNA nanoparticles at 37 ℃ and 5% CO2And (4) incubating for 2h and 4 h.
(5) Adherent cells were washed with PBS and stained with Prolong Gold antibody mount overnight at room temperature.
(6) The cells were stained with DAPI at room temperature for 5min and mounted.
(7) And taking a picture under a microscope and storing.
Second, experimental results
The results of the confocal microscope observation experiment are shown in FIG. 11. As can be seen in FIG. 11, the results of the cell binding and internalization experiments indicate that both the RNAh-Biotin-quasar670 and the RNAh-Biotin-quasar670-hdcp nanoparticles carry the targeting head, Biotin (Biotin), and are thus capable of binding to and internalizing into cells.
Flow cytometry detection
SGC7901 cells in RPMI1640+ 10% FBS + 1% PS medium at 37 ℃ and 5% CO2Culturing under the condition.
2x105Individual cells were incubated with 200nM and 400nM nanoparticles to be tested at 37 ℃ and 5% CO2Incubate for 2h, 3 replicates per sample.
1. After washing the cells with PBS, they were resuspended in PBS buffer and detected with FACS machine.
Receipts were collected and statistically analyzed.
The flow cytometer measurement results are shown in table 42:
table 42:
as can be seen from Table 42, the nanoparticles of RNAh-Biotin-quasar670-hdcp and RNAh-Biotin-quasar670 have a strong binding ability to SGC7901 cells.
Example 7
Testing the stability of 10-hydroxycamptothecin-containing drugs loaded on nucleic acid nanoparticles in serum
First, experimental material and experimental method
1. A sample to be tested: RNAh-Biotin-quasar670-hdcp nanoparticles prepared in example 5.
2. Experimental reagent:
RPMI-1640 medium (YS3160-500 mL); fetal Bovine Serum (FBS) (ExCellBio, FNA500-500 mL); Penicillin/Streptomycin (Penicilin/Streptomyces, PS) (Gibco,15140-122-100 mL); PBS buffer (Gibco, C20012500BT-500 mL); novexTMTris-Glycine NativeSample Buffer(2X)(Invitrogen,LC2673-20mL)；Novex TM8% 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 RPMI1640 medium containing 10% serum.
(2) Samples were taken after incubation at 37 ℃ for 10min, 1h, 12h, 36h, respectively.
(3) After quantification with NanoDrop, 200ng of RNAh-Biotin-quasar670-hdcp nanoparticles were added to the same volume of Tris-Glycine SDS Sample Buffer (2X) and mixed well.
(4) Take a block of Novex TM8% Tris-Glycine Mini gel, according to the sequence loading, set program 200V, 30min, start 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 of 8% non-denatured Gel (Coomassie Blue procedure) and figure 13 shows the results of electrophoresis of 8% non-denatured Gel (Stain Free Gel procedure). The result of the serum stability test shows that: at different time lengths of 0min, 10min, 1h, 12h and 36h, the sample bands of the RNAh-Biotin-quartz 670-hdcp nanoparticles have no obvious difference, which indicates that the RNAh-Biotin-quartz 670-hdcp 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-hdcp nanoparticles in SGC7901 cells
First, experimental material and experimental method
1. The sample to be detected is micromolecular 10-hydroxycamptothecin chemical drug and RNAh-Biotin-quasar670-hdcp nano-particles.
2. Experimental reagent:
cell Titer-Glo Luminescent Cell vitality Assay (Promega, G7572-100 mL); RPMI1640 medium (Gibco); PBS buffer (Gibco); fetal Bovine Serum (FBS) (excelBio, C20012500BT-500 mL); Penicillin/Streptomycin (penicilin/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 Envision2104Multilabel Reader (No. 01-094-.
4. The experimental method comprises the following steps:
(1) cell culture and plating
Cells were added to the corresponding basal medium separately10% FBS and 1% PS at 37 ℃ and 5% CO2Culturing under the condition. The cell density used for the experiment was above 80%. Cells were harvested, centrifuged at 1000rpm for 4 minutes, the medium resuspended, cell concentration adjusted, and added to a 96-well plate 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, 0.0023uM solutions; the administration of the cells is divided into micromolecular 10-hydroxycamptothecin chemical drugs and RNAh-Biotin-quasar670-hdcp nano-particles.
(3) Culturing after cell administration
The medicated cells were incubated at 37 deg.C and 5% CO2Cultured under the conditions for 72 hours.
(4) Detection kit for treating cells
The plate was brought to room temperature in advance and allowed 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 Envision2104Multilabel 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 (%)
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 nanoparticles carrying 10-hydroxycamptothecin, RNAh-Biotin-quasar670-hdcp, are significantly cytotoxic to SGC7901 cells and are unexpected: compared with the cell proliferation inhibition effect of the small molecule 10-hydroxycamptothecin drug, the cell proliferation inhibition effect of 5 mu M RNAh-Biotin-quasar670-hdcp on SGC7901 cells is more obvious, and the cell proliferation inhibition rate is improved by at least 25 percent (increased to 94.52 percent) on the basis that the cell inhibition rate after the small molecule 10-hydroxycamptothecin drug treatment is 75.43 percent.
To further confirm that the RNA nanoparticles not carrying 10-hydroxycamptothecin are not significantly cytotoxic to SGC7901 cells, the inventors further designed toxicity tests (drug administration gradients in the tests: 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)) for SGC7901 cells using RNAh-Biotin-FAM (FAM is a fluorescent label), and the results are shown in Table 45 and FIG. 15. IC from Table 4550As can be seen from the values in FIG. 15, the targeted fluorescent vector not carrying 10-hydroxycamptothecin is not significantly toxic to the experimental cells by itself.
Table 45:
assembly of nucleic acid nanoparticles
Example 9
1, 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 RNA single strands a, b and c at the same time according to a molar ratio of 1:1:1, and dissolving in DEPC water or TMS buffer solution;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off target bands, eluting in RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at low temperature under reduced pressure;
(5) 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:
① the RNA nanoparticles were diluted with ultrapure water according to the method of Table 55 below.
Table 55:
② mu.L (500ng) of the treated sample was mixed with 2. mu.L of 6 XDNA Loading Buffer, and the mixture was marked by ice-washing.
③ samples were run on 8% native PAGE gels at different incubation times and 12. mu.L of the treated samples were loaded in their entirety and programmed for 40min at 100V.
④ after the glue running, dyeing, placing on a horizontal shaking table for 30min, and taking pictures.
And (3) detection results:
the results of the native PAGE running gel of 7 sets of extended stretch-deformed + core short sequence RNA self-assembled products are shown in FIG. 16. Lanes 1 to 7 in FIG. 16 are, from left to right: 7 groups of extension segment deformation + core short sequence RNA self-assembly products R-15, R-16, R-17, R-18, R-19, R-20 and R-21.
The results in fig. 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) 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 MeasUre € ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results at 25 ℃ of 7 groups of extension segment deformation and core short sequence RNA nanoparticles are as follows:
table 56:
table 57:
table 58:
table 59:
table 60:
table 61:
table 62:
from the potential detection data described above, it can be seen that: the 7 groups of the extended segment deformation and core short sequence RNA nanoparticles have good stability, and further show that the nanoparticles formed by 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 the extended section deformation and core short sequence RNA nanoparticles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 64:
| name of reagent | Goods number | Manufacturer of the product |
| AE buffer | / | Takara |
| SYBR Green I dyes | / | Self-matching |
Table 65:
| name (R) | Model number | Manufacturer of the product |
| Real-Time System | CFX Connect | Bio-rad |
| Super clean bench | HDL | Beijing Dong gang haar Instrument manufacturing Co., Ltd |
The method comprises the following steps:
① samples were diluted with ultrapure water, and 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 at room temperature in dark for 30 min;
③ the detection is carried out on a computer, and the program is set to 20 ℃ for starting, the temperature is increased to 0.1-95 ℃ per second, and the reading is carried out once every 5 s.
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 a 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 × TBE Buffer (No RNAse) | / | Self-matching |
Table 76:
the method comprises the following steps:
① the DNA nanoparticles were diluted with ultrapure water according to the method of Table 77 below.
Table 77:
② mu.L (500ng) of the treated sample was mixed with 2. mu.L of 6 XDNA Loading Buffer, and the mixture was marked by ice-washing.
③ samples were run on 8% native PAGE gels at different incubation times and 12. mu.L of the treated samples were loaded in their entirety and programmed for 40min at 100V.
④ after the glue running, dyeing, placing on a horizontal shaking table for 30min, and taking pictures.
And (3) detection results:
the results of the native PAGE running gel of 7 sets of extended stretch-deformed + core short sequence DNA self-assembled 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.
The results in fig. 24 clearly show that the bands of the 7 sets of the products of the self-assembly of the DNA with the extended segment and the core short sequence are bright and clear, which indicates that the 7 sets of the DNA chains with the extended segment and the core short sequence complete the self-assembly and form a stable nanoparticle structure.
(2) 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 MeasUre € ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results at 25 ℃ of 7 groups of extension segment deformation and core short sequence DNA nanoparticles are as follows:
table 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 the extended section deformation and core short sequence DNA nano-particles have good stability, and further show that the nano-particles formed by self-assembly of the extended section deformation and the core short sequence DNA have a stable self-assembly structure.
(3) Particle size measurement
① preparing potential samples (7 groups of extension segment deformation + core short sequence DNA) to be placed in the sample cell, opening the sample cell cover of the instrument, and placing the instrument;
② opens the software, clicks the menu, and a manual measurement parameter setting dialog box appears;
③ setting software detection parameters;
④ click ok setting, appear measurement dialog, click Start, DLS measurements of hydrodynamic size of 7 sets of extended stretch variants + core short sequence RNA result 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 and core short sequence DNA nano-particles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 86:
| name of reagent | Goods number | Manufacturer of the product |
| AE buffer | / | Takara |
| SYBR Green I dyes | / | Self-matching |
Table 87:
| name (R) | Model number | Manufacturer of the product |
| Real-Time System | CFX Connect | Bio-rad |
| Super clean bench | HDL | Beijing Dong gang haar Instrument manufacturing Co., Ltd |
The method comprises the following steps:
② samples were diluted with ultrapure water, and 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 88:
② incubating at room temperature in dark for 30 min;
③ the detection is carried out on a computer, and the program is set to 20 ℃ for starting, the temperature is increased to 0.1-95 ℃ per second, and the reading is carried out once every 5 s.
And (3) detection results:
the TM values of 7 sets of extended stretch modified + core short sequence DNA nanoparticles are shown in FIG. 25 for the dissolution profile of D-8, FIG. 26 for the dissolution profile of D-9, FIG. 27 for the dissolution profile of D-10, FIG. 28 for the dissolution profile of D-11, FIG. 29 for the dissolution profile of D-12, FIG. 30 for the dissolution profile of D-13, and FIG. 31 for the dissolution profile of D-14.
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 × TBE Buffer (No RNAse) | / | Self-matching |
| Serum (FBS) | / | Excel |
| RPMI 1640 | / | GBICO |
Table 91:
the method comprises the following steps:
① preparing RNA nano particles to the concentration shown in the table below, diluting the prepared sample by the method shown in the table below, diluting the sample by 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
table 92:
② mixing 10 μ L of the treated sample with 2 μ L of 6 × DNA Loading Buffer, and labeling on ice;
③ taking 8% non-denaturing PAGE gel, applying a piece of gel to samples with different incubation times, completely applying 12 μ L of the treated samples, and setting the program to 100V for gel running for 40 min;
④ after the gum running, dyeing, placing on a horizontal shaking table, slowly shaking for 30min, and taking pictures.
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 0 min. From the results of the serum stability test, it can be seen that: the non-denatured gel fruits of 10min, 1h, 12h and 36h show that there is no obvious difference in the RNA nanoparticle sample bands at different times, which indicates that the RNA nanoparticles R-15 to R-21 are relatively stable in 1640 medium of 50% FBS without obvious degradation.
Example 12
The stability of 7 groups of extended length modified + core short sequence DNA nanoparticles in serum was characterized by non-denaturing PAGE.
The main reagents and instruments were as follows:
table 93:
| 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 |
| Serum (FBS) | / | Excel |
| RPMI 1640 | / | GBICO |
Table 94:
the method comprises the following steps:
② preparing DNA nanoparticles to the concentrations shown in the following table, diluting the prepared samples according to the method shown in the following table, diluting the samples by 5 tubes, and carrying out water bath on the diluted samples at 37 ℃ for different times (0, 10min, 1h, 12h and 36 h);
table 95:
② mixing 5 μ L of the treated sample with 1 μ L of 6 × DNA Loading Buffer, and labeling on ice;
③ taking 8% non-denaturing PAGE gel, applying a piece of gel to samples with different incubation times, applying 6 μ L of the treated samples, and setting the program to 100V for gel running for 40 min;
④ after the gum running, dyeing, placing on a horizontal shaking table, slowly shaking for 30min, and taking pictures.
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 0 min. From the results of the serum stability test, it can be seen that: the 10min, 1h, 12h and 36h non-denatured gel fruits showed no significant difference in the bands of the DNA nanoparticle samples at different times, indicating that the DNA nanoparticles D-8 to D-14 were relatively stable in 1640 medium with 50% FBS and had no significant degradation.
Nucleic acid nanoparticle-carried drug assay
Example 13
Doxorubicin mounting experiment:
according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), RNA nanoparticles formed by self-assembly of R-15, R-16, R-17, R-18, R-19, R-20 and R-21 in the previous example 9, and DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in example 10 were used as doxorubicin-carrying carriers, and the doxorubicin-carrying rates were respectively measured as follows:
the adriamycin loading rate of the RNA nano-particle R-15 is 20.5;
the adriamycin loading rate of the RNA nano-particle R-16 is 29.4;
the adriamycin loading rate of the RNA nano-particle R-17 is 30.9;
the adriamycin loading rate of the RNA nano-particle R-18 is 34.1;
the adriamycin loading rate of the RNA nano-particle R-19 is 27.1;
the adriamycin loading rate of the RNA nano-particle R-20 is 30.2;
the adriamycin loading rate of the RNA nano-particle R-21 is 20.1;
the adriamycin loading rate of the DNA nano-particle D-8 is 28.0;
the adriamycin loading rate of the DNA nano-particle D-9 is 27.9;
the adriamycin loading rate of the DNA nano-particle D-10 is 18.9;
the adriamycin loading rate of the DNA nano-particle D-11 is 26.8;
the adriamycin loading rate of the DNA nano-particle D-12 is 27.6;
the adriamycin loading rate of the DNA nano-particle D-13 is 31.8;
the adriamycin loading rate of the DNA nanoparticle D-14 was 32.
Flow cytometry (FACS) experiment for detecting cell binding capacity of DNA nanoparticles and carrier drug
Example 14
First, cell information
HepG2 (Source synergy cell bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), culture conditions at 37 ℃ and 5% CO2And saturation humidity.
Second, the object to be measured
Blank vector: d-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the aforementioned example 10 were self-assembled to form DNA nanoparticle carriers.
Carrier drug: according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the previous example 10 are used to carry doxorubicin, which is respectively marked as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin.
Third, main equipment, consumable
Table 96:
| manufacturer of the product | Model number | |
| Biological safety cabinet | Beijing Dong gang haar Instrument manufacturing Co Ltd | BSC-1360ⅡA2 |
| Low-speed centrifuge | Zhongke Zhongjia Instrument Co Ltd | SC-3612 |
| CO2Culture box | Thermo | 3111 |
| Inverted microscope | UOP | DSZ2000X |
| Flow cytometer | BD | BD FACSCaliburTM |
Four, main reagent
Table 97:
| name of reagent | Manufacturer of the product | Goods number | Remarks for note | |
| DMEM (Biotin free) | Providing all the | YS3160 | ||
| 1％BSA-PBS | Self-matching | － |
And fifthly, an experimental method:
1. adjusting the cell state to logarithmic phase, changing the culture medium to a biotin-free and folic acid-free culture medium, and placing the culture medium in an incubator at 37 ℃ for overnight incubation;
2. after incubation, cell suspension was collected by trypsinization, centrifuged at 1000rmp for 5min, adjusted in concentration, and 2X10 cells were collected5-5×105cells/EP tube, wash 2 times with 1 mL/tube of 1% BSA-PBS, and observe the tube bottom cells to prevent aspiration.
3. Dissolving the object to be tested, and diluting the object to be tested to the use concentration;
4. completely sucking cell supernatant, sequentially adding 100 mu L of corresponding samples into each tube, keeping out of the sun, and incubating for 2h at 37 ℃;
5. washed 2 times with 1% BSA-PBS; centrifuging at 1000rmp for 5 min;
6. finally, resuspending the cell pellet with 300. mu.L PBS and detecting it on flow machine (the blank vector used in this example was labeled by Quasar670, whereas doxorubicin in the vector drug was self-fluorescent and thus could be detected by FL4-APC and FL2-PE, respectively);
7. and (6) analyzing the data.
Sixth, experimental results
1. The results of the experiment are shown in the following table:
table 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 drug) and D-9 (blank vector), the binding rate is very high (88.6% -98.1%).
After incubation of HepG2 cells with D-10-adriamycin (vector drug) and D-10 (blank vector), the binding rate is high (89.4% -98.3%).
After incubation of HepG2 cells with D-11-adriamycin (vector medicine) and D-11 (blank vector), the binding rate is high (89.3% -97.8%).
After incubation of HepG2 cells with D-12-adriamycin (vector drug) and D-12 (blank vector), the binding rate is very high (94.6% -97.1%).
After incubation of HepG2 cells with D-13-adriamycin (vector drug) and D-13 (blank vector), the binding rate is high (89.6% -98.2%).
After incubation of HepG2 cells with D-14-adriamycin (vector drug) and D-14 (blank vector), the binding rate is very high (90.3% -98.3%).
Study of cytotoxicity of DNA nanoparticles and vector drugs in HepG2 cells
Example 15
The toxicity of the DNA nanoparticles and the carrier drug to HepG2 is detected by a CCK8 method.
First, main reagent
Table 99:
| name of reagent | Manufacturer of the product | Goods number |
| PBS | － | － |
| DMSO | SIGMA | D2650 |
| DMEM (Biotin free) | Providing all the drugs Zhida | 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 | Model number |
| 96-well cell culture plate | NEST | 701001 |
| Biological safety cabinet | Beijing Dong gang haar Instrument manufacturing Co Ltd | BSC-1360ⅡA2 |
| Low-speed centrifuge | Zhongke Zhongjia Instrument Co Ltd | SC-3612 |
| CO2Culture box | Thermo | 3111 |
| Inverted microscope | UOP | DSZ2000X |
| Enzyme-linked immunosorbent assay (ELISA) instrument | Shanghai Ou Ying laboratory Equipment Ltd | K3 |
Information on cells
HepG2 (Source synergy cell bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), culture conditions at 37 ℃ and 5% CO2And saturation humidity.
Fourth, experimental materials
1. Sample to be tested
Blank vector: the DNA nanoparticle carriers formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the foregoing example 10 are respectively denoted as: d-8, D-9, D-10, D-11, D-12, D-13 and D-14.
Carrier drug: according to the chemical method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the previous example 10 are used to carry doxorubicin, which is respectively marked as D-8-doxorubicin, D-9-doxorubicin, D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin and D-14-doxorubicin.
The original drug substance doxorubicin.
DMSO。
Fifth, the experimental procedure
1.HepG2 cells were harvested in the logarithmic growth phase, the Cell viability was 98.3% by trypan blue staining, and the cells were plated at 5000 cells/well in a volume of 100. mu.L/well in 8 96-well plates, 57 wells per plate, and incubated overnight at 37 ℃.
2. The samples to be tested were diluted and added as follows: removing 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 loaded | 10μM | 3.16μM | 1μM | 316nM | 100nM | 31.6nM | 10nM | 3.16nM | 1nM |
| Final concentration of empty vector | 1μM | 316nM | 100nM | 31.6nM | 10nM | 3.16nM | 1nM | 0.316nM | 0.1nM |
| Final concentration of parent doxorubicin | 10μM | 3.16μM | 1μM | 316nM | 100nM | 31.6nM | 10nM | 3.16nM | 1nM |
| DMSO(％) | 0.1 | 0.0316 | 0.01 | 0.00316 | 0.001 | 0.00036 | 0.0001 | 0.000036 | 0.00001 |
In this example, the drug-loaded and blank vehicles were each prepared as 100 μ M stock solutions in PBS and then diluted in complete medium (no biotin DMEM). The original doxorubicin was first prepared with DMSO as 100 μ M stock solution and then diluted with complete medium (no biotin DMEM). DMSO was directly diluted with complete medium (biotin-free DMEM).
3. Adding a sample to be detected, and putting a 96-well plate into 5% CO at 37 DEG C2Incubate in incubator for 72 h.
4. The kit was removed and thawed at room temperature, and 10. mu.L of CCK-8 solution was added to each well, or CCK8 solution was mixed with the medium at a ratio of 1:9 and then added to the wells at a rate of 100. mu.L/well.
5. The incubation is continued for 4h in the cell culture box, and the time is determined according to the experimental conditions such as the type of the cells, the density of the cells and the like.
6. Absorbance was measured at 450nm with a microplate reader.
7. And (3) calculating: cell viability (%) (OD experimental-OD blank) × 100%/(OD control-OD blank), IC calculated from GraphPad Prism 5.050。
Sixth, experimental results
Table 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 cells500.2725. mu.M, 0.05087. mu.M, 0.0386, 0.03955, 0.04271, 0.02294, 0.03017 and 0.03458, respectively; IC of DMSO on HepG2 cells50Is composed of>0.1 percent; IC of HepG2 cells acted on by D-8 (blank vector), D-9 (blank vector), D-10 (blank vector), D-11 (blank vector), D-12 (blank vector), D-13 (blank vector) and D-14 (blank vector)50Are all made of>1 μ M. It shows that compared with the pure blank vectors of D-8, D-9, D-10, D-11, D-12, D-13 and D-14, the original drug adriamycin of the small molecular drug and the drug-carrying D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin are toxic to HepG2 cells of HepG2 cell line, and the carried medicines of D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin have obvious synergistic effect compared with the original medicine of adriamycin.
Example 16
According to the chemical method of the mounting method of example 5 (except for special limitation, the method is the same as example 5), the DNA nanoparticles formed by self-assembly of D-10 and D-14 in the previous example 10 were used as the daunorubicin mounting carrier. The absorbance of daunorubicin at 492nm was measured using a microplate reader, and a standard curve was plotted (as shown in FIG. 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 drug containing 10-hydroxycamptothecin is formed by hanging the small molecular drug 10-hydroxycamptothecin on the nucleic acid nanoparticle carrier provided by the application, so that the delivery stability of the 10-hydroxycamptothecin can be improved, and under the condition that the nucleic acid nanoparticle carries a target head, on one hand, the 10-hydroxycamptothecin is delivered to a target cell in a targeted mode, the bioavailability of the drug is improved, on the other hand, toxic and side effects on non-target cells or tissues are reduced due to targeted delivery, the local drug concentration is reduced, and 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> 10-hydroxycamptothecin-containing medicine, preparation method, pharmaceutical composition and application thereof
<130>PN114935BYZD
<141>2019-09-30
<150>201811161967.2
<151>2018-09-30
<160>171
<170>SIPOSequenceListing 1.0
<210>1
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a1 sequence
<400>1
<210>2
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>mRNA
<222>(1)..(10)
<223> a1 sequence
<400>2
<210>3
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b1 sequence
<400>3
<210>4
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b1 sequence
<400>4
<210>5
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c1 sequence
<400>5
<210>6
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c1 sequence
<400>6
<210>7
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>7
<210>8
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>8
<210>9
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>9
<210>10
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>10
<210>11
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>11
<210>12
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>12
<210>13
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>13
<210>14
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>14
<210>15
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>15
<210>16
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>16
<210>17
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>17
<210>18
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>18
<210>19
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>19
<210>20
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>20
<210>21
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>21
<210>22
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>22
<210>23
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>23
<210>24
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>24
<210>25
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>25
<210>26
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>26
<210>27
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>27
<210>28
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>28
<210>29
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>29
<210>30
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>30
<210>31
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>31
<210>32
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>32
<210>33
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>33
<210>34
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>34
<210>35
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>35
<210>36
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>36
<210>37
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>37
<210>38
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>38
<210>39
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>39
<210>40
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>40
<210>41
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>41
<210>42
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>42
<210>43
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>43
<210>44
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>44
<210>45
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>45
<210>46
<211>10
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a sequence
<400>46
<210>47
<211>9
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b sequence
<400>47
<210>48
<211>12
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c sequence
<400>48
<210>49
<211>77
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(77)
<223> a sequence
<220>
<221>misc_feature
<222>(1)..(77)
<223> wherein M is U or T
<400>49
cgcgcgaaaa aacgcgcgaa aaaacgcgcg cccaccagcg mmccgggcgc gcgaaaaaac 60
gcgcgaaaaa acgcgcg 77
<210>50
<211>75
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(75)
<223> b sequence
<220>
<221>misc_feature
<222>(1)..(75)
<223> wherein M is U or T
<400>50
cgcgcgmmmm mmcgcgcgmm mmmmcgcgcg cccggmmcgc cgccagccgc cmmmmmmgcc 60
gccmmmmmmg ccgcc 75
<210>51
<211>78
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(78)
<223> c sequence
<220>
<221>misc_feature
<222>(1)..(78)
<223> wherein M is U or T
<400>51
ggcggcaaaa aaggcggcaa aaaaggcggc aggcggcama gcggmgggcg cgcgmmmmmm 60
cgcgcgmmmm mmcgcgcg 78
<210>52
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<220>
<221>modified_base
<222>(1)..(1)
<223>5'Biotin
<400>52
cgcgcgccca ccagcguucc gggcgccgc 29
<210>53
<211>27
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<220>
<221>modified_base
<222>(1)..(1)
<223>5'Biotin
<400>53
gcggcgcccg guucgccgcc aggcggc 27
<210>54
<211>31
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<220>
<221>modified_base
<222>(1)..(1)
<223>5'CY3
<400>54
gccgccaggc ggccauagcg gugggcgcgc g 31
<210>55
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>55
<210>56
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>56
<210>57
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>57
<210>58
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>58
<210>59
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>59
<210>60
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>60
<210>61
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>61
<210>62
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>62
<210>63
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>63
<210>64
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>64
<210>65
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>65
<210>66
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>66
<210>67
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>67
<210>68
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>68
<210>69
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>69
<210>70
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>70
<210>71
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>71
<210>72
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>72
<210>73
<211>10
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(10)
<223> a chain
<400>73
<210>74
<211>9
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(9)
<223> b chain
<400>74
<210>75
<211>12
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(12)
<223> c chain
<400>75
<210>76
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>76
cgcgcgccca ggagcguugg cgggcggcg 29
<210>77
<211>27
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>77
cgccgcccgc cuucgccgcc agccgcc 27
<210>78
<211>31
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>78
ggcggcaggc ggccauagcc cugggcgcgc g 31
<210>79
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>79
cgcgcgccca gcagcguucg cgggcggcg 29
<210>80
<211>27
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>80
cgccgcccgc guucgccgcc agccgcc 27
<210>81
<211>31
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>81
ggcggcaggc ggccauagcg cugggcgcgc g 31
<210>82
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>82
cgcgcgccca cgagcguugc ggggcggcg 29
<210>83
<211>27
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>83
cgccgccccg cuucgccgcc agccgcc 27
<210>84
<211>31
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>84
ggcggcaggc ggccauagcc gugggcgcgc g 31
<210>85
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>85
cgcgcgccca ggagcguugg cccgcggcg 29
<210>86
<211>27
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>86
cgccgcgggc cuucggggcc agccgcc 27
<210>87
<211>31
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>87
ggcggcaggc ccccauagcc cugggcgcgc g 31
<210>88
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>88
cgcgcgccca gcagcguucg ccccgccgc 29
<210>89
<211>27
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>89
gcggcggggc guucggcggc aggcggc 27
<210>90
<211>31
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>90
gccgccagcc gcccauagcg cugggcgcgc g 31
<210>91
<211>29
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>91
cgcgcgccca gcagcguucg gggcgccgc 29
<210>92
<211>28
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(28)
<223> b chain
<400>92
gcggcgcccc guucggccgg caggcggc 28
<210>93
<211>32
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(32)
<223> c chain
<400>93
gccgccagcc ggcccauagc gcugggcgcg cg 32
<210>94
<211>40
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(40)
<223> a chain
<400>94
cgcgcgcgag cguugcaaug acagauaagg aaccugcutt 40
<210>95
<211>36
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(36)
<223> b chain
<400>95
ggcagguucc uuaucuguca aagcuucggc ggcagc 36
<210>96
<211>23
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(23)
<223> c chain
<400>96
gcagccgccc auagccgcgc gcg 23
<210>97
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(39)
<223>EGFRapt
<400>97
gccttagtaa cgtgctttga tgtcgattcg acaggaggc 39
<210>98
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(41)
<223>PSMAapt
<400>98
gggccgaaaa agacctgact tctatactaa gtctacgtcc c 41
<210>99
<211>68
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(68)
<223> a chain
<400>99
cgcgcgccca ggagcgttgg cgggcggcgg ccttagtaac gtgctttgat gtcgattcga 60
caggaggc 68
<210>100
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>100
cgccgcccgc cttcgccgcc agccgcc 27
<210>101
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>101
ggcggcaggc ggccatagcc ctgggcgcgc g 31
<210>102
<211>68
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(68)
<223> a chain
<400>102
cgcgcgccca gcagcgttcg cgggcggcgg ccttagtaac gtgctttgat gtcgattcga 60
caggaggc 68
<210>103
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>103
cgccgcccgc gttcgccgcc agccgcc 27
<210>104
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>104
ggcggcaggc ggccatagcg ctgggcgcgc g 31
<210>105
<211>68
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(68)
<223> a chain
<400>105
cgcgcgccca cgagcgttgc ggggcggcgg ccttagtaac gtgctttgat gtcgattcga 60
caggaggc 68
<210>106
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>106
cgccgccccg cttcgccgcc agccgcc 27
<210>107
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>107
ggcggcaggc ggccatagcc gtgggcgcgc g 31
<210>108
<211>71
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(71)
<223> a chain
<400>108
cgcgcgccca ggagcgttgg cccgcggcgt gggccgaaaa agacctgact tctatactaa 60
gtctacgtcc c 71
<210>109
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>109
cgccgcgggc cttcggggcc agccgcc 27
<210>110
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>110
ggcggcaggc ccccatagcc ctgggcgcgc g 31
<210>111
<211>71
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(71)
<223> a chain
<400>111
cgcgcgccca gcagcgttcg ccccgccgct gggccgaaaa agacctgact tctatactaa 60
gtctacgtcc c 71
<210>112
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>112
gcggcggggc gttcggcggc aggcggc 27
<210>113
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>113
gccgccagcc gcccatagcg ctgggcgcgc g 31
<210>114
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>114
cgcgcgccca gcagcgttcg gggcgccgc 29
<210>115
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(20)
<223> b chain
<400>115
gcggcgcccc gttcggccgg caggcggc 28
<210>116
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(32)
<223> c chain
<400>116
gccgccagcc ggcccatagc gctgggcgcg cg 32
<210>117
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(29)
<223> a chain
<400>117
cgcgcgccca cgagcgttgc gggcgccgc 29
<210>118
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(27)
<223> b chain
<400>118
gcggcgcccg cttcggcggc aggcggc 27
<210>119
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(1)..(31)
<223> c chain
<400>119
gccgccagcc gcccatagcc gtgggcgcgc g 31
<210>120
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>120
gcggcgagcg gcgaggagcg uuggggccgg aggccgg 37
<210>121
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> b chain
<400>121
ccggccuccg gccccuucgg ggccagccgc c 31
<210>122
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>122
ggcggcaggc ccccauagcc cucgccgcuc gccgc 35
<210>123
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>123
gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37
<210>124
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>124
ccggccuccg gcccguucgc cgccagccgc c 31
<210>125
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>125
ggcggcaggc ggccauagcg cucgccgcuc gccgc 35
<210>126
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>126
gcggcgagcg gcgaggagcg uuggggccgg aggccgg 37
<210>127
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>127
ccggccuccg gccccuucgc cgccagccgc c 31
<210>128
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>128
ggcggcaggc ggccauagcc cucgccgcuc gccgc 35
<210>129
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>129
gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37
<210>130
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>130
ccggccuccg gcccguucgg cgccagccgc c 31
<210>131
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>131
ggcggcaggc gcccauagcg cucgccgcuc gccgc 35
<210>132
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>132
gcggcgagcg gcgagcagcg uucgggccgg aggccgg 37
<210>133
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>133
ccggccuccg gcccguucgg ccccagccgc c 31
<210>134
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>134
ggcggcaggg gcccauagcg cucgccgcuc gccgc 35
<210>135
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>135
gcggcgagcg gcgacgagcg uugcggccgg aggccgg 37
<210>136
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>136
ccggccuccg gccgcuucgc cgccagccgc c 31
<210>137
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>137
ggcggcaggc ggccauagcc gucgccgcuc gccgc 35
<210>138
<211>37
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>138
gcggcgagcg gcgacgagcg uugcggccgg aggccgg 37
<210>139
<211>31
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>139
ccggccuccg gccgcuucgg cgccagccgc c 31
<210>140
<211>35
<212>RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>140
ggcggcaggc gcccauagcc gucgccgcuc gccgc 35
<210>141
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>141
gcggcgagcg gcgaggagcg ttggggccgg aggccgg 37
<210>142
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>142
ccggcctccg gccccttcgg ggccagccgc c 31
<210>143
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>143
ggcggcaggc ccccatagcc ctcgccgctc gccgc 35
<210>144
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>144
gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37
<210>145
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>145
ccggcctccg gcccgttcgc cgccagccgc c 31
<210>146
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>146
ggcggcaggc ggccatagcg ctcgccgctc gccgc 35
<210>147
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>147
gcggcgagcg gcgaggagcg ttggggccgg aggccgg 37
<210>148
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>148
ccggcctccg gccccttcgc cgccagccgc c 31
<210>149
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>149
ggcggcaggc ggccatagcc ctcgccgctc gccgc 35
<210>150
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>150
gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37
<210>151
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>151
ccggcctccg gcccgttcgg cgccagccgc c 31
<210>152
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>152
ggcggcaggc gcccatagcg ctcgccgctc gccgc 35
<210>153
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>153
gcggcgagcg gcgagcagcg ttcgggccgg aggccgg 37
<210>154
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>154
ccggcctccg gcccgttcgg ccccagccgc c 31
<210>155
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>155
ggcggcaggg gcccatagcg ctcgccgctc gccgc 35
<210>156
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>156
gcggcgagcg gcgacgagcg ttgcggccgg aggccgg 37
<210>157
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>157
ccggcctccg gccgcttcgc cgccagccgc c 31
<210>158
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>158
ggcggcaggc ggccatagcc gtcgccgctc gccgc 35
<210>159
<211>37
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(37)
<223> a chain
<400>159
gcggcgagcg gcgacgagcg ttgcggccgg aggccgg 37
<210>160
<211>31
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> b chain
<400>160
ccggcctccg gccgcttcgg cgccagccgc c 31
<210>161
<211>35
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(35)
<223> c chain
<400>161
ggcggcaggc gcccatagcc gtcgccgctc gccgc 35
<210>162
<211>14
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(14)
<223> first extension segment
<400>162
<210>163
<211>14
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(14)
<223> first extension segment
<400>163
<210>164
<211>13
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(13)
<223> first extension segment
<400>164
<210>165
<211>13
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(13)
<223> first extension segment
<400>165
<210>166
<211>9
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(9)
<223> first extension segment
<400>166
<210>167
<211>9
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(9)
<223> first extension segment
<400>167
<210>168
<211>14
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(14)
<223> first extension segment
<400>168
<210>169
<211>14
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(14)
<223> first extension segment
<400>169
<210>170
<211>13
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(13)
<223> first extension segment
<400>170
<210>171
<211>13
<212>DNA/RNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(1)..(13)
<223> first extension segment
<400>171
Claims (20)
1. A drug containing 10-hydroxycamptothecin, which is characterized in that the drug comprises nucleic acid nanoparticles and 10-hydroxycamptothecin, and the 10-hydroxycamptothecin 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 a1 sequence or a sequence a1 sequence with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 sequence with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 sequence with at least one base insertion, deletion or substitution;
wherein the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively;
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' are provided.
2. The drug of claim 1, wherein 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 having at least one base insertion, deletion, or substitution.
3. The medicament of claim 1 or 2, wherein the base insertion, deletion or substitution occurs at:
(1) 1, 2, 4 or 5 bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(2) Between 8 th to 10 th bases from the 5' end of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and/or
(3) Between the 1 st to 3 rd bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(4) Between 6 th to 9 th bases from the 5' end of the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; and/or
(5) Between the 1 st to 4 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; and/or
(6) Between the 9 th to 12 th bases from the 5' end of the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.
4. The drug of claim 1 or 2, wherein the a sequence, the b sequence and the c sequence self-assemble into a structure represented by formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position is independently selected from C-G or G-C;
in the a sequence, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G;
in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C;
in the c sequence, the NNNN sequence from the 5 'end to the 3' end is CAUA or CATA;
preferably, the a sequence, the b sequence and the c sequence 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', respectively;
(2) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCGC-3', respectively;
(3) a sequence: 5'-CGAGCGUUGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCG-3', respectively;
(4) a sequence: 5'-GGAGCGUUGG-3' the flow of the air in the air conditioner,
b sequence: 5 '-CCUUCGGG-3',
c sequence: 5'-CCCCCAUAGCCC-3', respectively;
(5) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCGC-3', respectively;
(6) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGUUCGGCC-3',
c sequence: 5'-GGCCCAUAGCGC-3', respectively;
(7) a sequence: 5'-CGAGCGUUGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCCG-3', respectively;
(8) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,
b sequence: 5'-CCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCC-3', respectively;
(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', respectively;
(10) a sequence: 5'-CGAGCGTTGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3', respectively;
(11) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,
b sequence: 5'-CCTTCGGGG-3',
c sequence: 5'-CCCCCATAGCCC-3', respectively;
(12) a sequence: 5'-GCAGCGTTCG-3' the flow of the air in the air conditioner,
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', respectively;
(14) a sequence: 5'-CGAGCGTTGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCTTCGGCG-3',
c sequence: 5'-CGCCCATAGCCG-3' are provided.
5. The agent of claim 3, 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;
preferably, the first elongate section is selected from any one of the following:
(1): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3';
(2): a 3' end of the chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3';
(3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3';
(4): a 5' end of 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 chain: 5 '-TGGG-3';
(9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.
6. The agent of any one of claims 1 to 5, wherein the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any of the a, b, and c sequences, wherein the second extension is a Watson-Crick paired extension;
preferably, the second extension is an extended sequence of CG base pairs;
more preferably, the second extension is an extension sequence of 1-10 CG base pairs;
further preferably, the nucleic acid domain further comprises at least one set of second stretches:
a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3';
second group: a 3' end of the chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3';
third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.
7. The drug of claim 6, wherein the second extension is an extension comprising both CG base pairs and AT/AU base pairs, preferably the second extension is an extension of 2-50 base pairs;
preferably, 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; or the second extension is an extension sequence formed by alternating sequences of 1 CG base pair and 1 AT/AU base pair.
8. The medicament of any one of claims 1 to 7, 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;
preferably, the a sequence, the b sequence and the C sequence have 2' -F modifications on the C or U bases.
9. The drug according to any one of claims 1 to 8, wherein the 10-hydroxycamptothecin is physically and/or covalently attached to the nucleic acid nanoparticles, and the molar ratio of the 10-hydroxycamptothecin to the nucleic acid nanoparticles is 2 to 300:1, preferably 10 to 50:1, more preferably 15 to 25: 1.
10. The drug of any one of claims 1 to 9, wherein the nucleic acid nanoparticle further comprises a biologically active substance attached to the nucleic acid domain, wherein the biologically active substance is one or more of a target, a fluorescein, an interfering nucleic acid siRNA, a miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, a protein, a polypeptide, a flavonoid, glucose, natural salicylic acid, a mab, a vitamin, a phenolic lecithin, and a small molecule drug other than 10-hydroxycamptothecin.
11. The agent of claim 10, wherein the relative molecular weight of the nucleic acid domains is recorded as N1The total relative molecular weight of 10-hydroxycamptothecin and the bioactive substance is recorded as N2，N1/N21:1 or more; preferably, the biologically active substance is one or more of the target, the fluorescein and the miRNA,
wherein the target head is located on any sequence of the a sequence, the b sequence and the c sequence, preferably on the 5 'end or the 3' end of any sequence 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 an 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;
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.
12. The drug of claim 10, wherein the small molecule drug other than 10-hydroxycamptothecin is a drug containing any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, benzene ring groups, and acetamido groups; preferably, 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.
13. The drug according to claim 1, wherein the nucleic acid nanoparticles have a particle size of 1 to 100nm, preferably 5 to 50 nm; more preferably 10 to 30 nm; further preferably 10 to 15 nm.
14. A method for preparing a 10-hydroxycamptothecin-containing medicament, which is characterized by comprising the following steps:
providing a nucleic acid nanoparticle in a medicament according to any one of claims 1 to 13;
the 10-hydroxycamptothecin is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the drug containing the 10-hydroxycamptothecin is obtained.
15. The method of claim 14, wherein the step of attaching 10-hydroxycamptothecin by physical linkage comprises:
mixing and stirring the 10-hydroxycamptothecin, the nucleic acid nanoparticles and the first solvent to obtain a premixed system;
precipitating the premixed system to obtain the medicine containing 10-hydroxycamptothecin;
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 10-hydroxycamptothecin comprises;
precipitating the premixed system to obtain a precipitate;
washing the precipitate to remove impurities to obtain the medicine containing 10-hydroxycamptothecin;
more preferably, the precipitation is carried out under the temperature condition of less than 10 ℃ after the premixing system is mixed with absolute ethyl alcohol, so as to obtain the precipitate; further preferably, the precipitation is carried out at the temperature of 0-5 ℃ to obtain the precipitate;
more preferably, 6-12 times of volume of absolute ethyl alcohol is adopted to wash the precipitate to remove impurities, and the medicine containing 10-hydroxycamptothecin is obtained.
16. The method of claim 14, wherein the step of loading the 10-hydroxycamptothecin by covalent linkage comprises:
preparing 10-hydroxycamptothecin solution;
reacting the 10-hydroxycamptothecin solution with the amino outside the G ring of the nucleic acid nanoparticles under the mediation of formaldehyde to obtain a reaction system;
purifying the reaction system to obtain the medicine containing 10-hydroxycamptothecin;
preferably, the step of reacting comprises:
mixing the 10-hydroxycamptothecine solution with a paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under a dark condition to obtain the reaction system; 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.
17. The production method according to any one of claims 14 to 16, further comprising a step of producing the nucleic acid nanoparticle, which comprises: obtaining the nucleic acid domain by self-assembling single strands corresponding to the nucleic acid domain in the medicament of any one of claims 1 to 13;
preferably, 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 10 to 12 on the nucleic acid domain by means of physical linkage and/or covalent linkage.
18. The method according to claim 16, wherein the biologically active substance is immobilized by covalent bonding via solvent covalent bonding, linker covalent bonding or click bonding;
preferably, the solvent is covalently linked using a third solvent 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;
preferably, the linker is selected from the group consisting of disulfide bond, p-azido, bromopropyne, or PEG;
preferably, the click-to-link is performed by performing an alkynyl or azide modification on the bioactive substance precursor and the nucleic acid domain at the same time, and then by click-to-link;
preferably, the biologically active substance is linked in a click-link to the nucleic acid domain, 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.
19. A pharmaceutical composition comprising a 10-hydroxycamptothecin-containing drug of any one of claims 1 to 13.
20. Use of a 10-hydroxycamptothecin-containing medicament of any one of claims 1 to 13 in the manufacture of a medicament for the treatment of liver cancer, gastric cancer, head and neck cancer or leukemia.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018111619672 | 2018-09-30 | ||
| CN201811161967 | 2018-09-30 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110960684A true CN110960684A (en) | 2020-04-07 |
| CN110960684B CN110960684B (en) | 2022-05-20 |
Family
ID=70029808
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910945833.8A Active CN110960684B (en) | 2018-09-30 | 2019-09-30 | Medicine containing 10-hydroxycamptothecin, preparation method thereof, pharmaceutical composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110960684B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103403189A (en) * | 2011-06-08 | 2013-11-20 | 辛辛那提大学 | PRNA mutlivalent junction domain for use in stable multivalent RNA nanoparticles |
| CN104327141A (en) * | 2014-10-16 | 2015-02-04 | 上海交通大学 | RNA nanoparticle and application of RNA nanoparticle in preventing and curing stomach cancer |
| WO2016145008A2 (en) * | 2015-03-09 | 2016-09-15 | University Of Kentucky Research Foundation | Mirna for treatment of breast cancer |
| WO2016145005A1 (en) * | 2015-03-09 | 2016-09-15 | University Of Kentucky Research Foundation | Rna nanoparticles for brain tumor treatment |
| CN107614685A (en) * | 2015-04-17 | 2018-01-19 | 肯塔基大学研究基金会 | RNA nano particles and its application method |
-
2019
- 2019-09-30 CN CN201910945833.8A patent/CN110960684B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103403189A (en) * | 2011-06-08 | 2013-11-20 | 辛辛那提大学 | PRNA mutlivalent junction domain for use in stable multivalent RNA nanoparticles |
| CN104327141A (en) * | 2014-10-16 | 2015-02-04 | 上海交通大学 | RNA nanoparticle and application of RNA nanoparticle in preventing and curing stomach cancer |
| WO2016145008A2 (en) * | 2015-03-09 | 2016-09-15 | University Of Kentucky Research Foundation | Mirna for treatment of breast cancer |
| WO2016145005A1 (en) * | 2015-03-09 | 2016-09-15 | University Of Kentucky Research Foundation | Rna nanoparticles for brain tumor treatment |
| CN107614685A (en) * | 2015-04-17 | 2018-01-19 | 肯塔基大学研究基金会 | RNA nano particles and its application method |
Non-Patent Citations (2)
| Title |
|---|
| FENGMEI PI, PHD,ET AL.: "RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery", 《NANOMEDICINE: NANOTECHNOLOGY, BIOLOGY, AND MEDICINE》 * |
| SIJIN GUO,ET AL: "Methods for Construction and Characterization of Simple or Special Multifunctional RNA Nanoparticles Based on the 3WJ of Phi29 DNA Packaging Motor", 《METHODS》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110960684B (en) | 2022-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220226473A1 (en) | Nucleic acid nanocarrier drug and preparation method thereof, pharmaceutical composition and application thereof | |
| TWI743518B (en) | Nucleic acid nanoparticles, pharmaceutical composition thereof, medicine comprising doxorubicin and preparation method thereof | |
| CN110711253B (en) | Medicine containing adriamycin and preparation method thereof | |
| CN110960534B (en) | Medicine containing pentafluorouracil, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960683B (en) | Medicine containing methotrexate, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960530B (en) | Tacrine-containing medicine, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111150852B (en) | Cisplatin-containing medicine, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111096965B (en) | Medicine containing dihydroartemisinin, preparation method thereof, medicine composition and application thereof | |
| CN111053915B (en) | Lenalidomide-containing medicine, preparation method thereof, medicine composition and application | |
| CN110960684B (en) | Medicine containing 10-hydroxycamptothecin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960691B (en) | Gemcitabine-containing medicine, preparation method thereof, pharmaceutical composition and application | |
| CN110960689B (en) | Medicine containing daunorubicin, preparation method thereof, medicine composition and application thereof | |
| CN110960536B (en) | Aspirin-containing medicine, preparation method, pharmaceutical composition and application thereof | |
| CN111053765B (en) | Medicine containing paclitaxel, preparation method, pharmaceutical composition and application thereof | |
| CN111068065B (en) | Drug containing oxaliplatin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111097051B (en) | Medicine containing vincristine, preparation method thereof, medicine composition and application thereof | |
| CN110960690B (en) | Medicine containing epirubicin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111166894B (en) | Docetaxel-containing medicine, preparation method thereof, medicine composition and application | |
| CN111053919B (en) | Medicine containing idarubicin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111053914B (en) | Mitoxantrone-containing medicine, preparation method thereof, medicine composition and application thereof | |
| CN110960542B (en) | Medicament containing pirarubicin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111053913B (en) | Cytarabine-containing medicine, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110711254B (en) | Nucleic acid nanoparticles and pharmaceutical compositions comprising the same | |
| CN111084887B (en) | Flavone-containing medicine, preparation method thereof, medicine composition and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: Room 403, 4th Floor, Room 102206, Unit 6-12, 1st Floor, No. 8 Life Park Road, Life Science Park, Changping District, Beijing, China Patentee after: Baiyao Zhida (Beijing) nano Biotechnology Co.,Ltd. Country or region after: China Address before: 100080 8-1-1, floor 8, No. 21, Haidian South Road, Haidian District, Beijing Patentee before: Baiyao Zhida (Beijing) nano Biotechnology Co.,Ltd. Country or region before: China |