CN108743970A - 一种透明质酸修饰的线粒体靶向脂质体及其制备方法 - Google Patents
一种透明质酸修饰的线粒体靶向脂质体及其制备方法 Download PDFInfo
- Publication number
- CN108743970A CN108743970A CN201810579311.6A CN201810579311A CN108743970A CN 108743970 A CN108743970 A CN 108743970A CN 201810579311 A CN201810579311 A CN 201810579311A CN 108743970 A CN108743970 A CN 108743970A
- Authority
- CN
- China
- Prior art keywords
- liposome
- drug
- hyaluronic acid
- mitochondrially targeted
- loaded
- 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
- 239000002502 liposome Substances 0.000 title claims abstract description 141
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 77
- 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 title claims abstract description 75
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 75
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 55
- 229940079593 drug Drugs 0.000 claims abstract description 54
- 229960001378 dequalinium chloride Drugs 0.000 claims abstract description 25
- LTNZEXKYNRNOGT-UHFFFAOYSA-N dequalinium chloride Chemical compound [Cl-].[Cl-].C1=CC=C2[N+](CCCCCCCCCC[N+]3=C4C=CC=CC4=C(N)C=C3C)=C(C)C=C(N)C2=C1 LTNZEXKYNRNOGT-UHFFFAOYSA-N 0.000 claims abstract description 25
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 18
- 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 claims abstract description 8
- HAWSQZCWOQZXHI-FQEVSTJZSA-N 10-Hydroxycamptothecin Chemical compound C1=C(O)C=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 HAWSQZCWOQZXHI-FQEVSTJZSA-N 0.000 claims abstract description 5
- 229940009456 adriamycin Drugs 0.000 claims abstract description 4
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims abstract description 3
- 229960004768 irinotecan Drugs 0.000 claims abstract description 3
- 229910052697 platinum Inorganic materials 0.000 claims abstract description 3
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims abstract description 3
- 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 description 24
- 235000012000 cholesterol Nutrition 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 3
- 244000050510 Cunninghamia lanceolata Species 0.000 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 229940083466 soybean lecithin Drugs 0.000 claims description 3
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 229940099552 hyaluronan Drugs 0.000 claims description 2
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(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-MNSSHETKSA-N 0.000 claims description 2
- 230000036571 hydration Effects 0.000 claims description 2
- 238000006703 hydration reaction Methods 0.000 claims description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 claims description 2
- 229940067626 phosphatidylinositols Drugs 0.000 claims description 2
- 229910052698 phosphorus Inorganic materials 0.000 claims description 2
- 239000011574 phosphorus Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims 1
- 210000002969 egg yolk Anatomy 0.000 claims 1
- 229940067606 lecithin Drugs 0.000 claims 1
- 235000010445 lecithin Nutrition 0.000 claims 1
- 239000000787 lecithin Substances 0.000 claims 1
- 229930012538 Paclitaxel Natural products 0.000 abstract description 82
- 229960001592 paclitaxel Drugs 0.000 abstract description 82
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 abstract description 82
- 210000004881 tumor cell Anatomy 0.000 abstract description 37
- 206010028980 Neoplasm Diseases 0.000 abstract description 16
- 210000003470 mitochondria Anatomy 0.000 abstract description 15
- 230000006907 apoptotic process Effects 0.000 abstract description 6
- 230000036457 multidrug resistance Effects 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- 230000008685 targeting Effects 0.000 description 18
- 239000000243 solution Substances 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 12
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 11
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 229940107161 cholesterol Drugs 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 235000019441 ethanol Nutrition 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- PQMOXTJVIYEOQL-UHFFFAOYSA-N Cumarin Natural products CC(C)=CCC1=C(O)C(C(=O)C(C)CC)=C(O)C2=C1OC(=O)C=C2CCC PQMOXTJVIYEOQL-UHFFFAOYSA-N 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 102000001974 Hyaluronidases Human genes 0.000 description 5
- FSOGIJPGPZWNGO-UHFFFAOYSA-N Meomammein Natural products CCC(C)C(=O)C1=C(O)C(CC=C(C)C)=C(O)C2=C1OC(=O)C=C2CCC FSOGIJPGPZWNGO-UHFFFAOYSA-N 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 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 4
- 231100000582 ATP assay Toxicity 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 3
- 108010003272 Hyaluronate lyase Proteins 0.000 description 3
- 235000016408 Podocarpus macrophyllus Nutrition 0.000 description 3
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 3
- 244000162450 Taxus cuspidata Species 0.000 description 3
- 235000009065 Taxus cuspidata Nutrition 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 229960000956 coumarin Drugs 0.000 description 3
- 235000001671 coumarin Nutrition 0.000 description 3
- 229960002773 hyaluronidase Drugs 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108050009363 Hyaluronidases Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- PZNPLUBHRSSFHT-RRHRGVEJSA-N 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)COC(=O)CCCCCCCCCCCCCCC PZNPLUBHRSSFHT-RRHRGVEJSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101001105486 Homo sapiens Proteasome subunit alpha type-7 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 102100021201 Proteasome subunit alpha type-7 Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 190000008236 carboplatin Chemical compound 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006567 cellular energy metabolism Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 235000012754 curcumin Nutrition 0.000 description 1
- 229940109262 curcumin Drugs 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 1
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012738 dissolution medium Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229940108949 paclitaxel injection Drugs 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- -1 polyoxyethylene groups Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002633 protecting effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明公开了一种透明质酸修饰的线粒体靶向脂质体及其制备方法。本发明的线粒体靶向脂质体由载药脂质体和外层修饰的透明质酸组成,所述载药脂质体为包载有疏水性药物和地喹氯铵的脂质体,疏水性药物选自阿霉素、紫杉醇、10‑羟基喜树碱、伊利替康或顺铂中任一种。本发明的有益效果在于,透明质酸修饰的脂质体能够有效地靶向受体,增加肿瘤细胞对脂质体的摄取,并且进一步在肿瘤细胞内靶向细胞的线粒体,进而实现破坏线粒体的功能,促进肿瘤细胞的凋亡,抑制肿瘤细胞的生长,实现克服肿瘤细胞多药耐药性的能力。
Description
技术领域
本发明属于药物制剂技术领域,具体涉及一种透明质酸修饰的线粒体靶向脂质体及其制备方法。
背景技术
癌症作为现代医疗中难以治愈的疾病的之一,其中的一个重要原因在于肿瘤的多药耐药性导致抗肿瘤药物的效果大大降低。近年来,药物在肿瘤细胞内的靶向传递是一快迅速发展的领域,其中线粒体作为肿瘤细胞中保障细胞能量代谢并调节细胞程序性凋亡的细胞器,靶向线粒体能够促进细胞凋亡,有效地克服肿瘤细胞的多药耐药性,增强药物的抗肿瘤效果,逐渐成为一种有效地克服肿瘤细胞多药耐药性的一个研究方向。
紫杉醇(PTX)是从紫杉或红豆杉树中提取的一种具有广谱抗肿瘤活性的天然药物,属于紫杉烷类化合物,其外观为白色结晶粉末,具有高亲脂性,不溶于水(水中溶解度仅为0.3—0.5μg/ml),易溶于甲醇、氯仿、丙酮等有机溶剂。紫杉醇在肿瘤细胞内能够选择性的与β微管蛋白结合形成非功能性稳定微管束,进一步导致纺锤体的功能受到抑制,抑制细胞有丝分裂并最终导致肿瘤细胞的凋亡。紫杉醇对治疗癌症具有优异的治疗效果,是如今最常使用的广谱抗肿瘤药物之一,是治疗多种晚期癌症和难治疗癌症中最有效的药物之一,临床中适用于乳腺癌、非小细胞肺癌、卵巢癌等许多癌症。同时,在临床研究中证明紫杉醇与姜黄素、卡铂、阿霉素联合使用时能达到良好的抗肿瘤效果。
但是紫杉醇由于属于疏水性药物,所以其使用过程中会有诸多的弊端,现在使用广泛的紫杉醇注射剂采用聚氧乙烯蓖麻油(Cremoph or EL)和乙醇(50:50,v/v)作为溶剂以解决紫杉醇的溶解度问题,但是聚氧乙烯基蓖麻油会导致病人严重的过敏反应,不利于紫杉醇制剂的进一步使用。所以将紫杉醇包载在无毒副作用的脂质体中是一种非常理想的剂型。但是普通的紫杉醇脂质体无法实现对肿瘤部位的靶向,并且由于多药耐药性的影响,紫杉醇的抗肿瘤效果受到了极大的影响。
发明内容
为了克服现有技术的不足,本发明的目的在于提供涉及一种透明质酸修饰的线粒体靶向脂质体及其制备方法,其由包载有疏水性药物与地喹氯铵的脂质体与外层修饰的透明质酸组成,最终脂质体的粒径范围在40-500nm左右。
本发明的技术方案具体介绍如下。
本发明提供一种透明质酸修饰的线粒体靶向脂质体,其由载药脂质体和外层修饰的透明质酸组成,所述载药脂质体为包载有疏水性药物和地喹氯铵的脂质体。
本发明中,疏水性药物选自阿霉素、紫杉醇、10-羟基喜树碱、伊利替康或顺铂中任一种。
本发明中,其粒径范围在40-500nm之间。
本发明中,载药脂质体的原料配方包括磷脂、胆固醇、疏水性药物与地喹氯铵;其中:磷脂和胆固醇的摩尔比在50:50-98:2之间,疏水性药物占磷脂、胆固醇、疏水性药物与地喹氯铵总摩尔数的0.5-15mol%,地喹氯铵占磷脂、胆固醇、疏水性药物与地喹氯铵总摩尔数的0.1-30mol%。
本发明中,磷脂包括但不限于蛋黄卵磷脂、大豆磷脂、氢化大豆磷脂、磷脂酰乙醇胺和磷脂酰肌醇。
本发明中,载药脂质体的制备方法包括但不限于薄膜水化法、逆相蒸发法、冻融法和有机溶剂挥发法。
本发明中,透明质酸分子量在100-2000KDa之间,透明质酸与载药脂质体的质量比为1:3-1:200,透明质酸修饰载药脂质体的方法包括但不限于静电吸附与化学键连接。
本发明还提供一种上述的线粒体靶向脂质体的制备方法,首先制备包载有疏水性药物和地喹氯铵的载药脂质体,然后通过静电吸附作用在载药脂质体外层包裹一层带负电的透明质酸,得到线粒体靶向脂质体。
和现有技术相比,本发明的有益效果在于:
①本发明提供一种透明质酸修饰的线粒体靶向脂质体,静脉注射进入血液循环后,由于透明质酸包裹在脂质体外层,脂质体表现出负电性,能够有效地避免体内网状内皮系统的清除,增加脂质体在体内的循环时间,在长循环的基础上,透明质酸修饰的包载疏水性药物的脂质体在体内会由于EPR效应被动靶向肿瘤组织,在肿瘤部位实现药物的蓄积,增加肿瘤部位的药物浓度;
②脂质外修饰的透明质酸能够防止所包载药物的提前泄露,避免全身毒副作用;
③当脂质体到达肿瘤组织部位后,由于肿瘤细胞外会过表达受体,通过透明质酸对受体的靶向作用,能够有效地增加肿瘤细胞对脂质体的摄取,实现肿瘤细胞对脂质体的高度特异性摄取。
④透明质酸修饰的脂质体经内吞作用被肿瘤细胞摄取后,在肿瘤细胞溶酶体内由于透明质酸酶的大量表达,能够迅速的降解外层修饰的透明质酸,暴露出包载地喹氯铵的脂质体,脂质体此时由于地喹氯铵对线粒体的靶向作用,会介导脂质体靶向线粒体,同时将疏水性药物靶向进入线粒体中。在线粒体内疏水性药物能够破坏线粒体的功能,促进肿瘤细胞的凋亡,实现克服肿瘤细胞多药耐药性的能力。
本发明以紫杉醇脂质体为例,实验结果表明,该脂质体对A549和A549/Taxol细胞的靶向性,证实透明质酸对于CD44受体过度表达的A549细胞具有明显的靶向作用,并且该脂质体在透明质酸酶的作用下透明质酸会被降解,并在肿瘤细胞内实现对线粒体的靶向作用。并且通过MTT实验证明该脂质体能够有效抑制肿瘤细胞的生长,在肿瘤细胞内能够通过抑制肿瘤细胞ATP的生成促进肿瘤细胞的凋亡。
附图说明
图1:透明质酸修饰线粒体靶向紫杉醇脂质体的透射电镜图(A)以及紫杉醇脂质体(PTX-L)、包载地喹氯铵与紫杉醇的脂质体(PTX&DQA-L)、透明质酸修饰紫杉醇脂质体(HA-PTX-L)、透明质酸修饰线粒体靶向紫杉醇脂质体(HA-PTX&DQA)四种脂质体在一周内的粒径(B)、zeta电位(C)以及包封率(D)的变化情况。
图2:未包裹透明质酸(PTX&DQA-L)和包裹透明质酸的紫杉醇脂质体(HA-PTX&DQA)的体外释放实验,以及在加入2mg/ml透明质酸酶之后透明质酸修饰线粒体靶向紫杉醇脂质体的体外释放情况(HAase+HA-PTX-DQA)。
图3:透明质酸修饰线粒体靶向紫杉醇脂质体对A549/Taxol肿瘤细胞的特异性靶向。
图4:透明质酸修饰线粒体靶向紫杉醇脂质体在A549/Taxol肿瘤细胞内对细胞线粒体的特异性靶向。
图5:透明质酸修饰线粒体靶向紫杉醇脂质体对A549和A549/Taxol肿瘤细胞的抑制率以及IC50值。
图6:透明质酸修饰线粒体靶向紫杉醇脂质体对A549和A549/Taxol肿瘤细胞内ATP含量的抑制。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细说明。
实施例1
一种透明质酸修饰的线粒体靶向紫杉醇脂质体的制备方法
将蛋黄卵磷脂、胆固醇、紫杉醇与地喹氯铵按照摩尔比95:5:10:20溶解在氯仿:甲醇1:1的混合溶液中,用旋转蒸发仪将其蒸干成薄膜并持续真空干燥1h,然后加入0.02M磷酸盐缓冲溶液100ml,在40℃水浴条件下水化,并使脂质膜均匀分散,脂质体在40℃条件下水化1h。将水化完成的脂质体混悬液通过安装100nm聚碳酸酯膜的挤膜器,在50℃条件下挤膜10次。将活化的透明质酸按照质量比1:20逐滴加入制备完成的脂质体中,在摇床中反应过夜。通过粒径仪测定样品的粒径、zeta和PDI,通过高效液相测定脂质体的紫杉醇浓度并计算包封率和载药量。
实施例2
一种透明质酸修饰的线粒体靶向紫杉醇脂质体的制备方法
将氢化大豆磷脂HSPC、二油酰磷脂酰乙醇胺DOPE、胆固醇、紫杉醇与地喹氯铵按照摩尔比60:35:5:5:10溶解在氯仿:甲醇1:1的混合溶液中,用旋转蒸发仪将其蒸干成薄膜并持续真空干燥1h,然后加入0.02M磷酸盐缓冲溶液100ml,在40℃水浴条件下水化,并使脂质膜均匀分散,脂质体在40℃条件下水化1h。将水化完成的脂质体混悬液通过安装100nm聚碳酸酯膜的挤膜器,在50℃条件下挤膜10次。将活化的透明质酸按照质量比1:20逐滴加入制备完成的脂质体中,在摇床中反应过夜。通过粒径仪测定样品的粒径、zeta和PDI,通过高效液相测定脂质体的紫杉醇浓度并计算包封率和载药量。
实施例3
一种透明质酸修饰的线粒体靶向紫杉醇脂质体的制备方法
将大豆磷脂、胆固醇、紫杉醇与地喹氯铵按照摩尔比60:35:5:10:15溶解在氯仿:甲醇1:1的混合溶液中,用旋转蒸发仪将其蒸干成薄膜并持续真空干燥1h,然后加入0.02M磷酸盐缓冲溶液100ml,在40℃水浴条件下水化,并使脂质膜均匀分散,脂质体在40℃条件下水化1h。将水化完成的脂质体混悬液在均质机的作用下在50℃条件下300bar循环3次,在1100bar下均质10次。将活化的透明质酸按照质量比1:20逐滴加入制备完成的脂质体中,在摇床中反应过夜。通过粒径仪测定样品的粒径、zeta和PDI,通过高效液相测定脂质体的紫杉醇浓度并计算包封率和载药量。
实施例4
一种透明质酸修饰的线粒体靶向10-羟基喜树碱脂质体的制备方法
将蛋黄卵磷脂、胆固醇、10-羟基喜树碱与地喹氯铵按照摩尔比80:15:5:5:15溶解在氯仿:甲醇1:1的混合溶液中,用旋转蒸发仪将其蒸干成薄膜并持续真空干燥1h,然后加入0.02M磷酸盐缓冲溶液100ml,在40℃水浴条件下水化,并使脂质膜均匀分散,脂质体在40℃条件下水化1h。将水化完成的脂质体混悬液通过安装100nm聚碳酸酯膜的挤膜器,在50℃条件下挤膜10次。将活化的透明质酸按照质量比1:20逐滴加入制备完成的脂质体中,在摇床中反应过夜。通过粒径仪测定样品的粒径、zeta和PDI,通过高效液相测定脂质体的10-羟基喜树碱浓度并计算包封率和载药量
应用实施例
1.透明质酸修饰线粒体靶向紫杉醇脂质体的稳定性考察
为了考察透明质酸修饰对紫杉醇脂质体稳定性的影响,将按照实施例1-4的制备方法制备得到的透明质酸脂质体在4℃条件下保存一周,连续测定脂质体在一周之内的粒径、zeta电位以及紫杉醇包封率的变化情况。图1:图A中能够明显的看出在脂质体明显的包裹了一层透明质酸,说明透明质酸对线粒体靶向紫杉醇脂质体进行了有效地包载。图B、C、D中分别表明各种脂质体在4℃条件下都能够稳定的保存1周以上的时间,说明紫杉醇脂质体在一周内能够稳定的保存。
2.透明质酸修饰线粒体靶向脂质体的体外释药行为
为了考察透明质酸修饰的脂质体包载的紫杉醇与地喹氯铵在体外介质中的释药行为,采用恒温振荡摇床法进行体外释药实验的研究。取2ml制备的紫杉醇脂质体溶液,加入透析袋(截留分子量为8000-14000)内,两端夹紧后置25ml释放介质中(PBS,pH 7.4,含有0.5%Tween 80作为增溶剂),于37℃恒温摇床中以100r/min的速度持续震摇。另外配制相应浓度的紫杉醇溶液作为对照。分别在0,0.5,1,2,4,6,12,24,36,48h取出0.5ml透析液,同时补充相同体积的释放介质,48h时取出全部的脂质体溶液,直接破乳计算出紫杉醇的量。采用高效液相色谱测定,并计算紫杉醇和DQA的释放速率。释放百分率F%=At/A0×100%。其中F为累积释放量,At为累积释放量,A0为加入透析袋中药物总量。同时,为了进一步验证透明质酸对脂质体包载药物释药行为的影响,所以设置一组添加了2mg/ml透明质酸酶的紫杉醇脂质体与正常的透明质酸修饰脂质体进行比较。从图2中能够看出,包载PTX和DQA的脂质体在未修饰HA时,两种药物都能够快速释放进入介质,但是当脂质体体外包裹透明质酸层之后,两种药物的体外释放速率都会明显减少,其中地喹氯铵由于电性的原因释放速率会降低非常明显。当脂质体外层的透明质酸被透明质酸酶降解后,药物的释放水平会重新提高。说明透明质酸对脂质体中包载的紫杉醇和地喹氯铵能够起到很好的保护效果。
3.紫杉醇脂质体对A549肿瘤细胞的特异性靶向
3.1细胞对不同脂质体摄取能力的差异
为了确定HA修饰对A549/Taxol细胞过表达的CD44受体的靶向能力,将A549/Taxol细胞,以5×104接种于24孔板内,每孔加入含10%胎牛血清的RPMI 1640培养基1mL,置于37℃,5%CO2培养箱中培养。以香豆素标记脂质体,培养8h后,在不同的孔内依次加入香豆素溶液、脂质体和透明质酸修饰的脂质体,对照组加入空白培养基作为对照,于培养箱中共孵育2h。移除培养基,以4℃无菌PBS冲洗细胞,加入4%多聚甲醛固定细胞10min,用DAPI(2μg/ml)对细胞核染色5min,以4℃PBS洗细胞三遍,置于共聚焦显微镜下观察。如图3所示,从图D中能够发现,与香豆素溶液(B)以及普通脂质体(C)相比,透明质酸修饰的线粒体靶向紫杉醇脂质体(D)由于透明质酸的修饰明显的增强了对肿瘤细胞的靶向作用。图A为空白对照组。
3.2不同细胞对透明质酸修饰脂质体摄取能力的差异
为了进一步确定HA对A549/Taxol细胞过表达的CD44受体的靶向能力,将CD44受体表达存在差异的肿瘤细胞MCF-7与A549/Taxol,以5×104接种于24孔板内,每孔加入含10%胎牛血清的RPMI 1640培养基1mL,置于37℃,5%CO2培养箱中培养。培养8h后,加入香豆素标记的透明质酸修饰的脂质体,于培养箱中共孵育2h。移除培养基,以4℃无菌PBS冲洗细胞,加入4%多聚甲醛固定细胞10min,用DAPI(2μg/ml)对细胞核染色5min,以4℃PBS洗细胞三遍,置于共聚焦显微镜下观察。从图3E能够看出,当A549肿瘤细胞中加入过量透明质酸时,由于CD44受体的阻塞,透明质酸修饰脂质体的摄取也随之降低。
3.3HA抑制CD44受体,肿瘤细胞对透明质酸修饰脂质体的摄取能力的差异
为了进一步确定HA对A549/Taxol细胞过表达的CD44受体的靶向能力,将过表达CD44受体的A549/Taxol,以5×104接种于24孔板内,每孔加入含10%胎牛血清的RPMI 1640培养基1mL,置于37℃,5%CO2培养箱中培养。以香豆素标记脂质体,培养8h后,加入10mg/ml的透明质酸溶液,细胞继续培养1h,之后按照上述的操作方法在培养基中加入透明质酸修饰的脂质体,于培养箱中共孵育2h。移除培养基,以4℃无菌PBS冲洗细胞,加入4%多聚甲醛固定细胞10min,用DAPI(2μg/ml)对细胞核染色5min,以4℃PBS冲洗细胞三遍,置于共聚焦显微镜下观察。从图3F中能够看出,不表达CD44受体的MCF-7肿瘤细胞对透明质酸修饰线粒体靶向紫杉醇脂质体的摄取量低于A549/Taxol(F)。根据以上结果,表明透明质酸修饰的线粒体靶向紫杉醇脂质体能够有效的靶向过表达CD44受体的A549肿瘤细胞。
4.透明质酸修饰线粒体靶向紫杉醇脂质体对A549/Taxol细胞线粒体的靶向能力
为了确定HA修饰紫杉醇脂质体对A549/Taxol细胞线粒体的靶向能力,将过表达CD44受体的A549/Taxol细胞,以5×104个/孔接种于24孔板内,每孔加入含10%胎牛血清的RPMI 1640培养基1mL,置于37℃,5%CO2恒温培养箱中培养。以香豆素标记脂质体,培养8h后,在不同的孔内依次加入香豆素溶液、透明质酸修饰的脂质体以及透明质酸修饰线粒体靶向脂质体,对照组加入空白培养基作为对照,于培养箱中共孵育4h。细胞培养4h后,移除培养基,每孔加入200μl的线粒体红色荧光探针继续孵育0.5h。之后使用4℃无菌PBS冲洗细胞移除线粒体红色荧光探针,加入4%多聚甲醛固定细胞10min,用DAPI(2μg/ml)对细胞核染色5min,以4℃PBS洗细胞三遍,置于共聚焦显微镜下观察。从图4中可以看出,在线粒体靶向紫杉醇脂质体(D)与透明质酸修饰的紫杉醇脂质体(C)的比较中能够明显发现地喹氯铵的加入能够增加脂质体对肿瘤细胞线粒体的靶向作用,同时与香豆素溶液(B)相比,能够增加对药物的摄取。图A为空白对照组。
5.细胞毒性实验
线粒体靶向紫杉醇脂质体对肿瘤细胞的毒性实验使用MTT试剂方法进行测定。将A549和A549/Taxol细胞以5×103个/孔接种于96孔板内,培养12h后,将培养基更换为新鲜培养基并加入空白脂质体、紫杉醇溶液(PTX)、紫杉醇脂质体(PTX-L)、透明质酸紫杉醇(HA-PTX-L)以及透明质酸修饰线粒体靶向紫杉醇脂质体(HA-DQA&PTA),每种样品各设置7个浓度,紫杉醇浓度依次为0.001、0.01、0.1、1、10、20、40μg/mL,对照组加入空白培养基,每个样品平行五次。孵育72h后,加入MTT(5mg/Ml)20μL,继续孵育4h后,弃去培养液,加入DMSO150μL,摇床振摇5min使得DMSO充分溶解每孔中的甲瓒,用酶标仪测定在490nm的吸光度值。细胞存活率按照以下公式计算:
细胞存活率(%)=(ODtest–ODDMSO)/(ODcontrol–ODDMSO)×100。如图5所示,从实验结果中能够看出,与紫杉醇溶液、普通紫杉醇脂质体、透明质酸修饰的紫杉醇脂质体相比,透明质酸修饰的线粒体靶向紫杉醇脂质体对A549(A)和A549/Taxol(B)肿瘤细胞的抑制效果都是最强的,并且能够有效地降低紫杉醇对肿瘤细胞的IC50值(C)。
6.ATP含量测定
肿瘤细胞内ATP含量测定通过ATP含量测定试剂盒,通过磷钼酸比色法进行测定。将A549和A549/Taxol细胞以5×105个/孔接种于6孔板细胞中,每孔加入2ml培养基,培养24h后,更换各孔内的培养基并分别加入紫杉醇溶液(PTX)、紫杉醇脂质体(PTX-L)、透明质酸脂质体(HA-PTX-L)以及线粒体靶向紫杉醇脂质体(HA-PTX&DQA),最终紫杉醇的浓度都为10μg/mL并继续培养8.0h。8h后移除培养基,并用pH7.4PBS缓冲液冲洗细胞三次,使用胰酶消解细胞,在4℃1000g条件下离心收集肿瘤细胞到离心管内。然后在每个离心管内加入1ml酸性提取液,冰浴条件下超声波破碎1min(强度200W,超声2s,停1s),8000g4℃离心10min,取上清至另一离心管中,加入等体积的碱性提取液使之中和并混合均匀,在8000g4℃条件下离心10min,取上清,按照ATP含量测定试剂盒内说明书的方法加入相应体积的样溶液、显色剂溶液、肌酸激酶以及计算溶液到96孔板中,混合溶液在37℃水浴反应20min后,用酶标仪在700nm下测定各组的吸光度。如图6所示,从实验结果能能够明显看出,透明质酸修饰的线粒体靶向紫杉醇脂质体能够有效地抑制A549与A549/Taxol肿瘤细胞内ATP含量。
Claims (8)
1.一种透明质酸修饰的线粒体靶向脂质体,其特征在于:其由载药脂质体和外层修饰的透明质酸组成,所述载药脂质体为包载有疏水性药物和地喹氯铵的脂质体。
2.根据权利要求1所述的线粒体靶向脂质体,其特征在于,疏水性药物选自阿霉素、紫杉醇、10-羟基喜树碱、伊利替康或顺铂中任一种。
3.根据权利要求1所述的线粒体靶向脂质体,其特征在于,其粒径范围在40-500nm之间。
4.根据权利要求1所述的线粒体靶向脂质体,其特征在于,载药脂质体的原料配方包括磷脂、胆固醇、疏水性药物与地喹氯铵;其中:磷脂和胆固醇的摩尔比在50:50-98:2之间,疏水性药物占磷脂、胆固醇、疏水性药物与地喹氯铵总摩尔数的0.5-15mol%,地喹氯铵占磷脂、胆固醇、疏水性药物与地喹氯铵总摩尔数的0.1-30mol%。
5.根据权利要求4所述的线粒体靶向脂质体,其特征在于,磷脂包括但不限于蛋黄卵磷脂、大豆磷脂、氢化大豆磷脂、磷脂酰乙醇胺和磷脂酰肌醇。
6.根据权利要求1或4所述的线粒体靶向脂质体,其特征在于,载药脂质体的制备方法包括但不限于薄膜水化法、逆相蒸发法、冻融法和有机溶剂挥发法。
7.根据权利要求1所述的线粒体靶向脂质体,其特征在于,透明质酸分子量在100-2000KDa之间,透明质酸与载药脂质体的质量比为1:3-1:200,透明质酸修饰载药脂质体的方法包括但不限于静电吸附与化学键连接。
8.一种根据权利要求1所述的线粒体靶向脂质体的制备方法,其特征在于,首先制备包载有疏水性药物和地喹氯铵的载药脂质体,然后通过静电吸附作用在载药脂质体外层包裹一层带负电的透明质酸,得到线粒体靶向脂质体。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810579311.6A CN108743970B (zh) | 2018-06-07 | 2018-06-07 | 一种透明质酸修饰的线粒体靶向脂质体及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810579311.6A CN108743970B (zh) | 2018-06-07 | 2018-06-07 | 一种透明质酸修饰的线粒体靶向脂质体及其制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108743970A true CN108743970A (zh) | 2018-11-06 |
CN108743970B CN108743970B (zh) | 2021-09-24 |
Family
ID=63999233
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810579311.6A Active CN108743970B (zh) | 2018-06-07 | 2018-06-07 | 一种透明质酸修饰的线粒体靶向脂质体及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108743970B (zh) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110538330A (zh) * | 2019-10-12 | 2019-12-06 | 深圳大学 | 一种线粒体靶向传输co的药物及其制备方法 |
CN111773185A (zh) * | 2020-08-14 | 2020-10-16 | 上海市普陀区中心医院 | 一种透明质酸修饰的载蟾毒灵纳米脂质体及其制备方法和应用 |
CN111892668A (zh) * | 2020-07-03 | 2020-11-06 | 广东工业大学 | 一种化合物及其制备方法、荧光探针和抗肿瘤药物 |
CN112999159A (zh) * | 2021-03-22 | 2021-06-22 | 苏州艾和医药科技有限公司 | 一种ha介导的靶向双载药阳离子脂质体涂层及其制备方法 |
CN113368261A (zh) * | 2021-06-17 | 2021-09-10 | 苏州大学 | 一种非病毒载体及其制备方法与应用 |
CN114042147A (zh) * | 2021-10-22 | 2022-02-15 | 川北医学院附属医院 | 靶向调控线粒体呼吸链的微纳水凝胶微球及其制备与应用 |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20010001067A1 (en) * | 1997-09-08 | 2001-05-10 | Volkmar Weissig | Materials and methods for intracellular delivery of biologically active molecules |
CN101816629A (zh) * | 2009-02-26 | 2010-09-01 | 北京大学 | 一种双重靶向脂质体及其制备方法和应用 |
CN102133172A (zh) * | 2011-01-24 | 2011-07-27 | 北京大学 | 一种紫杉醇纳米胶束及其应用 |
CN102757555A (zh) * | 2011-04-29 | 2012-10-31 | 北京大学 | 地喹氯铵-聚乙二醇-二硬脂酰磷脂酰乙醇胺共轭化合物及其修饰的白藜芦醇脂质体 |
CN103585106A (zh) * | 2013-10-29 | 2014-02-19 | 北京化工大学 | 一种pH敏感性修饰脂质体及其制备方法 |
CN104825394A (zh) * | 2015-04-17 | 2015-08-12 | 北京大学 | 靶向肿瘤相关成纤维细胞的脂质体载药系统 |
CN104940040A (zh) * | 2015-07-09 | 2015-09-30 | 西安艾尔菲生物科技有限公司 | 透明质酸修饰的包封美白成分的脂质体及制备方法和应用 |
CN105018529A (zh) * | 2014-08-29 | 2015-11-04 | 上海交通大学 | 多功能多肽/脂质体/透明质酸组装的类病毒核酸载体 |
CN105055315A (zh) * | 2015-08-03 | 2015-11-18 | 四川大学 | 一种交联线粒体靶向阿霉素脂质体及其制备方法 |
CN105726485A (zh) * | 2016-03-24 | 2016-07-06 | 东华大学 | 一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法 |
-
2018
- 2018-06-07 CN CN201810579311.6A patent/CN108743970B/zh active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20010001067A1 (en) * | 1997-09-08 | 2001-05-10 | Volkmar Weissig | Materials and methods for intracellular delivery of biologically active molecules |
CN101816629A (zh) * | 2009-02-26 | 2010-09-01 | 北京大学 | 一种双重靶向脂质体及其制备方法和应用 |
CN102133172A (zh) * | 2011-01-24 | 2011-07-27 | 北京大学 | 一种紫杉醇纳米胶束及其应用 |
CN102757555A (zh) * | 2011-04-29 | 2012-10-31 | 北京大学 | 地喹氯铵-聚乙二醇-二硬脂酰磷脂酰乙醇胺共轭化合物及其修饰的白藜芦醇脂质体 |
CN103585106A (zh) * | 2013-10-29 | 2014-02-19 | 北京化工大学 | 一种pH敏感性修饰脂质体及其制备方法 |
CN105018529A (zh) * | 2014-08-29 | 2015-11-04 | 上海交通大学 | 多功能多肽/脂质体/透明质酸组装的类病毒核酸载体 |
CN104825394A (zh) * | 2015-04-17 | 2015-08-12 | 北京大学 | 靶向肿瘤相关成纤维细胞的脂质体载药系统 |
CN104940040A (zh) * | 2015-07-09 | 2015-09-30 | 西安艾尔菲生物科技有限公司 | 透明质酸修饰的包封美白成分的脂质体及制备方法和应用 |
CN105055315A (zh) * | 2015-08-03 | 2015-11-18 | 四川大学 | 一种交联线粒体靶向阿霉素脂质体及其制备方法 |
CN105726485A (zh) * | 2016-03-24 | 2016-07-06 | 东华大学 | 一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法 |
Non-Patent Citations (8)
Title |
---|
ŠTĚPÁN KOUDELKA: "Liposomal paclitaxel formulations", 《JOURNAL OF CONTROLLED RELEASE》 * |
XIU-YING LI ET AL: "Multifunctional liposomes loaded with paclitaxel and artemether for treatment of invasive brain glioma", 《BIOMATERIALS》 * |
YANG YU ET AL: "Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma", 《BIOMATERIALS》 * |
YONGFENG TIAN ET AL: "Overcoming drug-resistant lung cancer by paclitaxel-loaded hyaluronic acid-coated liposomes targeted to mitochondria", 《DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY》 * |
ZHENJIE WANG ET AL: "Nanopreparations for mitochondria targeting drug delivery system: Current strategies and future prospective", 《ASIAN JOURNAL OF PHARMACEUTICAL SCIENCES》 * |
宋彦峰,等: "线粒体靶向DQA-DOX连接物的制备及其逆转肿瘤耐药活性研究", 《西北药学杂志》 * |
武玉敏: "透明质酸修饰的载紫杉醇脂质体的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
韩立杰,等: "透明质酸修饰载紫杉醇靶向脂质体抑制脑肿瘤干细胞的初步评价", 《中国医院药学杂志》 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110538330A (zh) * | 2019-10-12 | 2019-12-06 | 深圳大学 | 一种线粒体靶向传输co的药物及其制备方法 |
CN110538330B (zh) * | 2019-10-12 | 2023-06-16 | 深圳大学 | 一种线粒体靶向传输co的药物及其制备方法 |
CN111892668A (zh) * | 2020-07-03 | 2020-11-06 | 广东工业大学 | 一种化合物及其制备方法、荧光探针和抗肿瘤药物 |
CN111892668B (zh) * | 2020-07-03 | 2022-07-12 | 广东工业大学 | 一种化合物及其制备方法、荧光探针和抗肿瘤药物 |
CN111773185A (zh) * | 2020-08-14 | 2020-10-16 | 上海市普陀区中心医院 | 一种透明质酸修饰的载蟾毒灵纳米脂质体及其制备方法和应用 |
CN112999159A (zh) * | 2021-03-22 | 2021-06-22 | 苏州艾和医药科技有限公司 | 一种ha介导的靶向双载药阳离子脂质体涂层及其制备方法 |
CN113368261A (zh) * | 2021-06-17 | 2021-09-10 | 苏州大学 | 一种非病毒载体及其制备方法与应用 |
CN114042147A (zh) * | 2021-10-22 | 2022-02-15 | 川北医学院附属医院 | 靶向调控线粒体呼吸链的微纳水凝胶微球及其制备与应用 |
CN114042147B (zh) * | 2021-10-22 | 2023-07-28 | 川北医学院附属医院 | 靶向调控线粒体呼吸链的微纳水凝胶微球及其制备与应用 |
Also Published As
Publication number | Publication date |
---|---|
CN108743970B (zh) | 2021-09-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108743970A (zh) | 一种透明质酸修饰的线粒体靶向脂质体及其制备方法 | |
Hong et al. | Novel ginsenoside-based multifunctional liposomal delivery system for combination therapy of gastric cancer | |
Zhou et al. | The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer | |
Wang et al. | Roles of ligand and TPGS of micelles in regulating internalization, penetration and accumulation against sensitive or resistant tumor and therapy for multidrug resistant tumors | |
JP7028774B2 (ja) | ギンセノシドを膜材料として有するリポソームならびにその調製および使用 | |
Li et al. | Metformin synergistically suppress tumor growth with doxorubicin and reverse drug resistance by inhibiting the expression and function of P-glycoprotein in MCF7/ADR cells and xenograft models | |
Ma et al. | Modulation of drug-resistant membrane and apoptosis proteins of breast cancer stem cells by targeting berberine liposomes | |
Lee et al. | Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft | |
RU2574926C9 (ru) | Липосомные композиции, используемые для доставки лекарственных средств | |
CN108420793A (zh) | 一种空白混合胶束及其制备方法和应用 | |
CN105142631A (zh) | 抗癌药物和用途 | |
Shi et al. | Oxidative stress-driven DR5 upregulation restores TRAIL/Apo2L sensitivity induced by iron oxide nanoparticles in colorectal cancer | |
US20150328234A1 (en) | Bufalin liposome, preparation method therefor and application thereof | |
KR20110132446A (ko) | 키나아제 단백질 결합 억제제 | |
WO2013075607A1 (zh) | 绿原酸的抗癌新用途 | |
Zhou et al. | pH and thermo dual stimulus-responsive liposome nanoparticles for targeted delivery of platinum-acridine hybrid agent | |
AU2018360559A1 (en) | Combination comprising at least one spliceosome modulator and at least one inhibitor chosen from BCL2 inhibitors, BCL2/BCLxL inhibitors, and BCLxL inhibitors and methods of use | |
CN104490786B (zh) | 一种靶向多功能双载药脂质体的制备方法和应用 | |
CN105287383A (zh) | 新型米托蒽醌脂质体联合化疗药物在抗肿瘤治疗中的应用 | |
CN109364026A (zh) | 生物素修饰的乳腺癌靶向脂质材料的制备和应用 | |
Xiao et al. | Fisetin and polymeric micelles encapsulating fisetin exhibit potent cytotoxic effects towards ovarian cancer cells | |
EP2723345A2 (en) | Composition comprising phosphatidylcholine as an active ingredient for attenuating toxicity of anticancer agent | |
Gopalakrishnan et al. | Encapsulation of a Ru (η 6-p-cymene) complex of the antibacterial drug trimethoprim into a polydiacetylene-phospholipid assembly to enhance its in vitro anticancer and antibacterial activities | |
CN102188439A (zh) | 一种程序性释放多种药物的纳米粒脂质体及其制备与应用 | |
Michel et al. | The role of catechin in electroporation of pancreatic cancer cells–Effects on pore formation and multidrug resistance proteins |
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 |