CN114642736B - Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof - Google Patents
Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof Download PDFInfo
- Publication number
- CN114642736B CN114642736B CN202011502081.7A CN202011502081A CN114642736B CN 114642736 B CN114642736 B CN 114642736B CN 202011502081 A CN202011502081 A CN 202011502081A CN 114642736 B CN114642736 B CN 114642736B
- Authority
- CN
- China
- Prior art keywords
- blood
- drug delivery
- delivery system
- brain barrier
- neutrophil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000012377 drug delivery Methods 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000004888 barrier function Effects 0.000 title claims description 12
- 210000001808 exosome Anatomy 0.000 claims abstract description 76
- 210000000440 neutrophil Anatomy 0.000 claims abstract description 69
- 230000008499 blood brain barrier function Effects 0.000 claims abstract description 54
- 210000001218 blood-brain barrier Anatomy 0.000 claims abstract description 53
- 239000003814 drug Substances 0.000 claims abstract description 31
- 206010018338 Glioma Diseases 0.000 claims abstract description 20
- 208000032612 Glial tumor Diseases 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims description 31
- 210000004027 cell Anatomy 0.000 claims description 27
- 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 23
- 239000000243 solution Substances 0.000 claims description 18
- 210000004556 brain Anatomy 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 14
- 210000003743 erythrocyte Anatomy 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 10
- 229960004679 doxorubicin Drugs 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 239000012224 working solution Substances 0.000 claims description 7
- 210000001185 bone marrow Anatomy 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 210000000170 cell membrane Anatomy 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000008188 pellet Substances 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 102000020897 Formins Human genes 0.000 claims description 2
- 108091022623 Formins Proteins 0.000 claims description 2
- 230000002934 lysing effect Effects 0.000 claims description 2
- 210000005259 peripheral blood Anatomy 0.000 claims description 2
- 239000011886 peripheral blood Substances 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 229940079593 drug Drugs 0.000 abstract description 16
- 206010061218 Inflammation Diseases 0.000 abstract description 12
- 230000004054 inflammatory process Effects 0.000 abstract description 12
- 208000015114 central nervous system disease Diseases 0.000 abstract description 9
- 239000003937 drug carrier Substances 0.000 abstract description 5
- 239000012466 permeate Substances 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 230000002757 inflammatory effect Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 18
- 206010028980 Neoplasm Diseases 0.000 description 17
- 230000000694 effects Effects 0.000 description 7
- 238000007917 intracranial administration Methods 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- PRQROPMIIGLWRP-UHFFFAOYSA-N N-formyl-methionyl-leucyl-phenylalanin Chemical compound CSCCC(NC=O)C(=O)NC(CC(C)C)C(=O)NC(C(O)=O)CC1=CC=CC=C1 PRQROPMIIGLWRP-UHFFFAOYSA-N 0.000 description 5
- 210000002865 immune cell Anatomy 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 210000003954 umbilical cord Anatomy 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 208000024827 Alzheimer disease Diseases 0.000 description 3
- 102000019034 Chemokines Human genes 0.000 description 3
- 108010012236 Chemokines Proteins 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 208000018737 Parkinson disease Diseases 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 238000011729 BALB/c nude mouse Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000012449 Kunming mouse Methods 0.000 description 2
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 2
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000001348 anti-glioma Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 230000035605 chemotaxis Effects 0.000 description 2
- 230000034994 death Effects 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
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 210000002414 leg Anatomy 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 2
- 229960004999 lycopene Drugs 0.000 description 2
- 235000012661 lycopene Nutrition 0.000 description 2
- 239000001751 lycopene Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 206010027191 meningioma Diseases 0.000 description 2
- 239000002539 nanocarrier Substances 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 208000010916 pituitary tumor Diseases 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 210000004927 skin cell Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- HAWSQZCWOQZXHI-FQEVSTJZSA-N 10-Hydroxycamptothecin Chemical compound C1=C(O)C=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 HAWSQZCWOQZXHI-FQEVSTJZSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 101710091342 Chemotactic peptide Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- XSVMFMHYUFZWBK-NSHDSACASA-N Rivastigmine Chemical compound CCN(C)C(=O)OC1=CC=CC([C@H](C)N(C)C)=C1 XSVMFMHYUFZWBK-NSHDSACASA-N 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- HPZOOQSXPMEJBV-ODCFVKFUSA-N Tirilazad mesylate Chemical group 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 1
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 1
- 229960003805 amantadine Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004781 brain capillary Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- PRQROPMIIGLWRP-BZSNNMDCSA-N chemotactic peptide Chemical compound CSCC[C@H](NC=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PRQROPMIIGLWRP-BZSNNMDCSA-N 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- -1 isovinblastine Chemical compound 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000010859 live-cell imaging Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 210000003668 pericyte Anatomy 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229960004136 rivastigmine Drugs 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
- 229960002930 sirolimus Drugs 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 208000023516 stroke disease Diseases 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 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 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Neurology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Botany (AREA)
- Heart & Thoracic Surgery (AREA)
- Psychology (AREA)
- Molecular Biology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Hospice & Palliative Care (AREA)
- Psychiatry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a blood-brain barrier drug delivery system, a preparation method and application thereof, wherein the blood-brain barrier drug delivery system comprises a neutrophil exosome and a drug entrapped in the neutrophil exosome; the medicine is a medicine for treating central nervous system diseases. The invention constructs a neutrophil exosome drug delivery system with good biocompatibility, which has biological characteristics similar to neutrophils, can efficiently permeate blood brain barrier, effectively respond to inflammation, targets inflammation parts, and is a drug carrier with great application prospect for treating glioma and other central nervous system diseases. The invention provides a new strategy with quite transformation application prospect for non-invasive inflammatory microenvironment targeted drug delivery of glioma and other central nervous system diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a blood-brain barrier drug delivery system, a preparation method and application thereof.
Background
Gliomas are the most common primary malignancy of the central nervous system, with a high degree of invasiveness and a very poor prognosis. Traditional surgical removal of tumors is greatly limited by their infiltration and invasiveness. Meanwhile, the blood brain barrier system formed by closely connecting brain capillary endothelial cells, pericytes, astrocyte peripheria and the like prevents toxic substances from invading the brain and prevents most of medicaments from entering the brain, so that glioma and therapeutic medicaments for a plurality of central system diseases (such as Parkinson disease, alzheimer disease and the like) are limited in application. Therefore, designing a safe carrier that can assist the drug to efficiently penetrate the blood brain barrier and effectively target the focus is an important research direction for glioma and intracranial disease treatment.
Currently, strategies for the blood brain barrier mainly include invasive and non-invasive, but most invasive methods present the risk of potential nerve damage and intracranial infection. With the development of nanotechnology, intracranial administration of nanocarriers of non-invasive blood-brain barrier has been rapidly developed. However, it is worth noting that most nanomaterials have the problems of undegraded accumulation in brain tissues, undefined degradation metabolic pathways and the like, and the nanomaterials have the problems of safety and stability in the tedious preparation process.
In recent years, with the continuous and deep knowledge of tumor inflammation microenvironment, targeting tumor inflammation microenvironment becomes a research hotspot. Research shows that the tumor inflammation microenvironment is characterized by the presence of leukocytes in both the supporting stroma and the tumor area. In tumor microenvironments, tumor cells and non-tumor cells (such as immune cells and stromal cells) may release cytokines and chemokines, etc. By responding to specific chemokines, different immune cell subsets migrate to the tumor microenvironment and modulate the immune response of the tumor.
Although more and more blood-permeable brain barrier drug delivery systems are being developed for glioma and brain disease treatment, such as: liposome, polymer/inorganic material nanoparticle, nano-carrier of dendrimer and other materials, self-assembled micelle and the like. However, the availability, safety, feasibility of large-scale preparation and stability of the carrier materials constituting the aforementioned drug delivery systems are still to be further improved and clarified, and most of the carrier materials are in laboratory research stage, so that clinical transformation is difficult. On the other hand, if immune cells are used as drug delivery vehicles, however, since chemotherapeutic drugs tend to have some toxicity to cells, and immune cells play a multifaceted role in tumor microenvironments, it makes direct use of immune cells as drug carriers challenging.
Exosomes are lipid bilayer extracellular vesicles of 30-150nm diameter secreted endogenously by cells, as nanovesicles of natural origin, which are considered as potential good endogenous drug carriers for drug delivery due to their nanosize effects, stability in circulation and ability to naturally penetrate physiological barriers.
CN111690600a discloses an engineering human umbilical cord mesenchymal stem cell exosome, a transdermal preparation and application, wherein the engineering human umbilical cord mesenchymal stem cell exosome extracting solution is obtained by culturing 2-5 generation human umbilical cord mesenchymal stem cells and then enriching; lycopene encapsulated in the exosome of the engineered human umbilical cord mesenchymal stem cells; the lycopene and the exosome of the engineering human umbilical cord mesenchymal stem cells act on the skin cells together, so that the newly generated cells of the epidermis and dermis layers under promotion of the exosome have better cell states, and the effects of improving the skin quality, preventing the skin cells from aging, recovering the normal structure and physiological functions of the skin and improving the skin state are achieved.
CN110652492a discloses a drug-loaded infrared exosome and application thereof, and a liver disease drug. The medicine-carrying erythrocyte exosome is obtained by introducing medicine for treating liver diseases into the erythrocyte exosome. The research shows that the erythrocyte exosomes have liver chemotaxis without any modification, the problem of exosome yield is solved by using the erythrocyte-derived exosomes, and the erythrocyte does not contain DNA and RNA, is safer than exosomes derived from other cells in clinical blood transfusion for decades, and has good application prospect.
CN110917173a discloses an active anti-inflammatory engineering exosome and a preparation method thereof, wherein the exosome takes an exosome derived from M2 type macrophages as a carrier, HAL is loaded into the carrier in an electroporation manner, and the average particle size of the exosome is 120-250nm. The exosomes can actively target to an inflammation position by utilizing the anti-inflammatory and anti-inflammatory properties of the vector, and can evade a plurality of biological barriers such as a mononuclear phagocyte system and the like.
Disclosure of Invention
Aiming at the defects of the existing intracranial administration technology, the invention aims to provide a blood-brain barrier drug delivery system, and a preparation method and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a blood-brain barrier drug delivery system comprising a neutrophil exosome and a drug entrapped in the neutrophil exosome; the medicine is a medicine for treating central nervous system diseases.
The invention constructs a neutrophil exosome drug delivery system with good biocompatibility, which has biological characteristics similar to neutrophils, can efficiently permeate blood brain barrier, effectively respond to inflammation, targets inflammation parts, and is a drug carrier with great application prospect for treating glioma and other central nervous system diseases. The invention takes doxorubicin as an anti-tumor model drug, and the neutrophil exosome drug-carrying system obtained by in-vivo evaluation test of a glioma in-situ transplantation model has very excellent anti-glioma effect.
Preferably, the central nervous system disorder comprises any one of glioma, pituitary tumor, meningioma, stroke, parkinson or alzheimer's disease.
The blood-permeable brain barrier drug delivery system disclosed by the invention can be used for targeted drug delivery of the central nervous system by utilizing the chemotaxis and blood-permeable brain barrier effect of neutrophil exosomes, and can be used for preventing or treating glioma, pituitary tumor, meningioma, cerebral apoplexy, parkinson or Alzheimer disease.
Preferably, the neutrophil exosomes comprise bone marrow or peripheral blood derived neutrophil exosomes.
Preferably, the central nervous system disease treatment drug comprises any one or a combination of at least two of doxorubicin, rapamycin, paclitaxel, docetaxel, hydroxycamptothecin, isovinblastine, amantadine or rivastigmine.
In a second aspect, the present invention provides a method for preparing a blood-brain barrier drug delivery system according to the first aspect, the method comprising the steps of:
and mixing the neutrophil exosome with the medicine, performing ultrasonic treatment, and incubating after the treatment is completed to recover the plasma membrane, so as to obtain the blood-permeable brain barrier medicine delivery system.
The preparation method of the blood-brain barrier drug delivery system is relatively simple to operate, is suitable for industrial production, and has a good transformation application prospect.
Preferably, the neutrophil exosomes are dissolved in a solvent at a concentration of 0.1-1.0mg/mL, for example 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL or 1.0mg/mL, etc., and other specific values within this range of values are selected and will not be described in detail herein. The concentration of neutrophil exosomes was measured using BCA kit.
Preferably, the solvent is a PBS solution. And the PBS solution of neutrophil exosomes was filtered with a 0.22 μm sterile filter before use.
Preferably, the drug is dissolved in a solvent at a concentration of 0.1-1.0mg/mL, for example, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1.0mg/mL, etc., and other specific values within this range of values are selected and will not be described in detail herein.
Preferably, the solvent is a PBS solution containing 5% DMSO and filtered through a 0.22 μm sterile filter.
Preferably, the mass ratio of the neutrophil exosome to the drug is 5:1-1:5, for example, 5:1, 2:1, 3:2, 1:1, 2:3, 1:2 or 1:5, and other specific point values within the numerical range can be selected, and will not be described in detail herein.
Preferably, the conditions of the ultrasound are: 15-25% amplitude, 4-10 cycles, 2-6s on, 1-4s off, and cooling the sample at 2-4deg.C for 1-5min for each cycle gap.
The ultrasonic conditions are specifically selected in the invention because the medicine can be more stably and efficiently packed in the neutrophil exosome without affecting the pharmacodynamic activity of the medicine.
The amplitude may be selected to be 15%, 20%, 25%, or the like; the number of the above cycles may be 4, 5, 6, 7, 8, 9 or 10; the on state may be maintained for 2s, 3s, 4s, 5s, 6s, or the like; the off state may be maintained for 1s, 2s, 3s, 4s, or the like; the cooling temperature can be 2 ℃, 3 ℃ or 4 ℃ and the like; the cooling time can be 1min, 2min, 3min, 4min, 5min, etc.; other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
Preferably, the incubation temperature is 35-40deg.C, such as 35deg.C, 36deg.C, 37deg.C, 38deg.C, 39deg.C or 40deg.C, etc., and the incubation time is 40-120min, such as 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min or 120min, etc., and other specific values within the above numerical ranges are selected, which will not be described in detail herein.
Preferably, after the incubation has ended, the sample is centrifuged (60-90) at (90000-120000) Xg for a min and washed with PBS.
The centrifugal force can be 90000g, 95000g, 100000g, 105000g, 110000g, 115000g or 120000g, the centrifugal time can be 60min, 70min, 80min or 90min, and other specific point values in the numerical ranges can be selected, and will not be described in detail here.
In the invention, the method for extracting the neutrophil exosome comprises the following steps:
(1) Culturing neutrophils with exosome-free serum, and centrifugally collecting supernatant;
(2) Centrifuging the supernatant obtained in the step (1) for 8-12 min at (800-1200) x g, centrifuging for 8-12 min at (1500-2500) x g, and centrifuging for 20-40 min at (9000-12000) x g to obtain supernatant;
(3) Filtering the supernatant obtained in the step (2), centrifuging (60-90) for a period of minutes at a concentration of (90000-120000) x g, and collecting the precipitate to obtain the neutrophil exosome.
The specific method is adopted to extract the neutrophil exosomes, so that the extraction rate is higher, and the extracted neutrophil exosomes can keep the physiological activity of the high-efficiency blood-brain-penetrating barrier and the inflammation trend to the greatest extent.
Preferably, the temperature of the culture in the step (1) is 36-38 ℃, such as 36 ℃, 37 ℃ or 38 ℃, and the like, and the time is 12-36 hours, such as 12 hours, 18 hours, 24 hours, 30 hours or 36 hours, and the like, and other specific point values in the numerical ranges can be selected, and will not be described in detail herein.
Preferably, the supernatant of step (2) is filtered with a 0.22 μm filter.
Preferably, after the precipitate is collected in the step (3), the precipitate is resuspended in PBS solution, and centrifuged at (90000-120000). Times.g for 60-90 min, and the precipitate is collected to obtain the neutrophil exosome.
In the present invention, the method for extracting neutrophils comprises the steps of:
(1') filtering the bone marrow with a 70 μm filter, centrifuging (400-500) ×g for 8-15 min, and re-suspending the pellet in PBS solution;
(2') lysing erythrocytes using an erythrocyte lysate, (400-500) ×g centrifuged (4-10) min, and resuspension of the pellet in PBS solution to obtain a single cell suspension;
(3') adding the single cell suspension into a mixed solution consisting of 58%,70% and 78% of Percoll working solution, (460-520). Times.g is centrifuged (25-40) for min, and the cells at the interface of 58% and 70% of Percoll working solution are taken to obtain the neutrophils.
The specific method for extracting the neutrophils enables the extraction rate to be higher, and the exosomes obtained by secretion of the extracted neutrophils can keep the physiological activities of high-efficiency blood-brain barrier permeation and inflammation trend to the greatest extent.
In a third aspect, the present invention provides the use of a blood-brain barrier drug delivery system according to the first aspect for the manufacture of a medicament for the prevention or treatment of a central nervous system disorder.
In a fourth aspect, the present invention provides the use of a blood-brain barrier drug delivery system according to the first aspect for the manufacture of a medicament for the prevention or treatment of intracranial tumors;
preferably, the intracranial tumor is a glioma.
Compared with the prior art, the invention has the following beneficial effects:
the invention constructs a neutrophil exosome drug delivery system with good biocompatibility, which has biological characteristics similar to neutrophils, can efficiently permeate blood brain barrier, effectively respond to inflammation, targets inflammation parts, and is a drug carrier with great application prospect for treating glioma and other central nervous system diseases. The invention takes doxorubicin as an anti-tumor model drug, and the neutrophil exosome drug-carrying system obtained by in-vivo evaluation test of a glioma in-situ transplantation model has very excellent anti-glioma effect.
Drawings
FIG. 1 is a transmission electron micrograph of the neutrophil exosomes prepared in example 1;
FIG. 2 is a transmission electron microscope image of the blood-brain barrier drug delivery system prepared in example 2;
FIG. 3 is a Western blot analysis of neutrophil exosomes prepared in example 1 and of the blood-brain barrier drug delivery system prepared in example 2;
FIG. 4 is a graph showing the results of observing the absorption of NEs-Exos/PKH67 of underlying C6 cells by a laser confocal microscope in the presence or absence of fMLP;
FIG. 5 is a live imaging view of the distribution of DiR fluorescence in tumor-bearing mice;
fig. 6 is a graph showing survival of each group of C6 glioma-bearing mice in example 6.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The Kunming mice referred to in the examples below were purchased from Chongqing medical university and BALB/c nude mice were purchased from Beijing Fukang Biotechnology Co., ltd. The reagents and raw materials required for other experiments were all available commercially.
Example 1
The method for extracting the neutrophil exosome in the embodiment comprises the following steps:
(1) Taking 25g of Kunming mice, killing the mice after anesthesia and neck breakage, putting the mice into 75% ethanol for sterilization for 5min, and transferring the mice into an ultra-clean workbench;
(2) Carefully separating leg skin of the mice with sterilized surgical equipment, cutting out whole thighs, putting into precooled PBS, peeling leg muscles of the mice, taking out femur and tibia, and putting into a culture dish containing RPMI 1640;
(3) Washing bone marrow with fresh appropriate amount of RPMI1640, filtering cells from bone marrow with 70 μm filter screen, centrifuging 450 Xg for 10min, and suspending precipitate in PBS solution;
(4) Using erythrocyte lysate to lyse erythrocytes, centrifuging for 5min at 450 Xg, and re-suspending the precipitate in PBS solution to obtain single cell suspension;
(5) Preparing a percoll stock solution by using 10 XPBS and a percoll cell separation solution in a volume ratio of 1:9, and preparing a percoll working solution with a volume ratio of 58%,70% and 78% by using 1 XPBS;
(6) Adding single cell suspension into mixed solution composed of 58%,70% and 78% of Percoll working solution, centrifuging for 30min at 490 Xg, and collecting cells at the interface of 58% and 70% of Percoll working solution to obtain neutrophils;
(7) Culturing neutrophils with exosome-free serum, and centrifugally collecting supernatant;
(8) Centrifuging the supernatant at 1000 Xg for 10min, centrifuging at 2000 Xg for 10min, and centrifuging at 10000 Xg for 30min to obtain supernatant;
(9) Filtering the supernatant, centrifuging at 100000 Xg for 70min, and collecting precipitate to obtain the neutrophil exosome.
Example 2
The preparation method of the drug delivery system for the blood-brain barrier comprises the following steps:
(1) Measurement of neutrophil exosome protein concentration using BCA kit, 0.4mg/mL of neutrophil exosome extracted according to the method of example 1 (exosome was diluted with 1 x PBS, used after filtration through 0.22 μm sterile filter) was added with an equivalent concentration of 0.4mg/mL of DOX solution (DOX was formulated with 1 x PBS containing 5% dmso);
(2) Placing the mixed solution of the neutrophil exosome and DOX into a centrifuge tube, and performing ultrasonic treatment in an ultrasonic cell disruption instrument; ultrasonic conditions: 20% amplitude, 6 cycles, 4s on, 2s off, cooling the sample on ice for 2min with each cycle gap;
(3) After the treatment was completed, the samples were incubated at 37℃for 1h to ensure recovery of the plasma membrane of NEs-Exos;
(4) After incubation at 37 ℃ is completed, the sample is centrifuged at 100000 Xg for 70min, washed with PBS, centrifuged again at 100000 Xg for 70min, and resuspended in PBS to obtain the blood-brain barrier drug delivery system.
Example 3
Examination of the neutrophil exosomes and the blood-brain barrier drug delivery systems prepared in example 1 and example 2 by transmission electron microscopy: the neutrophil exosomes or the blood-penetrating brain barrier drug delivery system were mixed with an equal amount of 4% paraformaldehyde and immobilized on a sample-carrying copper mesh (200 mesh, coated with a formaldehyde metal film). After 20min, the PBS was washed and the copper mesh was then placed on 1% glutaraldehyde drops for 5min. The copper mesh was then rinsed in ultra pure water. Each time for 2min, 8 times. Images were captured using a JEM 1200EX transmission electron microscope as shown in fig. 1 and 2, respectively, from which it can be seen that: the neutrophil exosomes appear as round cups or saucers (fig. 1), whereas the particle size of the DOX loaded neutrophil exosomes increases, but this morphology also conforms to the exosome morphology (fig. 2).
Western blot analysis was performed on neutrophil exosomes and the blood-brain barrier drug delivery systems prepared in example 1 and example 2: BCA measured protein concentration and Loading buffer was boiled for 10min after mixing. Using a 1.5mm SDS-PAGE gel, loading 40 mu L, and transferring to a 110V voltage for electrophoresis after 75V voltage reaches a separation position of a concentrated gel separation gel; transfer film was performed at 200mA for 90 min. After transfer, PVDF membranes were placed in a defatted protein solution prepared with 5% TBST, blocked for 2h, not washed after blocking, incubated overnight at 4℃with primary antibodies (CD 63, TSG101, ALIX), and washed 3 times with 1 XTBE on a shaker for 10min each. Incubation with the corresponding secondary antibody was carried out for 2h, and incubated at room temperature in 1 XTBST for 3 times each for 10min on a shaker. And (3) preparing ECL luminous liquid, mixing the liquid A and the liquid B in equal proportion, dripping the mixture onto the PVDF film, and developing. As shown in FIG. 3, the sample was shown to be rich in Exos-associated protein markers (TSG 101, alix and CD 63).
The transmission electron microscope, the particle size analysis and the western blotting result are combined to prove that the neutrophil exosome and the blood-brain barrier drug delivery system are successfully prepared.
Example 4
This example observes the response chemotactic peptide (fMLP) effect of the blood-brain barrier drug delivery system of the present invention, replaces the drug with PKH67 dye according to the method in example 2, loads PKH67 dye in the same loading manner, and establishes a transwell cell model to simulate the blood-brain barrier in vitro.
the transwell in vitro blood brain barrier model was established as follows: the matrigel is diluted by pre-cooled DMEM according to the ratio of 1:3, 50 mu L of diluted matrigel is absorbed and evenly spread on a basement membrane of an upper chamber of a 24-hole transwell chamber (bubbles are avoided), the liquid is spread fully through slight shaking, and after the glue spreading is completed, the transwell chamber is placed into an incubator. After 3h, the solution remaining in the chamber was carefully aspirated, and 30. Mu.L of serum-free DMEM medium was added to each well to hydrate the basement membrane for 30min. bEnd.3 cells were digested in advance and seeded in the upper chamber of a 24-well transwell chamber (1X 10) 5 ) Measurement of the TEER value of the monolayer bEnd.3 cells was performed using a resistance meter when the TEER value was greater than 300. Omega. Cm 2 The transwell chamber can be used as an in vitro permeabilization BBB assay. For detection of the blood-brain barrier permeabilized drug delivery System being carcinomatous after permeation through monolayer bEnd.3 cellsIn the case of uptake, a cell slide was plated and C6 cells were seeded in the lower chamber of a 24-well transwell chamber.
Experiments were performed with the addition of PKH67 loaded neutrophil exosomes (DMEM dilution) in the upper chamber and DMEM or DMEM diluted fMLP (100. Mu.M) in the lower chamber. Taking out the corresponding transwell cells after 4h, 8h and 12h respectively, sucking and removing liquid in the lower chamber, gently washing the climbing plate 3 times by using PBS, adding DAPI and incubating the cells for 15min, washing the cells 3 times by using PBS after incubation, and observing the absorption of the lower layer C6 cells to NEs-Exos/PKH67 under a laser confocal microscope.
The results are shown in FIG. 4: in the presence of fMLP, the uptake of neutrophil exosome drug-loaded particles by C6 cells increased over time, and reached the highest at 12h, with evident green fluorescence. In contrast, in the fMLP-free group, uptake of neutrophil exosome drug-loaded particles by lower chamber C6 cells was not apparent. The presence of fMLP increases the efficiency of penetration of the drug-loaded particles of the neutrophil exosomes through the transwell upper chamber, demonstrating the ability of the blood-brain barrier drug delivery system of the present invention to respond to chemokines similar to neutrophils.
Example 5
The present example explores the blood-brain barrier efficiency of the blood-brain barrier drug delivery system according to the present invention, and the method is as follows: modeling of tumor-bearing mice was performed using about 20g of male BALB/C nude mice as experimental model animals with C6-Luc glioma cells stably transfected with luciferase. Nude mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. Mice were fixed using a brain stereotactic apparatus, suspended in 2 μl PBS 2×10 6 The C6-Luc glioma cells were implanted into the right striatum (1.8 mm right, 1mm backward, 3mm downward with bregma as origin) of the mouse brain. The cell implantation process needs to slowly insert the needle, and the needle extraction process after implantation needs to be slowly carried out, so that the death of the mice caused by intracranial pressure mutation is avoided.
Neutrophil exosomes loaded with DiR fluorochromes were prepared as in example 2. The free DiR fluorochrome was administered by tail vein injection on day 15 after C6 cell seed tumor as a control. After injection was completed, the in vivo distribution of DiR fluorescence in tumor bearing mice (free DiR as control group) was observed and recorded under a small animal in vivo imager at set time points (30 min, 1h, 2h, 4h, 12h and 24 h).
The results are shown in FIG. 5: after 2h tail vein injection, diR loaded in neutrophil exosomes showed fluorescent signals in the brain of mice, and the fluorescence intensity of DiR in the brain was continuously increased with time. Whereas free DiR groups did not observe significant fluorescence in the brain during up to 24h of monitoring. Experimental results prove that the neutrophil exosome can effectively improve the blood-brain barrier efficiency of the wrapped medicine.
Example 6
The inhibition effect of the drug delivery system of the blood-brain barrier to glioma is explored in the embodiment, and the method is as follows: after the C6-Luc in situ glioma model was established as in example 5, the C6-Luc mice were randomly divided into 3 groups of 10 mice each according to the physiological saline group, the pure DOX group and the DOX-loaded neutrophil exosomes group. The corresponding medicines were injected into the tail vein on days 10, 12, 14, 16, 18, and 20, respectively, wherein the DOX amount was 3 mg.kg -1 . Death data of C6-Luc mice were recorded daily after brain engrafting of tumor cells, and Kaplan-Meier survival curves were plotted for 3 groups of tumor-bearing mice using GraphPad 7.00 software.
The results are shown in FIG. 6: the mice treated with the blood-brain-barrier drug delivery system of the present invention had significantly improved survival, with a median survival of 27 days, which was significantly longer than the free DOX group (22 days) and the physiological saline group (19 days), and these results confirmed that the blood-brain-barrier drug delivery system of the present invention was effective in inhibiting tumor growth and prolonging survival of mice.
The applicant states that the present invention is described by way of the above examples as a blood-brain barrier drug delivery system, and methods of making and using the same, but the present invention is not limited to, i.e., it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (17)
1. The blood-brain barrier drug delivery system is characterized by being prepared from a neutrophil exosome and doxorubicin entrapped in the neutrophil exosome; the blood-brain barrier drug delivery system is used for treating glioma;
the blood-brain barrier drug delivery system is prepared by a method comprising the following steps:
and mixing the neutrophil exosome with doxorubicin, performing ultrasonic treatment, and incubating after the treatment is finished to recover a plasma membrane to obtain the blood-brain-barrier drug delivery system.
2. The blood-permeable brain barrier drug delivery system of claim 1, wherein the neutrophil exosomes comprise bone marrow or peripheral blood derived neutrophil exosomes.
3. A method of preparing a blood-brain barrier drug delivery system according to claim 1 or 2, comprising the steps of:
and mixing the neutrophil exosome with doxorubicin, performing ultrasonic treatment, and incubating after the treatment is finished to recover a plasma membrane to obtain the blood-brain-barrier drug delivery system.
4. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the neutrophil exosomes are dissolved in the solvent at a concentration of 0.1-1.0 mg/mL.
5. The method of preparing a blood-brain barrier drug delivery system according to claim 4, wherein the solvent is a PBS solution and is filtered through a 0.22 μm sterile filter.
6. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the doxorubicin is dissolved in a solvent at a concentration of 0.1-1.0 mg/mL.
7. The method of preparing a blood-brain barrier drug delivery system according to claim 6, wherein the solvent is a PBS solution containing 5% dmso and filtered through a 0.22 μm sterile filter.
8. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the mass ratio of neutrophil exosomes to doxorubicin is 5:1-1:5.
9. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the ultrasound conditions are: 15-25% amplitude, 4-10 cycles, 2-6s cycles, 1-4s cycles, and cooling the sample at 2-4deg.C for 1-5min for each cycle gap.
10. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the incubation is performed at a temperature of 35-40 ℃ for a time of 40-120 min.
11. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the sample is centrifuged (60-90) at (90000-120000) ×g for a min after the incubation is completed, and washed with PBS.
12. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the method of extracting neutrophil exosomes comprises the steps of:
(1) Culturing neutrophils with exosome-free serum, and centrifugally collecting supernatant;
(2) Centrifuging the supernatant obtained in the step (1) for 8-12 min at (800-1200) x g, centrifuging for 8-12 min at (1500-2500) x g, and centrifuging for 20-40 min at (9000-12000) x g to obtain supernatant;
(3) Filtering the supernatant obtained in the step (2), centrifuging (60-90) for a period of minutes at a concentration of (90000-120000) x g, and collecting the precipitate to obtain the neutrophil exosome.
13. The method of claim 12, wherein the culturing in step (1) is performed at a temperature of 36-38 ℃ for a time of 12-36 h.
14. The method of preparing a blood-brain barrier drug delivery system according to claim 12, wherein the supernatant of step (2) is filtered with a 0.22 μm filter.
15. The method of claim 12, wherein the collecting the precipitate in step (3) is followed by re-suspending in PBS solution, and centrifuging (60-90) for a period of time of (90000-120000) ×g, and collecting the precipitate to obtain the neutrophil exosome.
16. The method of preparing a blood-brain barrier drug delivery system according to claim 12, wherein the method of extracting neutrophils comprises the steps of:
(1') filtering the bone marrow with a 70 μm filter, centrifuging (400-500) ×g for 8-15 min, and re-suspending the pellet in PBS solution;
(2') lysing erythrocytes using an erythrocyte lysate, (400-500) ×g centrifuged (4-10) min, and resuspension of the pellet in PBS solution to obtain a single cell suspension;
(3') adding the single cell suspension into a mixed solution consisting of 58%,70% and 78% of Percoll working solution, (460-520). Times.g is centrifuged (25-40) for min, and the cells at the interface of 58% and 70% of Percoll working solution are taken to obtain the neutrophils.
17. Use of a blood-permeable brain barrier drug delivery system according to claim 1 or 2 for the preparation of a medicament for the treatment of glioma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011502081.7A CN114642736B (en) | 2020-12-17 | 2020-12-17 | Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011502081.7A CN114642736B (en) | 2020-12-17 | 2020-12-17 | Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114642736A CN114642736A (en) | 2022-06-21 |
| CN114642736B true CN114642736B (en) | 2023-12-12 |
Family
ID=81991304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011502081.7A Active CN114642736B (en) | 2020-12-17 | 2020-12-17 | Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114642736B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115433720B (en) * | 2022-07-06 | 2023-04-18 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of fused extracellular vesicle analogue |
| CN116236460B (en) * | 2023-03-20 | 2024-07-23 | 苏州大学 | Rapamycin-loaded brain-targeted platelet-derived extracellular vesicles and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107812197A (en) * | 2014-09-20 | 2018-03-20 | 中国药科大学 | A kind of inflammation targeted neutrophil leucocyte delivery system and its application |
| WO2019139762A1 (en) * | 2018-01-09 | 2019-07-18 | Zen-Bio, Inc. | Exosome compositions and use thereof for joint disorders and diseases |
| CN110152015A (en) * | 2018-02-11 | 2019-08-23 | 上海市第六人民医院东院 | Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes |
| CN110699320A (en) * | 2019-11-26 | 2020-01-17 | 江苏大学 | Human peripheral blood neutrophil exosome and extraction method and application thereof |
| WO2020027467A1 (en) * | 2018-07-30 | 2020-02-06 | 주식회사 엑소코바이오 | Lyophilized preparation of stem cell-derived exosomes, and anti-inflammatory composition comprising same as active ingredient |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013106496A1 (en) * | 2012-01-10 | 2013-07-18 | modeRNA Therapeutics | Methods and compositions for targeting agents into and across the blood-brain barrier |
-
2020
- 2020-12-17 CN CN202011502081.7A patent/CN114642736B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107812197A (en) * | 2014-09-20 | 2018-03-20 | 中国药科大学 | A kind of inflammation targeted neutrophil leucocyte delivery system and its application |
| WO2019139762A1 (en) * | 2018-01-09 | 2019-07-18 | Zen-Bio, Inc. | Exosome compositions and use thereof for joint disorders and diseases |
| CN110152015A (en) * | 2018-02-11 | 2019-08-23 | 上海市第六人民医院东院 | Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes |
| WO2020027467A1 (en) * | 2018-07-30 | 2020-02-06 | 주식회사 엑소코바이오 | Lyophilized preparation of stem cell-derived exosomes, and anti-inflammatory composition comprising same as active ingredient |
| CN110699320A (en) * | 2019-11-26 | 2020-01-17 | 江苏大学 | Human peripheral blood neutrophil exosome and extraction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Tuying Yong等."Tumor exosome-based nanoparticles are efficient drug carriers for chemotherapy".Nat Commun..2019,第10卷(第1期),第1-16页. * |
| 汤雪莹 ; 刘敏 ; 宋艳志 ; 刘欣荣 ; 邓意辉 ; .中性粒细胞介导的药物递送系统在肿瘤靶向治疗中的应用.沈阳药科大学学报.2020,(01),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114642736A (en) | 2022-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109893515B (en) | Macrophage drug-loaded microparticle preparation and preparation method thereof | |
| CN106924755B (en) | Activated neutral particle cell membrane coated bionic nano particle and preparation method thereof | |
| CN109666695B (en) | Targeted integrin alphavbeta 3 exosome vector and preparation method and application thereof | |
| CN102302784B (en) | Tumor chemotherapeutic medicinal preparation and preparation method thereof | |
| WO2021174738A1 (en) | Bionic nanoparticle coated with mesenchymal stem cell membrane having surface overexpressing pd-l1 molecule, and preparation therefor and application thereof | |
| US10548853B2 (en) | Oncolytic virus formulation and preparation method thereof | |
| CN114642736B (en) | Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof | |
| CN107184987B (en) | Lipoic acid modified targeted integrin alpha v beta 3 nano-polypeptide carrier and preparation method and application thereof | |
| CN113209026A (en) | Tumor drug-carrying microparticle preparation and preparation method thereof | |
| CN107802646A (en) | A kind of anti-tumor medicine | |
| Lu et al. | Simultaneous inhibition of breast cancer and its liver and lung metastasis by blocking inflammatory feed-forward loops | |
| CN113355290B (en) | Anti-tumor engineered exosome, preparation method and application | |
| CN113633625B (en) | Nano-drug of hybrid membrane loaded oxidative phosphorylation inhibitor and preparation method thereof | |
| CN115089727B (en) | KC26 polypeptide modified milk exosome and preparation method and application thereof | |
| CN108542880B (en) | Method for constructing order-level targeted ischemic myocardial cell mitochondrion drug-loaded nano-micelle | |
| Cheng et al. | Enhanced tumor homing of pathogen-mimicking liposomes driven by R848 stimulation: a new platform for synergistic oncology therapy | |
| CN114225027B (en) | Genetically engineered liver cancer targeting cell membrane bionic nano microsphere and preparation method thereof | |
| CN108186772B (en) | Modification method of lemon exosomes | |
| CN114395531A (en) | Preparation method and application of bioactive substance or drug delivery microvesicle | |
| CN115068444B (en) | Macrophage membrane-coated liposome nanoparticle and preparation method thereof | |
| CN115814108A (en) | Engineered macrophage drug-loaded microparticle preparation for personalized tumor treatment and preparation method thereof | |
| CN115737826A (en) | Extracellular vesicle loaded with polydopamine nanoparticles and preparation method thereof | |
| CN112426537A (en) | Polypeptide nano micelle and preparation method and application thereof | |
| CN110585168A (en) | Application of utilizing cell surface vesicle as drug carrier | |
| CN114369575B (en) | Brain glioma cell-derived exosome without tumor promotion function, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |