CN111973758A - 一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 - Google Patents
一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 Download PDFInfo
- Publication number
- CN111973758A CN111973758A CN202010935242.5A CN202010935242A CN111973758A CN 111973758 A CN111973758 A CN 111973758A CN 202010935242 A CN202010935242 A CN 202010935242A CN 111973758 A CN111973758 A CN 111973758A
- Authority
- CN
- China
- Prior art keywords
- ptx
- dnase
- prodrug
- delivery system
- drug delivery
- 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
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 68
- 238000012377 drug delivery Methods 0.000 title claims abstract description 32
- 210000000440 neutrophil Anatomy 0.000 title claims abstract description 23
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000001276 controlling effect Effects 0.000 title claims abstract description 6
- 239000002105 nanoparticle Substances 0.000 claims abstract description 79
- 229940002612 prodrug Drugs 0.000 claims abstract description 59
- 239000000651 prodrug Substances 0.000 claims abstract description 59
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims abstract description 54
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims abstract description 54
- 108010039918 Polylysine Proteins 0.000 claims abstract description 34
- 229920000656 polylysine Polymers 0.000 claims abstract description 34
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 claims abstract description 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 19
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 claims abstract description 12
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- 238000001338 self-assembly Methods 0.000 claims abstract description 3
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 claims abstract 5
- 239000000243 solution Substances 0.000 claims description 45
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 37
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 26
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- 229920001223 polyethylene glycol Polymers 0.000 claims description 20
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 17
- 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 claims description 16
- 239000007995 HEPES buffer Substances 0.000 claims description 16
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 claims description 14
- 239000002202 Polyethylene glycol Substances 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000002390 rotary evaporation Methods 0.000 claims description 11
- 150000001540 azides Chemical class 0.000 claims description 10
- 235000019441 ethanol Nutrition 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 8
- 238000010898 silica gel chromatography Methods 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 238000000703 high-speed centrifugation Methods 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
- DLLMHEDYJQACRM-UHFFFAOYSA-N 2-(carboxymethyldisulfanyl)acetic acid Chemical compound OC(=O)CSSCC(O)=O DLLMHEDYJQACRM-UHFFFAOYSA-N 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 6
- 238000012650 click reaction Methods 0.000 claims description 6
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 6
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 6
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 6
- 229960005055 sodium ascorbate Drugs 0.000 claims description 6
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 6
- -1 succinimidyl carboxymethyl Chemical group 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 4
- 239000004698 Polyethylene Substances 0.000 claims description 4
- 125000000304 alkynyl group Chemical group 0.000 claims description 4
- 230000033228 biological regulation Effects 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000012044 organic layer Substances 0.000 claims description 4
- 229920000573 polyethylene Polymers 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000004873 anchoring Methods 0.000 claims description 2
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 2
- 125000002091 cationic group Chemical group 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 39
- 230000000694 effects Effects 0.000 abstract description 21
- 201000011510 cancer Diseases 0.000 abstract description 13
- 230000008685 targeting Effects 0.000 abstract description 10
- 230000002829 reductive effect Effects 0.000 abstract description 6
- 230000003834 intracellular effect Effects 0.000 abstract description 3
- 108091005804 Peptidases Proteins 0.000 abstract description 2
- 239000004365 Protease Substances 0.000 abstract description 2
- 230000009876 antimalignant effect Effects 0.000 abstract description 2
- 239000011258 core-shell material Substances 0.000 abstract description 2
- 239000011159 matrix material Substances 0.000 abstract description 2
- 239000002184 metal Substances 0.000 abstract description 2
- 229910052751 metal Inorganic materials 0.000 abstract description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 229930012538 Paclitaxel Natural products 0.000 description 113
- 229960001592 paclitaxel Drugs 0.000 description 113
- 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 description 113
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 33
- 102000001776 Matrix metalloproteinase-9 Human genes 0.000 description 18
- 102000016911 Deoxyribonucleases Human genes 0.000 description 17
- 108010053770 Deoxyribonucleases Proteins 0.000 description 17
- 229960003180 glutathione Drugs 0.000 description 16
- 239000003814 drug Substances 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 206010027476 Metastases Diseases 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 230000009401 metastasis Effects 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 238000011033 desalting Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 230000012292 cell migration Effects 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 239000002246 antineoplastic agent Substances 0.000 description 4
- 229940041181 antineoplastic drug Drugs 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000011260 co-administration Methods 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 239000002539 nanocarrier Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- VBVAVBCYMYWNOU-UHFFFAOYSA-N coumarin 6 Chemical compound C1=CC=C2SC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 VBVAVBCYMYWNOU-UHFFFAOYSA-N 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000012982 microporous membrane Substances 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000004890 Interleukin-8 Human genes 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 108010028275 Leukocyte Elastase Proteins 0.000 description 2
- 102000016799 Leukocyte elastase Human genes 0.000 description 2
- 102000029749 Microtubule Human genes 0.000 description 2
- 108091022875 Microtubule Proteins 0.000 description 2
- 102000003896 Myeloperoxidases Human genes 0.000 description 2
- 108090000235 Myeloperoxidases Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 210000004688 microtubule Anatomy 0.000 description 2
- RZJRJXONCZWCBN-UHFFFAOYSA-N octadecane Chemical compound CCCCCCCCCCCCCCCCCC RZJRJXONCZWCBN-UHFFFAOYSA-N 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000595920 Homo sapiens Plasminogen-like protein A Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102100035201 Plasminogen-like protein A Human genes 0.000 description 1
- 206010056342 Pulmonary mass Diseases 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229940126523 co-drug Drugs 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000008995 epigenetic change Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 102000017941 granulin Human genes 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 1
- 229940096397 interleukin-8 Drugs 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229940038384 octadecane Drugs 0.000 description 1
- 230000010494 opalescence Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000003668 pericyte Anatomy 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 125000002456 taxol group Chemical group 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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
- A61K47/6931—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 the material constituting the nanoparticle being a polymer
- A61K47/6935—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
-
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
-
- 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
- A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- 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
- A61K47/62—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 a protein, peptide or polyamino acid
-
- 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
- A61K47/62—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 a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- C12Y301/21001—Deoxyribonuclease I (3.1.21.1)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Nanotechnology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法。该智能递药系统是由胞内还原性谷胱甘肽响应的PTX前药纳米粒为核,可被金属基质蛋白酶剪切释放并串联穿膜肽的DNase Ⅰ接枝聚赖氨酸为壳,组成的“核‑壳”型DNase Ⅰ/PTX智能共递送载体。PTX前药纳米粒是利用前药分子PTX‑SS‑C18自组装形成,再对该纳米粒用聚赖氨酸进行包被;以MMP‑9底物肽串联穿膜肽为连接分子,将DNase Ⅰ蛋白接枝在包被于PTX前药纳米粒表面的聚赖氨酸氨基侧链上,构建智能药物递送系统,达到肿瘤微环境NETs调控与肿瘤细胞靶向的策略,联合提高抗恶性肿瘤效果。
Description
技术领域
本发明属于肿瘤及其微环境靶向与缓释给药系统技术领域,涉及一种肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统,具体涉及一种新型脱氧核糖核酸酶联合紫杉醇纳米粒的恶性肿瘤智能靶向纳米递释系统及其制备方法。
背景技术
随着科学水平和医疗技术的发展,非传染性疾病成为困扰人类健康的主要病种。其中,恶性肿瘤已在全球范围内成为仅次于心血管疾病的第二大致死病因。据世界卫生组织预测,2030年全球恶性肿瘤病人死亡人数将达到1200万,其中2/3将发生在中低收入国家。在我国,恶性肿瘤的发病率约为200/10万,每年新发病例在250万左右。恶性肿瘤给人类健康和生命造成极大威胁,给家庭及社会带来沉重的负担。因此,开发安全有效的抗肿瘤药物具有重大的社会意义和临床价值。
肿瘤微环境是在肿瘤进展过程中,由肿瘤细胞,基质细胞(成纤维细胞、炎性细胞、免疫细胞、周细胞、血管内皮细胞等),细胞外基质等共同组成的局部稳定环境,呈现出组织间液压升高与局部缺氧等病理性特征,为肿瘤的发生、发展、侵袭及转移等过程提供了必要的物质基础。一直以来,科学界对肿瘤靶向递药系统的研究,主要集中于肿瘤细胞靶向的开发。然而,由于肿瘤细胞的高度异质性,非常容易产生基因突变和表观遗传变化,导致耐药,临床获益非常有限。由于肿瘤微环境相对稳定,不容易耐药,引起了肿瘤靶向治疗研究者的高度关注。所以,靶向肿瘤细胞与调控肿瘤微环境相结合的治疗策略,成为肿瘤靶向研究领域新的热点。
肿瘤微环境被认为是“永不愈合伤口”与持续炎症状态,肿瘤组织通过释放趋化因子、细胞因子、过氧化氢等信号分子,将外周血中的中性粒细胞招募到肿瘤部位。肿瘤相关的中性粒细胞在肿瘤坏死因子α(TNF-α)、白介素8(IL-8)等细胞因子刺激下,形成NETs。NETs,即中性粒细胞胞外诱捕网(Neutrophil Extracellular Traps)是中性粒细胞受到外界刺激活化时释放到胞外的DNA骨架纤维网,同时包含组蛋白、髓过氧化物酶(MPO)、中性粒细胞弹性蛋白酶(NE)、组织蛋白酶G(CG)、以及基质金属蛋白酶9(MMP-9)等颗粒蛋白。研究表明:NETs作为肿瘤微环境的重要组成部分,在促进肿瘤细胞增殖、远处转移以及血管生成和阻碍肿瘤治疗中发挥着关键作用。
研究发现,脱氧核糖核酸酶Ⅰ(DNaseⅠ)可以通过降解NETs抑制恶性肿瘤的增殖并阻止向远处器官的转移,可以用于恶性肿瘤的治疗。然而,提高DNase I的体内循环寿命,增强其向肿瘤组织的渗透与滞留作用,是提高DNase I体内治疗肿瘤效果的关键所在。紫杉醇(Paclitaxel,PTX)是一线广谱抗肿瘤药物,疗效显著,具有很强的微管稳定功能,在临床上已经广泛用于乳腺癌、卵巢癌、肺癌和部分头颈癌的治疗。所以,将DNase I和PTX联合用药:DNaseⅠ可以高效降解肿瘤微环境NETs,从而抑制肿瘤细胞增殖,降低肿瘤转移和减少肿瘤血管新生,还可以疏通对肿瘤细胞的机械性屏障,促进PTX制剂向肿瘤内部的渗透;PTX直接作用于肿瘤细胞,通过稳定肿瘤细胞微管蛋白而杀死肿瘤细胞。二者相得益彰,同时靶向肿瘤的“土壤”和“种子”,发挥调控与重塑肿瘤微环境和直接杀死肿瘤细胞的协同治疗作用,最大限度提高肿瘤的治疗效果。
要实现DNaseⅠ与PTX两种药物的联合使用,选择合适的共递送载体显得尤为重要。共递送载体是药物递送领域的重要研究方向,目前共递送技术主要是将多种药物一起担载于脂质体、胶束、纳米粒等药物载体中,利用纳米载体将共载药物递送至同一作用部位,增强多种药物联用的治疗效果。然而,由于DNaseⅠ的靶点为肿瘤微环境中的NETs,而PTX的靶点为肿瘤细胞内的微管,二者靶部位不同导致现有共递释载体无法递送这两种药物至各自靶点发挥药效。
发明内容
鉴于目前仅针对肿瘤细胞的单一制剂疗效不理想,易引起耐药等缺陷,提供一种既靶向肿瘤细胞又能调控肿瘤微环境NETs的联合治疗方法,提高恶性肿瘤的治疗效果。
本发明的目的还在于,针对目前游离DNase I和PTX在治疗恶性肿瘤时存在的缺陷,提供一种智能靶向纳米药物递送载体及其制备方法,延长药物体内的循环时间,增加药物在肿瘤部位的蓄积,提高抗肿瘤疗效,降低毒副作用,达到靶向联合治疗增效减毒目的。
本发明的目的是,利用肿瘤微环境NETs中高表达MMP-9,以及肿瘤细胞内GSH浓度高的特殊条件,设计构建一种在肿瘤部位响应性治疗的智能载体。该智能载体能够能靶向至肿瘤微环境调控NETs,同时暴露的穿膜肽增加PTX前药纳米粒的细胞内吞作用,在肿瘤细胞内,以GSH为智能释药开关,响应病灶部位GSH介导的降解作用,使前药纳米粒在肿瘤细胞内发生裂解释放出PTX杀死肿瘤细胞,达到NETs降解与肿瘤细胞杀伤并策,联合提高恶性肿瘤的治疗效果。
本发明的目的通过以下技术方案实现的:
一种肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统,是由对GSH响应的PTX前药纳米粒、PTX前药纳米粒表面接枝的聚赖氨酸、琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮(NHS-PEG3500-N3)、MMP-9酶底物肽段与细胞穿膜肽串联氨基酸序列、4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Sulfo-SMCC)以及脱氧核糖核酸酶Ⅰ(DNaseⅠ)组成;其中,所述的PTX前药纳米粒和前药纳米粒表面接枝的聚赖氨酸通过电荷吸附作用实现包裹融合。
优选上述PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇(C18-OH)化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒NP/PTX,并将NP/PTX表面涂层上阳离子聚赖氨酸,PTX-SS-C18与聚赖氨酸的质量比为1:1~5,聚赖氨酸的分子量为70000~150000。
优选上述琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮(NHS-PEG3500-N3)中的聚乙二醇分子量为500-3500,MMP-9酶底物肽段与细胞穿膜肽串联后的氨基酸序列为GRKKRRQRRRPQPLGLAGGC,所述的DNaseⅠ是通过Sulfo-SMCC为连接分子化学接枝在串联的MMP-9酶底物肽段与细胞穿膜肽上。
优选上述PTX-SS-C18与DNaseⅠ的质量比为1:0.5~3。
具体的:本发明智能递药系统是由胞内还原性谷胱甘肽(GSH)响应的PTX前药纳米粒为核,可被金属基质蛋白酶(MMP-9)剪切释放并串联穿膜肽的DNaseⅠ接枝聚赖氨酸为壳,组成的“核-壳”型DNaseⅠ/PTX智能共递送载体。所述PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇(C18-OH)化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒,对该纳米粒用聚赖氨酸进行包被;以MMP-9底物肽串联穿膜肽(GRKKRRQRRRPQPLGLAGGC)为连接分子,将DNaseⅠ蛋白接枝在包被于PTX前药纳米粒表面的聚赖氨酸氨基侧链上,构建一种肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统。通过EPR效应,将DNase I递送至肿瘤微环境降解NETs,再将PTX高效递送至肿瘤细胞内杀死肿瘤细胞,达到肿瘤微环境NETs调控与肿瘤细胞靶向的策略,联合提高抗恶性肿瘤效果。
上述肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统的制备方法,将聚赖氨酸借助电荷吸附包被在PTX前药纳米粒表面,得到P-NP/PTX;利用Sulfo-SMCC为linker,将DNase I与N端炔基化的(Pro)GRKKRRQRRRPQPLGLAGGC肽进行接枝,得到多肽-蛋白偶联(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,再将该偶联物通过NHS-PEG3500-N3为linker,利用NHS端与P-NP/PTX表面游离氨基反应,另一端的叠氮基团与(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I偶联物N端的炔基发生点击化学反应,将(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I偶联物锚定在P-NP/PTX表面即组装成智能药物递送系统。
PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒NP/PTX;其中,PTX-SS-C18制备方法包括以下步骤:将1.0g二硫代甘醇酸与15mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应3h后,旋转蒸发去除乙酸和多余的乙酸酐,旋蒸后的产物溶于二氯甲烷,并加入1.49g的十八醇和65mg的DMAP,室温下搅拌反应15h后用1%HAc终止,有机层用无水硫酸钠干燥;粗产物经硅胶柱层析纯化后得到中间体HOOC-CH2-SS-CH2-COOC18;将中间体和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入200mgPTX,在避光条件下,继续室温反应10h;待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥;粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。
所述前药分子自组装形成PTX前药纳米粒NP/PTX的方法为采用乙醇注入法制备GSH响应的PTX前药纳米粒,具体为:称取5mgPTX-SS-C18溶于0.5mL无水乙醇,室温下将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得NP/PTX。
将制备好的PTX前药纳米粒与5mg/mL的聚赖氨酸溶液混合,借助电荷吸附,将聚赖氨酸包被在PTX前药纳米粒表面,多余的聚赖氨酸通过高速离心法去除,得到P-NP/PTX,PTX-SS-C18与聚赖氨酸的质量比为1:1~5。
上述智能药物递送系统组装过程具体如下:(1)首先,将P-NP/PTX用HEPES(pH7.4)重新分散,加入浓度为5mg/mL的NHS-PEG3500-N3溶液,室温搅拌2小时后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用;(2)将4mg DNaseI溶解于PBS(pH 7.4)中,加入0.5mg的Sulfo-SMCC,室温氮气保护下反应1小时;将5mg(Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES缓冲溶液中,加入上述DNase I溶液,室温氮气保护下搅拌反应4小时,得到(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物;(3)表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时后冷冻高速离心,水洗,即得。
本发明构建一种具有程序化释药能力的智能共递送载体,首先靶向肿瘤微环境NETs,到NETs部位后,释放出DNaseⅠ,降解NETs,然后智能载体再携载PTX渗透进入肿瘤细胞,在胞内再释放出PTX,是亟待解决的关键问题。MMP-9是NETs的重要组成部分,即NETs部位过表达MMP-9。所以,可以利用MMP-9对底物的酶切作用,触发DNase I在NETs部位的智能释放。还原性谷胱甘肽(GSH)在肿瘤细胞内的浓度(~2-10mM)是胞外和血浆中的(~2-10μM)1000倍。因此,GSH成为抗肿瘤药物胞内释放的“智能开关”。通过二硫键构建纳米递药载体(胶束、聚合物纳米粒、高分子结合物等),在模拟血液生理环境下载体稳定,而在模拟肿瘤细胞内GSH浓度下,载体解聚,释放出药物,显示出对GSH高度的响应性,有利于抗肿瘤药物在肿瘤细胞内的智能释放。
本发明的有益效果:该智能共递送药物载体集长循环、被动靶向、肿瘤微环境NETs部位MMP-9响应性释药、“原位暴露”式肿瘤细胞靶向、肿瘤胞内GSH响应性释药等多功能于一体。本发明构建的智能共递送载体,通过EPR效应聚集在肿瘤部位,有效延长药物在体内的半衰期,对NETs的降解效果显著提高,抑制了肿瘤细胞的增殖和远处转移。在NETs部位高表达的MMP-9对多肽响应性剪切,释放DNase I发挥效果,同时暴露的穿膜肽增加PTX前药纳米粒的细胞内吞作用,以GSH为智能释药开关,响应肿瘤细胞内GSH介导的降解作用,使前药纳米粒在肿瘤细胞内发生裂解,释放出PTX杀死肿瘤细胞。达到肿瘤微环境调控与肿瘤细胞杀伤并策,联合提高恶性肿瘤的治疗效果。
对本发明制备的用于治疗肿瘤的靶向纳米递药系统进行了体内外评价:
对本发明实施例方法制备的肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统进行了细胞毒性实验、细胞摄取实验、细胞迁移实验、体内药效学研究,结果表明,将DNase I锚定在纳米粒上后,显著增加了DNase I在肿瘤部位的蓄积,达到预期目的。而MMP-9响应性剪切之后,暴露出细胞穿膜肽,大大增加肿瘤细胞对药物的摄取,明显提高PTX的抗肿瘤效果,具体如下:
1)细胞摄取
前药纳米粒通过香豆素-6荧光标记后考察载体系统的细胞定性摄取情况,结果如图2所示,细胞对纳米粒的摄取具有浓度依赖性和时间依赖性,1μg/mL组的荧光强度明显强于0.5μg/mL组,2h组的荧光强度明显强于1h组。
2)细胞毒性实验
将体外诱导生成的NETs与A549细胞共培养在96孔板中,采用MTT法测定P-NP/PTX、游离DNase I和对MMP-9响应的纳米粒mP-NPS-DNase/PTX、对MMP-9不响应的纳米粒nP-NPS-DNase/PTX以及P-NP/PTX和游离DNase I共同给药对A549细胞的体外抑制情况,结果如图3所示,mP-NPS-DNase/PTX纳米粒组对A549的细胞毒作用与共同给药组没有显著的统计学差异,且明显强于其它对照组。
3)细胞迁移实验
将体外诱导生成的NETs与4T1细胞共培养于Transwell的上室中,分别给药P-NP/PTX、游离DNase I和mP-NPS-DNase/PTX、nP-NPS-DNase/PTX以及P-NP/PTX和游离DNase I共同给药,结果如图4所示,mP-NPS-DNase/PTX纳米粒组显著减少4T1细胞迁移至小室下层,抑制作用与共同给药组没有显著的统计学差异。
4)体内靶向性评价
皮下瘤小鼠分别尾静脉注射罗丹明-NHS标记的游离DNase I和mP-NPS-DNase/PTX,12h后的肿瘤组织切片荧光结果显示(图5),mP-NPS-DNase/PTX在肿瘤部位的荧光强度明显强于游离组,该结果表明将DNase I接枝在纳米粒上后能增加递药系统在肿瘤部位的主动靶向与蓄积。
5)体内抗肿瘤药效学评价
对BALB/c鼠乳房垫接瘤后分别给药mP-NPS-DNase/PTX、nP-NPS-DNase/PTX、P-NP/PTX、DNase I、P-NP/PTX与DNase I共同给药和生理盐水,通过肿瘤大小以及肺转移情况评价纳米载体的治疗效果。结果如图6所示,给药组小鼠体重与生理盐水组无明显差异,表明纳米载体生物相容性较好,无明显的毒副作用。通过图7可以发现,mP-NPS-DNase/PTX组的肿瘤明显小于其它对照组,对原发肿瘤的生长有较好的抑制作用。如图8所示,使用mP-NPS-DNase/PTX治疗后,肺结节数明显少于其它对照组,证明该纳米载体有效抑制了肿瘤的肺转移。
附图说明
图1为本发明共递送给药系统的透射电镜图,纳米粒呈规整的类球形。其中,图1A:NP/PTX,图1B:mP-NPS-DNase/PTX。
图2本发明中A549细胞对纳米粒的摄取荧光图,其中,图A和图C纳米粒浓度为0.5μg/mL,图B和图D纳米粒浓度为1μg/mL,图A、图B是A549细胞与P-NP/PTX共孵育1h的摄取情况,图C、图D是A549细胞与P-NP/PTX共孵育2h的摄取情况。
图3为本发明中A549细胞存活率柱状图。
图4为本发明中4T1细胞迁移率柱状图。
图5为本发明中游离DNase I和mP-NPS-DNase/PTX分别在肿瘤部位的蓄积。
图6为本发明中纳米粒给药组与生理盐水组小鼠体重随时间变化。
图7为本发明中各个给药组小鼠肿瘤大小比较。
图8为本发明中各个给药组肿瘤肺转移后形成结节的数量比较。
具体实施方式
下面结合具体实施例对本发明作进一步的阐述,具体实施例是在本发明的优选条件下进行。所述方法如无特别说明均为常规方法,所述原材料如无特别说明均能从公开商业途径而得。
肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统制备的步骤:
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseI,水洗2遍后,即得mP-NPS-DNase/PTX。实施例1:纳米粒理化性能表征及细胞迁移实验所用mP-NPS-DNase/PTX纳米粒的合成
将1.0g二硫代甘醇酸与15mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应3h。待反应完全后,旋转蒸发去除乙酸和多余的乙酸酐。旋蒸后的产物溶于二氯甲烷,并加入1.49g的十八醇及65mgDMAP,室温下搅拌反应15h后用1%HAc终止,有机层用无水硫酸钠干燥。粗产物经硅胶柱层析纯化后得到中间体HOOC-CH2-SS-CH2-COOC18。将HOOC-CH2-SS-CH2-COOC18和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入200mgPTX,在避光条件下,继续室温反应10h。待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥。粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。采用乙醇注入法制备GSH响应的PTX前药纳米粒。称取5mgPTX-SS-C18溶于0.5mL无水乙醇。室温下,将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得PTX前药纳米粒。
纳米粒溶液外观澄清,有明显的蓝色乳光。激光粒度分析表明,所得纳米粒子以106nm为有效直径呈正态分布,多分散性为0.113。扫描电镜下观察该纳米粒具有规整的球形外观,在溶液中分散良好,并具有较好的稳定性。
将制备好的PTX前药纳米粒与聚赖氨酸溶液混合,借助电荷吸附,将聚赖氨酸(分子量为70000~150000)包被在PTX前药纳米粒表面,多余的聚赖氨酸(分子量为70000~150000)通过高速离心法去除,得到P-NP/PTX,PTX-SS-C18与聚赖氨酸的质量比为1:1~5。将P-NP/PTX用HEPES(pH 7.4)重新分散,加入NHS-PEG3500-N3(购自于北京键凯科技有限公司),室温搅拌2小时后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用。PTX-SS-C18与NHS-PEG3500-N3(聚乙二醇分子量为500-3500)的质量比为1:2。
DNaseⅠ溶解于PBS(pH 7.4)中,加入Sulfo-SMCC,室温氮气保护下反应1小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的Sulfo-SMCC。Sulfo-SMCC与DNaseⅠ的质量比为1:1.5~7.5。将(Pro)GRKKRRQRRRPQPLGLAGGC多肽(购自于吉尔生化(上海)有限公司)溶解于HEPES(pH 7.0)缓冲溶液中,加入上述DNaseⅠ溶液,室温氮气保护下搅拌反应4小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的(Pro)GRKKRRQRRRPQPLGLAGGC,得到(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ多肽-蛋白偶联物,用HEPES(pH 7.4)缓冲液重新分散后,备用。Sulfo-SMCC与(Pro)GRKKRRQRRRPQPLGLAGGC的质量比为1:2.5。PTX-SS-C18与DNaseⅠ的质量比为1:3。
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,水洗2遍后,即得mP-NPS-DNase/PTX。
制得的mP-NPS-DNase/PTX外观呈规整的类球形,如图1B所示。该纳米粒对肿瘤细胞的生长有较好的抑制作用,如图3所示。通过细胞迁移实验,可发现该纳米粒能有效抑制肿瘤细胞的迁移,结果如图4所示。
实施例2:细胞定性摄取实验所用mP-NPS-DNase/PTX纳米粒的合成
PTX-SS-C18的合成步骤同实施例1。称取香豆素-6和5mg PTX-SS-C18溶于0.5mL无水乙醇。室温下,将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得香豆素-6标记的PTX前药纳米粒。
后续步骤同实施例1,得标记了香豆素-6的mP-NPS-DNase/PTX纳米粒。该纳米粒可被肿瘤细胞摄取,且摄取的量具有时间依赖性和浓度依赖性,如图2所示。
实施例3:体内药效学所用mP-NPS-DNase/PTX纳米粒的合成
将5.0g二硫代甘醇酸与75mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应3h。待反应完全后,旋转蒸发去除乙酸和多余的乙酸酐。旋蒸后的产物溶于二氯甲烷,并加入7.45g的十八醇及325mg DMAP,室温下搅拌反应15h后用1%HAc终止,有机层用无水硫酸钠干燥。粗产物经硅胶柱层析纯化后得到中间体HOOC-CH2-SS-CH2-COOC18。将HOOC-CH2-SS-CH2-COOC18和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入1.0g PTX,在避光条件下,继续室温反应10h。待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥。粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。采用乙醇注入法制备GSH响应的PTX前药纳米粒。称取48mg PTX-SS-C18溶于5mL无水乙醇。室温下,将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得PTX前药纳米粒。
将制备好的PTX前药纳米粒与144mg聚赖氨酸混合,借助电荷吸附,将聚赖氨酸包被在PTX前药纳米粒表面,多余的聚赖氨酸通过高速离心法去除,得到P-NP/PTX。将P-NP/PTX用HEPES(pH 7.4)重新分散,加入NHS-PEG3500-N3,室温搅拌2h后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用。PTX-SS-C18与NHS-PEG3500-N3的质量比为1:2。
将7.2mg DNaseⅠ溶解于PBS(pH 7.4)中,加入Sulfo-SMCC,室温氮气保护下避光反应1小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的Sulfo-SMCC。Sulfo-SMCC与DNase I的质量比为1:1.5。将(Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES(pH 7.0)缓冲溶液中,加入上述DNaseⅠ溶液,室温氮气保护下搅拌反应4小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的(Pro)GRKKRRQRRRPQPLGLAGGC,得到(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物,用HEPES(pH 7.4)缓冲液重新分散后,备用。Sulfo-SMCC与(Pro)GRKKRRQRRRPQPLGLAGGC的质量比为1:2。
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,水洗2遍后,即得mP-NPS-DNase/PTX。
制得的mP-NPS-DNase/PTX对小鼠的体重无明显影响,具有良好的安全性,如图6所示。该纳米粒抑制了原发肿瘤的生长,并且阻止了肿瘤向肺部的转移,肺结节数明显少于其他对照组,结果如图7、8所示。
实施例4:评估纳米粒在肿瘤部位聚积所用mP-NPS-DNase/PTX纳米粒的合成
表面聚乙二醇叠氮化P-NP/PTX的合成步骤同实施例3。
将7.2mgDNaseⅠ溶解于PBS(pH 7.4)中,加入0.1mg荧光染料罗丹明-NHS,室温下避光搅拌2h得DNaseⅠ-Rhodamine。向DNaseⅠ-Rhodamine溶液中加入Sulfo-SMCC,室温氮气保护下避光反应1小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的Sulfo-SMCC。Sulfo-SMCC与DNase I的质量比为1:1.5。将(Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES(pH7.0)缓冲溶液中,加入上述DNaseⅠ-Rhodamine溶液,室温氮气保护下搅拌反应4小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的(Pro)GRKKRRQRRRPQPLGLAGGC,得到(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I-Rhodamine多肽-蛋白偶联物,用HEPES(pH 7.4)缓冲液重新分散后,备用。Sulfo-SMCC与(Pro)GRKKRRQRRRPQPLGLAGGC的质量比为1:1.5。
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I-Rhodamine多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,水洗2遍后,即得接枝了荧光染料的mP-NPS-DNase/PTX。
制得的mP-NPS-DNase/PTX通过EPR效应聚积在肿瘤部位,其荧光强度明显亮于游离DNase I组,如图5所示。
实施例5:对MMP-9不响应的纳米粒nP-NPS-DNase/PTX的合成
MMP-9不响应是通过设计(Pro)GRKKRRQRRRPQPLGLAGGC肽中底物序列PLGLA全部用相应的D-型氨基酸取代得到。其余步骤同实施例3。
nP-NPS-DNase/PTX对肿瘤细胞的毒性以及抑制肿瘤细胞的能力均弱于mP-NPS-DNase/PTX,且体内药效实验结果均差于MMP-9响应性纳米粒mP-NPS-DNase/PTX。结果如图3、4、7、8所示。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征在于该纳米给药系统是由对GSH响应的PTX前药纳米粒、PTX前药纳米粒表面接枝的聚赖氨酸、琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮、MMP-9酶底物肽段与细胞穿膜肽串联氨基酸序列、4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐以及脱氧核糖核酸酶Ⅰ组成。
2.根据权利要求1所述的中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征是:所述PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒,并将PTX前药纳米粒表面涂层上阳离子聚赖氨酸,聚赖氨酸的分子量为70000~150000。
3.根据权利要求1所述的中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征是:所述的琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮中的聚乙二醇分子量为500-3500、MMP-9酶底物肽段与细胞穿膜肽串联后的氨基酸序列为GRKKRRQRRRPQPLGLAGGC,所述的DNaseⅠ是通过Sulfo-SMCC为连接分子化学接枝在串联的MMP-9酶底物肽段与细胞穿膜肽上。
4.根据权利要求2所述的中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征是:所述PTX-SS-C18与DNaseⅠ的质量比为1:0.15~3。
5.一种权利要求1所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征是:将聚赖氨酸借助电荷吸附包被在PTX前药纳米粒表面,得到P-NP/PTX;利用Sulfo-SMCC为linker,将DNaseⅠ与N端炔基化的(Pro)GRKKRRQRRRPQPLGLAGGC肽进行接枝,得到多肽-蛋白偶联(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ,再将该偶联物通过NHS-PEG3500-N3为linker,利用NHS端与P-NP/PTX表面游离氨基反应,另一端的叠氮基团与(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ偶联物N端的炔基发生点击化学反应,将(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ偶联物锚定在P-NP/PTX表面即组装成智能药物递送系统。
6.根据权利要求5所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征在于PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒NP/PTX;其中,PTX-SS-C18制备方法包括以下步骤:将1.0g二硫代甘醇酸与15mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应3h后,旋转蒸发去除乙酸和多余的乙酸酐,旋蒸后的产物溶于二氯甲烷,并加入1.49g的十八醇和65mg的DMAP,室温下搅拌反应15h后用1%HAc终止,有机层用无水硫酸钠干燥;粗产物经硅胶柱层析纯化后得到中间体HOOC-CH2-SS-CH2-COOC18;将中间体和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入200mgPTX,在避光条件下,继续室温反应10h;待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥;粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。
7.根据权利要求6所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征在于所述前药分子自组装形成PTX前药纳米粒NP/PTX的方法为采用乙醇注入法制备GSH响应的PTX前药纳米粒,具体为:
称取5mgPTX-SS-C18溶于0.5mL无水乙醇,室温下将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得NP/PTX。
8.根据权利要求7所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征在于:将制备好的PTX前药纳米粒与5mg/mL的聚赖氨酸溶液混合,借助电荷吸附,将聚赖氨酸包被在PTX前药纳米粒表面,多余的聚赖氨酸通过高速离心法去除,得到P-NP/PTX,PTX-SS-C18与聚赖氨酸的质量比为1:1~5。
9.根据权利要求5所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,所述的智能药物递送系统组装过程的特征在于:(1)首先,将P-NP/PTX用HEPES重新分散,加入浓度为5mg/mL的NHS-PEG3500-N3溶液,室温搅拌2小时后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用;(2)将4mgDNaseⅠ溶解于PBS中,加入0.5mg的Sulfo-SMCC,室温氮气保护下反应1小时;将5mg(Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES缓冲溶液中,加入上述DNaseⅠ溶液,室温氮气保护下搅拌反应4小时,得到(Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ多肽-蛋白偶联物;(3)表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时后冷冻高速离心,水洗,即得。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010935242.5A CN111973758B (zh) | 2020-09-08 | 2020-09-08 | 一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010935242.5A CN111973758B (zh) | 2020-09-08 | 2020-09-08 | 一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111973758A true CN111973758A (zh) | 2020-11-24 |
CN111973758B CN111973758B (zh) | 2022-07-26 |
Family
ID=73447743
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010935242.5A Expired - Fee Related CN111973758B (zh) | 2020-09-08 | 2020-09-08 | 一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111973758B (zh) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113238050A (zh) * | 2021-02-20 | 2021-08-10 | 吴炜 | 一种中性粒细胞胞外诱捕网的快速检测方法 |
CN114099417A (zh) * | 2021-11-16 | 2022-03-01 | 中国药科大学 | 中性粒细胞胞外杀菌网络响应性载药凝胶及其制法和使法 |
CN114224823A (zh) * | 2021-11-02 | 2022-03-25 | 南京医科大学 | 一种集化疗/光动力治疗/化学动力治疗“三位一体”的脑胶质瘤递药系统及其制备方法 |
CN114522219A (zh) * | 2022-02-23 | 2022-05-24 | 宜春学院 | 一种共递送聚合物前药及其制备方法和用途 |
CN115364244A (zh) * | 2022-09-06 | 2022-11-22 | 中国医学科学院生物医学工程研究所 | 一种搭便车中性粒细胞的药物递送体系及其制备方法和应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107875140A (zh) * | 2016-09-30 | 2018-04-06 | 复旦大学 | 一种双靶向药物递送系统及其在制备肿瘤治疗制剂中的应用 |
-
2020
- 2020-09-08 CN CN202010935242.5A patent/CN111973758B/zh not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107875140A (zh) * | 2016-09-30 | 2018-04-06 | 复旦大学 | 一种双靶向药物递送系统及其在制备肿瘤治疗制剂中的应用 |
Non-Patent Citations (2)
Title |
---|
BAOYAN WANG ET AL: "Improved anti-glioblastoma efficacy by IL-13Rα2 mediated copolymer nanoparticles loaded with paclitaxel", 《SCIENTIFIC REPORTS》 * |
YAN JIANG ET AL: "Enhanced Antiglioma Efficacy of Ultrahigh Loading Capacity Paclitaxel Prodrug Conjugate Self-Assembled Targeted Nanoparticles", 《APPL. MATER. INTERFACES》 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113238050A (zh) * | 2021-02-20 | 2021-08-10 | 吴炜 | 一种中性粒细胞胞外诱捕网的快速检测方法 |
CN114224823A (zh) * | 2021-11-02 | 2022-03-25 | 南京医科大学 | 一种集化疗/光动力治疗/化学动力治疗“三位一体”的脑胶质瘤递药系统及其制备方法 |
CN114224823B (zh) * | 2021-11-02 | 2023-12-05 | 南京医科大学 | 一种集化疗/光动力治疗/化学动力治疗“三位一体”的脑胶质瘤递药系统及其制备方法 |
CN114099417A (zh) * | 2021-11-16 | 2022-03-01 | 中国药科大学 | 中性粒细胞胞外杀菌网络响应性载药凝胶及其制法和使法 |
CN114522219A (zh) * | 2022-02-23 | 2022-05-24 | 宜春学院 | 一种共递送聚合物前药及其制备方法和用途 |
CN114522219B (zh) * | 2022-02-23 | 2024-03-26 | 宜春学院 | 一种共递送聚合物前药及其制备方法和用途 |
CN115364244A (zh) * | 2022-09-06 | 2022-11-22 | 中国医学科学院生物医学工程研究所 | 一种搭便车中性粒细胞的药物递送体系及其制备方法和应用 |
CN115364244B (zh) * | 2022-09-06 | 2024-04-12 | 中国医学科学院生物医学工程研究所 | 一种搭便车中性粒细胞的药物递送体系及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN111973758B (zh) | 2022-07-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111973758B (zh) | 一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 | |
Chen et al. | Recent advances in epsilon-poly-L-lysine and L-lysine-based dendrimer synthesis, modification, and biomedical applications | |
CN108478531A (zh) | 叶酸靶向还原敏感载药聚合物纳米胶束及其制备方法和应用 | |
US11478493B2 (en) | Fabrication and application of a hetero-targeted nano-cocktail with traceless linkers | |
CN102120036B (zh) | 生物降解的高分子键合Pt(IV)类抗癌药物纳米胶束及其制备方法 | |
CN107669632B (zh) | 药物载体、胶束、药物制剂、及其制备方法和用途 | |
US20160166693A1 (en) | Radiation enhanced macromolecular delivery of therapeutic agents for chemotherapy | |
CN108070025B (zh) | 一种细胞穿透肽和细胞穿透肽复合物及二者的应用 | |
US10428114B2 (en) | Type polypeptide targeting tumours | |
KR102065711B1 (ko) | 종양 인식형 광감각제-약물 접합체, 이의 제조방법 및 이를 포함하는 종양 예방 또는 치료용 약학 조성물 | |
CN107184987B (zh) | 一种硫辛酸修饰的靶向整合素αvβ3纳米多肽载体及其制备方法和应用 | |
JP2002512640A (ja) | 持続性治療薬の局所送達 | |
WO2012089768A1 (en) | System for the release of a therapeutic agent, pharmaceutical compositions containing it, the preparation and medical use thereof | |
CN107308457A (zh) | 一种具有肿瘤微环境响应性降解的深层穿透纳米递药系统 | |
CN113117095B (zh) | SP-AgNPs纳米材料及其和沙门氏菌联合在制备抗肿瘤药物中的应用 | |
KR102228272B1 (ko) | 항암 상승효과를 나타내는 종양세포 특이적 자기조립 나노약물 복합체 | |
Wang et al. | Luteinizing-hormone-releasing-hormone-containing biodegradable polymer micelles for enhanced intracellular drug delivery | |
CN113384554B (zh) | 一种药物递送载体及其制备方法和应用 | |
RU2451509C1 (ru) | Противоопухолевый препарат | |
KR102386477B1 (ko) | 신규한 세포 투과성 펩타이드 및 이의 용도 | |
CN115607680A (zh) | 金团簇-核酸适体及其衍生物组装体的制备及应用 | |
Pan et al. | Self‐Adaptive Nanoregulator to Mitigate Dynamic Immune Evasion of Pancreatic Cancer | |
WO2008121349A1 (en) | Tag and target delivery system | |
KR101818377B1 (ko) | 활성 산소종 관련 질환의 진단/치료용 디셀레나이드 가교결합을 함유한 블록 공중합체 및 이의 제조방법 | |
US20220071918A1 (en) | Mussel adhesive protein-based photothermal agent and photothermal- responsive adhesive nanoparticles |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220726 |