KR20220158984A - Tumor-targeted nanoparticles using CD47 positive cell membrane, uses thereof and method thereof - Google Patents
Tumor-targeted nanoparticles using CD47 positive cell membrane, uses thereof and method thereof Download PDFInfo
- Publication number
- KR20220158984A KR20220158984A KR1020210066684A KR20210066684A KR20220158984A KR 20220158984 A KR20220158984 A KR 20220158984A KR 1020210066684 A KR1020210066684 A KR 1020210066684A KR 20210066684 A KR20210066684 A KR 20210066684A KR 20220158984 A KR20220158984 A KR 20220158984A
- Authority
- KR
- South Korea
- Prior art keywords
- tumor
- cell membrane
- positive
- cancer
- nanoparticles
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 135
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 title claims abstract description 127
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 title claims abstract description 125
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 92
- 210000000170 cell membrane Anatomy 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 97
- 201000011510 cancer Diseases 0.000 claims abstract description 42
- 230000008685 targeting Effects 0.000 claims abstract description 22
- 238000012377 drug delivery Methods 0.000 claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 76
- 229960004679 doxorubicin Drugs 0.000 claims description 38
- 239000012528 membrane Substances 0.000 claims description 27
- 239000002246 antineoplastic agent Substances 0.000 claims description 23
- 102000001301 EGF receptor Human genes 0.000 claims description 21
- 108060006698 EGF receptor Proteins 0.000 claims description 21
- 210000002540 macrophage Anatomy 0.000 claims description 17
- 230000001093 anti-cancer Effects 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 210000004881 tumor cell Anatomy 0.000 claims description 6
- 206010057249 Phagocytosis Diseases 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 230000008782 phagocytosis Effects 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 claims description 3
- 208000032612 Glial tumor Diseases 0.000 claims description 2
- 206010018338 Glioma Diseases 0.000 claims description 2
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 206010039491 Sarcoma Diseases 0.000 claims description 2
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 claims description 2
- 201000010536 head and neck cancer Diseases 0.000 claims description 2
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 2
- 208000022679 triple-negative breast carcinoma Diseases 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 22
- 238000011282 treatment Methods 0.000 abstract description 15
- 229940079593 drug Drugs 0.000 abstract description 12
- 229940124597 therapeutic agent Drugs 0.000 abstract description 8
- 210000004369 blood Anatomy 0.000 abstract description 5
- 239000008280 blood Substances 0.000 abstract description 5
- 230000017531 blood circulation Effects 0.000 abstract description 4
- 241000699670 Mus sp. Species 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 18
- 238000010586 diagram Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 238000001727 in vivo Methods 0.000 description 12
- HPZOOQSXPMEJBV-ODCFVKFUSA-N Tirilazad mesylate Chemical compound CS(O)(=O)=O.O=C([C@@H]1[C@@]2(C)CC=C3[C@@]4(C)C=CC(=O)C=C4CC[C@H]3[C@@H]2C[C@H]1C)CN(CC1)CCN1C(N=1)=CC(N2CCCC2)=NC=1N1CCCC1 HPZOOQSXPMEJBV-ODCFVKFUSA-N 0.000 description 11
- 229960005395 cetuximab Drugs 0.000 description 10
- 238000012790 confirmation Methods 0.000 description 10
- 231100000135 cytotoxicity Toxicity 0.000 description 10
- 230000003013 cytotoxicity Effects 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 230000004614 tumor growth Effects 0.000 description 9
- 238000005538 encapsulation Methods 0.000 description 8
- 150000002632 lipids Chemical class 0.000 description 8
- 210000003734 kidney Anatomy 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 210000004072 lung Anatomy 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000003446 ligand Substances 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000004624 confocal microscopy Methods 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000002539 nanocarrier Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 101000863873 Homo sapiens Tyrosine-protein phosphatase non-receptor type substrate 1 Proteins 0.000 description 3
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102100029948 Tyrosine-protein phosphatase non-receptor type substrate 1 Human genes 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 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 2
- 239000012103 Alexa Fluor 488 Substances 0.000 description 2
- 239000004254 Ammonium phosphate Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 101710098610 Leukocyte surface antigen CD47 Proteins 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 2
- 235000019289 ammonium phosphates Nutrition 0.000 description 2
- 238000011394 anticancer treatment Methods 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- -1 cationic lipid Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000008004 cell lysis buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000007431 microscopic evaluation Methods 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 229960001592 paclitaxel Drugs 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical compound Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- BUOYTFVLNZIELF-UHFFFAOYSA-N 2-phenyl-1h-indole-4,6-dicarboximidamide Chemical compound N1C2=CC(C(=N)N)=CC(C(N)=N)=C2C=C1C1=CC=CC=C1 BUOYTFVLNZIELF-UHFFFAOYSA-N 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 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 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000087799 Koma Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 238000003917 TEM image Methods 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
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 208000012764 acute weight loss Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003095 anti-phagocytic effect Effects 0.000 description 1
- 229940124691 antibody therapeutics Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000008274 breast adenocarcinoma Diseases 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000000942 confocal micrograph Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000001094 effect on targets Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940082789 erbitux Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000000815 hypotonic solution Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- 208000011645 metastatic carcinoma Diseases 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000002034 xenobiotic effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Nanotechnology (AREA)
- Optics & Photonics (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
본 발명은 CD47 양성 세포막 유래 나노입자, 이의 용도 및 이의 제조방법에 관한 것으로, 구체적으로 CD47을 발현하도록 유전자 변형된 세포로부터 제조된 세포막 유래 나노입자에 치료제가 캡슐화되고 표면에 종양 표적 항체가 부착된 CD47 양성 세포막 유래 나노입자 및 이의 제조방법에 관한 것이다.The present invention relates to CD47-positive cell membrane-derived nanoparticles, uses thereof, and methods for producing the same. It relates to CD47-positive cell membrane-derived nanoparticles and a method for preparing the same.
최근, 질병 유전학에 대한 이해를 바탕으로 정밀 의학 개발에 많은 노력을 기울이고 있다. 표적 부위에 효과적인 치료제를 전달하기 위한 적절한 시스템은 정밀 의학의 전제 조건이다. 정확하고 효과적인 암 치료를 위해 다양한 유형의 전달 수단이 제안되고 테스트되고 있다. Recently, great efforts have been made to develop precision medicine based on an understanding of disease genetics. Appropriate systems for delivering effective therapeutic agents to target sites are a prerequisite for precision medicine. Various types of delivery vehicles are being proposed and tested for accurate and effective cancer treatment.
그 중에서 고분자 나노입자, 리포좀 및 금속 나노입자와 같은 합성 나노입자는 임상 응용을 위해 광범위하게 연구되고 있다. 이러한 나노입자는 유리 약물(free drug) 투여와 관련된 약점, 즉 고유한 독성, 불충분한 치료 활성 및 종양 특이성의 결여를 보완할 수 있다. 반면에, 나노입자는 생체이물(xenobiotic) 특성으로 인해 체내 면역계에 의해 쉽게 인식되어 혈액 순환에서 빠르게 제거되어 약동학적 특성의 손실을 초래한다. Among them, synthetic nanoparticles such as polymer nanoparticles, liposomes and metal nanoparticles have been extensively studied for clinical applications. These nanoparticles can compensate for the weaknesses associated with free drug administration: inherent toxicity, insufficient therapeutic activity, and lack of tumor specificity. On the other hand, due to their xenobiotic properties, nanoparticles are easily recognized by the body's immune system and rapidly removed from circulation, resulting in loss of pharmacokinetic properties.
최근, 세포막에서 유래한 다양한 나노입자가 연구되고 있다. 세포 유래 나노입자(Cell-derived nanoparticles, CDNs)는 생체적합성이 있고, 제조방법, 구성성분 및 조건에 따라 면역체계를 회피할 수 있다. CDNs는 원래 세포의 막 단백질과 지질 성분을 가지고 있기 때문에 원래 세포막의 특성을 공유한다. 따라서, 적혈구 원형질막(plasma membrane)에서 유래한 CDNs는 장기간 순환계(circulatory system)에 머무르기 때문에 흥미로운 연구로 관심을 끌었다.Recently, various nanoparticles derived from cell membranes have been studied. Cell-derived nanoparticles (CDNs) are biocompatible and can evade the immune system depending on the method of manufacture, composition and conditions. CDNs share the properties of native cell membranes because they contain protein and lipid components of the original cell membrane. Therefore, CDNs derived from the erythrocyte plasma membrane have attracted interest as an interesting study because they remain in the circulatory system for a long time.
적혈구의 장기간 순환은 원형질막에서 CD47 발현과 관련이 있다. CD47은 면역글로불린(immunoglobulin) superfamily에 속하는 인테그린 결합 단백질(integrin-associated protein, IAP)이다. 또한, 대식세포에서 신호 조절 단백질 알파(Signal-regulatory protein alpha, SIRPα)의 리간드로 알려져 있으며 “don’t eat me” 신호 역할을 한다. CD47과 SIRPα사이의 상호작용은 면역감시(immune surveillance)에서 벗어나게 한다. 또한, 종양세포 표면에 발현된 CD47은 종양 발생에 중요한 역할을 한다.Long-term circulation of erythrocytes is associated with CD47 expression at the plasma membrane. CD47 is an integrin-associated protein (IAP) belonging to the immunoglobulin superfamily. In addition, it is known as a ligand of signal-regulatory protein alpha (SIRPα) in macrophages and serves as a “don’t eat me” signal. The interaction between CD47 and SIRPα escapes immune surveillance. In addition, CD47 expressed on the surface of tumor cells plays an important role in tumor development.
항암제와 그 전달 시스템이 암 치료에 효과적이기 위해서는 적어도 다음 두 가지 조건을 만족시켜야 한다. 첫째는, 투여 후 혈액 내 약물 손실을 최소화하면서 해부학적 또는 면역학적 장벽에도 불구하고 표적 종양 조직에 도달해야 한다. 일반적으로 나노입자 캐리어는 소위 침투보존(enhanced permeability and retention, EPR) 효과로 수동 표적(passive targeting)을 통해 종양 조직에 선택적으로 축적되는 것으로 알려져 있다. 두 번째는, 약물은 종양 조직을 선택적으로 표적화하고 정상 조직에 영향을 최소화해야 한다. 능동 표적(Active targeting)은 높은 종양 특이적 약물 축적 및 낮은 비정상 세포독성을 유도하기 위한 암 치료의 진보된 전략이다. 한편 능동 종양 표적은 특이적 표적 리간드를 나노입자의 표면에 결합시켜 수행된다. For an anticancer drug and its delivery system to be effective in cancer treatment, at least the following two conditions must be satisfied. First, it must reach the target tumor tissue despite anatomical or immunological barriers while minimizing drug loss in the blood after administration. In general, it is known that nanoparticle carriers selectively accumulate in tumor tissues through passive targeting with a so-called enhanced permeability and retention (EPR) effect. Second, the drug should selectively target tumor tissue and have minimal effect on normal tissue. Active targeting is an advanced strategy in cancer treatment to induce high tumor-specific drug accumulation and low abnormal cytotoxicity. On the other hand, active tumor targeting is performed by binding a specific targeting ligand to the surface of the nanoparticle.
표피 성장인자 수용체(epidermal growth factor receptor, EGFR)는 대장암, 뇌암, 폐암 및 유방암을 포함한 다양한 암에서 과발현되거나 상향 조절된다는 것과 관련된 증거들이 많다. 따라서, 항-EGFR 항체 치료제는 암에서 EGFR 유도 신호를 방해하기 위해 개발되었으며, 현재 임상 환경에서 활용되고 있다. 또한, 세툭시맙(cetuximab)과 같은 EGFR 항체는 EGFR을 과발현하는 암세포의 능동 표적(Active targeting)을 위한 암 표적 리간드로 광범위하게 이용되고 있다.Evidence suggests that the epidermal growth factor receptor (EGFR) is overexpressed or upregulated in a variety of cancers, including colorectal, brain, lung and breast cancer. Accordingly, anti-EGFR antibody therapeutics have been developed to interfere with EGFR-induced signaling in cancer and are currently being utilized in clinical settings. In addition, EGFR antibodies such as cetuximab are widely used as cancer targeting ligands for active targeting of EGFR-overexpressing cancer cells.
이에 본 발명자들은 기존 약물 전달 시스템 분야에서 다양한 합성 나노입자들은 임상적 적용시 생체 내에서 탐식 세포에 의해 빠르게 제거되는 한계를 보이고 있으므로, 탐식작용을 피하는 것으로 알려진 “Don’t eat me” 신호 마커인 CD47을 세포막에서 과도한 양이 발현되도록 유전자 변형된 세포로부터 제조한 세포막 유래 나노입자에 치료제가 캡슐화되며 표면에 종양 표적 항체를 부착시킨 결과, 생체 적합성, 면역세포 회피성, 종양 표적능 등이 개선되어 항암치료 물질로서 유용하게 사용될 수 있음을 밝힘으로써, 본 발명을 완성하였다. Accordingly, the inventors of the present invention have shown that various synthetic nanoparticles in the field of existing drug delivery systems are rapidly removed by phagocytic cells in vivo during clinical application, so the “Don't eat me” signal marker known to avoid phagocytosis Therapeutic agents are encapsulated in cell membrane-derived nanoparticles prepared from cells genetically modified to express excessive amounts of CD47 in cell membranes, and as a result of attaching tumor-targeting antibodies to the surface, biocompatibility, immune cell evasion, and tumor-targeting ability are improved. By revealing that it can be usefully used as an anti-cancer treatment material, the present invention was completed.
본 발명의 목적은 치료제가 캡슐화되며 표면에 종양 표적 항체가 부착된 CD47 양성 세포막 유래 나노입자 및 이의 제조방법을 제공하기 위한 것이다.An object of the present invention is to provide a CD47-positive cell membrane-derived nanoparticle having a therapeutic agent encapsulated and a tumor-targeting antibody attached to the surface, and a method for preparing the same.
상기 목적을 달성하기 위하여, 본 발명은 In order to achieve the above object, the present invention
1) 세포막 유래 CD47 양성 나노입자;1) cell membrane-derived CD47-positive nanoparticles;
2) 상기 나노입자 표면에 부착된 종양 표적 항체; 및2) a tumor-targeting antibody attached to the nanoparticle surface; and
3) 상기 나노입자에 캡슐화된 항암제를 포함하는 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체를 제공한다.3) A CD47-positive-tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex comprising the anticancer agent encapsulated in the nanoparticle is provided.
또한, 본 발명은 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체를 포함하는 약물 전달체를 제공한다.In addition, the present invention provides a drug delivery system comprising a CD47-positive tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex.
아울러, 본 발명은 In addition, the present invention
1) CD47이 형질도입된 세포 유래 세포막에 초음파를 처리하여 CD47 양성 나노입자를 제조하는 단계;1) preparing CD47-positive nanoparticles by treating cell membranes derived from CD47-transduced cells with ultrasonic waves;
2) 상기 단계 1)의 CD47 양성 나노입자에 종양 표적 항체를 부착하는 단계; 및2) attaching the tumor-targeting antibody to the CD47-positive nanoparticles of step 1); and
3) 상기 단계 2)의 종양 표적 항체가 부착된 나노입자에 항암제 처리한 후, 42 내지 54시간 배양하여 상기 항암제를 캡슐화하는 단계를 포함하는 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체를 제조하는 방법을 제공한다.3) A CD47 positive-tumor target antibody-anti-cancer agent-cell membrane-derived nanoparticle complex comprising the step of encapsulating the anti-cancer agent by treating the nanoparticles to which the tumor-targeting antibody of step 2) is attached and culturing for 42 to 54 hours Provides a method for manufacturing.
본 발명의 CD47을 과발현하도록 유전자 변형된 세포로부터 제조된 세포막 유래 나노입자에 치료제가 캡슐화되고 표면에 종양 표적 항체가 부착된 CD47 양성 세포막 유래 나노입자는 종양 표적능 등이 우수하고, 특히 탐식작용을 회피하여 혈액에서 생체 적합성 및 혈액순환시간을 향상시켜 특정 암세포를 표적하여 약물을 전달하는 약물 전달체 또는 암 치료용 복합체로 유용하게 사용될 수 있다.CD47-positive cell membrane-derived nanoparticles in which a therapeutic agent is encapsulated in cell membrane-derived nanoparticles prepared from cells genetically modified to overexpress CD47 of the present invention and a tumor-targeting antibody is attached to the surface thereof are excellent in tumor targeting activity, etc., and in particular, phagocytosis It can be usefully used as a drug delivery system or complex for cancer treatment that delivers drugs by targeting specific cancer cells by improving biocompatibility and blood circulation time in the blood.
도 1은 CD47+iCDNs-DOX의 제조방법 및 특성을 나타낸 도이다:
(A) CD47+iCDNs-DOX 제조 과정 모식도;
(B) CDNs, CDNs-DOX, iCDNs 및 iCDNs-DOX의 전자현미경(× 80K) 이미지;
(C) 본 발명의 나노입자의 크기 및 표면전하; 및
(D) 나노 입자 크기를 10일 동안 모니터링한 결과.
도 2는 HEK293T 세포에 CD47을 형질감염시키는 모식도를 나타낸 도이다:
(A) CD47 암호화 플라스미드인 pCMV3-C-CD47GFPSpark의 구조; 및
(B) HEK293T 세포에 CD47을 형질감염시킨 후, 공초점 현미경(× 200)으로 관찰 결과.
도 3은 CDNs로의 독소루비신(DOX) 캡슐화의 효율성을 확인한 도이다:
(A) 분광광도법으로 DOX 탑재 효율 측정 결과; 및
(B) CDNs와 40 μg DOX를 다양한 시간동안 배양한 후, 탑재효율 결과.
도 4는 항-EGFR 항체와 CDNs의 결합을 gel filtration analysis(A) 및 SDS-PAGE(B)로 확인한 도이다.
도 5는 대식세포와 CDNs의 상호작용을 확인한 도이다:
(A) 세포 표면에 CD47-GFP 융합 단백질의 발현을 공초점현미경(x800)으로 확인한 이미지;
(B) 형질감염된 세포 용해물 및 CDNs의 Western blotting 결과;
(C) RAW 264.7 세포에 DiD 표지된 CD47-CDNs 또는 CD47+CDNs로 1 또는 4시간 동안 처리한 다음 유세포 분석 결과; 및
(D) 4시간 동안 CDNs과 함께 배양된 대식세포를 공초점 현미경(× 800)으로 확인한 이미지.
도 6은 MDA-MB-231 및 MDA-MB-453 세포에서 EGFR의 발현을 확인한 도이다.
도 7은 iCDNs의 EGFR-특이적 세포 결합 및 iCDNs-DOX의 종양 표적화 및 세포 독성을 확인한 도이다:
(A) MDA-MB-231 및 MDA-MB-453 세포에 Alexa fluor 488-labeled iCDNs를 처리한 다음 FACS analysis을 수행한 결과;
(B) MDA-MB-231 및 MDA-MB-453 세포에 iCDNs-DOX로 처리하고 DAPI로 염색한 후, 다양한 시점에서 공초점 현미경으로 확인한 이미지, PMT: photomultiplier tube; 및
(C) 세포 생존력 결과.
도 8은 이종 이식 마우스에서 iCDNs의 생체 분포를 확인한 도이다:
(A) 주요 장기의 형광 이미지;
(B) 주요 기관의 상대적 형광 강도; 및
(C) 및 (D) CDNs 및 iCDNs의 종양 대 간 비율(Tumor-to-liver ratios)
도 9는 CDNs-DOX에 의한 생체 내 종양 성장 억제를 확인한 도이다.
도 10은 CDNs-DOX로 처리된 마우스의 주요 기관의 조직 병리학 결과를 나타낸 도이다:
종양 사진의 검은색 점선은 괴사 부위(밝은 분홍색).1 is a diagram showing the preparation method and characteristics of CD47 + iCDNs-DOX:
(A) Schematic diagram of the CD47 + iCDNs-DOX manufacturing process;
(B) electron microscope (× 80K) images of CDNs, CDNs-DOX, iCDNs and iCDNs-DOX;
(C) size and surface charge of the nanoparticles of the present invention; and
(D) Results of nanoparticle size monitoring for 10 days.
Figure 2 is a schematic diagram showing the transfection of CD47 into HEK293T cells:
(A) Structure of the CD47 encoding plasmid, pCMV3-C-CD47GFPSpark; and
(B) After transfecting HEK293T cells with CD47, the result observed under a confocal microscope (× 200).
Figure 3 is a diagram confirming the efficiency of doxorubicin (DOX) encapsulation into CDNs:
(A) Measurement results of DOX loading efficiency by spectrophotometry; and
(B) Mounting efficiency results after incubating CDNs and 40 μg DOX for various times.
Figure 4 is a diagram confirming the binding of the anti-EGFR antibody and CDNs by gel filtration analysis (A) and SDS-PAGE (B).
Figure 5 is a diagram confirming the interaction between macrophages and CDNs:
(A) Images confirming the expression of the CD47-GFP fusion protein on the cell surface by confocal microscopy (x800);
(B) Western blotting results of transfected cell lysates and CDNs;
(C) Results of flow cytometry after treatment of RAW 264.7 cells with DiD-labeled CD47 - CDNs or CD47 + CDNs for 1 or 4 hours; and
(D) Confocal microscopy (× 800) image of macrophages cultured with CDNs for 4 hours.
Figure 6 is a diagram confirming the expression of EGFR in MDA-MB-231 and MDA-MB-453 cells.
Figure 7 is a diagram confirming EGFR-specific cell binding of iCDNs and tumor targeting and cytotoxicity of iCDNs-DOX:
(A) MDA-MB-231 and MDA-MB-453 cells treated with Alexa fluor 488-labeled iCDNs followed by FACS analysis;
(B) MDA-MB-231 and MDA-MB-453 cells treated with iCDNs-DOX and stained with DAPI, and confocal microscopy images at various time points, PMT: photomultiplier tube; and
(C) Cell viability results.
Figure 8 is a diagram confirming the biodistribution of iCDNs in xenotransplanted mice:
(A) Fluorescent images of major organs;
(B) Relative fluorescence intensity of major organs; and
(C) and (D) Tumor-to-liver ratios of CDNs and iCDNs
Figure 9 is a diagram confirming the inhibition of tumor growth in vivo by CDNs-DOX.
Figure 10 is a diagram showing the histopathological results of major organs of mice treated with CDNs-DOX:
The black dotted line in the tumor picture is the area of necrosis (light pink).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 1) 세포막 유래 CD47 양성 나노입자;The present invention relates to 1) cell membrane-derived CD47-positive nanoparticles;
2) 상기 나노입자 표면에 부착된 종양 표적 항체; 및2) a tumor-targeting antibody attached to the nanoparticle surface; and
3) 상기 나노입자에 캡슐화된 항암제를 포함하는 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체를 제공한다.3) A CD47-positive-tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex comprising the anticancer agent encapsulated in the nanoparticle is provided.
상기 단계 1)의 세포막은 CD47이 형질도입된 세포에서 분리된 세포막으로, CD47이 과발현된 세포막으로 제조된 나노입자는 항-식세포(phagocytic) 능력이 우수하여 면역세포에 대한 회피성을 나타낸다.The cell membrane in step 1) is a cell membrane separated from CD47-transduced cells, and the nanoparticles prepared from the CD47-overexpressed cell membrane have excellent anti-phagocytic ability and exhibit evasion to immune cells.
또한, 상기 2)의 종양 표적 항체는 표피 성장 인자 수용체(Epidermal growth factor receptor; EGFR)에 특이적으로 결합하는 항체로서, EGFR를 과발현하는 세포에 특이적으로 결합하는 것일 수 있고, 보다 구체적으로 EGFR를 과발현하는 암세포일 수 있으나, 이에 한정되는 것은 아니다.In addition, the tumor-targeting antibody of 2) is an antibody that specifically binds to epidermal growth factor receptor (EGFR), and may specifically bind to cells overexpressing EGFR, and more specifically EGFR It may be a cancer cell overexpressing, but is not limited thereto.
또한, 상기 3)의 상기 항암제는 종양 표적 항체가 특이적으로 결합하는 암세포에 대한 항암활성을 가지는 것이 바람직하고, 독소루비신(Doxorubicin)을 사용할 수 있으나, 이에 한정되는 것은 아니며, 표적 항체가 결합하는 암세포에 대한 치료 활성을 가지는 항암제는 모두 사용할 수 있으며, 상기 독소루비신은 일예로 기재한 것일 뿐, 본 발명이 상기 특정 항암제로 한정하는 것은 아니다.In addition, the anticancer agent of 3) preferably has anticancer activity against cancer cells to which the tumor target antibody specifically binds, and doxorubicin may be used, but is not limited thereto, and cancer cells to which the target antibody binds. All anticancer agents having therapeutic activity for may be used, and the doxorubicin is only described as an example, and the present invention is not limited to the specific anticancer agent.
또한, 상기 3)의 복합체는 종양 표적 항체로 인해 암세포에 특이적으로 결합하여 항암 활성을 가지며, 상기 암세포는 EGFR를 과발현하는 유방암, 두경부암, 전립선암, 폐암, 비호지킨 림프종, 신경아교종 및 육종 종양 세포로 이루어진 군으로부터 선택되는 것이 바람직하며, 삼중음성유방암 세포인 것이 가장 바람직하다.In addition, the complex of 3) has anticancer activity by specifically binding to cancer cells due to the tumor-targeting antibody, and the cancer cells overexpress EGFR in breast cancer, head and neck cancer, prostate cancer, lung cancer, non-Hodgkin's lymphoma, glioma and sarcoma It is preferably selected from the group consisting of tumor cells, most preferably triple negative breast cancer cells.
또한, 본 발명은 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체는 하기 제조방법으로 제조하는 것이 바람직하다:In addition, in the present invention, the CD47-positive tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex is preferably prepared by the following preparation method:
1) CD47이 형질도입된 세포 유래 세포막에 초음파를 처리하여 CD47 양성 나노입자를 제조하는 단계;1) preparing CD47-positive nanoparticles by treating cell membranes derived from CD47-transduced cells with ultrasonic waves;
2) 상기 단계 1)의 CD47 양성 나노입자에 종양 표적 항체를 부착하는 단계; 및2) attaching the tumor-targeting antibody to the CD47-positive nanoparticles of step 1); and
3) 상기 단계 2)의 종양 표적 항체가 부착된 나노입자에 항암제 처리한 후, 42 내지 54시간 배양하여 상기 항암제를 캡슐화하는 단계.3) encapsulating the anticancer agent by treating the nanoparticles to which the tumor-targeting antibody of step 2) is attached and culturing for 42 to 54 hours.
본 발명의 상기 단계 1)의 CD47 양성 나노입자는 말레이미드 모이어티(maleimide moieties)가 나노입자 표면에 노출된 것이 바람직하고, 단계 2)의 표적항체는 티올화된 항체이므로 상기 말레이미드 모이어티에 티올화된 종양 표적 항체가 결합된다.The CD47-positive nanoparticles of step 1) of the present invention preferably have maleimide moieties exposed on the nanoparticle surface, and since the target antibody of step 2) is a thiolated antibody, the maleimide moieties have thiol A localized tumor-targeting antibody is bound.
또한, 상기 단계 3)의 항암제는 인산염 구배(phosphate gradient)에 의해 나노입자로 캡슐화되며, 상기 항암제는 나노입자에 첨가한 후, 42 내지 54시간, 바람직하게는 46 내지 50시간, 가장 바람직하게는 48시간 배양되어야 하며, 상기 범위를 벗어날 경우, 항암제가 나노입자 내로 캡슐화되는 양이 현저히 낮아지므로 항암제를 첨가 후, 반드시 42 내지 54시간, 바람직하게는 46 내지 50시간 배양하여야 한다. 아울러, 상기 항암제가 본 발명의 복합체 내 유의적인 양으로 캡슐화되지 않으면 항암 효과를 나타낼 수 없으므로, 암 치료용으로 사용될 수 없다.In addition, the anticancer agent of step 3) is encapsulated into nanoparticles by a phosphate gradient, and the anticancer agent is added to the nanoparticles for 42 to 54 hours, preferably 46 to 50 hours, most preferably It should be cultured for 48 hours, and if it is out of the above range, since the amount of the anticancer agent encapsulated into the nanoparticles is significantly reduced, after adding the anticancer agent, it must be cultured for 42 to 54 hours, preferably 46 to 50 hours. In addition, since the anticancer agent cannot exhibit an anticancer effect unless it is encapsulated in a significant amount in the complex of the present invention, it cannot be used for cancer treatment.
또한, 상기 단계 3)의 나노입자 및 항암제는 100:5~10 몰비(molar ratio)로 혼합하는 것이 바람직하며, 상기 범위를 벗어날 경우, 나노입자의 탑재효율이 낮아 져 항암효과를 나타낼 수 없는 것이 당업자에게 자명한 사실이다.In addition, it is preferable to mix the nanoparticles and the anticancer agent in step 3) at a molar ratio of 100:5 to 10, and when out of the above range, the loading efficiency of the nanoparticles is lowered and the anticancer effect cannot be exhibited This is an obvious fact to those skilled in the art.
또한, 일반적으로 막 소포(membranous vesicle)에 치료제를 캡슐화하면 약물 자체와 비교하여 세포 독성을 감소되나, 본 발명의 제조 조건을 이용할 경우, 탑재된 약물의 효과가 유지되므로, 본 발명의 조건 및 방법을 벗어날 경우, 유의적인 항암 효과를 나타내지 못한다. In addition, encapsulation of a therapeutic agent in a membrane vesicle generally reduces cytotoxicity compared to the drug itself, but when the manufacturing conditions of the present invention are used, the effect of the loaded drug is maintained. Conditions and methods of the present invention If it is out of the range, it does not show significant anticancer effect.
또한, 상기 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체는 2000 nm 이하 직경을 가지는 것일 수 있다. 또한, 상기 복합체는 균일한 입도분포를 나타내며, 특징적인 입자의 크기 때문에 혈관이 매우 약하며 느슨한 구조를 갖는 암, 염증 등의 부위의 조직에 쉽게 혈관 내로 통과할 수 있다.In addition, the CD47-positive tumor-targeting antibody-anticancer agent-cell membrane-derived nanoparticle complex may have a diameter of 2000 nm or less. In addition, the composite exhibits a uniform particle size distribution, and because of the characteristic size of the particles, the blood vessels are very weak and can easily pass into the blood vessels in tissues of areas such as cancer and inflammation having a loose structure.
또한, 상기 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체의 제타 전위(zeta potential) 값이 -12 mV 내지 -15 mV로서, 음의 값을 가지므로, 서로 응집이 발생하지 않는다.In addition, since the CD47 positive-tumor target antibody-anti-cancer agent-cell membrane-derived nanoparticle complex has a zeta potential value of -12 mV to -15 mV, which is negative, aggregation does not occur.
본 발명의 구체적인 실시예에서, 본 발명자들은 CD47을 인코딩한 플라스미드를 HEK293T 세포에 형질 전환시키고 균질화 및 연속적인 원심 분리 과정을 사용하여 형질 전환된 세포로부터 CD47 양성 세포막 유래 나노입자(CD47+-CDNs)를 제조하였고, 제조된 세포막 유래 나노입자는 200 nm보다 작은 크기를 띠었고, 이는 투과 상승 및 저류 효과를 통해 종양에 수동 표적하기에 적합한 크기임을 확인하였다(도 1 내지 도 3 참조). In a specific embodiment of the present invention, the inventors transformed HEK293T cells with a plasmid encoding CD47 and obtained CD47-positive cell membrane-derived nanoparticles (CD47 + -CDNs) from the transformed cells using a homogenization and successive centrifugation process. was prepared, and the prepared cell membrane-derived nanoparticles had a size smaller than 200 nm, which was confirmed to be a suitable size for passive targeting to tumors through permeation increase and retention effects (see FIGS. 1 to 3).
또한, 본 발명자들은 CD47+-CDNs는 CDNs보다 RAW264.7 대식세포에 의해 적게 탐식되는 것을 확인하였고, 이 결과는 CD47+-CDNs이 대식세포에 의한 면역 반응을 피할 수 있으므로, 혈액에서 생체 적합성 및 혈액순환시간을 향상시킬 수 있음을 확인하였다(도 5 참조). In addition, the present inventors confirmed that CD47 + -CDNs were phagocytosed less by RAW264.7 macrophages than CDNs, and this result is that CD47 + -CDNs can avoid immune responses by macrophages, so they are biocompatible and It was confirmed that the blood circulation time could be improved (see FIG. 5).
또한, 본 발명자들은 독소루비신(DOX)은 인산염 농도 구배(Phosphate gradient method)으로 CD47+-CDNs에 포획하였고 그 포획율은 약 20%임을 확인하였다(도 4 참조). In addition, the present inventors confirmed that doxorubicin (DOX) was captured in CD47 + -CDNs by a phosphate gradient method, and the capture rate was about 20% (see FIG. 4).
또한, 본 발명자들은 표피성장인자수용체(EGFR)가 과발현된 암세포에 선택적으로 결합하기 위해 종양을 표적할 수 있는 항 EGFR 항체를 CD47+-CDNs에 접합시킨 후, 유세포 분석 결과, 항 EGFR 항체가 접합된 CD47+-CDNs(immuno-CD47+-CDNs; CD47+-iCDNs)은 EGFR 양성 MDA-MB-231 세포에 선택적으로 결합할 수 있었지만, EGFR 음성 MDA-MB-453 세포에는 결합할 수 없는 것을 확인하였다(도 6 및 도 7 참조). In addition, the present inventors conjugated an anti-EGFR antibody capable of targeting tumors to CD47 + -CDNs in order to selectively bind to epidermal growth factor receptor (EGFR) overexpressed cancer cells, and then, as a result of flow cytometry, the anti-EGFR antibody was conjugated CD47 + -CDNs (immuno-CD47 + -CDNs; CD47 + -iCDNs) were able to selectively bind to EGFR-positive MDA-MB-231 cells, but not to EGFR-negative MDA-MB-453 cells. (see FIGS. 6 and 7).
또한, 본 발명자들은 CD47+-iCDNs가 생체 내에서 CD47+-CDNs에 비해 종양 조직에 많이 축적되는 것을 종양이 이식된 쥐를 통해 확인하였다(도 8 참조). In addition, the present inventors confirmed that CD47 + -iCDNs were accumulated more in tumor tissues than CD47 + -CDNs in vivo in tumor-transplanted mice (see FIG. 8 ).
아울러, 본 발명자들은 공초점 현미경으로 CD47+-iCDNs-DOX가 EGFR 과발현 종양 세포의 세포질 내에 특이적으로 내재화되는 것을 독소루비신 형광을 검출함으로써 확인하였다(도 9 및 도 10 참조). In addition, the present inventors confirmed that CD47 + -iCDNs-DOX was specifically internalized into the cytoplasm of EGFR-overexpressing tumor cells by detecting doxorubicin fluorescence using a confocal microscope (see FIGS. 9 and 10).
따라서 본 발명의 항 EGFR 항체가 접합되어 있고 독소루비신을 포획한 CD47+-CDNs는 EGFR 과발현 암세포를 표적하여 약물을 전달하는 나노입자로 유용하게 사용될 수 있다.Therefore, CD47 + -CDNs conjugated with the anti-EGFR antibody of the present invention and capturing doxorubicin can be usefully used as nanoparticles for drug delivery by targeting EGFR-overexpressing cancer cells.
아울러, 본 발명은 본 발명의 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체를 포함하는 약물 전달체를 제공한다.In addition, the present invention provides a drug delivery system comprising the CD47-positive tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex of the present invention.
구체적으로, 본 발명의 CD47을 과발현하도록 유전자 변형된 세포로부터 제조된 세포막 유래 나노입자에 치료제가 캡슐화되고 표면에 종양 표적 항체가 부착된 CD47 양성 세포막 유래 나노입자는 종양 표적능 등이 우수하고, 특히 탐식작용을 회피하여 혈액에서 생체 적합성 및 혈액순환시간을 향상시켜 특정 암세포를 표적하여 약물을 전달하는 약물전달체로 유용하게 사용될 수 있다.Specifically, CD47-positive cell membrane-derived nanoparticles in which a therapeutic agent is encapsulated in cell membrane-derived nanoparticles prepared from cells genetically modified to overexpress CD47 of the present invention and a tumor-targeting antibody is attached to the surface have excellent tumor targeting ability, etc. It can be usefully used as a drug delivery system that delivers drugs by targeting specific cancer cells by improving biocompatibility and blood circulation time in the blood by avoiding phagocytosis.
본 발명의 조성물은 "약제학적으로 이용가능한 담체"를 포함할 수 있으며 약제학적으로 이용가능한 담체는 당업자에게 잘 알려진 여러 가지 인자에 따라 제조될 수 있는데, 예를 들면 본 발명의 복합체로 치료하고자 하는 질환 및 질병 또는 상태; 치료받을 개체, 나이, 크기 및 일반적인 상태; 조성물을 투여하는데 이용되는 경로, 예를 들어 비강, 구강, 안구, 국소, 경피 및 근육 등의 요인을 고려해야 하나, 이에 제한되지는 않는다. 일반적으로 경구 투여 경로 이외의 생리활성물질 투여에 이용되는 약제학적으로 이용가능한 담체에는 D5W, 덱스트로즈 및 생리학적 염을 용적의 5% 이내로 포함하는 수용액을 포함한다. 또한 약제학적으로 이용가능한 담체에는 보존제 및 항산화제와 같은 활성 성분들의 안정성을 보강시킬 수 있는 추가 성분들을 포함할 수 있다.The composition of the present invention may include a "pharmaceutically usable carrier", and the pharmaceutically usable carrier may be prepared according to various factors well known to those skilled in the art. disease and illness or condition; the individual to be treated, age, size and general condition; Factors such as, but not limited to, the route used to administer the composition, such as nasal, oral, ocular, topical, transdermal and intramuscular, should be considered. In general, pharmaceutically available carriers used for administration of bioactive substances other than oral administration routes include aqueous solutions containing D5W, dextrose and physiological salts in an amount of less than 5% by volume. In addition, pharmaceutically usable carriers may contain additional ingredients capable of enhancing the stability of active ingredients such as preservatives and antioxidants.
본 발명의 약제학적 조성물의 투여 경로는 적합한 모든 투여 경로가 이용될 수 있다. 또한, 본 발명의 약제학적 조성물의 투여량은 의도하는 치료에 유효한 용량으로 투여된다. 특정의 의학적 질환을 치료하거나 또는 이의 진행을 억제시키는데 요구되는 치료학적 유효량은 의학 분야에 공지된 예비임상 연구 및 임상 연구를 이용하여 당업자에 의해 용이하게 결정될 수 있다. 본 발명에서 상기 사용된 용어, "치료학적 유효량"은 임상의 또는 연구자가 목적하는, 특정 조직, 시스템, 및 동물 또는 인간의 생물학적 또는 의학적 반응을 유발시키는 활성 성분의 양을 말한다.Any route of administration suitable for the pharmaceutical composition of the present invention may be used. In addition, the dosage of the pharmaceutical composition of the present invention is administered in an effective dose for the intended treatment. The therapeutically effective amount required to treat or inhibit the progression of a particular medical condition can be readily determined by one skilled in the art using preclinical and clinical studies known in the medical arts. As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient that induces a biological or medical response in a particular tissue, system, and animal or human, which is desired by a clinician or researcher.
이하, 본 발명을 실시예 및 실험예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples and experimental examples.
단, 하기 실시예 및 실험예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 실시예 및 실험예에 의해 한정되는 것은 아니다.However, the following Examples and Experimental Examples specifically illustrate the present invention, and the content of the present invention is not limited by the Examples and Experimental Examples.
<실시예 1> 시료의 준비<Example 1> Preparation of samples
DSPE-PEG2000-maleimide(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000]은 Avanti Polar Lipid, Inc.(Alabaster, USA)에서 구입하였다. DMKE(O,O-dimyristyl-N-lysyl glutamate) 양이온 지질은 KOMA 바이오텍에서 구입하였다(서울, 대한민국). 마우스 CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP)는 Sino Biological사에서 구입하였다(베이징, 중국). DOX는 Sigma-Aldrich에서 구입하였다(St. Louis, MO, USA). 항-EGFR 항체(cetuximab, Erbitux®)는 Merck KgaA에서 구입하였다(Darmstadt, Germany). Martigel은 Corning에서 구입하였다(New York, NY, USA). DSPE-PEG 2000 -maleimide (1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000] was purchased from Avanti Polar Lipid, Inc. (Alabaster, USA). DMKE (O ,O-dimyristyl-N-lysyl glutamate) cationic lipid was purchased from KOMA Biotech (Seoul, Korea) Mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP) was purchased from Sino Biological (Beijing) , China) DOX was purchased from Sigma-Aldrich (St. Louis, MO, USA) Anti-EGFR antibody (cetuximab, Erbitux®) was purchased from Merck KgaA (Darmstadt, Germany) Martigel was purchased from Corning (New York, NY, USA).
<실시예 2> 세포주 및 세포 배양<Example 2> Cell line and cell culture
HEK293T(인간 배아 신장; CRL-3216TM), 인간 유방 선암종(Human breast adenocarcinoma) MDA-MB-231(HTB-26™), 인간 유방 전이성 암종(human breast metastatic carcinoma) MDA-MB-453(HTB-131™) 및 Raw 264.7(murine 대식세포, TIB-71TM) 세포주는 American Type Culture Collection(ATCC, Manassas, USA)에서 구입하였다. HEK293T (human embryonic kidney; CRL-3216 TM ), human breast adenocarcinoma MDA-MB-231 (HTB-26™), human breast metastatic carcinoma MDA-MB-453 (HTB- 131™) and Raw 264.7 (murine macrophage, TIB-71 ™ ) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, USA).
HEK293T 및 RAW 264.7 세포는 10% 소태아 혈청(FBS, Gibco, Carlsbad, CA, USA), 100 IU/mL 페니실린(Gibco) 및 100 μg/mL 스트렙토마이신(Gibco)이 보충된 Dulbecco의 변형된 Eagle 배지(DMEM, Gibco)에서 37℃, 95% 공기 및 5% CO2 상태에서 유지시켰다. MDA-MB-231 및 MDA-MB-453 세포는 10% 소태아 혈청, 100 IU/mL 페니실린 및 100 μg/mL 스트렙토마이신이 보충된 Leibovitz의 L-15 배지(Leibovitz's L-15)(Gibco)에서 37℃에서 CO2가 없는 상태에서 유지시켰다. HEK293T and RAW 264.7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 100 IU/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco). (DMEM, Gibco) at 37° C., 95% air and 5% CO 2 conditions. MDA-MB-231 and MDA-MB-453 cells were cultured in Leibovitz's L-15 (Gibco) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin. It was maintained in the absence of CO 2 at 37°C.
<실시예 3> 동물모델의 준비<Example 3> Preparation of animal model
본 발명의 모든 동물실험은 연세대학교 원주(YWCI-201805-006-01)의 Institutional Animal Care and Use Committee(IACUC)의 승인을 받았으며, 학교 지침 및 규정에 따른 프로토콜에 따라 수행하였다.All animal experiments of the present invention were approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Wonju (YWCI-201805-006-01), and were performed according to protocols in accordance with school guidelines and regulations.
<실시예 4> CD47<Example 4> CD47 ++ -세포 유래 나노입자(cell-derived nanoparticles; CDN)의 제조-Manufacture of cell-derived nanoparticles (CDN)
<4-1> CD47<4-1> CD47 ++ -CDNs의 제조-Manufacture of CDNs
CD47을 암호화하는 플라스미드를 상기 <실시예 2>에서 배양한 인간 배아 신장(HEK293T) 세포로 형질감염시켰다. The plasmid encoding CD47 was transfected into human embryonic kidney (HEK293T) cells cultured in <Example 2> above.
구체적으로, HEK293T는 60-mm 세포 배양 접시에서 융합(confluence) 정도가 80%될 때까지 배양하였고, 형질감염 전 pCD47-GFP 및 DMKE(1 : 3 중량비)를 30분 동안 Opti-MEM에서 혼합하였다. 그런 다음, 상기 세포에 혼합 형질감염 용액을 처리하고 48시간 동안 배양하였다. Specifically, HEK293T was cultured in a 60-mm cell culture dish until the degree of confluence reached 80%, and pCD47-GFP and DMKE (1:3 weight ratio) were mixed in Opti-MEM for 30 minutes before transfection. . Then, the cells were treated with the mixed transfection solution and cultured for 48 hours.
그런 다음, 원형질막을 추출하기 위해, pCD47로 형질감염된 상기 세포를 PBS(Sigma-Aldrich, St. Louis, MO, USA)에서 5 mM EDTA-4Na로 200 ×g에서 3분 동안 원심분리하고, 세포를 PBS로 세척하였다. 그럼 다음, 상기 세포를 4℃에서 20 mM Tris-HCl, 3mM MgCl2 및 10mM NaCl을 포함하는 저장액 완충액(hypotonic solution buffer)에 현탁한 다음, Dounce homogenizer를 사용하여 균질화하였다. 4℃에서 10분 동안 5,000 ×g에서 원심분리한 후, 상층액을 수집하고 4℃에서 30분간 15,000 ×g로 다시 원심분리 하였다. 상층액을 버리고 펠릿을 PBS(pH 7.4)에 분산시켰다. 현탁된 펠렛을 얼음 위에서 VibraCell VC50(Sonics & Materials Inc., Danbury, CT, USA)을 사용하여 22% amplitude에서 20초 동안 3회 초음파 처리하였다. Then, to extract the plasma membrane, the cells transfected with pCD47 were centrifuged with 5 mM EDTA-4Na in PBS (Sigma-Aldrich, St. Louis, MO, USA) at 200 × g for 3 minutes, and the cells were Washed with PBS. Then, the cells were suspended in a hypotonic solution buffer containing 20 mM Tris-HCl, 3
CD47+-세포 유래 나노입자인 CD47+-CDNs는 추가 실험을 위해 4℃에서 보관되었다.CD47 + -Cell-derived nanoparticles, CD47 + -CDNs, were stored at 4°C for further experiments.
<4-2> DSPE-PEG2000-maleimide 결합 CD47<4-2> DSPE-PEG2000-maleimide bound CD47 ++ -CDNs-CDNs
항체 접합을 위하여, DSPE-PEG2000-maleimide(1mg)를 N2 가스 하에서 건조하고 1 mL의 완충액으로 수화하여 미셀(micelle) 용액을 제조하였다. 그런 다음, 상기 미셀 용액(4 nmol 지질)을 상기 <4-1>에서 제조한 CD47+-CDNs(1 μmol 지질)에 첨가한 다음 얼음 위에서 10초 동안 3회 초음파 처리하였다. For antibody conjugation, DSPE-PEG2000-maleimide (1 mg) was dried under N 2 gas and hydrated with 1 mL of buffer to prepare a micelle solution. Then, the micellar solution (4 nmol lipid) was added to the CD47 + -CDNs (1 μmol lipid) prepared in <4-1> and then sonicated 3 times for 10 seconds on ice.
최종적으로, 상기 방법으로 Mal-CD47+-CDNs(DSPE-PEG2000-maleimide-결합된 CD47+-CDNs)을 제조하였다. Finally, Mal-CD47 + -CDNs (DSPE-PEG2000-maleimide-conjugated CD47 + -CDNs) were prepared by the above method.
<실시예 5> CDNs 내 독소루비신(Doxorubicin; DOX) 캡슐화<Example 5> Encapsulation of Doxorubicin (DOX) in CDNs
독소루비신을 상기 <실시예 4>에서 제조한 CDNs 내 캡슐화하기 위하여, 인산염 구배(phosphate gradient)를 이용하였다.To encapsulate doxorubicin in the CDNs prepared in <Example 4>, a phosphate gradient was used.
구체적으로, CDNs는 암모늄 인산염 버퍼(ammonium phosphate buffer; 300 mM, pH 7.4)에서 준비한 다음 PD-10 column(GE Healthcare, Chicago, IL, USA)을 통과하여 HEPES-EDTA buffer(25mM, 140mM NaCl, 2mM EDTA, pH 7.4)로 교환하였다. CDNs는 40 μg의 DOX의 존재 하에 37℃에서 다양한 기간 동안 배양하였고, 배양 후, PD-10 column을 통과하여 유리 DOX(free DOX)를 제거하였다. DOX의 캡슐화 효율은 480 nm에서 DOX의 흡광도를 측정하여 정량화하였다.Specifically, CDNs were prepared in ammonium phosphate buffer (300 mM, pH 7.4) and then passed through a PD-10 column (GE Healthcare, Chicago, IL, USA) to HEPES-EDTA buffer (25 mM, 140 mM NaCl, 2 mM). EDTA, pH 7.4). CDNs were cultured at 37°C for various periods of time in the presence of 40 μg of DOX, and after incubation, free DOX was removed by passing through a PD-10 column. The encapsulation efficiency of DOX was quantified by measuring the absorbance of DOX at 480 nm.
<실시예 6> 세툭시맙(cetuximab)이 결합된 CDNs-DOX의 제조<Example 6> Preparation of cetuximab-conjugated CDNs-DOX
반응성 티올기를 갖는 항체를 제조하기 위하여, 1 mg 세툭시맙을 HEPES-EDTA 버퍼(25mM HEPES, 140mM NaCl, 2mM EDTA, pH 7.4)에서 5 mg Traut 시약과 실온에서 1시간 동안 반응시켜 티올화시켰다. 그런 다음, PD-10 desalting column을 사용하여 미반응 Traut의 시약을 제거하였다. To prepare an antibody having a reactive thiol group, 1 mg of cetuximab was thiolated by reacting with 5 mg of Traut's reagent in HEPES-EDTA buffer (25 mM HEPES, 140 mM NaCl, 2 mM EDTA, pH 7.4) at room temperature for 1 hour. Then, unreacted Traut's reagent was removed using a PD-10 desalting column.
그런 다음, 상기 티올화된 항체를 0.2:1(항체: DSPE-PEG2000-maleimide) 몰비로 Mal-CD47+-CDNs-DOX에 첨가하고 혼합물을 4℃에서 16시간 동안 배양하였다. 비접합 항체는 HEPES-EDTA buffer(pH 7.4)에서 sepharose CL-4B column을 사용하여 제거시켰다. CDNs에 항체가 접합된 iCDNs는 SDS-PAGE에 의해 확인하였다.Then, the thiolated antibody was added to Mal-CD47 + -CDNs-DOX at a molar ratio of 0.2:1 (antibody: DSPE-PEG2000-maleimide) and the mixture was incubated at 4°C for 16 hours. Unconjugated antibodies were removed using a sepharose CL-4B column in HEPES-EDTA buffer (pH 7.4). Antibody-conjugated iCDNs to CDNs were identified by SDS-PAGE.
<실시예 7> CDNs 특성 확인<Example 7> Confirmation of CDNs characteristics
CDNs의 크기 및 표면 전하는 동적 광산란 제타-사이저(dynamic light scattering zeta-sizer; Nano-ZS90, Malvern Instruments Ltd. Malvern, UK)를 사용하여 측정하였고, 측정은 3회 반복하였다.The size and surface charge of CDNs were measured using a dynamic light scattering zeta-sizer (Nano-ZS90, Malvern Instruments Ltd. Malvern, UK), and the measurements were repeated three times.
또한, CDNs의 형태는 투과 전자 현미경(transmission electron microscopy; TEM)(JEM-2100F, JEOL Ltd, Tokyo, Japan)을 사용하여 관찰하였다. In addition, the morphology of CDNs was observed using a transmission electron microscopy (TEM) (JEM-2100F, JEOL Ltd, Tokyo, Japan).
구체적으로, CDNs 및 CDNs-DOX(1 nM)를 카르보닐(carbonyl) 코팅된 400 메쉬 구리 그리드에 로드하였다. 음성 염색을 위하여 10 μL의 2 % 우라닐 아세테이트를 그리드에 10분 동안 놓고, 제거한 다음 실온에서 10분 동안 건조시킨 후, TEM 이미지 촬영하였다.Specifically, CDNs and CDNs-DOX (1 nM) were loaded onto a carbonyl coated 400 mesh copper grid. For negative staining, 10 μL of 2% uranyl acetate was placed on the grid for 10 minutes, removed, dried at room temperature for 10 minutes, and TEM images were taken.
<실시예 8> 웨스턴 블랏팅 분석<Example 8> Western blotting analysis
MDA-MB-231 및 MDA-MB-453 세포의 EGFR 발현 수준을 웨스턴 블롯팅 분석으로 평가하였다. 세포를 6-웰 플레이트(웰당 2 × 105 개 세포)에 접종하고, 프로테아제 억제제(Thermo Scientific)와 함께 세포 용해 완충액(cell lysis buffer)(RIPA buffer, Thermo Scientific, MA, USA)을 사용하여 용해시켰다. 또한, 형질감염된 세포에서 CD47 발현을 분석하기 위하여, 세포 용해물 및 CDNs를 세포 용해 완충액에서 용해시켰다.EGFR expression levels of MDA-MB-231 and MDA-MB-453 cells were evaluated by Western blotting analysis. Cells were seeded in 6-well plates (2 × 10 5 cells per well) and lysed using cell lysis buffer (RIPA buffer, Thermo Scientific, MA, USA) with protease inhibitors (Thermo Scientific). made it In addition, to analyze CD47 expression in transfected cells, cell lysates and CDNs were lysed in cell lysis buffer.
구체적으로, 용해물을 10% SDS-PAGE로 분리한 후, 니트로 셀룰로오스 멤브레인(nitrocellulose membranes)으로 옮겼다. 멤브레인은 skim milk를 포함하는 TBST로 차단시킨 후, 항-EGFR rabbit 항체(Cell Signaling Technology, Danvers, USA) 또는 항-mouse CD47 항체(Invitrogen, Carlsbad, USA)의 1 : 1,000 희석액과 함께 배양하였다. 세척 후, 상기 멤브레인을 horseradish peroxidase-conjugated goat anti-rabbit 항체 또는 goat anti-rat 항체(Bethyl Laboratories, Montgomery, TX, USA)의 1:5000 희석으로 처리하고, 상기 멤브레인을 세척한 후, SuperSignal® West Pico(Thermo Scientific)을 처리하였다. 면역 반응 밴드는 Fusion Solo Chemidoc(Vilber Lourmat)를 사용하여 확인하였다.Specifically, the lysate was separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes. The membrane was blocked with TBST containing skim milk, and then incubated with a 1:1,000 dilution of anti-EGFR rabbit antibody (Cell Signaling Technology, Danvers, USA) or anti-mouse CD47 antibody (Invitrogen, Carlsbad, USA). After washing, the membrane was treated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-rat antibody (Bethyl Laboratories, Montgomery, TX, USA), and after washing the membrane, SuperSignal® West Pico (Thermo Scientific) was treated. Immunoreactive bands were identified using Fusion Solo Chemidoc (Vilber Lourmat).
<실시예 9> 세포 독성 분석<Example 9> Cytotoxicity assay
CDNs-DOX 및 iCDNs-DOX의 세포독성을 확인하기 위하여, MDA-MB-231 및 MDA-MB-453 세포를 96- 웰 플레이트(1×104 세포)에 접종하고 48시간 동안 배양하였다. To confirm the cytotoxicity of CDNs-DOX and iCDNs-DOX, MDA-MB-231 and MDA-MB-453 cells were seeded in a 96-well plate (1×10 4 cells) and cultured for 48 hours.
구체적으로, 상기 암세포에 100 μL의 무혈청 배양 배지에서 다양한 농도의 CDNs-DOX 및 iCDNs-DOX(0, 0.001, 0.01, 0.1, 1, 10 및 100 μM DOX/mL)을 처리한 다음 37℃에서 48 시간 동안 배양하였다. 그런 다음, 상기 세포에 37℃에서 2시간 동안 10μL의 Cell Counting Kit-8(CCK8) 용액(Dojindo Laboratories, Kumamoto, Japan)을 처리하였다. Infinite 200 Pro NanoQuant (TECAN)를 사용하여 450nm 파장에서 웰을 분석하였고, IC50(최대 억제 농도의 절반) 값은 GraphPad Prism 소프트웨어(GraphPad Software, Inc., San Diego, USA)를 사용하여 확인하였다.Specifically, the cancer cells were treated with various concentrations of CDNs-DOX and iCDNs-DOX (0, 0.001, 0.01, 0.1, 1, 10, and 100 μM DOX/mL) in 100 μL of serum-free culture medium, and then at 37° C. Incubated for 48 hours. Then, the cells were treated with 10 μL of Cell Counting Kit-8 (CCK8) solution (Dojindo Laboratories, Kumamoto, Japan) for 2 hours at 37°C. Wells were analyzed at 450 nm wavelength using an
<실시예 10> 시험관 내에서 대식세포 흡수 확인<Example 10> Confirmation of macrophage uptake in vitro
Raw 264.7 세포에 의한 CDNs 및 CD47+-CDNs의 세포 내 흡수를 FACS Calibur 유세포 분석기(BD Biosciences, San Jose, CA, USA) 및 공초첨 레이저 스캐닝 현미경(LSM 510; Zeiss, Heidenheim, Germany)으로 분석하였다. Intracellular uptake of CDNs and CD47 + -CDNs by Raw 264.7 cells was analyzed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and a confocal laser scanning microscope (LSM 510; Zeiss, Heidenheim, Germany). .
구체적으로, Raw 세포를 6-웰 플레이트에 웰당 2 × 105 세포 밀도로 접종하고 12 시간 동안 배양 하였다. CDNs 및 CD47+-CDNs는 3 μL의 VybrantTM Multicolor Cell-Labeling kit(Molecular Probes, Eugene, OR, USA)으로 제조업체의 프로토콜에 따라 37℃에서 15분 동안 형광 표지시켰다. 그런 다음, 1 μmol의 형광 CDNs 또는 CD47+-CDN으로 처리한 세포를 FACS Calibur 유세포 분석기(Becton Dickinson, San Jose, CA, USA)를 사용하여 다양한 시점에서 분석하였다. 동시에, 공초점 현미경(LSM 510; Zeiss, Heidenheim, Germany)으로 관찰하였다.Specifically, raw cells were seeded in a 6-well plate at a density of 2 × 10 5 cells per well and cultured for 12 hours. CDNs and CD47 + -CDNs were fluorescently labeled with 3 μL of the Vybrant ™ Multicolor Cell-Labeling kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer's protocol at 37°C for 15 minutes. Then, cells treated with 1 μmol of fluorescent CDNs or CD47 + -CDN were analyzed at various time points using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). At the same time, they were observed under a confocal microscope (LSM 510; Zeiss, Heidenheim, Germany).
<실시예 11> 시험관 내에서 iCDNs의 표적 세포 결합 확인<Example 11> Confirm target cell binding of iCDNs in vitro
<11-1> 유세포 분석<11-1> Flow cytometry
MDA-MB-231 및 MDA-MB-453 세포를 6-웰 플레이트에 접종하고 48시간 동안 배양하였다. 배양 후, 세포를 L-15(pH 7.4)로 2회 세척하고, 트립신-EDTA 용액(pH 7.4)을 처리하였다. 세포 펠릿(1 x 105 세포) 5 mL 폴리스티렌 둥근 바닥 튜브(Corning, New York, NY, USA)의 L-15에 현탁시켰다. Alexa Fluor 488(Invitrogen) 표지된 항-EGFR 항체-접합 CD47+-CDNs를 세포(튜브 당 50μM)에 첨가하고 계속 교반하면서 4℃에서 1시간 동안 배양하였다. 세포에 대한 immuno-CD47+-CDNs의 결합은 FACS Calibur 유세포 분석기(Becton Dickinson)로 분석하였다.MDA-MB-231 and MDA-MB-453 cells were seeded in 6-well plates and cultured for 48 hours. After culturing, the cells were washed twice with L-15 (pH 7.4) and treated with trypsin-EDTA solution (pH 7.4). Cell pellets (1 x 10 5 cells) were suspended in L-15 in 5 mL polystyrene round bottom tubes (Corning, New York, NY, USA). Alexa Fluor 488 (Invitrogen) labeled anti-EGFR antibody-conjugated CD47 + -CDNs were added to the cells (50 μM per tube) and incubated at 4° C. for 1 hour with continuous agitation. Binding of immuno-CD47 + -CDNs to cells was analyzed by FACS Calibur flow cytometer (Becton Dickinson).
<12-2> 공초점 현미경 분석<12-2> Confocal microscopy analysis
immuno-CD47+-CDNs-DOX(CD47+-iCDNs-DOX)의 세포 내 흡수를 확인하기 위하여, DOX를 포함하는 immuno-CD47+-CDNs를 세포(튜브 당 50 μM)에 첨가한 다음 37℃에서 0.5, 1, 4 또는 24시간 동안 무 혈청 배지에서 배양하였다. 배양 후, 세포를 차가운 PBS(pH 7.4)로 2회 세척하고, 2% 파라포름알데히드(paraformaldehyde)로 고정시켰다. 또한, 세포를 어둠 속에서 30분 동안 DAPI(4,6-diamidino-2-phenylindole) 용액(Vector Lab, Burlingame, CA, USA)으로 염색하고 슬라이드에 장착한 후, 공초점 현미경을 사용하여 슬라이드를 관찰하였다.To confirm intracellular uptake of immuno-CD47 + -CDNs-DOX (CD47 + -iCDNs-DOX), immuno-CD47 + -CDNs containing DOX were added to the cells (50 μM per tube) and then incubated at 37°C. Cultured in serum-free medium for 0.5, 1, 4 or 24 hours. After culturing, the cells were washed twice with cold PBS (pH 7.4) and fixed with 2% paraformaldehyde. In addition, cells were stained with DAPI (4,6-diamidino-2-phenylindole) solution (Vector Lab, Burlingame, CA, USA) for 30 minutes in the dark, mounted on slides, and then examined using a confocal microscope. Observed.
<실시예 13> 생체 내(<Example 13> In vivo ( in vivoin vivo ) 종양모델의 준비) Preparation of tumor model
생체 내 종양모델을 준비하기 위해, 6주령 암컷 BALB/c 누드 마우스(Orient Bio, Seongnam, Korea)의 유방지방조직(mammary fat pad)에 200 μL의 MDA-MB-231 세포(1×107)를 Matrigel(BD Biosciences)(1:1 volume ratio)과 혼합하여 피하주사하였다. To prepare an in vivo tumor model, 200 μL of MDA-MB-231 cells (1×10 7 ) were placed in mammary fat pads of 6-week-old female BALB/c nude mice (Orient Bio, Seongnam, Korea). was mixed with Matrigel (BD Biosciences) (1:1 volume ratio) and injected subcutaneously.
비니어 캘리퍼스(Vernier calipers)로 종양 크기를 측정한 후, 다음 식을 이용하여 종양 부피를 확인하였다(종양 부피 = (길이 × 높이2)/2). After measuring the tumor size with veneer calipers, the tumor volume was confirmed using the following equation (tumor volume = (length × height 2 )/2).
<실시예 14> iCDNs의 생체 내 분포 확인<Example 14> Confirmation of in vivo distribution of iCDNs
상기 <실시예 13>의 마우스의 종양 부피가 약 200 nm3에 도달하면 로다민 B(rhodamine B)가 표지된 CDNs, CD47+-CDNs 또는 immuno-CD47+-CDNs(10 mg lipid/kg)를 꼬리정맥을 통해 마우스에 주입하였다(n=3). 모든 마우스를 24 시간 후, 희생시키고 종양 조직을 포함한 주요 장기를 마우스에서 절개한 다음, Maestro 2 in vivo imaging system(Caliper Life Sciences, Hopkinton, MA, USA)을 사용하여 형광 강도를 측정하였다.When the tumor volume of the mouse of <Example 13> reached about 200 nm 3 , rhodamine B-labeled CDNs, CD47 + -CDNs or immuno-CD47 + -CDNs (10 mg lipid/kg) were added. Mice were injected through the tail vein (n=3). After 24 hours, all mice were sacrificed, and major organs including tumor tissue were dissected from the mice, and fluorescence intensity was measured using the
<실시예 15> iCDNs-DOX에 의한 생체 내 종양 성장 억제 효과 확인<Example 15> Confirmation of tumor growth inhibitory effect in vivo by iCDNs-DOX
iCDNs-DOX의 항암 효과에 대한 생체 내 평가를 위하여, <실시예 13>과 동일한 방법으로, 마우스에 MDA-MB-231 세포를 주입하고, 마우스 종양의 부피가 약 150 mm3에 도달하였을 때, 식염수, 유리 DOX, CD47-CDNs-DOX, CD47+CDNs-DOX 또는 CD47+iCDNs-DOX로 처리하였다. 처리는 2일 간격으로 3회 반복되었다(2.785 mg/kg DOX, n=4). 마우스의 종양 부피와 체중은 3일 간격으로 측정하였다.For in vivo evaluation of the anti-cancer effect of iCDNs-DOX, MDA-MB-231 cells were injected into mice in the same manner as in <Example 13>, and when the volume of the mouse tumor reached about 150 mm 3 , They were treated with saline, free DOX, CD47 - CDNs-DOX, CD47 + CDNs-DOX or CD47 + iCDNs-DOX. Treatment was repeated 3 times at 2-day intervals (2.785 mg/kg DOX, n=4). The tumor volume and body weight of the mice were measured every 3 days.
모든 마우스를 주사 후 39 일에 희생시키고 간, 심장, 신장, 폐 및 종양을 수집하고, 10 % 포르말린 용액으로 고정하고, 파라핀에 포매하고, 조직학적 분석을 위해 절개하였다. 절편된 조직은 H&E로 염색하고 현미경으로 관찰하였다.All mice were sacrificed 39 days after injection and livers, hearts, kidneys, lungs and tumors were collected, fixed in 10% formalin solution, embedded in paraffin, and dissected for histological analysis. The sectioned tissue was stained with H&E and observed under a microscope.
<실험예 1> iCDNs의 제조 <Experimental Example 1> Preparation of iCDNs
도 1A의 모식도에서와 같이, CDNs에 항-EGFR 항체 접합 및 항암 약물 독소루비신 캡슐화하는 과정을 나타내었다.As shown in the schematic diagram of FIG. 1A, the process of conjugating anti-EGFR antibodies to CDNs and encapsulating the anti-cancer drug doxorubicin is shown.
구체적으로, HEK293T 세포에 CD47을 코딩하는 플라스미드(pCMV3-C-CD47GFPSpark, 간단히 pCD47GFP)로 형질 감염시키고, 상기 형질 감염된 세포에서 CD47의 발현을 형광 현미경으로 확인하였다(도 2). 그런 다음, CD47을 발현하는 원형질막에 초음파 처리로 나노입자를 형성한 다음 DSPE-PEG2000-maleimide를 삽입하여 항체에 대한 커플링 모이어티(coupling moieties)를 제공하였다. Specifically, HEK293T cells were transfected with a plasmid encoding CD47 (pCMV3-C-CD47GFPSpark, simply pCD47GFP), and expression of CD47 in the transfected cells was observed under a fluorescence microscope (FIG. 2). Then, nanoparticles were formed in the plasma membrane expressing CD47 by sonication, and then DSPE-PEG2000-maleimide was inserted to provide coupling moieties for the antibody.
상기 나노입자 내 약물 캡슐화는 치료 약물용 나노 캐리어 개발에서 중요한 단계로, 본 발명에서는 DOX를 나노입자 내부와 외부에서 생성된 인산염 구배를 이용하여 CDNs에 효율적으로 캡슐화하였다(도 3). Drug encapsulation in the nanoparticles is an important step in the development of nanocarriers for therapeutic drugs. In the present invention, DOX was efficiently encapsulated in CDNs using phosphate gradients generated inside and outside the nanoparticles (FIG. 3).
구체적으로, CDNs와 DOX를 100:7의 지질:약물 몰비로 혼합한 다음, 실온에서 48시간 동안 배양했을 때, 구조적으로 안정한 CDNs 내부에 다량의 DOX를 캡슐화 할 수 있음을 확인하였다(19.6 % 로딩 효율). Specifically, when CDNs and DOX were mixed at a lipid:drug molar ratio of 100:7 and incubated at room temperature for 48 hours, it was confirmed that a large amount of DOX could be encapsulated inside structurally stable CDNs (19.6% loading). efficiency).
아울러, 티올화 세툭시맙 항체는 DOX를 함유하는 CDNs의 표면에 노출된 maleimide 말단에 접합되어 CD47+ iCDNs-DOX를 형성하였다(도 4). 한편, 이하, 본 발명의 실험예에서 다르게 명시하지 않는 CDNs는 CD47+ CDNs를 나타낸다.In addition, the thiolated cetuximab antibody was conjugated to the maleimide end exposed on the surface of CDNs containing DOX to form CD47 + iCDNs-DOX (FIG. 4). Meanwhile, hereinafter, CDNs unless otherwise specified in the experimental examples of the present invention represent CD47 + CDNs.
<실험예 2> CDNs의 물리 화학적 특성 확인<Experimental Example 2> Confirmation of physical and chemical properties of CDNs
TEM imaging(도 1B)에 나타낸 바와 같이, 본 발명의 CDNs, iCDNs, CDNs-DOX 및 iCDNs-DOX는 안정적인 소포 나노구조(vesicular nanostructure)를 나타냄을 확인하였다. 본 발명에서는 간단한 초음파 처리로 준비된 CDNs는 직경이 200nm 이하로 균일하게 분산되었고, 약간 음전하임을 확인하였다(-12.5 ~ -14.5mV)(도 1C). As shown in TEM imaging (FIG. 1B), it was confirmed that the CDNs, iCDNs, CDNs-DOX and iCDNs-DOX of the present invention exhibit a stable vesicular nanostructure. In the present invention, CDNs prepared by simple sonication were uniformly dispersed with a diameter of less than 200 nm and slightly negatively charged (-12.5 to -14.5 mV) (Fig. 1C).
한편, DOX 캡슐화 및 항체 접합은 소포 구조의 완전성에 영향을 미치지 않음을 확인하였다. 구체적으로, iCDNs-DOX는 10일간 4℃에서 보관하는 동안 심각한 크기 변화를 나타내지 않음을 확인하였다(도 1D).On the other hand, it was confirmed that DOX encapsulation and antibody conjugation did not affect the integrity of the vesicle structure. Specifically, it was confirmed that iCDNs-DOX did not show significant size change during storage at 4°C for 10 days (Fig. 1D).
<실험예 3> CDNs의 시험관내 대식세포 흡수 확인<Experimental Example 3> Confirmation of macrophage uptake of CDNs in vitro
공초점 현미경 분석을 통해, pCD47GFP로 형질감염된 HEK293T 세포는 CD47과 GFR의 융합 단백질이 원형질막에서 효율적으로 발현되었지만, 세포질에서는 발현하지 않음을 확인하였다(도 5A). 또한, 웨스턴블랏 분석을 통해 형질감염된 세포와 형질감염된 세포의 원형질막으로 만들어진 나노입자(CDNs)가 일시적으로 발현된 충분한 양의 CD47 단백질을 포함하고 있음을 확인하였다(도 5B).Confocal microscopic analysis confirmed that HEK293T cells transfected with pCD47GFP efficiently expressed the fusion protein of CD47 and GFR in the plasma membrane, but not in the cytoplasm (FIG. 5A). In addition, Western blot analysis confirmed that the transfected cells and nanoparticles (CDNs) made from the plasma membrane of the transfected cells contained a sufficient amount of transiently expressed CD47 protein (FIG. 5B).
대식세포 인식 측면에서 CD47-CDN 및 CD47+CDN을 비교하기 위해, 나노입자를 DiD 소수성 형광 염료(DiD hydrophobic fluorescence dye)로 표지한 다음 RAW 264.7 대식세포와 함께 배양하였다. To compare CD47 - CDN and CD47 + CDN in terms of macrophage recognition, nanoparticles were labeled with DiD hydrophobic fluorescence dye and then incubated with RAW 264.7 macrophages.
그 결과, 도 5C에 나타낸 바와 같이 유세포 분석을 통해 CD47-CDNs가 CD47+CDNs보다 대식세포에 의해 더 쉽게 흡수된다는 것을 확인하였다(도 5C). 동일한 실험 조건 하에서, CD47+CDNs와 함께 배양된 대식세포는 CD47-CDNs와 함께 배양된 세포에 비해 더 작은 형광 이동 및 더 낮은 평균 형광 강도를 나타냈으며, 시간이 지남에 따라 대식세포와의 상호작용이 더 낮아지는 것을 확인하였다.As a result, as shown in Figure 5C, it was confirmed through flow cytometry that CD47 - CDNs were more easily taken up by macrophages than CD47 + CDNs (Figure 5C). Under the same experimental conditions, macrophages cultured with CD47 + CDNs exhibited smaller fluorescence shifts and lower mean fluorescence intensity compared to cells cultured with CD47 − CDNs, and their interaction with macrophages over time It was confirmed that this is lower.
아울러, 도 5D에 나타낸 바와 같이 대식세포의 CDNs 흡수를 공초점 현미경으로 시각화하였다(도 5D). 4시간 동안 CD47-CDNs와 함께 배양된 대식세포는 CD47+CDNs와 함께 배양된 것보다 더 광범위하고 강한 형광을 나타냈다. In addition, as shown in Fig. 5D, uptake of CDNs by macrophages was visualized by confocal microscopy (Fig. 5D). Macrophages incubated with CD47 - CDNs for 4 hours showed more extensive and stronger fluorescence than those incubated with CD47 + CDNs.
따라서, 상기 실험을 통해 본 발명의 CD47+CDNs는 대식세포의 식균 작용을 유의적으로 회피하는 것을 확인하였다.Therefore, through the above experiments, it was confirmed that the CD47 + CDNs of the present invention significantly evaded the phagocytosis of macrophages.
<실험예 4> iCDNs-DOX의 시험관내 종양 표적 효과 확인<Experimental Example 4> Confirmation of in vitro tumor targeting effect of iCDNs-DOX
종양 표적화 CDNs인 항-EGFR immuno-CDNs(iCDNs) 제조를 위하여, DSPE-PEG2000-maleimide 및 DiD 형광 염료에 수화된 혼합물을 CDNs와 혼합한 후, 초음파 처리하였다. 또한, 항-EGFR 항체인 티올화 세툭시맙(Thiolated cetuximab)을 CDNs 표면에 노출시킨 maleimide 말단에 직접 결합시켜 iCDNs를 제조하였다. To prepare anti-EGFR immuno-CDNs (iCDNs), which are tumor-targeting CDNs, a mixture hydrated with DSPE-PEG2000-maleimide and DiD fluorescent dye was mixed with CDNs and subjected to sonication. In addition, iCDNs were prepared by directly binding an anti-EGFR antibody, thiolated cetuximab, to the maleimide end exposed on the CDNs surface.
iCDNs를 EGFR을 발현하는 표적 종양 MDA-MB-231 세포(도 6)에 처리하고, 이들의 상호작용을 cytometric 분석을 이용하여 확인하였다(도 7A)iCDNs were treated with target tumor MDA-MB-231 cells expressing EGFR (FIG. 6), and their interactions were confirmed using cytometric analysis (FIG. 7A).
그 결과, 도 7A에 나타낸 바와 같이, 표적 리간드가 결핍된 CDNs는 표적 MDA-MB-231 또는 대조군 MDA-MB-453 세포에 효과적으로 결합할 수 없었다. 그러나 본 발명의 iCDNs는 EGFR의 높은 발현을 나타내는 MDA-MB-231 세포에 대해 유의적으로 결합을 나타내었지만, MDA-MB-453 세포에는 그렇지 않음을 확인하였다. As a result, as shown in Fig. 7A, CDNs lacking the targeting ligand could not effectively bind to target MDA-MB-231 or control MDA-MB-453 cells. However, it was confirmed that the iCDNs of the present invention showed significant binding to MDA-MB-231 cells showing high expression of EGFR, but not to MDA-MB-453 cells.
따라서, 본 발명의 iCDNs가 EGFR 매개 상호작용을 통하여 표적 종양 세포를 특이적으로 인식할 수 있음을 확인하였다.Therefore, it was confirmed that the iCDNs of the present invention can specifically recognize target tumor cells through EGFR-mediated interactions.
<실험예 5> iCDNs의 종양 표적 약물 전달 효과 확인<Experimental Example 5> Confirmation of tumor-targeted drug delivery effect of iCDNs
iCDNs의 종양 표적 약물 전달을 확인하기 위하여, DOX를 인산염 구배 방법을 이용하여 CDNs에 캡슐화하였다. 최적 조건 하에서, DOX 및 CDNs(1mM 지질 : 40μg의 DOX)를 37℃에서 300 mM 암모늄 포스페이트의 존재 하에 4시간 동안 외소낭 배지(extravesicular medium)에서 배양하여, 가장 많은 양의 DOX(약 20%)를 CDNs로 캡슐화하였고, CDNs의 구조적 무결성(structural integrity)은 이후 안정적으로 유지됨을 확인하였다. 또한, 티올화 세툭시맙을 상기 동일한 방법으로 iCDNs-DOX 표면에 접합시켰다. To confirm the tumor-targeted drug delivery of iCDNs, DOX was encapsulated in CDNs using a phosphate gradient method. Under optimal conditions, DOX and CDNs (1 mM lipid: 40 μg of DOX) were cultured in extravesicular medium for 4 hours in the presence of 300 mM ammonium phosphate at 37°C, resulting in the highest amount of DOX (approximately 20%). was encapsulated into CDNs, and it was confirmed that the structural integrity of the CDNs was stably maintained thereafter. In addition, thiolated cetuximab was conjugated to the iCDNs-DOX surface in the same manner as above.
도 7B에 나타낸 바와 같이 공초점 현미경 분석을 통해 iCDNs-DOX로 처리된 MDA-MB-231 세포는 동일한 실험 조건에서 MDA-MB-453 세포보다 강한 DOX 형광을 나타냄을 확인하였다. 이는 iCDNs-DOX가 비표적 MDA-MB-453 세포보다 표적 MDA-MB-231 세포의 세포질에 DOX를 더 효과적으로 전달할 수 있음을 나타낸다.As shown in FIG. 7B , it was confirmed through confocal microscopic analysis that MDA-MB-231 cells treated with iCDNs-DOX showed stronger DOX fluorescence than MDA-MB-453 cells under the same experimental conditions. This indicates that iCDNs-DOX can more effectively deliver DOX to the cytoplasm of target MDA-MB-231 cells than non-target MDA-MB-453 cells.
따라서, iCDNs-DOX의 세툭시맙과 EGFR간의 상호작용을 통해 표적 세포에 DOX의 향상된 전달 효과를 나타냄을 확인하였다. Therefore, it was confirmed that the interaction between cetuximab and EGFR of iCDNs-DOX exhibits an enhanced delivery effect of DOX to target cells.
<실험예 6> iCDNs의 세포독성 확인<Experimental Example 6> Confirmation of cytotoxicity of iCDNs
MDA-MB-231 및 MDA-MB-453 세포에 대한 iCDNs-DOX의 세포 독성을 확인하기 위하여, 유리 DOX 및 비표적 CDNs-DOX의 세포 독성과 비교하였다. 일반적으로 막 소포(membranous vesicle)에 치료제를 캡슐화하면 약물 자체와 비교하여 세포 독성을 감소시키는 경향을 나타냄이 알려져 있다(Anti-EGFR lipid micellar nanoparticles co-encapsulating quantum dots and paclitaxel for tumor-targeted theranosis, Nanoscale 10(41) (2018) 19338-19350). 따라서, 약물 전달을 위해 캡슐화하였더라도, 표적 세포에 타겟 약물에 유의적인 효과를 나타내야 한다.To confirm the cytotoxicity of iCDNs-DOX on MDA-MB-231 and MDA-MB-453 cells, the cytotoxicity of free DOX and off-target CDNs-DOX was compared. In general, it is known that encapsulating a therapeutic agent in a membrane vesicle tends to reduce cytotoxicity compared to the drug itself (Anti-EGFR lipid micellar nanoparticles co-encapsulating quantum dots and paclitaxel for tumor-targeted theranosis, Nanoscale 10(41) (2018) 19338-19350). Therefore, even if encapsulated for drug delivery, the target drug should have a significant effect on target cells.
따라서, 본 발명의 iCDNs-DOX의 세포 독성을 CCK-8 분석을 통해 확인하였다.Therefore, the cytotoxicity of the iCDNs-DOX of the present invention was confirmed through CCK-8 assay.
구체적으로, 도 7C에 나타낸 바와 같이 iCDNs 또는 CDNs로의 캡슐화는 MDA-MB-453 세포에서 DOX의 세포 독성에 상당한 부정적 영향을 미침을 확인하였다(도 7C). MDA-MB-453 세포에서 유리 DOX의 IC50은 0.32±0.1 μM, CDNs-DOX 및 iCDNs-DOX의 IC50은 각각 3.92±1.46 μM 및 2.11±0.62 μM임을 확인하였다. Specifically, as shown in Fig. 7C, encapsulation with iCDNs or CDNs had a significant negative effect on the cytotoxicity of DOX in MDA-MB-453 cells (Fig. 7C). The IC 50 of free DOX in MDA-MB-453 cells was 0.32±0.1 μM, and the IC 50 of CDNs-DOX and iCDNs-DOX were 3.92±1.46 μM and 2.11±0.62 μM, respectively.
반면에, MDA-MB-231 세포에서 유리 DOX, CDNs-DOX, iCDNs-DOX의 IC50은 각각 4.05 ± 1.07 μM, 4.20 ± 0.95 μM 또는 3.77 ± 1.51 μM으로 본 발명의 캡슐화로 인한 영향이 없는 것을 확인하였다. On the other hand, the IC 50 of free DOX, CDNs-DOX and iCDNs-DOX in MDA-MB-231 cells were 4.05 ± 1.07 μM, 4.20 ± 0.95 μM or 3.77 ± 1.51 μM, respectively, indicating no effect due to the encapsulation of the present invention. Confirmed.
<실험예 7> 종양 이종 이식 마우스에서 iCDNs의 <Experimental Example 7> iCDNs in tumor xenograft mice in vivoin vivo 생체 분포 확인 Confirmation of biodistribution
iCDNs의 생체 분포 패턴을 확인하기 위하여, 마우스에 MDA-MB-231 세포를 이종 이식한 다음, DiD 표지된 CDNs 또는 iCDNs를 정맥주사하였다. 종양 조직 및 주요 기관(예: 간, 폐, 심장, 신장, 비장 및 혈액)을 수집하고, 주사 후 10분, 1시간, 4시간, 24시간 및 48시간에 이미징 시스템으로 형광 이미지를 촬영하였다. To confirm the biodistribution pattern of iCDNs, MDA-MB-231 cells were xenotransplanted into mice, and then DiD-labeled CDNs or iCDNs were intravenously injected. Tumor tissues and major organs (e.g., liver, lung, heart, kidney, spleen and blood) were collected and fluorescence images were taken with an imaging system at 10 minutes, 1 hour, 4 hours, 24 hours and 48 hours after injection.
그 결과, 도 8에 나타낸 바와 같이 iCDNs로 처리 된 마우스는 CDNs로 처리 된 마우스에 비해 오른쪽 유방 지방 패드의 종양 조직에서 상대적으로 더 강한 형광 신호를 보였고(도 8A), 모든 측정에서 iCDNs 처리시 강한 형광반응을 나타내었다. As a result, as shown in FIG. 8, the mice treated with iCDNs showed a relatively stronger fluorescence signal in the tumor tissue of the right mammary fat pad compared to the mice treated with CDNs (FIG. 8A), and in all measurements, the iCDNs treatment showed a stronger fluorescence signal. A fluorescence reaction was shown.
또한, 생체 내 이미징 분석 후, 주요 장기(간, 폐, 신장, 비장 및 종양)를 마우스에서 절개한 후, 생체외 형광 이미지 확인하였다(도 8B). In addition, after in vivo imaging analysis, major organs (liver, lung, kidney, spleen, and tumor) were dissected from the mouse, and ex vivo fluorescence images were confirmed (FIG. 8B).
그 결과, 도 8B에 나타낸 바와 같이 동일 처리시점에서 iCDNs 처리군은 CDNs 처리군보다 종양에서 더 강한 형광 신호를 나타냄을 확인하였다.As a result, as shown in FIG. 8B, it was confirmed that the iCDNs-treated group exhibited a stronger fluorescence signal in the tumor than the CDNs-treated group at the same treatment time point.
또한 CDNs 처리군의 종양 형광 신호는 주사 후 10분에 낮았고 4시간 후에 가장 높았으며 신호가 점차 소멸되었으나, 본 발명의 iCDNs 그룹의 종양 형광 신호는 주입 후 10분에도 강했고 48시간 동안 높은 수준의 형광 강도를 유지됨을 확인하였다. In addition, the tumor fluorescence signal of the CDNs-treated group was low at 10 minutes after injection and the highest at 4 hours, and the signal gradually disappeared. It was confirmed that the strength was maintained.
CDN과 iCDN의 종양 표적성을 비교하기 위하여 각 측정에서 종양 대 간 비율(tumor-to-liver ratio, TLR)을 계산하였다(도 8D). To compare the tumor targeting of CDN and iCDN, the tumor-to-liver ratio (TLR) was calculated for each measurement (Fig. 8D).
그 결과, 도 8D에 나타낸 바와 같이 iCDNs 그룹의 TLR은 CDNs 그룹의 TLR보다 높았고, iCDNs의 개선된 TLR은 혈관 내 순환으로부터의 효율적인 혈관외유출(extravasation)과 효율적인 EGFR 매개 내포작용(endocytosis)으로 활성 타겟 세포를 인식할 수 있음을 확인하였다.As a result, as shown in FIG. 8D, the TLR of the iCDNs group was higher than that of the CDNs group, and the improved TLR of the iCDNs was active through efficient extravasation from the intravascular circulation and efficient EGFR-mediated endocytosis. It was confirmed that the target cells could be recognized.
<실험예 8> iCDNs-DOX에 의한 생체내 종양 성장 억제<Experimental Example 8> Inhibition of in vivo tumor growth by iCDNs-DOX
iCDNs-DOX의 항암 치료 효능을 평가하기 위하여, 도 9A에 나타낸 바와 같이 MDA-MB-231 종양을 보유한 마우스에 CD47+iCDNs-DOX를 3일 간격으로 세 번 정맥 주사하고, 종양 성장을 39일 동안 확인하였다. 또한, 유리 DOX, CD47-CDNs-DOX 및 CD47+CDNs-DOX을 처리한 마우스의 종양 성장과 비교하였다.To evaluate the anti-cancer therapeutic efficacy of iCDNs-DOX, as shown in Figure 9A, CD47 + iCDNs-DOX was intravenously injected three times every 3 days to mice bearing MDA-MB-231 tumors, and tumor growth was monitored for 39 days. Confirmed. In addition, tumor growth in mice treated with free DOX, CD47 - CDNs-DOX and CD47 + CDNs-DOX was compared.
그 결과 도 9B에 나타낸 바와 같이, CD47+iCDNs-DOX는 치료군 중에서 종양 성장을 가장 효과적으로 억제하는 것을 확인하였다. 한편, 비-표적 CD47+CDNs-DOX가 유리 DOX 및 CD47-CDNs-DOX보다 더 강한 종양 성장 억제를 보였음에도 불구하고, 종양 표적 CD47+iCDNs-DOX는 가장 효과적으로 종양 성장을 억제시켰고, 마지막 날에 절제된 종양의 무게는 CD47+iCDNs-DOX가 현저한 종양 억제 효과를 나타냄을 확인하였다(도 9C 및 9D). 한편, 유리 DOX 처리 또는 CD47-CDNs-DOX 처리 마우스는 유사한 종양 무게(각각 0.613 ± 0.240 mg 및 0.593 ± 0.240 mg)를 나타냄을 확인하였다. As a result, as shown in FIG. 9B, it was confirmed that CD47 + iCDNs-DOX inhibited tumor growth most effectively among the treatment groups. On the other hand, although non-targeted CD47 + CDNs-DOX showed stronger tumor growth inhibition than free DOX and CD47 - CDNs-DOX, tumor-targeted CD47 + iCDNs-DOX inhibited tumor growth most effectively and at the last day The weight of the excised tumors confirmed that CD47 + iCDNs-DOX exhibited significant tumor suppression effects (FIGS. 9C and 9D). On the other hand, it was confirmed that mice treated with free DOX or CD47 - CDNs-DOX showed similar tumor weights (0.613 ± 0.240 mg and 0.593 ± 0.240 mg, respectively).
결론적으로, CD47+iCDNs-DOX 처리된 마우스의 종양 무게는 비표적 CD47+CDNs-DOX 처리된 마우스보다 낮았고, 이러한 결과는 CDN 표면에 노출된 면역 세포-회피 CD47 단백질과 종양 표적화 세툭시맙 분자가 종양 세포의 세포질로 DOX의 세포 내 전달을 상승적으로 향상시켜, 암세포의 나노 캐리어 시스템의 항암 치료 효능을 높일 수 있음을 확인하였다.In conclusion, the tumor weight of CD47 + iCDNs-DOX-treated mice was lower than that of non-targeting CD47 + CDNs-DOX-treated mice, suggesting that the immune cell-evacuating CD47 protein exposed on the CDN surface and the tumor-targeting cetuximab molecule It was confirmed that the anticancer treatment efficacy of the nanocarrier system of cancer cells can be increased by synergistically enhancing the intracellular delivery of DOX into the cytoplasm of tumor cells.
한편, 도 9E에 나타낸 바와 같이, 본 발명의 세포막 나노 캐리어 시스템 자체는 치료 중 심각한 생체 내 독성을 나타내지 않는다.On the other hand, as shown in Figure 9E, the cell membrane nanocarrier system itself of the present invention does not show significant in vivo toxicity during treatment.
구체적으로, DOX를 포함하는 본 발명의 막 나노입자로 처리된 모든 마우스는 급성 체중 감소를 나타내지 않았고, 이러한 결과를 통해 본 발명의 막 나노 입자 시스템이 세포독성이 없어, 임상적으로 안정하게 사용될 수 있음을 확인하였다(도 9E).Specifically, all mice treated with the DOX-containing membrane nanoparticles of the present invention did not show an acute weight loss, and through these results, the membrane nanoparticle system of the present invention has no cytotoxicity and can be used clinically safely. It was confirmed that there was (FIG. 9E).
<실험예 9> CD47<Experimental Example 9> CD47 ++ iCDNs-DOX로 처리 된 마우스의 조직학적 분석Histological analysis of mice treated with iCDNs-DOX
종양 성장 측정의 마지막 날 심장, 신장, 간, 폐, 종양 조직 등 주요 기관 및 조직을 절제하고, 조직학적 분석을 수행하였다. On the last day of tumor growth measurement, major organs and tissues such as heart, kidney, liver, lung, and tumor tissue were resected and histological analysis was performed.
유리 DOX는 고용량에서 심장 독성을 유발할 수 있다는 점이 잘 알려져 있으나, 식염수 처리된 대조군 마우스와 비교하여, 유리 DOX, CD47-CDNs-DOX, CD47+ CDNs-DOX 및 CD47+iCDNs-DOX가 처리된 모든 마우스 그룹(DOX 처리량 2.8mg/kg)에서 심장 조직에 유의한 형태학적 변화가 나타나지 않았고, 간, 신장 및 폐의 절편된 조직에서도 유의적인 변화가 나타나지 않음을 확인하였다. Although it is well known that free DOX can induce cardiotoxicity at high doses, all mice treated with free DOX, CD47 - CDNs-DOX, CD47 + CDNs-DOX and CD47 + iCDNs-DOX compared to saline-treated control mice. It was confirmed that there was no significant morphological change in heart tissue in the group (DOX treatment amount of 2.8 mg/kg), and no significant change was also shown in the sectioned tissues of liver, kidney and lung.
그러나, 종양조직에서는 CD47-CDNs-DOX, CD47+CDNs-DOX 또는 CD47+iCDNs-DOX로 처리된 마우스의 종양 조직에서 광범위한 괴사 영역이 관찰된 반면, 식염수 또는 유리 DOX 처리된 마우스는 조직 전체의 불규칙한 핵산 위치와 많은 혈관을 보이므로 종양의 전형적인 조직을 나타내었다.However, extensive areas of necrosis were observed in tumor tissues of mice treated with CD47 - CDNs-DOX, CD47 + CDNs-DOX, or CD47 + iCDNs-DOX, whereas in saline or free DOX-treated mice, irregular throughout the tissue was observed. Nucleic acid localization and many blood vessels were present, indicating a typical tissue of a tumor.
따라서, 본 발명의 나노입자 표면에 노출된 종양 표적화 세툭시맙 및 CD47 분자는 표적 종양 조직의 성장 억제에 대해 상승적(또는 부가적)이고 유의적인 효과를 나타내므로, CD47 및 항암 리간드를 노출하는 세포 유래 나노 캐리어가 표적 암 치료를 위한 새로운 약물 전달 시스템으로 사용될 수 있음을 확인하였다.Thus, the tumor-targeting cetuximab and CD47 molecules exposed on the surface of the nanoparticles of the present invention exhibit a synergistic (or additive) and significant effect on the growth inhibition of target tumor tissue, thus cells exposing CD47 and anti-cancer ligands. It was confirmed that the derived nanocarriers can be used as a new drug delivery system for targeted cancer treatment.
Claims (16)
2) 상기 나노입자 표면에 부착된 종양 표적 항체; 및
3) 상기 나노입자에 캡슐화된 항암제를 포함하는 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체.
1) cell membrane-derived CD47-positive nanoparticles;
2) a tumor-targeting antibody attached to the nanoparticle surface; and
3) A CD47-positive tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex comprising an anticancer agent encapsulated in the nanoparticle.
The CD47-positive-tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex according to claim 1, wherein the cell membrane is a cell membrane transduced with CD47.
The CD47-positive-tumor-targeting antibody-anticancer agent-cell membrane-derived nanoparticle complex according to claim 1, wherein the CD47-positive nanoparticles avoid phagocytosis of macrophages.
The CD47-positive-tumor targeting antibody-anticancer agent-cell membrane-derived nanoparticle complex according to claim 1, wherein the target antibody specifically binds to an epidermal growth factor receptor.
The CD47-positive-tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex according to claim 1, wherein the complex specifically binds to cancer cells.
The method of claim 5, wherein the cancer cells are selected from the group consisting of breast cancer, head and neck cancer, prostate cancer, lung cancer, non-Hodgkin's lymphoma, glioma and sarcoma tumor cells nanoparticle complexes.
The CD47 positive-tumor targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex according to claim 5, wherein the cancer cells are triple-negative breast cancer cells.
The CD47-positive-tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex according to claim 1, wherein the anticancer agent has anticancer activity against cancer cells to which the tumor target antibody specifically binds.
[Claim 9] The CD47-positive tumor-targeting antibody-anti-cancer agent-cell membrane-derived nanoparticle complex according to claim 8, wherein the anti-cancer agent is doxorubicin.
A drug delivery system comprising a CD47-positive tumor-targeting antibody-anti-cancer drug-cell membrane-derived nanoparticle complex.
2) 상기 단계 1)의 CD47 양성 나노입자에 종양 표적 항체를 부착하는 단계; 및
3) 상기 단계 2)의 종양 표적 항체가 부착된 나노입자에 항암제 처리한 후, 42 내지 54시간 배양하여 상기 항암제를 캡슐화하는 단계를 포함하는 CD47양성-종양 표적항체-항암제-세포막 유래 나노입자 복합체를 제조하는 방법.
1) preparing CD47-positive nanoparticles by treating cell membranes derived from CD47-transduced cells with ultrasonic waves;
2) attaching the tumor-targeting antibody to the CD47-positive nanoparticles of step 1); and
3) A CD47 positive-tumor target antibody-anti-cancer agent-cell membrane-derived nanoparticle complex comprising the step of encapsulating the anti-cancer agent by treating the nanoparticles to which the tumor-targeting antibody of step 2) is attached and culturing for 42 to 54 hours How to manufacture.
The method of claim 11, wherein the CD47-positive nanoparticles of 1) have maleimide moieties exposed on the surface of the nanoparticles. How to.
The method of claim 11, wherein the target antibody in step 2) is a thiolated antibody attached to the nanoparticle.
The method of claim 11, wherein the anticancer agent of step 3) is added to the nanoparticles and then cultured for 46 to 50 hours.
The method of claim 11, wherein the nanoparticle and the anticancer agent of step 3) are mixed at a molar ratio of 100: 5 to 10 to prepare a CD47-positive-tumor targeting antibody-anticancer agent-cell membrane-derived nanoparticle complex Way.
The method of claim 11, wherein the anticancer agent of step 4) is encapsulated into nanoparticles by a phosphate gradient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210066684A KR102659839B1 (en) | 2021-05-25 | 2021-05-25 | Tumor-targeted nanoparticles using CD47 positive cell membrane, uses thereof and method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210066684A KR102659839B1 (en) | 2021-05-25 | 2021-05-25 | Tumor-targeted nanoparticles using CD47 positive cell membrane, uses thereof and method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20220158984A true KR20220158984A (en) | 2022-12-02 |
| KR102659839B1 KR102659839B1 (en) | 2024-04-22 |
Family
ID=84418013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210066684A KR102659839B1 (en) | 2021-05-25 | 2021-05-25 | Tumor-targeted nanoparticles using CD47 positive cell membrane, uses thereof and method thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102659839B1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116286996A (en) * | 2023-03-24 | 2023-06-23 | 暨南大学附属第一医院（广州华侨医院） | Medicine carrier targeting CXCL16+ macrophages and preparation method and application thereof |
| CN116617408A (en) * | 2023-03-31 | 2023-08-22 | 江南大学附属医院 | Microorganism and functional nucleic acid co-delivery system and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120002942A (en) * | 2010-07-01 | 2012-01-09 | 포항공과대학교 산학협력단 | Microvesicles derived from the protoplast of cells and use thereof |
| KR20190037163A (en) * | 2017-09-28 | 2019-04-05 | 주식회사 탠덤 | Novel plasma membrane-based vesicle used for treating cancer |
-
2021
- 2021-05-25 KR KR1020210066684A patent/KR102659839B1/en active IP Right Grant
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120002942A (en) * | 2010-07-01 | 2012-01-09 | 포항공과대학교 산학협력단 | Microvesicles derived from the protoplast of cells and use thereof |
| KR20190037163A (en) * | 2017-09-28 | 2019-04-05 | 주식회사 탠덤 | Novel plasma membrane-based vesicle used for treating cancer |
Non-Patent Citations (5)
| Title |
|---|
| Acta Pharmaceutica Sinica B, 11(8), p.2096-2113, 2021.02.01. * |
| Anti-EGFR lipid micellar nanoparticles co-encapsulating quantum dots and paclitaxel for tumor-targeted theranosis, Nanoscale 10(41) (2018) 19338-19350 |
| Biomaterials, 128, p.69-83, 2017. * |
| International Journal of Nanomedicine, 12, p.2759-2767, 2017. * |
| Lipid-coated polymeric nanoparticles for cancer drug delivery, Biomater Sci 3(7) (2015) 923-36. |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116286996A (en) * | 2023-03-24 | 2023-06-23 | 暨南大学附属第一医院（广州华侨医院） | Medicine carrier targeting CXCL16+ macrophages and preparation method and application thereof |
| CN116617408A (en) * | 2023-03-31 | 2023-08-22 | 江南大学附属医院 | Microorganism and functional nucleic acid co-delivery system and preparation method and application thereof |
| CN116617408B (en) * | 2023-03-31 | 2023-12-08 | 江南大学附属医院 | Microorganism and functional nucleic acid co-delivery system and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR102659839B1 (en) | 2024-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kim et al. | Dual-targeting immunoliposomes using angiopep-2 and CD133 antibody for glioblastoma stem cells | |
| Peng et al. | Herceptin-conjugated paclitaxel loaded PCL-PEG worm-like nanocrystal micelles for the combinatorial treatment of HER2-positive breast cancer | |
| Kim et al. | Anti-EGF receptor aptamer-guided co-delivery of anti-cancer siRNAs and quantum dots for theranostics of triple-negative breast cancer | |
| Byeon et al. | Doxorubicin-loaded nanoparticles consisted of cationic-and mannose-modified-albumins for dual-targeting in brain tumors | |
| Belhadj et al. | Multifunctional targeted liposomal drug delivery for efficient glioblastoma treatment | |
| Zong et al. | Enhanced glioma targeting and penetration by dual-targeting liposome co-modified with T7 and TAT | |
| Schneider et al. | Minimizing the non-specific binding of nanoparticles to the brain enables active targeting of Fn14-positive glioblastoma cells | |
| Liu et al. | Doxorubicin delivered using nanoparticles camouflaged with mesenchymal stem cell membranes to treat colon cancer | |
| He et al. | LRP1-mediated pH-sensitive polymersomes facilitate combination therapy of glioblastoma in vitro and in vivo | |
| Cui et al. | Dual-target peptide-modified erythrocyte membrane-enveloped PLGA nanoparticles for the treatment of glioma | |
| Lee et al. | Nano-sized metabolic precursors for heterogeneous tumor-targeting strategy using bioorthogonal click chemistry in vivo | |
| Choi et al. | Targeted antitumor efficacy and imaging via multifunctional nano-carrier conjugated with anti-HER2 trastuzumab | |
| Ni et al. | PSMA-targeted nanoparticles for specific penetration of blood-brain tumor barrier and combined therapy of brain metastases | |
| Ying et al. | Macrophage membrane-biomimetic adhesive polycaprolactone nanocamptothecin for improving cancer-targeting efficiency and impairing metastasis | |
| Zhang et al. | Near infrared-activatable biomimetic nanogels enabling deep tumor drug penetration inhibit orthotopic glioblastoma | |
| KR102659839B1 (en) | Tumor-targeted nanoparticles using CD47 positive cell membrane, uses thereof and method thereof | |
| Qi et al. | Combined integrin α v β 3 and lactoferrin receptor targeted docetaxel liposomes enhance the brain targeting effect and anti-glioma effect | |
| Duan et al. | Vincristine-loaded and sgc8-modified liposome as a potential targeted drug delivery system for treating acute lymphoblastic leukemia | |
| US20170258718A1 (en) | Pharmaceutical composition, preparation and uses thereof | |
| Sun et al. | Targeting epirubicin plus quinacrine liposomes modified with DSPE-PEG2000-C (RGDfK) conjugate for eliminating invasive breast cancer | |
| Ye et al. | Enhanced therapeutic efficacy of LHRHa-targeted brucea javanica oil liposomes for ovarian cancer | |
| Cui et al. | Gather wisdom to overcome barriers: well-designed nano-drug delivery systems for treating gliomas | |
| Zhang et al. | HAase-sensitive dual-targeting irinotecan liposomes enhance the therapeutic efficacy of lung cancer in animals | |
| Huang et al. | Antibody-activated trans-endothelial delivery of mesoporous organosilica nanomedicine augments tumor extravasation and anti-cancer immunotherapy | |
| Liu et al. | Targeted delivery of irinotecan to colon cancer cells using epidermal growth factor receptor-conjugated liposomes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| AMND | Amendment | ||
| X701 | Decision to grant (after re-examination) | ||
| GRNT | Written decision to grant |