CN116370643A - Nanometer assembly, preparation method and application thereof - Google Patents
Nanometer assembly, preparation method and application thereof Download PDFInfo
- Publication number
- CN116370643A CN116370643A CN202310419155.8A CN202310419155A CN116370643A CN 116370643 A CN116370643 A CN 116370643A CN 202310419155 A CN202310419155 A CN 202310419155A CN 116370643 A CN116370643 A CN 116370643A
- Authority
- CN
- China
- Prior art keywords
- nano
- assembly
- temperature
- pnd
- polymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims description 16
- 229920000642 polymer Polymers 0.000 claims abstract description 58
- 239000003805 procoagulant Substances 0.000 claims abstract description 29
- 229920000388 Polyphosphate Polymers 0.000 claims abstract description 14
- 239000001205 polyphosphate Substances 0.000 claims abstract description 14
- 235000011176 polyphosphates Nutrition 0.000 claims abstract description 14
- 239000011258 core-shell material Substances 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 66
- 238000006243 chemical reaction Methods 0.000 claims description 50
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 44
- 239000007788 liquid Substances 0.000 claims description 42
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 40
- 229910052757 nitrogen Inorganic materials 0.000 claims description 33
- 239000002246 antineoplastic agent Substances 0.000 claims description 31
- 238000007710 freezing Methods 0.000 claims description 30
- 230000008014 freezing Effects 0.000 claims description 30
- 239000003814 drug Substances 0.000 claims description 22
- 239000012265 solid product Substances 0.000 claims description 22
- 229910052786 argon Inorganic materials 0.000 claims description 20
- 229940044683 chemotherapy drug Drugs 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 19
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- 239000012986 chain transfer agent Substances 0.000 claims description 16
- SJIXRGNQPBQWMK-UHFFFAOYSA-N 2-(diethylamino)ethyl 2-methylprop-2-enoate Chemical compound CCN(CC)CCOC(=O)C(C)=C SJIXRGNQPBQWMK-UHFFFAOYSA-N 0.000 claims description 14
- 238000001338 self-assembly Methods 0.000 claims description 11
- 238000006116 polymerization reaction Methods 0.000 claims description 10
- 229960004316 cisplatin Drugs 0.000 claims description 9
- 239000003999 initiator Substances 0.000 claims description 8
- 230000000973 chemotherapeutic effect Effects 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 7
- 229940127089 cytotoxic agent Drugs 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 5
- 239000000178 monomer Substances 0.000 claims description 5
- 230000000259 anti-tumor effect Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 74
- 208000005189 Embolism Diseases 0.000 abstract description 16
- 210000004204 blood vessel Anatomy 0.000 abstract description 15
- 238000001879 gelation Methods 0.000 abstract description 10
- 230000001965 increasing effect Effects 0.000 abstract description 8
- 210000004369 blood Anatomy 0.000 abstract description 7
- 239000008280 blood Substances 0.000 abstract description 7
- 230000002378 acidificating effect Effects 0.000 abstract description 6
- 230000008685 targeting Effects 0.000 abstract description 6
- 230000015556 catabolic process Effects 0.000 abstract description 5
- 238000006731 degradation reaction Methods 0.000 abstract description 5
- 230000007935 neutral effect Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 230000001737 promoting effect Effects 0.000 abstract description 4
- 206010021143 Hypoxia Diseases 0.000 abstract description 3
- 208000001435 Thromboembolism Diseases 0.000 abstract description 3
- 230000007954 hypoxia Effects 0.000 abstract description 3
- 230000004060 metabolic process Effects 0.000 abstract description 3
- 230000001976 improved effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 56
- 230000000694 effects Effects 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 23
- 241000699670 Mus sp. Species 0.000 description 20
- 238000000429 assembly Methods 0.000 description 20
- 239000000499 gel Substances 0.000 description 19
- 238000011282 treatment Methods 0.000 description 19
- 230000006399 behavior Effects 0.000 description 17
- 230000000052 comparative effect Effects 0.000 description 17
- 210000004881 tumor cell Anatomy 0.000 description 15
- 206010027476 Metastases Diseases 0.000 description 14
- 230000009401 metastasis Effects 0.000 description 14
- 230000006907 apoptotic process Effects 0.000 description 13
- 210000004443 dendritic cell Anatomy 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 241000283973 Oryctolagus cuniculus Species 0.000 description 12
- 208000007536 Thrombosis Diseases 0.000 description 12
- 229940079593 drug Drugs 0.000 description 12
- 210000004072 lung Anatomy 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 230000015271 coagulation Effects 0.000 description 7
- 238000005345 coagulation Methods 0.000 description 7
- 201000007270 liver cancer Diseases 0.000 description 7
- 208000014018 liver neoplasm Diseases 0.000 description 7
- 238000007920 subcutaneous administration Methods 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 108090000190 Thrombin Proteins 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 210000001165 lymph node Anatomy 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000035800 maturation Effects 0.000 description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 6
- 229960004072 thrombin Drugs 0.000 description 6
- 230000007704 transition Effects 0.000 description 6
- 108010073385 Fibrin Proteins 0.000 description 5
- 102000009123 Fibrin Human genes 0.000 description 5
- 229940041181 antineoplastic drug Drugs 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 229950003499 fibrin Drugs 0.000 description 5
- 230000004614 tumor growth Effects 0.000 description 5
- 230000002792 vascular Effects 0.000 description 5
- YPCMISLZCVOUJB-UHFFFAOYSA-N 4-cyano-4-methyl-5-phenyl-5-sulfanylidenepentanoic acid Chemical group OC(=O)CCC(C)(C#N)C(=S)C1=CC=CC=C1 YPCMISLZCVOUJB-UHFFFAOYSA-N 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 230000008595 infiltration Effects 0.000 description 4
- 238000001764 infiltration Methods 0.000 description 4
- 210000003141 lower extremity Anatomy 0.000 description 4
- -1 nitro cisplatin Chemical compound 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical group N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 206010056342 Pulmonary mass Diseases 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 230000011748 cell maturation Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000003073 embolic effect Effects 0.000 description 3
- 230000010102 embolization Effects 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 239000011261 inert gas Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000002601 intratumoral effect Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002114 nanocomposite Substances 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 230000000171 quenching effect Effects 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 229910001961 silver nitrate Inorganic materials 0.000 description 3
- 239000008279 sol Substances 0.000 description 3
- 230000006641 stabilisation Effects 0.000 description 3
- 238000011105 stabilization Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 101100339431 Arabidopsis thaliana HMGB2 gene Proteins 0.000 description 2
- 102100029968 Calreticulin Human genes 0.000 description 2
- 108700010013 HMGB1 Proteins 0.000 description 2
- 101150021904 HMGB1 gene Proteins 0.000 description 2
- 206010019695 Hepatic neoplasm Diseases 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 206010061216 Infarction Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920000037 Polyproline Polymers 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000005975 antitumor immune response Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000010523 cascade reaction Methods 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 210000002434 celiac artery Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 230000003090 exacerbative effect Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 2
- 230000007574 infarction Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000001746 injection moulding Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000010526 radical polymerization reaction Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000005747 tumor angiogenesis Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 description 1
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 108010080865 Factor XII Proteins 0.000 description 1
- 102000000429 Factor XII Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000270295 Serpentes Species 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000010109 chemoembolization Effects 0.000 description 1
- 238000002512 chemotherapy Methods 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
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000000547 effect on apoptosis Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 description 1
- 229960001025 iohexol Drugs 0.000 description 1
- 208000037906 ischaemic injury Diseases 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 238000002638 palliative care Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000009991 scouring Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001732 thrombotic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000002417 xiphoid bone Anatomy 0.000 description 1
Images
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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- 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
- A61K31/74—Synthetic polymeric materials
- A61K31/80—Polymers containing hetero atoms not provided for in groups A61K31/755 - A61K31/795
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
-
- 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
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- 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
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Neurosurgery (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The nano assembly provided by the invention has a core-shell structure, comprises an inner core composed of a procoagulant and a chemotherapeutics and an outer shell composed of a thermosensitive nano polymer, has good temperature sensitivity and excellent gelation behavior, can be converted into high-strength gel, can effectively increase the strength of the coagulated blood, can effectively inhibit the degradation of the coagulated blood, is sensitive to pH, is beneficial to the release of polyphosphate in blood vessels with neutral pH values, thereby triggering thromboembolism, aggravating anaerobic metabolism of tumor hypoxia, increasing the acidic environment of tumor sites, and further promoting the release of the chemotherapeutics by the acidic pH values of the tumor sites, so that the targeting is improved.
Description
Technical Field
The invention relates to the technical field of antitumor drugs, in particular to a nano assembly and a preparation method and application thereof.
Background
Hepatocellular carcinoma (HCC, abbreviated as liver cancer) is a serious disease seriously harming human health, and has extremely high malignancy, rapid invasive growth, easy recurrence after treatment, and survival rate of less than 5% in 5 years, which is called as "king in cancer". The first country of liver cancer in the world has morbidity of about 55% of the world, mortality is the second place of malignant tumor, and morbidity and mortality still have continuous rising trend. Among the various treatments, such as ablation, resection, grafting, and trans-arterial chemoembolization (TACE), TACE is the treatment of choice for advanced HCC.
TACE can be guided by imaging equipment, and vascular embolic materials and therapeutic drugs can be delivered to tumor target blood vessels in a super-selective way through an elongated cannula, so that the effects of blocking intratumoral blood flow, releasing drugs and targeting therapy are achieved. The method aims to "starve" the tumor by depriving it of oxygen and nutrients, thereby inducing ischemic injury and necrosis, and thereby inhibiting tumor growth. However, embolic materials also face a "flow-embolization" contradiction: the high-strength embolism is resistant to blood flow scouring and can prevent vascular recanalization. However, the existing embolization agent clinically used is used for sealing the peripheral vascular network of liver cancer with abundant and high heterogeneity through physical viscosity, so complete and thorough embolization is generally difficult to realize, and thus, TACE treatment has a plurality of problems of non-radical palliative treatment such as incomplete radical cure, easy recurrence, low long-term survival rate and the like of tumors.
In recent years, the use of spontaneously induced thrombosis in the body for the embolization treatment of tumors has become of increasing interest. Studies have shown that a tiny clot can block the nutrient supply to the entire blood vessel and cause thousands of cells to "avalanche" die. The treatment mode of the tumor vascular infarction (TumorVessel Infarction, TVI) has the following advantages: 1) The curative effect of the tumor is good: the TVI not only aims at the tumor angiogenesis, but also can seal the established tumor angiogenesis with high efficiency, and has more excellent curative effect on radical tumor than the angiogenesis inhibitor; 2) The curative effect is quick and durable: research shows that tumor cells begin to die within hours after TVI treatment, and that intratumoral coagulum can exist for a long time; 3) Intratumoral specific coagulation: unlike normal blood vessels, which are generally non-thrombotic environments, tumors are considered to be a "hard to heal wound" with a hypersensitive procoagulant microenvironment. However, in TVI treatment, the selection of coagulation proteins, the requirement of targeting, and the inability of the formed clot to maintain effectively for a long time remain a major issue to be addressed.
Disclosure of Invention
The invention aims to provide a nano assembly and a preparation method and application thereof, and the nano assembly provided by the invention has good temperature sensitivity and excellent gelation behavior, can be converted into high-strength gel, can effectively increase the strength of a coagulated blood block, can inhibit degradation of the coagulated blood block for a long time, is sensitive to pH, is beneficial to release of polyphosphate in blood vessels with neutral pH values, thereby triggering thromboembolism, exacerbating anaerobic metabolism of tumor hypoxia, increasing the acidic environment of tumor sites, and further promoting release of chemotherapeutic drugs by acidic pH values of tumor sites, thereby improving targeting.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a nano assembly, which has a core-shell structure, and comprises an inner core composed of a procoagulant and a chemotherapeutic drug and an outer shell composed of a temperature-sensitive nano polymer, wherein the mass ratio of the procoagulant, the chemotherapeutic drug and the temperature-sensitive nano polymer in the nano assembly is (10-75): 4:80;
the temperature-sensitive nano polymer is prepared by taking N-isopropyl acrylamide and diethylaminoethyl methacrylate as monomers through a RAFT method.
Preferably, the procoagulant is a polyphosphate having a degree of polymerization of 25 to 250.
Preferably, the chemotherapeutic agent is a nitrated cisplatin or tetravalent platinum chemotherapeutic agent.
Preferably, the mass ratio of procoagulant, chemotherapeutic drug and temperature sensitive nano-polymer in the nano-assembly is 10:4:80.
preferably, the polymerization degree of the temperature-sensitive nano polymer is 100-250.
The invention also provides a preparation method of the nano assembly, which comprises the following steps: and (3) mixing the solution of the chemotherapeutic medicine and the solution of the temperature-sensitive nano polymer, dropwise adding the solution of the procoagulant, and performing self-assembly reaction to obtain the nano assembly.
Preferably, the temperature-sensitive nano polymer is prepared by adopting a RAFT method, and comprises the following steps of:
(1) Mixing N-isopropyl acrylamide and an organic solvent, sequentially performing first liquid nitrogen freezing, first vacuumizing and first argon introducing, and then performing second liquid nitrogen freezing to obtain a first solid product;
(2) Mixing the first solid product obtained in the step (1) with a chain transfer agent, sequentially performing third liquid nitrogen freezing, second vacuumizing and second argon introducing, and performing fourth liquid nitrogen freezing to obtain a second solid product;
(3) Mixing the second solid product obtained in the step (2) with an initiator, sequentially carrying out third vacuumizing and third argon introducing, and then carrying out a first RAFT reaction (namely, a reversible addition fragmentation chain transfer (RAFT) free radical polymerization) to obtain a macromolecular chain transfer agent intermediate, namely, macro-CTA;
(4) And (3) mixing the solution of Macro-CTA and deoxidized diethylaminoethyl methacrylate obtained in the step (3), and then performing a second RAFT reaction to obtain the temperature-sensitive nano polymer poly (N-isopropylacrylamide-b-diethylaminoethyl methacrylate).
Preferably, the temperature of the first RAFT reaction in the step (3) is 65-75 ℃, and the time of the first RAFT reaction is 6-24 hours.
Preferably, after the second RAFT reaction in step (4) is completed, the method further includes: and sequentially dialyzing and freeze-drying the product of the second RAFT reaction to obtain the temperature-sensitive nano polymer.
The invention also provides an application of the nano-assembly prepared by the technical scheme or the nano-assembly prepared by the preparation method in preparing antitumor drugs.
The nano assembly provided by the invention has good temperature sensitivity and sol-gel phase transition behavior, has the characteristic of shear thinning, can realize drug delivery through needle tube or microcatheter push injection due to the fluidity and high dispersion stability of the nano assembly, and simultaneously has good application in clinical TAE treatment; the nano-assembly can realize gelation under normal human body temperature, and is converted into high-strength gel, so that the slow release of procoagulants and chemotherapeutics is realized. The strength of the blood clot can be effectively increased, the degradation of the blood clot is inhibited, and the hydrophobic interface after the gel can effectively activate the blood platelet so as to enhance the coagulation behavior of the procoagulant; the chemotherapeutic medicine of the nano assembly can promote apoptosis of tumor cells and activate ICD effect, and can improve the immune microenvironment of tumor parts and activate immune response through infiltration of lymphocytes and activation of dendritic cells, thereby inhibiting metastasis of tumors. The nano-assembly loaded with the procoagulant and the chemotherapeutic drug realizes the cooperative treatment of embolism-chemotherapy-immunotherapy, and has great clinical liver cancer treatment potential. Experimental results show that the PND-Pt-PolyP nano assembly prepared in the embodiment 1 of the invention has good assembly behavior and presents a core-shell structure; because the PND-Pt-PolyP nano-assembly prepared in the embodiment 1 has temperature sensitive property, the PND-Pt-PolyP nano-assembly is easy to inject at room temperature, the grid structure formed after gelation can effectively release the medicine slowly, and meanwhile, the high-strength gel can also effectively plug blood vessels; the procoagulant PolyP released by the nano-assembly prepared in example 1 can effectively promote the generation of FXII and thrombin, and increase the strength and stability of fibrin; the chemotherapeutic drug Pt of the nano-assembly prepared in the embodiment 1 can promote apoptosis of tumor cells and promote ICD effect, and induce curing of DC. The nano-assembly prepared in the embodiment 1 has an embolism-chemotherapy-immunity cooperative treatment mode, and can effectively inhibit tumor growth, reduce tumor metastasis and enhance anti-tumor immune response of tumor parts; meanwhile, the PND-Pt-PolyP nanometer assembly can realize the physical embolism of the temperature-sensitive nanometer polymer and the synergistic embolism treatment of the coagulation embolism caused by the PolyP on the VX2 transplanted tumor rabbit liver cancer model, and has good clinical potential.
Drawings
FIG. 1 is a schematic flow chart of the PND-Pt-PolyP nano-assembly prepared according to example 1 of the present invention;
FIG. 2 is an electron microscope image of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention;
FIG. 3 is a graph showing particle size and potential of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention;
FIG. 4 is a graph showing drug release of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention;
FIG. 5 is a graph showing the phase transition behavior of the temperature-sensitive sol gel of PND-Pt-PolyP in example 1, PND-Pt in comparative example 2 and PND-PolyP in comparative example 4 of the present invention;
FIG. 6 is a graph showing the effect trend of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention on FXII production;
FIG. 7 is a graph showing the effect trend of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention on thrombin generation;
FIG. 8 is a graph showing the trend of the effect of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention on fibrin;
FIG. 9 is a scanning electron microscope image of blood clots of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention;
FIG. 10 is a graph showing the effect of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention on apoptosis of 4T1 tumor cells;
FIG. 11 is an ICD effect graph of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention on 4T1 tumor cells;
FIG. 12 is a graph showing the effect trend of PND-Pt-PolyP nano-assemblies prepared in example 1 of the present invention on DC cell maturation;
FIG. 13 is a schematic representation of tumors tested in six mice for 4T1 subcutaneous tumors in accordance with the present invention;
FIG. 14 is a graph showing the relative tumor growth rate of 4T1 subcutaneous tumors tested in six mice in accordance with the present invention;
FIG. 15 is a graph showing the change in tumor weight of 4T1 subcutaneous tumors in six mice tested in accordance with the present invention;
FIG. 16 is a diagram showing immunofluorescence of tumor tissue from a 4T1 subcutaneous tumor of six mice tested in accordance with the present invention;
FIG. 17 is a graph of the number of lung nodules of a lung metastasis model of a test seven mice in accordance with the present invention;
FIG. 18 is a graph of lung weights of a model of lung metastasis in seven mice tested in accordance with the present invention;
FIG. 19 is a graph showing the infiltration of CD8+ cells in paraneoplastic lymph nodes tested in seven mice lung metastasis models in accordance with the present invention;
FIG. 20 is a graph showing maturation of DC cells in paraneoplastic lymph nodes tested in seven mice lung metastasis models in accordance with the present invention;
fig. 21 is a DSA and H & E staining of a rabbit VX 2-transplanted tumor liver cancer model at various times in a test eight according to the invention.
Detailed Description
The invention provides a nano assembly, which has a core-shell structure, and comprises an inner core composed of a procoagulant and a chemotherapeutic drug and an outer shell composed of a temperature-sensitive nano polymer, wherein the mass ratio of the procoagulant, the chemotherapeutic drug and the temperature-sensitive nano polymer in the nano assembly is (10-75): 4:80;
The temperature-sensitive nano polymer is prepared by taking N-isopropyl acrylamide and diethylaminoethyl methacrylate as monomers through a RAFT method.
In the present invention, the procoagulant is preferably a polyphosphate; the degree of polymerization of the polyphosphate is preferably 25 to 250, more preferably 45.
In the present invention, the chemotherapeutic agent is a nitrated cisplatin or tetravalent platinum chemotherapeutic agent.
In the invention, the mass ratio of procoagulant, chemotherapeutic medicine and temperature sensitive nano polymer in the nano assembly is preferably (10-75) 4:80, more preferably 10:4:80. the invention controls the mass ratio of procoagulant, chemotherapeutics and temperature sensitive nano polymer in the nano assembly in the above range to obtain the nano assembly with better performance.
In the present invention, the polymerization degree of the temperature-sensitive nano-polymer is preferably 100 to 250, more preferably 220. The polymerization degree of the temperature-sensitive nano polymer is controlled in the range, so that the temperature-sensitive sol-gel phase transition behavior is better.
The nano assembly provided by the invention has good temperature sensitivity and excellent gelation behavior, can be converted into high-strength gel, can effectively increase the strength of a coagulated blood block, can inhibit the degradation of the coagulated blood block for a long time, is sensitive to pH, is beneficial to the release of polyphosphate in blood vessels with neutral pH value, thereby triggering thromboembolism, exacerbating anaerobic metabolism of tumor hypoxia, increasing the acidic environment of tumor parts, and further promoting the release of chemotherapeutic drugs by the acidic pH value of tumor parts, so that the targeting is improved.
The invention also provides a preparation method of the nano assembly, which comprises the following steps: and (3) mixing the solution of the chemotherapeutic medicine and the solution of the temperature-sensitive nano polymer, dropwise adding the solution of the procoagulant, and performing self-assembly reaction to obtain the nano assembly.
In the invention, when the chemotherapeutic drug is nitro cisplatin, the preparation method of the solution of the chemotherapeutic drug comprises the following steps: the ratio of the mass is 1:2, mixing cisplatin with silver nitrate, adding water for dissolution, stirring overnight at room temperature in the dark, and centrifuging to remove precipitated silver nitrate, thereby obtaining supernatant which is a solution of a chemotherapeutic drug (or a nitrocisplatin solution). In the present invention, the nitrocisplatin solution is preferably stored in a dark, 4 ℃ environment.
In the invention, the temperature-sensitive nano polymer is preferably prepared by adopting a RAFT method, and comprises the following steps of:
(1) Mixing N-isopropyl acrylamide and an organic solvent, sequentially performing first liquid nitrogen freezing, first vacuumizing and first argon introducing, and then performing second liquid nitrogen freezing to obtain a first solid product;
(2) Mixing the first solid product obtained in the step (1) with a chain transfer agent, sequentially performing third liquid nitrogen freezing, second vacuumizing and second argon introducing, and performing fourth liquid nitrogen freezing to obtain a second solid product;
(3) Mixing the second solid product obtained in the step (2) with an initiator, sequentially carrying out third vacuumizing and third argon introducing, and then carrying out a first RAFT reaction to obtain a macromolecular chain transfer agent intermediate Macro-CTA;
(4) And (3) mixing the solution of the macromolecular chain transfer agent intermediate Macro-CTA obtained in the step (3) and the deoxidized diethylaminoethyl methacrylate, and then carrying out a second RAFT reaction to obtain the temperature-sensitive nano polymer poly (N-isopropyl acrylamide-b-diethylaminoethyl methacrylate).
The invention preferably mixes N-isopropyl acrylamide and an organic solvent, then sequentially carries out first liquid nitrogen freezing, first vacuumizing and first argon introducing, and then carries out second liquid nitrogen freezing to obtain a first solid product.
In the invention, the preparation of the temperature-sensitive nano polymer is preferably carried out in a sealed tetrafluoro-joint gate grinding reaction tube.
In the present invention, the organic solvent is preferably DMSO. In the present invention, the ratio of the amount of the substance of N-isopropylacrylamide to the volume of the organic solvent is preferably (15 to 25) mmol: (8-12) mL. In the present invention, the mixing of the N-isopropylacrylamide and the organic solvent is preferably performed under stirring.
In the present invention, the first liquid nitrogen is frozen for a period of preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the first liquid nitrogen for freezing to fully solidify the liquid and prevent the liquid from splashing when vacuumizing.
In the present invention, the time of the first vacuuming is preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the first vacuumizing to fully suck the oxygen in the bottle, thereby preventing the quenching of the reaction by the oxygen.
In the present invention, the time for the first argon gas to be introduced is preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the first argon gas to fully charge inert gas, and maintains the anaerobic environment of the reaction. In the invention, thawing after freezing the liquid nitrogen is performed under the water bath condition; the temperature of the water bath is preferably 15 to 35 ℃, more preferably 30 ℃. The invention can defrost under the water bath condition to defrost quickly.
In the present invention, the second liquid nitrogen is frozen for a period of preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the second liquid nitrogen for freezing to fully solidify the liquid and prevent the liquid from splashing during the vacuumizing.
After the first solid product is obtained, the invention preferably mixes the first solid product with the chain transfer agent, sequentially carries out third liquid nitrogen freezing, second vacuumizing and second argon introducing, and then carries out fourth liquid nitrogen freezing to obtain the second solid product.
In the present invention, the chain transfer agent is preferably 4-cyano-4- (thiobenzoyl) pentanoic acid. In the present invention, the ratio of the amounts of the substances of N-isopropylacrylamide and 4-cyano-4- (thiobenzoyl) pentanoic acid is preferably 200:1. the invention controls the ratio of the amounts of N-isopropyl acrylamide and 4-cyano-4- (thiobenzoyl) valeric acid in the above range to synthesize a polymer with a specific structure and good temperature-sensitive property.
In the present invention, the time for freezing the third liquid nitrogen is preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the second liquid nitrogen for freezing to fully solidify the liquid and prevent the liquid from splashing during the vacuumizing. .
In the present invention, the time of the second vacuuming is preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the second vacuumizing to fully suck the oxygen in the bottle, thereby preventing the quenching of the reaction by the oxygen.
In the present invention, the second argon gas is preferably introduced for 3 to 8 minutes, more preferably for 4 to 6 minutes. The invention uses the second argon gas to fully charge inert gas, so as to maintain the anaerobic environment of the reaction. In the invention, thawing after freezing the liquid nitrogen is performed under the water bath condition; the temperature of the water bath is preferably 15-35 ℃, more preferably 30 ℃. The invention can defrost under the water bath condition to defrost quickly.
In the present invention, the time for freezing the fourth liquid nitrogen is preferably 5 minutes. The invention uses the fourth liquid nitrogen for freezing so as to fully solidify the liquid and prevent the liquid from splashing when vacuumizing.
After the second solid product is obtained, the invention preferably mixes the second solid product with an initiator, sequentially performs third vacuumizing and third introducing argon, and then performs reversible addition fragmentation chain transfer free radical polymerization (first RAFT reaction) reaction to obtain a macromolecular chain transfer agent intermediate (Macro-CTA).
In the present invention, the initiator is preferably azobisisobutyronitrile. In the present invention, the ratio of the amounts of the substances of the N-isopropylacrylamide and the initiator is preferably 2000:1. the invention controls the ratio of the amounts of N-isopropyl acrylamide and initiator in the above range to fully initiate the RAFT reaction.
In the present invention, the third vacuuming time is preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention utilizes the third vacuumizing to fully suck the oxygen in the bottle, thereby preventing the quenching of the reaction by the oxygen.
In the present invention, the third argon introduction time is preferably 3 to 8 minutes, more preferably 4 to 6 minutes. The invention uses the third argon inlet to fully charge inert gas, so as to maintain the anaerobic environment of the reaction. In the invention, thawing after freezing the liquid nitrogen is performed under the water bath condition; the temperature of the water bath is preferably 15-35 ℃, more preferably 30 ℃. The invention can defrost under the water bath condition to defrost quickly.
In the present invention, the temperature of the first RAFT reaction is preferably 65 to 75 ℃, more preferably 70 ℃; the time of the first RAFT reaction is preferably 6 to 24 hours, more preferably 24 hours. The invention controls the temperature and time of the first RAFT reaction in the above range to fully react to obtain macromolecular chain transfer agent Macro-CTA (i.e. pNIPAM).
After obtaining the intermediate, the invention preferably mixes the intermediate and the solution of diethylaminoethyl methacrylate, and then carries out a second RAFT reaction to obtain the temperature-sensitive nano polymer poly (N-isopropyl acrylamide-b-diethylaminoethyl methacrylate).
In the present invention, the solution of diethylaminoethyl methacrylate is preferably subjected to an oxygen removal treatment before use. In the present invention, the solvent used for the solution of diethylaminoethyl methacrylate is preferably DMSO. In the present invention, the ratio of the amounts of the substances of N-isopropylacrylamide and diethylaminoethyl methacrylate is preferably (20 to 200): 1. the invention controls the ratio of the amounts of N-isopropyl acrylamide and diethylaminoethyl methacrylate in the above range to simultaneously impart temperature-sensitive property and positive surface charge to the polymer.
In the present invention, the mixing of the intermediate and the solution of diethylaminoethyl methacrylate is preferably performed under sealed conditions. In the present invention, the temperature of the second RAFT reaction is preferably 65 to 75 ℃, more preferably 70 ℃; the time of the second RAFT reaction is preferably 6 to 24 hours, more preferably 24 hours. The temperature and time of the second RAFT reaction are controlled in the above range, so that the temperature-sensitive nano polymer { namely the polymer p (NIPAM-b-DEAM) or PND } is obtained through sufficient polymerization.
After the second RAFT reaction is completed, the product of the second RAFT reaction is preferably dialyzed and freeze-dried in sequence to obtain the temperature-sensitive nano polymer.
In the present invention, the molecular weight cut-off of the dialysis bag used for dialysis is preferably 3000 to 10000Da, more preferably 3500Da; the dialysis time is preferably 6 to 8 days. In the present invention, the freeze-drying is preferably performed by freeze-drying the dialyzed dialysate at-40 ℃ for 48 hours.
After the temperature-sensitive nano polymer is obtained, the temperature-sensitive nano polymer and ultrapure water are preferably mixed to obtain a temperature-sensitive nano polymer solution.
After the solution of the chemotherapeutic medicine and the solution of the temperature-sensitive nano polymer are obtained, the invention mixes the solution of the chemotherapeutic medicine and the solution of the temperature-sensitive nano polymer, and then drops the solution of the procoagulant for self-assembly reaction to obtain the nano assembly.
In the invention, the mixture of the solution of the chemotherapeutic drug and the solution of the temperature-sensitive nano polymer is preferably stirred at room temperature for 45-55 h, more preferably 48h. The invention controls the mixing time in the above range so as to fully assemble the chemotherapeutic drug and the temperature-sensitive nano polymer.
In the present invention, the rate of the dropping is preferably 10 to 30mL/h, more preferably 20mL/h. The invention controls the dropping speed in the above range so as to ensure that the dropping speed is slowly and fully assembled and avoid aggregation.
In the present invention, the temperature of the self-assembly reaction is preferably room temperature; the self-assembly reaction time is preferably 22 to 26 hours, more preferably 24 hours. The invention controls the temperature and time of self-assembly reaction in the above range to avoid the polymer forming sol at low temperature or gel at high temperature to influence the assembly behavior.
In the present invention, the nano-assembly is preferably stored in an environment of 4 ℃.
The invention also provides an application of the nano-assembly prepared by the technical scheme or the nano-assembly prepared by the preparation method in preparing antitumor drugs.
In the present invention, the antitumor drug is preferably an antitumor drug for embolism-chemotherapy-immunity cooperative treatment.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The nano assembly has a core-shell structure and comprises an inner core composed of a procoagulant and a chemotherapeutics and an outer shell composed of a temperature-sensitive nano polymer, wherein the mass ratio of procoagulant, chemotherapeutics and the temperature-sensitive nano polymer in the nano assembly is 10:4:80;
the chemotherapeutic drug is nitro cisplatin, and the procoagulant is polyphosphate with a polymerization degree of 45;
the preparation method of the nano assembly comprises the following steps: weighing 300mg of PND freeze-dried powder, dissolving in 2.625mL of ultrapure water, fully dissolving to obtain PND solution, adding 0.725mL of 21mg/mL of nitrocisplatin solution, stirring at room temperature for 48h until the solution is changed from white clear to yellow clear, taking 900 mu LPND-Pt solution, dripping 100 mu L of 100mg/mL of PolyP solution, stirring at room temperature, performing self-assembly reaction for 24h to obtain a nano assembly, marking as PND-Pt-PolyP10, and storing in a 4-DEG refrigerator;
The temperature-sensitive nano polymer PND is prepared by taking N-isopropyl acrylamide and diethylaminoethyl methacrylate as monomers through a RAFT method, and comprises the following steps:
(1) Accurately weighing a monomer NIPAM (2.26 g,20 mmol), adding into a tetrafluoro-portal reaction tube with a magnetic stirrer, accurately weighing DMSO (10 mL), adding into the reaction tube, stirring on a constant-temperature magnetic stirrer to completely dissolve NIPAM, transferring into a liquid nitrogen bath, performing first liquid nitrogen freezing for 4min, performing first vacuumizing for 5min by a vacuum pump, stirring in a hot water bath, and performing first argon filling for 5min; after the first argon is introduced, freezing by using second liquid nitrogen to obtain a solid first solid product;
(2) Adding a chain transfer agent 4-cyano-4- (thiobenzoyl) valeric acid (27.9 mg,0.1 mmol) into the first solid product obtained in the step (1), stirring, sequentially performing third liquid nitrogen freezing, second vacuumizing and second argon-introducing circulation operation, and performing fourth liquid nitrogen freezing after the second argon-introducing operation is completed to obtain a solid second solid product;
(3) And (3) adding an initiator AIBN (2.6 mg,0.01 mmol) which is accurately weighed into the second solid product obtained in the step (2), and then sequentially carrying out third vacuumizing and third argon-introducing circulation operation. After the reaction solution is completely melted, placing the reaction tube in an oil bath pot at 70 ℃ for a first RAFT reaction for 24 hours to obtain an intermediate;
(4) Adding a deoxidized diethylaminoethyl methacrylate solution (0.785 ml,2 mmol) into the intermediate obtained in the step (3), sealing the pinholes by vacuum gel, continuously placing in an oil bath pot at 70 ℃ for a second RAFT reaction for 24 hours, transferring to a dialysis bag (molecular weight cut-off 3500 Da) after the reaction is completed, dialyzing for 7d, and finally freeze-drying the dialysate at-40 ℃ for 4 days8h to obtain the temperature-sensitive nano polymer PND 200-20 Freeze-dried powder.
The preparation method of the solution of the nitro cisplatin comprises the following steps of;
the molar ratio is 1:2 to a clean 50mL round bottom flask was added cisplatin (300 mg,1 mmol) and silver nitrate (340 mg,2 mmol), dissolved in water, stirred overnight at room temperature in the absence of light, followed by centrifugation to remove the precipitated AgCl to give a supernatant as a nitrocisplatin solution, which was then assayed for concentration by ICP-OES and stored in the absence of light at 4 ℃ environment;
the solution of PolyP is an aqueous solution of polyphosphate having a degree of polymerization of 45.
Fig. 1 is a schematic flow chart of the preparation of a nano-assembly according to embodiment 1 of the present invention, specifically: firstly, respectively preparing chemotherapeutic drugs of nitro cisplatin and temperature-sensitive nano polymer, preparing procoagulant of polyphosphate, and obtaining a nano assembly through self-assembly reaction of the three.
Example 2
The nano-assembly was prepared according to the method of example 1, except that the added mass of PolyP was 75mg, and the prepared nano-assembly was designated PND-Pt-PolyP75.
Comparative example 1
Nitrocisplatin, designated Pt, was prepared as in example 1.
Comparative example 2
PND-Pt was prepared according to the method of example 1, except that 300mg of PND lyophilized powder was weighed and dissolved in 2.625mL of ultra-pure water, after sufficient dissolution, a PND solution was obtained, and then 0.725mL of nitrocisplatin solution at a concentration of 21mg/mL was added, and stirred at room temperature for 48 hours until the solution was changed from white clear transparent to yellow clear transparent, to obtain PND-Pt.
Comparative example 3
Pt-PolyP was prepared in the same manner as in example 1, except that 0.725mL of a solution of nitrocisplatin at a concentration of 21mg/mL was added to 2.625mL of an ultrapure water solution, 100. Mu.L of a solution of PolyP at a concentration of 100mg/mL was added dropwise, and the mixture was stirred at room temperature to conduct self-assembly reaction for 24 hours, to obtain Pt-PolyP.
Comparative example 4
The nano-assembly was prepared according to the method of example 1, except that the mass of the added PolyP was 1mg, and the prepared nano-assembly was designated PND-Pt-PolyP1.
Comparative example 5
The nano-assembly was prepared as in example 1 except that 100. Mu.L of a solution of PolyP at a concentration of 100mg/mL was added dropwise to a 900. Mu.L ND solution (88.9 mg/mL) as in example 1, and the self-assembly reaction was carried out with stirring at room temperature for 24 hours, which was designated PND-PolyP.
Characterization of test one, PND-Pt-PolyP10
The PND-Pt-PolyP10 nano-assembly prepared in example 1 and the assembly prepared in comparative example were diluted to 1mg/mL according to polymer concentration, the particle size and potential were measured by electron microscopy using a dynamic light scattering particle sizer, and element Mapping was performed to obtain an electron microscopy chart as shown in FIG. 2, wherein a is Pt-Poly, b is PND-Pt, c is PND-Pt-PolyP1 and d is PND-Pt-PolyP10 in the first row of FIG. 2, and the second behavior element Mapping chart (indicating the positions of different elements in the nano-assembly) of FIG. 2, and the particle size and potential analysis chart are shown in FIG. 3.
As can be seen from fig. 2, in the absence of polymer stabilization, nitrocisplatin and PolyP are in an aggregated state, and after stabilization by adding PND, the morphology of the prepared nanomaterial is gradually changed from a dispersed spherical structure to a uniform core-shell structure with different addition amounts of PolyP. The results of the elemental Mapping graph show that both Pt and PolyP are in the nano-assembly at the core position, while the polymer forms a stable shell.
As is clear from FIG. 3, pt and PolyP are aggregated without stabilizing the polymer, aggregate with a particle size of 4000nm is formed, the potential is about-30 mV, PND-Pt prepared in comparative example 1 is reduced to about-180 nm, the potential is about 15mV due to the action of the cationic nanogel, the potential is gradually reduced with the addition of polyphosphate PolyP, at the same time, when the addition amount of PolyP is 1, the charge of the assembly is neutral, particles are unstable to form aggregation, the particle size is increased to 400nm, the addition of PolyP is continued from 15 to 75, the particles tend to be stable, the particle size is maintained at about 180nm, and the potential is gradually increased to about-15 mV to about-22 mV due to the addition of the negatively charged polyphosphate. Therefore, the PND-Pt-PolyP10 prepared in the embodiment 1 has better stability, forms a core-shell structure, can effectively load Pt and PolyP into the core, and is beneficial to the stability and slow release of the medicine.
Testing the drug release behavior of PND-Pt-PolyP10 nanocomposites prepared in example 1
The PND-Pt-PolyP10 nano-assembly prepared in example 1 was tested for drug release under different conditions as follows:
(1) Configuring a release medium: PBS release buffers with pH values of 7.4 and 6.5 are prepared by different proportions of sodium dihydrogen phosphate and disodium hydrogen phosphate.
(2) 200. Mu.L of the PND-Pt-PolyP10 nano-assembly solution prepared in example 1 was placed in dialysis bags with a molecular weight cut-off of 3500Da, sealed with clamps, each set was set in 3 parallel, placed in 37℃to gel it, and then the dialysis bags were immersed in 50mL centrifuge tubes which had been stabilized to 37℃and filled with 30mL of release solution, shaken in a shaker at 37℃and a rotational speed of 180rpm.
(3) At predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h), 1mL of release solution was taken and 1mL of blank release solution was replenished. The released solution was examined for the content of Pt and P by ICP-OES to obtain a drug release profile of PND-Pt-PolyP10 nano-assembly prepared in example 1, as shown in FIG. 4.
As can be seen from fig. 4, for the PND-Pt-PolyP10 nano-assembly prepared in example 1, pt and PolyP showed different release behavior at different pH, pt was released at acidity, while PolyP was released slowly at pH 6.5 and faster at neutral pH 7.4. The accumulated release amounts of the two drugs respectively reach 80% (Pt, pH 6.5) and 65% (PolyP, pH 7.4). The PND-Pt-PolyP10 nano-assembly prepared in the embodiment 1 has pH sensitivity to release of Pt and PolyP, and improves targeting property of the PND-Pt-PolyP10 nano-assembly.
Test three, sol gel phase transition behavior of PND-Pt-PolyP10 nano-Assembly prepared in example 1
PND-Pt-PolyP10 nano-assemblies in example 1, PND prepared in example 1 and PND-Pt prepared in comparative example 2, PND-PolyP prepared in comparative example 5 is prepared into a solution with concentration of 80mg/mL, and the temperature-sensitive sol-gel phase transition behavior of the PND-Pt-PolyP is characterized by an advanced rotary rheometer, so that a temperature-sensitive sol-gel phase transition behavior diagram is obtained, and is shown in FIG. 5. From fig. 5, it is apparent that, due to the thermosensitive properties of the cationic thermosensitive nano-polymer, PND-Pt-PolyP10 nano-assemblies, PND-Pt and PND-PolyP all have good gelation behavior, the gelation temperature is about 35 ℃, and as the temperature increases, the elastic modulus of the nano-assembly material increases from 10Pa at room temperature to 1000Pa (8% PND-Pt-PolyP 10) after gelation, indicating that the PND-Pt-PolyP10 nano-assembly in example 1 has excellent gelation behavior, not only can maintain long-term release of the drug, but also can perform effective occlusion of tumor site blood vessels. Meanwhile, the injection molding agent has good shear thinning property, and the shear viscosity is obviously reduced along with the increase of the shear rate, so that the injection molding agent is easy to inject.
In vitro procoagulant studies to test four, PND-Pt-PolyP10 nanocomposites
The PND-Pt-PolyP10 nano-assemblies of example 1 were mixed with plasma, the chromogenic substrate for factor XII (S-2302) and thrombin (S-2238) were added separately, and the trend of the effect of the material on FXII, thrombin and fibrin formation was investigated by detection of the absorbance at 405nm by a microplate reader, as shown in FIGS. 6, 7 and 8, respectively.
From fig. 6 to 8, it can be seen that the PND-Pt-PolyP10 nano-assembly prepared in example 1 can significantly promote the production of FXII (fig. 6) and thrombin (fig. 7), can increase the turbidity of fibrin and can improve the stability of fibrin structure (fig. 8);
meanwhile, after the PND-Pt-PolyP10 nano-assembly is blended with whole blood, the mixture is placed at 37 ℃ for stabilization for 10min, then liquid nitrogen quick freezing is carried out, the influence of materials on the blood clot structure is observed after freeze drying, and a scanning electron microscope image of the PND-Pt-PolyP10 nano-assembly prepared in example 1 on the blood clot is shown in figure 9. As can be seen from fig. 9, the porous structure of the nano-assembly Cheng Xianchu gel prepared in example 1 can facilitate the slow release of the drug, while the pure blood clot presents a large number of red blood cells, the morphology is complete and dispersed, after the two are compounded, the aggregated red blood cells can be filled in the gel holes, and the whole structure is more compact, which indicates that the gel formed by the PND-Pt-PolyP10 nano-assembly prepared in example 1 can stabilize the structure of the blood clot, facilitate the long-term maintenance of the blood clot, and inhibit the degradation of the blood clot.
Test five, PND-Pt-PolyP10 nano-assemblies for in vitro immune activation
The effect of PND-Pt-PolyP10 nano-assemblies prepared in example 1 on tumor cell apoptosis was investigated, and the specific steps were as follows: 4T1 cells with good growth state are digested and collected and then spread into a 24-hole plate, 7 ten thousand cells are spread on each dish, and an incubator is incubated overnight to enable the cells to adhere to the wall; the old culture media in 24 well plates were aspirated according to Pt (prepared in comparative example 1), PND-Pt (prepared in comparative example 2) and PND-Pt-PolyP10 (prepared in example 1) solutions each having a Pt concentration of 20ug/mL, 1mL of the prepared Pt, PND-Pt and PND-Pt-PolyP10 solutions were added to each group of 3 wells, and the blank group was filled with the whole culture media without drugs, and after 24 hours, stained according to an apoptosis kit, and the cell fractions at different apoptosis periods were observed using a flow cytometer to obtain a trend graph showing the effect on apoptosis of 4T1 tumor cells as shown in fig. 10. As can be seen from fig. 10, in which apoptosis is mainly chemotherapy of Pt, pt-containing groups have a better ability to induce apoptosis of tumor cells, and in which PND-Pt-PolyP10 nano-assemblies are effective in promoting early apoptosis and late apoptosis of tumor cells.
The effect of the PND-Pt-PolyP10 nano-assembly prepared in example 1 on tumor cell immunogenic death (ICD effect) was investigated, and the specific procedure was as follows: the well-grown 4T1 cells were digested and collected, plated in 24-well plates, 7-thousand cells were plated on each plate, and incubated overnight in an incubator to adhere, pt (prepared in comparative example 1), PND-Pt (prepared in comparative example 2) and PND-Pt-PolyP10 (prepared in example 1) solutions were prepared at a Pt concentration of 20. Mu.g/mL, old medium in the 24-well plates was aspirated, 1mL of the prepared Pt, PND-Pt, PND-Pt-PolyP10 solution (wherein the concentrations of the respective components were PND: 400. Mu.g/mL, pt: 20. Mu.g/mL, polyP: 50. Mu.g/mL), 3 wells per group, and a blank group was added with the whole medium containing no drug. After 24 hours, expression of CRT and HMGB1 was measured by a flow cytometer, and the amount of ATP secreted from the supernatant was measured by an ATP kit, to obtain an ICD effect map of the nano-assembly prepared in example 1 on 4T1 tumor cells. The results of FIG. 11 show that the Pt-containing groups can well induce the release of HMGB1, increase the expression of CRT, promote the secretion of ATP and promote the ICD effect caused by materials, thereby activating the immune system and carrying out the immunotherapy of tumors.
The effect of PND-Pt-PolyP10 nano-assemblies on Dendritic Cell (DC) maturation was investigated as follows: 4T1 cells and DC2.4 cells with good growth state are digested and collected and respectively paved into 24 pore plates, 7 ten thousand cells are paved on each dish, and the incubator is incubated overnight to enable the cells to adhere to the walls. To a 4T1 cell plate, pt (prepared in comparative example 1), PND-Pt (prepared in comparative example 2) and PND-Pt-PolyP10 (prepared in example 1) solutions were prepared at a Pt concentration of 20. Mu.g/mL, respectively, old medium in 24 well plates was aspirated, 1mL of the prepared above-mentioned Pt, PND-Pt and PND-Pt-PolyP10 solutions (wherein the concentrations of the respective components were PND: 400. Mu.g/mL, pt: 20. Mu.g/mL, polyP: 50. Mu.g/mL) were added, 3 wells each, and the whole medium containing no drug was added to the blank group. After 12h, the drug-containing medium was aspirated, the treated 4T1 cells were collected by digestion, resuspended in 1mL medium, the old medium of DC2.4 was replaced, and the cells were cultured together for 24h, after which the cells were collected by digestion, the maturation of the DC cells was analyzed by flow cytometry to obtain a trend graph of the effect on DC cell maturation, and the results are shown in FIG. 12. As can be seen from fig. 12, pt-containing groups all have the ability to promote DC maturation, mainly because Pt promotes tumor cell apoptosis and produces ICD effects, wherein PND-Pt-PolyP10 nano-assemblies can significantly increase maturation of DC cells, thereby increasing presentation of tumor-associated antigens, enhancing tumor immune responses.
Test of anti-tumor Effect of PND-Pt-PolyP10 nano-Assembly prepared in six example 1
BALB/c females were purchased for six weeks of age and weighing between 16 and 19 g. After one week of laboratory animal house adaptive feeding, the mice were shaved with hairs around their right hind limbs, 4T1 cells were cultured, and the cells were cultured until the number of cells was sufficientAnd in log phase, cells were collected by digestion and centrifugation. After washing once with PBS, re-suspended with PBS to prepare 10 7 The cell suspension/mL was placed in an ice box for use. A syringe was used to subcutaneously inject 100 μl of cell suspension over the right hind limb of each mouse. And (5) continuing feeding after injection. The tumor volume calculation formula is: v= (l×w≡2)/2, where V represents the tumor volume, L represents the long diameter of the tumor, and W represents the short diameter. To the extent that the tumor grows to 100mm 3 About, mice were randomly divided into 5 groups of 5 mice each, each intratumorally dosed with 50 μl: (1) physiological saline; (2) nitrocisplatin; (3) a temperature sensitive nano polymer PND; (4) PND-Pt; (5) PND-Pt-PolyP10; wherein the PND concentration in each group was 80mg/mL, the Pt concentration was 4mg/mL, and the PolyP concentration was 10mg/mL. During the experiment, the body weight and tumor volume of the mice were measured every two days, the mice were sacrificed on day 14 after dosing, the tumors were dissected, and the isolated tumors were weighed and photographed, fixed with 4% paraformaldehyde, sectioned with paraffin embedded, HE stained, ki67 and TUNEL immunofluorescent stained. The results are shown in fig. 6, the free nitrocisplatin group is basically dead on the third day due to the strong side effect of Pt, while the PND-Pt-PolyP nano-assembly significantly inhibited the side effect of Pt, and can significantly inhibit the growth of tumor, and as a result, the tumor relative growth rate graph of the mouse 4T1 subcutaneous tumor is shown in fig. 14, the tumor weight change graph of the mouse 4T1 subcutaneous tumor is shown in fig. 15, and the tumor tissue immunofluorescence graph of the mouse 4T1 subcutaneous tumor is shown in fig. 16.
By immunofluorescent staining of tumor tissue, the result shows that PND-Pt-PolyP10 nano-assembly can significantly increase apoptosis of tumor cells, and simultaneously release of PolyP can trigger coagulation cascade reaction of tumor blood vessels, induce aggregation of blood platelets, and the long-acting embolism result leads to destruction of blood vessels of tumor tissue, so that PND-Pt-PolyP10 nano-assembly has the best anti-tumor effect.
Testing of anti-tumor metastasis Effect of seven, PND-Pt-PolyP10 nano-assemblies
BALB/c females were purchased for six weeks of age with weights between 16-19g and after one week of laboratory animal house adaptive rearing, the mice were shaved with clean hair around the right hind limbs. Culturing 4T1 thinCells, cells were harvested by digestion and centrifugation until the number of cells was sufficient and in log phase. After washing once with PBS, re-suspended with PBS to prepare 10 7 The cell suspension/mL was placed in an ice box for use. A syringe was used to subcutaneously inject 100 μl of cell suspension over the right hind limb of each mouse. And (5) continuing feeding after injection. The tumor volume calculation formula is: v= (l×w≡2)/2, where V represents the tumor volume, L represents the long diameter of the tumor, and W represents the short diameter. To the extent that the tumor grows to 100mm 3 Left and right, mice were randomly divided into 5 groups of 5 animals each, each intratumorally dosed: (1) physiological saline; (2) nitrocisplatin; (3) a temperature sensitive nano polymer PND; (4) PND-Pt; (5) PND-Pt-PolyP10; wherein the PND concentration in each group was 80mg/mL, the Pt concentration was 4mg/mL, and the PolyP concentration was 10mg/mL. Simultaneous intravenous injection of 4T1 cells (100. Mu.L, 10 6 /mL). Mice were sacrificed 21 days later, lung tissue was dissected, lung nodules and weights were recorded, paraneoplastic lymph nodes were dissected, and CD8 was observed by flow cytometry + The number of lung nodules obtained from the mice lung metastasis model is shown in FIG. 17, the lung weight of the mice lung metastasis model is shown in FIG. 18, and CD8 in the paraneoplastic lymph nodes of the mice lung metastasis model + The cell infiltration status is shown in FIG. 19, and the DC cell maturation status in the paraneoplastic lymph nodes of the mouse lung metastasis model is shown in FIG. 20;
from FIGS. 17 to 20, it can be seen that the number of pulmonary nodules was significantly reduced in mice, and metastasis was inhibited in the tumor with CD8 in the paraneoplastic lymph nodes, by treatment with PND-Pt-PolyP10 nanoeggregates (FIGS. 17 and 18) + The result shows that the PND-Pt-PolyP10 nano-assembly prepared in the embodiment 1 can effectively trigger immune response of tumor, enhance infiltration of lymphocytes and maturation of DC cells, and inhibit metastasis of tumor cells.
Test eight, evaluation of in vivo embolic Effect of PND-Pt-PolyP10 nanocomposites
Taking New Zealand white rabbits, anesthetizing experimental rabbits planted with VX2 tumor on the hind legs by 10% chloral hydrate (2.5 ml/mg), preparing skin, conventionally sterilizing, taking fish-like tumor tissue with better activity, and slowing down by using sterile phosphoric acid Flushing with saline solution (PBS), and cutting into 1mm size with sterile ophthalmic scissors 3 Left and right tumor blocks are put into Hanks' liquid for standby. Fasted, water-fed laboratory rabbits for 12 hours are weighed and then are subjected to intraperitoneal injection according to the volume of 1.4-1.7ml/kg, and modified as appropriate. After conventional skin preparation, the fully anesthetized experimental rabbits were bound on a homemade rabbit plate, placed on a sterile operating table, sterilized and spread, a longitudinal incision of about 3cm was made along the lower abdominal midline of the xiphoid process of the rabbits, the liver was fully exposed, a incision was made with sterile ophthalmic forceps on the left outer lobe of the liver (proximal hepatic sickle ligament), VX2 tumor mass was placed, and filled with a previously sheared gelatin sponge strip of 1cm size, the liver was returned to the abdominal cavity after the incision was thoroughly stopped, gentamicin (2-4 ten thousand IU) was injected into the wound, and ampicillin (1-2 ten thousand IU) was injected three consecutive days post-operation after suturing the wound. After 17 days, conventional disinfection towel is laid, the successfully placed rabbit is placed on a table, the rabbit is cannulated to the main trunk of the celiac artery of the rabbit under the guidance of a 4F Cobra catheter and a guide wire through a vascular sheath, the guide wire is withdrawn, a proper amount of mixed solution of heparinized saline and iohexol contrast agent (300 mg I/ml) is injected, a proper amount of heparinized saline is injected after 'smoking' verification to wash the catheter, and the catheter is connected with a high-pressure injector, so that celiac artery radiography is performed at the flow rate of 1ml/s, the total amount of 3ml and the pressure of 200 Kpa. Then through the super selection of microcatheter, the opening of the catheter is placed in the blood supply vessel of the tumor, and the PND-Pt-PolyP nano assembly is injected after the contrast confirmation of the flow rate of 0.5ml/s, the total amount of 2ml and the pressure of 200Kpa, so as to obtain DSA images and H at different times of the rabbit VX2 transplanted tumor liver cancer model &E staining pattern, as shown in fig. 21. As can be seen from FIG. 21, before injecting PND-Pt-PolyP10 nano-assembly, tumor blood vessel under DSA presents spherical structure and multi-polarized branches (FIG. 21 a), after injecting PND-Pt-PolyP10 to plug tumor, tumor blood vessel can be plugged in 10min, and can not be developed under DSA (FIG. 21 b), good plug effect is still presented after four hours (FIG. 21 c), and tumor is peeled off for H after 4 hours&E staining (figure 21 d) shows that the tumor blood vessel and the tumor tissue have a large number of red blood cells aggregated, because the released PolyP can effectively cause coagulation cascade reaction of the tumor blood vessel to generate thrombus, and meanwhile, due to the physical barrier effect of gel, the long-acting embolism of blood clots can be maintained, so that better liver cancer treatment effect is achieved.
In summary, the PND-Pt-PolyP10 nano-assembly prepared in example 1 has good assembly behavior and shows a core-shell structure; because the PND-Pt-PolyP10 nano assembly prepared in the embodiment 1 has temperature sensitive property, the PND-Pt-PolyP10 nano assembly is easy to inject at room temperature, the grid structure formed after gelation can effectively release the medicine slowly, and meanwhile, the high-strength gel can also effectively plug blood vessels; the procoagulant PolyP released by the nano-assembly prepared in example 1 can effectively promote the generation of FXII and thrombin, and increase the strength and stability of fibrin; the chemotherapeutic drug Pt of the nano-assembly prepared in the embodiment 1 can promote apoptosis of tumor cells and promote ICD effect, and induce curing of DC. The nano-assembly prepared in the embodiment 1 has an embolism-chemotherapy-immunity cooperative treatment mode, and can effectively inhibit tumor growth, reduce tumor metastasis and enhance anti-tumor immune response of tumor parts; meanwhile, on a VX2 transplanted tumor rabbit liver cancer model, PND-Pt-PolyP10 nano-assemblies can realize the physical embolism of temperature-sensitive nano-polymers and the synergistic embolism treatment of coagulation embolism caused by PolyP, and have good clinical potential.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The nanometer assembly is characterized by having a core-shell structure, and comprising an inner core composed of a procoagulant and a chemotherapeutic drug and an outer shell composed of a temperature-sensitive nanometer polymer, wherein the mass ratio of the procoagulant, the chemotherapeutic drug and the temperature-sensitive nanometer polymer in the nanometer assembly is (10-75): 4:80;
the temperature-sensitive nano polymer is prepared by taking N-isopropyl acrylamide and diethylaminoethyl methacrylate as monomers through a RAFT method.
2. The nano-assembly according to claim 1, wherein the procoagulant is a polyphosphate having a degree of polymerization of 25 to 250.
3. The nano-assembly of claim 1, wherein the chemotherapeutic agent is a nitrated cisplatin or tetravalent platinum chemotherapeutic agent.
4. The nano-assembly of claim 1, wherein the mass ratio of procoagulant, chemotherapeutic drug and temperature sensitive nano-polymer is 10:4:80.
5. The nano-assembly according to claim 1, wherein the temperature-sensitive nano-polymer has a degree of polymerization of 100 to 250.
6. The method for preparing the nano-assembly according to any one of claims 1 to 5, comprising the steps of: and (3) mixing the solution of the chemotherapeutic medicine and the solution of the temperature-sensitive nano polymer, dropwise adding the solution of the procoagulant, and performing self-assembly reaction to obtain the nano assembly.
7. The preparation method of claim 6, wherein the temperature-sensitive nano-polymer is prepared by a RAFT method, and comprises the following steps:
(1) Mixing N-isopropyl acrylamide and an organic solvent, sequentially performing first liquid nitrogen freezing, first vacuumizing and first argon introducing, and then performing second liquid nitrogen freezing to obtain a first solid product;
(2) Mixing the first solid product obtained in the step (1) with a chain transfer agent, sequentially performing third liquid nitrogen freezing, second vacuumizing and second argon introducing, and performing fourth liquid nitrogen freezing to obtain a second solid product;
(3) Mixing the second solid product obtained in the step (2) with an initiator, sequentially carrying out third vacuumizing and third argon introducing, and then carrying out a first RAFT reaction to obtain a macromolecular chain transfer agent intermediate Macro-CTA;
(4) And (3) mixing the solution of the macromolecular chain transfer agent intermediate Macro-CTA obtained in the step (3) and the deoxidized diethylaminoethyl methacrylate, and then carrying out a second RAFT reaction to obtain the temperature-sensitive nano polymer poly (N-isopropyl acrylamide-b-diethylaminoethyl methacrylate).
8. The method according to claim 7, wherein the temperature of the first RAFT reaction in the step (3) is 65 to 75 ℃, and the time of the first RAFT reaction is 6 to 24 hours.
9. The method of claim 7, wherein after the completion of the second RAFT reaction in step (4), further comprising: and sequentially dialyzing and freeze-drying the product of the second RAFT reaction to obtain the temperature-sensitive nano polymer.
10. Use of a nano-assembly according to any one of claims 1 to 5 or a nano-assembly prepared by a method according to any one of claims 6 to 9 for the preparation of an anti-tumour agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310419155.8A CN116370643A (en) | 2023-04-19 | 2023-04-19 | Nanometer assembly, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310419155.8A CN116370643A (en) | 2023-04-19 | 2023-04-19 | Nanometer assembly, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116370643A true CN116370643A (en) | 2023-07-04 |
Family
ID=86980534
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310419155.8A Pending CN116370643A (en) | 2023-04-19 | 2023-04-19 | Nanometer assembly, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116370643A (en) |
-
2023
- 2023-04-19 CN CN202310419155.8A patent/CN116370643A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9089512B2 (en) | Active scaffolds for on-demand drug and cell delivery | |
| JPS6025926A (en) | Blood vessel embolism forming composition | |
| Wang et al. | Photo-responsive hydrogel facilitates nutrition deprivation by an ambidextrous approach for preventing cancer recurrence and metastasis | |
| JP2010516349A (en) | Method for modifying myocardial infarction progression | |
| CN104382918B (en) | A kind of Evacet thermo-sensitive gel for locally injected into tumor | |
| US9687552B2 (en) | Association of poly(N-acryloylglycinamide) with at least one active principle | |
| WO2021179843A1 (en) | Anti-tumor nano adjuvant based on vesicle formed by cross-linked biodegradable polymer, preparation method therefor and use thereof | |
| CN105462142A (en) | Thermo-sensitive type interpenetrating polymer network hydrogel material and preparation method and application thereof | |
| KR20220125798A (en) | Systems and pharmaceutical compositions for treatment by direct injection of a targeted cell population | |
| CN113332241A (en) | Small micelle nano-drug and preparation method and application thereof | |
| CN101926821B (en) | Drug composition for targeted release of trace elements and preparation method and application thereof | |
| Liu et al. | Thermosensitive selenium hydrogel boosts antitumor immune response for hepatocellular carcinoma chemoradiotherapy | |
| Suhar et al. | Hyaluronan and elastin-like protein (HELP) gels significantly improve microsphere retention in the myocardium | |
| KR102192908B1 (en) | Method for manufacturing controlled releasable DDS device using thermosensitive hydrogel | |
| CN107550879A (en) | A kind of preparation method of gelfoam drug bearing microsphere | |
| CN108186571A (en) | Reversible crosslink asymmetry vesica is preparing the application in treating acute leukemia drug | |
| WO2023246336A1 (en) | Preparation for bionic injectable polypeptide hydrogel and use thereof | |
| CN116370643A (en) | Nanometer assembly, preparation method and application thereof | |
| US11419892B2 (en) | Antimicrobial platelet-like particles | |
| WO2023072161A1 (en) | Composition containing mesenchymal stem cells and hydrogel, and use thereof | |
| KR101132673B1 (en) | Ubiquitous Injectable, Thermo-Reversible Gel for Hydrophobic and Hydrophilic Drug Delivery | |
| KR101898816B1 (en) | Parenteral Drug Delivery System based on low Molecular Polysaccharides | |
| Li et al. | ROS-responsive EPO nanoparticles ameliorate ionizing radiation-induced hematopoietic injury | |
| CN104324032B (en) | Antituberculotics three compound recipe microsphere vascular targeting thromboembolism slow releasing agent and its production and use | |
| Xie et al. | A thermosensitive Pickering gel emulsion with a high oil–water ratio for long-term X-ray imaging and permanent embolization of arteries |
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 |