CN111053765B - Medicine containing paclitaxel, preparation method, pharmaceutical composition and application thereof - Google Patents
Medicine containing paclitaxel, preparation method, pharmaceutical composition and application thereof Download PDFInfo
- Publication number
- CN111053765B CN111053765B CN201910969109.9A CN201910969109A CN111053765B CN 111053765 B CN111053765 B CN 111053765B CN 201910969109 A CN201910969109 A CN 201910969109A CN 111053765 B CN111053765 B CN 111053765B
- Authority
- CN
- China
- Prior art keywords
- sequence
- nucleic acid
- paclitaxel
- chain
- drug
- 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.)
- Active
Links
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 194
- 239000003814 drug Substances 0.000 title claims abstract description 186
- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 173
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 title claims abstract description 173
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 229940079593 drug Drugs 0.000 title claims description 136
- 239000002105 nanoparticle Substances 0.000 claims abstract description 303
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 198
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 192
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 192
- 230000008685 targeting Effects 0.000 claims abstract description 31
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 278
- 238000000034 method Methods 0.000 claims description 68
- 239000000126 substance Substances 0.000 claims description 58
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 56
- 230000000975 bioactive effect Effects 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 239000002679 microRNA Substances 0.000 claims description 31
- 238000012986 modification Methods 0.000 claims description 31
- 230000004048 modification Effects 0.000 claims description 29
- 239000002904 solvent Substances 0.000 claims description 28
- 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 26
- 206010028980 Neoplasm Diseases 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 22
- 239000002953 phosphate buffered saline Substances 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 229940088623 biologically active substance Drugs 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 21
- 239000002245 particle Substances 0.000 claims description 19
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 18
- 108091070501 miRNA Proteins 0.000 claims description 18
- 229920002866 paraformaldehyde Polymers 0.000 claims description 18
- 239000002244 precipitate Substances 0.000 claims description 18
- 230000001376 precipitating effect Effects 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 229940126586 small molecule drug Drugs 0.000 claims description 15
- 235000020958 biotin Nutrition 0.000 claims description 13
- 239000011616 biotin Substances 0.000 claims description 13
- 229960002685 biotin Drugs 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 13
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 11
- 108020004459 Small interfering RNA Proteins 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 125000000304 alkynyl group Chemical group 0.000 claims description 10
- 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 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
- 206010006187 Breast cancer Diseases 0.000 claims description 9
- 208000026310 Breast neoplasm Diseases 0.000 claims description 9
- 206010033128 Ovarian cancer Diseases 0.000 claims description 9
- 206010061535 Ovarian neoplasm Diseases 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
- 239000000203 mixture Substances 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 235000013824 polyphenols Nutrition 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 150000001540 azides Chemical class 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- 239000002243 precursor Substances 0.000 claims description 7
- 229940088594 vitamin Drugs 0.000 claims description 7
- 229930003231 vitamin Natural products 0.000 claims description 7
- 235000013343 vitamin Nutrition 0.000 claims description 7
- 239000011782 vitamin Substances 0.000 claims description 7
- 125000003277 amino group Chemical group 0.000 claims description 6
- 235000019152 folic acid Nutrition 0.000 claims description 6
- 239000011724 folic acid Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 6
- 108091023037 Aptamer Proteins 0.000 claims description 5
- 102000053642 Catalytic RNA Human genes 0.000 claims description 5
- 108090000994 Catalytic RNA Proteins 0.000 claims description 5
- 108020004422 Riboswitch Proteins 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000001404 mediated effect Effects 0.000 claims description 5
- 229920001184 polypeptide Polymers 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 108091092562 ribozyme Proteins 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- XXFUZSHTIOFGNV-UHFFFAOYSA-N 1-bromoprop-1-yne Chemical compound CC#CBr XXFUZSHTIOFGNV-UHFFFAOYSA-N 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 4
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims description 4
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims description 4
- 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 4
- 229930003935 flavonoid Natural products 0.000 claims description 4
- 150000002215 flavonoids Chemical class 0.000 claims description 4
- 235000017173 flavonoids Nutrition 0.000 claims description 4
- 229960000304 folic acid Drugs 0.000 claims description 4
- 230000002452 interceptive effect Effects 0.000 claims description 4
- 229940067606 lecithin Drugs 0.000 claims description 4
- 239000000787 lecithin Substances 0.000 claims description 4
- 235000010445 lecithin Nutrition 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
- 150000002989 phenols Chemical class 0.000 claims description 4
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 4
- 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
- 125000000852 azido group Chemical group *N=[N+]=[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
- 239000008103 glucose Substances 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 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
- 238000003756 stirring Methods 0.000 claims description 3
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 claims description 3
- 239000002390 adhesive tape Substances 0.000 claims 7
- 239000000758 substrate Substances 0.000 claims 7
- 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
- 238000003780 insertion Methods 0.000 abstract description 20
- 230000037431 insertion Effects 0.000 abstract description 20
- 238000012217 deletion Methods 0.000 abstract description 14
- 230000037430 deletion Effects 0.000 abstract description 14
- 238000006467 substitution reaction Methods 0.000 abstract description 14
- 230000008901 benefit Effects 0.000 abstract description 5
- 108020004414 DNA Proteins 0.000 description 263
- 238000001338 self-assembly Methods 0.000 description 131
- 210000004027 cell Anatomy 0.000 description 114
- 238000001514 detection method Methods 0.000 description 71
- 239000000047 product Substances 0.000 description 67
- 239000000523 sample Substances 0.000 description 64
- 238000011068 loading method Methods 0.000 description 50
- 239000013598 vector Substances 0.000 description 48
- 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 46
- 238000001962 electrophoresis Methods 0.000 description 46
- 229940009456 adriamycin Drugs 0.000 description 37
- 238000011534 incubation Methods 0.000 description 35
- 238000004090 dissolution Methods 0.000 description 31
- 229960004679 doxorubicin Drugs 0.000 description 31
- 239000000499 gel Substances 0.000 description 30
- 230000000694 effects Effects 0.000 description 27
- 238000005259 measurement Methods 0.000 description 27
- 239000003153 chemical reaction reagent Substances 0.000 description 26
- 210000002966 serum Anatomy 0.000 description 26
- 238000002474 experimental method Methods 0.000 description 24
- 230000002829 reductive effect Effects 0.000 description 20
- 238000009739 binding Methods 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 239000000872 buffer Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 238000000246 agarose gel electrophoresis Methods 0.000 description 12
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 239000000969 carrier Substances 0.000 description 11
- 239000012091 fetal bovine serum Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 239000002539 nanocarrier Substances 0.000 description 11
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 11
- 239000012634 fragment Substances 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 238000001816 cooling Methods 0.000 description 9
- 238000007865 diluting Methods 0.000 description 9
- 239000012160 loading buffer Substances 0.000 description 9
- 229910021642 ultra pure water Inorganic materials 0.000 description 9
- 239000012498 ultrapure water Substances 0.000 description 9
- 238000004113 cell culture Methods 0.000 description 8
- 230000000295 complement effect Effects 0.000 description 8
- 238000005520 cutting process Methods 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 8
- 238000010438 heat treatment Methods 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 239000002086 nanomaterial Substances 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
- 238000002835 absorbance Methods 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 238000004043 dyeing Methods 0.000 description 7
- 239000012149 elution buffer Substances 0.000 description 7
- 239000011259 mixed solution 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
- 210000001519 tissue Anatomy 0.000 description 7
- 231100000419 toxicity Toxicity 0.000 description 7
- 230000001988 toxicity Effects 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
- 201000011510 cancer Diseases 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method 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
- 238000006731 degradation reaction Methods 0.000 description 6
- 238000003384 imaging method Methods 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
- 238000011160 research Methods 0.000 description 6
- 125000002091 cationic group Chemical group 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 239000012470 diluted sample Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 238000009830 intercalation Methods 0.000 description 5
- 230000002687 intercalation Effects 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 239000013603 viral vector Substances 0.000 description 5
- 108060006698 EGF receptor Proteins 0.000 description 4
- 102000001301 EGF receptor Human genes 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
- 239000012980 RPMI-1640 medium Substances 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
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- -1 diterpene alkaloid compound Chemical class 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 231100000252 nontoxic Toxicity 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
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 4
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 3
- 101000655352 Homo sapiens Telomerase reverse transcriptase Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 102000019197 Superoxide Dismutase Human genes 0.000 description 3
- 108010012715 Superoxide dismutase Proteins 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 229940045799 anthracyclines and related substance Drugs 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 239000004744 fabric Substances 0.000 description 3
- 201000010536 head and neck cancer Diseases 0.000 description 3
- 208000014829 head and neck neoplasm Diseases 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 239000000700 radioactive tracer Substances 0.000 description 3
- 239000011550 stock solution Substances 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
- 201000004384 Alopecia Diseases 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
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 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 2
- 108091060592 XDNA Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 238000012054 celltiter-glo Methods 0.000 description 2
- 230000008859 change 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
- 230000006378 damage Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 229940014144 folate Drugs 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 230000002163 immunogen 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
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 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
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000012265 solid product Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 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 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 229940126585 therapeutic drug Drugs 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
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 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
- 102000053602 DNA Human genes 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 241000713666 Lentivirus Species 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
- 206010028116 Mucosal inflammation Diseases 0.000 description 1
- 201000010927 Mucositis 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
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 101710163270 Nuclease 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
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 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
- 206010039966 Senile dementia Diseases 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 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
- 230000001154 acute effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 230000004071 biological effect Effects 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
- 238000004364 calculation method Methods 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 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
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 238000013270 controlled release 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
- 238000012258 culturing Methods 0.000 description 1
- 238000013211 curve analysis Methods 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
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process 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
- 229930002995 diterpene alkaloid Natural products 0.000 description 1
- 239000002552 dosage form Substances 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
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 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
- 210000005205 gut mucosa Anatomy 0.000 description 1
- 208000024963 hair loss Diseases 0.000 description 1
- 230000003676 hair loss Effects 0.000 description 1
- 238000003306 harvesting Methods 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
- 230000008629 immune suppression Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 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
- 208000032839 leukemia Diseases 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 230000007246 mechanism Effects 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
- 239000007758 minimum essential medium Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 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
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229940049954 penicillin Drugs 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
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000035755 proliferation Effects 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
- 238000000611 regression analysis Methods 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
- 235000009566 rice Nutrition 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000007675 toxicity by organ Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 231100000583 toxicological profile Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- 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
- 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
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)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Optics & Photonics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application provides a medicine containing paclitaxel, a preparation method, a pharmaceutical composition and application thereof. The medicine comprises nucleic acid nanoparticles and paclitaxel, and the paclitaxel 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 medicine containing paclitaxel provided by the application has the advantages that the nucleic acid structure domain of the medicine is modified by the target head, the targeting property is good, the paclitaxel 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 paclitaxel, a preparation method, a pharmaceutical composition and application thereof.
Background
Paclitaxel, also known as taxol, taxol and tertin, is the best natural anticancer drug found at present, and has been widely used for treating breast cancer, ovarian cancer, partial head and neck cancer and lung cancer in clinic. Paclitaxel as a diterpene alkaloid compound with anticancer activity has a novel and complex chemical structure, wide and remarkable biological activity, a novel and unique action mechanism and a scarce natural resource, so that the paclitaxel is greatly favored by botanicals, chemists, pharmacologists and molecular biologists, and becomes an anticancer star which draws attention in the next half of the 20 th century and a research focus.
Currently, antineoplastic drugs, including paclitaxel, must be administered at high doses of chemotherapeutic drugs in order to achieve effective therapeutic levels at the tumor site, but systemic administration of high doses may damage healthy normal cells and cause adverse effects in a range of tissues and organs. These adverse effects include immune system suppression (myelosuppression), inflammation and ulcers of the gut mucosa (mucositis), hair loss (alopecia) and organ-specific toxicity, such as cardiotoxicity and neurotoxicity. In order to avoid the adverse reactions, a tumor local administration mode needs to be used for replacing the traditional systemic administration mode so as to achieve the effects of increasing the tumor local drug concentration and reducing the systemic drug concentration. Therefore, how to achieve such local drug delivery and in vitro controlled release has become a focus of cancer chemotherapy research.
In order to reduce the side effect caused by poor targeting of the active ingredients of the medicine, the medicine delivery carrier is produced, and the function of the carrier is mainly to carry the active ingredients of the medicine and deliver the active ingredients into blood or tissue cells to treat diseases. There are a variety of approaches to achieve targeted delivery of different drugs. And is implemented by an instrument or apparatus, such as a gene gun, an electroporator, etc. The methods do not need to use a gene vector, but the transfection efficiency is generally low, the operation is complex, and the damage to tissues is large. It is also mediated by viral vectors such as adenovirus and lentivirus, which have high in vitro transfection activity, but have the disadvantages of immunogenicity and susceptibility to mutation, and thus, the in vivo delivery brings huge safety hazards. And non-viral vectors, especially biodegradable polymer materials are used for realizing the targeted transportation of the medicine. The 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 paclitaxel is one of the difficulties in solving the limited clinical application of the existing paclitaxel drug.
Disclosure of Invention
The main objective of the present application is to provide a drug containing paclitaxel, its preparation method, pharmaceutical composition and application, so as to improve the delivery reliability of paclitaxel drug.
In order to achieve the above objects, according to one aspect of the present application, there is provided a paclitaxel-containing drug, which comprises a nucleic acid nanoparticle and paclitaxel, and the paclitaxel is suspended on the nucleic acid nanoparticle; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence a, a sequence b and a sequence c, the sequence a comprises a sequence a1 or a sequence a1 with at least one base insertion, deletion or substitution, the sequence b comprises a sequence b1 or a sequence b1 with at least one base insertion, deletion or substitution, and the sequence c comprises a sequence c1 or a sequence c1 with at least one base insertion, deletion or substitution; wherein, the sequence of a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3', respectively; b1 is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3'; the sequence of c1 is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3' 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 to form a structure shown in a formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position are each independently selected from C-G or G-C; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; among the c sequences, the NNNN sequence in the 5 'to 3' direction is CAUA or CATA.
Further, the sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5'-CCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGCCG-3', c sequence: 5'-CGGCCAUAGCGC-3'; (3) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGCCG-3', c sequence: 5'-CGGCCAUAGCCG-3'; (4) a sequence: 5'-GGAGCGUUGG-3', sequence b: 5 '-CCUUCGGG-3', c sequence: 5'-CCCCCAUAGCCC-3'; (5) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCG-3', c sequence: 5'-CGCCCAUAGCGC-3', respectively; (6) a sequence: 5'-GCAGCGUUCG-3', sequence b: 5'-CGUUCGGCC-3', c sequence: 5'-GGCCCAUAGCGC-3'; (7) a sequence: 5'-CGAGCGUUGC-3', sequence b: 5'-GCUUCGGCG-3', c sequence: 5'-CGCCCAUAGCCG-3'; (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'; (11) a sequence: 5'-GGAGCGTTGG-3', sequence b: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3'; (12) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3', respectively; (13) a sequence: 5'-GCAGCGTTCG-3', sequence b: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3'; (14) a sequence: 5'-CGAGCGTTGC-3', sequence b: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3'; (15) a sequence: 5'-CGAGCGTTCC-3', respectively; b sequence: 5 '-GGTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3' is added.
Further, the nucleic acid domain also comprises a first extension segment, wherein the first extension segment is a Watson-Crick paired extension segment, and the first extension segment is positioned at the 5 'end and/or the 3' end of any one of the sequence a, the sequence b and the sequence c; preferably, the first elongate section is selected from any one of the following: (1): a 5' end of chain: 5' -CCCA-3', 3' end of c chain: 5 '-UGGG-3'; (2): a 3' end of chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of chain: 5' -CCCG-3', 3' end of c strand: 5 '-CGGG-3'; (5): a 5' end of chain: 5' -CCCC-3', 3' end of c strand: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'; (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of the chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3'; (9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.
Further, the nucleic acid domain also comprises a second extension segment, the second extension segment is positioned at the 5 'end and/or the 3' end of any sequence in the sequence a, the sequence b and the sequence c, and the second extension segment is a Watson-Crick paired extension segment; preferably, the second extension is an extension of a CG base pair; more preferably, the second extension is an extension sequence of 1-10 CG base pairs.
Further, the nucleic acid domain further comprises at least one set of second stretches: a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' -end of c chain: 5 '-CGCGCG-3'; second group: a 3' end of chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3'; third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.
Further, the second extension is an extension sequence containing both CG base pairs and AT/AU base pairs, and preferably the second extension is an extension sequence of 2-50 base pairs.
Further, the second extension segment is an extension sequence formed by alternately arranging a sequence of continuous 2-8 CG base pairs and a sequence of continuous 2-8 AT/AU base pairs; alternatively, the second extension is an extension in which a sequence of 1 CG base pairs alternates with a sequence of 1 AT/AU base pairs.
Further, bases, ribose and phosphate in the sequence a, the sequence b and the sequence c have at least one modifiable site, and any modifiable site is modified by any one of the following modification linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups; preferably, the sequence a, sequence b and sequence C have 2' -F modifications at the C or U bases.
Further, paclitaxel is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode, and the molar ratio of the paclitaxel 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 paclitaxel.
Further, the relative molecular weight of the nucleic acid domains is denoted as N 1 The total relative molecular weight of paclitaxel and biologically active substance is denoted as N 2 ，N 1 /N 2 ≥1:1。
Further, the bioactive substance is one or more of a target, fluorescein and miRNA, wherein the target is located on any sequence of a, b and c sequences, preferably the 5' end or the 3' end of any sequence of a, b and c, or is inserted between GC bonds of the nucleic acid structure domain, the miRNA is anti-miRNA, the fluorescein is modified on the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence; preferably, the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
Further, the small molecule drug except paclitaxel is a drug containing any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
Further, the protein is one or more of SOD, survivin, hTERT, EGFR and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
Further, the particle size of the nucleic acid nanoparticles is 1-100 nm, preferably 5-50 nm; more preferably 10 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 paclitaxel-containing drug, comprising the steps of: providing the nucleic acid nanoparticle described above; the paclitaxel is carried on the nucleic acid nanoparticles by means of physical connection and/or covalent connection, so as to obtain the drug containing the paclitaxel.
Further, the step of carrying paclitaxel by means of physical attachment comprises: mixing and stirring paclitaxel, nucleic acid nanoparticles and a first solvent to obtain a premix system; precipitating the premixed system to obtain a medicine containing paclitaxel; 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 paclitaxel-containing drug comprises: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the medicine containing paclitaxel; 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 paclitaxel; 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 paclitaxel is obtained.
Further, the step of carrying paclitaxel by means of covalent attachment comprises: preparing a paclitaxel solution; enabling the paclitaxel solution to react with the amino outside the G ring of the nucleic acid nanoparticles under the mediated action of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the medicine containing the paclitaxel; preferably, the step of reacting comprises: mixing the paclitaxel solution, a paraformaldehyde solution and nucleic acid nanoparticles, and reacting under the condition of keeping out of the sun to obtain a reaction system; the concentration of the preferable paraformaldehyde solution is preferably 3.7-4 wt%, the preferable paraformaldehyde solution is a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
Further, the preparation method further comprises a step of preparing a nucleic acid nanoparticle, which comprises: obtaining a nucleic acid domain by self-assembling the single strand corresponding to the nucleic acid domain; preferably, after obtaining the nucleic acid domain, the method of making further comprises: the bioactive substances are carried on the nucleic acid structural domain in a physical connection and/or covalent connection mode, and then the nucleic acid nano-particles are obtained.
Further, in the process of carrying the bioactive substances in a covalent connection mode, carrying is carried out through solvent covalent connection, linker covalent connection or click link; preferably, the solvent is a third solvent used in the covalent attachment as the attachment medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, the linker is selected from the group consisting of disulfide bond, p-azido, bromopropyne, or PEG; preferably, click-linking is performed by alkynyl or azido modification of the biologically active substance precursor and the nucleic acid domain simultaneously, followed by click-linking.
Further, when the biologically active substance is linked to the nucleic acid domain in a click-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 paclitaxel-containing drugs described above.
According to a fourth aspect of the present application, there is also provided the use of any one of the above paclitaxel-containing drugs in the preparation of a medicament for the treatment of a tumor.
Further, the tumor is breast cancer or ovarian cancer.
According to a fifth aspect of the present application, there is also provided a method for preventing and/or treating a tumor, the method comprising: providing any one of the above paclitaxel-containing drugs or pharmaceutical compositions; administering an effective amount of the above paclitaxel-containing drug or pharmaceutical composition to a patient with a tumor.
Further, the tumor is breast cancer or ovarian cancer.
The paclitaxel-containing medicament provided by the application comprises nucleic acid nanoparticles and paclitaxel, and the paclitaxel is carried on the nucleic acid nanoparticles in a physical connection and/or covalent connection mode. In the nucleic acid nanoparticle, the three sequences or the variant sequences thereof provided by the application can be contained, so that not only the nucleic acid domains can be formed by self assembly, but also paclitaxel can be connected to any 5 'end and/or 3' end of the three chains as a carrier, or the paclitaxel can be stably inserted between the chains of the nucleic acid domains. According to the application, the micromolecular drug paclitaxel is hung on the nucleic acid nanoparticles, the internal hydrophobicity, the external hydrophilicity and the base stacking effect of the nucleic acid nanoparticles are utilized, the coating effect is achieved on the paclitaxel, the paclitaxel cannot be dissolved within a certain time due to the coating effect or covalent connection, and the delivery stability is improved. In addition, when the nucleic acid structure domain is modified by a target head, the target head has better targeting property, can stably deliver the paclitaxel and has high reliability; meanwhile, the contact chance of the paclitaxel with non-target cells or tissues can be reduced, and the toxic and side effects are reduced.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, are included to provide a further understanding of the application, and the description of the exemplary embodiments and illustrations of the application are intended to explain the application and are not intended to limit the application. In the drawings:
FIG. 1 shows the result of electrophoresis detection of RNA nanoparticles formed by self-assembly in example 1 of the present application;
FIG. 2 shows the result of electrophoresis detection of DNA nanoparticles formed by self-assembly in example 1 of the present application;
FIG. 3 shows the result of 2% agarose gel electrophoresis detection of the first 7 groups of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present application;
FIG. 4 shows the result of 4% agarose gel electrophoresis detection of the first 7 groups of short-sequence RNA nanoparticles formed by self-assembly in example 2 of the present application;
FIG. 5 shows the result of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;
FIG. 6 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence RNA nanoparticles formed by self-assembly in example 3 of the present application;
FIG. 7 shows the result of 2% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;
FIG. 8 shows the results of 4% agarose gel electrophoresis detection of 7 sets of conventional sequence DNA nanoparticles formed by self-assembly in example 4 of the present application;
FIG. 9 shows the result of 2% agarose gel electrophoresis detection of DNA nanoparticles of group 8 and group 9 formed by self-assembly in example 4 of the present application;
FIG. 10 shows a TEM image of self-assembled DNA nanoparticles D-7 of the present application in example 4;
FIG. 11a shows a standard curve of paclitaxel absorbance during the RNA nanoparticle loading rate detection in example 5 of the present application;
FIG. 11b is a graph showing the standard curve of paclitaxel absorbance during the DNA nanoparticle loading rate measurement in example 5 of the present application;
FIG. 12 detection of the DNA-Bio-EGFRApt-Cy 5-paclitaxel nanoparticles by electrophoresis after incubation in serum for various periods of time in example 7 of the present application;
FIG. 13 shows the results of detecting the inhibition of U87MG cell proliferation by the small molecule drugs paclitaxel and RNAh-Biotin-quasar 670-paclitaxel nanoparticles in example 8 of the present application; and
FIGS. 14a to 14d show the results of detecting that DNAh-Bio-EGFRatt-Cy 5-paclitaxel nanoparticles inhibit SKOV3 cell proliferation in example 9, wherein FIG. 14a shows the inhibition of small molecule drug paclitaxel on SKOV3 cell proliferation, FIG. 14b shows the inhibition of DNAh-Bio-EGFRatt-Cy 5-paclitaxel (targeting agent) on SKOV3 cell proliferation, FIG. 14c shows the inhibition of DNAh-Bio-EGFRatt-Cy 5 (targeting fluorescent vector) on SKOV37 cell proliferation, and FIG. 14d shows the inhibition of DMSO blank control on SKOV3 cell proliferation;
FIG. 15 shows the result of non-denaturing PAGE gel electrophoresis detection of 7 sets of modified-segment + core short-sequence RNA self-assembly products in example 10 of the present invention;
FIG. 16 shows the dissolution curve of the RNA nanoparticle R-15 in example 10 of the present invention;
FIG. 17 shows the dissolution curve of the RNA nanoparticle R-16 in example 10 of the present invention;
FIG. 18 shows the dissolution curve of the RNA nanoparticle R-17 in example 10 of the present invention;
FIG. 19 shows the dissolution curve of the RNA nanoparticle R-18 in example 10 of the present invention;
FIG. 20 shows the dissolution curve of RNA nanoparticle R-19 in example 10 of the present invention;
FIG. 21 shows the dissolution curve of the RNA nanoparticle R-20 in example 10 of the present invention;
FIG. 22 shows the dissolution curve of RNA nanoparticle R-21 in example 10 of the present invention;
FIG. 23 shows the result of non-denaturing PAGE gel electrophoresis detection of 7 sets of modified-segment + core short-sequence DNA self-assembly products in example 11 of the present invention;
FIG. 24 shows a dissolution curve of DNA nanoparticle D-8 in example 11 of the present invention;
FIG. 25 shows a dissolution curve of the DNA nanoparticle D-9 in example 11 of the present invention;
FIG. 26 is a graph showing a dissolution curve of DNA nanoparticle D-10 in example 11 of the present invention;
FIG. 27 shows the dissolution curve of the DNA nanoparticle D-11 in example 11 of the present invention;
FIG. 28 is a graph showing the dissolution curve of the DNA nanoparticle D-12 in example 11 of the present invention;
FIG. 29 is a graph showing the dissolution curve of DNA nanoparticle D-13 in example 11 of the present invention;
FIG. 30 is a graph showing a dissolution curve of DNA nanoparticle D-14 in example 11 of the present invention;
FIG. 31 shows the result of electrophoresis detection of RNA nanoparticle R-15 in example 12 after incubation in serum for various times;
FIG. 32 shows the result of electrophoresis detection of RNA nanoparticle R-16 in example 12 after incubation in serum for various times;
FIG. 33 shows the result of electrophoresis detection of RNA nanoparticle R-17 in example 12 after incubation in serum for various times;
FIG. 34 shows the result of electrophoresis detection of RNA nanoparticle R-18 in example 12 after incubation in serum for various times;
FIG. 35 shows the result of electrophoresis detection of RNA nanoparticle R-19 in example 12 after incubation in serum for various times;
FIG. 36 shows the result of electrophoresis detection of the RNA nanoparticle R-20 in example 12 of the present invention after incubation in serum for various periods of time;
FIG. 37 shows the result of electrophoresis detection of the RNA nanoparticle R-21 of example 12 after incubation in serum for various periods of time;
FIG. 38 shows the result of electrophoresis detection of DNA nanoparticle D-8 in example 13 of the present invention after incubation in serum for various times;
FIG. 39 shows the results of electrophoresis detection of the DNA nanoparticle D-9 of example 13 of the present invention after incubation in serum for various periods of time;
FIG. 40 shows the results of electrophoresis detection of the DNA nanoparticle D-10 of example 13 of the present invention after incubation in serum for various periods of time;
FIG. 41 shows the result of electrophoresis detection of DNA nanoparticle D-11 in example 13 of the present invention after incubation in serum for various times;
FIG. 42 shows the results of electrophoresis detection of DNA nanoparticle D-12 in example 13 of the present invention after incubation in serum for various periods of time;
FIG. 43 shows the result of electrophoresis detection of DNA nanoparticle D-13 in example 13 of the present invention after incubation in serum for various times;
FIG. 44 shows the result of electrophoresis detection of DNA nanoparticle D-14 in example 13 after incubation in serum for various times; and
FIGS. 45a, 45b, 45c, 45D and 45e show cell viability curves corresponding to 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 16 of the present invention;
FIG. 46 shows a standard curve of daunorubicin absorbance used in the mounting rate measurement process of example 17.
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, DNAh or blank vector: refers to a blank nucleic acid nanoparticle vector, such as RNAh or DNAh, that does not contain any biologically active substance.
Targeting vectors: refers to a nucleic acid nanoparticle vector containing a targeting tip but not containing a fluorescent substance, such as Biotin-RNAh or Biotin-DNAh.
A fluorescent carrier: refers to a nucleic acid nanoparticle vector containing a fluorescent substance but not containing a targeting moiety, such as Cy3-RNAh or Cy 3-DNAh.
Targeting fluorescent vector: refers to a nucleic acid nanoparticle vector containing a target and a fluorescent substance, such as RNAh-Biotin-FAM or DNAh-Biotin-FAM.
Targeting drugs: refers to nucleic acid nanoparticle vector containing target head, fluorescent substance and chemical drug, such as RNAh-Biotin-quasar 670-paclitaxel or DNAh-Biotin-quasar 670-paclitaxel.
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 those used as vectors, are self-assembled by RNA strands at present, and very few self-assembly nanoparticles adopt a form of RNA strand and DNA strand combination, but do not adopt pure DNA strands to realize self-assembly.
In order to provide a novel RNA nanoparticle carrier which is highly reliable and can be autonomously assembled, the present applicant has compared and improved existing RNA nanoparticles, developed a series of novel RNA nanoparticles, and further tried to use pure DNA strands for self-assembly in view of improvement of applicability and cost reduction, and unexpectedly found that not only self-assembly into DNA nanoparticles can be achieved by changing these DNA strands, but also the performance is as excellent as that of RNA nanoparticles. Moreover, the self-assembly of DNA nanoparticles also has the advantages of low price and easy operation. Experiments prove that the improved RNA nanoparticles and DNA nanoparticles can be used for carrying various medicaments and stably exist in serum; further experiments verify that the carrier can carry the medicine into cells, and the carrier is nontoxic to the cells. And the carrier carrying the medicine can play a role in relieving and treating the corresponding diseases.
On the basis of the above research results, the applicant proposed the technical solution of the present application. The application provides a medicine containing paclitaxel, which comprises nucleic acid nanoparticles and paclitaxel, wherein the paclitaxel is carried on the nucleic acid nanoparticles; the nucleic acid nanoparticle comprises a nucleic acid domain, wherein the nucleic acid domain comprises a sequence, a sequence and c sequence, the a sequence comprises a1 sequence or a sequence of a1 sequence with at least one base insertion, deletion or substitution, the b sequence comprises a sequence of b1 sequence or a sequence of b1 sequence with at least one base insertion, deletion or substitution, and the c sequence comprises a sequence of c1 sequence or a sequence of c1 sequence with at least one base insertion, deletion or substitution; wherein, the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3'; 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 medicine containing paclitaxel provided by the application comprises nucleic acid nanoparticles and paclitaxel, and the paclitaxel is carried on the nucleic acid nanoparticles. The nucleic acid nanoparticle can be used as a carrier in which paclitaxel is linked to any 5 'end and/or 3' end of the three strands or can be stably inserted between strands of the nucleic acid domain, as well as a nucleic acid domain formed by self-assembly by including the three sequences or their variants. The drug containing the paclitaxel provided by the application is characterized in that the small molecule drug paclitaxel is carried on the nucleic acid nanoparticles, and the nucleic acid nanoparticles have hydrophobicity inside and hydrophilicity outside and stacking effect of basic groups, so that the drug is equivalent to a coating effect on the paclitaxel, and the paclitaxel cannot be dissolved within a certain time due to coating or covalent connection, so that the delivery stability is improved. In addition, when the nucleic acid structure domain is modified by a target head, the target head has better targeting property, can stably deliver the paclitaxel and has high reliability; meanwhile, the contact probability of the paclitaxel with non-target cells or tissues can be reduced, and the toxic and side effects are reduced.
The self-assembly refers to a technique in which basic structural units spontaneously form an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds. The self-assembly process is not a simple superposition of weak interaction forces (wherein, the weak interaction force refers to hydrogen bonds, van der waals force, electrostatic force, hydrophobic force and the like) among a large number of atoms, ions or molecules, but a plurality of individuals are simultaneously and spontaneously connected in parallel and are combined together to form a compact and ordered whole body, and the self-assembly process is a complex synergistic action of the whole body.
The generation of self-assembly requires two conditions: self-contained power and guidance. The kinetics of self-assembly refers to the synergistic effect of weak interaction forces between molecules, which provides energy for molecular self-assembly. The direction of self-assembly refers to the complementarity of the molecules in space, that is, the occurrence of self-assembly requires the rearrangement of the molecules to be satisfied in the size and direction of space.
The DNA nanotechnology is a mode of molecular self-assembly from bottom to top, and spontaneously forms a stable structure from a molecular conformation as a starting point based on the physical and chemical properties of nucleic acid molecules, following the strict base pairing principle of nucleic acids. A plurality of DNA fragments are connected together in a correct sequence in vitro, and a sub-assembly structure is established through a base complementary pairing principle, so that a complex multilevel structure is formed finally. Unlike DNA, RNA can be structured beyond the limitations of the double helix. RNA can form a series of different base pairs with at least two hydrogen bonds between the base pairs. The different bases can be divided into two types, including standard Watton-Crick base pair type and non-Watton-Crick base pair type, so that the RNA can form a large number of and various types of circulating structure modules, and the modules are basic units forming the tertiary structure of the folded RNA. RNA nanotechnology can take advantage of these naturally occurring 3D modules and their predictable interactions, where many biologically active RNA structures can have atomic-level resolution, such as ribosomes, various classes of ribozymes, and natural RNA aptamers present in riboswitches. One advantageous feature of RNA nanotechnology is that structures can be designed that are comparable in size and complexity to natural RNA species. The unique assembly properties of RNA within the native RNA complex can also be exploited.
In the nucleic acid nanoparticles, the nucleic acid nanoparticles comprise three sequences shown by the sequences SEQ ID NO.1, SEQ ID NO. 3 and SEQ ID NO. 5 or sequences after variation thereof, or three sequences shown by the sequences SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6 or sequences after variation thereof, and the nucleic acid nanoparticles can be formed by self-assembly.
The nanoparticles formed by self-assembly of SEQ ID NO.1, SEQ ID NO. 3 and SEQ ID NO. 5 are RNA nanoparticles, and the nanoparticles formed by self-assembly of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6 are DNA nanoparticles. In a preferred embodiment, when the nucleic acid nanoparticle is an RNA nanoparticle, at least one of the sequences a, b, and c comprises at least one base insertion, deletion, or substitution. The specific position and the base type of the variant sequence in the RNA nano-particle can be improved into the nano-particle for improving the drug loading capacity or the stability according to the requirement on the premise of realizing self-assembly.
In order to make the nucleic acid nanoparticles have relatively higher stability and further make the drugs obtained by paclitaxel hanging more stable, when base insertion, deletion or substitution is carried out on the sequences shown in SEQ ID NO 1/2, SEQ ID NO 3/4 and/or SEQ ID NO 5/6, base insertion, deletion or substitution can be carried out on the base at certain specific positions of the sequences, so that the mutated sequences can be self-assembled into nanoparticles as the original sequences on one hand, and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of homology with the original sequences on the other hand, so that the nanoparticles formed by self-assembling the sequences have the same drug loading characteristics and similar stability, and paclitaxel can be well hung and delivered.
In a preferred embodiment, the above base insertion, deletion or substitution occurs at: (1) 1 or 2 between the 1 st, 2 nd, 4 th and 5 th bases from the 5' end of the a sequence shown in SEQ ID NO; and/or (2) between 8 th to 10 th bases from the 5' end of the sequence a shown in SEQ ID NO.1 or 2; and/or (3) among 1 to 3 bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (4) between 6 th and 9 th bases from the 5' end of the b sequence shown in SEQ ID NO. 3 or 4; and/or (5) among bases 1 to 4 from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6; and/or (6) between bases 9 to 12 from the 5' end of the c sequence shown in SEQ ID NO. 5 or 6.
In the preferred embodiment, the base positions with variations are defined as the non-classical Watson-Crick paired base positions or the bulge unpaired base positions in the nanostructure formed by the sequences shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, thereby not affecting the formation of these bulges or loop structures, thereby maintaining the flexibility and tension of the nanostructure formed by the sequences and helping to maintain the stability of the nanostructure as a carrier.
In order to further improve the stability of the nucleic acid nanoparticles and further improve the stability of the drug formed after paclitaxel loading, in a preferred embodiment, the a sequence, the b sequence and the c sequence are self-assembled into a structure shown in formula (1):
wherein W-C represents Watson-Crick pairing, N and N ' represent non-Watson-Crick pairing, each W-C at any position is independently selected from C-G or G-C, and the two bases at the 5' end and 3' end of each of at least two of the a, b, and C sequences are not complementary; in the sequence a, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G; in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C; among the c sequences, the NNNN sequence in the 5 'to 3' direction is CAUA or CATA.
In the preferred embodiment, the sequences a, b and C form a nucleic acid domain having the formula (1) by self-assembly, wherein the bases at the other positions except for the non-Watson-Crick base pairs defined by N and N' form a classical Watson-Crick pair, and the bases of the Watson-Crick pair are selected from G-C or C-G base pairs. The nucleic acid nanostructure is more stable because the force of hydrogen bonds between G-C or C-G base pairs is greater than the force of hydrogen bonds between A-U/T or U/T-A base pairs. And a bulge or loop structure formed by non-Watson-Crick pairing base brings larger tension to the nucleic acid nano-carrier, so that the adaptability of the nucleic acid nano-carrier to microenvironment change is stronger, and the stability of the nucleic acid nano-particle is higher.
In the nanoparticles having the structure of formula (1), the specific sequence composition of the a sequence, the b sequence and the c sequence is not particularly limited as long as the structure can be formed. From the viewpoint of self-assembly of nucleic acid sequences, in order to further improve the efficiency of self-assembly of the three sequences into the nanoparticle having the structure of formula (1), 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 one sequence is independent, self-complementary pairing is not performed to form a secondary structure; (2) one end of any two sequences is complementary and matched to form a double chain, and the other end is not complementary and matched to form a Y-shaped or T-shaped structure. The principle of the base selection is to make the two ends of any 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, and the non-Watson-Crick pairing endows the nano-particles with greater flexibility and is also beneficial to improving the stability of the nano-particles in the face of change of microenvironment.
In a preferred embodiment, the sequence a, the sequence b and the sequence c are any one of the following groups: (1) a sequence (SEQ ID NO: 7): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 8): 5'-CCUUCGCCG-3', c sequence (SEQ ID NO: 9): 5'-CGGCCAUAGCCC-3', respectively; (2) a sequence (SEQ ID NO: 10): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 11): 5'-CGUUCGCCG-3', c sequence (SEQ ID NO: 12): 5'-CGGCCAUAGCGC-3'; (3) a sequence (SEQ ID NO: 13): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 14): 5 '-GCUUCGCCGCCG-3', c sequence (SEQ ID NO: 15): 5'-CGGCCAUAGCCG-3', respectively; (4) a sequence (SEQ ID NO: 16): 5'-GGAGCGUUGG-3', b sequence (SEQ ID NO: 17): 5 '-CCUUCGGG-3', c sequence (SEQ ID NO: 18): 5'-CCCCCAUAGCCC-3'; (5) a sequence (SEQ ID NO: 19): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 20): 5'-CGUUCGGCG-3', c sequence (SEQ ID NO: 21): 5'-CGCCCAUAGCGC-3', respectively; (6) a sequence (SEQ ID NO: 22): 5'-GCAGCGUUCG-3', b sequence (SEQ ID NO: 23): 5'-CGUUCGGCC-3', c sequence (SEQ ID NO: 24): 5'-GGCCCAUAGCGC-3'; (7) a sequence (SEQ ID NO: 25): 5'-CGAGCGUUGC-3', b sequence (SEQ ID NO: 26): 5'-GCUUCGGCG-3', c sequence (SEQ ID NO: 27): 5'-CGCCCAUAGCCG-3', respectively; (8) a sequence (SEQ ID NO: 28): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 29): 5'-CCTTCGCCG-3', c sequence (SEQ ID NO: 30): 5'-CGGCCATAGCCC-3'; (9) a sequence (SEQ ID NO: 31): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 32): 5'-CGTTCGCCG-3', c sequence (SEQ ID NO: 33): 5'-CGGCCATAGCGC-3'; (10) a sequence (SEQ ID NO: 34): 5'-CGAGCGTTGC-3', b sequence (SEQ ID NO: 35): 5'-GCTTCGCCG-3', c sequence (SEQ ID NO: 36): 5'-CGGCCATAGCCG-3', respectively; (11) a sequence (SEQ ID NO: 37): 5'-GGAGCGTTGG-3', b sequence (SEQ ID NO: 38): 5'-CCTTCGGGG-3', c sequence (SEQ ID NO: 39): 5'-CCCCCATAGCCC-3'; (12) a sequence (SEQ ID NO: 40): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 41): 5'-CGTTCGGCG-3', c sequence (SEQ ID NO: 42): 5'-CGCCCATAGCGC-3'; (13) a sequence (SEQ ID NO: 43): 5'-GCAGCGTTCG-3', b sequence (SEQ ID NO: 44): 5'-CGTTCGGCC-3', c sequence (SEQ ID NO: 45): 5'-GGCCCATAGCGC-3', respectively; (14) a sequence (SEQ ID NO: 46): 5'-CGAGCGTTGC-3', b sequence (SEQ ID NO: 47): 5'-GCTTCGGCG-3', c sequence (SEQ ID NO: 48): 5'-CGCCCATAGCCG-3', respectively; (15) a sequence (SEQ ID NO: 175): 5'-CGAGCGTTCC-3'; b sequence (SEQ ID NO: 176): 5 '-GGTTCGCCG-3', c sequence (SEQ ID NO: 177): 5'-CGGCCATAGCCG-3' are provided.
The nucleic acid nanoparticles formed by self-assembly of the fifteen groups of sequences have higher stability and higher self-assembly efficiency.
The nucleic acid nanoparticles mentioned above can be not only self-assembled into shapes, but also have the ability to carry or carry paclitaxel drugs. Depending on the position of G-C or C-G base pairs in the nucleic acid nanoparticles described above, the amount of paclitaxel carried may also vary.
In order to make the nucleic acid domain capable of carrying more paclitaxel and bioactive substances (see the description of bioactive substances 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 chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3'; (3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3'; (4): a 5' end of chain: 5' -CCCG-3', 3' end of c chain: 5 '-CGGG-3'; (5): a 5' end of the chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3'; (6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3'. (7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3'; (8): a 5' end of 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), c chain 5' end: 5'-GGCGGCAGG-3' (SEQ ID NO: 167); (13): a 5' end of chain: 5'-GCGGCGAGCGGCGA-3' (SEQ ID NO:168), the 3' end of the c-chain: 5'-TCGCCGCTCGCCGC-3' (SEQ ID NO: 169); (14): a 3' end of chain: 5'-GGCCGGAGGCCGG-3' (SEQ ID NO:170), 5' end of b chain: 5'-CCGGCCTCCGGCC-3' (SEQ ID NO: 171).
The first extension not only increases the length of any one or more of the three sequences forming the nucleic acid nanostructure, but also the first extension composed of GC bases further improves the stability of the formed nanoparticles. Moreover, the first extension segment composed of the sequence also keeps higher self-assembly activity and efficiency of the sequence a, the sequence b and the sequence c.
From the viewpoint of the size of the formed nucleic acid nanoparticles and the stability thereof when transported in vivo as a drug delivery vehicle, it is desirable to be able to transport the drug while trying not to be filtered out by the kidney until reaching the target cells. In a preferred embodiment, the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any of the a sequence, the b sequence and the c sequence, the second extension being a Watson-Crick paired extension; more preferably, the second extension is an extension of a CG base pair; further preferably, the second extension is an extension sequence of 1-10 CG base pairs.
In a preferred embodiment, the above-mentioned nucleic acid domain further comprises at least one set of second stretches: a first group: a 5' end of 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 on itself.
It is noted that the 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 relationship of the OR in AT/AU base "is that, specifically, the second extension is an extended sequence containing both CG base pairs and AT base pairs, or the second extension is an extended sequence containing both CG base pairs and AU base pairs.
More specifically, the sequences a, b and c after adding the above second extension may be the following sequences, respectively:
sequence a is (SEQ ID NO: 49):
b is (SEQ ID NO: 50):
sequence c is (SEQ ID NO: 51):
m in the sequence a, the sequence b and the sequence c is U or T, and when M is T, the synthesis cost of the sequences is greatly reduced.
In practical application, the specific arrangement positions of the CG base pairs and the extended sequences of AT/AU base pairs can be reasonably adjusted according to actual needs. In a more preferred embodiment, the second extension is an extension sequence formed by alternating a sequence of 2 to 8 CG base pairs and a sequence of 2 to 8 AT/AU base pairs; or the second extension is an extension sequence formed by alternating a sequence of 1 CG base pair and a sequence of 1 AT/AU base pair.
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 following systemic injection; 3) toxicity and adverse immune response. Currently, these three challenges have been largely overcome: 1) 2 '-fluoro (2' -F) or 2 '-O-methyl (2' -OMe) 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 paclitaxel can be carried by physical linkage and/or covalent linkage. When paclitaxel is simultaneously linked to the nucleic acid domain by physical insertion and covalent linkage, the physical insertion is usually inserted between GC base pairs, and the preferred number of insertion sites is 1-100: the ratio of 1 was inserted. When covalent linkage is used, paclitaxel usually reacts with the amino group outside the G ring to form covalent linkage. More preferably, the molar ratio between paclitaxel and nucleic acid nanoparticles is 2-300: 1, preferably 2-290: 1, more preferably 2-29: 1, further preferably 10-50: 1, and most preferably 15-25: 1.
In addition to the nucleic acid nanoparticles serving as a delivery vehicle for paclitaxel in the paclitaxel-containing drug provided by the present application, 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 paclitaxel.
In order to improve the efficiency of the nucleic acid nanoparticles in loading and carrying the biologically active substance to be loaded, the relative molecular weights of the nucleic acid domains and the relative molecular weights of paclitaxel and the biologically active substance should preferably be matched. In a preferred embodiment, the relative molecular weight of the nucleic acid domains is denoted as N 1 The total relative molecular weight of paclitaxel and bioactive substance is denoted as N 2 ，N 1 /N 2 1:1 or more; preferably, the biologically active substance is a targeting, a fluorescein, an interfering nucleic acid siRNA, a miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, a drug (generally interpreted as a small molecule drug, i.e., a chemically synthesized drug), a protein, a polypeptide, a flavonoid, a grapeOne or more of sugar, natural salicylic acid, monoclonal antibody, vitamins, phenols and lecithin.
The paclitaxel-containing drugs of the present application have different performance optimizations depending on the type of bioactive substance specifically loaded. For example, when the bioactive substance is biotin or folic acid, it can act to target the paclitaxel-containing drug, e.g., specifically to cancer cells. When the bioactive substance is fluorescein, it acts to provide a luminescent tracer effect to the nucleic acid nanoparticles, such as may be one or more of FAM, CY3, CY5, or Quasar670, and the like. When the bioactive substances are certain siRNA, miRNA, protein, polypeptide, RNA antibody and micromolecule drugs except paclitaxel, the drug containing paclitaxel can become a new product with specific treatment effect, such as a drug 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 covalently linked to any one of the sequences a, b and c by a linker, wherein the linker can be selected from disulfide bond, p-azido, bromopropyne or PEG. As used herein, "any sequence" is a base at any position of any sequence of a, b, and c sequences, and it is more convenient to attach the sequence to the 5 'end or the 3' end, and the application is more extensive. Folate modification can be either physical intercalation mode of ligation or physical intercalation + covalent ligation.
The fluorescein 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 of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence according to actual needs. When the anti-miR-21 is synthesized at all three positions, the inhibition effect of the anti-miR-21 on the miR-21 is relatively stronger.
When the bioactive substances capable of being carried are other small-molecule drugs except paclitaxel, the drugs include, but are not limited to, drugs for treating liver cancer, stomach cancer, lung cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, melanoma, leukemia, senile dementia, ankylosing spondylitis, malignant lymphoma, bronchial cancer, rheumatoid arthritis, HBV hepatitis B, multiple myeloma, pancreatic cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, esophageal cancer, oral cancer and lupus erythematosus according to the types of diseases which can be treated by different drugs; preferably, the head and neck cancer is brain cancer, neuroblastoma or glioblastoma.
When the bioactive substance capable of being carried is a small molecule drug other than paclitaxel, the bioactive substance includes, but is not limited to, drugs containing any one or more of the following groups according to the difference of the molecular structure of the drug or the difference of characteristic groups of the drug: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
In a preferred embodiment, the protein is one or more of an antibody or aptamer to SOD (superoxide dismutase), Survivin (Survivin), hTERT (human telomerase reverse transcriptase), EGFR (epidermal growth factor receptor), PSMA (prostate specific membrane antigen); the vitamins are levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
In a preferred embodiment, the particle size of the nucleic acid nanoparticles is 1 to 100nm, preferably 5 to 50nm, more preferably 10 to 30nm, and 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 paclitaxel-containing drug, which comprises the following steps: providing any one of the nucleic acid nanoparticles described above; the paclitaxel is carried on the nucleic acid nanoparticles by means of physical connection and/or covalent connection, so as to obtain the drug containing the paclitaxel.
When physical attachment is used, paclitaxel is usually inserted between the GC base pairs by physical intercalation. When covalent linkage is used, paclitaxel usually reacts with the amino group outside the G ring to form covalent linkage. The medicine containing paclitaxel prepared by the method has better targeting property after being modified by the target head, can stably deliver paclitaxel and has high reliability.
In a preferred embodiment, the step of loading paclitaxel by physical attachment comprises: mixing and stirring paclitaxel, nucleic acid nanoparticles and a first solvent to obtain a premixed system; precipitating the premixed system to obtain the medicine containing the paclitaxel. The dosage of paclitaxel and nucleic acid nanoparticles can be adjusted according to the variation of the loading amount, which can be understood by those skilled in the art and will not be described herein.
In order to improve the efficiency and stability of physical connection, the amount of paclitaxel 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 paclitaxel-containing drug comprises: precipitating the premixed system to obtain a precipitate; washing the precipitate to remove impurities to obtain the medicine containing paclitaxel. More preferably, the premix system is mixed with absolute ethyl alcohol and then precipitated at a temperature of less than 10 ℃ to obtain a precipitate, and still more preferably, the precipitate is precipitated at a temperature of 0 to 5 ℃ to obtain a precipitate. More preferably, anhydrous ethanol with the volume 6-12 times that of the precipitate is adopted to wash and remove impurities, and the medicine containing the paclitaxel is obtained.
In a preferred embodiment, the step of loading paclitaxel by covalent attachment comprises: preparing a paclitaxel solution; enabling the paclitaxel solution to react with the amino outside the G ring of the nucleic acid nanoparticles under the mediated action of formaldehyde to obtain a reaction system; purifying the reaction system to obtain the medicine containing the paclitaxel.
Preferably, the step of reacting comprises: and mixing the paclitaxel solution, the paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under the condition of keeping out of the sun to obtain a reaction system. The paraformaldehyde solution can release formaldehyde small molecules so as to participate in the chemical reaction. In order to improve the reaction efficiency, the concentration of the paraformaldehyde solution is preferably 3.7-4 wt%, the paraformaldehyde solution is preferably a solution formed by mixing paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
In a preferred embodiment, the following reaction can occur:
in the above preparation method, the nucleic acid nanoparticles may be prepared by a self-assembly form such as: (1) mixing RNA or DNA single strands a, b and c at the same time, and dissolving in DEPC water or TMS buffer solution; (2) heating the mixed solution to 80 ℃/95 ℃ (wherein the RNA assembly temperature is 80 ℃, and the DNA assembly temperature is 95 ℃), keeping for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min; (3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃; (4) cutting a target strip, eluting in an RNA/DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at a low temperature under reduced pressure to obtain a self-assembly product, namely a nucleic acid structural domain, thereby obtaining the nucleic acid nanoparticles.
In order to provide the paclitaxel-containing drug with other functions according to practical requirements, in a preferred embodiment, after obtaining the nucleic acid domain, the preparation method further comprises: the bioactive substances mentioned above are loaded on the nucleic acid domain by means of physical linkage and/or covalent linkage, so as to obtain the nucleic acid nanoparticle. The biologically active substance may also be attached by physical and/or covalent attachment. Forms of covalent attachment include, but are not limited to, mounting by solvent covalent attachment, linker covalent attachment, or click linkage; preferably, the solvent is a third solvent used in the covalent attachment as the attachment medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS, and glacial acetic acid; preferably, linker is selected from disulfide bond, p-azido, bromopropyne or PEG; preferably, click-linking is performed by alkynyl or azide modification of the biologically active substance precursor and the nucleic acid domain at the same time and then by click-linking.
The above classification does not mean that a certain bioactive substance is linked to a nucleic acid domain in only one manner. Instead, some bioactive substances may be linked to the nucleic acid domain by physical intercalation, by covalent linkage, or by click linkage. However, for a particular biologically active substance, there may be only one type of attachment, or there may be multiple types of attachment, but there may be some type of attachment that has a beneficial utility.
In the above 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: 1, and inserting.
Specifically, depending on the species of the bioactive substance, the length of the a, b, and c sequences forming the nucleic acid domains in the nucleic acid nanoparticles, and the number of GC-complementary base pairs therein, the molar ratio of the bioactive substance to the nucleic acid domains can be rationally selected for physical intercalation.
In a preferred embodiment, when the bioactive substance and the nucleic acid domain are physically intercalated and covalently linked, the molar ratio of the bioactive substance physically intercalated and linked to the drug covalently linked is 1-200: 1. the connection mode is suitable for anthracycline and acridine medicines. The proportion of the drugs connected in different connection modes is not limited to the range, and the drugs can be effectively suspended, have no toxic effect on cells and can be effectively released after reaching a target.
When the bioactive substance precursor and the nucleic acid structural domain are simultaneously subjected to alkynyl or azide modification and connected in a click-to-link mode, different click-to-links are selected according to different structural changes of the medicament. And the attachment position may be changed correspondingly according to the structure of the active material, which can be understood by those skilled in the art.
In a preferred embodiment, when the biologically active substance is linked to the nucleic acid domain in a click-link fashion, the site of the biologically active substance precursor for the alkynyl or azide modification is selected from the group consisting of hydroxyl, carboxyl, sulfhydryl or amino, and the site of the nucleic acid domain for the alkynyl or azide modification is selected from the group consisting of amino, imino or hydroxyl.
When the nucleic acid domain is bound to a drug, the nucleic acid domain is water-soluble, and many drugs have poor water-solubility, and when the nucleic acid domain is bound to the drug, the water-solubility is improved. When the drugs are anthracyclines, the drugs are covalently bound to the nucleic acid domain via an-NH bond on the nucleotide guanosine (the-NH group is hundreds of times more active than other groups that may covalently bind to the drug under appropriate pH conditions), thereby forming a drug-loaded nucleic acid domain. Therefore, depending on the size of the specific drug molecule and the number of GC base pairs in the a-, b-and c-sequences of the specifically designed nucleic acid domain, a binding reaction is performed with a theoretical supersaturation binding amount of 1.1 to 1.3 times, and a maximum of 35 to 45 drugs can be bound to one nucleic acid domain. When the drug has 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.
In a typical embodiment, there is also provided a pharmaceutical composition comprising any one of the nucleic acid nanoparticles described above. In the drug containing the nucleic acid nanoparticles, the nucleic acid domain can be modified by a targeting head of a targeted cell to achieve good targeting, and meanwhile, the corresponding therapeutic drug and/or tracer molecule can be carried, so that the therapeutic drug and/or tracer molecule can be stably delivered, and the reliability is high.
According to a third aspect of the present application, there is also provided a pharmaceutical composition comprising any one of the paclitaxel-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 a fourth aspect of the present application, there is also provided the use of any one of the above paclitaxel-containing drugs in the preparation of a medicament for the treatment of a tumor. Further, the tumor is breast cancer or ovarian cancer. The specific application can be to improve the medicament per se on the basis of the medicament of the application to obtain a new medicament, or to prepare the medicament of the application serving as a main active ingredient into a preparation with a proper dosage form and the like.
According to a fifth aspect of the present application, there is also provided a method of preventing and/or treating a tumor, the method comprising: providing any one of the above paclitaxel-containing drugs or pharmaceutical compositions; administering an effective amount of the above paclitaxel-containing drug or pharmaceutical composition to a patient with a tumor. Further, the tumor is breast cancer or ovarian cancer.
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, e.g., reduction of breast or ovarian cancer, at dosages and for periods of time necessary. In a particular embodiment, the dosage may be adjusted to provide the optimum therapeutically responsive dosage, and the therapeutically effective amount may vary depending on the following factors: the disease state, age, sex, weight of the individual and the ability of the formulation to elicit a desired response in the individual. A therapeutically effective amount is also meant to include an amount by which the beneficial effect of the treatment exceeds its toxic or detrimental effects. A prophylactically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention or inhibition of the development of acute breast or ovarian cancer. A prophylactically effective amount can be determined according to the description of a therapeutically effective amount above. For any particular subject, specific dosages may be adjusted over time according to the individual need and the professional judgment of the person to whom they are administered.
It should be noted that the nucleic acid nanoparticles formed by self-assembly of the sequences or variants of the sequences provided herein can also be used as basic building blocks, and can be further polymerized to form polymers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., according to practical requirements.
The advantageous effects of the present application will be further described below with reference to specific examples.
Assembly of nucleic acid nanoparticles
Example 1
One, RNA and DNA nanoparticle vector:
(1) the three polynucleotide base sequences that make up the RNA nanoparticles are shown in table 1:
table 1:
(2) three polynucleotide base sequences of DNA nanoparticles
DNA has the same sequence as that of the RNA, except that U is replaced by T. Wherein the molecular weight of 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 strands a, b and c of the RNA nanoparticles and DNA nanoparticles were synthesized by Competition Biotechnology, Inc. (Shanghai).
II, self-assembly experiment steps:
(1) mixing 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 and 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 larger than that of the single chain after the molecular weight is assembled, so that the position of a strip lags behind the a chain and the b chain, the actual situation is consistent with the theory, and the stable composite structure is formed by the self-assembly of the RNA single chains, and the RNA nano-particles are formed.
The results of the electrophoretic detection of the DNA self-assembly products are shown in FIG. 2. In fig. 2, lanes 1 to 3 are, from left to right: a chain, b chain, DNA self-assembly product. As can be seen from the figure, the bands of the DNA self-assembly products are bright and clear, and are single bands, which proves that the DNA single strands form a stable composite structure through self-assembly, and form DNA nanoparticles.
In this example, it was verified by gel electrophoresis that: the sequences a, b and c including RNA core sequence SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 can be successfully self-assembled into RNA nano-particles. Sequences a, b, and c, including the DNA core sequence SEQ ID NO 2, SEQ ID NO 4, and SEQ ID NO 6, can also successfully self-assemble into DNA nanoparticles.
The sequences a, b and c of the RNA nanoparticles and the DNA nanoparticles include various extension sequences (including drug-loading binding sequences) that facilitate the function of loading the nucleic acid domains, and a targeting head or fluorescein linked to the nucleic acid domains, in addition to the core sequence forming the nucleic acid domains. It can be seen that the presence of substances other than these core sequences does not affect the formation of nucleic acid domains and the successful self-assembly of nucleic acid nanoparticles. The self-assembled nucleic acid nanoparticles can have a targeting type under the guidance of a target head, and the fluorescein can enable the nucleic acid nanoparticles to have visibility and traceability.
Example 2
One, 7 groups of short sequence RNA nano-particle carriers:
(1)7 groups of three polynucleotide base sequences composing the RNA nanoparticle are respectively shown in tables 2 to 8:
table 2: r-1
Table 3: r-2
Table 4: r-3
Table 5: r-4
Table 6: r-5
Table 7: r-6
Table 8: r-7
The single strands of the 7 groups of short-sequence RNA nanoparticle carriers are synthesized by the corporation of Venezetian Biotechnology (Shanghai).
II, self-assembly experiment steps:
(1) mixing and dissolving the RNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting a target band, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a short-sequence RNA self-assembly product;
(5) electrophoretic analysis detection and laser scanning observation;
(6) and (4) measuring the molecular weight.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis picture of 7 groups of short sequence RNA self-assembly products is shown in FIG. 3. Lanes 1 to 7 in FIG. 3 are, from left to right: short sequences R-1, R-2, R-3, R-4, R-5, R-6 and R-7.
The 4% agarose gel electrophoresis 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) Measurement of electric potential
The measuring method comprises the following steps: preparing a potential sample (a self-assembly product is dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking to finish setting, appearing a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 7 groups of short sequence RNA nanoparticles are shown in tables 9 to 15 below:
table 9:
table 10:
table 11:
table 12:
table 13:
table 14:
table 15:
from the potential detection data described above, it can be seen that: the 7 groups of short sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the short sequence RNAs have more stable self-assembly structures.
This example shows that: the different combinations of the core sequences a, b and c can form the RNA nano-particle with the nucleic acid 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 groups of three polynucleotide base sequences constituting the RNA nanoparticles are respectively shown in tables 16 to 22:
table 16: r-8
Table 17: r-9
Table 18: r-10
Table 19: r-11
Table 20: r-12
Table 21: r-13
Table 22: r-14 (in the following a chain)uGAcAGAuAAGGAAccuGcudTdTAs survivin siRNA)
The 7 groups of conventional sequence RNA nanoparticle vectors are synthesized by commissioned Suzhou Jima company, wherein the sequences a, b and C in R-8 to R-14 are respectively extended RNA oligonucleotide sequences formed by adding extension segments on the basis of the sequences a, b and C in R-1 to R-7, targeting module fragments are not extended, and C/U base 2' F modification is carried out (the enzyme cutting resistance and stability are enhanced). In addition, a Survivin (Survivin) siRNA nucleic acid interference therapeutic fragment is modified in the RNA nanoparticle R-14, specifically, a sense strand of Survivin siRNA is extended at the 3 'end of the a strand (see the 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 and dissolving the RNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off a target strip, eluting in an RNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and evaporating at a low temperature under reduced pressure;
(5) electrophoretic analysis detection and laser scanning observation;
(6) and (4) measuring the potential.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis of 7 sets of conventional sequence RNA self-assembly products is shown in FIG. 5. Lanes 1 to 7 in FIG. 5 are, from left to right: the self-assembly products of the conventional sequence RNA are R-8, R-9, R-10, R-11, R-12, R13 and R-14.
FIG. 6 shows the electrophoresis of 4% agarose gel of 7 sets of conventional sequence RNA self-assembly products. Lanes 1 to 7 in FIG. 6 are, from left to right: the self-assembly products of the conventional sequence RNA are R-8, R-9, R-10, R-11, R-12, R13 and R-14.
As can be seen from the results of FIGS. 5 and 6, it can be clearly seen that the bands of the 7 sets of conventional sequence RNA self-assembly products are all bright and clear single bands, indicating that the 7 sets of conventional sequences can self-assemble into the nano-structure. Wherein, after a section of Survivin siRNA nucleic acid interference treatment fragment is modified in the conventional sequence RNA self-assembly product R-14, the self-assembly structure still has a stable self-assembly structure, which also indicates that the nucleic acid nano-particle can carry a nucleic acid drug and has the function of a delivery carrier of the nucleic acid drug.
(2) Determination of potential
The measuring method comprises the following steps: preparing a potential sample (self-assembly product dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening the software, clicking the menu measurei @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the results of the potential measurements for 7 sets of conventional sequence RNA nanoparticles are shown in tables 23 to 29 below:
table 23:
table 24:
table 25:
table 26:
table 27:
table 28:
table 29:
from the potential detection data described above, it is found that: the 7 groups of conventional sequence RNA self-assembly products have good stability, and further show that the nanoparticles formed by self-assembly of the conventional sequence RNA have a stable self-assembly structure.
This example shows that: on the basis of RNA core sequences of different combinations, the addition of the extension segment can also successfully self-assemble into RNA nanoparticles with stable structure. Meanwhile, the added extension fragment enables the RNA nanoparticles to have excellent drug-loading performance (see example 5 in particular).
Example 4
First, first 7 groups of conventional sequence DNA nanoparticle vectors:
(1) the base sequences of three polynucleotides constituting the DNA nanoparticles of 9 groups are shown in tables 30 to 36:
part a of the table has extended the EGFRApt or PSMAApt (A9L) head:
EGFRapt(SEQ ID NO：97)：GCCTTAGTAACGTGCTTTGATGTCGATTCGACAGGAGGC；
PSMAapt(A9L，SEQ ID NO：98)：
GGGCCGAAAAAGACCTGACTTCTATACTAAGTCTACGTCCC。
table 30: d-1
Table 31: d-2
Table 32: d-3
Table 33: d-4
Table 34: d-5
Table 35: d-6
Table 36: d-7
The single strands of the 7 sets of conventional sequence DNA nanoparticles were synthesized by hong, sozhou entrusted, where:
d-1 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined section below) to the core sequence (8) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCC-3') described previously;
d-2 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRatt target head (see underlined section below) to the core sequence (9) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGCCG-3', c sequence: 5'-CGGCCATAGCGC-3') described previously;
d-3 is a regular-sequence DNA nanoparticle formed after adding an extended sequence comprising the EGFRept target head (see underlined section below) to the core sequence (10) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGCCG-3', c sequence: 5'-CGGCCATAGCCG-3') described above;
d-4 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined section below) to the core sequence (11) (a sequence: 5'-GGAGCGTTGG-3', b sequence: 5'-CCTTCGGGG-3', c sequence: 5'-CCCCCATAGCCC-3') described above;
d-5 is a regular-sequence DNA nanoparticle formed after adding an extension sequence comprising a PSMAapt target head (see underlined section below) to the core sequence (12) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCG-3', c sequence: 5'-CGCCCATAGCGC-3') described previously;
d-6 is the core sequence (13) (a sequence: 5'-GCAGCGTTCG-3', b sequence: 5'-CGTTCGGCC-3', c sequence: 5'-GGCCCATAGCGC-3') added with an extension sequence not containing the targeting structure; the formed conventional sequence DNA nanoparticles;
d-7 is an extension sequence which does not contain a targeting structure and is added to the core sequence (14) (a sequence: 5'-CGAGCGTTGC-3', b sequence: 5'-GCTTCGGCG-3', c sequence: 5'-CGCCCATAGCCG-3') described above; and forming the conventional sequence DNA nano-particles.
In addition, single-stranded sequences for forming the 8 th set of DNA nanoparticles and single-stranded sequences of the 9 th set of DNA nanoparticles were synthesized.
a chain: (SEQ ID NO:172:)
The front three bases of the 5' end and the rear three bases of the 3' end are respectively subjected to thio modification, the 5' end is connected with Biotin, and the bold part is an EGFRApt sequence;
b chain (SEQ ID NO: 173:): 5'-GCGCCCGGTTCGCCGCCAGCCGCCGC-3', respectively carrying out sulfo-modification on the first three bases at the 5 'end and the last three bases at the 3' end;
c chain (SEQ ID NO: 174:): 5'-GCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCG-3', respectively; the first three bases of the 5' end and the last three bases of the 3' end are respectively subjected to sulfo modification, and the 3' end is connected with Cy5 fluorescent label.
Wherein, group 9 is the DNA nanoparticles formed after adding the extension sequence on the basis of the core sequence (15) described above. The specific sequence is as follows:
chain a (SEQ ID NO: 178:):
the front three bases of the 5' end and the rear three bases of the 3' end are subjected to thio modification respectively, and the 5' end is connected with Biotin;
b chain (SEQ ID NO: 179:):
the first three bases of the 5 'end and the last three bases of the 3' end are respectively subjected to sulfo-modification;
chain c (SEQ ID NO: 180:):
the first three bases of the 5' end and the last three bases of the 3' end are respectively subjected to sulfo modification, and the 5' end is connected with Cy5 fluorescent label.
II, self-assembly experiment steps:
(1) mixing and dissolving the DNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 95 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off a target band, eluting in a DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a conventional sequence DNA self-assembly product;
(5) electrophoretic analysis detection and laser scanning observation;
(6) detecting the potential;
(7) measuring the particle size;
(8) and (5) observing by using a transmission electron microscope.
Third, self-assembly experimental results
(1) Results of electrophoresis
The 2% agarose gel electrophoresis of the first 7 sets of conventional sequence DNA self-assembly products is shown in FIG. 7. Lanes 1 to 7 in FIG. 7 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.
The electrophoresis picture of 4% agarose gel of the first 7 groups of conventional sequence DNA self-assembly products is shown in FIG. 8. Lanes 1 to 7 in FIG. 8 are, from left to right: the self-assembly products of the conventional sequence DNA are D-1, D-2, D-3, D-4, D-5, D-6 and D-7.
The 2% agarose gel electrophoresis picture of the self-assembly products of the sequence DNAs of groups 8 and 9 is shown in FIG. 9. The lanes in FIG. 9 are from right to left: the single strands of group 8, a, and DNA self-assembly products D-8 and D-9.
As can be seen from the results of FIG. 7, FIG. 8 and FIG. 9, it can be clearly seen that the bands of the self-assembly products of the 9 groups of conventional sequence DNAs are bright and clear, indicating that the self-assembly of the 9 groups of conventional sequence DNA strands is completed, and a stable nanoparticle structure is formed. Wherein, the two groups of self-assembly structures D-6 and D-7 carry EGFRatt or PSMAaptt target heads, the molecular weight is slightly lower, the position of the strip is obviously more ahead than that of other strips, the actual condition and the theoretical condition completely conform to each other, and the stability of the self-assembly structures is further proved.
This example shows that: when various functional extension fragments are added on the basis of different DNA core sequence combinations or are simultaneously connected with a target head, the DNA nano-particles can be successfully assembled, and the DNA nano-particles also have the performances of drug loading, cell targeting, visual tracking and the like.
(2) Measurement of electric potential
The determination method comprises the following steps: preparing a potential sample (self-assembly product dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking to finish setting, appearing a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results of 3 groups of conventional sequence DNA nanoparticles are shown in tables 37 to 39 below:
table 37:
table 38:
table 39:
from the potential detection data described above, it is found that: the 3 groups of conventional sequence DNA self-assembly products have good stability, and further show that the nanoparticles formed by the self-assembly of the conventional sequence DNA have a stable self-assembly structure.
(3) Particle size measurement
1. Preparing a potential sample (a conventional sequence DNA self-assembly product D-7) and putting the potential sample into a sample cell, opening a sample cell cover of an instrument, and putting the instrument into the instrument;
2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
3. setting software detection parameters;
4. then click on the ok setting, the measurement dialog box appears, click Start, DLS measurements of hydrodynamic size of self-assembled product D-7 result in table 40 below:
table 40:
(4) observation result of transmission electron microscope
And (3) carrying out transmission electron microscope irradiation on the conventional sequence DNA self-assembly product D-7, and comprising the following steps:
1. a drop of sample is suspended on a 400-mesh carbon-coated copper net for 1 minute at room temperature;
2. sucking the liquid by filter paper;
3. dyeing for 1 minute by using 2% uranium acetate;
4. sucking dry by filter paper, and drying at room temperature;
5. JEM-1400 was observed by 120kv using a transmission electron microscope and photographed.
The result is shown in FIG. 10, from which it is apparent that the conventional sequence DNA self-assembly product D-7 is an integral structure and can be clearly seen to have a T-shaped structure.
Example 5
Paclitaxel Loading experiment
Carrying out chemical method mounting:
first, experimental material and experimental method
1. Experimental materials and reagents:
(1) nucleic acid nanoparticles: similar to the RNA nanoparticles in example 1, except that the fluorescent label on the c-strand is Cy 5.
(2) DEPC water: biyun Tian.
(3) PBS buffer: cellgro.
(4) 4% Paraformaldehyde
(5) Paclitaxel (Epirarunixon).
(6) Chloroform: and (4) carrying out north transformation.
(7) Anhydrous ethanol: and (6) north transformation.
2. The experimental method comprises the following steps:
(1) paclitaxel (1.354 μmoL) was precisely weighed and dissolved in DEPC water (1.0mL) and PBS buffer (1.25mL), 4% paraformaldehyde aqueous solution (0.25mL) was added thereto and mixed with ice-water bath cooling, and the mixture was mixed with RNA nanoparticles (33.84nmoL) and reacted at 4 ℃ for 72 hours in the dark.
(2) Taking 10 mu L of reaction solution to dilute by 10 times, taking 50 mu M paclitaxel aqueous solution and 310 ng/mu L RNA nano-particles as controls, and carrying out HPLC analysis according to the equal volume injection. The reaction conversion can be judged to be basically complete according to the peak area ratio of each component.
(3) The reaction mixture was extracted with chloroform (10mL x3), followed by addition of 25mL of absolute ethanol, mixing, and then sufficiently precipitating the product by keeping the mixture at 4 ℃ in the dark (4 hours). Centrifugation (4000/min) and transfer of the supernatant, washing of the solid product with ethanol (50mL) again, and evaporation of the solvent at low temperature under reduced pressure to give a dark red solid product.
(4) And (3) calculating the mounting rate:
1. preparing a paclitaxel-absolute ethyl alcohol standard solution with a known concentration: 2uM, 4uM, 6uM, 8uM, 10uM, each 100 ul;
2. dissolving paclitaxel-RNAh granules in 100ul PBS;
3. placing the standard solution and paclitaxel-RNAh particles in a PCR plate, heating at 85 deg.C for 5min, and cooling to room temperature;
4. measuring the absorbance of the paclitaxel at 233nm by using a microplate reader, drawing a standard curve, and calculating to obtain the molar concentration of the paclitaxel in the carried product;
5. measuring the absorbance of RNA at 260nm by using a spectrophotometer to obtain the mass concentration of RNAh particles in each sample;
6. and calculating the mounting rate according to the measured molar concentration of the paclitaxel and the mass concentration of the RNAh particles.
The standard curve of paclitaxel carried by RNA nucleic acid nanoparticles is shown in FIG. 11a, and the specific process is calculated as follows:
C RNAh -1＝32.9ug/ml，M RNAh ≈30000，100ul；C paclitaxel -1＝12.8uM，100ul；
C RNAh -2＝58.5ug/ml，M RNAh ≈30000，100ul；C Paclitaxel -2＝24.03uM，100ul；
The average value is taken to obtain that the carrying rate of the taxol-RNAh nucleic acid nano-particles is about 12, which indicates that about 12 taxol molecules can be carried on each nucleic acid nano-particle carrier.
(II) experiments on DNA nucleic acid nanoparticle Loading
The mounting method and the mounting rate are calculated in the same way as the RNA nucleic acid nanoparticles, and the specific nucleic acid nanoparticles used are as follows: DNAh-Bio-EFGRapt-Cy5, wherein three strands of DNAh are respectively:
a chain: (SEQ ID NO:172:)
b chain (SEQ ID NO: 173:): 5'-GCGCCCGGTTCGCCGCCAGCCGCCGC-3', respectively carrying out thio modification on the first three bases (italic parts) of the 5' end and the last three bases (3 ') of the 5' end;
c chain (SEQ ID NO: 174:): 5'-GCGGCGGCAGGCGGCCATAGCCGTGGGCGCGCG-3', respectively; the first three bases of the 5' end and the last three bases of the 3' end (italic parts) are respectively modified by sulfo, and the 3' end is connected with Cy5 fluorescent label.
The standard curve of paclitaxel carried by DNA nucleic acid nanoparticles is shown in FIG. 11b, and the specific calculation process is as follows:
C DNAh -1＝32.9ug/ml，M DNAh ≈39500，100ul；C paclitaxel -1＝12.8uM，100ul；
C DNAh -2＝58.5ug/ml，M DNAh ≈39500，100ul；C Paclitaxel -2＝24.03uM，100ul；
The average value is taken to obtain the loading rate of the taxol-DNAh nano-particles to be about 12, which shows that each DNA rice particle carrier can load about 12 taxol.
Example 5 shows that both the RNA nanoparticles (of example 1) and the DNA nanoparticles with the extension fragment, the targeting head and the fluorescein have the function of drug loading, and the small molecule drug paclitaxel can be loaded by means of covalent linkage (paraformaldehyde-solvent covalent).
Example 6
Flow cytometry experiment for detecting cell binding capacity of drug-loaded DNA nanoparticles
First, cell information
SK-OV-3 (from ATCC, cat # HTB-77) in MEM + 10% FBS at 37 deg.C and 5% CO 2 And saturation humidity.
Second, the object to be measured
Targeting drugs: DNAh-Bio-EGFRApt-Cy 5-paclitaxel (the product of the loading of DNA nanoparticles in example 5).
A fluorescent carrier: DNAh-Bio-EGFRapt-Cy 5.
Third, equipment and consumables (see table 41)
Watch 41
Reagent (see table 42)
Table 42:
and fifthly, an experimental method:
1. adjusting the cell state to logarithmic phase, changing the culture medium to a biotin-free and folic acid-free culture medium, and placing the culture medium in an incubator at 37 ℃ for overnight incubation;
2. dissolving a to-be-detected object, and preparing a to-be-detected object stock solution;
3. digesting, collecting single cell suspension, counting, and adjusting cell density to 2 × 10 5 mL, planting 1 mL/well into 24-well plate;
4. adding the substances to be detected into corresponding cell pores respectively, shaking and mixing to obtain final concentrations of 0.1 μ M, 0.2 μ M and 0.4 μ M;
5. incubating the cell plate in an incubator at 37 ℃ for 2 hours;
6. after incubation is finished, collecting cell suspension by trypsinization;
7. centrifuging to collect cell precipitate, and washing twice with PBS;
8. finally, 300 mu L PBS is used for resuspending the cell sediment, and the detection is carried out on a flow type machine;
9. fluorescent carrier or paclitaxel detection channel: wavelength of excitation light: 488nm, emission light channel: 560 nm;
10. and (6) analyzing the data.
Sixthly, experimental results (see Table 43)
Table 43:
as can be seen from Table 43, the paclitaxel targeting agent DNAh-Bio-EFGRapt-Cy 5-paclitaxel was able to bind to SK-OV-3 cells with nearly one hundred percent binding rate; the fluorescent vector DNAh-Bio-EFGRapt-Cy5 can be combined with SK-OV-3 cells, and the combination rate is one hundred percent.
Example 7
Detection of stability of DNAh-Bio-EGFRApt-Cy 5-paclitaxel nanoparticles in serum
Experimental materials, reagents and equipment
1. Experimental Material
DNAh-Bio-EGFRApt-Cy 5-paclitaxel (same as example 6) at a concentration of 1000.0. mu.g/ml.
2. Experimental reagent
6 XDNA sample buffer (TSJ010, engine biology), 100bp DNA molecular marker (TSJ010, engine biology); 10000 × SolarGelRed nucleic acid dye (E1020, solarbio); 8% non-denaturing polyacrylamide gel (self-formulated); 1 × TBE Buffer (No RNase) (self-mix); serum (FBS) (Excel); RPMI1640 (GBICO).
Electrophoresis apparatus (PowerPac Basic, Bio-rad), vertical electrophoresis tank (Mini PROTEAN Tetra Cell, Bio-rad), decolorizing shaker (TS-3D, orbital shaker), gel imager (Tanon 3500, Tanon).
Second, Experimental methods
(1) Taking 2 mu L of DNAh-Bio-EGFRApt-Cy 5-paclitaxel nanoparticles, diluting the 2 mu L of the nanoparticles with 50% FBS1640 and 6 mu L of RPMI1640 culture medium until the concentration reaches 200 mu g/ml, respectively diluting the diluted nanoparticles in 5 tubes, and carrying out water bath on the diluted samples at 37 ℃ for different times (0, 10min, 1h, 12h and 36 h).
(2) The treated sample 10. mu.l was mixed with 2. mu.l of 6 XDNA Loading Buffer, and the mixture was labeled by ice-wash.
(3) 8% Native PAGE is taken, nanoparticle samples with different incubation times are coated with a gel, the loading amount is 12 mu L/hole/sample, and the program electrophoresis is set at 90-100V for 50 min.
(4) And after the electrophoresis is finished, dyeing, placing the mixture on a horizontal shaking table for 30min, and photographing and imaging.
Third, experimental results
The results of native PAGE gel electrophoresis are shown in FIG. 12, wherein 1 represents 0min, 2 represents 10min, 3 represents 1h, 4 represents 12h, and 5 represents 36 h. The target band of DNAh-Bio-EGFRApt-Cy 5-paclitaxel nanoparticle is about 200bp, and it can be seen from FIG. 12 that DNAh-Bio-EGFRApt-Cy 5-paclitaxel nanoparticle is basically stable when incubated at 37 ℃.
Example 8
Study of cytotoxicity of RNAh-Bio-670-paclitaxel nanoparticles in U87MG cells
First, experimental material and experimental method
1. Experimental materials:
a sample to be tested: small molecule drugs paclitaxel and RNAh-Bio-670-paclitaxel nanoparticles (note: RNAh-Bio-670-paclitaxel nanoparticles are nanoparticles formed by performing Biotin modification on the 5 'ends of the a chain and the b chain and performing quasar670 fluorescein modification on the 3' end of the c chain, and then carrying paclitaxel (carrying by the chemical method in example 5) prepared by the self-assembly method in example 1).
Preparing the concentration of the medicine:
ready-to-use reagents were prepared in corresponding volume containers and quantified to 10uM with PBS.
2. The experimental reagent:
EMEM medium (Gibco); fetal Bovine Serum (FBS) (ExCell Bio, FNA500-500 mL); Penicillin/Streptomycin (Penicilin/Streptomyces, PS) (Gibco,15140-122-100 mL); PBS buffer (Gibco, C20012500BT-500 mL); Trypsin-EDTA (Stemcell,07901-500 mL); DMSO (Sigma, D5879-1L); CellTiter-Glo Luminescent Cell vitality Assay kit (CTG) (Promega, G7572-100 mL).
3. An experimental instrument:
inverted Microscope (Inverted Microscope) (Olympus IX71, No. 112A-1); 96-well Plate Reader (96-well Plate Reader) (Molecular Devices, Flexstation 3); perkin Elmer Envision 2104Multilabel Reader (No. 01-094-.
4. The experimental method comprises the following steps:
1) cell culture and plating
U87MG was added to EMEM basal medium with 10% FBS and 1% PS, respectively, at 37 ℃ and 5% CO 2 Culturing 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 96-well plates in a volume of 90 μ L of 5000 cells, 4 wells per group.
2) Gradient drug concentration formulation and administration
After 24 hours, compound solutions were transferred to each well at 200nM per sample, 4 replicates.
Solvent control ═ DMSO
Medium (untreated) control cells only without Compound treatment
Blank control No cells used for Instrument zeroing
3) Post-administration culture of cells
The medicated cells were incubated at 37 deg.C and 5% CO 2 Cultured under the conditions for 72 hours.
4) Detection kit for treating cells
The plate was brought to room temperature in advance and allowed to stand for 30 minutes. Add 100. mu.L CellTiter-
The reagents were mixed on a shaker for 2 minutes to facilitate cell lysis. Values were read and recorded using a Perkin Elmer Envision 2104Multilabel Reader instrument.
5) Acquiring and processing experimental data
The obtained experimental data were analyzed using excel software and curve analysis was fitted using GraphPad Prism 5 software.
II, experimental results:
table 44: cell viability (%)
| Cell lines | Time of treatment | Paclitaxel | RNAh-Bio-670-Taxol |
| U87MG | 72h | 56.74 | 76.23 |
The results are shown in Table 44 and FIG. 13, and it can be seen from Table 44 and FIG. 13 that the nanoparticles of paclitaxel and RNAh-Bio-670-paclitaxel have significant inhibitory effect on the proliferation of U87MG cells, and it is unexpected that: at a concentration of 10. mu.M, the inhibition rates of the two drugs on cells were 23.77% and 43.26%, respectively. As can be seen, the RNAh-Bio-670-paclitaxel nanoparticles have stronger inhibitory activity on cell proliferation, so that the dosage of the drug can be obviously reduced, and the toxic and side effects can be reduced.
Further, in order to confirm that the targeted fluorescent vector itself has no significant toxicity to U87MG cells, the present application further designed toxicity experiments of RNAh-Bio-FAM targeted fluorescent vector to U87MG cells, and compared with the toxicity of small molecule chemical drug Cisplatin (cissplatin) to U87MG cells, the results showed that the fluorescent vector itself has no significant toxicity to U87MG cells (data not shown).
Example 9
Cytotoxicity of DNAh-Bio-EGFRApt-Cy 5-paclitaxel nanoparticles in SKOV3 cells, respectively
First, experimental material
1. Cell information (see table 45):
table 45:
2. samples to be tested (see table 46):
table 46:
3. consumables and equipment (see table 47):
table 47:
4. reagents (see table 48):
table 48:
II, an experimental method:
1) harvesting cells in logarithmic growth phase, taking a small amount of cells, and staining and counting the cells by trypan blue to ensure that the cell activity reaches more than 98%;
2) cell density was adjusted to 2.22X 10 with growth medium 4 /mL；
3) Planting 90 mu L/well cell suspension into a 96-well plate, wherein the number of cells in each well in the plate is 2000;
4) placing the planted cell plate in an incubator at 37 ℃ for overnight incubation;
5) compound was diluted 3.16-fold in gradient from 9 concentration points;
6) taking out the cell culture plate, adding 10 mu L/hole of 10X concentration drug working solution into corresponding holes of the cell culture plate, making three multiple holes for each concentration, and obtaining the final action concentration of the drug shown in the following table 49;
table 49:
7) placing the cell culture plate in an incubator to continue incubation for 96 hours;
8) mixing CellTiter
The AQueous One Solution reagent is put in room temperature for meltingMelting in water bath at 90 min or 37 deg.C, and balancing at room temperature for 30 min;
9) add 20. mu.L/well CellTiter to cell culture plate
An AQueous One Solution reagent;
10) placing the cell culture plate in an incubator at 37 ℃ for further incubation for 3 hours;
11) OD of each well in the cell plate was read with microplate reader 490 A value;
12) and (4) processing and analyzing data.
The data were graphically processed using GraphPad Prism 5.0 software to calculate IC50, data were subjected to sigmoidal nonlinear regression analysis to match the appropriate dose-effect curves. The survival rate was calculated as follows, and IC50 was automatically calculated in GraphPad Prism 5.0.
Cell viability (%) - (OD) Hole to be tested –OD Blank control )/(OD Negative control -OD Blank control )x 100％。
Third, the experimental results (see Table 50, FIGS. 14a to 14d)
Table 50:
as can be seen from table 50 and fig. 14a, 14b, 14c, and 14d, for SKOV3 cell line, compared with the single DNAh targeted fluorescent vector, the small molecule drug paclitaxel and DNAh drug-loaded particle DNAh-Bio-EGFRapt-Cy 5-paclitaxel are toxic to SKOV3 cells, IC50 of paclitaxel and paclitaxel-Bio-EGFRapt-DNAh acting on SKOV3 cells are respectively <0.001 μ M and 16.05 μ M, and IC50 of DNAh-Bio-EGFRapt-Cy5 and DMSO acting on SKOV3 cells is >1 μ M and > 1%, respectively.
Assembly of nucleic acid nanoparticles
Example 10
One, 7 groups of extended segment deformation + core short sequence RNA nano particle carriers:
(1)7 sets of three polynucleotide base sequences which form the RNA nano-particle with the extension segment deformed and the core short sequence:
table 51: r-15:
table 52: r-16:
table 53: r-17:
table 54: r-18:
table 55: r-19:
table 56: r-20:
table 57: r-21:
II, self-assembly testing:
(1) mixing RNA single strands a, b and c at the same time according to a molar ratio of 1:1:1, and dissolving in DEPC water or TMS buffer solution;
(2) heating the mixed solution to 80 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off 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 and detection and laser scanning observation.
Third, self-assembly test results
(1) Electrophoretic detection
The main reagents and instruments were as follows:
table 58:
| name of reagent | Goods number | Manufacturer of the |
| 6×DNA Loading buffer | TSJ010 | Organisms of Onychidae |
| 20bp DNA Ladder | 3420A | TAKARA |
| 10000 SolarGelRed nucleic acid dye | E1020 | solarbio |
| 8% non-denaturing PAGE gel | / | Self-matching |
| 1 × TBE Buffer (No RNAse) | / | Self-matching |
Table 59:
the method comprises the following steps:
the RNA nanoparticles were diluted with ultrapure water according to the method of Table 60 below.
Table 60:
| the measured concentration (μ g/mL) | |
| R-15 | 165.937 |
| R-16 | 131.706 |
| R-17 | 144.649 |
| R-18 | 164.743 |
| R-19 | 126.377 |
| R-20 | 172.686 |
| R-21 | 169.455 |
② mixing 10 microliter (500ng) of the treated sample with 2 microliter of 6 multiplied by DNA Loading Buffer, operating on ice and marking.
Taking 8% non-denaturing PAGE gel, coating a piece of gel on samples with different incubation times, and completely loading 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.
And fourthly, dyeing after glue running is finished, placing the dyed fabric on a horizontal shaking table for 30min, and photographing and imaging.
And (3) detection results:
the results of native PAGE gel for 7 sets of extended stretch-degenerate + core short sequence RNA self-assembly products are shown in FIG. 15. Lanes 1 to 7 in FIG. 15 are, from left to right: 7 groups of extension segment deformation + core short sequence RNA self-assembly products R-15, R-16, R-17, R-18, R-19, R-20 and R-21.
The results in fig. 15 clearly show that the bands of the 7 sets of extended stretch-deformed + core short sequence RNA self-assembly products are bright and clear, which indicates that the 7 sets of extended stretch-deformed + core short sequence RNA strands complete self-assembly and form a stable nanoparticle structure.
(2) Determination of potential
The determination method comprises the following steps: preparing a potential sample (self-assembly product dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking the setting of finishing the determination, generating a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results at 25 ℃ of 7 groups of extended segment deformation + core short sequence RNA nanoparticles are as follows:
table 61:
table 62:
table 63:
table 64:
table 65:
table 66:
table 67:
from the potential detection data described above, it is found that: the 7 groups of the extended segment deformation and core short sequence RNA nanoparticles have good stability, and further show that the nanoparticles formed by self-assembly of the extended segment deformation and the core short sequence RNA have a stable self-assembly structure.
(3) Particle size measurement
1. Preparing a potential sample (7 groups of extension sections are deformed and core short sequence RNA is added) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
2. opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
3. setting software detection parameters;
4. then click on the confirmed setting, a measurement dialog box appears, and Start is clicked, and the results of DLS measurement values of hydrodynamic sizes of 7 groups of extended stretch variants + core short sequence RNAs are as follows:
table 68:
| average particle diameter (nm) | |
| R-15 | 6.808 |
| R-16 | 6.978 |
| R-17 | 7.592 |
| R-18 | 7.520 |
| R-19 | 6.936 |
| R-20 | 7.110 |
| R-21 | 6.720 |
(4) TM value detection
And (3) detecting the TM values of the 7 groups of extended section deformation + core short sequence RNA nanoparticles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 69:
| name of reagent | Goods number | Manufacturer of the product |
| AE buffer | / | Takara |
| SYBR Green I dyes | / | Self-matching |
Table 70:
| name(s) | Model number | Manufacturer of the product |
| Real-Time System | CFX Connect | Bio-rad |
| Super clean bench | HDL | BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd. |
The method comprises the following steps:
after diluting the sample with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1: 200 dilution) to give a final volume of 20. mu.L, at the following dilution concentrations:
table 71:
② incubating for 30min at room temperature in dark place;
and thirdly, detecting on a computer, wherein the program is set to be 20 ℃, the temperature is increased to 0.1-95 ℃ per second, and the reading is carried out once every 5 seconds.
And (3) detection results:
the TM values of 7 sets of extended stretch modified + core short sequence RNA nanoparticles are shown in the following, wherein the dissolution curve of R-15 is shown in FIG. 16, the dissolution curve of R-16 is shown in FIG. 17, the dissolution curve of R-17 is shown in FIG. 18, the dissolution curve of R-18 is shown in FIG. 19, the dissolution curve of R-19 is shown in FIG. 20, the dissolution curve of R-20 is shown in FIG. 21, and the dissolution curve of R-21 is shown in FIG. 22. Because of the specificity of the RNA sample, the temperature corresponding to 1/2RFUmax within the range of 20-90 ℃ is taken as the Tm value of the sample in the detection.
Table 72:
| TM value (. degree. C.) | |
| R-15 | 57.5℃ |
| R-16 | 53.8℃ |
| R-17 | 55.2℃ |
| R-18 | 54.5℃ |
| R-19 | 54.0℃ |
| R-20 | 59.5℃ |
| R-21 | 65.0℃ |
The TM values of 7 groups of extension segment deformation and core short sequence RNA nanoparticles are higher, which indicates that the self-assembly product has good structural stability.
Example 11
The first and the 7 groups of the extended segment deformation + core short sequence DNA nano particle carriers:
(1)7 groups of three polynucleotide base sequences which form the extension segment deformation + core short sequence DNA nano-particles:
table 73: d-8:
table 74: d-9:
table 75: d-10:
table 76: d-11:
table 77: d-12:
table 78: d-13:
table 79: d-14:
II, self-assembly testing:
(1) mixing and dissolving the DNA single strands a, b and c in DEPC water or TMS buffer solution at the same time according to the molar ratio of 1:1: 1;
(2) heating the mixed solution to 95 ℃, keeping the temperature for 5min, and then slowly cooling to room temperature at the speed of 2 ℃/min;
(3) loading the product on 8% (m/V) native PAGE gel and electrophoretically purifying the complex at 100V in TBM buffer at 4 ℃;
(4) cutting off a target band, eluting in a DNA elution buffer solution at 37 ℃, precipitating with ethanol overnight, and volatilizing at low temperature under reduced pressure to obtain a DNA self-assembly product;
(5) electrophoretic analysis detection and laser scanning observation;
(6) detecting the potential;
(7) detecting the particle size;
(8) and (5) detecting a TM value.
Third, self-assembly test results
(1) The main reagents and instruments for electrophoretic detection are as follows:
table 80:
| name of reagent | Goods number | Manufacturer(s) of |
| 6×DNA Loading buffer | TSJ010 | Organisms of Onychidae |
| 20bp DNA Ladder | 3420A | TAKARA |
| 10000 SolarGelRed nucleic acid dye | E1020 | solarbio |
| 8% non-denaturing PAGE gel | / | Self-matching |
| 1 × TBE Buffer (No RNAse) | / | Self-matching |
Table 81:
the method comprises the following steps:
the DNA nanoparticles were diluted with ultrapure water according to the method of the following Table 82.
Table 82:
② mixing 10 microliter (500ng) of the treated sample with 2 microliter of 6 multiplied by DNA Loading Buffer, operating on ice and marking.
Taking 8% non-denaturing PAGE gel, coating a piece of gel on samples with different incubation times, and completely loading 12 mu L of processed samples, and setting the program to run gel for 40min at 100V.
And fourthly, dyeing after glue running is finished, placing the dyed fabric on a horizontal shaking table for 30min, and photographing and imaging.
And (3) detection results:
the results of native PAGE gel of 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are shown in FIG. 23. Lanes 1 to 7 in FIG. 23 are, from left to right: 7 groups of extension segment deformation + core short sequence DNA self-assembly products D-8, D-9, D-10, D-11, D-12, D-13 and D-14.
It can be clearly seen from the results of fig. 23 that the bands of the 7 sets of extended stretch-deformed + core short sequence DNA self-assembly products are bright and clear, which indicates that the 7 sets of extended stretch-deformed + core short sequence DNA strands complete self-assembly and form a stable nanoparticle structure.
(2) Determination of potential
The determination method comprises the following steps: preparing a potential sample (a self-assembly product is dissolved in ultrapure water) and putting the potential sample into a sample cell, opening a sample cell cover of the instrument and putting the instrument into the sample cell;
opening software, clicking a menu measurere @ ManUal, and presenting a ManUal measurement parameter setting dialog box;
setting software detection parameters;
then clicking to finish setting, appearing a measurement dialog box, and clicking Start to Start;
and (3) measuring results: the potential detection results at 25 ℃ of 7 groups of extension segment deformation and core short sequence DNA nanoparticles are as follows:
table 83:
table 84:
table 85:
table 86:
table 87:
table 88:
table 89:
from the potential detection data described above, it is found that: the 7 groups of the extended section deformation and core short sequence DNA nano-particles have good stability, and further show that the nano-particles formed by self-assembly of the extended section deformation and the core short sequence DNA have a stable self-assembly structure.
(3) Particle size measurement
Firstly, preparing a potential sample (7 groups of extension segment deformation and core short sequence DNA) to be placed in a sample cell, opening a sample cell cover of an instrument, and placing the instrument;
opening software, clicking a menu, and displaying a manual measurement parameter setting dialog box;
setting software detection parameters;
and clicking the setting after determination, generating a measurement dialog box, clicking Start, and obtaining the DLS measurement values of the hydrodynamic sizes of 7 groups of the extended segment variants and the core short sequence RNA as follows:
table 90:
| average particle diameter (nm) | |
| D-8 | 7.460 |
| D-9 | 7.920 |
| D-10 | 7.220 |
| D-11 | 7.472 |
| D-12 | 6.968 |
| D-13 | 7.012 |
| D-14 | 6.896 |
(4) TM value detection
And (3) detecting the TM values of the 7 groups of extension segment deformation and core short sequence DNA nano-particles by adopting a dissolution curve method, wherein the sample is consistent with the potential sample.
Reagents and instrumentation were as follows:
table 91:
| name of reagent | Goods number | Manufacturer of the product |
| AE buffer | / | Takara |
| SYBR Green I dyes | / | Self-matching |
Table 92:
| name(s) | Type number | Manufacturer of the product |
| Real-Time System | CFX Connect | Bio-rad |
| Super clean bench | HDL | BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd. |
The method comprises the following steps:
after diluting the sample with ultrapure water, 5. mu.g of the diluted sample was mixed with 2. mu.L of SYBR Green I dye (1: 200 dilution) to a final volume of 20. mu.L, at the following dilution concentrations:
table 93:
② incubating for 30min at room temperature in dark place;
and thirdly, detecting on a computer, setting a program to start at 20 ℃, raising the temperature to between 0.1 and 95 ℃ per second, and reading once every 5 seconds.
And (3) detection results:
the TM values of 7 sets of extended length modified + core short sequence DNA nanoparticles are shown in the following, and the dissolution profile of D-8 is shown in FIG. 24, the dissolution profile of D-9 is shown in FIG. 25, the dissolution profile of D-10 is shown in FIG. 26, the dissolution profile of D-11 is shown in FIG. 27, the dissolution profile of D-12 is shown in FIG. 28, the dissolution profile of D-13 is shown in FIG. 29, and the dissolution profile of D-14 is shown in FIG. 30.
Table 94:
| TM value (. degree. C.) | |
| D-8 | 48.5 |
| D-9 | 52.5 |
| D-10 | 54.5～55.0 |
| D-11 | 48.7 |
| D-12 | 51.5 |
| D-13 | 51.0 |
| D-14 | 49.2 |
As can be seen from the dissolution curves of 7 sets of extended stretch deformation + core short sequence DNA nanoparticles shown in FIGS. 24 to 30, 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 12
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 95:
| name of reagent | Goods number | Manufacturer(s) of |
| 6×DNA Loading buffer | TSJ010 | Organisms of Onychidae |
| 20bp DNA Ladder | 3420A | TAKARA |
| 10000 SolarGelRed nucleic acid dye | E1020 | solarbio |
| 8% non-denaturing PAGE gel | / | Self-matching |
| 1 XTBE Buffer (No RNase) | / | Self-matching |
| Serum (FBS) | / | Excel |
| RPMI 1640 | / | GBICO |
Table 96:
the method comprises the following steps:
firstly, preparing the RNA nano-particles into the concentration shown in the following table, then diluting the prepared sample according to the method shown in the following table, diluting the sample by 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
table 97:
mixing 10 mu L of the treated sample with 2 mu L of 6 multiplied DNA Loading Buffer, operating on ice and marking;
thirdly, 8% non-denaturing PAGE gel is taken, samples with different incubation times are coated with a piece of gel, all samples processed by 12 mu L are loaded, and the procedure of 100V gel running is set for 40 min;
fourthly, dyeing is carried out after glue running is finished, the dyeing is placed on a horizontal shaking table to be slowly oscillated for 30min, and photographing and imaging are carried out.
The electrophoresis detection result of R-15 is shown in FIG. 31, the electrophoresis detection result of R-16 is shown in FIG. 32, the electrophoresis detection result of R-17 is shown in FIG. 33, the electrophoresis detection result of R-18 is shown in FIG. 34, the electrophoresis detection result of R-19 is shown in FIG. 35, the electrophoresis detection result of R-20 is shown in FIG. 36, and the electrophoresis detection result of R-21 is shown in FIG. 37. In fig. 31 to 37, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and 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 13
The stability of 7 groups of extended length modified + core short sequence DNA nanoparticles in serum was characterized by non-denaturing PAGE.
The main reagents and instruments were as follows:
table 98:
| name of reagent | Goods number | Manufacturer(s) of | |
| 6×DNA Loading buffer | TSJ010 | Organisms of Onychidae | |
| 20bp DNA Ladder | 3420A | TAKARA | |
| 10000 × SolarGelRed nucleusAcid | E1020 | solarbio | |
| 8% native PAGE gel | / | Self-matching | |
| 1 × TBE Buffer (No RNAse) | / | Self-matching | |
| Serum (FBS) | / | Excel | |
| RPMI 1640 | / | GBICO |
TABLE 99:
the method comprises the following steps:
preparing DNA nano particles into the concentration shown in the following table, diluting the prepared sample according to the method shown in the following table, diluting the sample by 5 tubes, and carrying out water bath on the diluted sample at 37 ℃ for different time (0, 10min, 1h, 12h and 36 h);
table 100:
mixing 5 mu L of the treated sample with 1 mu L of 6 multiplied by DNA Loading Buffer, and marking by operating on ice;
thirdly, 8% non-denaturing PAGE gel is taken, samples with different incubation times are coated with a piece of gel, all samples processed by 6 mu L are loaded, and the procedure of 100V gel running is set for 40 min;
and fourthly, dyeing after the glue running is finished, placing the dyed fabric on a horizontal shaking table to slowly oscillate for 30min, and taking pictures for imaging.
The electrophoresis detection result of D-8 is shown in FIG. 38, the electrophoresis detection result of D-9 is shown in FIG. 39, the electrophoresis detection result of D-10 is shown in FIG. 40, the electrophoresis detection result of D-11 is shown in FIG. 41, the electrophoresis detection result of D-12 is shown in FIG. 42, the electrophoresis detection result of D-13 is shown in FIG. 43, and the electrophoresis detection result of D-14 is shown in FIG. 44. In fig. 38 to 44, lanes from left to right are M: marker; 1: 36 h; 2: 12 h; 3: 1 h; 4: 10 min; 5: and 0 min. From the results of the serum stability test, it can be seen that: the non-denatured gel fruits of 10min, 1h, 12h and 36h showed no significant difference in the DNA nanoparticle sample bands at different times, indicating that the DNA nanoparticles D-8 to D-14 were relatively stable in 1640 medium of 50% FBS with no significant degradation.
Nucleic acid nanoparticle-carried drug assay
Example 14
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 10, 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 11 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 15
First, cell information
HepG2 (Source synergy cell bank), DMEM + 10% FBS + 1% double antibody (gibco, 15140-122), culture conditions at 37 ℃ and 5% CO 2 And saturation humidity.
Second, the object to be measured
Blank vector: 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 11.
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 11 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 101:
four, main reagent
Table 102:
| name of reagent | Manufacturer of the product | Goods number | Remarks for note | |
| DMEM (Biotin free) | Providing all the | YS3160 | ||
| 1％BSA-PBS | Self-matching | － |
And fifthly, an experimental method:
1. adjusting the cell state to logarithmic phase, changing the culture medium to a biotin-free and folic acid-free culture medium, and placing the culture medium in an incubator at 37 ℃ for overnight incubation;
2. after the incubation is finished, pancreatin is eliminatedCollecting cell suspension, centrifuging at 1000rmp for 5min, adjusting concentration, and collecting 2 × 10 5 -5×10 5 cells/EP tube, wash 2 times with 1 mL/tube of 1% BSA-PBS, and observe the tube bottom cells to prevent aspiration.
3. Dissolving the object to be tested, and diluting the object to be tested to the use concentration;
4. completely sucking cell supernatant, sequentially adding 100 mu L of corresponding samples into each tube, keeping out of the sun, and incubating for 2h at 37 ℃;
5. washed 2 times with 1% BSA-PBS; centrifuging at 1000rmp for 5 min;
6. finally, resuspending the cell pellet with 300. mu.L PBS and detecting it on flow machine (the blank vector used in this example was labeled by Quasar670, whereas doxorubicin in the vector drug was self-fluorescent and thus could be detected by FL4-APC and FL2-PE, respectively);
7. and (6) analyzing the data.
Sixth, experimental results
1. The results of the experiment are shown in the following table:
table 103:
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 16
The toxicity of the DNA nanoparticles and the carrier drug to HepG2 is detected by a CCK8 method.
First, main reagent
Table 104:
| name of reagent | Manufacturer of the product | Goods number |
| PBS | － | － |
| DMSO | SIGMA | D2650 |
| DMEM (Biotin free) | All-medicinal Zhida Providence | YS3160 |
| FBS | Excell Bio | FSP500 |
| Double antibody | gibco | 15140-122 |
| Pancreatin | gibco | 25200-056 |
| CCK8 kit | Biyuntian (a Chinese character) | C0038 |
Second, main consumables and instrument
Table 105:
| 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 |
| CO 2 Culture box | Thermo | 3111 |
| Inverted microscope | UOP | DSZ2000X |
| Enzyme mark instrument | SHANGHAI OYIN EXPERIMENT EQUIPMENT Co.,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% CO 2 And saturation humidity.
Fourth, experimental materials
1. Sample to be tested
Blank vector: the DNA nanoparticle carriers formed by self-assembly of D-8, D-9, D-10, D-11, D-12, D-13 and D-14 in the foregoing example 11 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 11 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 the original culture medium, adding 100 μ L culture medium of samples to be tested with different concentrations, and repeating the wells for 3 times.
Table 106:
| number of 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, each of the drug-loaded and blank vehicles was first prepared as a 100 μ M stock solution in PBS and then diluted in complete medium (biotin-free DMEM). The technical doxorubicin is prepared into a stock solution of 100 μ M with DMSO and then diluted with complete medium (biotin-free DMEM). DMSO was directly diluted with complete medium (biotin-free DMEM).
3. Adding a sample to be detected, and putting a 96-well plate into 5% CO at 37 DEG C 2 Incubate in incubator for 72 h.
4. The kit was removed and thawed at room temperature, and 10. mu.L of CCK-8 solution was added to each well, or CCK8 solution was mixed with the medium at a ratio of 1:9 and then added to the wells at a rate of 100. mu.L/well.
5. 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 5.0 50 。
Sixth, experimental results
Table 107:
and (4) conclusion:
as can be seen from the above table and FIGS. 45a, 45b, 45c, 45D, and 45e, the IC of D-10-doxorubicin, D-11-doxorubicin, D-12-doxorubicin, D-13-doxorubicin, and D-14-doxorubicin on HepG2 cells 50 0.2725. mu.M, 0.05087. mu.M, 0.0386, 0.03955, 0.04271, 0.02294, 0.03017 and 0.03458, respectively; IC of DMSO on HepG2 cells 50 Is composed of>0.1 percent; IC of HepG2 cells acted on by D-8 (blank vector), D-9 (blank vector), D-10 (blank vector), D-11 (blank vector), D-12 (blank vector), D-13 (blank vector) and D-14 (blank vector) 50 Are all made of>1 μ M. It shows that compared with the pure blank vectors of D-8, D-9, D-10, D-11, D-12, D-13 and D-14, the original drug adriamycin of the small molecular drug and the drug-carrying D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin are toxic to HepG2 cells of HepG2 cell line, and the carried medicines of D-8-adriamycin, D-9-adriamycin, D-10-adriamycin, D-11-adriamycin, D-12-adriamycin, D-13-adriamycin and D-14-adriamycin have obvious synergistic effect compared with the original medicine of adriamycin.
Example 17
According to the chemical method of mounting in example 5 (the same method as in example 5 except for specific limitations), DNA nanoparticles formed by self-assembly of D-10 and D-14 in the previous example 11 were used as daunorubicin mounting vectors. The absorbance of daunorubicin at 492nm was measured with a microplate reader, and a standard curve was plotted (as shown in FIG. 46).
The daunorubicin carrying rates are respectively measured as follows:
the daunorubicin loading rate of the DNA nano-particles D-10 is 24.0;
the daunorubicin loading rate of the DNA nanoparticle D-14 was 25.1.
From the above description, it can be seen that the above-described embodiments of the present application achieve the following technical effects: the present application provides a series of nucleic acid nanoparticle carriers with thermodynamic stability, chemical stability, high loading rate, and combinable multiple modules. The 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 paclitaxel containing medicine is formed by hanging the small molecular medicine paclitaxel on the nucleic acid nanoparticle carrier provided by the application, so that the delivery stability of the paclitaxel can be improved, and under the condition that the nucleic acid nanoparticle carries a target head, on one hand, the paclitaxel is delivered to a target cell in a targeted manner, so that the bioavailability of the medicine is improved, on the other hand, the toxic and side effects on non-target cells or tissues are reduced due to the targeted delivery, the local administration concentration is reduced, and the toxic and side effects caused by high administration 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> paclitaxel-containing medicine, preparation method, pharmaceutical composition and application thereof
<130> PN114938BYZD
<141> 2019-09-30
<150> 201811205135.6
<151> 2018-10-16
<160> 180
<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 sequences
<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 sequences
<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 sequences
<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 sequences
<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 sequences
<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 sequences
<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 sequences
<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
<210> 172
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(65)
<223> a chain
<400> 172
cgcgcgccca cgagcgttcc gggcgcgcct tagtaacgtg ctttgatgtc gattcgacag 60
gaggc 65
<210> 173
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(26)
<223> b chain
<400> 173
gcgcccggtt cgccgccagc cgccgc 26
<210> 174
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(33)
<223> c chain
<400> 174
gcggcggcag gcggccatag ccgtgggcgc gcg 33
<210> 175
<211> 10
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(10)
<223> a sequence
<400> 175
<210> 176
<211> 9
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(9)
<223> b sequences
<400> 176
<210> 177
<211> 12
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12)
<223> c sequence
<400> 177
<210> 178
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(34)
<223> a chain
<400> 178
cgcgcgcgcc cacgagcgtt ccgggcgccg ccgc 34
<210> 179
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(33)
<223> b chain
<400> 179
gcggcggcgc ccggttcgcc gccagccgcc gcc 33
<210> 180
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(36)
<223> c chain
<400> 180
ggcggcggca ggcggccata gccgtgggcg cgcgcg 36
Claims (47)
1. A drug comprising paclitaxel, wherein the drug comprises a nucleic acid nanoparticle and paclitaxel, and paclitaxel is suspended on the nucleic acid nanoparticle;
the nucleic acid nanoparticle comprises a nucleic acid domain comprising a sequence a comprising a variation of the sequence a1, a sequence b comprising a variation of the sequence b1, and a sequence c comprising a variation of the sequence c 1;
wherein the sequence a1 is SEQ ID NO: 1: 5'-CCAGCGUUCC-3' or SEQ ID NO: 2: 5'-CCAGCGTTCC-3';
the b1 sequence is SEQ ID NO: 3: 5 '-GGUUCGCCG-3' or SEQ ID NO: 4: 5 '-GGTTCGCCG-3';
the c1 sequence is SEQ ID NO: 5'-CGGCCAUAGCGG-3' or SEQ ID NO: 6: 5'-CGGCCATAGCGG-3';
the sequence a, the sequence b and the sequence c self-assemble to form a structure shown in formula (1):
wherein W-C represents a Watson-Crick pair, N and N' represent non-Watson-Crick pairs, and W-C at any position are each independently selected from C-G or G-C;
in the a sequence, the first N from the 5' end is A, the second N is G, the third N is U or T, and the fourth N is any one of U, T, A, C or G;
in the b sequence, the first N 'from the 5' end is any one of U, T, A, C or G; the second N 'is U or T, and the third N' is C;
in the c sequence, the NNNN sequence along the direction from the 5 'end to the 3' end is CAUA or CATA;
the sequence a, the sequence b and the sequence c are any one of the following groups:
(1) a sequence: 5'-GGAGCGUUGG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCC-3', respectively;
(2) a sequence: 5'-GCAGCGUUCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCGC-3', respectively;
(3) a sequence: 5'-CGAGCGUUGC-3' the flow of the air in the air conditioner,
b sequence: 5'-GCUUCGCCG-3',
c sequence: 5'-CGGCCAUAGCCG-3';
(4) a sequence: 5'-GGAGCGUUGG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5 '-CCUUCGGG-3',
c sequence: 5'-CCCCCAUAGCCC-3', respectively;
(5) a sequence: 5'-GCAGCGUUCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCGC-3';
(6) a sequence: 5'-GCAGCGUUCG-3' 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', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-GCUUCGGCG-3',
c sequence: 5'-CGCCCAUAGCCG-3', respectively;
(8) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,
b sequence: 5'-CCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCC-3', respectively;
(9) a sequence: 5'-GCAGCGTTCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGTTCGCCG-3',
c sequence: 5'-CGGCCATAGCGC-3';
(10) a sequence: 5'-CGAGCGTTGC-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-GCTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3';
(11) a sequence: 5'-GGAGCGTTGG-3' the flow of the air in the air conditioner,
b sequence: 5'-CCTTCGGGG-3',
c sequence: 5'-CCCCCATAGCCC-3', respectively;
(12) a sequence: 5'-GCAGCGTTCG-3', and the adhesive tape is used for adhering the film to a substrate,
b sequence: 5'-CGTTCGGCG-3',
c sequence: 5'-CGCCCATAGCGC-3', respectively;
(13) a sequence: 5'-GCAGCGTTCG-3' the flow of the air in the air conditioner,
b sequence: 5'-CGTTCGGCC-3',
c sequence: 5'-GGCCCATAGCGC-3', 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', respectively;
(15) a sequence: 5'-CGAGCGTTCC-3', respectively;
b sequence: 5 '-GGTTCGCCG-3',
c sequence: 5'-CGGCCATAGCCG-3' are provided.
2. The agent of claim 1, further comprising a first extension in the nucleic acid domain, wherein the first extension is a Watson-Crick paired extension located 5 'and/or 3' to any of the a, b, and c sequences.
3. The medicament of claim 2, wherein the first elongate section is selected from at least any one of the group consisting of:
(1): a 5' end of chain: 5' -CCCA-3', 3' end of c strand: 5 '-UGGG-3';
(2): a 3' end of the chain: 5' -GGG-3', 5' end of b chain: 5 '-CCC-3';
(3): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-UGG-3';
(4): a 5' end of chain: 5' -CCCG-3', 3' end of c chain: 5 '-CGGG-3';
(5): a 5' end of the chain: 5' -CCCC-3', 3' end of c chain: 5 '-GGGG-3';
(6): b 3' end of strand: 5' -CCC-3', 5' -end of c chain: 5 '-GGG-3';
(7): b 3' end of strand: 5' -CCG-3', the 5' end of the c chain: 5 '-CGG-3';
(8): a 5' end of chain: 5' -CCCA-3', 3' end of c chain: 5 '-TGGG-3';
(9): b 3' end of strand: 5' -CCA-3', 5' end of c chain: 5 '-TGG-3'.
4. The agent of any one of claims 1 to 3, wherein the nucleic acid domain further comprises a second extension located 5 'and/or 3' to any one of the a, b, and c sequences, wherein the second extension is a Watson-Crick paired extension.
5. The agent of claim 4, wherein said second extension is an extension of CG base pairs.
6. The drug of claim 5, wherein the second extension is an extension of 1-10 CG base pairs.
7. The agent of claim 4, wherein said nucleic acid domain further comprises at least one second set of extensions of:
a first group: a 5' end of the chain: 5' -CGCGCG-3 ', 3' end of c chain: 5 '-CGCGCG-3';
second group: a 3' end of chain: 5' -CGCCGC-3 ', 5' -end of b chain: 5 '-GCGGCG-3';
third group: b 3' end of strand: 5' -GGCGGC-3 ', 5' -end of c chain: 5 '-GCCGCC-3'.
8. The agent of claim 4, wherein said second extension is an extended sequence comprising both CG base pairs and AT/AU base pairs.
9. The agent of claim 8, wherein the second extension is an extended sequence of 2 to 50 base pairs.
10. The drug of claim 8, wherein the second extension is an extension in which a sequence of 2 to 8 CG base pairs in succession alternates with a sequence of 2 to 8 AT/AU base pairs in succession; or
The second extension segment is an extension sequence formed by alternating sequences of 1 CG base pair and 1 AT/AU base pair.
11. The agent according to any one of claims 1 to 3, wherein the bases, ribose and phosphate in the a sequence, the b sequence and the c sequence have at least one modifiable site, and any of the modifiable sites is modified by any one of the following modifying linkers: -F, methyl, amino, disulfide, carbonyl, carboxyl, mercapto and aldehyde groups.
12. The agent of claim 11, wherein the sequence a, the sequence b and the sequence C have 2' -F modifications at the C or U bases.
13. The drug according to any one of claims 1 to 3, wherein the paclitaxel is loaded on the nucleic acid nanoparticles in a form of physical linkage and/or covalent linkage, and the molar ratio between the paclitaxel and the nucleic acid nanoparticles is 2-300: 1.
14. The drug according to claim 13, wherein the molar ratio between paclitaxel and the nucleic acid nanoparticles is 10-50: 1.
15. The drug according to claim 14, wherein the molar ratio between paclitaxel and the nucleic acid nanoparticles is 15-25: 1.
16. The drug of any one of claims 1 to 3, wherein the nucleic acid nanoparticle further comprises a biologically active substance attached to the nucleic acid domain, wherein the biologically active substance is one or more of a target, a fluorescein, an interfering nucleic acid siRNA, a miRNA, a ribozyme, a riboswitch, an aptamer, an RNA antibody, a protein, a polypeptide, a flavonoid, glucose, natural salicylic acid, a mab, a vitamin, a phenolic lecithin, and a small molecule drug other than paclitaxel.
17. The agent of claim 16, wherein the relative molecular weight of the nucleic acid domains is recorded as N 1 The total relative molecular weight of paclitaxel and the biologically active substance is recorded as N 2 ，N 1 / N 2 ≥1:1。
18. The drug of claim 16, wherein the biologically active substance is one or more of the target, the fluorescein, and the miRNA,
wherein the target head is located on any sequence of the a sequence, the b sequence and the c sequence.
19. The drug of claim 18, wherein the targeting moiety is located at the 5 'end or 3' end of any of the a sequence, the b sequence, the c sequence, or is inserted between GC bonds of the nucleic acid domains,
the miRNA is an anti-miRNA, the fluorescein is modified at the 5' end or the 3' end of the anti-miRNA, and the miRNA is located at any one or more of the 3' end of the a sequence, the 5' end and the 3' end of the c sequence.
20. The drug of claim 19, wherein the target head is folic acid or biotin, the fluorescein is any one or more of FAM, CY5 and CY3, and the anti-miRNA is anti-miR-21.
21. The drug according to claim 16, wherein the small molecule drug other than paclitaxel is a drug containing any one or more of the following groups: amino groups, hydroxyl groups, carboxyl groups, mercapto groups, phenyl ring groups, and acetamido groups.
22. The medicament of claim 16, wherein the protein is one or more of SOD, survivin, hTERT, EGFR, and PSMA; the vitamin is levo-C and/or esterified C; the phenols are tea polyphenols and/or grape polyphenols.
23. The drug according to claim 1, wherein the nucleic acid nanoparticles have a particle size of 1 to 100 nm.
24. The drug of claim 23, wherein the nucleic acid nanoparticles have a particle size of 5 to 50 nm.
25. The drug of claim 24, wherein the nucleic acid nanoparticles have a particle size of 10-30 nm.
26. The drug of claim 25, wherein the nucleic acid nanoparticles have a particle size of 10-15 nm.
27. A process for the preparation of a paclitaxel-containing drug, said process comprising the steps of:
providing a nucleic acid nanoparticle in a medicament according to any one of claims 1 to 26;
and (3) carrying the paclitaxel on the nucleic acid nanoparticles by means of physical connection and/or covalent connection to obtain the drug containing the paclitaxel.
28. The method of claim 27, wherein the step of attaching paclitaxel by physical attachment comprises:
mixing and stirring the paclitaxel, the nucleic acid nanoparticles and a first solvent to obtain a premixed system;
and precipitating the premixed system to obtain the medicine containing the paclitaxel.
29. The method according to claim 28, wherein the first solvent is one or more selected from the group consisting of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
30. The method of claim 28, wherein the step of precipitating the pre-mixed system to obtain the paclitaxel-containing drug comprises:
precipitating the premixed system to obtain a precipitate;
and washing the precipitate to remove impurities to obtain the medicine containing the paclitaxel.
31. The method according to claim 30, wherein the precipitation is carried out at a temperature of less than 10 ℃ after the premix system is mixed with absolute ethanol to obtain the precipitate.
32. The method according to claim 31, wherein the precipitation is performed at a temperature of 0 to 5 ℃ to obtain the precipitate.
33. The preparation method according to claim 31, wherein the precipitate is washed with 6-12 times of anhydrous ethanol to remove impurities, thereby obtaining the paclitaxel-containing drug.
34. The method of claim 27, wherein the step of entrapping paclitaxel by covalent attachment comprises:
preparing a paclitaxel solution;
reacting the paclitaxel solution with the G ring exoamino of the nucleic acid nanoparticles under the mediated action of formaldehyde to obtain a reaction system;
purifying the reaction system to obtain the medicine containing the paclitaxel.
35. The method of claim 34, wherein the step of reacting comprises:
and mixing the paclitaxel solution, a paraformaldehyde solution and the nucleic acid nanoparticles, and reacting under the condition of keeping out of the sun to obtain the reaction system.
36. The method according to claim 35, wherein the concentration of the paraformaldehyde solution is 3.7 to 4 wt%.
37. The method according to claim 35, wherein the paraformaldehyde solution is a mixture of paraformaldehyde and a second solvent, and the second solvent is one or more of DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
38. The production method according to any one of claims 27 to 37, further comprising a step of producing the nucleic acid nanoparticle, which comprises: the nucleic acid domain is obtained by self-assembling single strands corresponding to the nucleic acid domain in the nucleic acid nanoparticle in the medicament according to any one of claims 1 to 26.
39. The method of claim 38, wherein after obtaining the nucleic acid domain, the method further comprises: the nucleic acid nanoparticle is obtained by mounting a bioactive substance in the drug according to any one of claims 27 to 37 on the nucleic acid domain by means of physical and/or covalent attachment.
40. The method of claim 38, wherein the biologically active substance is covalently attached by solvent covalent attachment, linker covalent attachment, or click linkage.
41. The method of claim 40, wherein the solvent covalent bonding uses a third solvent as a bonding medium, and the third solvent is selected from one or more of paraformaldehyde, DCM, DCC, DMAP, Py, DMSO, PBS and glacial acetic acid.
42. The method of claim 40, wherein the linker is selected from the group consisting of disulfide bond, p-azido group, bromopropyne, and PEG.
43. The method of claim 40, wherein the click-through linkage is performed by modifying the biologically active substance precursor and the nucleic acid domain with an alkynyl or azide at the same time and then by click-through linkage.
44. The method of claim 40, wherein the biologically active substance is click-linked to the nucleic acid domain, the site of the biologically active substance precursor for the alkynyl or azido modification is selected from the group consisting of a 2 ' hydroxyl, a carboxyl or an amino group, and the site of the nucleic acid domain for the alkynyl or azido modification is selected from the group consisting of a G exocyclic amino, a 2 ' -hydroxyl, an A amino or a 2 ' -hydroxyl group.
45. A pharmaceutical composition comprising the paclitaxel-containing pharmaceutical according to any of claims 1 to 26.
46. Use of a paclitaxel-containing medicament according to any of claims 1 to 26 in the manufacture of a medicament for the treatment of tumors.
47. The use of claim 46, wherein the tumor is breast cancer or ovarian cancer.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018112051356 | 2018-10-16 | ||
| CN201811205135 | 2018-10-16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111053765A CN111053765A (en) | 2020-04-24 |
| CN111053765B true CN111053765B (en) | 2022-07-26 |
Family
ID=70297531
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910969109.9A Active CN111053765B (en) | 2018-10-16 | 2019-10-12 | Medicine containing paclitaxel, preparation method, pharmaceutical composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111053765B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116785445B (en) * | 2023-07-14 | 2024-07-16 | 百药智达（北京）纳米生物技术有限公司 | Targeting chemical medicine and preparation method thereof, pharmaceutical composition and application of targeting chemical medicine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5278051A (en) * | 1991-12-12 | 1994-01-11 | New York University | Construction of geometrical objects from polynucleotides |
| US5386020A (en) * | 1991-01-10 | 1995-01-31 | New York University | Multiply connected, three-dimensional nucleic acid structures |
| CN1390253A (en) * | 1999-11-13 | 2003-01-08 | 默克专利股份公司 | High order nucleic acid based structures |
| WO2012094586A2 (en) * | 2011-01-07 | 2012-07-12 | Peixuan Guo | Synthetic non-covalently self-assembling bipartite prna chimeras |
| 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 |
-
2019
- 2019-10-12 CN CN201910969109.9A patent/CN111053765B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5386020A (en) * | 1991-01-10 | 1995-01-31 | New York University | Multiply connected, three-dimensional nucleic acid structures |
| US5278051A (en) * | 1991-12-12 | 1994-01-11 | New York University | Construction of geometrical objects from polynucleotides |
| CN1390253A (en) * | 1999-11-13 | 2003-01-08 | 默克专利股份公司 | High order nucleic acid based structures |
| WO2012094586A2 (en) * | 2011-01-07 | 2012-07-12 | Peixuan Guo | Synthetic non-covalently self-assembling bipartite prna chimeras |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Design and Development of Nanosized DNA Assemblies in Polypod-like Structures as Efficient Vehicles for Immunostimulatory CpG Motifs to Immune Cells;Kohta Mohri等;《ACSNANO》;20120621;第6卷(第7期);第5931-5940页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111053765A (en) | 2020-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220226473A1 (en) | Nucleic acid nanocarrier drug and preparation method thereof, pharmaceutical composition and application thereof | |
| CN110711253B (en) | Medicine containing adriamycin and preparation method thereof | |
| US20210393785A1 (en) | Nucleic acid nanocarrier drug and preparation method thereof | |
| EP3821911A1 (en) | Nucleic acid nanoparticles, pharmaceutical composition comprising same, drug comprising doxorubicin and preparation method therefor | |
| CN111150852B (en) | Cisplatin-containing medicine, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111053765B (en) | Medicine containing paclitaxel, preparation method, pharmaceutical composition and application thereof | |
| EP3926047A1 (en) | Nucleic acid nano-carrier drug, preparation method therefor, and 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 | |
| CN110960530B (en) | Tacrine-containing medicine, preparation method thereof, pharmaceutical composition and application thereof | |
| CN111053913B (en) | Cytarabine-containing medicine, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960683B (en) | Medicine containing methotrexate, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960534B (en) | Medicine containing pentafluorouracil, 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 | |
| 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 | |
| CN111053914B (en) | Mitoxantrone-containing medicine, preparation method thereof, medicine composition and application thereof | |
| 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 | |
| CN111084887B (en) | Flavone-containing medicine, preparation method thereof, medicine composition and application | |
| CN110960542B (en) | Medicament containing pirarubicin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960689B (en) | Medicine containing daunorubicin, preparation method thereof, medicine composition and application thereof | |
| CN110960690B (en) | Medicine containing epirubicin, preparation method thereof, pharmaceutical composition and application thereof | |
| CN110960536B (en) | Aspirin-containing medicine, preparation method, pharmaceutical composition and application thereof |
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 |
|
| CP03 | Change of name, title or address |