CN117883596A - T of hypoxia response 1 -T 2 Switchable MRI contrast agent, preparation method and application thereof - Google Patents
T of hypoxia response 1 -T 2 Switchable MRI contrast agent, preparation method and application thereof Download PDFInfo
- Publication number
- CN117883596A CN117883596A CN202211228073.7A CN202211228073A CN117883596A CN 117883596 A CN117883596 A CN 117883596A CN 202211228073 A CN202211228073 A CN 202211228073A CN 117883596 A CN117883596 A CN 117883596A
- Authority
- CN
- China
- Prior art keywords
- contrast agent
- hypoxia
- mri contrast
- nitroimidazole
- reaction
- 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.)
- Pending
Links
- 206010021143 Hypoxia Diseases 0.000 title claims abstract description 119
- 230000007954 hypoxia Effects 0.000 title claims abstract description 113
- 239000002616 MRI contrast agent Substances 0.000 title claims abstract description 101
- 230000004044 response Effects 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 76
- 229920002125 Sokalan® Polymers 0.000 claims abstract description 59
- 239000002105 nanoparticle Substances 0.000 claims abstract description 57
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims abstract description 49
- 150000003384 small molecules Chemical class 0.000 claims abstract description 41
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000004584 polyacrylic acid Substances 0.000 claims abstract description 39
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 35
- 235000018417 cysteine Nutrition 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 26
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims abstract description 9
- 239000002872 contrast media Substances 0.000 claims description 64
- 238000006243 chemical reaction Methods 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 49
- -1 2-nitroimidazole small molecule Chemical class 0.000 claims description 37
- 238000003384 imaging method Methods 0.000 claims description 35
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 33
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 18
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 claims description 16
- 229940031182 nanoparticles iron oxide Drugs 0.000 claims description 16
- YZEUHQHUFTYLPH-UHFFFAOYSA-N 2-nitroimidazole Chemical compound [O-][N+](=O)C1=NC=CN1 YZEUHQHUFTYLPH-UHFFFAOYSA-N 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 229910052742 iron Inorganic materials 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 12
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 11
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 11
- SYFMSLVOHQZYEB-UHFFFAOYSA-N 2-nitro-1-(oxiran-2-ylmethyl)imidazole Chemical compound [O-][N+](=O)C1=NC=CN1CC1OC1 SYFMSLVOHQZYEB-UHFFFAOYSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 9
- 238000000502 dialysis Methods 0.000 claims description 8
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 claims description 8
- LTEKQAPRXFBRNN-UHFFFAOYSA-N piperidin-4-ylmethanamine Chemical compound NCC1CCNCC1 LTEKQAPRXFBRNN-UHFFFAOYSA-N 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- 239000007987 MES buffer Substances 0.000 claims description 6
- 239000013067 intermediate product Substances 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 230000020874 response to hypoxia Effects 0.000 claims description 3
- 239000012298 atmosphere Substances 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 2
- 125000000524 functional group Chemical group 0.000 claims description 2
- 150000002505 iron Chemical class 0.000 claims description 2
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical group O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical group O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000002243 precursor Substances 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 15
- 230000002776 aggregation Effects 0.000 abstract description 13
- 238000004220 aggregation Methods 0.000 abstract description 13
- 230000001146 hypoxic effect Effects 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000011534 incubation Methods 0.000 description 38
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 32
- 102000004459 Nitroreductase Human genes 0.000 description 32
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 32
- 108020001162 nitroreductase Proteins 0.000 description 32
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 31
- 238000000338 in vitro Methods 0.000 description 29
- 238000002595 magnetic resonance imaging Methods 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 26
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 18
- 210000001519 tissue Anatomy 0.000 description 18
- 238000000108 ultra-filtration Methods 0.000 description 17
- 241000699670 Mus sp. Species 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 13
- 238000003786 synthesis reaction Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000011056 performance test Methods 0.000 description 9
- 230000008859 change Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 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 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 7
- 230000005540 biological transmission Effects 0.000 description 7
- 239000007810 chemical reaction solvent Substances 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 230000005291 magnetic effect Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 210000003462 vein Anatomy 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000011725 BALB/c mouse Methods 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 230000003213 activating effect Effects 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 239000012074 organic phase Substances 0.000 description 6
- 238000010992 reflux Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 4
- 210000002889 endothelial cell Anatomy 0.000 description 4
- 229960003180 glutathione Drugs 0.000 description 4
- 230000003278 mimic effect Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000012429 reaction media Substances 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000003606 umbilical vein Anatomy 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229910052688 Gadolinium Inorganic materials 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000002059 diagnostic imaging Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052762 osmium Inorganic materials 0.000 description 2
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000002390 rotary evaporation Methods 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
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000002344 surface layer Substances 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- KUNMIWQQOPACSS-UHFFFAOYSA-N 1-nitroimidazole Chemical compound [O-][N+](=O)N1C=CN=C1 KUNMIWQQOPACSS-UHFFFAOYSA-N 0.000 description 1
- JFDMLXYWGLECEY-UHFFFAOYSA-N 2-benzyloxirane Chemical compound C=1C=CC=CC=1CC1CO1 JFDMLXYWGLECEY-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 101710141544 Allatotropin-related peptide Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001808 coupling effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000002405 nuclear magnetic resonance imaging agent Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000005426 pharmaceutical component Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- BITYAPCSNKJESK-UHFFFAOYSA-N potassiosodium Chemical compound [Na].[K] BITYAPCSNKJESK-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- 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
-
- 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
- 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
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
-
- 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
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Nanotechnology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a T with hypoxia response 1 ‑T 2 Switchable MRI contrast agents, methods of making and uses thereof. The T is 1 ‑T 2 The switchable MRI contrast agent comprises ferric oxide nanoparticles of which the surfaces are modified with polyacrylic acid, and hypoxia sensitive small molecules and cysteine which are modified on the surfaces of the ferric oxide nanoparticles in a connecting way, so that the response aggregation capability of the tiny ferric oxide nanoparticles to hypoxia is endowed. The MRI contrast agent provided by the invention takes the tiny ferric oxide nano particles as the carrier, can specifically respond and initiate aggregation in the hypoxic region of the tumor microenvironment, and realizes T in the tumor region 1 Contrast signal to T 2 The contrast signal is switched, so that the interference of a background signal can be obviously reduced, the high-accuracy diagnosis of tumor tissues is realized, and the method has the advantages of high sensitivity, selectivity, good biocompatibility and the like in the tumor diagnosis; the preparation method is simple, free of danger and universal.
Description
Technical Field
The invention relates to a novel MRI diagnostic contrast agent, in particular to a T with hypoxia response 1 -T 2 Switchable MRI contrast agent, specific preparation method and application thereof, such as detection of various tumors and related detection of early diagnosis of tumors, belonging to medicineThe technical field of preparation of the agent.
Background
Cancer is one of the major causes of death in humans. The World Health Organization (WHO) has shown in 2019 that cancer occupies the first or second of the cause leaderboards in more than 2/3 of the countries as a result of statistics of cause of death of the global population before 70 years of age. How to realize early and accurate diagnosis of cancer becomes a key place for treating cancer. MRI is one of the most widely used image diagnosis means in clinic, and has the advantages of no ionizing radiation, no penetration depth limitation, high spatial resolution and the like because of being a non-invasive imaging mode, and plays an extremely important role in clinical tumor diagnosis at present. The current clinical common MRI contrast agent is T 1 Gadolinium-based small molecules of the type. However, the small molecular MRI contrast agent has the problems of short blood circulation time, lack of specificity to tumor tissues, potential toxicity of gadolinium ions and the like, so that the small molecular MRI contrast agent is difficult to safely realize high-efficiency diagnosis of the tumor tissues. There is therefore an urgent need to develop new generation of safe and efficient T 1 The type MRI contrast agent is used for accurate diagnosis of cancer.
Iron Oxide Nanoparticles (IONPs) have been extensively studied for use as T 2 MRI contrast agents of the type and exhibiting long blood circulation times, low biotoxicity and variable modification of the surface, as such, several IONPs are certified by the United states Food and Drug Administration (FDA) to be useful as T 2 MRI contrast medium, when IONPs are smaller than 4nm, the ratio of the disordered magnetic area of the surface layer of the nano-particle is larger than that of the magnetic area of the inner core, the inner core can only generate weak magnetic moment, and the surface layer contains a large amount of paramagnetic Fe with unpaired electrons 3+ Plays the leading role and overall exhibits T 1 Contrast enhancement effect. The IONPs at this time are called Extremely Small Iron Oxide Nanoparticles (ESIONPs), which are different from T 2 Traditional IONPs as a new generation T for MRI contrast agents 1 A type MRI contrast agent. At present, ESIONPs with the thickness of 3nm are synthesized, and after PEG layers are modified on the surfaces, the relaxation rate r of the ESIONPs is higher than that of the PEG layers 1 Even exceeds the commercial Gd-DTPA to 4.77mM -1 s -1 But is still unavoidable somewhat faster due to the too small sizeDefects in rapid metabolism, and lack of specificity in imaging. Based on magnetic coupling effect, the ESIONPs can realize T when changing from a single particle dispersion state to a multiparticulate aggregation state 1 To T 2 The transition of the type MRI contrast agent, i.e. the realization of a bright to dark contrast enhanced signal switching, greatly increases the specificity of the diagnosis, and as the particle size becomes larger after aggregation, the enrichment of the contrast agent at the tumor is greatly increased, which not only greatly prolongs the imaging window time, but also reduces the amount of contrast agent.
For tumor tissue, some physiological parameters in the tumor microenvironment, including pH, glutathione (GSH), reactive Oxygen Species (ROS), hypoxia, and enzymes, etc., are often different from normal tissues and cells. Among them, the extracellular weak acid environment (pH-6.5-7.0) and the intracellular high GSH concentration (GSH-0.5-10 mM) in tumor tissues are the most common characteristics, and researchers have developed a variety of intelligent response drug delivery and image diagnosis systems for tumor tissues by utilizing the characteristics of the two tumor microenvironments. Some researchers modify slightly acid-sensitive polymers on the surfaces of very small ferric oxide particles so as to realize the contrast agent from T under the action of weak acid in tumor microenvironment 1 Weighting MRI signals to T 2 Weighted MRI signal transitions through this tumor site-specific T 1 -T 2 The interference of the background signal can be greatly reduced. However, most of these very small iron oxide particles are synthesized by a high temperature pyrolysis method, which is complicated in steps, and has a certain risk at a high temperature of 200-300 , and most of these pH or GSH responsive aggregated polymers are synthesized by ATRP or RAFT, which is not a green synthesis means, and suffers from the drawbacks of complicated steps and low yield.
Disclosure of Invention
The main purpose of the invention is to provide a T with high sensitivity, simple preparation and green hypoxia response 1 -T 2 A switchable MRI contrast agent and a preparation method thereof, which overcome the defects of the prior art.
Another object of the invention is to provide a hypoxia responseT of (2) 1 -T 2 Use of switchable MRI contrast agents.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a T with hypoxia response 1 -T 2 A switchable MRI contrast agent comprising:
iron oxide nanoparticles of surface-modified polyacrylic acid;
and connecting the hypoxia-sensitive small molecule and cysteine modified on the surface of the ferric oxide nano particle.
In some preferred embodiments, the hypoxia sensitive small molecule comprises a 2-nitroimidazole small molecule.
In some preferred embodiments, the T 1 -T 2 Switchable MRI contrast agents can be specifically aggregated in response to hypoxic environments within tumor microenvironments to achieve T 1 Contrast agent to T 2 Switching of the type contrast agent.
The embodiment of the invention also provides a T of the hypoxia response 1 -T 2 A method of preparing a switchable MRI contrast agent comprising:
providing monodisperse tiny ferric oxide nano particles, and modifying polyacrylic acid on the surface of the nano particles to prepare the ferric oxide nano particles with the surface modified polyacrylic acid;
Connecting a hypoxia sensitive small molecule and cysteine on the surface of the ferric oxide nano particle to prepare the hypoxia response T 1 -T 2 Switchable MRI contrast agents.
The embodiment of the invention also provides the T of the hypoxia response 1 -T 2 Use of a switchable MRI contrast agent for the preparation of a product with tumor detection function.
Further, an embodiment of the present invention provides a composition for contrast, including: t of the aforementioned hypoxia response 1 -T 2 A switchable MRI contrast agent; and, pharmaceutically acceptable adjuvants.
Further, an embodiment of the present invention provides a non-medical imaging method, which includes: to be manufacturedThe shadow subject takes the T of the hypoxia response 1 -T 2 A switchable MRI contrast agent or contrast composition, and performing contrast.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides T of hypoxia response 1 The T2 switchable MRI contrast agent takes the tiny ferric oxide nano particles as the carrier, can specifically respond and initiate aggregation in the hypoxia region of the tumor microenvironment, and realizes T in the tumor region 1 Contrast signal to T 2 The contrast signal is switched, so that the interference of a background signal can be obviously reduced, the high-accuracy diagnosis of tumor tissues is realized, and the method has the advantages of high sensitivity, selectivity, good biocompatibility and the like in tumor diagnosis. The enrichment of the nano particles in the tumor area is further increased along with the size enlargement, the blood reflux perfusion is reduced, and the imaging window time is further prolonged; the preparation method is simple, free of danger and universal.
Description of the drawings:
in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.
FIGS. 1a and 1b are T of the hypoxia response in an exemplary embodiment of the invention 1 -T 2 Synthesis of switchable MRI contrast agents and tumor hypoxia induced nanoparticle contrast agent aggregation to achieve T 1 -T 2 Schematic application diagram and synthetic diagram of signal switching.
FIG. 2 is a transmission electron microscope image of ESIONPs-PAA nanoparticles in example 1 of the present invention.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of a small molecule 1 synthesized in example 1 of the present invention.
FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of a small molecule 2 (i.e., a 2-nitroimidazole small molecule) synthesized in example 1 of the present invention.
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of a small molecule 3 synthesized in comparative example 1 of the present invention.
FIG. 6a is a transmission electron micrograph of a hypoxia-responsive MRI contrast agent (HR-ESIONPs) of example 1 of the present invention, and FIG. 6b is a transmission electron micrograph of HR-ESIONPs after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase for 4h in vitro simulated hypoxia conditions.
FIG. 7 is the longitudinal relaxation rate (r) of the MRI contrast agent (HR-ESIONPs) of example 1 and the MRI contrast agent (NHR-ESIONPs) of comparative example 1 before and after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro under simulated hypoxia conditions for 4 hours 1 ) And transverse relaxation rate (r) 2 ) Is a comparison of the figures.
FIG. 8 shows the results of the incubation of MRI contrast agent (HR-ESIONPs) with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in example 1 of the present invention before and after 4h of incubation under in vitro simulated hypoxia conditions 1 Weighted solution imaging map.
FIG. 9 shows the results of the incubation of MRI contrast agent (HR-ESIONPs) with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in example 1 of the present invention before and after 4h of incubation under in vitro simulated hypoxia conditions 2 Weighted solution imaging map.
FIG. 10 is the reciprocal transverse relaxation time (1/T) of MRI contrast agent (HR-ESIONPs) incubated with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase under in vitro simulated hypoxia conditions in example 1 of the present invention 2 ) Graph of change over time.
FIG. 11 is a T of an MRI contrast agent (NHR-ESIONPs) of comparative example 1 of the present invention before and after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro to mimic hypoxia conditions for 4 hours 1 Weighted solution imaging map.
FIG. 12 is a graph showing the in vitro simulation of the hypoxic conditions of the MRI contrast agent (NHR-ESIONPs) and reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in comparative example 1 of the present inventionT before incubation and after incubation for 4h 2 Weighted solution imaging map.
FIG. 13 is a graph showing cytotoxicity test of MRI contrast agent (HR-ESIONPs) in Human Umbilical Vein Endothelial Cells (HUVEC) and mouse breast cancer cells (4T 1) in example 1 of the present invention.
FIG. 14 is a graph of tissue toxicity test of MRI contrast agents (HR-ESIONPs) in BALB/c mice in example 1 of the present invention.
FIG. 15 shows the in vivo T-results of the MRI contrast agent (HR-ESIONPs) of example 1 and the MRI contrast agent (NHR-ESIONPs) of comparative example 1 in tumor-bearing mice transplanted with 4T1 cells of the present invention 2 Weighted MRI imaging images.
FIGS. 16a and 16b are in vivo T of MRI contrast agent (HR-ESIONPs) of example 1 and MRI contrast agent (NHR-ESIONPs) of comparative example 1 of the present invention in tumor-bearing mice transplanted with 4T1 cells 2 Quantitative average signal intensity analysis plot after weighted MRI imaging.
FIG. 17a is a biological transmission electron microscopy image of tumor tissue after injection of MRI contrast agent (HR-ESIONPs) into a 4T1 tumor-bearing mouse by tail vein for 6h in example 1 of the present invention; FIG. 17b is a biological transmission electron microscopy image of tumor tissue after injection of MRI contrast agent (NHR-ESIONPs) into 4T1 tumor-bearing mice by tail vein for 6h in example 1 of the present invention.
Detailed Description
Aiming at a plurality of defects in the prior art, the inventor of the present invention can put forward the technical proposal of the invention through long-term research and a large number of practices. The technical scheme, the implementation process, the principle and the like are further explained as follows. It should be understood, however, that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described in the following (embodiments) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
As one aspect of the technical scheme of the invention, the invention relates to a T applied to high-sensitivity hypoxia response 1 -T 2 A switchable MRI contrast agent comprising:
as core tiny ferric oxide nano particles, a large amount of hydrophilic polyacrylic acid (PAA) is modified on the surface, so that huge chemical modification potential is provided;
and, ligating a hypoxia sensitive small molecule and a cysteine (Cys) modified to the outer layer of the iron oxide nanoparticle, thereby imparting the response aggregation capability of the very small iron oxide nanoparticle to hypoxia.
In some preferred embodiments, the T 1 -T 2 The switchable MRI contrast agent contains 0.5-1.0wt% of hypoxia sensitive small molecules and 21.5-30.0wt% of cysteine.
In some preferred embodiments, the source of the hypoxia-sensitive small molecule is a 2-nitroimidazole small molecule or the like, but is not limited thereto.
Further, the molar ratio of cysteine to hypoxia sensitive small molecule is greater than 20:1.
further, the polyacrylic acid has an average number average molecular weight of 2000.
In some preferred embodiments, the very small iron oxide nanoparticles have a particle size of less than 5nm. The synthesis of the extremely small ferric oxide nano particles is simple, the extremely small ferric oxide nano particles can be directly finished in a water phase, the synthesized nano particles have extremely good particle size uniformity, and as the extremely small ferric oxide nano particles with uniform particle size grow in the synthesis process, a layer of PAA is coated on the surface, the extremely good water solubility and biocompatibility of the extremely small ferric oxide nano particles are provided, a large number of carboxyl groups are reserved, the further potential of further modification of the extremely small ferric oxide nano particles is further provided, and the extremely small ferric oxide nano particles have important significance for subsequent research.
Further, the initial state of the very small iron oxide nanoparticles should be monodisperse.
Further, the iron oxide nanoparticle should be surface-modified with more than 50wt% of functional groups that can be re-modified, for example, preferably at least any one of carboxyl groups, amino groups, and the like.
In some preferred embodiments, the T 1 -T 2 Switchable MRI contrast agents capable of specifically responding to hypoxic conditions within a tumor microenvironmentAggregation of T 1 Contrast agent to T 2 Switching of the type contrast agent.
In some preferred embodiments, the MRI contrast agent of the present invention is first monodisperse very small iron oxide nanoparticles, which are themselves T 1 The surface of the contrast agent is provided with rich carboxyl groups, so that the contrast agent further modifies the capability of hypoxia-sensitive 2-nitroimidazole micromolecule and cysteine (Cys), and the capability of responding to aggregation is provided, and finally the MRI contrast agent (HR-ESIONPs) can specifically respond to the tumor hypoxia environment to aggregate, thereby realizing the MRI signal from T 1 To T 2 As particularly shown in fig. 1 b.
More specifically, referring to FIG. 1a, an embodiment of the present invention provides a T of hypoxia response 1 -T 2 Switchable MRI contrast agent mainly comprising tiny ferric oxide particles as a carrier and used as a T 1 The contrast agent has the advantages of rapid renal clearance, easy water dissolution and good biocompatibility due to the ultra-small size and PAA protection of the outer layer after synthesis, and is prepared by sequentially connecting a certain amount of cysteine (Cys) and 2-nitroimidazole hypoxia-sensitive small molecules on the surface of the tiny ferric oxide nano particles through simple amidation reaction to obtain the MRI contrast agent HR-ESIONPs, the integral particle size, good water dispersibility and biosafety of the tiny ferric oxide nano particles are not changed, but the contrast agent can make sensitive response to the hypoxia environment caused in the tumor growth process, and the transition of the tiny ferric oxide nano particles from a monodisperse state to an aggregation state is induced, from the MRI signal point of view, namely, from T 1 Conversion of contrast signals to T 2 Transition of the type contrast signal (transition from bright signal to dark signal). And as the size becomes larger, the enrichment of the nano particles in the tumor area is further increased, the blood reflux perfusion is reduced, and the imaging window time is further prolonged.
Experiments show that the embodiment of the invention provides a T of hypoxia response 1 -T 2 Longitudinal relaxation rate r of switchable MRI contrast agent before and after response 1 From 19.17mM -1 s -1 Becomes as follows18.10mM -1 s -1 Transverse relaxation rate r 2 From 31.81mM -1 s -1 Becomes 141.21mM -1 s -1 R of it 2 /r 1 The change of the ratio also indicates that the contrast agent is changed from T in the hypoxia environment 1 Conversion to T 2 The contrast agent can effectively reduce the interference of background signals during tumor diagnosis and improve the specificity and selectivity of the contrast agent. In addition, the contrast agent has good biocompatibility and extremely low biotoxicity.
As another aspect of the present invention, it relates to a T of hypoxia response 1 -T 2 The preparation method of the switchable MRI contrast agent comprises the following steps:
providing monodisperse tiny ferric oxide nano particles, and modifying polyacrylic acid on the surface of the nano particles to prepare the ferric oxide nano particles with the surface modified polyacrylic acid;
connecting a hypoxia sensitive small molecule and cysteine on the surface of the ferric oxide nano particle to prepare the hypoxia response T 1 -T 2 Switchable MRI contrast agents.
In a preferred embodiment, the preparation method specifically comprises the following steps:
(1) Providing tiny iron oxide nanoparticles with monodispersion of less than 5nm, and simultaneously modifying polyacrylic acid (PAA) on the surface, which can be named ESIONPs-PAA;
(2) Preparing a hypoxia-sensitive 2-nitroimidazole small molecule;
(3) The surface of the extremely small ferric oxide nano-particles is connected with 2-nitroimidazole micromolecules and cysteine (Cys) for preparing a nano-particle contrast agent which specifically responds to the tumor hypoxia microenvironment, is named as HR-ESIONPs, and is used for specifically responding to the tumor hypoxia microenvironment to realize accurate diagnosis.
The invention provides a T of hypoxia response 1 -T2 switchable MRI contrast agent preparation comprising synthesis of esio nps-PAA with particle size of about 3.6nm, modification of hypoxia sensitive 2-nitroimidazole small molecule and cysteine (Cys) on the outer surface of esio nps-PAA to achieve functionalization.
In one embodiment, step (1) comprises: and (3) reacting a mixed reaction system containing polyacrylic acid and an iron-containing precursor at 100-110 for 2-2.5h to obtain the surface-modified polyacrylic acid ferric oxide nano particles.
Further, the step (1) specifically includes: and (3) dissolving polyacrylic acid in water, heating to 100-110 , then rapidly adding a mixed solution of ferric salt and ferrous salt and an ammonia water solution to form a mixed reaction system, reacting for 2-2.5h at 100-110 , cooling to room temperature after the reaction is finished, and dialyzing with a dialysis bag with the molecular weight cutoff of 10000-14000 for purification.
Further, the iron salt may be ferric chloride hexahydrate, and the ferrous salt may be ferrous sulfate heptahydrate or the like, but is not limited thereto.
In a preferred embodiment, step (1) specifically comprises: dissolving PAA in deionized water, heating to reflux state, and rapidly adding FeCl 3 6H 2 O and FeSO 4 7H 2 And (3) refluxing and stirring the mixed solution of O and 28% ammonia water solution at 100-110 for about 2-2.5h to obtain PAA modified extremely small iron oxide nano particles (ESIONPs-PAA).
Further, the polyacrylic acid (PAA) has an average number average molecular weight of 2000; the polyacrylic acid is dissolved in water, the concentration of the PAA is controlled to be 4.0mg/mL as much as possible.
Further, the concentration of the ferric salt in the mixed solution is 500mmol/L, and the concentration of the ferrous salt is 250mmol/L.
Further, the FeCl 3 6H 2 O and FeSO 4 7H 2 The molar amounts of O and 28% ammonia solution also need to be precisely controlled, and in a 20mL system, 0.4mL of 500mmol/L FeCl needs to be added 3 6H 2 O and 250mmol/L FeSO 4 7H 2 O, and 6mL of 28% aqueous ammonia.
Further, the final purification was dialyzed in deionized water using a dialysis bag.
Further, the molecular weight of the dialysis bag is 10000-14000.
In some embodiments, the method of preparing a 2-nitroimidazole small molecule in step (2) comprises:
mixing 4- (aminomethyl) piperidine and benzaldehyde, dissolving in a first solvent, and stirring at 120-130 for reaction for 36-48h to obtain an intermediate product;
and (3) dissolving the intermediate product and 1- (2, 3-epoxypropyl) -2-nitroimidazole in a second solvent in protective atmosphere, stirring and reacting for 72-84h, then adding hydrochloric acid solution, and continuing stirring and reacting for 4-5h at 40-45 to prepare the 2-nitroimidazole micromolecule.
The synthesis of the 2-nitroimidazole small molecule provided by the invention also comprises a two-step method, compared with other reported ROS (reactive oxygen species) and pH-responsive polymers, the synthesis is much simpler, and the irreversible combination of the molecule and cysteine (Cys) is the result of the response to the hypoxia environment.
Further, the first solvent may include toluene, and the second solvent may include ethanol, but is not limited thereto.
In a preferred embodiment, the preparation method of the 2-nitroimidazole small molecule specifically comprises the following steps: 4- (aminomethyl) piperidine and benzaldehyde are mixed and dissolved in toluene and reacted at 120-130 for 36-48 hours to obtain an intermediate product, which can be called small molecule 1. Dissolving the small molecule 1 and 1- (2, 3-epoxypropyl) -2-nitroimidazole in ethanol, and reacting for 72-84 hours at 120-130 to obtain the 2-nitroimidazole small molecule.
Furthermore, the toluene and the ethanol are super-dry solvents for removing water in advance.
Further, the molar ratio of 4- (aminomethyl) piperidine to benzaldehyde is substantially 1:1.
further, the molar ratio of the intermediate (small molecule 1) to 1- (2, 3-epoxypropyl) -2-nitroimidazole needs to be greater than 1:1.
further, in the above purification, the means for removing by-products and impurity molecules is basically multiple extraction, and the means for finally removing the organic solvent is the use of a rotary evaporator and a vacuum drying oven.
In some embodiments, step (3) comprises:
activating carboxyl groups of ferric oxide nano particles of surface modified polyacrylic acid by adopting 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 2-3h, adding cysteine for reaction for 18-24h, continuously using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation for 2-3h, adding the 2-nitroimidazole micromolecule for reaction for 18-24h, and obtaining the T with hypoxia response of the surface-linked cysteine and 2-nitroimidazole micromolecule 1 -T 2 Switchable MRI contrast agents.
In a preferred embodiment, the step (3) specifically includes: firstly, activating carboxyl of ESIONPs-PAA, then sequentially adding cysteine (Cys) and 2-nitroimidazole small molecules to finally obtain the hypoxia-responsive tiny ferric oxide nanoparticle contrast agent (HR-ESIONPs).
Further, here, the activation of carboxyl group uses 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
Further, the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is essentially 1:1.
further, the first carboxyl activation is first followed by cysteine (Cys) ligation, and after removal of excess unreacted Cys, the second carboxyl activation is performed, and after activation, the 2-nitroimidazole small molecule is ligated.
Further, removal of unreacted small molecules was performed using ultrafiltration tubing.
Further, the molar ratio of cysteine (Cys) to 2-nitroimidazole small molecule needs to be greater than 20:1.
further, the reaction solvent used in the activation of the carboxyl group is MES buffer having a pH of 5.8-6.0 and a concentration of 40-60 mmol/L.
Further, the reaction solvent for cysteine (Cys) ligation is deionized water.
Further, the reaction solvent for the connection of the 2-nitroimidazole small molecules is PBS buffer solution with pH value of 7.2-7.4.
Further, all reactions can be completed at room temperature.
As one of more specific embodiments of the present invention, the preparation method may include the steps of:
(1) Preparation of ESIONPs-PAA: 4.0mg/mL of PAA was dissolved in 20mL of deionized water, nitrogen was first bubbled for 1h to remove dissolved oxygen therein, then warmed to 100deg.C to bring it to reflux, followed by rapid addition of 0.4mL of 500mM FeCl 3 6H 2 O and 250mM FeSO 4 7H 2 O, and 6mL of 28% aqueous ammonia solution. Then the reaction is reacted for 2 hours at 100 , after the reaction is finished, the reaction is cooled to room temperature, and the reaction is dialyzed for a plurality of days by a dialysis bag with the molecular weight cut-off of 14000 for purification. Finally, the tiny ferric oxide nano particles with the surfaces modified by PAA, namely ESIONPs-PAA, are obtained.
(2) Preparation of 2-nitroimidazole small molecule (by 2-step synthesis): first, equimolar amounts of 4- (aminomethyl) piperidine and benzaldehyde were mixed and dissolved in a certain amount of anhydrous toluene. The reaction mixture was stirred at 120for 36h and the solvent was removed to finally give a yellow oily liquid designated small molecule 1. The small molecules 1 and 1- (2, 3-epoxypropyl) -2-nitroimidazole were then dissolved in 10mL of ultra-dry ethanol. The reaction mixture was stirred under nitrogen at 120for 72 hours to allow sufficient reaction, cooled to room temperature, concentrated, and then added with hydrochloric acid. The reaction was stirred at 40for 4 hours, and after the completion of the reaction, a small amount of water was added to dilute the reaction solution, and the mixture was extracted with methylene chloride. The aqueous phase was collected, then the pH was adjusted to 11 with NaOH solution and extracted again with dichloromethane. The organic phase was collected, washed twice with saturated sodium chloride solution and dried overnight in anhydrous magnesium sulfate. After filtration, the solvent was removed and dried under vacuum overnight to give a butter-like 2-nitroimidazole small molecule, designated small molecule 2. Wherein, the feeding mole ratio of the small molecule 1 to the 1- (2, 3-epoxypropyl) -2-nitroimidazole is required to be more than 1:1.
(3) Preparation of hypoxia-responsive contrast Agents (HR-ESIONPs): the carboxyl groups of esiofps-PAA were first activated with equimolar amounts of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 2h (the reaction solvent was 50mM MES buffer at ph=6.0), then the excess activating reagent was removed by ultrafiltration using an ultrafiltration tube, followed by adding cysteine (Cys) for 24h (the reaction solvent was deionized water), after removing the excess cysteine by ultrafiltration using an ultrafiltration tube, the carboxyl groups were further activated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 2h (the reaction solvent was 50mM MES buffer at ph=6.0), then the excess activating reagent was removed by ultrafiltration using an ultrafiltration tube, followed by adding 2-nitroimidazole small molecules for 24h (the reaction solvent was PBS buffer at ph=7.4), and finally the surface-linked cysteine and 2-nitroimidazole small molecules were obtained after removing the excess 2-nitroimidazole small molecules by ultrafiltration using an ultrafiltration tube (the small-size of PBS buffer solution).
In the preparation step of ESIONPs-PAA, the average molecular weight of PAA selected by the invention is 2000, so that the PAA can be dialyzed by a dialysis bag with any molecular weight cutoff of more than 2000 to remove the redundant unreacted PAA.
Wherein, in the preparation step of the 2-nitroimidazole small molecule, for the treatment of the anhydrous toluene, sodium and potassium are required to be stirred with commercially available analytically pure toluene overnight in advance, and then the distillation operation is performed the next day to obtain the anhydrous toluene. In the synthesis of the first step of micromolecule 1, the solvent anhydrous toluene is excessive, and a large amount of water is generated due to the reaction of 4- (aminomethyl) piperidine and benzaldehyde, so that water generated in the reaction process is required to be continuously separated by adopting water separation equipment, the reaction efficiency of the reaction is improved as much as possible, and the requirement of green chemistry is met. In the case of ultra-dry ethanol, since the solvent amount should be kept small, commercially available ultra-dry ethanol is directly purchased, after the reaction, the reaction solution is concentrated to about 1mL after cooling to room temperature, 2mL (1.2N) hydrochloric acid solution is further added, stirring is continued at 40for 4 hours, after the reaction is completed, a small amount of water is added to dilute the reaction solution, the mixture is extracted with methylene chloride (4X 15 mL), the aqueous phase is collected, and the pH is adjusted to 11 with NaOH solution (1M). The mixture was extracted again with dichloromethane (4X 15 mL) and the organic phase was collected. Further, the collected organic phases were washed twice with saturated sodium chloride (315 mL) and dried overnight with anhydrous magnesium sulfate. After filtration, the solvent was removed by rotary evaporation and then dried in vacuo.
In the preparation step of the hypoxia response contrast agent (HR-ESIONPs), the ultrafiltration tube can remove excessive unreacted small molecules, wherein the ultrafiltration tube is selected from the ultrafiltration tubes with the molecular weight cutoff of 10000, and is usually repeatedly washed and ultrafiltered for 3 times by using water, and the rotating speed is selected to be 2000rpm multiplied by 20min.
Compared with the prior art, the method for preparing the contrast agent has simple and convenient process and low cost. It can reach the tumor by the high permeability and retention effect (EPR effect) of the tumor and respond to the hypoxic environment of the tumor microenvironment to realize the MRI signal from T 1 To T 2 Is used for the accurate diagnosis of tumors. Since hypoxia is one of the significant features of many solid tumors and other common diseases such as atherosclerosis, the contrast agent prepared by the invention has high sensitivity response capability to hypoxia, provides possibility of being applied to detection of various disease models, and is a general hypoxia response T 1 -T2 switchable MRI contrast agent.
Yet another aspect of embodiments of the invention also provides T of the hypoxia response 1 -T 2 Use of a switchable MRI contrast agent.
For example, embodiments of the present invention provide T for the hypoxia response 1 -T 2 Use of a switchable MRI contrast agent for the preparation of a product with tumor detection function.
Preferably, the concentration of the product imaged in the mouse is 0.1mmol kg -1
For example, embodiments of the present invention provide a contrast composition comprising T in response to hypoxia 1 -T 2 A switchable MRI contrast agent; and, pharmaceutically acceptable adjuvants.
Further, the pharmaceutically acceptable adjuvant may also be a diluent or the like.
For example, embodiments of the present invention provide a pharmaceutical composition comprising T in response to hypoxia 1 -T 2 A switchable MRI contrast agent; anda pharmaceutically acceptable carrier.
Further, the pharmaceutical composition may further comprise pharmaceutical components such as therapeutic drugs, tracer molecules, etc.
For example, an embodiment of the present invention provides a non-medical imaging method, comprising: taking the T of the hypoxia response to an object to be imaged 1 -T 2 A switchable MRI contrast agent or contrast composition, and performing contrast.
In order to make the technical solution and advantages of the present invention more clearly explained, the technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and the accompanying drawings, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The implementation conditions used in the following examples may be further adjusted according to actual needs, and the implementation conditions not specified are generally those in routine experiments.
Example 1
T for hypoxia response in the present example 1 -T 2 The preparation method of the switchable MRI contrast agent comprises the following steps:
step one: 4.0mg/mL of PAA with a number average molecular weight of 2000 was dissolved in 20mL of deionized water and placed in a 100mL three-necked round bottom flask, nitrogen was first bubbled for 1-2h to remove dissolved oxygen therefrom, then the temperature was raised to 100deg.C to bring it into reflux, followed by rapid addition of 0.4mL of 500mM FeCl 3 6H 2 O and 250mM FeSO 4 7H 2 O, and 6mL of 28% aqueous ammonia solution. Then the reaction is reacted for 2 hours at 100 , after the reaction is finished, the reaction is cooled to room temperature, and dialysis is carried out for a plurality of days by using a dialysis bag with the molecular weight cutoff of 10000-14000 so as to remove the redundant unreacted PAA. Finally, the tiny ferric oxide nano particles with the surfaces modified by PAA, namely ESIONPs-PAA, are obtained. As shown in the transmission electron microscope chart of FIG. 2, the obtained PAA had a uniform particle size and an average particle size of 3.6nm. Determination using ICPThe concentration of Fe is ready for use.
Step two: 4- (aminomethyl) piperidine (2.96 mL,4.6 mmol) and benzaldehyde (2.56 mL,24.6 mmol) were mixed and dissolved in a large amount of anhydrous toluene which had been dehydrated with sodium potassium beforehand, and placed in a 250mL single neck round bottom flask. The reaction mixture was stirred at 120 for 36 hours, and since a large amount of water was generated during the reaction, it was necessary to remove the reaction water using a condensation water removal device, and after the completion of the reaction, residual toluene was removed using a rotary evaporator, followed by vacuum drying overnight to obtain a yellow oily liquid. Named small molecule 1. As shown in the nuclear magnetic diagram of FIG. 3, the correct synthesis of small molecule 1 was confirmed. The synthetic route can be represented by the following chemical equation:
Step three: small molecule 1 (317 mg,1.77 mmol) and 1- (2, 3-epoxypropyl) -2-nitroimidazole (250 mg,1.55 mmol) were dissolved in 10mL ultra-dry ethanol and placed in a 50mL single neck round bottom flask. The reaction mixture was stirred well at 120for 72 hours under nitrogen atmosphere to allow sufficient reaction, after completion of the reaction, cooled to room temperature, the reaction solution was concentrated to about 0.9-1.1mL, then 2mL (1.2N) of hydrochloric acid solution was added to acidify, stirring was continued at 40for 4 hours, after completion of the reaction, a small amount of water was added to dilute the reaction solution, the mixture was extracted with methylene chloride (4X 15 mL), the aqueous phase was collected, and the pH of the collected aqueous phase was adjusted to about 11 with NaOH solution (1M). The mixture was extracted again with dichloromethane (4X 15 mL) and the organic phase was collected. The collected organic phases were washed three times with saturated sodium chloride solution (3X 15 mL), and the organic phases were collected and then dried overnight with anhydrous magnesium sulfate. After filtration, the solvent was removed by rotary evaporation and then dried in vacuo to give the 2-nitroimidazole small molecule, designated small molecule 2. As shown in the nuclear magnetic resonance chart of FIG. 4, the correct synthesis of small molecule 2 was confirmed. The synthetic route can be represented by the following chemical equation:
step four: the ESIONPs-PAA obtained in the first step (quantitative 3.2mg of Fe) was first dissolved in 8-10mL of MES buffer (pH=6.0, 50 mM), and then the surface carboxyl groups of the ESIONPs-PAA were activated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (30.4 mg, 160. Mu. Mol) and N-hydroxysuccinimide (18.4 mg, 160. Mu. Mol) for 2-3 hours. Then using ultrafiltration tube to remove superfluous activating reagent for three times, re-dissolving the nanoparticle solution on the ultrafiltration tube in 8-10mL deionized water, adding cysteine (19.2 mg,0.16 mmol) to stir and react for 24h at room temperature, then using ultrafiltration tube to remove superfluous cysteine for three times, re-dissolving the nanoparticle solution on the ultrafiltration tube in 8-10mL MES buffer, repeating the above carboxyl activating operation, re-dissolving the nanoparticle solution on the upper nanoparticle solution in 8-10mL PBS buffer (pH=7.2-7.4), adding 2-nitroimidazole micromolecules (287 mg,5 mu mol) to stir and react for 24h at room temperature, and finally obtaining the hypoxia response contrast agent (HR-ESIONPs) after removing superfluous 2-nitroimidazole micromolecules by using ultrafiltration tube. As shown in fig. 6a, the particle size and dispersibility of the synthesized hypoxia response nanoparticle contrast agent are not changed, and the synthesized hypoxia response nanoparticle contrast agent still has good monodispersity, and the synthetic route can be shown in fig. 1 b.
Example 2
T for hypoxia response in the present example 1 -T 2 The preparation method of the switchable MRI contrast agent comprises the following steps:
step one: this step is similar to step one of example 1, except that: the reaction was carried out at 110for 2.5 hours, and 4.0mg/mL of PAA having a molecular weight of about 2000 was used as the reaction medium, and 0.4mg/mL of 500mM FeCl having a molecular weight of 2000 was used as the reaction medium 3 6H 2 O and 250mM FeSO 4 7H 2 The mixed aqueous solution of O and 6mL of 28% aqueous ammonia solution were modified to 0.4mL of 50mM FeCl 3 6H 2 O and 25mM FeSO 4 7H 2 O, and 6mL of a 2.8% aqueous ammonia solution.
Step two: this step is similar to step two in example 1, except that: the reaction temperature was 130and the reaction time was 40 hours.
Step three: this step is similar to step three in example 1, except that: stirring and reacting for 84 hours, then adding hydrochloric acid solution, continuously stirring and reacting for 5 hours at 45 , and adjusting the molar ratio of the small molecule 1 to the 1- (2, 3-epoxypropyl) -2-nitroimidazole to be 2:1, anhydrous magnesium sulfate was dried overnight and changed to anhydrous sodium sulfate overnight.
Step four: this step is similar to step four in example 1, except that: the amount of ESIONPs-PAA obtained in the first step was taken to be 4.5mg of Fe, and then the surface carboxyl groups of ESIONPs-PAA were activated using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (36.5 mg, 192. Mu. Mol) and N-hydroxysuccinimide (22.1 mg, 192. Mu. Mol) for 3-4 hours. The addition of 2-nitroimidazole small molecule changed the weight from 287mg to 205mg. Cysteine is added for reaction for 18 hours, and 2-nitroimidazole micromolecule is added for reaction for 20 hours.
Example 3
T for hypoxia response in the present example 1 -T 2 The preparation method of the switchable MRI contrast agent comprises the following steps:
step one: this step is similar to step one of example 1, except that: the reaction was carried out at 105for 2.2 hours, and 4.0mg/mL of PAA having a molecular weight of 2000 was used as the reaction medium, and 0.8mg/mL of PAA having a molecular weight of 2000 and 0.4mL of 500mM FeCl were used as the reaction medium 3 6H 2 O and 250mM FeSO 4 7H 2 O was modified to 0.4mL of 100mM FeCl in a 6mL aqueous 28% ammonia solution 3 6H 2 O and 50mM FeSO 4 7H 2 Mixed aqueous solution of O and 6mL of 5.6% aqueous ammonia solution.
Step two: this step is substantially the same as step two in example 1, except that: the reaction temperature was 125and the reaction time was prolonged to 48 hours.
Step three: this step is similar to step three in example 1, except that: after acidification by the addition of 2mL (1.2N) of hydrochloric acid solution, the reaction was continued with stirring at 43for 5h.
Step four: this step is similar to step four in example 1, except that: the amount of ESIONPs-PAA obtained in the first step was taken to be 4.0mg of Fe, and then the surface carboxyl groups of ESIONPs-PAA were activated using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (33.4 mg, 176. Mu. Mol) and N-hydroxysuccinimide (20.24 mg, 176. Mu. Mol) for 3-4 hours. The amount of cysteine added was changed from 19.2mg to 21.12mg, and stirred at room temperature for 36-48 hours to effect a sufficient reaction. Cysteine is added for reaction for 20 hours, and 2-nitroimidazole micromolecule is added for reaction for 18 hours.
Comparative example 1:
this control was essentially the same as example 1, except that (2, 3-epoxypropyl) benzene (208 mg,1.55 mmol) was used in step three instead of 1- (2, 3-epoxypropyl) -2-nitroimidazole (250 mg,1.55 mmol) therein, and the final control small molecule was designated small molecule 3, as shown in the nuclear magnetic resonance chart of FIG. 5, which demonstrates the correct synthesis of small molecule 3. The synthetic route can be represented by the following chemical equation:
in the fourth step, small molecule 3 (220 mg,5 mu mol) is used for replacing small molecule 2 (2-nitroimidazole small molecule) in the second step, and finally the MRI contrast agent without hypoxia response is obtained and is named as NHR-ESIONPs.
Performance test one:
testing the MRI contrast agent product obtained in example 1 of the present invention with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase on a 0.5T MRI tester under in vitro simulated hypoxia conditions, longitudinal relaxation time T before and after incubation for 4h 1 T and T 1 A weighted imaging method of operation comprising:
preparing the two groups of samples with iron concentration of 0.02mM, 0.04mM, 0.08mM, 0.16mM and 0.32mM respectively, and performing linear fitting by taking the iron ion concentration (mM) as an abscissa and taking the reciprocal of the longitudinal relaxation time as an ordinate after testing on an MRI tester with 0.5T to obtain the MRI contrast agent, the reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and the nitroreductase of the invention with transverse relaxation rates of 19.17mM before and after incubation under the in vitro simulated hypoxia condition -1 s -1 And 18.10mM -1 s -1 FIG. 7 shows the longitudinal relaxation rates (r) of the MRI contrast agent (HR-ESIONPs) of example 1 and the MRI contrast agent (NHR-ESIONPs) of comparative example 1 before and after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro under simulated hypoxia conditions for 4h 1 ) And transverse relaxation rate (r) 2 ) It can be seen that the contrast agent of the invention has a slight decrease in its longitudinal relaxation rate after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase under in vitro simulated hypoxia conditions.
As shown in FIG. 8, the MRI contrast agent (HR-ESIONPs) of example 1 was shown to be T before and after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro to mimic hypoxia conditions for 4 hours 1 Weighted solution imaging with T at different concentrations 1 As can be seen from the weighted imaging, the contrast agent obtained in example 1 of the present invention has a T 1 The contrast effect was significantly brighter before incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro under simulated hypoxia conditions than after incubation for 4 h.
Remarks: t (T) 1 The weighted imaging parameters are set as: echo Time (TE) =134 ms, repetition Time (TR) =500 ms, number of Scans (NS) =1.
And II, performance test:
test of the MRI contrast agent product obtained in example 1 of the present invention with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase on a 0.5T MRI tester under in vitro simulated hypoxia conditions, transverse relaxation time T before and after incubation for 4h 2 T and T 2 A weighted imaging method of operation comprising:
preparing the two groups of samples with iron concentration of 0.02mM, 0.04mM, 0.08mM, 0.16mM and 0.32mM respectively, testing on a MRI tester with 0.5T, and performing linear fitting by taking the iron ion concentration (mM) as an abscissa and taking the reciprocal of the transverse relaxation time as an ordinate to obtain the MRI contrast agent, the reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and the nitroreductase of the invention to simulate hypoxia in vitroLongitudinal relaxation rates before and after incubation were 31.81mM, respectively -1 s -1 And 141.21mM -1 s -1 As shown in FIG. 7, it can be seen that the contrast agent of the present invention has a significant increase in its transverse relaxation rate after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase under in vitro simulated hypoxia conditions. And the ratio (r 2 /r 1 ) The ratio of the transverse relaxation rate to the longitudinal relaxation rate before incubation is 1.66, the ratio of the transverse relaxation rate to the longitudinal relaxation rate after incubation is 7.80, and the ratio change shows that the contrast agent obtained by the embodiment can respond under the condition of simulating hypoxia, and the contrast agent is prepared from the following materials 1 Conversion of contrast agent to T 2 Contrast agents of the type.
As shown in FIG. 9, the MRI contrast agent (HR-ESIONPs) of example 1 was shown to be T before and after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro to mimic hypoxia conditions for 4 hours 2 Weighted solution imaging with T at different concentrations 2 As can be seen from the weighted imaging, the contrast agent obtained in example 1 of the present invention has a T 2 The contrast effect was significantly darker than after 4h incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase before incubation under in vitro simulated hypoxia conditions.
Remarks: t (T) 2 The weighted imaging parameters are set as: echo Time (TE) =334 ms, repetition Time (TR) =2000 ms, number of Scans (NS) =1.
And (3) performance test:
testing the MRI contrast agent product obtained according to the invention with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase on a 0.5T MRI tester for different time periods of incubation with transverse relaxation (1/T) under in vitro simulated hypoxia conditions 2 ) The operation method comprises the following steps:
the contrast agent (40. Mu.g of Fe) obtained in this example was dissolved in 1mL of PBS solution (pH=7.4) together with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase, incubated at 37for 0h, respectively, Measurement of transverse relaxation time T with a 0.5T pulse Nuclear magnetic resonance imager at time points of 1h, 2h, 3h, 4h and 5h 2 The reciprocal transverse relaxation time (1/T) 2 ) The change in value with time (as shown in FIG. 10, for the MRI contrast agent (HR-ESIONPs) of example 1) was measured as the reciprocal transverse relaxation time (1/T) of incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro to mimic hypoxia conditions 2 ) Change over time).
Inverse transverse relaxation time (1/T) 2 ) The value gradually increases to about 2 hours, and the transverse relaxation time reciprocal rate (1/T 2 ) The surface of the prepared tiny ferric oxide nano-particles modified with the hypoxia response small molecules is increased from about 12.5 to about 25 in 0h, which shows that the transverse relaxation rate of the tiny ferric oxide nano-particles can be obviously increased under the action of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase. The change in the reciprocal transverse relaxation time rate is derived from aggregation of extremely small iron oxide nanoparticles, and increases the non-uniformity of the magnetic field, thereby resulting in an increase in the reciprocal transverse relaxation time rate. And after 2h, little increase in transverse relaxation rate occurred, indicating that the hypoxia aggregation reaction can be completed within 2 h.
Remarks: t (T) 2 The weighted imaging parameters are set as: echo Time (TE) =334 ms, repetition Time (TR) =2000 ms, number of Scans (NS) =1.
And (4) performance test:
testing the MRI contrast agent product obtained in comparative example 1 of the present invention with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase on a 0.5T MRI tester under in vitro simulated hypoxia conditions, longitudinal relaxation time T before and after incubation for 4h 1 T and T 1 A weighted imaging method of operation comprising:
preparing the two groups of samples with iron concentration of 0.01mM, 0.02mM, 0.04mM, 0.08mM and 0.16mM respectively, testing on a MRI tester with 0.5T, and performing linear fitting by taking the iron ion concentration (mM) as an abscissa and the reciprocal of the longitudinal relaxation time as an ordinate to obtain the MRI contrast agent and the reduced nicotinoyl of the inventionThe transverse relaxation rates of the amine adenine dinucleotide phosphate (NADPH) and nitroreductase before and after incubation under in vitro simulated hypoxia conditions were 18.19mM, respectively -1 s -1 And 25.28mM -1 s -1 As shown in FIG. 7, it can be seen that the contrast agent of the present invention showed a slight increase in longitudinal relaxation rate after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase under in vitro simulated hypoxia conditions.
As shown in FIG. 11, the MRI contrast agent (NHR-ESIONPs) of comparative example 1 was incubated with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase for 4 hours before and after incubation under in vitro simulated hypoxia conditions 1 Weighted solution imaging with T at different concentrations 1 As can be seen from the weighted imaging, the contrast agent obtained in comparative example 1 of the present invention has T 1 The contrast effect was almost the same as the brightness after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro under simulated hypoxia conditions.
Remarks: t (T) 1 The weighted imaging parameters are set as: echo Time (TE) =134 ms, repetition Time (TR) =500 ms, number of Scans (NS) =1.
Performance test five:
test of the MRI contrast agent product obtained in comparative example 1 of the present invention with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase on a 0.5T MRI tester under in vitro simulated hypoxia conditions, transverse relaxation time T before and after incubation for 4h 2 T and T 2 A weighted imaging method of operation comprising:
preparing the two groups of samples with iron concentration of 0.01mM, 0.02mM, 0.04mM, 0.08mM and 0.16mM respectively, and performing linear fitting by taking the iron ion concentration (mM) as an abscissa and taking the reciprocal of the transverse relaxation time as an ordinate after testing on an MRI tester with 0.5T to obtain the MRI contrast agent, the reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and the nitroreductase of the invention, wherein the longitudinal relaxation rates before and after incubation under the in vitro simulated hypoxia condition are 29.5mM respectively -1 s -1 And 63.27mM -1 s -1 As shown in FIG. 7, it can be seen that the contrast agent of the present invention has a slight increase in its transverse relaxation rate after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase under in vitro simulated hypoxia conditions. And the ratio (r 2 /r 1 ) Is a gold standard for judging the type of the contrast agent, the ratio of the transverse relaxation rate to the longitudinal relaxation rate before incubation is 1.62, the ratio of the transverse relaxation rate to the longitudinal relaxation rate after incubation is 2.50, and the slight improvement is probably due to some influence of biological enzymes in the contrast agent, and the contrast agent obtained in the comparison example can not respond under the condition of simulating hypoxia as can be seen from the change of the ratio, and the contrast agent obtained in the comparison example is characterized by that 1 Conversion of contrast agent to T 2 Contrast agents of the type.
As shown in FIG. 12, the MRI contrast agent (NHR-ESIONPs) of comparative example 1 was incubated with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase for 4 hours before and after incubation under in vitro simulated hypoxia conditions 2 Weighted solution imaging with T at different concentrations 2 As can be seen from the weighted imaging, the contrast agent obtained in comparative example 1 of the present invention has T 2 The contrast effect was almost the same as the brightness after incubation with reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and nitroreductase in vitro under simulated hypoxia conditions.
Remarks: t (T) 2 The weighted imaging parameters are set as: echo Time (TE) =334 ms, repetition Time (TR) =2000 ms, number of Scans (NS) =1.
Performance test six:
the method for detecting toxicity of the contrast agent obtained in the embodiment 1 of the invention to normal cells and cancer cells comprises the following steps:
the cytotoxicity of the contrast agent obtained in this example was measured on human umbilical vein endothelial cells (HUVEC cells) and mouse breast cancer cells (4T 1 cells) using CCK-8.
In the following, HUVEC cells were seeded at a density of 5000 cells per well in a 96-well plate of 100. Mu.L, and the 96-well plate was placed in CO 2 In an incubator, the cells were cultured at 37for 24 hours. The contrast agent of inventive example 1 was then dissolved inIn complete medium, and in gradient dilution of multiple concentrations (8 different concentrations of 0.06-1.00mM were made according to the invention), the cells were incubated for 24h in wells of 96-well plates, then 100. Mu.L of 10% CCK-8 solution was added to each well and incubated for 3h in a cell incubator. Measurement of absorbance OD at 450nm with an ELISA 450nm . Each contrast agent concentration (referred to as the experimental group) was performed in 4 replicates for the control group. The relative viability of the cells was calculated from the absorbance values. Blank, i.e., complete medium without cells, control, i.e., cells cultured without material.
Cytotoxicity testing of 4T1 cells was essentially the same as described above.
Cell relative survival (%) = 100 (experimental OD-blank OD)/(control OD-blank OD)
As shown in FIG. 13, which is a graph of cytotoxicity test of the MRI contrast agent (HR-ESIONPs) in Human Umbilical Vein Endothelial Cells (HUVEC) and mouse breast cancer cells (4T 1) in example 1, both 4T1 and HUVEC cell viability showed higher activity levels. Even at an iron concentration of 1mM, the cell viability of both 4T1 cells and Human Umbilical Vein Endothelial Cells (HUVECs) was above 98%. These results indicate that the cytotoxicity of the contrast agent obtained in example 1 of the present invention is negligible.
Performance test seven:
the method for detecting the toxicity of the biological tissues of the MRI contrast agent obtained in the embodiment 1 of the invention comprises the following steps:
the tissue toxicity of the MRI contrast agent obtained in this example in normal BALB/c mice (4-6 weeks, 20 g) was determined by hematoxylin-eosin staining (H & E staining).
Four week old BALB/c mice were divided into two groups, the experimental and control groups:
150. Mu.L of a physiological saline solution containing the contrast agent obtained in example 1 was injected into the tail vein of the first group, wherein the iron ion injection concentration was 0.1mM/kg;
The second group was injected with 150 l of physiological saline as a control group;
after 3 days of raising under normal conditions, the cervical dislocation is killed, and the heart, liver, spleen, lung and kidney are collected for H & E section staining and observed by microscopic photographing.
As shown in FIG. 14, in the tissue toxicity test chart of the MRI contrast agent (HR-ESIONPs) in the BALB/c mice in example 1, the obtained MRI contrast agent in example 1 has less damage to the tissues of various organs, and the high concentration sample has no obvious improvement. In particular, hepatocytes in liver sections were relatively normal and did not have any signs of inflammatory response. Pulmonary fibrosis was also not observed in the lung sections. No tissue necrosis was observed for all other slice samples. The MRI contrast agent obtained in the embodiment 1 is a biological safe contrast agent and has wide biological application prospect.
Performance test eight:
in vivo MRI imaging experiments with the contrast agent obtained in example 1 of the present invention, the method of operation included:
a batch of BALB/c mice (4-6 weeks old, 20 g) under SPF conditions was first purchased from a company. 4T1 cells were selected as subcutaneous tumor model cells. Culturing and dispersing in PBS solution uniformly to obtain density of 2X10 6 Each Balb/c mouse was then subcutaneously injected with 100. Mu.L of the PBS solution in the armpit and incubated for 7-10 days to successfully establish a tumor model. The obtained tumor-bearing mice can be used for subsequent in vivo experiments. All animal experiments followed the protocol approved by the national academy of sciences animal protection research.
Successfully constructed 4T 1 Tumor-bearing mice were divided into two groups: the experimental group (HR-ESIONPs) and the control group (NHR-ESIONPs) were respectively used for hypoxia response. First, 100. Mu.L of 29% Ulatin solution was intraperitoneally injected, and after both groups of mice entered deep anesthesia, a blank scan before contrast medium injection was performed. Then, the experimental group was injected with HR-ESIONPs through the tail vein, the control group was injected with NHR-ESIONPs through the tail vein, and the iron ion dose of both groups was 0.1mmol/kg. Then fixing the mice, placing the mice in a 1.5T micro magnetic resonance imaging instrument, and carrying out T at a plurality of time points of 1h, 2h, 3h, 4h, 5h and 6h after injection 2 And (5) weighting and developing.
As shown in FIG. 15, after two groups of contrast agents were injected from the tail vein into tumor-bearing mice at an injection amount of 0.1mmol/kg, the changes with time were followedThe two groups exhibited significantly different imaging enhancement effects. In order to further intuitively analyze the change in the signal intensity ratio (signal intensity after injection and before injection) of the tumor region in the MRI image, the signal intensity is represented by a gray value determined by image J software. Wherein the brightness of the MRI image of the tumor part of the HR-ESIONPs group is reduced along with the increase of time, and the MRI image shows obvious T 2 The dark signal is enhanced. FIGS. 16a and 16b are in vivo T-cell responses of MRI contrast agent (HR-ESIONPs) of example 1 and MRI contrast agent (NHR-ESIONPs) of comparative example 1 in tumor-bearing mice transplanted with 4T1 cells 2 Quantitative average signal intensity analysis plot after weighted MRI imaging. In addition, it can be seen that the signal intensity ratio gradually decreases and reaches 512.9% at the time point of 6h (as shown in fig. 16 a). However, the brightness of the MRI image of the NHR-ESIONPs group tumor was significantly improved at 1h-4h, the signal intensity ratio reached 122.+ -. 3.7% (as shown in FIG. 16 b), and then gradually decreased to about 100% of the initial level at the 6h time point. According to the MRI test results, the magnetic resonance effect of the experimental group is better than that of the control group, which shows that the extremely small ferric oxide nanoparticle contrast agent with 2-nitroimidazole micromolecules and cysteine connected on the surface has sensitive hypoxia responsiveness and can selectively activate T at the tumor part 2 MRI signal, consisting of T 1 Contrast agent switching to T 2 Contrast agent, improving specificity and selectivity of tumor diagnosis.
In conclusion, the invention provides a T with hypoxia response 1 -T 2 Switchable MRI contrast agent has high specificity and selectivity to tumor site, and can selectively activate T at tumor site 2 MRI signal, consisting of T 1 Contrast agent switching to T 2 The contrast agent improves the specificity and selectivity of tumor detection, thereby realizing excellent imaging contrast performance. And has good biocompatibility and low toxicity, and can be rapidly metabolized from the body.
Remarks: t (T) 2 The weighted mouse imaging parameters were set to te=60.86 ms, tr=3000 ms, matrix=512 256,slice thickness =0.27 mm.
Performance test nine:
the method for operating the intratumoral TEM experiment of the contrast agent obtained in the embodiment 1 of the invention comprises the following steps:
two BALB/c mice (4-6 weeks old, 20 g) under SPF conditions were first purchased from a company. 4T1 cells were selected as subcutaneous tumor model cells. Culturing and dispersing in PBS solution uniformly to obtain density of 2X10 6 Each Balb/c mouse was then subcutaneously injected with 100. Mu.L of the PBS solution in the armpit and incubated for 7-10 days to successfully establish a tumor model. The obtained tumor-bearing mice can be used for subsequent in vivo experiments. All animal experiments followed the protocol approved by the national academy of sciences animal protection research.
One mouse was used in the experimental group and one mouse was used in the control group, 150. Mu.L of PBS solution of HR-ESIONPs and NHR-ESIONPs, each having an iron ion dose of 0.1mmol/kg, was injected into the tail vein, and then after 6 hours of injection, the two mice were sacrificed for cervical dislocation, and tumors of the two mice were obtained. Then cutting to obtain tissue with the size of 1-2mm 3 Immediately after the materials are taken, putting the obtained material into 2.5% glutaraldehyde fixing solution precooled at 4 and then putting the obtained material into a refrigerator at 4 for fixing and preserving overnight. The next day the fixative was poured off and the samples were rinsed three times with 0.1m phosphate buffer, ph=7.0, 15min each; fixing the sample with 1% osmium acid solution for 1-2h; carefully remove the osmium acid waste solution, rinse the sample three times with 0.1m phosphate buffer, ph=7.0, 15min each; dehydrating the sample with ethanol solutions of gradient concentration (including 30%,50%,70%,80%,90% and 95% concentration), each concentration being treated for 15min, and then 100% ethanol for 20min; finally, the mixture is transited to pure acetone for 20min. Treating the sample with a mixture of embedding medium and acetone (V/v=1/1) for 1h; treating the sample with a mixture of embedding medium and acetone (V/v=3/1) for 3h; treating the sample with pure embedding medium overnight; embedding the sample subjected to the permeation treatment, and heating at 70 overnight to obtain the embedded sample. Slicing the sample in an ultrathin slicing machine to obtain 70-90nm slices, respectively dyeing the slices for 5-10min by using a lead citrate solution and a 50% ethanol saturated solution of uranyl acetate, and airing the slices to observe in a transmission electron microscope.
As shown in FIG. 17a, the biological transmission electron microscope image of the tumor tissue after the injection of the MRI contrast agent (HR-ESIONPs) into the 4T1 tumor-bearing mice for 6 hours in example 1, the experimental group injected with the HR-ESIONPs showed a remarkable aggregation state in the tumor, and as shown in FIG. 17b, the biological transmission electron microscope image of the tumor tissue after the injection of the MRI contrast agent (NHR-ESIONPs) into the 4T1 tumor-bearing mice for 6 hours in example 1, the control group injected with the NHR-ESIONPs remained in a dispersed state in the tumor, and the response aggregation capacity of the HR-ESIONPs to the hypoxic tumor was verified.
In addition, the inventor also uses other raw materials listed above and other process conditions to replace various raw materials and corresponding process conditions in the embodiment 1 to perform corresponding tests, and the obtained magnetic resonance imaging contrast agent is ideal in biocompatibility, safety, relaxation rate and imaging contrast performance.
It should be noted that, in this document, an element defined by the phrase "including " generally does not exclude that there are additional identical elements in a step, a process, a method or an experimental apparatus including the element.
It should be noted that the above-mentioned specific embodiments of the present invention do not limit the protection scope of the present invention. Any of various other corresponding changes and modifications made according to the technical idea of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. T of hypoxia response 1 -T 2 A switchable MRI contrast agent comprising:
iron oxide nanoparticles of surface-modified polyacrylic acid;
and connecting the hypoxia-sensitive small molecule and cysteine modified on the surface of the ferric oxide nano particle.
2. The T of the hypoxia response of claim 1 1 -T 2 A switchable MRI contrast agent characterized by: the hypoxia sensitive small molecule comprises a 2-nitroimidazole small molecule; and/or the molar ratio of cysteine to hypoxia sensitive small molecule is greater than 20:1, a step of; and/or, the polyacrylic acid is flatThe average number average molecular weight was 2000.
3. The T of the hypoxia response of claim 1 1 -T 2 A switchable MRI contrast agent characterized by: the T is 1 -T 2 The switchable MRI contrast agent contains 0.5-1.0wt% of hypoxia sensitive small molecules and 21.5-30.0wt% of cysteine;
and/or, the particle size of the iron oxide nanoparticles is less than 5nm; and/or the surface of the iron oxide nanoparticle is modified with more than 50wt% of a re-modified functional group, preferably at least any one of carboxyl and amino;
and/or, the T 1 -T 2 Switchable MRI contrast agent can be specifically aggregated in response to hypoxia environment within 4-6h in tumor microenvironment to realize T 1 Contrast agent to T 2 Switching of the type contrast agent.
4. A T of hypoxia response as defined in any one of claims 1-3 1 -T 2 A method for preparing a switchable MRI contrast agent, comprising:
providing monodisperse tiny ferric oxide nano particles, and modifying polyacrylic acid on the surface of the nano particles to prepare the ferric oxide nano particles with the surface modified polyacrylic acid;
Connecting a hypoxia sensitive small molecule and cysteine on the surface of the ferric oxide nano particle to prepare the hypoxia response T 1 -T 2 Switchable MRI contrast agents.
5. The method according to claim 4, comprising: reacting a mixed reaction system containing polyacrylic acid and an iron-containing precursor at 100-110 for 2-2.5h to prepare the surface-modified polyacrylic acid ferric oxide nano-particles;
preferably, the preparation method specifically comprises the following steps: dissolving polyacrylic acid in water, heating to 100-110 , then rapidly adding a mixed solution of ferric salt and ferrous salt and an ammonia water solution to form a mixed reaction system, reacting for 2-2.5h at 100-110 , cooling to room temperature after the reaction is finished, and dialyzing with a dialysis bag with the molecular weight cutoff of 10000-14000 for purification;
preferably, the polyacrylic acid has an average number average molecular weight of 2000; the concentration of the polyacrylic acid dissolved in water is 4.0mg/mL;
particularly preferably, the concentration of the ferric salt in the mixed solution is 500mmol/L, and the concentration of the ferrous salt is 250mmol/L;
particularly preferably, the iron salt is ferric chloride hexahydrate and the ferrous salt is ferrous sulfate heptahydrate.
6. The method of manufacturing according to claim 4, wherein: the hypoxia-sensitive small molecules comprise 2-nitroimidazole small molecules, and preferably, the preparation method of the 2-nitroimidazole small molecules comprises the following steps:
Mixing 4- (aminomethyl) piperidine and benzaldehyde, dissolving in a first solvent, and stirring at 120-130 for reaction for 36-48h to obtain an intermediate product;
dissolving the intermediate product and 1- (2, 3-epoxypropyl) -2-nitroimidazole in a second solvent in a protective atmosphere, stirring and reacting for 72-84 hours, then adding hydrochloric acid solution, and continuing stirring and reacting for 4-5 hours at 40-45 to prepare the 2-nitroimidazole micromolecule;
preferably, the first solvent comprises toluene and the second solvent comprises ethanol;
preferably, the molar ratio of 4- (aminomethyl) piperidine to benzaldehyde is 1:1, a step of;
preferably, the molar ratio of the intermediate product to 1 (2, 3-epoxypropyl) -2-nitroimidazole is greater than 1:1.
7. the method of manufacturing according to claim 6, comprising:
the carboxyl groups of the ferric oxide nano particles of the surface modified polyacrylic acid are activated for 2 to 3 hours by adopting 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, then cysteine is added for reaction for 18 to 24 hours, and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are continuously usedImide is activated for 2-3h, and then the 2-nitroimidazole micromolecule is added for reaction for 18-24h, so that T with surface connected with the hypoxia response of cysteine and the 2-nitroimidazole micromolecule is obtained 1 -T 2 A switchable MRI contrast agent; preferably, the solvents used in the activation of carboxyl groups are MES buffer solutions with the pH value of 5.8-6.0 and the concentration of 40-60mmol/L, the solvents used in the reaction with cysteine are water, and the solvents used in the reaction with 2-nitroimidazole small molecules are PBS buffer solutions with the pH value of 7.2-7.4; preferably, the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1:1, a step of; preferably, the molar ratio of cysteine to 2-nitroimidazole small molecule is greater than 20:1.
8. a T of hypoxia response as defined in any one of claims 1 to 3 1 -T 2 Use of a switchable MRI contrast agent for the preparation of a product with tumor detection function.
9. A composition for contrast, characterized by comprising: a T of hypoxia response as defined in any one of claims 1 to 3 1 -T 2 A switchable MRI contrast agent; and, a pharmaceutically acceptable adjuvant, preferably, the pharmaceutically acceptable adjuvant includes a diluent.
10. A method of imaging a non-medical object, comprising: administering to a subject to be imaged a T of hypoxia response as defined in any one of claims 1 to 3 1 -T 2 A switchable MRI contrast agent or the composition for contrast according to claim 9, and performing contrast.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211228073.7A CN117883596A (en) | 2022-10-09 | 2022-10-09 | T of hypoxia response 1 -T 2 Switchable MRI contrast agent, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211228073.7A CN117883596A (en) | 2022-10-09 | 2022-10-09 | T of hypoxia response 1 -T 2 Switchable MRI contrast agent, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117883596A true CN117883596A (en) | 2024-04-16 |
Family
ID=90639842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211228073.7A Pending CN117883596A (en) | 2022-10-09 | 2022-10-09 | T of hypoxia response 1 -T 2 Switchable MRI contrast agent, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117883596A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118121729A (en) * | 2024-04-29 | 2024-06-04 | 中国科学院苏州纳米技术与纳米仿生研究所 | PH-GSH sequential response T2-T1-T2 switching contrast agent and preparation method and application thereof |
-
2022
- 2022-10-09 CN CN202211228073.7A patent/CN117883596A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118121729A (en) * | 2024-04-29 | 2024-06-04 | 中国科学院苏州纳米技术与纳米仿生研究所 | PH-GSH sequential response T2-T1-T2 switching contrast agent and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11324841B2 (en) | Metal oxide nanoparticle-based magnetic resonance imaging contrast agent with a central cavity | |
| US20170216463A1 (en) | Magnetic Nanoparticles | |
| Gómez-Vallejo et al. | PEG-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney MRI contrast agents | |
| CN103212093B (en) | A kind of have cell targeted magnetic Nano material and biomedical applications thereof | |
| US9457104B2 (en) | Hydrophilic nanoparticles surface-modified with monosaccharide phosphate or monosaccharide phosphate derivatives, its colloidal solution and use thereof | |
| Hu et al. | High-performance nanostructured MR contrast probes | |
| CN110585237B (en) | Nano diagnosis and treatment agent and preparation method and application thereof | |
| JPH06506468A (en) | Melanin image enhancer | |
| US20090317327A1 (en) | Aqueous Dispersion of Superparamagnetic Single-Domain Particles, Production and Use Thereof in Diagnosis and Therapy | |
| KR20090119867A (en) | Mri t1 contrasting agent comprising manganese oxide nanoparticle | |
| CN102526769A (en) | Double-developer for CT and MRI simultaneously and preparation method thereof | |
| CN109626439B (en) | Metal-doped ferrite nano material, preparation method of magnetic nano particles containing metal-doped ferrite nano material and application of magnetic nano particles | |
| CN117883596A (en) | T of hypoxia response 1 -T 2 Switchable MRI contrast agent, preparation method and application thereof | |
| CN101444630A (en) | Method for preparing high magnetic resonance sensitivity ferroferric oxide nano-particle with tumor-targeting function | |
| CN106421823A (en) | Preparation method of amphoteric ion modified ultra-fine iron oxide particles | |
| Liang et al. | Synthesis of NaYF4: Yb, Er upconversion nanoparticle-based optomagnetic multifunctional composite for drug delivery system | |
| Arkaban et al. | Imaging and therapeutic capabilities of the AuNPs@ MnCO3/Mn3O4, coated with PAA and integrated with folic acid, doxorubicin and propidium iodide for murine breast cancer | |
| CN111388668B (en) | Magnetic composite nano material and preparation method and application thereof | |
| GB2568434A (en) | Polymer-metal oxide complex, preparation method therefor, and applications | |
| CN103083688A (en) | Ferrate magnetic nanocomposite with core-shell structure, preparation method and application thereof | |
| JP2015508796A (en) | Superparamagnetic nanoparticles with PEG-substituted α-hydroxyphosphonate outer shell | |
| CN112870387B (en) | Magnetic nano-drug carrier and preparation method and application thereof | |
| CN113616815B (en) | pH responsive T 1 /T 2 Switching type MRI contrast agent and preparation method and application thereof | |
| CN114344489A (en) | Multi-modal FePt @ Fe3O4Nano contrast agent and preparation method and application thereof | |
| Yao et al. | Multifunctional ferritin nanocages for bimodal imaging and targeted delivery of doxorubicin into cancer cells |
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 |