CN112423738A - 通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体及其制造方法 - Google Patents
通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体及其制造方法 Download PDFInfo
- Publication number
- CN112423738A CN112423738A CN201880094068.7A CN201880094068A CN112423738A CN 112423738 A CN112423738 A CN 112423738A CN 201880094068 A CN201880094068 A CN 201880094068A CN 112423738 A CN112423738 A CN 112423738A
- Authority
- CN
- China
- Prior art keywords
- nanoparticle
- lipid
- reactive group
- nanoparticles
- liposome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 265
- 150000002632 lipids Chemical class 0.000 title claims abstract description 118
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 21
- 238000012986 modification Methods 0.000 title description 8
- 230000004048 modification Effects 0.000 title description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 43
- 239000002502 liposome Substances 0.000 claims abstract description 36
- 230000012202 endocytosis Effects 0.000 claims abstract description 21
- 239000012528 membrane Substances 0.000 claims abstract description 16
- 238000010298 pulverizing process Methods 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 15
- 201000010099 disease Diseases 0.000 claims abstract description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 13
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 13
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 8
- 239000002131 composite material Substances 0.000 claims description 30
- 230000015572 biosynthetic process Effects 0.000 claims description 13
- 150000003904 phospholipids Chemical class 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 230000009918 complex formation Effects 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 239000002246 antineoplastic agent Substances 0.000 claims description 6
- 230000001093 anti-cancer Effects 0.000 claims description 3
- 230000002209 hydrophobic effect Effects 0.000 claims description 3
- 230000002147 killing effect Effects 0.000 claims description 3
- 230000000149 penetrating effect Effects 0.000 claims description 2
- 238000001415 gene therapy Methods 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 18
- 230000035515 penetration Effects 0.000 abstract description 13
- 108020004459 Small interfering RNA Proteins 0.000 description 38
- 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 21
- 239000003112 inhibitor Substances 0.000 description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- 102000009027 Albumins Human genes 0.000 description 12
- 108010088751 Albumins Proteins 0.000 description 12
- 230000004700 cellular uptake Effects 0.000 description 11
- 229960004679 doxorubicin Drugs 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000008055 phosphate buffer solution Substances 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 230000030279 gene silencing Effects 0.000 description 8
- 238000012226 gene silencing method Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000000975 dye Substances 0.000 description 7
- 239000007850 fluorescent dye Substances 0.000 description 7
- 230000007246 mechanism Effects 0.000 description 7
- -1 microbubbles Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 description 5
- 229960001076 chlorpromazine Drugs 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 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 description 4
- 230000006395 clathrin-mediated endocytosis Effects 0.000 description 4
- 238000004624 confocal microscopy Methods 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000034701 macropinocytosis Effects 0.000 description 4
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000000527 sonication Methods 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000003570 cell viability assay Methods 0.000 description 3
- 230000006895 clathrin independent endocytosis Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 229920002477 rna polymer Polymers 0.000 description 3
- LWTDZKXXJRRKDG-KXBFYZLASA-N (-)-phaseollin Chemical compound C1OC2=CC(O)=CC=C2[C@H]2[C@@H]1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-KXBFYZLASA-N 0.000 description 2
- YZOUYRAONFXZSI-SBHWVFSVSA-N (1S,3R,5R,6R,8R,10R,11R,13R,15R,16R,18R,20R,21R,23R,25R,26R,28R,30R,31S,33R,35R,36R,37S,38R,39S,40R,41S,42R,43S,44R,45S,46R,47S,48R,49S)-5,10,15,20,25,30,35-heptakis(hydroxymethyl)-37,39,40,41,42,43,44,45,46,47,48,49-dodecamethoxy-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontane-36,38-diol Chemical compound O([C@@H]([C@H]([C@@H]1OC)OC)O[C@H]2[C@@H](O)[C@@H]([C@@H](O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3O)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O3)O[C@@H]2CO)OC)[C@H](CO)[C@H]1O[C@@H]1[C@@H](OC)[C@H](OC)[C@H]3[C@@H](CO)O1 YZOUYRAONFXZSI-SBHWVFSVSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 229920000858 Cyclodextrin Polymers 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical group ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 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
- XSDQTOBWRPYKKA-UHFFFAOYSA-N amiloride Chemical compound NC(=N)NC(=O)C1=NC(Cl)=C(N)N=C1N XSDQTOBWRPYKKA-UHFFFAOYSA-N 0.000 description 2
- 229960002576 amiloride Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000001045 blue dye Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- ARQRPTNYUOLOGH-UHFFFAOYSA-N chcl3 chloroform Chemical compound ClC(Cl)Cl.ClC(Cl)Cl ARQRPTNYUOLOGH-UHFFFAOYSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- QDERNBXNXJCIQK-UHFFFAOYSA-N ethylisopropylamiloride Chemical compound CCN(C(C)C)C1=NC(N)=C(C(=O)N=C(N)N)N=C1Cl QDERNBXNXJCIQK-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000003211 trypan blue cell staining Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 1
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- 101710163504 Phaseolin Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- DLPNSGYBXBYABV-UHFFFAOYSA-N [Br-].[Br-].S1C(C)=C(C)[NH+]=C1N1N(C=2C=CC=CC=2)[NH2+]C(C=2C=CC=CC=2)=N1 Chemical compound [Br-].[Br-].S1C(C)=C(C)[NH+]=C1N1N(C=2C=CC=CC=2)[NH2+]C(C=2C=CC=CC=2)=N1 DLPNSGYBXBYABV-UHFFFAOYSA-N 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- WHPMALGCHJRYKZ-UHFFFAOYSA-N pentanedial Chemical compound O=CCCCC=O.O=CCCCC=O WHPMALGCHJRYKZ-UHFFFAOYSA-N 0.000 description 1
- 229960004065 perflutren Drugs 0.000 description 1
- LWTDZKXXJRRKDG-UHFFFAOYSA-N phaseollin Natural products C1OC2=CC(O)=CC=C2C2C1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-UHFFFAOYSA-N 0.000 description 1
- 229950004354 phosphorylcholine Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Images
Classifications
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- A61K47/6905—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 colloid or an emulsion
- A61K47/6911—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 colloid or an emulsion the form being a liposome
-
- 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
- A61K47/6905—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 colloid or an emulsion
- A61K47/6919—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 colloid or an emulsion the form being a ribbon or a tubule cochleate
-
- 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
- A61K47/6925—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 microcapsule, nanocapsule, microbubble or nanobubble
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
-
- 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
- A61K9/5005—Wall or coating material
- A61K9/5015—Organic compounds, e.g. fats, sugars
-
- 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
- A61K9/5089—Processes
-
- 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
- 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
- A61K9/5192—Processes
-
- 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
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dispersion Chemistry (AREA)
- Nanotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及一种通过被摄取到细胞内部而用于对疾病进行治疗的纳米粒子复合体及其制造方法,尤其涉及一种可以通过利用稳定性高且生物相容性优秀的基于脂类的物质对纳米粒子表面进行改性而提升细胞内摄取效率,可以通过在纳米粒子表面的一部分结合管状形态的脂质结构体而实现内吞乃至直接贯通细胞膜,还可以有效地被球状体形态的肿瘤细胞摄取,而且可以通过采用在将基于脂质的脂质体(如微泡、膜泡等)与纳米粒子结合之后施加机械性的力量而粉碎脂质体并借此在纳米粒子表面形成脂质结构体的自顶向下(Top‑down)方式而轻易地实现大规模生产的纳米粒子复合体及其制造方法。
Description
技术领域
本发明涉及一种通过被摄取到细胞内部而用于对疾病进行治疗的纳米粒子复合体及其制造方法,尤其涉及一种可以通过利用稳定性高且生物相容性优秀的基于脂类的物质对纳米粒子表面进行改性而提升细胞内摄取效率,可以通过在纳米粒子表面的一部分结合管状形态的脂质结构体而实现内吞乃至直接贯通细胞膜,还可以有效地被球状体形态的肿瘤细胞摄取,而且可以通过采用在将基于脂质的脂质体(如微泡、膜泡等)与纳米粒子结合之后施加机械性的力量而粉碎脂质体并借此在纳米粒子表面形成脂质结构体的自顶向下(Top-down)方式而轻易地实现大规模生产的纳米粒子复合体及其制造方法。
背景技术
在药物传递领域,细胞内摄取效率是达成药物传递功效的重要尺度。因此,开发出了很多用于提升细胞内摄取效率的新技术。作为一实例,如下述专利文献所记载,拟通过在纳米粒子上以化学方式结合如细胞渗透肽等而提升纳米粒子的细胞内摄取效率。
<专利文献>
公开专利公报第10-2017-0040748号(2017.04.13.公开)“包含多嵌段多肽的药物载体以及自组装纳米结构体”
但是,现有的用于提升细胞内摄取效率的技术并不能达成充分的效果,具有因为所结合的物质轻易地发生分解而导致的稳定性下降的问题。
发明内容
发明所要解决的问题
本发明的目的在于提供一种可以通过利用稳定性高且生物相容性优秀的基于脂类的物质对纳米粒子表面进行改性而显著提升细胞内摄取效率的纳米粒子复合体及其制造方法。
此外,本发明的目的在于提供一种可以通过在纳米粒子表面的一部分结合管状形态的脂质结构体而实现内吞(100至200nm大小的粒子被摄取到细胞内部的机理)乃至直接贯通细胞膜的纳米粒子复合体及其制造方法。
此外,本发明的目的在于提供一种可以借助于组织穿透力(tissue penetration)使得纳米粒子有效地被球状体形态的肿瘤细胞摄取的纳米粒子复合体。
此外,本发明的目的在于提供一种可以通过采用在将基于脂质的脂质体(如微泡、膜泡等)与纳米粒子结合之后施加机械性的力量而粉碎脂质体并借此在纳米粒子表面形成脂质结构体的自顶向下(Top-down)方式,而不是直接将脂质结构体附着在纳米粒子上而轻易地实现大规模生产的纳米粒子复合体及其制造方法。
用于解决问题的方案
为了达成如上所述的目的,本发明通过如下所述构成的实施例实现。
在本发明的一实施例中,适用本发明的纳米粒子复合体,其特征在于,包括:纳米粒子,通过被摄取到细胞内部而用于对疾病进行治疗;以及,基于脂质的脂质结构体,通过结合到上述纳米粒子的外表面一部分而提升纳米粒子的细胞内摄取效率。
在本发明的另一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述纳米粒子的直径为100至300nm,上述脂质结构体的长度为50至300nm而宽度为3至20nm。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述脂质结构体为外侧面包括亲水性的脂质头基而内部包括疏水性的脂质尾端的整体长度较长的管状形态。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述纳米粒子包括第1反应基,上述脂质结构体包括与纳米粒子的第1反应基化学结合的第2反应基,通过第1反应基与第2反应基的化学结合而将脂质结构体结合到纳米粒子。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述纳米粒子的外表面包括第1反应基,上述管状形态的脂质结构体的一端包括第2反应基,通过第1反应基与第2反应基的化学结合而将管状形态的脂质结构体结合到上述纳米粒子的外表面。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述纳米粒子是由担载有药物或可以对疾病进行治疗的物质构成。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述纳米粒子复合体不仅可以实现内吞,还可以通过直接贯通细胞膜而被摄取到细胞内部。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述脂质结构体可以将上述纳米粒子的组织穿透力提升至球状体形态的肿瘤细胞的组织内部。
在本发明的又一实施例中,适用本发明的纳米粒子复合体,其特征在于:上述纳米粒子包括抗癌剂,可以提升对肿瘤细胞的杀灭效率。
在本发明的又一实施例中,适用本发明的纳米粒子复合体的制造方法,其特征在于,包括:形成包括第1反应基的纳米粒子的纳米粒子形成步骤;形成包括与上述第1反应基化学结合的第2反应基的基于磷脂质的微小尺寸的脂质体的脂质体形成步骤;通过对上述纳米粒子与脂质体进行混合而使得第1反应基与第2反应基相互结合并借此形成纳米粒子被结合到脂质体外侧面的脂质体-纳米粒子复合体的脂质复合体形成步骤;以及,通过向在上述脂质复合体形成步骤中形成的脂质体-纳米粒子复合体施加机械性的力量而粉碎脂质体并借此形成结合到纳米粒子的外表面一部分的脂质结构体,从而制造出纳米粒子复合体的粉碎形成步骤;上述脂质体为微泡或膜泡,上述纳米粒子通过被摄取到细胞内部而用于对疾病进行治疗,上述脂质结构体可以提升纳米粒子的细胞内摄取效率。
在本发明的又一实施例中,适用本发明的纳米粒子复合体的制造方法,其特征在于:在上述粉碎形成步骤中,通过向脂质体-纳米粒子复合体施加机械性的力量并维持一段时间而对脂质体进行粉碎并使得构成脂质体的磷脂质发生重组,从而形成结合到纳米粒子的管状形态的脂质结构体。
发明效果
本发明可以通过如上所述的本实施例达成如下所述的效果。
本发明可以通过利用稳定性高且生物相容性优秀的基于脂类的物质对纳米粒子表面进行改性而显著提升细胞内摄取效率。
此外,本发明可以通过在纳米粒子表面的一部分结合管状形态的脂质结构体而实现内吞(100至200nm大小的粒子被摄取到细胞内部的机理)乃至直接贯通细胞膜。
此外,本发明可以借助于组织穿透力(tissue penetration)使得纳米粒子有效地被球状体形态的肿瘤细胞摄取。
此外,本发明可以通过采用在将基于脂质的脂质体(如微泡、膜泡等)与纳米粒子结合之后施加机械性的力量而粉碎脂质体并借此在纳米粒子表面形成脂质结构体的自顶向下(Top-down)方式,而不是直接将脂质结构体附着在纳米粒子上而轻易地实现大规模生产。
附图说明
图1是适用本发明之一实施例的纳米粒子复合体的模式图。
图2是适用本发明之一实施例的纳米粒子复合体的透射电子显微镜(TEM)图。
图3是适用本发明之一实施例的纳米粒子复合体的冷冻透射电子显微镜(Cyro-TEM)图。
图4是适用本发明之一实施例的与纳米粒子复合体结合的脂质结构体的冷冻透射电子显微镜(Cyro-TEM)图。
图5是对用于确认适用本发明之一实施例的纳米粒子复合体的细胞摄取效率的利用流式细胞仪(Flow cytometry)的分析结果进行图示的图。
图6以及图7是用于确认适用本发明之一实施例的纳米粒子复合体的细胞摄取效率的共聚焦显微镜(confocal microscope)图。
图8是对用于确认利用内吞抑制剂(Endocytosis Inhibitor)进行处理之后的适用本发明之一实施例的纳米粒子复合体的细胞摄取效率的利用流式细胞仪(Flowcytometry)的分析结果进行图示的图。
图9是对用于确认适用本发明之一实施例的纳米粒子复合体作为抗癌剂载体的功效的细胞存活率检测(cell viability assay)结果进行图示的图。
图10以及图11是对用于确认适用本发明之一实施例的纳米粒子复合体作为药物载体的功效的基因沉默(gene silencing)结果进行图示的示意图。
图12是用于确认适用本发明之一实施例的纳米粒子复合体在球状体肿瘤细胞模型中的细胞摄取效率的共聚焦显微镜(confocal microscope)图。
附图标记说明:
1:纳米粒子;
2:脂质结构体;
21:脂质头基;
22:脂质尾端。
具体实施方式
接下来,将参阅附图对适用本发明的通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体及其制造方法进行详细的说明。除非另有明确的定义,否则在本说明书中使用的所有术语的含义与具有本发明所属技术领域之一般知识的技术人员所通常理解的相应术语的一般含义相同,当与在本说明书中使用的术语的含义冲突时,将遵循在本说明书中使用的定义。此外,对于可能会导致本发明的要旨变得不清晰的公知功能以及构成,将省略与其相关的详细说明。在整个说明书中,当记载为某个部分“包括”某个构成要素时,除非另有明确的相反记载,否则并不是排除其他构成要素,而是表示还可以包括其他构成要素。
接下来,将参阅图1至图12对适用本发明之一实施例的通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体进行详细的说明,上述纳米粒子复合体,包括:纳米粒子1,通过被摄取到细胞内部而用于对疾病进行治疗;以及,基于脂质的脂质结构体2,通过结合到上述纳米粒子1的外表面一部分而提升纳米粒子1的细胞内摄取效率。
上述纳米粒子1是通过被摄取到细胞内部而用于对疾病进行治疗的构成,是由担载有药物或可以对疾病进行治疗的物质构成,可以使用通过被摄取到细胞内部而用于对疾病进行治疗的现有的各种纳米粒子,例如,可以使用担载有药物(包括小干扰核糖核酸(siRNA)的概念)的白蛋白纳米粒子、担载有药物的生物降解性高分子纳米粒子、对特定蛋白质的表达进行抑制的小干扰核糖核酸(siRNA)纳米粒子等。上述纳米粒子可以采用如100至300nm的直径以及球形的形态。上述纳米粒子可以包括与脂质结构体结合的化学反应基(以下称之为“第1反应基”)。例如,第1反应基可以是包括如硫醇基、胺基、氨基、羧基等的化合物。
上述脂质结构体2是通过与纳米粒子1的外表面一部分结合而提升纳米粒子1的细胞内摄取效率的基于脂质的结构体,上述脂质结构体的长度可以是40至300nm而宽度可以是3至20nm,可以在上述纳米粒子的外表面结合一个以上。此外,上述脂质结构体可以包括与纳米粒子的第1反应基化学结合的化学反应基(以下称之为“第2反应基”)。例如,第2反应基可以是包括如硫醇基、胺基、氨基、羧基等的化合物。
上述脂质结构体可以是如外侧面包括亲水性的脂质头基21而内部包括疏水性的脂质尾端22的整体长度较长的管状形态,可以通过位于管状形态的脂质结构体一端的第2反应基与上述纳米粒子的第1反应基的化学结合而将管状形态的脂质结构体与上述纳米粒子1的外表面结合。例如,当纳米粒子为白蛋白纳米粒子且在上述脂质结构体上形成N-羟基琥珀酰亚胺(NHS,N-hydroxysuccinimide)反应基时,可以通过N-羟基琥珀酰亚胺-胺(NHS-amine)反应将上述纳米粒子1与脂质结构体2结合。本发明的可以通过利用稳定性高且生物相容性优秀的基于脂类的物质对纳米粒子表面进行改性而显著提升细胞内摄取效率,而此时100至200nm大小的纳米粒子被摄取到细胞内部的机理为内吞,上述纳米粒子复合体可以通过在纳米粒子表面的一部分结合管状形态的脂质结构体而实现内吞乃至直接贯通细胞膜,还可以有效地被球状体形态的肿瘤细胞摄取,因此可以适用于生体模型。
接下来,将对适用本发明之另一实施例的纳米粒子复合体的制造方法进行说明。上述纳米粒子复合体的制造方法,包括:形成包括第1反应基的纳米粒子的纳米粒子形成步骤;形成包括与上述第1反应基化学结合的第2反应基的基于脂质的微小尺寸的脂质体(如微泡、膜泡等)的脂质体形成步骤;通过对上述纳米粒子与脂质体进行混合而使得第1反应基与第2反应基相互结合并借此形成纳米粒子被结合到脂质体外侧面的脂质体-纳米粒子复合体的脂质复合体形成步骤;以及,通过向在上述脂质复合体形成步骤中形成的脂质体-纳米粒子复合体施加机械性的力量(mechanical force)而粉碎脂质体并借此形成结合到纳米粒子的外表面一部分的脂质结构体的粉碎形成步骤。
上述纳米粒子形成步骤,是以通过被摄取到细胞内部而用于对疾病进行治疗的纳米粒子包括第1反应基的方式形成上述纳米粒子的步骤,可以使用现有的各种制造纳米粒子的方法,例如在担载有药物的白蛋白纳米粒子中,可以将存在于白蛋白的胺基作为第1反应基使用,而在对特定蛋白质的表达进行抑制的小干扰核糖核酸(siRNA)纳米粒子中,可以通过向小干扰核糖核酸(siRNA)涂布附着有胺的玻尿酸而在小干扰核糖核酸(siRNA)纳米粒子上形成第1反应基。
上述脂质体形成步骤,是形成包括与上述第1反应基化学结合的第2反应基的基于脂质的微小尺寸的脂质体(如微泡、膜泡等)的步骤,例如,微泡可以由脂质(例如,磷脂质)形成且在内部填充有气体,在上述微泡的外侧面包括第2反应基。在内部填充有气体且由磷脂质构成的膜泡形态的微小尺寸的微泡可以通过现有的制造方法进行制造,例如可以通过将与第2反应基结合的磷脂质和没有与第2反应基结合的磷脂质在有机溶剂中按照一定的比例进行混合而形成脂质膜并将上述脂质膜溶解到容器中之后注入气体的方式形成。
上述脂质复合体形成步骤,是通过对上述纳米粒子与脂质体进行混合而使得第1反应基与第2反应基相互结合并借此形成在脂质的外侧面结合有纳米粒子的脂质体-纳米粒子复合体的步骤。
上述粉碎形成步骤,是通过向在上述脂质复合体形成步骤中形成的脂质体-纳米粒子复合体施加机械性的力量(mechanical force)而粉碎脂质体并借此形成结合到纳米粒子的外表面一部分的脂质结构体的步骤。在利用如超声波设备等向脂质体-纳米粒子复合体施加机械性的力量并维持一定时间时,脂质体将被粉碎且构成脂质体的磷脂质将发生重组,从而形成结合到纳米粒子的管状形态的脂质结构体。本发明可以通过采用在将基于脂质的脂质体与纳米粒子结合之后施加机械性的力量而粉碎脂质体并借此在纳米粒子表面形成脂质结构体的自顶向下(Top-down)方式,而不是直接将脂质结构体附着在纳米粒子上而轻易地实现大规模生产。
接下来,将结合实施例对本发明进行更为详细的说明。但是,下述内容只是用于对本发明进行更为详细的说明,本发明的权利要求范围并不因此而受到限定。
<实施例1>纳米粒子复合体的制造
1.纳米粒子(NPs)的形成
在蒸馏水中溶解白蛋白(Human serum albumin)至20mg/mL的浓度之后,利用0.2MNaOH将pH调节至8而制备出白蛋白溶液,然后将100%乙醇以1mL/min的速度向上述白蛋白溶液进行滴定。接下来,添加4%的戊二醛(glutaraldehyde)10μL并在遮光条件下使乙醇挥发一整夜,然后在以13200rpm、10min条件执行离心分离之后利用移液管去除没有被粒子化的白蛋白,接下来在利用磷酸盐缓冲溶液(PBS)再分散并以3000rpm、5min的条件执行离心分离之后利用移液管获取除微丸(micropellet)之外的上层液(纳米粒子(NPs))(此外,在执行荧光试验时,将满足需要的荧光染料(dye)和纳米粒子在常温下进行一整夜的反应之后以13200rpm、10min条件执行离心分离,接下来利用移液管去除没有发生反应的荧光染料(dye),然后利用磷酸盐缓冲溶液(PBS)再分散后使用)。
2.膜泡的形成
将脂质即1,2-双硬脂酰基-SN-丙三基-3-磷酸胆碱(DSPC,1,2-distearoyl-sn-glycero-3-phosphocholine)和1,2-双硬脂酰基-SN-丙三基-3-磷脂酰乙醇胺-N-聚乙二醇琥珀酰亚胺酯(DSPE-PEG-NHS2000,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol succinimidyl ester)以9.25:0.75的摩尔比进行混合并在氯仿(Chloroform)中溶解至10mg/ml的浓度之后以1mg/ml分别添加100ul至液相色谱药瓶(lc vial)中,接下来在利用氮气挥发氯仿(chloroform)之后利用干燥器在真空下干燥1小时以上而形成脂质膜(lipid film)。通过向脂质膜药瓶(lipid filmvial)投入1ml的自动磷酸盐缓冲溶液(auto PBS)而形成脂质溶液,接下来通过将装有脂质溶液的高效液相色谱药瓶(HPLC vial)投入到温度为55℃以上的水中而使得脂质溶液的温度达到55℃以上,然后通过水浴声波器(bath sonic)中执行15秒左右的声波降解(sonication)(此时,将浸泡在热水中的过程以及声波降解(sonication)执行过程重复执行3次左右)而形成膜泡。
3.利用脂质的微泡的形成
将装有通过实施例1至实施例2的过程获取到的结果物的药瓶(vial)填充全氟丙烷气体(C3F8 gas)30秒,接下来利用药瓶搅拌器(Vial Mixel)进行45秒的搅拌(mixing),从而形成包括N-羟基琥珀酰亚胺(NHS)反应基的基于脂质的微泡。
4.纳米粒子复合体(Directional LT-NPs)的形成
(1)将在实施例1的1中形成的纳米粒子投入到膜泡溶液(通过将在实施例1的2中形成的膜泡以1mg/ml的浓度混合到磷酸盐缓冲溶液(PBS)中而形成)之后,为了诱导N-羟基琥珀酰亚胺-胺(NHS-amine)反应而在室温(RT)条件下进行2小时的反应(此时,形成膜泡-纳米粒子复合体(Liposome-NPs)),接下来利用超声波设备以2W、1MHz、占空比(dutycycle)100%的条件施加5分钟以上的机械性力量(mechanical force)。接下来,为了确保被粉碎的膜泡可以充分形成脂质结构体,在室温(RT)条件下执行1小时以上的培养(incubation),从而形成在纳米粒子的外表面一部分结合有脂质结构体的纳米粒子复合体(Directional LT-NPs)。
(2)除了利用在实施例1的3中形成的微泡替代膜泡之外,以与实施例1的4中的(1)相同的其他条件形成在纳米粒子的外表面一部分结合有脂质结构体的纳米粒子复合体(Directional LT-NPs)。
<实施例2>纳米粒子复合体的特性确认
1.利用透射电子显微镜(TEM)对在实施例1的1中形成的纳米粒子(NPs)以及在实施例1的4中的(2)形成的纳米粒子复合体(Directional LT-NPs)进行测定,其结果如图2所示,并利用冷冻透射电子显微镜(Cyro-TEM)对上述纳米粒子复合体以及脂质结构体进行测定,其结果分别如图3以及图4所示。
2.通过图2可以确认纳米粒子的表面平滑,而纳米粒子复合体则因为脂质结构体附着在纳米粒子的表面而呈现出不平滑的表面。此外,通过图3可以确认在纳米粒子的表面附着有管状形态的脂质结构体,通过图4可以明确确认上述脂质结构体为管状形态。此外,通过图3以及图4可以确认纳米粒子的直径为100至300nm,上述脂质结构体的长度为50至300nm而宽度为3至20nm,上述脂质结构体可以在上述纳米粒子的外表面结合一个以上。
<实施例3>纳米粒子复合体的细胞摄取效率评估
1.为了对在实施例1的1中形成的纳米粒子(NPs)和在实施例1的4中的(2)形成的纳米粒子复合体(Directional LT-NPs)的细胞摄取效率进行评估而利用流式细胞仪(Flowcytometry)进行分析,其结果如图5所示,并利用共聚焦显微镜进行图像化,其结果如图6所示。利用流式细胞仪的分析是通过利用Alexa 488荧光染料(dye)标记的纳米粒子以及纳米粒子复合体对A549细胞(1×104)进行处理的方式执行,而利用共聚焦显微镜的分析是通过利用Cy5.5荧光染料(dye)标记的纳米粒子以及纳米粒子复合体对利用DAPI对核进行染色并利用鬼笔环肽(phalloidin)对细胞骨骼(cytoskeleton)进行染色的A549细胞(1×105)进行处理的方式执行。
2.此外,为了对在实施例1的1中形成的纳米粒子(NPs)和在实施例1的4中的(1)形成的膜泡-纳米粒子复合体(Liposome-NPs)、纳米粒子复合体(Directional LT-NPs)的细胞摄取效率进行评估而利用共聚焦显微镜进行图像化,其结果如图7所示。利用共聚焦显微镜的分析是通过利用Alexa 555荧光染料(dye)标记的纳米粒子、膜泡-纳米粒子复合体以及纳米粒子复合体对利用DAPI对核进行染色的A549细胞(1×105)进行处理的方式执行。
3.通过图5可以确认纳米粒子复合体的细胞摄取功效明显高于纳米粒子,通过图6可以确认在利用纳米粒子复合体替代纳米粒子时在细胞内呈现出了更多的红色,在利用荧光图像的试验中也可以确认到与图5相同的结果。此外,通过图7可以确认纳米粒子复合体的细胞摄取功效明显高于纳米粒子或膜泡-纳米粒子复合体,借此可以确认除了由脂质构成的微泡之外,通过膜泡或其他脂质球形体也可以形成纳米粒子复合体。
<实施例4>利用内吞抑制剂(Endocytosis Inhibitor)进行处理之后的纳米粒子复合体的细胞摄取效率评估
1.对于利用内吞抑制剂(Endocytosis Inhibitor)进行处理之后的细胞,对在实施例1的1中形成的纳米粒子(NPs)和在实施例1的4中的(2)形成的纳米粒子复合体(Directional LT-NPs)的细胞摄取效率进行了评估。众所周知,因为200nm大小的纳米粒子大体上是通过巨胞饮(macropinocytosis)、非网格蛋白依赖性内吞(clathrin-independent endocytosis)、网格蛋白依赖性内吞(clathrin-dependent endocytosis)等三种机理实现细胞内摄取,因此在选定对其进行抑制(inhibition)的抑制剂(inhibitor)之后首先分别或同时对A549细胞(1×104)进行1小时的处理,接下来利用Alexa 488荧光染料(dye)标记的纳米粒子以及纳米粒子复合体进行3小时的处理并利用流式细胞仪进行测定。以纳米粒子为基准对测定结果进行标准化(normalize),其结果如图8所示。作为巨胞饮抑制剂(Macropinocytosis inhibitor)选定5-(N-甲基-N-异丙基)阿米洛利(EIPA,5-(N-Ethyl-N-isopropyl)amiloride)(浓度为25ug/ml)对Na+/H+交换(exchange)机理进行抑制,作为网格蛋白依赖性内吞抑制剂(Clathrin-dependent endocytosis inhibitor)选定氯丙嗪(CPZ,chlorpromazine)(浓度为20ug/ml)对抑制被膜小窝形成(inhibitsclathrin-coated pit formation)进行抑制,作为非网格蛋白依赖性内吞抑制剂(Clathrin-independent endocytosis inhibitor)选定甲基-β-环糊精(MβCD,methyl-β-cyclodextrin)(浓度为3mg/ml)对胆固醇依赖型内吞过程(cholesterol-dependentendocytic process)进行抑制。
2.通过图8可以确认纳米粒子(NPs)因为抑制剂(Inhibitor)即氯丙嗪(CPZ)以及5-(N-甲基-N-异丙基)阿米洛利(EIPA)而导致细胞摄取功效的大幅降低,而在同时利用三种类型的抑制剂(inhibitor)对所有内吞(endocytosis)机理进行抑制时几乎无法达成纳米粒子的细胞摄取,还可以确认而纳米粒子复合体(Directional LT-NPs)的细胞摄取功效与纳米粒子相比优秀约350%左右,而且在同时利用三种类型的抑制剂(inhibitor)对所有内吞(endocytosis)机理进行抑制时的细胞摄取功效也明显比纳米粒子优秀。借此,可以确认纳米粒子复合体不仅可以实现内吞(endocytosis),还可以通过直接穿透(directpenetration)的方式直接穿透细胞膜。
<实施例5>纳米粒子复合体作为抗癌剂载体的功效评估
1.除了在向白蛋白溶液添加混合有阿霉素(doxorubicin)的溶液进行反应之后执行乙醇滴定直至混合溶液变浑浊之外,按照与实施例1的1以及实施例1的4中的(2)相同的方式执行,从而形成含阿霉素的纳米粒子、含阿霉素的纳米粒子复合体,将具有抗癌抗性的乳腺癌细胞株即MCF-7/ADR接种到孔板(well plates)中,接下来分别利用包含阿霉素(100mM,DOX)、含阿霉素的纳米粒子(含100mM的阿霉素)以及含阿霉素的纳米粒子复合体(含100mM的阿霉素)的培养基在37℃下进行6小时的培养,然后利用一般培养基(normalmedia)进行48小时的培养并执行细胞存活率(Cell vialbilty)检测,其结果如图9所示。细胞存活率(Cell viability)检测是利用噻唑蓝比色法(MTT assay)以及台盼蓝染色排除法(trypan blue dye exclusion method),而细胞存活率(Cell viability)是通过对包含0.4%台盼蓝染料(trypan blue dye)的细胞进行培养并利用纽巴氏血细胞计数器(Neubauer hemocytometer)进行计数的方式确定。在噻唑蓝比色法(MTT assay)中使用96孔板(96-well plates)和1.5mg/ml的噻唑蓝试剂(MTT reagent)(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide),在利用15ul的噻唑蓝试剂(MTT reagent)进行2小时的培养之后向各个孔(well)添加200μl的二甲基亚砜(DMSO),所产生的培养板(Resulting culture plates)是利用盘式分析仪(plate reader)(Bio Tek Instruments,Inc,Winooski,VT,USA)在570nm下进行测定。
2.通过图9可以确认,在利用相同浓度的阿霉素对乳腺癌细胞进行处理时,在使用纳米粒子复合体的情况下,在一般癌细胞和具有对抗癌剂的抗癌抗性的细胞中都能够提升抗癌剂的细胞杀灭效果。
<实施例6>纳米粒子复合体作为基因药物载体的评估
1.在将白蛋白(Human serum albumin)以40mg/mL的浓度溶解到0.1mM的4-羟乙基哌嗪乙磺酸(HEPES)和0.01mM的乙二胺四乙酸(EDTA)之后投入硫醇改性的血管内皮生长因子小干扰核糖核酸双链体(thiol-modify VEGF siRNA duplex)(5'修饰),接下来以1mL/min的速度滴定100%乙醇直至白蛋白与小干扰核糖核酸(siRNA)混合的溶液变浑浊在遮光条件下使乙醇挥发一整夜,然后在以13200rpm、10min的条件执行离心分离之后利用移液管去除没有被粒子化的白蛋白或未反应的药物,接下来在利用磷酸盐缓冲溶液(PBS)再分散并以3000rpm、5min的条件执行离心分离之后利用移液管获取除微丸(micropellet)之外的上层液(担载有小干扰核糖核酸的纳米粒子(siRNA NPs))。上述硫醇改性的血管内皮生长因子小干扰核糖核酸双链体(thiol-modify VEGF siRNA duplex)的正义(sense)为5'-AUGUGAAUGCAGACCAAAGAA-3'(SEQ ID NO.1)而反义(antisense)为5'-thiol-UUCUUUGGUCUGCAUUCACAU-3'(SEQ ID NO.2)。
2.将在实施例6的1中形成的担载有小干扰核糖核酸(siRNA)的纳米粒子投入到微泡溶液(通过将在实施例1的3中形成的微泡以1mg/ml的浓度混合到磷酸盐缓冲溶液(PBS)中而形成)之后,在室温(RT)条件下进行2小时的反应,接下来利用超声波设备以2W、1MHz、占空比(duty cycle)100%的条件施加5分钟以上的机械性力量(mechanical force)。接下来,为了确保被粉碎的微泡可以充分形成脂质结构体,在室温(RT)条件下执行1小时以上的培养(incubation),从而形成在担载有小干扰核酸(siRNA)的纳米粒子的外表面一部分结合有脂质结构体的担载有小干扰核糖核酸(siRNA)的纳米粒子复合体(Directional siRNALT-NPs)。
3.对实施例6的1以及2中的担载有小干扰核糖核酸的纳米粒子(siRNA NPs)、担载有小干扰核糖核酸(siRNA)的纳米粒子复合体(Directional siRNA LT-NPs)的基因沉默(Gene silencing)进行确认,其结果如图9以及图10所示。基因沉默(Gene silencring)的确认是分别利用担载有小干扰核糖核酸的纳米粒子(siRNA NPs)、担载有小干扰核糖核酸(siRNA)的纳米粒子复合体(Directional siRNA LT-NPs)对MCF-7细胞(1×105)进行3小时的处理并执行24小时的培养之后提取出信使核糖核酸(mRNA),并利用相同的方法对细胞执行了聚合酶链式反应(PCR)。在聚合酶链式反应凝胶阻滞试验(PCR gel retardationassay)中,基因条带(gene bands)是利用GelRed核酸染料(GelRed Nucleic Acid stain)进行染色并通过GelDoc成像设备(GelDoc imaging device)进行视觉化。在图10中,对利用ImagePro对基因条带(gene band)的强度(intensity)进行相对定量的值进行了图示。
4.通过图9以及图10可以确认,与使用担载有小干扰核糖核酸的纳米粒子(siRNANPs)时相比,使用担载有小干扰核糖核酸(siRNA)的纳米粒子复合体(Directional siRNALT-NPs)时的基因沉默(gene silencing)效率明显更加优秀,表明纳米粒子复合体可以有效地作为基因药物载体使用。
<实施例7>在肿瘤细胞球状体(Tumor cell spheroid)中的功效评估1.肿瘤细胞球状体(Tumor cell spheroid)与一般的粘附性(adherent)癌细胞不同,是利用3D培养基(3D culture)进行增殖,而且因为如上所述的球状体(spheroid)是癌细胞在培养基上漂浮(floating)生长,因此会导致癌细胞的聚集生长。因为已经有研究结果指出聚集生长的形态会模拟(mimic)细胞外基质(Extracellular matrix,ECM),因此为了在体外(in vitro)对基于脂质表面改性的组织穿透力(tissue penetration)与否进行确认而在上述模型下进行了试验。
2.球状体(Spheroid)细胞的形成
在将10g的聚(2-羟乙基甲基丙烯酸酯)(Poly(2-hydroxyethyl methacrylate))10g添加到1L的100%无水乙醇(pure ethanol)中并在60℃下进行溶解之后,将3.3ml的溶解的聚(2-羟乙基甲基丙烯酸酯)(Poly(2-hydroxyethyl methacrylate))以100phi为基准均匀地分散到整个板(plate)中并通过进行24小时的干燥而执行涂布(coating),接下来将MCF7细胞接种到在上述过程中准备的板(plate)上并维持5天而获得了形成球状体(spheroid)的细胞。
3.利用纳米粒子复合体对球状体(Spheroid)细胞进行处理
(1)为了对在实施例1的1中形成的纳米粒子(NPs)和在实施例1的4中的(2)形成的纳米粒子复合体(Directional LT-NPs)的球状体(Spheroid)细胞摄取效率进行评估而利用共聚焦显微镜进行图像化,其结果如图12所示。利用Alexa 555荧光染料(dye)标记的纳米粒子以及纳米粒子复合体对利用DAPI对核进行染色的在实施例7的2中形成的球状体(Spheroid)形态的MCF-7细胞(1×105)进行处理并在经过3小时之后利用共聚焦显微镜进行测定。
(2)通过图11可以确认纳米粒子复合体的细胞摄取效率明显高于纳米粒子,借此可以得知不仅是在单纯的体外(in vitro)环境下,在模拟(mimic)体内(in vivo)的环境下纳米粒子复合体的摄取效率也非常优秀,因此可以确认纳米粒子复合体在组织中具有优秀的穿透效率。
在上述内容中,申请人对本发明的多种实施例进行了说明,但是如上所述的实施例只是实现本发明之技术思想的一实施例,可以实现本发明之技术思想的任意变更例或修改例都应该解释为包含在本发明的权利要求范围之内。
<110> 西江大学校产学协力团
<120> 通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体及其制造方法
<130> PDAHJ-17179
<160> 2
<170> KoPatentIn 3.0
<210> 1
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> 正义siRNA
<400> 1
augugaaugc agaccaaaga a 21
<210> 2
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> 反义siRNA
<400> 2
uucuuugguc ugcauucaca u 21
Claims (11)
1.一种纳米粒子复合体,其特征在于,包括:
纳米粒子,通过被摄取到细胞内部而用于对疾病进行治疗;以及,基于脂质的脂质结构体,通过结合到上述纳米粒子的外表面一部分而提升纳米粒子的细胞内摄取效率。
2.根据权利要求1所述的纳米粒子复合体,其特征在于:
上述纳米粒子的直径为100至300nm,上述脂质结构体的长度为50至300nm而宽度为3至20nm。
3.根据权利要求1所述的纳米粒子复合体,其特征在于:
上述脂质结构体为外侧面包括亲水性的脂质头基而内部包括疏水性的脂质尾端的整体长度较长的管状形态。
4.根据权利要求2所述的纳米粒子复合体,其特征在于:
上述纳米粒子包括第1反应基,上述脂质结构体包括与纳米粒子的第1反应基化学结合的第2反应基,通过第1反应基与第2反应基的化学结合而将脂质结构体结合到纳米粒子。
5.根据权利要求3所述的纳米粒子复合体,其特征在于:
上述纳米粒子的外表面包括第1反应基,上述管状形态的脂质结构体的一端包括第2反应基,通过第1反应基与第2反应基的化学结合而将管状形态的脂质结构体结合到上述纳米粒子的外表面。
6.根据权利要求3所述的纳米粒子复合体,其特征在于:
上述纳米粒子是由担载有药物或可以对疾病进行治疗的物质构成。
7.根据权利要求3所述的纳米粒子复合体,其特征在于:
上述纳米粒子复合体不仅可以实现内吞,还可以通过直接贯通细胞膜而被摄取到细胞内部。
8.根据权利要求3所述的纳米粒子复合体,其特征在于:
上述脂质结构体可以提升上述纳米粒子的球状体细胞内摄取效率。
9.根据权利要求3所述的纳米粒子复合体,其特征在于:
上述纳米粒子包括抗癌剂或基因治疗剂,可以提升具有抗癌抗性的肿瘤细胞的杀灭效率或细胞的基因治疗效率。
10.一种纳米粒子复合体的制造方法,其特征在于,包括:
形成包括第1反应基的纳米粒子的纳米粒子形成步骤;形成包括与上述第1反应基化学结合的第2反应基的基于磷脂质的微小尺寸的脂质体的脂质体形成步骤;通过对上述纳米粒子与脂质体进行混合而使得第1反应基与第2反应基相互结合并借此形成纳米粒子被结合到脂质体外侧面的脂质体-纳米粒子复合体的脂质复合体形成步骤;以及,通过向在上述脂质复合体形成步骤中形成的脂质体-纳米粒子复合体施加机械性的力量而粉碎脂质体并借此形成结合到纳米粒子的外表面一部分的脂质结构体,从而制造出纳米粒子复合体的粉碎形成步骤;
上述脂质体为微泡或膜泡,
上述纳米粒子通过被摄取到细胞内部而用于对疾病进行治疗,上述脂质结构体可以提升纳米粒子的细胞内摄取效率。
11.根据权利要求10所述的纳米粒子复合体的制造方法,其特征在于:
在上述粉碎形成步骤中,通过向脂质体-纳米粒子复合体施加机械性的力量并维持一段时间而对脂质体进行粉碎并使得构成脂质体的磷脂质发生重组,从而形成结合到纳米粒子的管状形态的脂质结构体。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR20180063163 | 2018-06-01 | ||
KR10-2018-0063163 | 2018-06-01 | ||
PCT/KR2018/010309 WO2019231051A1 (ko) | 2018-06-01 | 2018-09-04 | 지질을 이용한 표면 개질을 통해 세포 내 섭취 효율을 향상시킨 나노입자 복합체 및 이의 제조방법 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112423738A true CN112423738A (zh) | 2021-02-26 |
CN112423738B CN112423738B (zh) | 2023-06-20 |
Family
ID=68697624
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201880094068.7A Active CN112423738B (zh) | 2018-06-01 | 2018-09-04 | 通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体及其制造方法 |
Country Status (6)
Country | Link |
---|---|
US (1) | US11865211B2 (zh) |
EP (1) | EP3808344A4 (zh) |
JP (1) | JP7160385B2 (zh) |
KR (2) | KR102180630B1 (zh) |
CN (1) | CN112423738B (zh) |
WO (1) | WO2019231051A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114173768A (zh) * | 2019-05-10 | 2022-03-11 | 西江大学校产学协力团 | 疾病治疗用纳米粒子复合体及其制造方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20230171408A (ko) * | 2022-06-13 | 2023-12-20 | (주) 멥스젠 | 아포지단백질을 포함하는 하이브리드 나노입자의 합성 방법 |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101903018A (zh) * | 2007-10-17 | 2010-12-01 | 韩国科学技术院 | 用于递送核酸基因的ldl样阳离子纳米微粒、其制备方法以及使用其递送核酸基因的方法 |
CN101970687A (zh) * | 2007-11-09 | 2011-02-09 | 东北大学 | 用于体内基因输送的自组装胶束样纳米颗粒 |
JP2011184406A (ja) * | 2010-03-10 | 2011-09-22 | Nippon Telegr & Teleph Corp <Ntt> | 量子ドット複合体、量子ドット複合体含有ベシクル及びこれらの製造方法 |
CN104755108A (zh) * | 2012-10-25 | 2015-07-01 | 松岗大学研究基金会 | 结合含有药物的纳米粒子的超声波造影剂及其制造方法 |
WO2015153805A2 (en) * | 2014-04-01 | 2015-10-08 | Children's Hospital Los Angeles | Targeted polymerized nanoparticles for cancer treatment |
US20170042824A1 (en) * | 2014-04-16 | 2017-02-16 | Trustees Of Boston University | Gm3 functionalized nanoparticles |
CN106832435A (zh) * | 2017-01-22 | 2017-06-13 | 福建农林大学 | 一种莲子淀粉‑脂质复合物纳米颗粒的加工方法 |
CN107412181A (zh) * | 2017-07-05 | 2017-12-01 | 张琦 | 一种两亲性白芨胶骨架控制脂质纳米粒释放的制备方法 |
WO2018064350A1 (en) * | 2016-09-30 | 2018-04-05 | Eriochem Usa, Llc | Apo-e modified lipid nanoparticles for drug delivery to targeted tissues and therapeutic methods |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5863670B2 (ja) | 2010-01-19 | 2016-02-17 | ノースウェスタン ユニバーシティ | 核酸および/または他の構成要素を含有している合成ナノ構造体 |
JP2014101273A (ja) * | 2011-02-28 | 2014-06-05 | Tokyo Medical And Dental Univ | 脂質構造体の製造方法 |
KR101085203B1 (ko) * | 2011-04-01 | 2011-11-21 | 서울대학교산학협력단 | 의약 전달용 인지질 나노입자 |
US9750819B2 (en) | 2012-05-23 | 2017-09-05 | Ohio State Innovation Foundation | Lipid nanoparticle compositions and methods of making and methods of using the same |
KR101488822B1 (ko) * | 2012-08-02 | 2015-02-04 | (주)아이엠지티 | 암의 진단 및 치료를 위한 마이크로버블-나노리포좀 복합체 |
KR101487088B1 (ko) | 2012-10-25 | 2015-01-27 | 서강대학교산학협력단 | 약물을 함유한 나노입자가 결합된 초음파 조영제 및 이의 제조방법 |
KR20160045074A (ko) | 2013-07-30 | 2016-04-26 | 베네밀크 오와이 | 수유 반추동물용 사료 |
KR101686145B1 (ko) | 2015-05-18 | 2016-12-13 | 한국과학기술원 | 인도시아닌 그린-리포좀 복합체를 포함하는 암 치료용 조성물 |
WO2017041033A1 (en) * | 2015-09-04 | 2017-03-09 | Brinker C Jeffrey | Mesoporous silica nanoparticles and supported lipid bi-layer nanoparticles for biomedical applications |
KR101837537B1 (ko) | 2015-10-02 | 2018-03-14 | 한양대학교 에리카산학협력단 | 다중 약물의 효율적 세포내 전달을 위한 다중 블럭 폴리펩타이드를 포함하는 약물전달체 및 자가조립 나노구조체 |
EP3411082B1 (en) * | 2016-02-01 | 2023-06-21 | Trust Bio-Sonics Inc. | Lipid microbubbles and process of making thereof |
US20210177756A1 (en) * | 2019-12-13 | 2021-06-17 | Lawrence Livermore National Security, Llc | Nanotube-vesicle compositions and uses thereof |
-
2018
- 2018-09-04 US US17/056,402 patent/US11865211B2/en active Active
- 2018-09-04 CN CN201880094068.7A patent/CN112423738B/zh active Active
- 2018-09-04 WO PCT/KR2018/010309 patent/WO2019231051A1/ko unknown
- 2018-09-04 JP JP2020566223A patent/JP7160385B2/ja active Active
- 2018-09-04 EP EP18920326.8A patent/EP3808344A4/en not_active Withdrawn
-
2019
- 2019-05-10 KR KR1020190054779A patent/KR102180630B1/ko active IP Right Grant
-
2020
- 2020-10-07 KR KR1020200129172A patent/KR102180631B1/ko active IP Right Grant
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101903018A (zh) * | 2007-10-17 | 2010-12-01 | 韩国科学技术院 | 用于递送核酸基因的ldl样阳离子纳米微粒、其制备方法以及使用其递送核酸基因的方法 |
CN101970687A (zh) * | 2007-11-09 | 2011-02-09 | 东北大学 | 用于体内基因输送的自组装胶束样纳米颗粒 |
JP2011184406A (ja) * | 2010-03-10 | 2011-09-22 | Nippon Telegr & Teleph Corp <Ntt> | 量子ドット複合体、量子ドット複合体含有ベシクル及びこれらの製造方法 |
CN104755108A (zh) * | 2012-10-25 | 2015-07-01 | 松岗大学研究基金会 | 结合含有药物的纳米粒子的超声波造影剂及其制造方法 |
WO2015153805A2 (en) * | 2014-04-01 | 2015-10-08 | Children's Hospital Los Angeles | Targeted polymerized nanoparticles for cancer treatment |
US20170042824A1 (en) * | 2014-04-16 | 2017-02-16 | Trustees Of Boston University | Gm3 functionalized nanoparticles |
WO2018064350A1 (en) * | 2016-09-30 | 2018-04-05 | Eriochem Usa, Llc | Apo-e modified lipid nanoparticles for drug delivery to targeted tissues and therapeutic methods |
CN106832435A (zh) * | 2017-01-22 | 2017-06-13 | 福建农林大学 | 一种莲子淀粉‑脂质复合物纳米颗粒的加工方法 |
CN107412181A (zh) * | 2017-07-05 | 2017-12-01 | 张琦 | 一种两亲性白芨胶骨架控制脂质纳米粒释放的制备方法 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114173768A (zh) * | 2019-05-10 | 2022-03-11 | 西江大学校产学协力团 | 疾病治疗用纳米粒子复合体及其制造方法 |
Also Published As
Publication number | Publication date |
---|---|
US20210308050A1 (en) | 2021-10-07 |
CN112423738B (zh) | 2023-06-20 |
US11865211B2 (en) | 2024-01-09 |
JP2021533082A (ja) | 2021-12-02 |
KR102180630B1 (ko) | 2020-11-19 |
KR20200118400A (ko) | 2020-10-15 |
JP7160385B2 (ja) | 2022-10-25 |
KR20190137687A (ko) | 2019-12-11 |
EP3808344A1 (en) | 2021-04-21 |
KR102180631B1 (ko) | 2020-11-19 |
EP3808344A4 (en) | 2022-03-30 |
WO2019231051A1 (ko) | 2019-12-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yan et al. | Mitochondria-targeted tetrahedral DNA nanostructures for doxorubicin delivery and enhancement of apoptosis | |
Yezhelyev et al. | Proton-sponge coated quantum dots for siRNA delivery and intracellular imaging | |
Robertson et al. | pH-sensitive tubular polymersomes: formation and applications in cellular delivery | |
Chen et al. | Smart multifunctional nanostructure for targeted cancer chemotherapy and magnetic resonance imaging | |
Spillmann et al. | Multifunctional liquid crystal nanoparticles for intracellular fluorescent imaging and drug delivery | |
US20090317802A1 (en) | Compositions and Methods to Monitor RNA Delivery to Cells | |
Sigot et al. | Targeted cellular delivery of quantum dots loaded on and in biotinylated liposomes | |
Park et al. | Multimodal magnetic nanoclusters for gene delivery, directed migration, and tracking of stem cells | |
Kotmakçı et al. | Extracellular vesicles as natural nanosized delivery systems for small-molecule drugs and genetic material: steps towards the future nanomedicines | |
Vilaça et al. | Surface functionalization of zeolite-based drug delivery systems enhances their antitumoral activity in vivo | |
Vighi et al. | Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter | |
Lee et al. | DNA amplification in neutral liposomes for safe and efficient gene delivery | |
Luo et al. | Live‐cell imaging of octaarginine‐modified polymer dots via single particle tracking | |
CN112423738A (zh) | 通过利用脂质的表面改性提升细胞内摄取效率的纳米粒子复合体及其制造方法 | |
AU2006280519A1 (en) | A nanoparticle suitable for delivery of a biomolecule into or out of a membrane enclosed cell or cell organelle | |
Wang et al. | Permeabilization-tolerant plasma membrane imaging reagent based on amine-rich glycol chitosan derivatives | |
Huang et al. | Research advances of engineered exosomes as drug delivery carrier | |
Versluis et al. | Coiled coil driven membrane fusion between cyclodextrin vesicles and liposomes | |
US20160361266A1 (en) | Modified silica shell particles, and methods of making and using the same | |
WO2020098754A1 (zh) | 疏水性纳米生物探针 | |
Ou et al. | Micro cell vesicle technology (mCVT): A novel hybrid system of gene delivery for hard-to-transfect (HTT) cells | |
CN114173768A (zh) | 疾病治疗用纳米粒子复合体及其制造方法 | |
Xu et al. | Cell Nucleus Penetration by Quantum Dots Induced by Nuclear Staining Organic Fluorophore and UV‐Irradiation | |
KR102565700B1 (ko) | 지질을 이용한 표면 개질을 통해 조직 투과성을 향상시킨 안약 제제용 나노입자 복합체 | |
KR20170131212A (ko) | 아노이키스 내성 암세포의 화학요법을 위한 나노입자 |
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 | ||
TR01 | Transfer of patent right |
Effective date of registration: 20240628 Address after: Seoul, South Kerean Patentee after: Yingsi Biopharmaceutical Co.,Ltd. Country or region after: Republic of Korea Address before: Seoul, South Kerean Patentee before: SOGANG University RESEARCH FOUNDATION Country or region before: Republic of Korea |