WO2024119373A1 - Use of aggregation-induced light-emitting engineered mitochondrion in preparation of medicament for treating cancer - Google Patents
Use of aggregation-induced light-emitting engineered mitochondrion in preparation of medicament for treating cancer Download PDFInfo
- Publication number
- WO2024119373A1 WO2024119373A1 PCT/CN2022/136952 CN2022136952W WO2024119373A1 WO 2024119373 A1 WO2024119373 A1 WO 2024119373A1 CN 2022136952 W CN2022136952 W CN 2022136952W WO 2024119373 A1 WO2024119373 A1 WO 2024119373A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dcpy
- aggregation
- mitochondria
- mito
- engineered
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 97
- 210000003470 mitochondria Anatomy 0.000 title claims abstract description 77
- 201000011510 cancer Diseases 0.000 title claims abstract description 55
- 230000002776 aggregation Effects 0.000 title claims abstract description 41
- 238000004220 aggregation Methods 0.000 title claims abstract description 41
- 239000003814 drug Substances 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000000463 material Substances 0.000 claims abstract description 20
- 238000011282 treatment Methods 0.000 claims description 37
- 238000004020 luminiscence type Methods 0.000 claims description 27
- 230000014509 gene expression Effects 0.000 claims description 21
- 229940079593 drug Drugs 0.000 claims description 10
- 108010090931 Proto-Oncogene Proteins c-bcl-2 Proteins 0.000 claims description 9
- 102000013535 Proto-Oncogene Proteins c-bcl-2 Human genes 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 108090000397 Caspase 3 Proteins 0.000 claims description 8
- 102000003952 Caspase 3 Human genes 0.000 claims description 8
- 230000002438 mitochondrial effect Effects 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 230000005775 apoptotic pathway Effects 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- YOBTXORLVXZWSR-UHFFFAOYSA-M 1,4-dimethylpyridin-1-ium;iodide Chemical compound [I-].CC1=CC=[N+](C)C=C1 YOBTXORLVXZWSR-UHFFFAOYSA-M 0.000 claims description 3
- UJOBWOGCFQCDNV-UHFFFAOYSA-N Carbazole Natural products C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 claims description 3
- DMBHHRLKUKUOEG-UHFFFAOYSA-N N-phenyl aniline Natural products C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 150000003904 phospholipids Chemical class 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 3
- HZNVUJQVZSTENZ-UHFFFAOYSA-N 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(C#N)=C(C#N)C1=O HZNVUJQVZSTENZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 71
- 230000000975 bioactive effect Effects 0.000 abstract description 7
- 231100000433 cytotoxic Toxicity 0.000 abstract description 2
- 230000001472 cytotoxic effect Effects 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 230000037353 metabolic pathway Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 22
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 21
- 239000002953 phosphate buffered saline Substances 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 18
- 108091006296 SLC2A1 Proteins 0.000 description 11
- 230000001225 therapeutic effect Effects 0.000 description 11
- 238000002560 therapeutic procedure Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 235000015277 pork Nutrition 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 7
- 230000006907 apoptotic process Effects 0.000 description 7
- 230000034659 glycolysis Effects 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 6
- 230000004888 barrier function Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000011275 oncology therapy Methods 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241000699660 Mus musculus Species 0.000 description 5
- 230000006536 aerobic glycolysis Effects 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000010354 integration Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 230000035515 penetration Effects 0.000 description 5
- 230000002195 synergetic effect Effects 0.000 description 5
- 238000004435 EPR spectroscopy Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000035806 respiratory chain Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- 238000011729 BALB/c nude mouse Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- 230000001640 apoptogenic effect Effects 0.000 description 3
- 230000005880 cancer cell killing Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000002296 dynamic light scattering Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000004065 mitochondrial dysfunction Effects 0.000 description 3
- 238000012900 molecular simulation Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000009702 cancer cell proliferation Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000004898 mitochondrial function Effects 0.000 description 2
- 210000001700 mitochondrial membrane Anatomy 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 238000005424 photoluminescence Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000000700 radioactive tracer Substances 0.000 description 2
- 238000001959 radiotherapy 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
- 238000010186 staining Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- VCUVETGKTILCLC-UHFFFAOYSA-N 5,5-dimethyl-1-pyrroline N-oxide Chemical compound CC1(C)CCC=[N+]1[O-] VCUVETGKTILCLC-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 101000911513 Homo sapiens Uncharacterized protein FAM215A Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 101710180313 Protease 3 Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 102100026728 Uncharacterized protein FAM215A Human genes 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000006567 cellular energy metabolism Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- FDPIMTJIUBPUKL-UHFFFAOYSA-N dimethylacetone Natural products CCC(=O)CC FDPIMTJIUBPUKL-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001362 electron spin resonance spectrum Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000006571 energy metabolism pathway Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000002496 iodine Chemical class 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000005641 tunneling Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the aggregation-induced emission material is DCPy.
- the present invention provides a method for preparing aggregation-induced luminescence engineered mitochondria and its application as a cancer treatment drug.
- the cancer treatment drug can specifically be a microwave power therapy sensitizer, and the raw materials or reagents designed by the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified. Therefore, compared with the current technology, the present invention provides a microwave therapy sensitizer with a simple synthesis method and good therapeutic effect, which can produce a strong cancer cell killing effect, and can be used for the synergistic treatment of cancer with microwave power and mitochondria, has no toxic side effects, and has good application prospects.
- FIG8 is a fluorescence image of mitochondria labeled with DCPy red probe (10 ⁇ g/mL) after separation.
- TEM Transmission electron microscopy
- a field emission gun of a Tecnai G2S-Twin UV-visible absorption spectra were performed using a U-3100 spectrophotometer (Hitachi).
- Photoluminescence (PL) spectra were performed using an Edinburgh F900 fluorescence spectrometer with a xenon arc lamp.
- In vivo fluorescence imaging was performed by In vivo Smart-LF was obtained.
- Flow cytometry was used to analyze the flow cytometer (BD Accuri C6).
- DCPy and 200 ⁇ g/mL Mito lysate were mixed in PBS solution, ultrasonicated for 10 minutes, stirred for 1 hour, and the excess cell suspension was filtered into a Millipore tube (MWCO 100 kDa;), centrifuged at 3500 rpm for 20 minutes, and the resulting precipitate was washed several times.
- the optimal loading ratio between DCPy and Mito was determined to be 1:9 by measuring UV absorption, in w/w units.
- DCPy on mitochondria was imaged by confocal microscopy (Leica, Germany).
- PANC-1 cells were cultured in culture dishes at 4 ⁇ 10 4 cells mL-1 for 2 days.
- DCPy and Mito@DCPy were then added to the cells for culture.
- the cells were collected after removing the unbound nanoparticles and washing with fresh culture buffer and then observed by flow cytometry or stained with Hoechst and further imaged by confocal fluorescence.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Zoology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Provided is use of an aggregation-induced light-emitting engineered mitochondrion in the preparation of a medicament for treating cancer. The aggregation-induced light-emitting engineered mitochondrion is a mitochondrion modified by an aggregation-induced light-emitting material. The aggregation-induced light-emitting material is DCPy. Provided is a microwave sensitizer based on an engineered bioactive mitochondrion which has the capacity to efficiently generate cytotoxic ROS and is capable of regulating the metabolic pathway of cancer cells. The sensitizer can be used for synergistically treating cancer by means of microwave power and the mitochondrion.
Description
本发明涉及癌症药物领域，特别涉及聚集诱导发光工程线粒体在制备治疗癌症的药物中的应用。The present invention relates to the field of cancer medicine, and in particular to the application of aggregation-induced luminescence engineered mitochondria in the preparation of medicines for treating cancer.
癌症作为威胁人类健康的重大疾病之一，一直受到研究者的高度重视。随着发病率和死亡率的不断增加，癌症已经成为一个重大的全球公共卫生问题。目前癌症主要的治疗方式有手术，化疗，放疗，免疫治疗等，但是这些传统的治疗方式也存在着一些弊端，比如手术易复发，放疗因辐射抵抗的问题使得放疗效果不理想，化疗因药物选择性差会带来严重的副作用等。因此，生物治疗等新兴的癌症治疗方式为了解决现有技术中存在的问题也在不断涌现。Cancer, as one of the major diseases threatening human health, has always been highly valued by researchers. With the continuous increase in morbidity and mortality, cancer has become a major global public health issue. At present, the main treatments for cancer include surgery, chemotherapy, radiotherapy, immunotherapy, etc. However, these traditional treatments also have some disadvantages, such as surgery is prone to recurrence, radiotherapy has unsatisfactory effects due to radiation resistance, and chemotherapy has serious side effects due to poor drug selectivity. Therefore, emerging cancer treatments such as biological therapy are constantly emerging to solve the problems existing in existing technologies.
微波动态癌症疗法与传统癌症疗法相比，具有组织穿透深、肿瘤消融量大、手术痛苦小、副作用小等优点，可以在肿瘤区域产生ROS，并诱导癌细胞凋亡。在微波治疗中，适合的增敏剂是治疗顺利发挥效果的基础。在微波治疗中，不适当的增敏剂，如一些金属离子的毒性或高浓度增敏剂，可能会引起严重的副作用。聚合诱导发光(AIE)由唐本忠院士团队于2001年首次提出。由于聚合诱导发光剂(AIEgen)在聚合状态下具有独特的发光特性以及在细胞毒性ROS生成能力方面表现出的高效率，其作为微波动力疗法的增敏剂运用于癌症治疗领域，可以使微波动力治疗达到预期的杀伤癌细胞和治疗效果。Compared with traditional cancer therapy, microwave dynamic cancer therapy has the advantages of deep tissue penetration, large tumor ablation volume, less surgical pain, and fewer side effects. It can generate ROS in the tumor area and induce apoptosis of cancer cells. In microwave therapy, suitable sensitizers are the basis for the smooth effect of treatment. In microwave therapy, inappropriate sensitizers, such as the toxicity of some metal ions or high concentrations of sensitizers, may cause serious side effects. Aggregation-induced emission (AIE) was first proposed by the team of Academician Tang Benzhong in 2001. Because the aggregation-induced emission agent (AIEgen) has unique luminescence properties in the aggregated state and exhibits high efficiency in the generation of cytotoxic ROS, it is used as a sensitizer for microwave dynamic therapy in the field of cancer treatment, which can enable microwave dynamic therapy to achieve the expected killing of cancer cells and therapeutic effects.
此外，除了增敏剂的作用外，癌细胞中线粒体功能失调导致的Bcl-2蛋白的过度表达会提高癌细胞的抗氧化能力，削弱癌细胞中ROS的破坏能力，从而使癌细胞的存活率明显提高，削弱了微波治疗的效果。此外，功能失调的线粒体可以通过Bcl-2蛋白的过度表达影响癌细胞的凋亡途径，从而维持癌细胞的增殖。它们还影响癌细胞的新陈代谢，使其能够打开类似缺氧的途径，导致细胞凋亡的减少和治疗的阻力。因此，线粒体 功能失调将大大削弱微波治疗的效果，这是一个亟待解决的问题。因此，开发一种整合AIEgen增敏剂(DCPy)和生物活性线粒体的AIE工程生物活性线粒体(AEBM)用于微波动力协同治疗癌症，将具有良好的应用前景。In addition to the effects of sensitizers, overexpression of Bcl-2 protein caused by mitochondrial dysfunction in cancer cells will increase the antioxidant capacity of cancer cells and weaken the destructive ability of ROS in cancer cells, thereby significantly improving the survival rate of cancer cells and weakening the effect of microwave therapy. In addition, dysfunctional mitochondria can affect the apoptotic pathway of cancer cells through overexpression of Bcl-2 protein, thereby maintaining the proliferation of cancer cells. They also affect the metabolism of cancer cells, enabling them to open hypoxia-like pathways, leading to a decrease in apoptosis and resistance to treatment. Therefore, mitochondrial dysfunction will greatly weaken the effect of microwave therapy, which is an urgent problem to be solved. Therefore, the development of an AIE-engineered bioactive mitochondria (AEBM) that integrates AIEgen sensitizers (DCPy) and bioactive mitochondria for microwave-powered synergistic cancer treatment will have good application prospects.
发明内容Summary of the invention
针对现有技术中的缺陷，本发明提出了聚集诱导发光工程线粒体在制备治疗癌症的药物中的应用，所述聚集诱导发光工程线粒体通过整合AIEgen增敏剂(DCPy)和生物活性线粒体，用于微波动力协同治疗癌症。In view of the defects in the prior art, the present invention proposes the use of aggregation-induced emission engineered mitochondria in the preparation of drugs for treating cancer. The aggregation-induced emission engineered mitochondria integrate AIEgen sensitizers (DCPy) and biologically active mitochondria for microwave-powered synergistic treatment of cancer.
本发明提供聚集诱导发光工程线粒体在制备治疗癌症的药物中的应用；所述聚集诱导发光工程线粒体为聚集诱导发光材料修饰的线粒体。The present invention provides the use of aggregation-induced luminescence engineered mitochondria in the preparation of drugs for treating cancer; the aggregation-induced luminescence engineered mitochondria are mitochondria modified with aggregation-induced luminescence materials.
进一步的，所述聚集诱导发光材料为DCPy。Furthermore, the aggregation-induced emission material is DCPy.
进一步的，所述DCPy分子被嵌入线粒体的磷脂双分子层中。Furthermore, the DCPy molecules are embedded in the phospholipid bilayer of mitochondria.
进一步的，所述聚集诱导发光工程线粒体的制备方法如下：Furthermore, the preparation method of the aggregation-induced luminescence engineered mitochondria is as follows:
S1：7-(二苯胺)-9-乙基-9H-(咔唑)-2-碳醛溶液在乙醇中加入1,4-二甲基碘化吡啶和哌啶，室温下回流3小时；当冷却到室温时，产品收集，洗涤，冷冻干燥，得到红色碘盐固体；S1: 1,4-dimethylpyridinium iodide and piperidine were added to a solution of 7-(diphenylamine)-9-ethyl-9H-(carbazole)-2-carbaldehyde in ethanol and refluxed at room temperature for 3 hours; when cooled to room temperature, the product was collected, washed, and freeze-dried to obtain a red iodized salt solid;
S2：用丙酮溶解所述红色碘盐固体，滴加六氟磷酸钾溶液；将制备好的混合物进一步充分搅拌纯化得到聚集诱导发光材料；S2: dissolving the red iodized salt solid with acetone, and adding potassium hexafluorophosphate solution dropwise; further stirring and purifying the prepared mixture to obtain an aggregation-induced luminescence material;
S3：将所述聚集诱导发光材料和线粒体裂解液与PBS溶液充分混合，超声，搅拌；把多余的细胞悬液过滤进入微孔管，离心，洗涤沉淀，得到聚集诱导发光工程线粒体。S3: The aggregation-induced luminescence material and mitochondrial lysate are fully mixed with the PBS solution, ultrasonicated, and stirred; the excess cell suspension is filtered into a microporous tube, centrifuged, and the precipitate is washed to obtain the aggregation-induced luminescence engineered mitochondria.
进一步的，所述聚集诱导发光工程线粒体使Bcl-2蛋白表达减少，pro-caspase3蛋白表达增加，从而重新激活凋亡途径。Furthermore, the aggregation-induced luminescence engineered mitochondria reduce the expression of Bcl-2 protein and increase the expression of pro-caspase3 protein, thereby reactivating the apoptosis pathway.
进一步的，所述聚集诱导发光工程线粒体具有生成活性氧的能力。Furthermore, the aggregation-induced luminescence engineered mitochondria have the ability to generate reactive oxygen species.
进一步的，所述药物为微波增敏剂。Furthermore, the drug is a microwave sensitizer.
本发明还提供聚集诱导发光工程线粒体在治疗癌症中的应用，所述聚集诱导发光工 程线粒体为聚集诱导发光材料修饰的线粒体。The present invention also provides the use of aggregation-induced luminescence engineered mitochondria in the treatment of cancer, wherein the aggregation-induced luminescence engineered mitochondria are mitochondria modified with aggregation-induced luminescence materials.
进一步的，所述聚集诱导发光材料为DCPy。Furthermore, the aggregation-induced emission material is DCPy.
进一步的，所述应用中需要使用微波照射。Furthermore, the application requires the use of microwave irradiation.
综上，与现有技术相比，本发明达到了以下技术效果：In summary, compared with the prior art, the present invention achieves the following technical effects:
本发明提供了聚集诱导发光工程线粒体的制备方法以及其作为癌症治疗药物的应用。所述癌症治疗药物具体可为一种微波动力治疗增敏剂，其发明设计的原料或试剂均为普通市售产片，涉及到的操作如无特殊说明均为本领域常规操作。因此，相较于目前的技术，本发明提供了一种合成方法简单，治疗效果良好的微波治疗增敏剂，能够产生很强的癌细胞杀伤效果，并可用于微波动力与线粒体的协同治疗癌症，无毒副作用，具有良好的应用前景。The present invention provides a method for preparing aggregation-induced luminescence engineered mitochondria and its application as a cancer treatment drug. The cancer treatment drug can specifically be a microwave power therapy sensitizer, and the raw materials or reagents designed by the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified. Therefore, compared with the current technology, the present invention provides a microwave therapy sensitizer with a simple synthesis method and good therapeutic effect, which can produce a strong cancer cell killing effect, and can be used for the synergistic treatment of cancer with microwave power and mitochondria, has no toxic side effects, and has good application prospects.
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.
图1为本发明AIE工程生物活性线粒体的制备及其在微波动力癌症治疗中的应用。GLUT1：葡萄糖转运体1，MDT：微波动力癌症治疗，OXPHOS：氧化磷酸化。Figure 1 shows the preparation of AIE-engineered bioactive mitochondria of the present invention and its application in microwave-powered cancer therapy. GLUT1: glucose transporter 1, MDT: microwave-powered cancer therapy, OXPHOS: oxidative phosphorylation.
图2为(A,B)DCPy整合前后的线粒体TEM图像。比例尺为0.5μm。(C)呼吸链中DCPy修饰前后，分离的线粒体的复合物I的生物活性。(D)Mito、DCPy和Mito@DCPy的UV-Vis曲线和(E)荧光曲线。(F)DCPy与线粒体膜整合的分子模拟。(G)在微波辐射下，H2DCF-DA与或不与DCPy和Mito@DCPy在PBS中的525nm处的荧光强度随时间的变化。(H)-OH和(I)O的EPR图谱。-OH和(I)1O2，是由Mito、DCPy和Mito@DCPy样品产生。Figure 2 shows (A, B) TEM images of mitochondria before and after DCPy integration. The scale bar is 0.5 μm. (C) Biological activity of complex I of isolated mitochondria before and after DCPy modification in the respiratory chain. (D) UV-Vis curves and (E) fluorescence curves of Mito, DCPy and Mito@DCPy. (F) Molecular simulation of DCPy integration into mitochondrial membrane. (G) Changes in the fluorescence intensity of H2DCF-DA with or without DCPy and Mito@DCPy at 525 nm in PBS over time under microwave irradiation. EPR spectra of (H)-OH and (I)O. -OH and (I)1O2 are generated by Mito, DCPy and Mito@DCPy samples.
图3为(A)细胞对DCPy和Mito@DCPy的吸收。DCPy和Mito@DCPy在不同时间点的荧光图像。蓝色，Hoechst；红色，DCPy。(B)细胞共聚焦实验。(b-e)PANC-1细胞用以下染色剂染色的共聚焦图像：(b)Hoechst(c1)Mito-Tracker，(c2)ER-Tracker，(c3)Lyso-Tracker，(d1-d3)Mito@DCPy。(e1-e3)合并的共聚焦图像(c和d板)。(f1-f3) 散点图图像代表c和d图像之间的校正因子指数。比例尺为20μm。Figure 3 shows (A) cellular uptake of DCPy and Mito@DCPy. Fluorescence images of DCPy and Mito@DCPy at different time points. Blue, Hoechst; red, DCPy. (B) Cell confocal experiment. (b-e) Confocal images of PANC-1 cells stained with the following stains: (b) Hoechst (c1) Mito-Tracker, (c2) ER-Tracker, (c3) Lyso-Tracker, (d1-d3) Mito@DCPy. (e1-e3) Merged confocal images (c and d panels). (f1-f3) Scatter plot images represent the correction factor index between c and d images. Scale bar is 20 μm.
图4(A)与PBS、DCPy、Mito和Mito@DCPy共同培养后的细胞存活，有/无微波。(B)通过Western blotting检测四种不同处理下的原钙化酶3蛋白表达水平。(C)与DMEM、DCPy、Mito和Mito@DCPy共同培养时细胞的GLUT1浓度。(D)与DMEM、DCPy、Mito和Mito@DCPy共同培养时细胞的乳糖产量。Figure 4 (A) Cell survival after co-culture with PBS, DCPy, Mito, and Mito@DCPy, with/without microwaves. (B) Protocalcitonin 3 protein expression levels under four different treatments detected by Western blotting. (C) GLUT1 concentration of cells co-cultured with DMEM, DCPy, Mito, and Mito@DCPy. (D) Lactose production of cells co-cultured with DMEM, DCPy, Mito, and Mito@DCPy.
图5(A)注射后24小时，通过生物成像进行原发器官和肿瘤的图像。T代表肿瘤；K代表肾脏；Lu代表肺部；S代表脾脏；Li代表肝脏；H代表心脏；(B)通过平均荧光强度对DCPy和Mito@DCPy在实验性裸鼠体内的生物分布进行半定量分析。(C)给予PBS、DCPy、Mito、MW、Mito@DCPy和Mito@DCPy、5分钟MW后，实验性裸鼠的肿瘤体积变化。(D)给予PBS、DCPy、Mito、MW、Mito@DCPy和Mito@DCPy，5分钟MW后实验性裸鼠的体重变化。(E)给予PBS、Mito@DCPy L、Mito@DCPy L+B、Mito@DCPy MW和Mito@DCPy MW+B后裸鼠的肿瘤生长曲线。(F)实验小鼠在Mito@DCPy L、Mito@DCPy L+B、Mito@DCPy MW和Mito@DCPy MW+B后的体重变化(L代表白光，B代表3毫米猪肉屏障)。Figure 5 (A) Images of primary organs and tumors by bioimaging 24 hours after injection. T represents tumor; K represents kidney; Lu represents lung; S represents spleen; Li represents liver; H represents heart; (B) Semi-quantitative analysis of the biodistribution of DCPy and Mito@DCPy in experimental nude mice by mean fluorescence intensity. (C) Changes in tumor volume of experimental nude mice after administration of PBS, DCPy, Mito, MW, Mito@DCPy and Mito@DCPy, MW for 5 minutes. (D) Changes in body weight of experimental nude mice after administration of PBS, DCPy, Mito, MW, Mito@DCPy and Mito@DCPy, MW for 5 minutes. (E) Tumor growth curves of nude mice after administration of PBS, Mito@DCPy L, Mito@DCPy L+B, Mito@DCPy MW and Mito@DCPy MW+B. (F) Body weight changes of experimental mice after Mito@DCPy L, Mito@DCPy L+B, Mito@DCPy MW and Mito@DCPy MW+B (L represents white light and B represents 3 mm pork barrier).
图6为本发明线粒体的蛋白质浓度与分离的线粒体数量之间存在正相关。FIG. 6 shows that there is a positive correlation between the protein concentration of the mitochondria of the present invention and the number of isolated mitochondria.
图7(A)PBS中的Mito和(B)PBS中的Mito@DCPy的DLS大小分布。FIG. 7 DLS size distribution of (A) Mito in PBS and (B) Mito@DCPy in PBS.
图8为DCPy红色探针(10μg/mL)标记的线粒体分离后的荧光图像。FIG8 is a fluorescence image of mitochondria labeled with DCPy red probe (10 μg/mL) after separation.
图9为不同浓度的分离线粒体培养后的细胞存活率。FIG. 9 shows the cell survival rate after incubation with different concentrations of isolated mitochondria.
图10为本发明Mito、DCPy和Mito@DCPy的Zeta电位。FIG. 10 shows the Zeta potential of Mito, DCPy and Mito@DCPy of the present invention.
图11为原钙蛋白酶3的表达水平(WB检测)。在微波照射(5瓦，5分钟)之前，用DMEM、Mito、DCPy和Mito@DCPy对PANC-1细胞进行了6小时的培养。然后，PANC-1被培养了24小时。得到不同条件下处理24小时后的细胞裂解液。Figure 11 shows the expression level of protocalpain 3 (WB detection). PANC-1 cells were cultured for 6 hours with DMEM, Mito, DCPy and Mito@DCPy before microwave irradiation (5 watts, 5 minutes). Then, PANC-1 was cultured for 24 hours. Cell lysates were obtained after 24 hours of treatment under different conditions.
图12为Bcl-2蛋白的表达水平(WB检测)。用DMEM、Mito、DCPy和Mito@DCPy对PANC-1细胞进行了24小时的处理。Figure 12 shows the expression level of Bcl-2 protein (WB detection). PANC-1 cells were treated with DMEM, Mito, DCPy and Mito@DCPy for 24 hours.
图13为通过Western blotting检测Bcl-2蛋白的表达水平。Figure 13 shows the expression level of Bcl-2 protein detected by Western blotting.
图14为注射后1小时，通过生物成像进行原发器官和肿瘤的图像。T代表肿瘤；K代表肾脏；Lu代表肺部；S代表脾脏；Li代表肝脏；H代表心脏；(B)基于(A)的 半定量生物分布在Balb/c裸体小鼠。Figure 14 shows images of primary organs and tumors by bioimaging 1 hour after injection. T represents tumor; K represents kidney; Lu represents lung; S represents spleen; Li represents liver; H represents heart; (B) Semi-quantitative biodistribution of (A) in Balb/c nude mice.
图15为本发明各种处理后第15天肿瘤的H&E染色。FIG. 15 is H&E staining of the tumor on day 15 after various treatments of the present invention.
图16为本发明对各自治疗后第15天从小鼠身上剥离的肿瘤进行TUNEL检测。绿色荧光代表由TUNEL检测试剂盒染色的凋亡细胞。蓝色荧光代表被DAPI染色的细胞核。Figure 16 shows the TUNEL detection of tumors peeled off from mice on the 15th day after treatment. Green fluorescence represents apoptotic cells stained by TUNEL detection kit. Blue fluorescence represents cell nuclei stained by DAPI.
图17为本发明从不同组的心脏、肾脏、脾脏、肺脏、肝脏获得组织学数据(所有面板的比例尺为1毫米)。FIG. 17 shows histological data obtained from different groups of heart, kidney, spleen, lung, and liver in the present invention (the scale bar in all panels is 1 mm).
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
最近的研究发现，在癌细胞中，由代谢物(如乳酸等)调控的Bcl-2家族蛋白的表达被活跃的糖酵解所促进，以维持生存。因此，随着Bcl-2的表达减少，对癌症的治疗将更有效地杀死癌细胞。此外，线粒体功能失调可能导致对细胞凋亡和生存的抵抗，以及缺氧样途径的激活，从而形成肿瘤。基于上述事实，将正常细胞中的功能性线粒体移植到癌细胞中，让癌细胞恢复正常的线粒体功能，可能对癌症治疗有帮助。为达到更好的治疗效果，将正常细胞中的线粒体功能化后移植到癌细胞，让癌细胞恢复正常的线粒体功能，可以加强治疗的效果。Recent studies have found that in cancer cells, the expression of Bcl-2 family proteins regulated by metabolites (such as lactate, etc.) is promoted by active glycolysis to maintain survival. Therefore, as the expression of Bcl-2 decreases, cancer treatment will be more effective in killing cancer cells. In addition, mitochondrial dysfunction may lead to resistance to apoptosis and survival, as well as activation of hypoxia-like pathways, leading to tumor formation. Based on the above facts, transplanting functional mitochondria from normal cells into cancer cells to restore normal mitochondrial function in cancer cells may be helpful for cancer treatment. In order to achieve better therapeutic effects, functionalizing mitochondria in normal cells and transplanting them into cancer cells to restore normal mitochondrial function in cancer cells can enhance the effect of treatment.
实施例1实验耗材和方法Example 1 Experimental consumables and methods
(1)材料和试剂(1) Materials and reagents
本发明中使用的青霉素-链霉素、胎牛血清(简称FBS)、磷酸盐缓冲盐水(简称PBS)和DMEM均购自Gibco Life Technologies。二甲基亚砜和丙酮购自Aladdin。CCK-8购自中国的Beyotime公司。本发明中使用的其他材料和试剂都是分析试剂级的。Penicillin-streptomycin, fetal bovine serum (FBS), phosphate buffered saline (PBS), and DMEM used in the present invention were purchased from Gibco Life Technologies. Dimethyl sulfoxide and acetone were purchased from Aladdin. CCK-8 was purchased from Beyotime, China. Other materials and reagents used in the present invention were of analytical reagent grade.
(2)实验仪器(2) Experimental instruments
透射电子显微镜(TEM)使用Tecnai G2S-Twin的场发射枪进行记录。紫外-可见光 吸收光谱分析是用U-3100分光光度计(日立)实现的。爱丁堡F900荧光光谱仪用氙气弧光灯进行光致发光(PL)光谱分析。体内荧光成像由
In vivo Smart-LF获得。流动细胞的分析是通过流式细胞仪(BD Accuri C6)获得的。
Transmission electron microscopy (TEM) was recorded using a field emission gun of a Tecnai G2S-Twin. UV-visible absorption spectra were performed using a U-3100 spectrophotometer (Hitachi). Photoluminescence (PL) spectra were performed using an Edinburgh F900 fluorescence spectrometer with a xenon arc lamp. In vivo fluorescence imaging was performed by In vivo Smart-LF was obtained. Flow cytometry was used to analyze the flow cytometer (BD Accuri C6).
(3)本发明的聚集诱导材料的制备(3) Preparation of the aggregation inducing material of the present invention
7-(二苯胺)-9-乙基-9H-(咔唑)-2-碳醛(1.28mmol,0.5g)溶液在15mL乙醇中加入1,4-二甲基碘化吡啶(1.16mmol,0.27g)和哌啶(1滴)在室温下回流3小时。当冷却到室温时，产品收集，洗涤3次，冷冻干燥，得到红色碘盐固体(80％，0.56g)，用丙酮溶解固体产物，滴加20mL KPF
6溶液。将制备好的混合物进一步充分搅拌25分钟纯化得到最终产物DCPy(99％，0.57g)，也就是聚集诱导发光材料。
7-(Diphenylamine)-9-ethyl-9H-(carbazole)-2-carbonaldehyde (1.28mmol, 0.5g) solution was added to 15mL ethanol, 1,4-dimethylpyridinium iodide (1.16mmol, 0.27g) and piperidine (1 drop) were refluxed at room temperature for 3 hours. When cooled to room temperature, the product was collected, washed 3 times, and freeze-dried to obtain a red iodine salt solid (80%, 0.56g). The solid product was dissolved in acetone and 20mL KPF 6 solution was added dropwise. The prepared mixture was further stirred for 25 minutes and purified to obtain the final product DCPy (99%, 0.57g), which is the aggregation-induced emission material.
(4)线粒体的提取(4) Mitochondrial extraction
按照制造商Beyotime的说明书，从Balb/c小鼠的肾脏问题中提取纯线粒体。为了量化提取的线粒体，用MitoTracker对肾脏细胞进行染色，MitoTracker被加入到DMEM培养基中并孵化30分钟。通过结合荧光强度检测和BCA蛋白测定(Beyotime)，计算出提取的线粒体的平均数量。Pure mitochondria were extracted from the kidney problems of Balb/c mice according to the manufacturer's instructions, Beyotime. To quantify the extracted mitochondria, kidney cells were stained with MitoTracker, which was added to the DMEM medium and incubated for 30 minutes. The average number of extracted mitochondria was calculated by combining fluorescence intensity detection and BCA protein assay (Beyotime).
(5)DCPy修饰的线粒体(Mito@DCPy)的制备(5) Preparation of DCPy-modified mitochondria (Mito@DCPy)
将0.01～10mg/mL的DCPy和200μg/mL的Mito裂解液在PBS溶液中混合均匀，超声处理10分钟，搅拌1小时，将多余的细胞悬浮液过滤到Millipore管中(MWCO 100kDa；)，用离心机在3500rpm下离心20分钟，并将所得沉淀物洗涤数次。通过测量紫外线吸收，确定DCPy和Mito之间的最佳装载比例为1:9，单位为w/w。0.01-10 mg/mL DCPy and 200 μg/mL Mito lysate were mixed in PBS solution, ultrasonicated for 10 minutes, stirred for 1 hour, and the excess cell suspension was filtered into a Millipore tube (MWCO 100 kDa;), centrifuged at 3500 rpm for 20 minutes, and the resulting precipitate was washed several times. The optimal loading ratio between DCPy and Mito was determined to be 1:9 by measuring UV absorption, in w/w units.
(6)Mito@DCPy的生物测定(6) Bioassay of Mito@DCPy
按照制造商的说明书(北京太阳神科技有限公司)检测呼吸链中复合物I的活性。The activity of complex I in the respiratory chain was detected according to the manufacturer's instructions (Beijing Sun God Technology Co., Ltd.).
(7)细胞培养(7) Cell culture
PANC-1的细胞在含有10％FBS和其他抗性成分的DMEM中培养，在37℃的条件下，使用5％的二氧化碳。PANC-1 cells were cultured in DMEM containing 10% FBS and other antimicrobial components at 37°C in the presence of 5% carbon dioxide.
(8)体外ROS生成的电子顺磁共振(EPR)和荧光检测(8) Electron paramagnetic resonance (EPR) and fluorescence detection of ROS generation in vitro
为了检测ROS的产生，将DCPy或DMSO中的Mito@DCPy加入DCFH-DA中，并记录所产生的荧光。为了检测-OH和1O2，分别加入DCPy、Mito和Mito@DCPy的DMPO。实验方法包括微波照射后检测ROS；通过EPR记录-OH和1O2的信号。To detect the generation of ROS, DCPy or Mito@DCPy in DMSO was added to DCFH-DA, and the generated fluorescence was recorded. To detect -OH and 1O2, DMPO of DCPy, Mito, and Mito@DCPy were added, respectively. The experimental method included the detection of ROS after microwave irradiation; the signals of -OH and 1O2 were recorded by EPR.
(9)细胞存活率(9) Cell survival rate
在微波照射(5瓦，5分钟)之前，用Mito(0.01毫克/毫升)、PBS、DCPy(0.01毫 克/毫升)和Mito@DCPy(0.1毫克/毫升)分别处理PANC-1细胞6小时。辐照后PANC-1培养24小时，用标准的CCK8检测法计算细胞的相对活力。PANC-1 cells were treated with Mito (0.01 mg/mL), PBS, DCPy (0.01 mg/mL), and Mito@DCPy (0.1 mg/mL) for 6 h before microwave irradiation (5 W, 5 min). PANC-1 cells were cultured for 24 h after irradiation, and the relative viability of cells was calculated using a standard CCK8 assay.
(10)细胞摄取(10) Cellular uptake
DCPy在线粒体上的位置通过共聚焦显微镜(Leica，德国)进行成像。PANC-1细胞以4×10
4个细胞mL-1在培养皿中培养2天。然后将DCPy和Mito@DCPy加入细胞中进行培养。在去除非结合的纳米颗粒并用新鲜的培养缓冲液清洗后收集细胞，然后用流式细胞仪观察或用Hoechst染色，进一步用共聚焦荧光成像。
The location of DCPy on mitochondria was imaged by confocal microscopy (Leica, Germany). PANC-1 cells were cultured in culture dishes at 4 × 10 4 cells mL-1 for 2 days. DCPy and Mito@DCPy were then added to the cells for culture. The cells were collected after removing the unbound nanoparticles and washing with fresh culture buffer and then observed by flow cytometry or stained with Hoechst and further imaged by confocal fluorescence.
(11)糖酵解试验(11) Glycolysis test
用Glycolysis Cell-based Assay Kit测量培养液中的乳酸浓度。未处理的细胞作为对照，然后分别加入分离的Mito、DCPy和Mito@DCPy，孵育24小时后，分别收集每孔的上清液，并根据说明书进行分析。The lactate concentration in the culture medium was measured using the Glycolysis Cell-based Assay Kit. Untreated cells were used as controls, and then isolated Mito, DCPy, and Mito@DCPy were added, respectively. After incubation for 24 h, the supernatant of each well was collected and analyzed according to the instructions.
(12)蛋白质印迹和酶联免疫吸附试验(12) Western blotting and enzyme-linked immunosorbent assay
按照制造商Beyotime的说明书获得不同条件下24小时处理后的细胞裂解液，20μL在80～200V电压下进行SDS-PAGE(12％凝胶)。转移的PVDF膜被0.2％的I-Block缓冲液封锁。分离的样品与兔单克隆抗体β-肌动蛋白、原细胞***酶3和Bcl-2进行孵化，然后与相应的二抗进行孵化。荧光信号用增强化学发光(ECL)检测器成像。酶联免疫吸附法用于定量测量葡萄糖转运体-1在其蛋白水平上的表达模式。葡萄糖转运体-1的浓度是通过使用商业ELISA试剂盒，按照制造商的说明书进行测量。Cell lysates after 24 h treatment under different conditions were obtained according to the manufacturer’s instructions of Beyotime, and 20 μL was subjected to SDS-PAGE (12% gel) at 80–200 V. The transferred PVDF membrane was blocked with 0.2% I-Block buffer. The separated samples were incubated with rabbit monoclonal antibodies to β-actin, protease 3, and Bcl-2, followed by incubation with the corresponding secondary antibodies. The fluorescence signals were imaged using an enhanced chemiluminescence (ECL) detector. Enzyme-linked immunosorbent assay was used to quantitatively measure the expression pattern of glucose transporter-1 at its protein level. The concentration of glucose transporter-1 was measured by using a commercial ELISA kit according to the manufacturer’s instructions.
(13)动物模型(13) Animal models
动物材料选自广东省医学实验动物中心。Balb/c雄性健康裸鼠，5周龄。随后的小鼠实验严格按照实验指南和机构规范进行，参考指南。SIAT的实验动物护理指南。PANC-1肿瘤模型的建立：将PANC-1细胞皮下注射到Balb/c裸鼠体内，统一注射部位在小鼠右背。在体内实验中，当肿瘤体积增长到约100mm
3时，观察治疗效果。
Animal materials were selected from Guangdong Medical Laboratory Animal Center. Balb/c male healthy nude mice, 5 weeks old. Subsequent mouse experiments were carried out in strict accordance with the experimental guidelines and institutional regulations, referring to the guidelines for laboratory animal care of SIAT. Establishment of PANC-1 tumor model: PANC-1 cells were subcutaneously injected into Balb/c nude mice, with the injection site uniformly located on the right back of the mice. In the in vivo experiment, the therapeutic effect was observed when the tumor volume grew to approximately 100 mm3 .
(14)体内抗肿瘤效果(14) Anti-tumor effect in vivo
将患有PANC-1肿瘤的小鼠分为6大组，每组5只。PBS(100μL)；DCPy(100μL，20mg/kg)；Mito(100μL，20mg/kg)；微波5W，5分钟+PBS(100μL)；微波5W，5分钟+DCPy(100μL，20mg/kg)；微波5W，5分钟+Mito@DCPy(20mg/kg，100μL)。将不同的溶液注入这些小鼠体内，并将小鼠培养12小时，在微波下对肿瘤部位进行照射。每隔3天，记录小鼠的肿瘤大小和体重的变化。在第15天终止它们进行组织学分析。在微波治疗深部肿瘤的实验中，将PANC-1肿瘤宿主Balb/c裸鼠分为五个不同的小组，其中 每组包括5只。对照组。PBS(100μL)；Mito@DCPy(100μL，20mg/kg)+(白光5分钟，每平方厘米90mW光强)；Mito@DCPy(100μL，20mg/kg)+(白光5分钟，每平方厘米90mW光强，3mm Pork屏障)。Mito@DCPy(100μL)+(微波5W，5分钟)；Mito@DCPy(100μ，20mg/kg)+(微波5W，5分钟，3mm Pork屏障)。12小时后，将微波相关组的肿瘤部位在微波下照射，每隔三天记录小鼠肿瘤形态的变化情况。Mice bearing PANC-1 tumors were divided into 6 large groups, with 5 mice in each group. PBS (100μL); DCPy (100μL, 20mg/kg); Mito (100μL, 20mg/kg); Microwave 5W, 5 minutes + PBS (100μL); Microwave 5W, 5 minutes + DCPy (100μL, 20mg/kg); Microwave 5W, 5 minutes + Mito@DCPy (20mg/kg, 100μL). Different solutions were injected into these mice, and the mice were cultured for 12 hours, and the tumor sites were irradiated under microwaves. Every 3 days, the changes in tumor size and body weight of the mice were recorded. They were terminated on the 15th day for histological analysis. In the experiment of microwave treatment of deep tumors, PANC-1 tumor host Balb/c nude mice were divided into five different groups, each of which included 5 mice. Control group. PBS (100 μL); Mito@DCPy (100 μL, 20 mg/kg) + (white light for 5 minutes, 90 mW per square centimeter); Mito@DCPy (100 μL, 20 mg/kg) + (white light for 5 minutes, 90 mW per square centimeter, 3 mm Pork barrier). Mito@DCPy (100 μL) + (microwave 5W, 5 minutes); Mito@DCPy (100 μ, 20 mg/kg) + (microwave 5W, 5 minutes, 3 mm Pork barrier). After 12 hours, the tumor sites of the microwave-related group were irradiated under microwaves, and the changes in the tumor morphology of the mice were recorded every three days.
(15)统计学分析(15) Statistical analysis
所有的结果都以平均值±SD表示。除非另有说明，每个实验均以一式三份进行。采用t检验进行验证。(**p<0.01,*p<0.05,***p<0.001)。All results are presented as mean ± SD. Unless otherwise stated, each experiment was performed in triplicate. Student's t-test was used for validation. (**p<0.01, *p<0.05, ***p<0.001).
实施例2结果和分析Example 2 Results and Analysis
正常的小鼠肝脏提供了本发明所需的健康线粒体。本发明使用BCA蛋白检测试剂盒和流式细胞仪，通过分析收集到的标记的线粒体颗粒来弄清线粒体数量和蛋白含量之间的关联。从结果来看，线粒体蛋白浓度和分离的线粒体数量之间存在良好的关系(图6)。本发明还应用动态光散射(DLS)和透射电子显微镜(TEM)来评估DCPy整合前后线粒体的大小。结果显示，DCPy修饰并不影响线粒体结构的完整性，因为自由线粒体和线粒体@DCPy的流体力学直径没有明显差异(图2A，B，6)。此外，通过拍摄线粒体(图2A)和线粒体@DCPy(图2B)的TEM图像表明，DCPy修饰并没有导致线粒体的变形和解体。为了产生能量，线粒体利用OXPHOS用酶氧化营养物质，释放的能量用于ATP合成。在呼吸链中，OXPHOS过程包含四个呼吸链复合体(I-IV)和ATP生成。为了确定分离的Mito和Mito@DCPy的生物活性，本发明选择测试复合物I的酶活性，它是还原分子进入OXPHOS过程的主要入口。结果显示，与分离的Mito相比，Mito@DCPy表现出强大的酶活性，而且酶活性随着浓度的增加而增强(图2C)。上述结果都表明，DCPy修饰具有生物相容性，因为它没有破坏线粒体原有的生物活性和结构整合。本发明还实施了紫外-可见吸收和荧光光谱来分析Mito@DCPy的光学特性。如图2D、E所示，Mito@DCPy的吸收带很宽，在450nm处达到峰值，宽而最大强度的发射带在650nm处。此外，本发明通过分子模拟来确定DCPy分子如何与线粒体膜结合。分子模拟的结果显示，DCPy分子被嵌入磷脂双分子层中(图2F)。在磷酸盐缓冲盐水(PBS)中，DCPy的Zeta电位为+18.1mV(图9)，因此可以推断，其正电荷特性有利于通过静电作用吸附负的Mito。Mito的Zeta电位为-10.8mV，而Mito@DCPy的表面电荷相对减少，为-5.1mV，表明DCPy被加载到Mito上(图9)。此外，图2G表明，DCPy和Mito@DCPy在MW 辐射下表现出良好的ROS生成效率。本发明还通过应用非常可靠的EPR(电子顺磁共振)来分析ROS的产生，成功地证实了Mito@DCPy强大的ROS生成能力，因为ROS具有较短的寿命和高的化学活性(图2H，I)。因此，人工修饰的Mito@DCPy明显含有生物成分和合成的MW致敏剂的治疗效果。Normal mouse liver provides healthy mitochondria required by the present invention. The present invention uses a BCA protein detection kit and a flow cytometer to analyze the collected labeled mitochondrial particles to clarify the relationship between the number of mitochondria and protein content. From the results, there is a good relationship between the mitochondrial protein concentration and the number of isolated mitochondria (Figure 6). The present invention also uses dynamic light scattering (DLS) and transmission electron microscopy (TEM) to evaluate the size of mitochondria before and after DCPy integration. The results show that DCPy modification does not affect the integrity of mitochondrial structure, because there is no significant difference in the hydrodynamic diameter of free mitochondria and mitochondria@DCPy (Figure 2A, B, 6). In addition, by taking TEM images of mitochondria (Figure 2A) and mitochondria@DCPy (Figure 2B), it is shown that DCPy modification does not cause deformation and disintegration of mitochondria. In order to generate energy, mitochondria use OXPHOS to oxidize nutrients with enzymes, and the energy released is used for ATP synthesis. In the respiratory chain, the OXPHOS process includes four respiratory chain complexes (I-IV) and ATP generation. In order to determine the biological activity of isolated Mito and Mito@DCPy, the present invention chooses to test the enzyme activity of complex I, which is the main entrance for reduced molecules to enter the OXPHOS process. The results showed that compared with isolated Mito, Mito@DCPy exhibited strong enzyme activity, and the enzyme activity increased with increasing concentration (Figure 2C). The above results all indicate that DCPy modification is biocompatible because it does not destroy the original biological activity and structural integration of mitochondria. The present invention also implements UV-visible absorption and fluorescence spectroscopy to analyze the optical properties of Mito@DCPy. As shown in Figures 2D and E, the absorption band of Mito@DCPy is very wide, reaching a peak at 450nm, and the wide and maximum intensity emission band is at 650nm. In addition, the present invention determines how DCPy molecules bind to mitochondrial membranes by molecular simulation. The results of molecular simulation show that DCPy molecules are embedded in the phospholipid bilayer (Figure 2F). In phosphate buffered saline (PBS), the Zeta potential of DCPy is +18.1mV (Figure 9), so it can be inferred that its positive charge characteristics are conducive to the adsorption of negative Mito through electrostatic action. The Zeta potential of Mito is -10.8mV, while the surface charge of Mito@DCPy is relatively reduced, which is -5.1mV, indicating that DCPy is loaded on Mito (Fig. 9). In addition, Fig. 2G shows that DCPy and Mito@DCPy show good ROS generation efficiency under MW radiation. The present invention also successfully confirms the powerful ROS generation ability of Mito@DCPy by applying highly reliable EPR (electron paramagnetic resonance) to analyze the generation of ROS, because ROS has a shorter life span and high chemical activity (Fig. 2H, I). Therefore, the artificially modified Mito@DCPy obviously contains the therapeutic effect of biological components and synthetic MW sensitizers.
在确定分离出的线粒体具有生物活性后，本发明接着研究了Mito@DCPy的细胞调节作用。结果表明，游离的Mito可以通过内吞作用、巨噬细胞和膜纳米管的隧道作用实现细胞间的转移。因此，选择人胰腺癌细胞(PANC-1)作为目标细胞系来研究细胞与线粒体的相互作用。用特异性的线粒体追踪剂DCPy对分离出的Mito进行染色。如图8所示，线粒体追踪剂DCPy显示的红色荧光信号清晰而强烈，这表明DCPy可以有效地标记线粒体。用Mito@DCPy培养后，PANC-1细胞中DCPy的红色荧光在最初1小时内没有明显增加，荧光强度低于DCPy处理的细胞(图3a)。孵化4小时后，细胞中游离的DCPy的荧光强度与Mito@DCPy的荧光强度相当，表明DCPy和Mito@DCPy在4小时内都被有效地内化到PANC-1细胞中。由于线粒体的尺寸是亚微米级的，因此就细胞吸收率而言，线粒体@DCPy与自由DCPy分子相比要慢一些。此外，为了检查Mito@DCPy在活体癌细胞中的亚细胞位置(图3b)，Mito@DCPy和不同类型的商业细胞器特异性荧光染色剂被用来共同染色PANC-1细胞。结果显示，MitoTracker Green的绿色信号和Mito@DCPy的红色信号之间有很强的共位，而LysoTracker Green和ERTracker Green的绿色信号之间有轻微的共位(其中结果显示Mito的皮尔逊系数为0.860，内质网为0.675，(溶酶体为0.689)。上述研究表明，Mito@DCPy进入了PANC-1细胞，并在线粒体中广泛积累。After determining that the isolated mitochondria have biological activity, the present invention then studied the cell regulation effect of Mito@DCPy. The results showed that free Mito can achieve intercellular transfer through endocytosis, macrophages and membrane nanotube tunneling. Therefore, human pancreatic cancer cells (PANC-1) were selected as the target cell line to study the interaction between cells and mitochondria. The isolated Mito was stained with a specific mitochondrial tracer DCPy. As shown in Figure 8, the red fluorescence signal displayed by the mitochondrial tracer DCPy is clear and strong, indicating that DCPy can effectively mark mitochondria. After incubation with Mito@DCPy, the red fluorescence of DCPy in PANC-1 cells did not increase significantly within the first hour, and the fluorescence intensity was lower than that of DCPy-treated cells (Figure 3a). After incubation for 4 hours, the fluorescence intensity of free DCPy in the cells was comparable to that of Mito@DCPy, indicating that both DCPy and Mito@DCPy were effectively internalized into PANC-1 cells within 4 hours. Since the size of mitochondria is submicron, mitochondria@DCPy is slower than free DCPy molecules in terms of cellular absorption rate. Furthermore, to examine the subcellular location of Mito@DCPy in living cancer cells (Figure 3b), Mito@DCPy and different types of commercial organelle-specific fluorescent stains were used to co-stain PANC-1 cells. The results showed that there was a strong colocalization between the green signal of MitoTracker Green and the red signal of Mito@DCPy, while there was a slight colocalization between the green signals of LysoTracker Green and ERTracker Green (the results showed that the Pearson coefficients for Mito were 0.860, 0.675 for the endoplasmic reticulum, and 0.689 for the lysosome). The above studies indicate that Mito@DCPy entered PANC-1 cells and accumulated extensively in mitochondria.
因此，本发明调查了Mito对癌细胞增殖的影响。随着添加到PANC-1细胞中的游离线粒体浓度的增加，细胞增殖逐渐被抑制，揭示了分离出的健康线粒体被癌细胞吸收后对细胞增殖有抑制作用(图9)。在使用DCPy的MDT中，由于活性氧的有效产生，MW照射下的癌细胞被杀死约80％，而在无微波组中，对癌细胞的杀伤力有限，表明DCPy的暗毒性可以忽略不计(图6b)。在没有微波的情况下，纯Mito@DCPy也能杀死癌细胞，表明活性Mito的抗癌影响仍然存在。此外，在含有Mito@DCPy的MDT中，用微波照射导致癌细胞的死亡最为明显。Therefore, the present invention investigates the effect of Mito on cancer cell proliferation. As the concentration of free mitochondria added to PANC-1 cells increased, cell proliferation was gradually inhibited, revealing that the isolated healthy mitochondria had an inhibitory effect on cell proliferation after being absorbed by cancer cells (Figure 9). In the MDT using DCPy, due to the effective production of reactive oxygen species, about 80% of cancer cells under MW irradiation were killed, while in the microwave-free group, the lethality to cancer cells was limited, indicating that the dark toxicity of DCPy was negligible (Figure 6b). In the absence of microwaves, pure Mito@DCPy can also kill cancer cells, indicating that the anti-cancer effect of active Mito still exists. In addition, in the MDT containing Mito@DCPy, the death of cancer cells caused by microwave irradiation was most obvious.
然后，本发明研究了有丝***抑制癌细胞生长和增强MDT的机制。众多证据表明，有氧糖酵解的主要原因可能是癌细胞中线粒体的缺陷。此外，癌细胞中有氧糖酵解的增加可能进一步促进癌细胞的侵袭和转移，以及肿瘤微环境的酸化。然而，通过移植正常的线粒体来抑制癌症中的有氧糖酵解是一个可行的选择，从而不仅促进药物敏感性的提 高，还能有效地减少癌细胞的生长。癌细胞中有氧糖酵解的最终产物是L-乳酸，由于其浓度可以直接表明糖酵解的程度，因此可以通过测量来确定糖酵解的程度。因此，收集细胞培养基，并测量用Mito和Mito@DCPy处理的细胞培养物中的L-乳酸浓度。如图4a所示，在用Mito@DCPy和Mito处理的PANC-1细胞中，L-乳酸的产生被有效减少，表明细胞的糖酵解在生物活性Mito的控制下被削弱。此外，本发明还分析了葡萄糖转运体1(GLUT1)的表达，它是一种单体蛋白，可以在整个哺乳动物细胞的质膜上运输葡萄糖。图4C中ELISA的结果显示，与未处理的细胞相比，用DCPy处理的细胞中GLUT1的表达没有明显变化，而用Mito和Mito@DCPy培养的细胞中GLUT1的表达明显下降。这些发现表明，通过将内源性的Mito移植到癌细胞中，可以将原本浪费的细胞能量代谢途径糖酵解转变为正常的OXPHOS，同时降低GLUT1的表达，从而减少葡萄糖的摄取，表现出突出的抗癌效果。Then, the present invention studied the mechanism by which Mito inhibits cancer cell growth and enhances MDT. Numerous evidences indicate that the main cause of aerobic glycolysis may be the defect of mitochondria in cancer cells. In addition, the increase of aerobic glycolysis in cancer cells may further promote the invasion and metastasis of cancer cells, as well as the acidification of the tumor microenvironment. However, it is a viable option to inhibit aerobic glycolysis in cancer by transplanting normal mitochondria, thereby not only promoting the improvement of drug sensitivity, but also effectively reducing the growth of cancer cells. The final product of aerobic glycolysis in cancer cells is L-lactic acid, and since its concentration can directly indicate the extent of glycolysis, the extent of glycolysis can be determined by measurement. Therefore, the cell culture medium was collected and the concentration of L-lactic acid in the cell culture treated with Mito and Mito@DCPy was measured. As shown in Figure 4a, in PANC-1 cells treated with Mito@DCPy and Mito, the production of L-lactic acid was effectively reduced, indicating that the glycolysis of the cells was weakened under the control of bioactive Mito. In addition, the present invention also analyzed the expression of glucose transporter 1 (GLUT1), which is a monomeric protein that can transport glucose across the plasma membrane of mammalian cells. The results of ELISA in Figure 4C showed that the expression of GLUT1 in cells treated with DCPy did not change significantly compared with untreated cells, while the expression of GLUT1 in cells cultured with Mito and Mito@DCPy was significantly decreased. These findings suggest that by transplanting endogenous Mito into cancer cells, the originally wasteful cellular energy metabolism pathway glycolysis can be converted to normal OXPHOS, while reducing the expression of GLUT1, thereby reducing glucose uptake, showing a prominent anti-cancer effect.
此外，还分析了不同处理后PANC-1细胞中凋亡相关蛋白的表达。在本发明的研究***中，研究了由Bcl-2蛋白系以及caspase-3组成的凋亡级联激活的米托相关凋亡途径。作为一种抗凋亡蛋白，Bcl-2在癌细胞中的过量表达不仅使癌细胞表现出抗凋亡特性，帮助癌细胞增殖和防御，而且还使其具有抗氧化特性，保护癌细胞免受氧化损伤。值得注意的是，在图4F中，发现自由生活的米托舱和基于DCPy的MDT可以潜在地减少PANC-1细胞中Bcl-2的表达。一般来说，作为下游核心效应物的caspase-3通常以procaspase的形式存在，由pro-caspase3形成的cleaved-caspase-3可以诱导细胞凋亡并在裂解后激活凋亡级联。由于Bcl-2可以抑制caspase级联的激活，Mito@DCPy引起的Bcl-2的明显下调诱发了pro-caspase-3表达的明显下降(图4F)。这些结果表明，健康的Mito@DCPy移植不仅可以自行下调Bcl-2的过度表达，下调pro-caspase-3的表达，还可以增强MW照射产生ROS的治疗效果，使MDT的治疗效率更加协同。In addition, the expression of apoptosis-related proteins in PANC-1 cells after different treatments was analyzed. In the research system of the present invention, the Mito-related apoptotic pathway activated by the apoptotic cascade composed of the Bcl-2 protein system and caspase-3 was studied. As an anti-apoptotic protein, the overexpression of Bcl-2 in cancer cells not only makes cancer cells exhibit anti-apoptotic properties, helps cancer cell proliferation and defense, but also makes them have antioxidant properties, protecting cancer cells from oxidative damage. It is worth noting that in Figure 4F, it was found that free-living Mito compartments and DCPy-based MDT can potentially reduce the expression of Bcl-2 in PANC-1 cells. In general, caspase-3, as a downstream core effector, usually exists in the form of procaspase, and cleaved-caspase-3 formed by pro-caspase3 can induce cell apoptosis and activate the apoptotic cascade after cleavage. Since Bcl-2 can inhibit the activation of the caspase cascade, the significant downregulation of Bcl-2 caused by Mito@DCPy induced a significant decrease in the expression of pro-caspase-3 (Figure 4F). These results indicate that healthy Mito@DCPy transplants can not only downregulate the overexpression of Bcl-2 and downregulate the expression of pro-caspase-3 by themselves, but also enhance the therapeutic effect of ROS generation by MW irradiation, making the therapeutic efficiency of MDT more synergistic.
随后，在上述体外实验结果的前提下，本发明进行了动物实验，研究Mito@DCPy作为微波增敏剂在体内实施的可能性。首先，对携带PANC-1肿瘤的小鼠进行了Mito@DCPy微波增敏剂的体内生物分布的评估(图5A)。在注射后的适当时间点，使用小动物生物成像***观察Mito@DCPy在动物体内的明亮荧光信号的生物分布图像。此外，在24小时后处决了这组小鼠，并收集了组织成像所需的材料：其主要器官和肿瘤样本(图6B，C)。在肿瘤中，Mito@DCPy组的信号比DCPy组强，这可能是对Mito@DCPy优越靶向能力的进一步验证。为了研究Mito@DCPy的治疗能力，实验中随机选取了有以下实验性肿瘤的实验小鼠：(1)PBS(0.1毫升)；(2)DCPy(20毫克公斤-1，0.1 毫升)；(3)Mito(20毫克公斤-1，0.1毫升)；(4)PBS(0.1毫升)+(微波5W，300秒)；(5)DCPy(20毫克公斤-1，0.1毫升)+(微波5W，300秒)；(6)Mito@DCPy(20毫克公斤-1，0.1毫升)+(微波5W，300秒)。在注射各种治疗剂12小时后，小鼠(治疗4,5,6)肿瘤区被照成MW(5W，300s)。在15天的治疗中，记录了各种治疗中的肿瘤体积和体重。在图5C，D中，肿瘤的体积迅速扩大，在所有四个处理(PBS，DCPy，Mito和MW)中，肿瘤的生长趋势几乎相同，表明与对照组相比，DCPy、Mito单独或MW都不能延缓肿瘤的生长趋势。可能是由于DCPy的浓度有限，在DCPy+MW治疗中可以观察到更好的治疗效果(仅针对MDT)。令人鼓舞的是，Mito@DCPy+微波治疗的肿瘤抑制效率最高。在这个实验中，得到了与体外实验相同的结果：PANC-1肿瘤在治疗后被切除，15天后没有发现复发。为了检查肿瘤的生殖活性，采用免疫组化染色、苏木精染色和曙红染色试验。与其他治疗方法相比，Mito@DCPy+微波组显示出最明显的肿瘤组织凋亡，如图(14，15)所示。这些体内结果令人信服地验证了Mito@DCPy通过与MDT和工程生物活性线粒体的协同处理，抑制了PANC-1癌细胞的增殖并引发了肿瘤组织的凋亡。此外，在此期间，由于所有实验组的体重都在缓慢增加，因此没有出现明显的副作用(图5C)。本发明对主要器官进行了H&E染色，结果进一步证实，Mito@DCPy具有良好的生物安全性(图S16)。此外，为了进一步评估Mito@DCPy对深部肿瘤的治疗能力，对患有肿瘤的实验小鼠随机进行了以下实验。(1)PBS(100μL)；(2)白光照射(90mW cm-2，300s)+Mito@DCPy(100μL，20mg kg-1)；(3)白光照射(90mW cm-2，300s)+Mito@DCPy(20mg kg-1，100μL)+(3mm猪肉屏障)。(4)微波照射(5W，300s)+Mito@DCPy(100μL)；(5)微波照射(5W，300s)+Mito@DCPy(20mg kg-1，100μL)+(3mm猪肉屏障)。注射后12小时，小鼠的肿瘤区接受以下处理：90mW cm-2的白光照射300s(处理2,3)，5W的MW照射300s(处理4,5)。15天的疗程后，记录不同处理下的肿瘤体积和体重。在图4E中，用Mito@DCPy和白光照射处理的小鼠对肿瘤生长表现出明显的抑制作用。通过对正位肿瘤的真实建模，为了增强穿透深度，肿瘤表面覆盖了3毫米厚的外猪肉。结果发现，虽然Mito@DCPy+MW+猪肉的肿瘤生长抑制率近似退步到4.4％左右，但在Mito@DCPy+白光+猪肉组中，可以预见几乎可以忽略的抑制效果，说明MW的穿透深度要优于白光。因此，可以得出结论，Mito@DCPy+MW增强的抗癌效果是由于纳米粒子的肿瘤保留率升高，MW的渗透深度，以及MW诱导的MDT。综上所述，Mito@DCPy MW增敏剂介导的MW动态癌症治疗效果不仅表现出对肿瘤生长的良好抑制，而且毒性也很小。Subsequently, based on the above in vitro experimental results, the present invention conducted animal experiments to study the feasibility of Mito@DCPy as a microwave sensitizer in vivo. First, the in vivo biodistribution of the Mito@DCPy microwave sensitizer was evaluated in mice carrying PANC-1 tumors (Figure 5A). At appropriate time points after injection, a small animal bioimaging system was used to observe the biodistribution images of the bright fluorescent signals of Mito@DCPy in the animal body. In addition, this group of mice was executed after 24 hours, and the materials required for tissue imaging were collected: their major organs and tumor samples (Figure 6B, C). In the tumor, the signal of the Mito@DCPy group was stronger than that of the DCPy group, which may be a further verification of the superior targeting ability of Mito@DCPy. To investigate the therapeutic ability of Mito@DCPy, mice bearing the following experimental tumors were randomly selected: (1) PBS (0.1 ml); (2) DCPy (20 mg kg-1, 0.1 ml); (3) Mito (20 mg kg-1, 0.1 ml); (4) PBS (0.1 ml) + (microwave 5W, 300 sec); (5) DCPy (20 mg kg-1, 0.1 ml) + (microwave 5W, 300 sec); (6) Mito@DCPy (20 mg kg-1, 0.1 ml) + (microwave 5W, 300 sec). Twelve hours after injection of various therapeutic agents, the tumor areas of mice (treatments 4, 5, and 6) were irradiated with MW (5W, 300 s). During the 15-day treatment, the tumor volume and body weight in various treatments were recorded. In Figure 5C, D, the tumor volume expanded rapidly, and the growth trend of the tumor was almost the same in all four treatments (PBS, DCPy, Mito and MW), indicating that DCPy, Mito alone or MW could not delay the growth trend of the tumor compared with the control group. Probably due to the limited concentration of DCPy, a better therapeutic effect can be observed in the DCPy+MW treatment (only for MDT). Encouragingly, the tumor inhibition efficiency of Mito@DCPy+microwave treatment was the highest. In this experiment, the same results as in the in vitro experiment were obtained: PANC-1 tumors were resected after treatment, and no recurrence was found after 15 days. To examine the reproductive activity of the tumor, immunohistochemical staining, hematoxylin staining and eosin staining tests were used. Compared with other treatments, the Mito@DCPy+microwave group showed the most obvious tumor tissue apoptosis, as shown in Figures (14, 15). These in vivo results convincingly verified that Mito@DCPy inhibited the proliferation of PANC-1 cancer cells and triggered apoptosis of tumor tissues through synergistic treatment with MDT and engineered bioactive mitochondria. In addition, during this period, no obvious side effects occurred because the body weight of all experimental groups was slowly increasing (Figure 5C). The present invention performed H&E staining on the main organs, and the results further confirmed that Mito@DCPy has good biosafety (Figure S16). In addition, in order to further evaluate the therapeutic ability of Mito@DCPy on deep tumors, the following experiments were randomly performed on experimental mice with tumors. (1) PBS (100μL); (2) white light irradiation (90mW cm-2, 300s) + Mito@DCPy (100μL, 20mg kg-1); (3) white light irradiation (90mW cm-2, 300s) + Mito@DCPy (20mg kg-1, 100μL) + (3mm pork barrier). (4) Microwave irradiation (5W, 300s) + Mito@DCPy (100μL); (5) Microwave irradiation (5W, 300s) + Mito@DCPy (20mg kg-1, 100μL) + (3mm pork barrier). 12 hours after injection, the tumor area of the mice received the following treatments: 90mW cm-2 white light irradiation for 300s (treatment 2, 3), 5W MW irradiation for 300s (treatment 4, 5). After a 15-day course of treatment, the tumor volume and body weight under different treatments were recorded. In Figure 4E, mice treated with Mito@DCPy and white light irradiation showed a significant inhibitory effect on tumor growth. Through realistic modeling of orthotopic tumors, in order to enhance the penetration depth, the tumor surface was covered with a 3 mm thick outer pork barrier. The results showed that although the tumor growth inhibition rate of Mito@DCPy+MW+pork regressed to approximately 4.4%, in the Mito@DCPy+white light+pork group, an almost negligible inhibitory effect could be foreseen, indicating that the penetration depth of MW was superior to that of white light. Therefore, it can be concluded that the enhanced anticancer effect of Mito@DCPy+MW is due to the increased tumor retention rate of nanoparticles, the penetration depth of MW, and the MDT induced by MW. In summary, the MW dynamic cancer therapy effect mediated by the Mito@DCPy MW sensitizer not only showed good inhibition of tumor growth, but also had low toxicity.
综上，为了解决PDT对深部肿瘤的低渗透性和治疗效果，本发明设计了一种基于AIE工程生物活性线粒体的新型微波动力癌症疗法。在不影响线粒体的完整性和生物活性的前提下，本发明设计的有机小分子DCPy可以在线粒体的膜上快速标记，同时。此外，固定在线粒体上的DCPy的松散的分子内包装和受限的移动有助于光敏剂产生明亮的荧光和高效的ROS。微波的深层穿透力也可以使该疗法在深层次上对肿瘤有效。内化后，健康的Mito可以通过降低GLUT1的表达来减少葡萄糖的吸收，从而将癌细胞原本浪费的有氧糖酵解能量代谢途径改变为正常的OXPHOS。此外，移植的Mito影响了Bcl-2蛋白和pro-caspase3蛋白的表达，使前者减少，后者增加，从而重新激活了凋亡途径。基于DCPy的MW疗法由于Mito促进细胞凋亡的作用而实现了对癌细胞的增强杀伤。合成分子和分离的活细胞器之间的整合产生了新的协同作用，以解决复杂的生物问题。In summary, in order to solve the low permeability and therapeutic effect of PDT on deep tumors, the present invention designs a new microwave-powered cancer therapy based on AIE-engineered bioactive mitochondria. Without affecting the integrity and bioactivity of mitochondria, the organic small molecule DCPy designed by the present invention can be quickly labeled on the membrane of mitochondria, and at the same time. In addition, the loose intramolecular packaging and restricted movement of DCPy fixed on mitochondria help the photosensitizer to produce bright fluorescence and efficient ROS. The deep penetration of microwaves can also make this therapy effective for tumors at a deep level. After internalization, healthy Mito can reduce glucose absorption by reducing the expression of GLUT1, thereby changing the aerobic glycolysis energy metabolism pathway that was originally wasted by cancer cells to normal OXPHOS. In addition, the transplanted Mito affects the expression of Bcl-2 protein and pro-caspase3 protein, reducing the former and increasing the latter, thereby reactivating the apoptotic pathway. DCPy-based MW therapy achieves enhanced killing of cancer cells due to the effect of Mito in promoting cell apoptosis. The integration between synthetic molecules and isolated living organelles produces new synergies to solve complex biological problems.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
- 聚集诱导发光工程线粒体在制备治疗癌症的药物中的应用，其特征在于，所述聚集诱导发光工程线粒体为聚集诱导发光材料修饰的线粒体。The application of aggregation-induced luminescence engineered mitochondria in the preparation of drugs for treating cancer is characterized in that the aggregation-induced luminescence engineered mitochondria are mitochondria modified with aggregation-induced luminescence materials.
- 根据权利要求1所述的应用，其特征在于，所述聚集诱导发光材料为DCPy。The use according to claim 1, characterized in that the aggregation-induced emission material is DCPy.
- 根据权利要求2所述的应用，其特征在于，所述DCPy分子被嵌入线粒体的磷脂双分子层中。The use according to claim 2 is characterized in that the DCPy molecule is embedded in the phospholipid bilayer of mitochondria.
- 根据权利要求1所述的应用，其特征在于，所述聚集诱导发光工程线粒体的制备方法如下：The use according to claim 1, characterized in that the preparation method of the aggregation-induced luminescence engineered mitochondria is as follows:S1：7-(二苯胺)-9-乙基-9H-(咔唑)-2-碳醛溶液在乙醇中加入1,4-二甲基碘化吡啶和哌啶，室温下回流3小时；当冷却到室温时，产品收集，洗涤，冷冻干燥，得到红色碘盐固体；S1: 1,4-dimethylpyridinium iodide and piperidine were added to a solution of 7-(diphenylamine)-9-ethyl-9H-(carbazole)-2-carbaldehyde in ethanol and refluxed at room temperature for 3 hours; when cooled to room temperature, the product was collected, washed, and freeze-dried to obtain a red iodized salt solid;S2：用丙酮溶解所述红色碘盐固体，滴加六氟磷酸钾溶液；将制备好的混合物进一步充分搅拌纯化得到聚集诱导发光材料；S2: dissolving the red iodized salt solid with acetone, and adding potassium hexafluorophosphate solution dropwise; further stirring and purifying the prepared mixture to obtain an aggregation-induced luminescence material;S3：将所述聚集诱导发光材料和线粒体裂解液与PBS溶液充分混合，超声，搅拌；把多余的细胞悬液过滤进入微孔管，离心，洗涤沉淀，得到聚集诱导发光工程线粒体。S3: The aggregation-induced luminescence material and mitochondrial lysate are fully mixed with the PBS solution, ultrasonicated, and stirred; the excess cell suspension is filtered into a microporous tube, centrifuged, and the precipitate is washed to obtain the aggregation-induced luminescence engineered mitochondria.
- 根据权利要求1所述的应用，其特征在于，所述聚集诱导发光工程线粒体使Bcl-2蛋白表达减少，pro-caspase3蛋白表达增加，从而重新激活凋亡途径。The use according to claim 1 is characterized in that the aggregation-induced luminescence engineered mitochondria reduce the expression of Bcl-2 protein and increase the expression of pro-caspase3 protein, thereby reactivating the apoptosis pathway.
- 根据权利要求1所述的应用，其特征在于，所述聚集诱导发光工程线粒体具有生成活性氧的能力。The use according to claim 1 is characterized in that the aggregation-induced luminescence engineered mitochondria have the ability to generate reactive oxygen species.
- 根据权利要求1所述的应用，其特征在于，所述药物为微波增敏剂。The use according to claim 1, characterized in that the drug is a microwave sensitizer.
- 聚集诱导发光工程线粒体在治疗癌症中的应用，其特征在于，所述聚集诱导发光工程线粒体为聚集诱导发光材料修饰的线粒体。The application of aggregation-induced luminescence engineered mitochondria in the treatment of cancer is characterized in that the aggregation-induced luminescence engineered mitochondria are mitochondria modified with aggregation-induced luminescence materials.
- 根据权利要求8所述的应用，其特征在于，所述聚集诱导发光材料为DCPy。The use according to claim 8, characterized in that the aggregation-induced emission material is DCPy.
- 根据权利要求8所述的应用，其特征在于，所述应用中需要使用微波照射。The use according to claim 8 is characterized in that microwave irradiation is required in the application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2022/136952 WO2024119373A1 (en) | 2022-12-06 | 2022-12-06 | Use of aggregation-induced light-emitting engineered mitochondrion in preparation of medicament for treating cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2022/136952 WO2024119373A1 (en) | 2022-12-06 | 2022-12-06 | Use of aggregation-induced light-emitting engineered mitochondrion in preparation of medicament for treating cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024119373A1 true WO2024119373A1 (en) | 2024-06-13 |
Family
ID=91378449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/136952 WO2024119373A1 (en) | 2022-12-06 | 2022-12-06 | Use of aggregation-induced light-emitting engineered mitochondrion in preparation of medicament for treating cancer |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2024119373A1 (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160299078A1 (en) * | 2013-12-03 | 2016-10-13 | The Hong Kong University Of Science And Technology | Specific Detection and Quantification of Cardiolipin and Isolated Mitochondria by Positively Charged AIE Fluorogens and Method of Manufacturing Thereof |
| WO2016165487A1 (en) * | 2015-04-13 | 2016-10-20 | The Hong Kong University Of Science And Technology | Real-time monitoring mitophagy process by fluorescent photostable mitochondrial specific bioprobe with aie characteristics |
| CN109313135A (en) * | 2016-07-21 | 2019-02-05 | 香港科技大学 | Aggregation-induced emission body for imaging cancerous |
| CN110407736A (en) * | 2018-04-27 | 2019-11-05 | 香港科技大学 | The preparation and application of near-infrared compound with strong two-photon absorption |
| CN113956292A (en) * | 2021-10-29 | 2022-01-21 | 南京师范大学 | Mitochondrial targeting compound with aggregation-induced emission property and synthesis method and application thereof |
| CN113975391A (en) * | 2021-10-27 | 2022-01-28 | 深圳先进技术研究院 | Self-oxygen-supply photosensitizer and preparation method and application thereof |
-
2022
- 2022-12-06 WO PCT/CN2022/136952 patent/WO2024119373A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160299078A1 (en) * | 2013-12-03 | 2016-10-13 | The Hong Kong University Of Science And Technology | Specific Detection and Quantification of Cardiolipin and Isolated Mitochondria by Positively Charged AIE Fluorogens and Method of Manufacturing Thereof |
| WO2016165487A1 (en) * | 2015-04-13 | 2016-10-20 | The Hong Kong University Of Science And Technology | Real-time monitoring mitophagy process by fluorescent photostable mitochondrial specific bioprobe with aie characteristics |
| CN109313135A (en) * | 2016-07-21 | 2019-02-05 | 香港科技大学 | Aggregation-induced emission body for imaging cancerous |
| CN110407736A (en) * | 2018-04-27 | 2019-11-05 | 香港科技大学 | The preparation and application of near-infrared compound with strong two-photon absorption |
| CN113975391A (en) * | 2021-10-27 | 2022-01-28 | 深圳先进技术研究院 | Self-oxygen-supply photosensitizer and preparation method and application thereof |
| CN113956292A (en) * | 2021-10-29 | 2022-01-21 | 南京师范大学 | Mitochondrial targeting compound with aggregation-induced emission property and synthesis method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| LIU JINGJING, LIU XINGANG, WU MIN, QI GUOBIN, LIU BIN: "Engineering Living Mitochondria with AIE Photosensitizer for Synergistic Cancer Cell Ablation", NANO LETTERS, AMERICAN CHEMICAL SOCIETY, US, vol. 20, no. 10, 14 October 2020 (2020-10-14), US , pages 7438 - 7445, XP093178577, ISSN: 1530-6984, DOI: 10.1021/acs.nanolett.0c02778 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Mitochondria-based aircraft carrier enhances in vivo imaging of carbon quantum dots and delivery of anticancer drug | |
| Xu et al. | Chemo-photodynamic therapy with light-triggered disassembly of theranostic nanoplatform in combination with checkpoint blockade for immunotherapy of hepatocellular carcinoma | |
| CN113456613B (en) | Construction and application of near-infrared light activated macrophage-nano prodrug targeted drug delivery system | |
| Shao et al. | A smart multifunctional nanoparticle for enhanced near-infrared image-guided photothermal therapy against gastric cancer | |
| US11369425B2 (en) | Lactoferrin-conjugated nanoparticle complex and use thereof | |
| KR20180114517A (en) | Phamaceutical composition for treating cancer | |
| Feng et al. | An acid-responsive MOF nanomedicine for augmented anti-tumor immunotherapy via a metal ion interference-mediated pyroptotic pathway | |
| Yu et al. | Near-infrared photoactivatable semiconducting polymer nanocomplexes with bispecific metabolism interventions for enhanced cancer immunotherapy | |
| Zhou et al. | Inspired heat shock protein alleviating prodrug enforces immunogenic photodynamic therapy by eliciting pyroptosis | |
| Zhang et al. | Research progress in the synthesis and biological application of quantum dots | |
| Jiao et al. | Tumor cell-derived extracellular vesicles for breast cancer specific delivery of therapeutic P53 | |
| Jiang et al. | Peptide Supramolecular Assembly‐Instructed In Situ Self‐Aggregation for Stratified Targeting Sonodynamic Therapy Enhancement of AIE Luminogens | |
| Wang et al. | B16 membrane-coated vesicles for combined photodynamic therapy and immunotherapy shift immune microenvironment of melanoma | |
| Zhang et al. | Biocompatible 5-Aminolevulinic Acid/Au nanoparticle-loaded ethosomal vesicles for in vitro transdermal synergistic photodynamic/photothermal therapy of hypertrophic scars | |
| Zhang et al. | Moderating hypoxia and promoting immunogenic photodynamic therapy by HER-2 nanobody conjugate nanoparticles for ovarian cancer treatment | |
| Yu et al. | AIEgens/Mitochondria Nanohybrids as Bioactive Microwave Sensitizers for Non‐Thermal Microwave Cancer Therapy | |
| Wang et al. | Autophagy responsive intra-intercellular delivery nanoparticles for effective deep solid tumor penetration | |
| Raza et al. | Engineered tumor microvesicles modified by SP94 peptide for arsenic trioxide targeting drug delivery in liver cancer therapy | |
| Li et al. | Nuclear PARP1‐Targeted Photosensitizer as a Dual‐Mode DNA‐Damaging Agent and Immune Activator for Tumor Ablation | |
| WO2024119373A1 (en) | Use of aggregation-induced light-emitting engineered mitochondrion in preparation of medicament for treating cancer | |
| US20230248850A1 (en) | Gold molecular clusters and methods of using same for near-infrared imaging | |
| Shao et al. | Integration of activation by hypoxia and inhibition resistance of tumor cells to apoptosis for precise and augmented photodynamic therapy | |
| CN114432264B (en) | Composite nano material based on ferrocene and hypericin, preparation method and application | |
| CN115040494B (en) | Multifunctional nano vesicle of ginsenoside-modified co-carried multielement complex, and preparation method and application thereof | |
| Wang et al. | Functionalized Tumor Cell Membrane-Camouflaged Photo-Activatable Nanoparticle for Spatiotemporal Antitumor Therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22967534 Country of ref document: EP Kind code of ref document: A1 |