CN109554397A - Nano particle and preparation method thereof - Google Patents
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- CN109554397A CN109554397A CN201710883289.XA CN201710883289A CN109554397A CN 109554397 A CN109554397 A CN 109554397A CN 201710883289 A CN201710883289 A CN 201710883289A CN 109554397 A CN109554397 A CN 109554397A
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- 239000002105 nanoparticle Substances 0.000 title claims abstract description 115
- 238000002360 preparation method Methods 0.000 title claims abstract description 47
- 239000007788 liquid Substances 0.000 claims abstract description 102
- 239000000243 solution Substances 0.000 claims abstract description 83
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 46
- 239000000376 reactant Substances 0.000 claims abstract description 42
- 239000000203 mixture Substances 0.000 claims abstract description 27
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 23
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 claims abstract description 16
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 claims abstract description 16
- 239000002245 particle Substances 0.000 claims abstract description 16
- 230000001376 precipitating effect Effects 0.000 claims abstract description 14
- 239000004094 surface-active agent Substances 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- 239000006185 dispersion Substances 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 9
- 239000011259 mixed solution Substances 0.000 claims abstract description 6
- 239000000725 suspension Substances 0.000 claims abstract description 5
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 claims abstract 5
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 claims abstract 5
- 229960004502 levodopa Drugs 0.000 claims abstract 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 10
- 239000010452 phosphate Substances 0.000 claims description 10
- -1 polyoxyethylenes Polymers 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 8
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 6
- 239000011575 calcium Substances 0.000 claims description 6
- 229910052791 calcium Inorganic materials 0.000 claims description 6
- 159000000007 calcium salts Chemical class 0.000 claims description 6
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 6
- 235000011180 diphosphates Nutrition 0.000 claims description 6
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical group 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 239000011574 phosphorus Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 238000007792 addition Methods 0.000 claims 1
- 210000000232 gallbladder Anatomy 0.000 claims 1
- 238000009938 salting Methods 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 238000001890 transfection Methods 0.000 abstract description 31
- 210000004027 cell Anatomy 0.000 description 121
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 36
- 206010006187 Breast cancer Diseases 0.000 description 35
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- 230000030279 gene silencing Effects 0.000 description 21
- 238000012360 testing method Methods 0.000 description 20
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 18
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 18
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 18
- 229960003638 dopamine Drugs 0.000 description 18
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 18
- 239000012528 membrane Substances 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- 210000001744 T-lymphocyte Anatomy 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 239000008187 granular material Substances 0.000 description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 108010074708 B7-H1 Antigen Proteins 0.000 description 5
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- 108090000695 Cytokines Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 229960005069 calcium Drugs 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 229940048084 pyrophosphate Drugs 0.000 description 4
- 230000002103 transcriptional effect Effects 0.000 description 4
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
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- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102100039496 Choline transporter-like protein 4 Human genes 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 101000889282 Homo sapiens Choline transporter-like protein 4 Proteins 0.000 description 2
- 101000998146 Homo sapiens Interleukin-17A Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 102000003814 Interleukin-10 Human genes 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 102100033461 Interleukin-17A Human genes 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 2
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000003012 bilayer membrane Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 239000000571 coke Substances 0.000 description 2
- 235000019700 dicalcium phosphate Nutrition 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 235000019820 disodium diphosphate Nutrition 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- GYQBBRRVRKFJRG-UHFFFAOYSA-L disodium pyrophosphate Chemical compound [Na+].[Na+].OP([O-])(=O)OP(O)([O-])=O GYQBBRRVRKFJRG-UHFFFAOYSA-L 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 235000019837 monoammonium phosphate Nutrition 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229940048086 sodium pyrophosphate Drugs 0.000 description 2
- VWDWKYIASSYTQR-YTBWXGASSA-N sodium;dioxido(oxo)azanium Chemical compound [Na+].[O-][15N+]([O-])=O VWDWKYIASSYTQR-YTBWXGASSA-N 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 2
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 2
- MEYZYGMYMLNUHJ-UHFFFAOYSA-N tunicamycin Natural products CC(C)CCCCCCCCCC=CC(=O)NC1C(O)C(O)C(CC(O)C2OC(C(O)C2O)N3C=CC(=O)NC3=O)OC1OC4OC(CO)C(O)C(O)C4NC(=O)C MEYZYGMYMLNUHJ-UHFFFAOYSA-N 0.000 description 2
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- JUNWLZAGQLJVLR-UHFFFAOYSA-J calcium diphosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])(=O)OP([O-])([O-])=O JUNWLZAGQLJVLR-UHFFFAOYSA-J 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- 229940043256 calcium pyrophosphate Drugs 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- MWKXCSMICWVRGW-UHFFFAOYSA-N calcium;phosphane Chemical compound P.[Ca] MWKXCSMICWVRGW-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 235000019821 dicalcium diphosphate Nutrition 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 101150054448 pdl-1 gene Proteins 0.000 description 1
- QLFFCLRSMTUBEZ-UHFFFAOYSA-N phosphoric acid;sodium Chemical compound [Na].[Na].OP(O)(O)=O QLFFCLRSMTUBEZ-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
Abstract
The invention discloses a kind of nano particles and preparation method thereof: organic solvent being mixed with surfactant, obtains A liquid；First reactant solution is mixed with functional mass solution, obtains B liquid；Second reactant solution is mixed with functional mass solution, obtains C liquid；Second reactant is capable of forming precipitating after contacting with the first reactant；Mix A liquid, B liquid obtains D liquid；A liquid, C liquid are mixed, and DOPA solution is added into gained mixed solution and mixes, obtains E liquid；D liquid is added in E liquid, gained mixture is centrifuged, precipitating is collected, obtains monofilm nano particle；The dispersion liquid of monofilm nano particle is prepared, and the mixture of DOPC, cholesterol is added into dispersion liquid, or the mixture of DOTAP, cholesterol is added, the particle in gained suspension particle solution is duplicature nano particle.The present invention improves transfection efficiency of the nano particle to target cell of carrying function substance by substep mixed organic solvents, surfactant, the first reactant solution, functional mass solution.
Description
Technical field
The present invention relates to technical field of life science, more particularly to a kind of nano particle and preparation method thereof.
Background technique
Nano particle is the nano material that a kind of Intermediate Gray has cavity, small-molecule drug, siRNA, antibody etc. can be wrapped
It wraps up in again inside cavity, imported into target cell.Til cell (T cell) is a kind of lymphocyte, by the cancerous tissue of patient and
The T cell of patient is cultivated together, is then allowed this Lymphocyte expansion to feed back in patient body later, is existing kill cancer cell
Technology.But the surface of cancer cell can have a kind of albumen for being called PD-L1, the albumen of this PD-L1 can be with the PD- of T cell
1 albumen combines together, will inhibit killing of the T cell to cancer cell.Therefore, it is desirable to T cell energy Efficient killing effect cancer cell it is necessary to
Trying every possible means can the PD-L1 reduction of the PD-1 and cancer cell of T cell.
Presently mainly respectively by PD-L1, PD-1 have the functional mass of inhibiting effect by carrier transfect to T cell,
Cancer cell, to realize that the PD-1 of T cell and the PD-L1 of cancer cell are reduced.But the generally existing transfection efficiency of current carrier is low
The shortcomings that.
Summary of the invention
Based on this, it is necessary to transfect inefficient problem to suspension cell for existing nano particle, provide a kind of efficient
Nano particle and preparation method thereof.
An object of the present invention is to provide a kind of preparation method of nano particle, includes the following steps:
Organic solvent is mixed with surfactant, obtains A liquid；
First reactant solution is mixed with functional mass solution, obtains B liquid；
Second reactant solution is mixed with functional mass solution, obtains C liquid；Second reactant and first reactant
Precipitating is capable of forming after contact；
Mix the A liquid, the B liquid obtains D liquid；
The A liquid, the C liquid are mixed, and the chloroformic solution mixing of DOPA (dopamine) is added into gained mixed solution,
Obtain E liquid；
The D liquid is added in the E liquid, gained mixture is centrifuged, precipitating is collected, obtains monofilm nano particle；
The dispersion liquid of monofilm nano particle is prepared, and DOPC (1,2- dioleoyl phosphatide is added into the dispersion liquid
Phatidylcholine), the mixture of cholesterol, or DOTAP (2- dioleoyl hydroxypropyl -3-N, N, N- trimethylammonium), cholesterol is added
Mixture, the particle in gained suspension particle solution is duplicature nano particle.
In wherein some embodiments, first reactant solution is calcium salt soln, and second reactant solution is
Phosphate solution or pyrophosphate solution, first reactant, the calcium of the second reactant, phosphorus amount of substance ratio be (25~
400): 1.The embodiment of the present invention can obtain by the quantity ratio of control calcium phosphorus and obtain nanoparticle core, uneven particle well
Size is uniform and has good stability, and is not susceptible to agglomerate.If have exceeded the embodiment of the present application restriction or can not shape
At nanoparticle core or it is to be formed by so granular core to agglomerate one, is not easy to carry out subsequent wrapping operation.
In wherein some embodiments, the concentration of the calcium salt soln is 4.5~5.5M；The phosphate solution or coke
The concentration of phosphate solution is 45~55mM.The embodiment of the present invention, can be further by using 4.5~5.5M calcium chloride solution
The homogeneity for the precipitating that the first reactant generates in conjunction with the second reactant is improved, and then improves the homogeneity of nano particle.
In wherein some embodiments, the calcium salt soln includes that calcium chloride solution, calcium nitrate solution, calcium gluconate are molten
Liquid；The phosphate solution includes that dipotassium hydrogen phosphate solution, ammonium dibasic phosphate solution, ammonium dihydrogen phosphate, calcium monohydrogen phosphate are molten
Liquid, calcium phosphate solution, sodium dihydrogen phosphate, disodium phosphate soln, sodium radio-phosphate,P-32 solution；The pyrophosphate solution includes coke
Calcium phosphate solution, sodium acid pyrophosphate solution, sodium pyrophosphate solution.
In wherein some embodiments, the concentration of DOPA is 15~25mg/ml, the DOPA solution in the DOPA solution
Addition volume and the A liquid, C liquid mixed solution volume ratio be (70~80): 1.
In wherein some embodiments, the solvent of the DOPA solution includes chloroform, methylene chloride, ethyl acetate, four
Hydrogen furans.
In wherein some embodiments, the DOPC, cholesterol mixture in, DOPC, cholesterol mass ratio be 1:
(2.5~3.5)；The DOTAP, cholesterol mixture in, DOTAP, cholesterol mass ratio be 2:(2.5~3.5).
In wherein some embodiments, in the preparation of the A liquid, organic solvent is hexamethylene, benzene, toluene, normal heptane, four
Any one or more of chlorination carbon；Surfactant includes polyoxyethylenes (5) nonylplenyl ether；Organic solvent and surface are living
Property agent mixing volume ratio be 80:20~50:50.The application is screened by organic solvent, surfactant screens, it is therefore an objective to be drawn
Enter grease system, the advantages of grease system is to obtain that suspension is high and the first reactant for being protected is in conjunction with the second reactant
The precipitating of generation improves stability, so that being less likely to occur to agglomerate between particle.
In wherein some embodiments, the A liquid, B liquid amount ratio be (50~200): 1；The dosage of the A liquid, C liquid
Than for (50~200): 1；The amount ratio of E liquid and D liquid is (1~3): (1~3).
In wherein some embodiments, the dispersion liquid for preparing monofilm nano particle is specifically by the monofilm
Nano particle is dispersed in chloroform, methylene chloride, ethyl acetate or tetrahydrofuran.
In wherein some embodiments, the functional mass in the functional mass solution includes functional nucleic acid sequence
(dsDNA, siRNA etc.), protein antibody, drug molecule.
In wherein some embodiments, the functional mass in the functional mass solution includes functional nucleic acid sequence.
It is a further object of the present invention to provide a kind of nano particle, which is obtained by above-mentioned preparation method.
Compared with prior art, the embodiment of the present invention has the advantages that
The embodiment of the present invention is molten by substep mixed organic solvents, surfactant, the first reactant solution, functional mass
Liquid, the second reactant solution, then superscribe double-layer outer membrane again, improve the nano particle of carrying function substance to target cell
Transfection efficiency.Specifically, the embodiment of the present invention passes through substep mixed organic solvents, surfactant, the first reactant solution, function
Energy substance solution, the second reactant solution, so that the nanometer formed after the first reactant, the second reactant carrying function substance
Grain core is small and uniform, is not susceptible to agglomerate, and after wrapping dopamine, DOPC or DOTAP as core, gained nano particle is produced
Product partial size, which can be controlled, to be further improved hydrophily on the outside of film while lesser scale (20nm or so), wrapping, increases
Dispersibility, the stability of grain, are not susceptible to agglomerate, and also enhance the affinity with cell surface, are easy to be absorbed by cell,
Increase transfection efficiency.
Detailed description of the invention
Figure 1A is transmission electron microscope (TEM) photo of the nano particle of preparation of the embodiment of the present invention；Figure 1B is this hair
Dynamic light scattering (Dynamic Light Scattering) map of the nano particle of bright embodiment preparation；
Fig. 2A is cellular uptake rate of the nano particle of preparation of the embodiment of the present invention under different concentration；Fig. 2 B is this hair
Cellular uptake rate of the nano particle of bright embodiment preparation in the progradation with the time；
Fig. 3 A is that the nano particle of preparation of the embodiment of the present invention transfects the PD1mRNA level of cell after til cell；Fig. 3 B is
The PD1 protein level of cell after the transfection til cell of the nano particle of preparation of the embodiment of the present invention；Fig. 3 C is the embodiment of the present invention
After the transfection cell of the nano particle of preparation, using the cell of the flow cytomery PD1 positive；
Fig. 4 A is that the nano particle of preparation of the embodiment of the present invention transfects the PDL1mRNA transcription water of cell after breast cancer cell
It is flat；Fig. 4 B is that the nano particle of preparation of the embodiment of the present invention transfects the PDL1 protein expression level of cell after breast cancer cell；Figure
After transfection cell of the 4C for the nano particle of preparation of the embodiment of the present invention, using the cell of the flow cytomery PDL1 positive；
Fig. 5 be different proportion, different silencing degree til cell the lethality of breast cancer cell is tested；
Fig. 6 A, Fig. 6 B are that transfection influences test result figure to til cell subgroup respectively；Before Fig. 6 C, Fig. 6 D are respectively transfection
Til cell generates cell factor variation test result figure afterwards；
Fig. 7 is the structural schematic diagram that the embodiment of the present invention prepares gained nano particle.
Specific embodiment
Nano particle and preparation method thereof of the invention is described in further detail below in conjunction with specific embodiment.
In order to be more clearly understood that technology contents of the invention, spy lifts following embodiment and is described in detail.It should be understood that this
It is a little that examples are only for illustrating the present invention and not for limiting the scope of the present invention.Actual conditions are not specified in the following example
Experimental method, usually according to normal condition, for example (,) Sambrook et al., molecular cloning: laboratory manual (New York:
ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to item proposed by manufacturer
Part.Used various common chemical reagent, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention
The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed
Any and all combinations of project.
In order to enable technical solution of the present invention is clearer, illustrates it can be readily appreciated that showing to it, it should be noted that
Protection scope of the present invention is not limited only to content described in following each examples.
Embodiment 1, the nano particle for containing silencing siRNA (PD1 of silencing til cell) and preparation method thereof
The preparation of 1.1 nano particles
This part provides a kind of preparation method of nano particle, includes the following steps:
Prepare A liquid: hexamethylene is mixed with polyoxyethylenes (5) nonylplenyl ether, the amount ratio of the two is 70:30 volume
Than；Hexamethylene at this could alternatively be benzene, toluene, normal heptane, carbon tetrachloride；
Prepare B liquid: (volume used is 100 μ to 5M calcium chloride solution (volume used is 75 μ L) with 100 μM of siRNA solution
L it) mixes, and 50 μ l of ultrapure water is added；In other examples, calcium chloride here can also change the sour calcium solution of work or grape
Saccharic acid calcium solution；
Prepare C liquid: 50mM disodium hydrogen phosphate (volume used is 75 μ L) and 100 μM of siRNA (volume used is 100 μ L)
Mixing, and 50 μ l of ultrapure water is added；In other embodiments, the disodium phosphate soln at this can change that make dipotassium hydrogen phosphate molten
Liquid, ammonium dibasic phosphate solution, ammonium dihydrogen phosphate, calcium hydrogen phosphate solution, calcium phosphate solution, sodium dihydrogen phosphate, phosphoric acid
Disodium hydrogen solution, sodium radio-phosphate,P-32 solution, calcium pyrophosphate solution, sodium acid pyrophosphate solution or sodium pyrophosphate solution；
Prepare D liquid: taking A liquid a (volume used is 15ml), 150 μ l B liquid are added, stir 20min；
Prepare E liquid: taking A liquid a (volume used is 15ml), 150 μ l C liquid are added, stir 5min, DOPA is then added
Chloroformic solution (concentration of DOPA is 20mg/ml, and the volume of the solution is 200 μ L), and continue stir 15min；The step
Chloroform also could alternatively be methylene chloride, ethyl acetate or tetrahydrofuran；
It prepares single layer membrane granule: E liquid is drop by drop entered into D liquid, stir 20 minutes, gained is precipitated as monofilm nanometer
Particle, that is, the core that the first reactant, the second reactant, function siRNA are formed only are enclosed with one layer of DOPA outside；In the step
In, in order to enable final nano particle size is more uniform, the rate of addition of E liquid is controlled in 1~3ml/min herein；The step
Suddenly further include the steps that cleaning monofilm nano particle, collect；Specifically include: the step of cleaning includes: to E
Liquid, D liquid mixture in volume ratio be 1:1 amount be added 10ml ethyl alcohol, continue stir 5 points of min, under the conditions of 10,000g
It is centrifuged 20min, supernatant is abandoned, stays precipitating；Add 10ml ethyl alcohol into precipitating, 20min be centrifuged under 10,000g, then abandon supernatant,
Stay precipitating；The collected step includes: that 1ml chloroform is added to collect monofilm nano particle；
It prepares the double-deck membrane granule: the above-mentioned monofilm nano particle being prepared being distributed in the chloroform of 1ml, by gained
Mixture (the volume ratio of dispersion liquid and 100 μ l DOPC, the mixture (volume ratio 1:3) of cholesterol or DOTAP, cholesterol
To be evaporated chloroform and unbonded DOPC or DOTAP, cholesterol in Rotary Evaporators in 2:3), remainder is double
Tunic particle is the purpose product implemented of the present invention, with phosphate buffer (PBS pH=7.4) by the double-deck membrane granule.
The structural schematic diagram of bilayer membrane granule manufactured in the present embodiment is shown in Fig. 7, the first reactant, the second reactant, function
The core for the core nano particle that siRNA is formed, DOPA formation include the inner membrance outside core, and DOPC, DOTAP form outer membrane.
In above-mentioned preparation method, PD1siRNA:
Positive-sense strand: 5 '-AGACCUUGAUACUUUCAAAdTsdT-3',
Antisense strand: 5 '-UUUGAAAGUAUCAAGGUCUdTsdT-3 '；
The nano particle that the present embodiment preparation method obtains: pattern is fine, and size is between controlled range, particle without viscous
It is attached to together, is uniformly dispersed, see Figure 1A；The size of nano-grain can be uniformly dispersed in 20nm or so, test again
The suitable size and good dispersibility for having demonstrate,proved particle, are shown in Figure 1B.
1.2 transfection til cells
This part is related to the nano particle transfection til cell being prepared using 1.1, includes the following steps:
Step 1, with 1.5 × 105The density in a til cell/hole is inoculated with 6 orifice plates, is placed in incubator (condition: 37 DEG C, 5%
CO2) overnight, remove cell culture medium；
Step 2: being separately added into the fresh cell culture containing 1.1 nano particles into the processed 6 orifice plates of step 1
Base is stood under the conditions of 37 DEG C；Wherein, nano particle has wrapped up siRNA (amount are as follows: the siRNA total amount of addition subtracts not of 50nM
Wrap up the amount into the siRNA of nano particle)；
Step 3: the culture medium in the processed 6 orifice plates of step 2 is discarded, cleaned with fresh phosphoric buffer (PBS)
Three times, the nano particle not absorbed by til cell is washed away.
The present embodiment is in order to realize optimal cellular uptake effect, and fresh cell culture medium contains embodiment 1 in step 2
The concentration of nano particle is set as three concentration ranks, i.e. 20nM, 40nM, 80nM, detects til cell under three concentrations of nanoparticles
Rate is taken to the feeding of nano particle.
As a result see Fig. 2A, it can be seen that when the content of nano particle is 40nM in the medium, til cell pair
The uptake rate of nano particle can reach 65% or so.
In the case where obtaining preferred concentrations of nanoparticles, also to the preferred concentration, (content of nano particle is the present embodiment
Duration is preferably absorbed under 40nM) to be tested.
As a result Fig. 2 B is seen, it can be seen that the til cell that 2h can be realized 65% or so is transfected.
1.3 are transfected the mRNA transcriptional level of PD1 gene in til cell, protein expression level detection
Referring to above-mentioned 1.2, following til cell processing is set:
A, the til cell of normal condition not being transfected is labeled as " TILs "；
B, with the til cell for the nano particle transfection for containing 40nM control siRNA, it is labeled as " TILs+LCP+C-
siRNA-40nM"；
Wherein, the sequence of siRNA is compareed are as follows: 5'-UUCUCCGAACGUGUCACGUTT-3'；
C, the til cell transfected under 20nM concentration with the present embodiment nano particle is labeled as " TILs+LCP+siRNA-
20nM"；
D, the til cell transfected under 40nM concentration with the present embodiment nano particle is labeled as " TILs+LCP+siRNA-
40nM"；
E, the til cell transfected under 80nM concentration with the present embodiment nano particle is labeled as " TILs+LCP+siRNA-
80nM"；
F, the til cell transfected under 160nM concentration with the application nano particle is labeled as " TILs+LCP+siRNA-
160nM”。
The testing result of the transcriptional level of mRNA is shown in Fig. 3 A, as the concentration of nano particle gradually increases from 20nM to 160nM
Greatly, the transcription of PD1 drops to 15% or so from 86%.As it can be seen that efficient PD1siRNA can be transferred to TIL thin for nano particle
In born of the same parents, thus expression of the silencing PD1 in til cell.
The testing result of the expression of PD1 albumen is shown in Fig. 3 B in til cell.When the concentration of nano particle is 40nM,
The expression quantity of PD1 albumen just significantly reduces.As it can be seen that when concentrations of nanoparticles is 40nM, so that it may effectively reduce PD1 albumen
In the expression of til cell.
The testing result of PD1 positive cell is shown in Fig. 3 C after transfection, can see arrow institute according to flow cytomery figure
Movement has occurred in the position of finger, peak value, this just illustrates that PD1 positive cell reduces in til cell, therefore illustrates that nano particle can
With efficient transfection til cell.
Embodiment 2, the nano particle for containing silencing siRNA (PDL1 of silencing breast cancer cell MCF7) and its preparation side
Method
The preparation of 2.1 nano particles (referring to embodiment 1)
This part provides a kind of preparation method of nano particle, includes the following steps:
Prepare A liquid: hexamethylene is mixed with polyoxyethylenes (5) nonylplenyl ether, the amount ratio of the two is 70:30 volume
Than；
Prepare B liquid: (volume used is 100 μ to 5M calcium chloride solution (volume used is 75 μ L) with 100 μM of siRNA solution
L it) mixes, and 50 μ l of ultrapure water is added；
Prepare C liquid: 50mM disodium hydrogen phosphate (volume used is 75 μ L) and 100 μM of siRNA (volume used is 100 μ L)
Mixing, and 50 μ l of ultrapure water is added；
Prepare D liquid: taking A liquid a (volume used is 15ml), 150 μ l B liquid are added, stir 20min；
Prepare E liquid: taking A liquid a (volume used is 15ml), 150 μ l C liquid are added, stir 5min, DOPA is then added
Chloroformic solution (concentration of DOPA is 20mg/ml, and the volume of the solution is 200 μ L), and continue stir 15min；
It prepares single layer membrane granule: E liquid is drop by drop entered into D liquid, stir 20 minutes, gained is precipitated as monofilm nanometer
Particle, that is, the core that the first reactant, the second reactant, function siRNA are formed only are enclosed with one layer of DOPA outside；In the step
In, in order to enable final nano particle size is more uniform, the rate of addition of E liquid is controlled in 1~3ml/min herein；The step
Suddenly further include the steps that cleaning monofilm nano particle, collect；Specifically include: the step of cleaning includes: to E
Liquid, D liquid mixture in volume ratio be 1:1 amount be added 10ml ethyl alcohol, continue stir 5 points of min, under the conditions of 10,000g
It is centrifuged 20min, supernatant is abandoned, stays precipitating；Add 10ml ethyl alcohol into precipitating, 20min be centrifuged under 10,000g, then abandon supernatant,
Stay precipitating；The collected step includes: that 1ml chloroform is added to collect monofilm nano particle；
It prepares the double-deck membrane granule: the above-mentioned monofilm nano particle being prepared being distributed in the chloroform of 1ml, by gained
Mixture (the volume ratio of dispersion liquid and 100 μ l DOPC, the mixture (volume ratio 1:3) of cholesterol or DOTAP, cholesterol
To be evaporated chloroform and unbonded DOPC or DOTAP, cholesterol in Rotary Evaporators in 2:3), remainder is double
Tunic particle is the purpose product implemented of the present invention, with phosphate buffer (PBS pH=7.4) by the double-deck membrane granule.
The structural schematic diagram of bilayer membrane granule manufactured in the present embodiment is shown in Fig. 7, the first reactant, the second reactant, function
The core for the core nano particle that siRNA is formed, DOPA formation include the inner membrance outside core, and DOPC, DOTAP form outer membrane.
In above-mentioned preparation method, the sequence of siRNA is PD-L1siRNA:
Positive-sense strand: 5'-AGACGUAAGCAGUGUUGAAdTsdT-3',
Antisense strand: 5 '-UUCAACACUGCUUACGUCUdTsdT-3 '；
The nano particle that this part preparation method obtains is same as Example 1: pattern is fine, size in controlled range,
It is not adhered to together between grain, is uniformly dispersed, sees Figure 1A；The size of nano-grain can divide in 20nm or so
It dissipates uniformly, demonstrates the suitable size and good dispersibility of particle again, see Figure 1B.
2.2 transfection breast cancer cells
The nano particle being prepared using 2.1 transfects breast cancer cell, includes the following steps:
Step 1, with 1.5 × 105The density in a breast cancer cell/hole is inoculated with 6 orifice plates, be placed in incubator (condition: 37 DEG C,
5%CO2) overnight, remove cell culture medium；
Step 2: being separately added into the fresh of the nano particle containing above-mentioned 2.1 preparation into the processed 6 orifice plates of step 1
Cell culture medium stands 4h under the conditions of 37 DEG C；Wherein, nano particle has wrapped up siRNA (amount are as follows: the siRNA of addition of 40nM
Total amount subtracts the amount that do not wrap up into the siRNA of nano particle)；
Step 3: the culture medium in the processed 6 orifice plates of step 2 is discarded, cleaned with fresh phosphoric buffer (PBS)
Three times, the nano particle not absorbed by breast cancer cell is washed away.
2.3 are transfected the mRNA transcriptional level of PDL1 gene in breast cancer cell, protein expression level detection
Referring to above-mentioned 2.2, following breast cancer cell processing is set:
A, the breast cancer cell of normal condition not being transfected is labeled as " Blank Control "；
B, the breast cancer transfected under 40nM concentrations of nanoparticles with the nano particle for containing 40nM control siRNA is thin
Born of the same parents are labeled as " LCP+C-siRNA-40nM "；
Wherein, the sequence of siRNA is compareed are as follows: 5'-UUCUCCGAACGUGUCACGUTT-3'；
C, the breast cancer cell transfected under 10nM concentration with the nano particle of the present embodiment is labeled as " 10nM "；
D, the breast cancer cell transfected under 20nM concentration with the nano particle of the present embodiment is labeled as " 20nM "；
E, the breast cancer cell transfected under 40nM concentration with the nano particle of the present embodiment is labeled as " 40nM ".
The testing result of the transcriptional level of mRNA is shown in Fig. 4 A, as the concentration of nano particle gradually increases from 10nM to 40nM
Greatly, the transcription of PD1 drops to 20% or so from 80%.As it can be seen that PDL1siRNA efficient can be transferred to mammary gland by nano particle
In cancer cell, thus expression of the silencing PDL1 in breast cancer cell.
The testing result of protein expression level is shown in Fig. 4 B, inventors have found that when the concentration of nano particle is 40nM, PDL1
Apparent reduction just has occurred in the expression quantity of albumen.It can be seen that when the concentration of nano particle is 40nM, so that it may be effectively reduced
Expression of the PDL1 albumen in breast cancer cell.
The testing result of PDL1 positive cell is shown in Fig. 4 C after transfection, can see arrow institute according to flow cytomery figure
Movement has occurred in the position of finger, peak value, this just illustrates the Leukopenia of the PDL1 positive in breast cancer cell, therefore illustrates nanometer
Particle can efficient transfection breast cancer cell.
Embodiment 3, cell killing efficiency test
Til cell involved in the present embodiment embodiment 1 is mixed with the breast cancer cell MCF7 that embodiment 2 is related to, and is surveyed
Til cell is tried to the lethality of breast cancer cell MCF7.
The til cell of selection includes: the til cell of high expression PD1 not being transfected, is labeled as " PD1+ "；It is received by 40nM
The til cell for the low expression PD1 that rice grain transfected is labeled as " PD1- ".
The breast cancer cell MCF7 of selection includes: the breast cancer cell of high expression PDL1 not being transfected, is labeled as " PDL1
+"；The breast cancer cell of the low expression PDL1 transfected by 40nM nano particle is labeled as " PDL1- "；
The breast cancer cell MCF7 of the til cell of selection, selection is mixed with different cell weight ratios, amounts to 12
Different mixed processings includes the following:
Quantity is than PD1+/PDL1+, PD1-/PDL1+, PD1+/PDL1-, PD1-/PDL1- for 10:1；
Quantity is than PD1+/PDL1+, PD1-/PDL1+, PD1+/PDL1-, PD1-/PDL1- for 30:1；
Quantity is than PD1+/PDL1+, PD1-/PDL1+, PD1+/PDL1-, PD1-/PDL1- for 100:1；
The test of cell killing efficiency is detected by Lactic dehydrogenase detection kit.Use CytoTox
Non-Radioactive Cytotoxicity Assay (Promega, WI) carries out standard lactic dehydrogenase enzyme r e lease inspection in 4 hours
It surveys.Method is as follows:
1. by breast cancer cell to be checked (silencing breast cancer cell MCF7KAnd non-silencing breast cancer cell MCF7) with 1 ×
104The density in every hole is inoculated on 96 orifice plates, after culture 18 hours, culture medium is changed into without phenol red, containing 5% fetal calf serum
1640 culture medium of RPMI (Gibco-BRL) continues culture 6 hours.
2. to til cell: by til cell (silencing til cell TILKAnd non-silencing til cell TIL) cultivated in CNE-2
It is cultivated 24 hours in base, then collects cell, without phenol red, the 1640 culture medium (Gibco- of RPMI containing 5% fetal calf serum
BRL cell is resuspended in).
3. taking 50 μ L cell suspensions, til cell is added in the hole containing breast cancer cell according to above-mentioned different proportion, is incubated
After educating 4 hours, 96 orifice plates are centrifuged (250 × g, 10 minutes), takes 50 μ L supernatants to be added in another 96 orifice plate, is mentioned according to manufacturer
The guide for use of confession carries out subsequent operation.
Killing-efficiency calculation formula: % killing-efficiency=(experimental result-breast cancer cell basal release value-til cell base
Plinth release value)/(kill cell after mixing and discharge peak value-breast cancer cell basal release value) × 100.
Test result refers to Fig. 5, through the figure it is found that under different ratios, the processing killing of PD1-/PDL1-
Effect be it is best, have extra high killing-efficiency.
Embodiment 4, cytokine test
Firstly, the present embodiment contains siRNA nano particle referring to the preparation of 1 preparation method of embodiment, 40nM concentration is then used
Nano particle transfect til cell.
Respectively (i.e. to the til cell (til cell before silencing is labeled as TILs) before transfection, the til cell after transfection
The til cell of silencing is labeled as TILsK) subgroup detected, detection method is conventional method in that art.Testing result please join
See Fig. 6 A and Fig. 6 B；Fig. 6 A is the til cell after silencing, and Fig. 6 B is the til cell before silencing, according to Fig. 6 A and Fig. 6 B it is found that
After silencing PD1 and before, til cell subgroup does not change.Illustrate that entire silencing process does not have the cell subsets of til cell
It influences.
Secondly, the present embodiment is referring to the step 1.1 and step 1.2 acquisition 40nM concentration nano particle transfection in embodiment 1
Til cell, then with the til cell (TILs after being transfectedK) and MCF7, MCF7K, tri- kinds of cells of CTL6 co-culture, and altogether
The cytokine-expressing amount of culture front and back test transfection front and back, the method for test cell factor expression amount are that this field is routinely grasped
Make.Testing result refers to Fig. 6 C, Fig. 6 D, is transfected the IL17 of til cell, IL10, INF γ, INF alpha expression amount increased,
Especially INF γ is dramatically increased, and illustrates that the killing ability of the til cell after being transfected is remarkably reinforced.
It should be noted that proving by many experiments, above-described embodiment can in the range of parameters described below limits
Realize: first reactant solution is calcium salt soln, and second reactant solution is phosphate solution or pyrophosphate
Solution, first reactant, the calcium of the second reactant, phosphorus amount of substance ratio be (25~400): 1；The calcium salt soln it is dense
Degree is 4.5~5.5M；The concentration of the phosphate solution or pyrophosphate solution is 45~55mM；In the DOPA solution
The concentration of DOPA is 15~25mg/ml, the volume ratio of the addition volume of the DOPA solution and the A liquid, the mixed solution of C liquid
For (70~80): 1；The DOPC, cholesterol mixture in, DOPC, cholesterol mass ratio be 1:(2.5~3.5)；It is described
DOTAP, cholesterol mixture in, DOTAP, cholesterol mass ratio be 2:(2.5~3.5)；In the preparation of the A liquid, have
Solvent is any one or more of hexamethylene, benzene, toluene, normal heptane, carbon tetrachloride；Surfactant includes polyoxy for second
Alkene (5) nonylplenyl ether；The volume ratio that organic solvent is mixed with surfactant is 80:20~50:50；The A liquid, B liquid
Amount ratio is (50~200): 1；The A liquid, C liquid amount ratio be (50~200): 1；The amount ratio of E liquid and D liquid be (1~
3): (1~3).
Comparative example 1
(1) nano particle for containing silencing siRNA (PD1 of silencing til cell) that this comparative example uses is by such as
Lower preparation method obtains:
Step 1, hexamethylene 10.5ml, polyoxyethylenes (5) nonylplenyl ether 4.5ml, 5M calcium chloride solution is (used
Volume is 50 μ L), 100 μM of siRNA solution (volume used is 66.7 μ L), 33.3 μ L of ultrapure water mixing, obtain mixture I；
Step 2, by hexamethylene 10.5ml, polyoxyethylenes (5) nonylplenyl ether 4.5ml, 50mM disodium hydrogen phosphate (institute
50 μ L with volume), 100 μM of siRNA solution (volume used is 66.7 μ L), 33.3 μ L of ultrapure water mixing, obtain mixture II；
Mixture I is instilled mixture II with the speed of 1~3ml/min, stirred 20 minutes, gained is precipitated as by step 3
Nano particle.
Step 1, in step 2, the volume ratio of hexamethylene and polyoxyethylenes (5) nonylplenyl ether is 70:30, package
The sequence of siRNA, concentration (50nM) are the same as embodiment 1.
The nano particle of this comparative example preparation is single-layer membrane structure, and granular size is uneven, and partial size is greater than 20nm, dispersibility compared with
Difference.
By the Concentration Modulation 40nM of gained nano particle, til cell is transfected referring to 1.2 parts in embodiment 1, when different
Between section sample, detect transfection efficiency.It the results are shown in Table 1.According to table 1, propulsion of 1 nano particle of comparative example with the time, feeding
Take rate variation unobvious, 8 hours corresponding feeding of transfection take rate not high.
Table 1
| 0.25h | 2h | 4h | 6h | 8h | |
| Feeding takes rate % | 2.17±0.21 | 25.43±6.31 | 31.21±4.47 | 43.53±3.28 | 43.57±4.32 |
(2) nano particle for containing silencing siRNA (PDL1 of silencing breast cancer cell MCF7) that this comparative example uses
Preparation method referring to above-mentioned (1) in this comparative example.
By the Concentration Modulation 40nM of gained nano particle, breast cancer cell MCF7 is transfected referring to 2.2 parts in embodiment 1,
It is sampled in different time sections, detects transfection efficiency.It the results are shown in Table 2.According to table 2,1 nano particle of comparative example is with the time
It promotes, feeding takes rate variation unobvious, and 8 hours corresponding feeding of transfection take rate not high.
Table 2
(3) killing-efficiency is tested: taking above-mentioned (1) transfection 2h to obtain til cell, above-mentioned (2) transfection 2h acquisition breast cancer thin
Born of the same parents MCF7 carries out lethality test referring to the test method of above-described embodiment 3, if tri- ratios of 10:1,30:1,100:1, as a result
It is shown in Table 3.According to table 3, since transfection efficiency is not high, after 1 nano particle of comparative example transfects til cell, breast cancer cell MCF7,
The two killing-efficiency is substantially less than embodiment.
Table 3
| Killing-efficiency % | PD1-/PDL1- |
| 10:1 | 22.11±1.50 |
| 30:1 | 30.06±4.40 |
| 100:1 | 48.09±6.84 |
(4) cytokine test: referring to embodiment 4 method, with above-mentioned (1) obtain the til cell being transfected with
MCF7、MCF7K, tri- kinds of cells of CTL6 co-culture, and the cytokine-expressing amount before and after test transfection before and after co-cultivation.Detection
It the results are shown in Table 4.
Table 4
| Unit pg/ml | IL17 | IL10 | INFγ | TNFα |
| TIL/CTL4 | 23±3.55 | 15.00±4.00 | 17.33±3.61 | 5.33±1.15 |
| TIL/MCF7K | 309.67±90.73 | 56.33±7.51 | 772.67±10.50 | 81.22±6.11 |
| TIL/MCF7 | 269.00±33.05 | 27.67±10.21 | 414.33±22.30 | 58.33±4.73 |
| TILK/CTL4 | 23.00±7.55 | 13.00±4.00 | 23.67±28.88 | 13.11±3.46 |
| TILK/MCF7K | 509.67±90.73 | 66.33±7.51 | 923.67±48.39 | 91.67±9.45 |
| TILK/MCF7 | 369.00±30.05 | 32.67±10.21 | 595.67±24.68 | 86.13±4.58 |
According to table 4, since transfection efficiency is not high, gained intercellular cytokine-expressing amount in transfection front and back is unobvious.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of nano particle, which comprises the steps of:
Organic solvent is mixed with surfactant, obtains A liquid；
First reactant solution is mixed with functional mass solution, obtains B liquid；
Second reactant solution is mixed with functional mass solution, obtains C liquid；Second reactant is contacted with first reactant
After be capable of forming precipitating；
Mix the A liquid, the B liquid obtains D liquid；
The A liquid, the C liquid are mixed, and DOPA solution is added into gained mixed solution and mixes, obtains E liquid；
The D liquid is added in the E liquid, gained mixture is centrifuged, precipitating is collected, obtains monofilm nano particle；
The dispersion liquid of monofilm nano particle is prepared, and the mixture of DOPC, cholesterol, Huo Zhejia are added into the dispersion liquid
Enter the mixture of DOTAP, cholesterol, the particle in gained suspension particle solution is duplicature nano particle.
2. the preparation method of nano particle according to claim 1, which is characterized in that first reactant solution is calcium
Salting liquid, second reactant solution are phosphate solution or pyrophosphate solution, first reactant, the second reaction
The calcium of object, the amount of substance ratio of phosphorus are (25~400): 1.
3. the preparation method of nano particle according to claim 2, which is characterized in that the concentration of the calcium salt soln is
4.5~5.5M；The concentration of the phosphate solution or pyrophosphate solution is 45~55mM.
4. the preparation method of nano particle according to claim 1, which is characterized in that DOPA's is dense in the DOPA solution
Degree is 15~25mg/ml, the additions volume of the DOPA solution and the A liquid, C liquid mixed solution volume ratio for (70~
80): 1.
5. the preparation method of nano particle according to claim 1, which is characterized in that the mixing of the DOPC, cholesterol
In object, DOPC, cholesterol mass ratio be 1:(2.5~3.5)；The DOTAP, cholesterol mixture in, DOTAP, gallbladder are solid
The mass ratio of alcohol is 2:(2.5~3.5).
6. the preparation method of nano particle according to claim 1, which is characterized in that in the preparation of the A liquid, You Jirong
Agent is any one or more of hexamethylene, benzene, toluene, normal heptane, carbon tetrachloride；Surfactant includes polyoxyethylenes
(5) nonylplenyl ether；The volume ratio that organic solvent is mixed with surfactant is 80:20~50:50.
7. the preparation method of nano particle according to claim 1, which is characterized in that the A liquid, B liquid amount ratio be
(50~200): 1；The A liquid, C liquid amount ratio be (50~200): 1；The amount ratio of E liquid and D liquid is (1~3): (1~3).
8. the preparation method of nano particle according to claim 1, which is characterized in that the function in the functional mass solution
Energy substance includes functional nucleic acid sequence, protein antibody, drug molecule.
9. the preparation method of nano particle according to claim 8, which is characterized in that the function in the functional mass solution
Energy substance includes functional nucleic acid sequence.
10. a kind of nano particle, which is characterized in that obtained by according to claim 1-9 described in any item preparation methods.
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| CN111569061A (en) * | 2020-05-06 | 2020-08-25 | 吴延恒 | Nano material for nucleic acid vaccine enhancer |
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|---|---|---|---|---|
| CN101239711A (en) * | 2007-02-05 | 2008-08-13 | 张桂英 | Method for preparing gene transmission vector nano calcium phosphate |
| US20120201872A1 (en) * | 2009-08-03 | 2012-08-09 | The University Of North Carolina At Chapel Hill | Liposomes comprising a calcium phosphate-containing precipitate |
| CN105878047A (en) * | 2014-12-23 | 2016-08-24 | 广州暨南大学医药生物技术研究开发中心 | Preparation method and application of fibroblast growth factor covering lipide calcium phosphate nanoparticles |
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| WO2014021678A1 (en) * | 2012-08-02 | 2014-02-06 | (주)아이엠지티 | Microbubble-nanoliposome complex for cancer diagnosis and treatment |
| JP6590802B2 (en) * | 2013-11-06 | 2019-10-16 | ザ ユニバーシティ オブ シカゴＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃｈｉｃａｇｏ | Nanoscale transporter for delivery or co-delivery of chemotherapeutic drugs, nucleic acids and photosensitizers |
| CN105168151B (en) * | 2015-08-07 | 2017-10-24 | 哈尔滨工业大学 | A kind of preparation method of isotope of redox-sensitive type nano target carrier |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101239711A (en) * | 2007-02-05 | 2008-08-13 | 张桂英 | Method for preparing gene transmission vector nano calcium phosphate |
| US20120201872A1 (en) * | 2009-08-03 | 2012-08-09 | The University Of North Carolina At Chapel Hill | Liposomes comprising a calcium phosphate-containing precipitate |
| CN105878047A (en) * | 2014-12-23 | 2016-08-24 | 广州暨南大学医药生物技术研究开发中心 | Preparation method and application of fibroblast growth factor covering lipide calcium phosphate nanoparticles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111569061A (en) * | 2020-05-06 | 2020-08-25 | 吴延恒 | Nano material for nucleic acid vaccine enhancer |
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