CN113372904B - 一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法 - Google Patents
一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法 Download PDFInfo
- Publication number
- CN113372904B CN113372904B CN202110638001.9A CN202110638001A CN113372904B CN 113372904 B CN113372904 B CN 113372904B CN 202110638001 A CN202110638001 A CN 202110638001A CN 113372904 B CN113372904 B CN 113372904B
- Authority
- CN
- China
- Prior art keywords
- dna
- motif
- sequence
- aunp
- imaging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 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 title claims abstract description 84
- 229960003180 glutathione Drugs 0.000 title claims abstract description 42
- 238000003384 imaging method Methods 0.000 title claims abstract description 35
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 34
- 230000004044 response Effects 0.000 title claims abstract description 20
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 15
- 238000002560 therapeutic procedure Methods 0.000 title claims abstract description 10
- 238000010276 construction Methods 0.000 title claims abstract description 9
- 239000010931 gold Substances 0.000 claims abstract description 24
- 239000003504 photosensitizing agent Substances 0.000 claims abstract description 24
- 102100034256 Mucin-1 Human genes 0.000 claims abstract description 18
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910052737 gold Inorganic materials 0.000 claims abstract description 18
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 18
- 108091023037 Aptamer Proteins 0.000 claims abstract description 17
- 239000002105 nanoparticle Substances 0.000 claims abstract description 17
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims abstract description 15
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 claims abstract description 10
- 230000000295 complement effect Effects 0.000 claims abstract description 7
- 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 description 50
- 230000002195 synergetic effect Effects 0.000 claims description 9
- SURLGNKAQXKNSP-DBLYXWCISA-N chlorin Chemical group C\1=C/2\N/C(=C\C3=N/C(=C\C=4NC(/C=C\5/C=CC/1=N/5)=CC=4)/C=C3)/CC\2 SURLGNKAQXKNSP-DBLYXWCISA-N 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 229940025294 hemin Drugs 0.000 claims description 2
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 229940009456 adriamycin Drugs 0.000 claims 4
- 238000011262 co‐therapy Methods 0.000 claims 3
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 37
- 108020004414 DNA Proteins 0.000 abstract description 35
- 238000011282 treatment Methods 0.000 abstract description 20
- 239000002246 antineoplastic agent Substances 0.000 abstract description 15
- 229940044683 chemotherapy drug Drugs 0.000 abstract description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 59
- 229960004679 doxorubicin Drugs 0.000 description 21
- 239000000243 solution Substances 0.000 description 20
- 238000001069 Raman spectroscopy Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 14
- 229940079593 drug Drugs 0.000 description 13
- 239000003814 drug Substances 0.000 description 13
- ZKSVYBRJSMBDMV-UHFFFAOYSA-N 1,3-diphenyl-2-benzofuran Chemical compound C1=CC=CC=C1C1=C2C=CC=CC2=C(C=2C=CC=CC=2)O1 ZKSVYBRJSMBDMV-UHFFFAOYSA-N 0.000 description 10
- 238000002428 photodynamic therapy Methods 0.000 description 10
- 230000008685 targeting Effects 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 8
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 7
- 239000002585 base Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 6
- 239000001509 sodium citrate Substances 0.000 description 6
- 238000012377 drug delivery Methods 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000002073 fluorescence micrograph Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 108010008707 Mucin-1 Proteins 0.000 description 3
- 238000001530 Raman microscopy Methods 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.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 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- HPZOOQSXPMEJBV-ODCFVKFUSA-N Tirilazad mesylate Chemical compound CS(O)(=O)=O.O=C([C@@H]1[C@@]2(C)CC=C3[C@@]4(C)C=CC(=O)C=C4CC[C@H]3[C@@H]2C[C@H]1C)CN(CC1)CCN1C(N=1)=CC(N2CCCC2)=NC=1N1CCCC1 HPZOOQSXPMEJBV-ODCFVKFUSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 238000010226 confocal imaging Methods 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 229960002918 doxorubicin hydrochloride Drugs 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000007306 functionalization reaction Methods 0.000 description 2
- 239000002539 nanocarrier Substances 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000000379 polymerizing effect Effects 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- PXEZTIWVRVSYOK-UHFFFAOYSA-N 2-(3,6-diacetyloxy-2,7-dichloro-9h-xanthen-9-yl)benzoic acid Chemical compound C1=2C=C(Cl)C(OC(=O)C)=CC=2OC2=CC(OC(C)=O)=C(Cl)C=C2C1C1=CC=CC=C1C(O)=O PXEZTIWVRVSYOK-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 238000003332 Raman imaging Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- QZPSXPBJTPJTSZ-UHFFFAOYSA-N aqua regia Chemical compound Cl.O[N+]([O-])=O QZPSXPBJTPJTSZ-UHFFFAOYSA-N 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000000701 chemical imaging Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000003749 cleanliness Effects 0.000 description 1
- 238000013267 controlled drug release Methods 0.000 description 1
- 238000011254 conventional chemotherapy Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 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
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- -1 sulfhydryl compound Chemical class 0.000 description 1
- 238000000479 surface-enhanced Raman spectrum Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- 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
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/18—Metal complexes
- C09K2211/188—Metal complexes of other metals not provided for in one of the previous groups
Abstract
本发明公开了一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法,包括金纳米粒子、DNA Y‑motif,DNA Y‑motif包括DNA S1、DNA S2和MUC1适体DNA S3,DNA序列S1分别与DNA序列S2和MUC1适体DNA S3碱基互补配对杂交成Y形结构,S1序列5'端的巯基连接在金纳米粒子表面构成AuNP Y‑motif,S2序列5'端修饰光敏剂,S2序列3'端修饰双模式成像基团且靠近金纳米粒子表面,通过调整双模式成像基团与金纳米粒子之间的距离,实现荧光‑表面增强拉曼散射双模式肿瘤细胞成像。其以适体为靶向,利用内源性物质DNA构建携带化疗药物及光敏剂的纳米探针,实现CT‑PDT的协同治疗。
Description
技术领域:
本发明属于肿瘤细胞协同治疗及光谱成像领域,具体涉及一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法,将化疗药物与光敏剂的共同递送实现了化疗(CT)与光动力治疗(PDT)的协同治疗及双光谱(荧光光谱、表面增强拉曼散射光谱)肿瘤细胞成像分析。
背景技术:
谷胱甘肽(GSH)是一种巯基化合物,在细胞中GSH含量的异常变化直接关系到一些疾病的产生,如肿瘤、神经疾病、艾滋病等。GSH已经成为公认的肿瘤细胞标志物,在许多恶性肿瘤中明显高于正常细胞。因此,对GSH的监测及成像研究是癌症早期诊断的迫切需要。GSH在癌细胞内含量为2-10mM,健康细胞为2-10μM,肿瘤微环境内GSH的含量大约是正常组织的100-1000倍,GSH水平的显著差异使得GSH应答的纳米载体成为靶向细胞内药物传递的首选。其中,二硫键作为典型的还原敏感键被广泛应用于肿瘤特异性刺激应答纳米载药探针的设计中。
作为一种非侵入性医疗技术,PDT已经成为一种可靠的癌症治疗方式。越来越多的研究旨在利用无害的光敏剂和可见光在PDT中消融恶性肿瘤。PDT还可引起急性炎症和白细胞浸润,从而促进免疫反应。PDT依赖于光敏剂(PSs)产生的活性氧(ROS),与常规化疗相比,PDT可提供额外的组织选择性,这是因为预先确定的PSs积累和光照放置的原因,治疗区域的大小取决于光照射区域的大小,确保对邻近健康组织的副作用较小。化疗药物有强烈的毒副作用,限制了它的应用。为了克服这些局限性,人们开发了一系列靶向能力好、治疗效率高、毒副作用小的靶向纳米平台,实现了肿瘤的特异性治疗,提高了疗效。适体、短肽和小分子,最近成为有前途的靶向配体设计新型药物传递***。由于它们能特异性识别肿瘤细胞中过表达的不同细胞表面靶点,因此,靶向给药控释***的构建对药物靶向供给在癌症治疗中的安全性至关重要。PDT有助于克服化疗药物的多药耐药。因此,一种既能实现光敏剂与化疗药物的有效协同递送,又能降低化疗的脱靶毒性的药物递送***在癌症治疗中具有很大的潜力。二氢卟吩E6(Chlorin e6,Ce6)作为传统的光敏剂常用与肿瘤的PDT治疗。
目前的光学传感器主要依靠单一的信号依赖性分析,容易受到一些物质的干扰。相比之下,基于双信号的光学传感器,如比率传感器,具有较高的精度。然而,更重要的是,使用不同类型信号的双模式光学传感器更有利于提高灵敏度和精度,具有相当大的应用前景。近年来,基于表面增强拉曼散射(SERS)和荧光的双信号光学传感器在检测领域得到了广泛的发展。例如,谭团队最近开发了基于Cy3和金纳米粒子的荧光-SERS开关纳米探针来监测细胞的动态行为和检测多种miRNAs。张与其同事制备了基于SERS和荧光信号的细胞色素C激活双模受体传感器,该传感器由DNA修饰的Au纳米三角形和Cy5修饰的互补链自组装而成。因此利用SESR技术与荧光技术构建大量的纳米平台策略,用来实现对肿瘤的诊疗一体化,提高了肿瘤检测的精准度,增强了肿瘤治疗效果。
发明内容:
基于上述情况,本发明目的在于设计一种基于DNA的纳米探针携带化疗药物及光敏剂以肿瘤微环境响应进行药物释放,将光敏剂与化疗药物有效协同递送,实现肿瘤的CT-PDT协同治疗,提高肿瘤治疗效率以及肿瘤细胞的成像分析。
为了实现上述目的,本发明涉及的用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针,包括金纳米粒子、DNA Y-motif,DNA Y-motif包括DNA S1、DNA S2和MUC1适体DNAS3,DNA序列S1分别与DNA序列S2和MUC1适体DNA S3碱基互补配对杂交成Y形结构,其中,S1序列为5'-SH-GCT ACG ATA ACG ACG AAA GGA TCA ACT G-3',S2序列为5'-CCA GGG AATCCA TCG TCG SSA TCG TAG C-3',S3序列为5'-ACA CGG CAG TTG ATC CTT TGG ATA CCCTGG CGT GT-3',S1序列5'端的巯基连接在金纳米粒子表面构成AuNP Y-motif,S2序列5'端修饰光敏剂,S2序列3'端修饰双模式成像基团且靠近金纳米粒子表面,通过调整双模式成像基团与金纳米粒子之间的距离,实现荧光-表面增强拉曼散射双模式肿瘤细胞成像。
所述用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针到达细胞表面时,MUC1适体与MUC1蛋白结合,促进了肿瘤细胞对纳米探针的内吞,进入细胞后,GSH将双硫键切断后,DNA-Y-motif完全解链释放光敏剂,实现肿瘤微环境响应的精准靶向治疗及光动力治疗。AuNPs-Y-motif探针中的双模式成像基团紧靠AuNPs会产生强烈的拉曼信号,荧光猝灭,当GSH切断双硫键时双模式成像基团会远离AuNPs使荧光信号恢复,即肿瘤细胞内GSH与AuNP-Y-motif探针作用引起Y-motif的构象变化至使双模式成像基团产生拉曼信号减弱荧光信号增强的变化,实现了肿瘤细胞的表面增强拉曼散射光谱成像分析。
具体地,所述双模式成像基团包括但不限于Cy3、Cy5和ROX中的一种。
具体地,所述光敏剂包括但不限于光敏剂二氢卟吩E6(Ce6)、亚甲基蓝或hemin。
进一步地,所述用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针,还包括化疗药物,所述化疗药物***到DNA Y-motif碱基对中。具体地,化疗药物包括但不限于阿霉素(DOX)。此时,探针在携带光敏剂的同时也携带化疗药物,通过共同递送光敏剂和化疗药物来实现内源性物质控制药物释放,实现肿瘤细胞的CT-PDT联合治疗。
本发明涉及的用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针的构建方法,具体包括以下步骤:
(1)DNA S2序列5'端修饰光敏剂,3'端修饰双模式成像基团,将DNA S1、DNA S2和MUC1适体DNA S3退火处理,然后在冰浴中冷却备用;
(2)冷却后的DNA S1与AuNPs振动混合,使DNA S1通过共价金-硫醇键固定在AuNPs表面,形成AuNPs-S1;
(3)将AuNPs-S1重新分散在PBS中,加入步骤(1)处理后的DNA S2以及mucin-1适体DNA S3,在室温下震荡,充分反应后,mucin-1适体DNA S3和DNA S2分别与DNA S1碱基互补配对杂交成Y形结构,得到探针AuNP-Y-motif;
(4)将化疗药物与AuNP-Y-motif混合,使化疗药物***到GC碱基对中。
相对于现有技术,本发明所述的GSH响应纳米探针用于肿瘤成像和靶向协同治疗具有以下优势:
(1)通过适体的靶向作用,利用内源性物质DNA构建携带化疗药物及光敏剂的纳米探针,实现了光敏剂与化疗药物的有效协同递送,提高肿瘤的靶向治疗效率,且该纳米探针制备简单、花费低。
(2)使用荧光、拉曼双模式光学信号对肿瘤细胞进行成像,更有利于提高灵敏度和精确度,克服了单信号策略中复杂环境的干扰。
附图说明:
图1为本发明的原理示意图。
图2为实施例1所述的AuNPs的透射电镜图(A)和探针的紫外光谱图(B)、动态光散射粒径图(C)、拉曼表征图(D)。
图3为实施例1所述的不同浓度的GSH(a-f:0,1,2,3,4,5mM)与AuNP-Y-motif探针响应释放DOX(A)及Cy3(B)的荧光光谱图。
图4为实施例1中不同照射时间下所述的AuNP-Y-motif探针的体外1O2产生检测图。
图5为实施例1所述的AuNP-Y-motif探针的细胞内1O2产生检测图,其中DCF表示DCF的荧光图,Bright表示白光图,Merge表示荧光图与白光图的叠加图。
图6为实施例1所述的细胞荧光成像图,其中Cy3表示Cy3的荧光图,DOX表示DOX的荧光图,Bright表示白光图,Merge表示荧光图与白光图的叠加图。
图7为实施例1所述的细胞拉曼成像图,其中,从左到右每一列分别为HeLa细胞的拉曼成像图,HeLa细胞的拉曼成像与细胞白光像的叠加图,细胞的白光像以及对应细胞内Cy3拉曼信号图。
图8为实施例1所述的探针对肿瘤细胞活性的检测图。
具体实施方式:
下面结合实施例及附图来详细说明本发明。
实施例1
本实施例中所述双模式成像基团采用Cy3,光敏剂采用Ce6。
1、金纳米颗粒的合成
取乙醇2L,再称量氢氧化钾(KOH)500g,将KOH溶于2L乙醇中,配成碱缸用于后续实验。每10天补加一定量的乙醇与KOH。本文以AuNPs作为载体,其合成方法采用传统的柠檬酸还原法制备(也称一步还原法),具有还原性的柠檬酸钠可以把氯金酸溶液中的Au3+还原成Au的单质,Au单质将会进一步的聚合生长,在柠檬酸钠的调控下(控制柠檬酸钠和氯金酸的体积比例)进而聚合成不同粒径的金纳米粒子。在制备过程中所有使用的玻璃器皿、磁子等先在碱缸中浸泡12h,然后清洗干净,在烘箱内烘干,再将烘干后的玻璃器皿用王水溶液中浸泡24h,清洗干净,然后烘干。这样可以有效的避免的外界因素导致的胶体不稳定性,保证了在接下来的AuNPs的合成不受污染,胶体状态更加稳定。先配制1%的HAuCl4溶液,再取1%的HAuCl4溶液1mL,定容至100mL容量瓶来获得0.01%的HAuCl4溶液,取柠檬酸钠用二次水溶解,获得1%的柠檬酸钠溶液。搭建回流装置,之后倒入0.01%的HAuCl4溶液,打开搅拌并加热,当溶液达到微沸状态时(有较大气泡产生)快速加入柠檬酸三钠溶液,此时观察溶液颜色,当逐渐变为酒红色时,为使溶液充分反应获得稳定的AuNPs溶液,持续加热搅拌回流30min,之后关闭加热,继续搅拌,使其自然恢复到室温,最后将冷却好的AuNPs溶液放冰箱4℃保存。最后,所有实验玻璃器皿都再放回碱缸中浸泡,以备下一次使用,保证其清洁度。
2、AuNP-Y-motif探针的制备
2.1信标的DNA序列,具体信息如表1。
表1实验使用DNA序列
S2的构建过程具体为:首先在S2序列5'端修饰Ce6,3'端修饰Cy3,通过优化碱基互补配对的链熔温度,在近Cy3一端***了GSH响应的双硫键,GSH响应后该DNA Y-motif可以解链释放药物及Cy3。
2.2AuNP-Y-motif探针的制备
为成功制备AuNP-Y-motif探针,首先将硫醇修饰DNA S1(1.0μM,50.0μL)在95℃水浴中退火5min,避免碱基错配,然后在冰浴中冷却30min后使用。冷却后的S1与1mL的AuNPs于混合10mL小烧杯中,在37℃振荡器中轻轻摇动约16h。16h后,在随后的盐老化过程中,将200.0μL 0.05M NaCl溶液缓慢均匀的滴加至混合物中,继续摇动6h,6h后再将200.0μL0.1M NaCl溶液缓慢均匀的滴加入混合物中再摇动6h,以增强探针的稳定性。二次加盐6h后,在10 000rpm下离心30min,然后再洗涤2次,去除游离多余的S1序列,至此,S1链通过共价金-硫醇键固定在AuNPs表面。随后,将洗涤2遍的沉淀重新分散在PBS中(0.1M,pH值7.4),然后DNA S2(1μM,50μL)以及mucin-1适体序列(S3,1μM,50μL)如上所述同样进行退火,将退火冷却后的S2、S3加入重新分散的AuNP-S1中,并在室温下震荡6h。6h后可获得探针AuNP-Y-motif,将该探针与2μL 10mM盐酸阿霉素可DOX混合,在室温下震荡过夜,这样盐酸阿霉素可以完全***到GC碱基对。如前所述,将混合物离心洗涤,然后重悬于PBS中,这样便获得了负载DOX及光敏剂Ce6的AuNP-Y-motif,用于后续实验。制备好的探针用拉曼检测Cy3信号来验证其制备成功。
3、AuNP-Y-motif探针的表征
用UV、TEM、DLS和拉曼对AuNP-Y-motif探针进行表征。如图2-A的TEM图像显了AuNPs的尺寸和形貌,AuNPs的平均直径约为25nm,呈球体状,分散均匀。DLS测量进一步表明,当表面修饰DNA Y-motif结构时,AuNPs的水合粒径直径增大(图2-B)。UV-vis进一步显示功能化导致AuNPs的紫外吸收产生轻微红移(图2-C)。AuNPs经核酸链修饰后在526nm处的特征吸收红移至529nm,这是因为功能化改变了AuNPs周围环境的介电常数造成的。Cy3分子是一种拉曼信号分子,由于Cy3分子接近AuNPs,因此可以检测到强烈的Cy3SERS信号。SERS光谱在1586cm-1(Cy3的特征峰)处显示出较强的拉曼信号(图2-D)。以上所有这些表征结果都证实了AuNP-Y-motif探针的成功制备。
4、AuNP-Y-motif探针与GSH响应的荧光分析
准确配制不同浓度的GSH溶液(0,1,2,3,4,5mM),使其与AuNP-Y-motif探针反应6h。反应结束后,将不同浓度的GSH与AuNP-Y-motif探针混合溶液离心(10000rpm,30min),测定上清液中Cy3和DOX的荧光强度。
然后,我们对不同浓度的GSH与AuNP-Y-motif反应的药物释放行为进行了探究,如图3-A、B所示,在上清液中可以成功的检测到DOX和Cy3的荧光信号,GSH浓度在0–5mM的范围内,DOX和Cy3的荧光强度随着GSH浓度的增加有显著的上升,由此可以得出GSH浓度越高,DOX的释放量越大。
5、HeLa的细胞培养
HeLa细胞使用DMEM培养液(10%的牛血清白蛋白、1%的青霉素链霉素)培养的,在37℃下含有5%的二氧化碳的100%的湿润气体中的培养箱中进行培养。
6、AuNP-Y-motif探针的体外1O2产生检测
用1,3-二苯异苯呋喃(DPBF)测定了体内AuNP-Y-motif的1O2生成能力,将50μLAuNP-Y-motif加入50μg/mL DPBF溶液中。混合物经650nm灯照射不同时间。照射后,在403nm激发波长下记录485nm处的荧光强度。以不含AuNP-Y-motif的DPBF溶液为对照。
如图4-A、B所示,DPBF、DPBF+光照、DPBF+AuNP-Y-motif,都未能引起DPBF的荧光减弱,而AuNP-Y-motif与DPBF在650nm的激光照射下,随着激光照射时间从0到20min的延长,DPBF的荧光强度迅速下降,说明在光照下有ROS生成至使DPBF被氧化,其荧光强度随之减弱。结果表明,AuNP-Y-motif纳米探针在体外溶液中具有良好的生成1O2的能力。
DCFH-DA是一种细胞内ROS-sensitive探针,可以特异性被细胞内产生的1O2氧化,转变为具有强荧光信号的2,7-二氯荧光素(DCF)。采用2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)检测细胞内1O2生成能力。将AuNP-Y-motif与HeLa细胞孵育。培养4h后,清洗细胞2次,再取10μM DCFH-DA与HeLa细胞共孵育30min,清洗细胞2次。然后,在650nm波长下,照射培养皿细胞20min。未加入AuNP-Y-motif、未光照的培养皿细胞作为对照组。20min后在共聚焦激光扫描显微镜上,激发波长为488nm,发射波长采集范围为510~540nm。
如图5所示,只有DCFH-DA的HeLa细胞(5a)中未见DCF的荧光,有DCFH-DA且激光照射的HeLa细胞(5b)中DCF荧光可以忽略,不使用激光照射的DCFH-DA和AuNP-Y-motif处理的HeLa细胞(5c)可以观察到轻微的DCF荧光。相比之下,用DCFH-DA与AuNP-Y-motif孵育的HeLa细胞经激光照射(5d)后显示出强烈的绿色荧光,证实在癌细胞中有效生成1O2。
7、HeLa细胞的荧光成像
将HeLa细胞分入皿中培养24h,然后将20μL AuNP-Y-motif溶液与1mL培养液混合再加入到共聚焦培养皿中培养。37℃孵育不同时间后,用PBS洗涤3次,以减少背景荧光。然后进行共聚焦成像,激发光为514nm,从带通滤波器560~590nm处采集Cy3的荧光发射,用激发波长488nm,采集波长范围为590~650nm处采集DOX的荧光发射。
研究了肿瘤细胞中AuNP-Y-motif靶向药物释放的能力,对HeLa细胞进行了荧光共聚焦成像分析。如图6所示,AuNP-Y-motif与细胞孵1h后,在GSH的作用下,在细胞质中成功检测了Cy3和微弱的DOX的荧光信号。证实DOX可成功的从AuNP-Y-motif中被释放出来,随着培养时间的延长,进入肿瘤细胞的AuNP-Y-motif探针含量逐渐升高,Cy3和DOX的荧光强度也逐渐增强。在孵育了6h后,可以观察到在细胞核内出现明显的红色荧光,这表明DOX在细胞核中积累,从而与染色体结合杀死癌细胞。这表明了,DOX被成功封装在AuNP-Y-motif探针中,并实现了肿瘤细胞内GSH控制的药物释放。总之,GSH刺激响应纳米平台的开发实现了在肿瘤细胞内激活成像和药物控制释放。
8、HeLa细胞的拉曼成像
将HeLa细胞接种于SERS基底金片上,在培养箱内培养24h,然后用PBS清洗一遍,将AuNP-Y-motif探针与HeLa细胞孵育不同时间(1h、2h、3h)。随后,利用PBS去除游离的AuNP-Y-motif,在雷尼绍Invia拉曼显微镜上进行成像,50×物镜,10%的激光强度,用633nm的HeNe激光器,曝光时间为5s,拉曼信号的检测范围从1700cm-1到600cm-1范围收集。
图7中可以看到,在1h时可以在HeLa细胞中观察到强烈的Cy3的SERS信号,随着时间的推移Cy3的拉曼信号逐渐削弱。结果表明,由于MUC1的靶向作用,AuNP-Y-motif探针经内吞作用进入细胞,在最初拉曼信号最强,随着时间增加,大量探针与GSH作用,至使Cy3标记的寡核苷酸从AuNP-Y-motif上释放,拉曼信号逐渐降低。
9、AuNP-Y-motif探针的治疗研究
用CCK-8法测定AuNP-Y-motif探针对HeLa细胞和HEK293T细胞的细胞活性。将2种细胞接种在96孔板中,并生长24h。然后用不同浓度的AuNP-Y-motif(0.1-0.9nM)孵育16h后,用PBS洗涤细胞,在650nm波长下,照射20min。未经激光照射的细胞作为对照。之后,每一个小孔加10μL CCK-8溶液,孵育3.5h,用酶标仪测定每孔在450nm处的光密度(OD)。为了进一步研究不同的治疗方法的效果,将HeLa细胞与游离的DOX、游离的Ce6、仅装载DOX的AuNP-Y-motif(AuNP-Y-motif/DOX)、仅装载Ce6的AuNP-Y-motif(AuNP-Y-motif/Ce6)和AuNP-Y-motif(装载两种药物)孵育,然后通过上述处理对细胞进行测量。细胞存活率按细胞毒性检测试剂盒描述计算。
用CCK-8细胞活性实验探究了AuNP-Y-motif的CT-PDT对肿瘤细胞的协同治疗作用。首先,探索了特定的细胞靶向肿瘤治疗能力,选择过表达MUC1受体的HeLa细胞作为靶模型,以HEK293T细胞为对照组,评估AuNP-Y-motif副作用及治疗作用。如图8-A、B所示,AuNP-Y-motif(不含药物)对HeLa细胞和HEK293T细胞的毒性可以忽略,表明AuNP-Y-motif具有良好的生物相容性和无毒性。然而,随着AuNP-Y-motif(负载药物)浓度的增加和激光照射20min,HeLa细胞的活力显著下降。在相同条件下,与对照组相比,非靶标HEK293T细胞的存活率较高,说明AuNP-Y-motif对HeLa细胞有一定的毒性作用,并实现了药物的特异性释放。同时,这也解释了非靶向HEK293T细胞对AuNP-Y-motif的摄取效率较低,而AuNP-Y-motif对靶向的HeLa细胞具有良好的选择性,这归因于HEK293T细胞MUC1受体的低表达。
在HeLa细胞内进一步测试不同药物治疗的细胞毒性,如图8-C相对细胞活性显示,AuNP-Y-motif装载DOX与Ce6组(8C-e)的化疗-PDT协同治疗作用对肿瘤细胞生长的抑制作用明显高于对照组(Control)、游离的Ce6组(8C-a)、游离DOX组(8C-b)、AuNP-Y-motif只装载DOX组(8C-c)、AuNP-Y-motif只装载Ce6组(8C-d)。该结果表明具有靶向性的纳米载体的两种治疗作用要远优于非靶向的无载体的单一药物治疗策略。
序列表
<110> 青岛科技大学
<120> 一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法
<130> 2021
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
shgctacgat aacgacgaaa ggatcaactg 30
<210> 2
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccagggaatc catcgtcgss atcgtagc 28
<210> 3
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
acacggcagt tgatcctttg gataccctgg cgtgt 35
Claims (4)
1.一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针,其特征在于,包括金纳米粒子、DNA Y-motif和阿霉素,DNA Y-motif包括DNA S1、DNA S2和MUC1适体DNA S3,DNAS1序列分别与DNA S2序列和MUC1适体DNA S3序列碱基互补配对杂交成Y形结构,其中,DNAS1序列为5'-SH-GCT ACG ATA ACG ACG AAA GGA TCA ACT G-3',DNA S2序列为5' -CCAGGG AAT CCA TCG TCG SSA TCG TAG C -3',DNA S3序列为5'- ACA CGG CAG TTG ATC CTTTGG ATA CCC TGG CGT GT -3',DNA Y-motif通过DNA S1序列5'端的巯基连接在金纳米粒子表面构成AuNP-Y-motif,DNA S2序列5'端修饰光敏剂,DNA S2序列3'端修饰双模式成像基团且靠近金纳米粒子表面,通过调整双模式成像基团与金纳米粒子之间的距离,实现荧光-表面增强拉曼散射双模式肿瘤细胞成像;阿霉素***到AuNP-Y-motif中的GC碱基对中。
2.根据权利要求1所述的用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针,其特征在于,所述双模式成像基团为Cy3、Cy5和ROX中的一种。
3.根据权利要求2所述的用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针,其特征在于,所述光敏剂为二氢卟吩E6、亚甲基蓝或hemin。
4.一种权利要求3所述用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针的构建方法,具体包括以下步骤:
(1)DNA S2序列5'端修饰光敏剂,3'端修饰双模式成像基团,将DNA S1、DNA S2和MUC1适体DNA S3退火处理,然后在冰浴中冷却备用;
(2)冷却后的DNA S1与金纳米粒子振动混合,使DNA S1通过共价金-硫醇键固定在金纳米粒子表面,形成AuNP-S1;
(3)将AuNP-S1重新分散在PBS中,加入步骤(1)处理后的DNA S2以及MUC1 适体DNA S3,在室温下震荡,充分反应后,MUC1适体DNA S3和DNA S2分别与DNA S1碱基互补配对杂交成Y形结构,得到AuNP-Y-motif;
(4)将阿霉素与AuNP-Y-motif混合,使阿霉素***到AuNP-Y-motif中的GC碱基对中。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110638001.9A CN113372904B (zh) | 2021-06-08 | 2021-06-08 | 一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110638001.9A CN113372904B (zh) | 2021-06-08 | 2021-06-08 | 一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113372904A CN113372904A (zh) | 2021-09-10 |
CN113372904B true CN113372904B (zh) | 2022-08-23 |
Family
ID=77576598
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110638001.9A Active CN113372904B (zh) | 2021-06-08 | 2021-06-08 | 一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113372904B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114574591B (zh) * | 2022-05-06 | 2022-07-29 | 南京邮电大学 | 一种癌症诊疗一体化纳米试剂及其制备方法和应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009075817A1 (en) * | 2007-12-06 | 2009-06-18 | Minerva Biotechnologies Corporation | Method for treating cancer using interference rna |
EP2858630A4 (en) * | 2012-06-07 | 2016-02-24 | Harvard College | NANOTHERAPEUTIC PRODUCTS FOR DRUG TARGETING |
CN106932371B (zh) * | 2017-03-09 | 2020-03-10 | 青岛科技大学 | 一种细胞内谷胱甘肽的荧光成像方法 |
CN111643673B (zh) * | 2020-07-08 | 2022-09-13 | 福建医科大学孟超肝胆医院(福州市传染病医院) | 一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物及其应用 |
-
2021
- 2021-06-08 CN CN202110638001.9A patent/CN113372904B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN113372904A (zh) | 2021-09-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Cell membrane-coated porphyrin metal–organic frameworks for cancer cell targeting and O2-evolving photodynamic therapy | |
Fan et al. | Intranuclear biophotonics by smart design of nuclear-targeting photo-/radio-sensitizers co-loaded upconversion nanoparticles | |
KR102081666B1 (ko) | 암 치료용 약학 조성물 | |
Zhang et al. | pH-driven targeting nanoprobe with dual-responsive drug release for persistent luminescence imaging and chemotherapy of tumor | |
CN111150853B (zh) | 一种肿瘤联合治疗药物载体的制备及应用 | |
Zhao et al. | Multifunctional magnetic nanoparticles for simultaneous cancer near-infrared imaging and targeting photodynamic therapy | |
JP7055881B2 (ja) | 新規光増感剤複合ナノ多機能材料の調製及びその使用 | |
US11369683B2 (en) | Systems and methods for targeted imaging and ablation of cardiac cells | |
Liang et al. | A supramolecular nanovehicle toward systematic, targeted cancer and tumor therapy | |
CN104395471B (zh) | Ided纳米结构在核酸技术中的用途 | |
CN113372904B (zh) | 一种用于肿瘤成像和靶向协同治疗的谷胱甘肽响应纳米探针及其构建方法 | |
Zhang et al. | Research progress in the synthesis and biological application of quantum dots | |
Appidi et al. | A plasmon-enhanced fluorescent gold coated novel lipo-polymeric hybrid nanosystem: synthesis, characterization and application for imaging and photothermal therapy of breast cancer | |
Zeng et al. | Highly efficient Chemo/Photothermal therapy alleviating tumor hypoxia against cancer and attenuate liver metastasis in vivo | |
CN112826943B (zh) | 一种蛋白纳米载体、负载有靶向物质的载体及制备方法和应用 | |
Yang et al. | Photocontrolled chondrogenic differentiation and long-term tracking of mesenchymal stem cells in vivo by upconversion nanoparticles | |
Wu et al. | A transformable gold nanocluster aggregate-based synergistic strategy for potentiated radiation/gene cancer therapy | |
Adriouach et al. | Squalene-PEG: Pyropheophorbide-a nanoconstructs for tumor theranostics | |
Kang et al. | Applications of nanocomposites based on zeolitic imidazolate framework-8 in photodynamic and synergistic anti-tumor therapy | |
Zhao et al. | Specific photothermal killing of cancer cells by RNase-conjugated glyco-gold nanoparticles | |
CN110917349A (zh) | 一种碗状isp复合功能性纳米粒子及其制备方法和应用 | |
EP4101471A1 (en) | Nanoparticles for cancer treatment | |
CN113117078B (zh) | 一种肿瘤治疗新药物AuNCs@GTTN及其制备方法和应用 | |
Eshghi et al. | Synthesis and characterisation of new designed protoporphyrin-stabilised gold nanoparticles for cancer cells nanotechnology-based targeting | |
CN109289048B (zh) | 一种肿瘤血管阻断协同光治疗试剂及其合成方法与应用 |
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 |