CN115960305A - Nano gel, nano medicine based on nano gel, preparation method and application thereof - Google Patents
Nano gel, nano medicine based on nano gel, preparation method and application thereof Download PDFInfo
- Publication number
- CN115960305A CN115960305A CN202211633371.4A CN202211633371A CN115960305A CN 115960305 A CN115960305 A CN 115960305A CN 202211633371 A CN202211633371 A CN 202211633371A CN 115960305 A CN115960305 A CN 115960305A
- Authority
- CN
- China
- Prior art keywords
- nano
- nanogel
- drug
- tumor
- monomer
- 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.)
- Granted
Links
- 239000003814 drug Substances 0.000 title claims abstract description 147
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 229940079593 drug Drugs 0.000 claims abstract description 142
- 239000000178 monomer Substances 0.000 claims abstract description 92
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 38
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 37
- 239000002245 particle Substances 0.000 claims abstract description 34
- 239000004094 surface-active agent Substances 0.000 claims abstract description 24
- 230000001105 regulatory effect Effects 0.000 claims abstract description 21
- 229940044683 chemotherapy drug Drugs 0.000 claims abstract description 17
- 239000003999 initiator Substances 0.000 claims abstract description 14
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 13
- 238000001179 sorption measurement Methods 0.000 claims abstract description 12
- 230000000977 initiatory effect Effects 0.000 claims abstract description 7
- 239000008346 aqueous phase Substances 0.000 claims abstract description 5
- 230000004044 response Effects 0.000 claims description 30
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.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 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 claims description 24
- 229960002918 doxorubicin hydrochloride Drugs 0.000 claims description 22
- 239000002246 antineoplastic agent Substances 0.000 claims description 20
- YQIGLEFUZMIVHU-UHFFFAOYSA-N 2-methyl-n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C(C)=C YQIGLEFUZMIVHU-UHFFFAOYSA-N 0.000 claims description 17
- 238000011068 loading method Methods 0.000 claims description 17
- 229960004657 indocyanine green Drugs 0.000 claims description 16
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims description 16
- 230000009467 reduction Effects 0.000 claims description 13
- DJVKJGIZQFBFGS-UHFFFAOYSA-N n-[2-[2-(prop-2-enoylamino)ethyldisulfanyl]ethyl]prop-2-enamide Chemical compound C=CC(=O)NCCSSCCNC(=O)C=C DJVKJGIZQFBFGS-UHFFFAOYSA-N 0.000 claims description 12
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 11
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 150000003384 small molecules Chemical class 0.000 claims description 8
- 229940041181 antineoplastic drug Drugs 0.000 claims description 7
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 7
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 claims description 6
- 230000001276 controlling effect Effects 0.000 claims description 5
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 3
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 claims description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 3
- XHZPRMZZQOIPDS-UHFFFAOYSA-N 2-Methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(C)(C)NC(=O)C=C XHZPRMZZQOIPDS-UHFFFAOYSA-N 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 3
- 229940067606 lecithin Drugs 0.000 claims description 3
- 239000000787 lecithin Substances 0.000 claims description 3
- 235000010445 lecithin Nutrition 0.000 claims description 3
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 claims description 3
- 229960003105 metformin Drugs 0.000 claims description 3
- SWPMNMYLORDLJE-UHFFFAOYSA-N n-ethylprop-2-enamide Chemical compound CCNC(=O)C=C SWPMNMYLORDLJE-UHFFFAOYSA-N 0.000 claims description 3
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 3
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 106
- 239000000499 gel Substances 0.000 abstract description 33
- 230000035515 penetration Effects 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 16
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 abstract description 10
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 abstract description 10
- 210000002744 extracellular matrix Anatomy 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 7
- 230000004888 barrier function Effects 0.000 abstract description 6
- 235000019589 hardness Nutrition 0.000 description 115
- 239000000243 solution Substances 0.000 description 60
- 229910021642 ultra pure water Inorganic materials 0.000 description 32
- 239000012498 ultrapure water Substances 0.000 description 32
- 210000004027 cell Anatomy 0.000 description 24
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 17
- 229940043267 rhodamine b Drugs 0.000 description 17
- 238000004132 cross linking Methods 0.000 description 16
- 238000001514 detection method Methods 0.000 description 14
- 239000012071 phase Substances 0.000 description 14
- 230000004043 responsiveness Effects 0.000 description 14
- 229910052799 carbon Inorganic materials 0.000 description 13
- 238000009826 distribution Methods 0.000 description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 238000001035 drying Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 9
- 210000004881 tumor cell Anatomy 0.000 description 9
- 210000004204 blood vessel Anatomy 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 239000006185 dispersion Substances 0.000 description 8
- 238000000108 ultra-filtration Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 7
- 238000002296 dynamic light scattering Methods 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 230000005540 biological transmission Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 230000004700 cellular uptake Effects 0.000 description 6
- 239000006059 cover glass Substances 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 238000004043 dyeing Methods 0.000 description 6
- 230000002708 enhancing effect Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 6
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 229910052786 argon Inorganic materials 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 230000035699 permeability Effects 0.000 description 5
- 239000012679 serum free medium Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- 206010006187 Breast cancer Diseases 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 238000011067 equilibration Methods 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000000089 atomic force micrograph Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000007720 emulsion polymerization reaction Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 210000003462 vein Anatomy 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
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- HPZOOQSXPMEJBV-ODCFVKFUSA-N Tirilazad mesylate Chemical compound CS(O)(=O)=O.O=C([C@@H]1[C@@]2(C)CC=C3[C@@]4(C)C=CC(=O)C=C4CC[C@H]3[C@@H]2C[C@H]1C)CN(CC1)CCN1C(N=1)=CC(N2CCCC2)=NC=1N1CCCC1 HPZOOQSXPMEJBV-ODCFVKFUSA-N 0.000 description 2
- 206010054094 Tumour necrosis Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000000973 chemotherapeutic effect Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 210000003141 lower extremity Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 2
- 239000002539 nanocarrier Substances 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229920002818 (Hydroxyethyl)methacrylate Polymers 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical group [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 description 1
- 229940099500 cystamine Drugs 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000002828 effect on organs or tissue Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000012854 evaluation process Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000005395 methacrylic acid group Chemical group 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- KYKDOARTYFGHPC-UHFFFAOYSA-N n-propan-2-ylpropanamide Chemical compound CCC(=O)NC(C)C KYKDOARTYFGHPC-UHFFFAOYSA-N 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000007903 penetration ability Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Images
Classifications
-
- 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
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of nano-drug preparations, and particularly relates to a nano-gel, a nano-drug based on the nano-gel, and a preparation method and application of the nano-gel. The nanogel is obtained by initiating a monomer to carry out polymerization reaction in an aqueous phase by an initiator under the condition that the monomer exists in a cross-linking agent and a surfactant. The hardness of the nanogel can be regulated and controlled by changing the molar ratio of the cross-linking agent to the monomer, and the hydrated particle size of the nanogel can be regulated and controlled by the using amount of the surfactant. The series of nano gels can interact with anti-tumor chemotherapy drugs in an electrostatic adsorption mode to realize drug carrying, and series of nano drugs with different hardness can be obtained. Experiments prove that the series of nano-drugs can be targeted to tumor parts, wherein the soft nano-drugs can obviously overcome the physical barrier formed by the tumor extracellular matrix, realize higher tumor enrichment and deeper deep penetration, and further obtain better anti-tumor curative effect.
Description
Technical Field
The invention belongs to the technical field of nano-drug preparations, and particularly relates to a nano-gel, a nano-drug based on the nano-gel, and a preparation method and application of the nano-gel.
Background
The nano-drug enhances the delivery efficiency of the small-molecule chemotherapy drug and the application of tumor therapy by enhancing the permeation and retention effects for decades of development history, however, the nano-drug still faces the obstruction of abnormal tumor mechanics microenvironment after reaching the tumor site. Abnormal tumor mechanics microenvironment is a common feature of solid tumors such as breast cancer, liver cancer, pancreatic cancer and the like, and is represented by tortuous and irregular tumor vessels, dense tumor extracellular matrix, high interstitial fluid pressure and high tumor solid stress. The main reason for this mechanical microenvironment is the rapid proliferation of tumor cells. The dense tumor extracellular matrix seriously hinders the ability of the nano-drug to penetrate through tumor blood vessels and penetrate into the interior of tumors to interact with tumor cells, and limits the anti-tumor efficacy of the nano-drug. Therefore, overcoming the physical barrier inside the tumor is the key to enhance the anti-tumor efficacy of the nano-drug.
Although the physicochemical properties of the nano-drugs such as particle size, morphology, charge, surface modification, etc. have been gradually optimized to enhance the antitumor efficacy, these optimizations are not sufficient to enhance the deep penetration ability of the nano-drugs. The mechanical properties of nano-drugs, especially hardness, have been shown to be closely linked to plasma half-life, tumor enrichment, and cellular uptake. Some researches believe that the soft nano-drug has stronger deformation capability, can easily penetrate through gaps with the diameter smaller than the self particle size through extrusion, and can more easily penetrate through the tumor vessel wall and gaps of tumor extracellular matrix to realize deep tumor penetration. However, other studies (such as adv. Mater.,2015,27,1402-1407, acs nano,2015,9,9912-9921, acs nano,2019,13, 7676-7689) suggest that the hard nano-drug is more beneficial to be taken up by tumor cells to exert the effect of killing the tumor cells, so that the development of the nano-drug which can realize the enrichment of the deep tumor part in the tumor part and can be taken up by the tumor cells efficiently and is designed on the basis of the mechanical property of the nano-carrier has great application potential in the aspect of enhancing the anti-tumor curative effect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a nanogel, a nano-drug based on the nanogel, a preparation method and an application thereof, aiming at overcoming the physical barrier formed by tumor extracellular matrix and solving the technical problems of low tumor enrichment, weak tumor deep penetration capability, limited anti-tumor curative effect and the like of the traditional carried chemotherapeutic drug.
In order to achieve the purpose, the invention provides a nanogel with adjustable hardness, which is obtained by initiating a monomer to carry out polymerization reaction in a water phase by an initiator in the presence of a cross-linking agent and a surfactant; the hardness of the nanogel can be regulated by changing the molar ratio of the cross-linking agent to the monomer, and the average hydrated particle size of the nanogel can be regulated by the using amount of the surfactant.
Preferably, the monomer comprises one or more of a temperature-responsive monomer, a pH-responsive monomer, and a reduction-responsive monomer; the temperature response type monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid.
Preferably, the crosslinking agent is one or more of N, N ' -bis (acryloyl) cystamine, N ' -methylene bisacrylamide, and N, N ' -vinyl bisacrylamide; the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide.
Preferably, the monomers include pH-responsive monomers and temperature-responsive monomers; the feeding molar ratio of the pH response type monomer to the temperature response type monomer is (3-8) to 100; the feeding molar ratio of the cross-linking agent to the temperature response type monomer is (1-20) to 100; the mass ratio of the initiator to the temperature response type monomer is (5-15): 550.
Preferably, the surfactant is one or more of sodium lauryl sulfate, sodium lauryl sulfate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (20-35): 550.
Preferably, the average hydrated particle size of the nanogel in PBS buffer solution at 37 ℃ is 150-300 nm, more preferably 180-240 nm, and the surface charge distribution is-13.5 mV to-23.8 mV.
Preferably, the nanogel has a Young's modulus of 79.0 to 439.2kPa.
Preferably, the reaction temperature of the polymerization reaction is 70-85 ℃, and the reaction time is 4-8 h.
According to another aspect of the present invention, a preparation method of the nanogel is provided, wherein a monomer is obtained by initiating a polymerization reaction of the monomer in an aqueous phase through an initiator in the presence of a cross-linking agent and a surfactant, the hardness of the nanogel is regulated and controlled by regulating a molar ratio of the cross-linking agent to the monomer, and the average hydrated particle size of the nanogel is regulated and controlled by regulating and controlling the amount of the surfactant.
According to another aspect of the invention, the nano-drug based on the nanogel comprises the nanogel and a water-soluble small-molecule anti-tumor chemotherapeutic drug loaded on the nanogel through electrostatic adsorption.
Preferably, the water-soluble small molecule antitumor drug is selected from doxorubicin hydrochloride, metformin and indocyanine green; the drug loading rate of the water-soluble micromolecule antitumor drug in the nano-drug is 2-10%.
Preferably, the average hydrated particle size of the nano-drug in a PBS buffer solution at 37 ℃ is 150-300 nm, and more preferably 180-240 nm.
Preferably, when the molar ratio of the cross-linking agent to the temperature-responsive monomer is controlled to be (1-6): 100, more preferably (1-3): 100, the young modulus of the nanogel is 20-150 kPa, more preferably 50-100 kPa, and the nano-drug corresponding to the nanogel carrier in the hardness range has better tumor deep penetration, cell uptake and anti-tumor effect.
According to another aspect of the invention, the application of the nano-drug in preparing the drug for treating the tumor is provided.
In general, the above technical solution conceived by the present invention has the following advantages compared to the prior art
Has the beneficial effects that:
(1) The invention provides a nanogel with adjustable hardness (Young modulus), and a series of nanogels with different hardnesses can be obtained by regulating and controlling the feeding molar ratio of a cross-linking agent and a monomer. The nanogel is used as a nano carrier with a unique network structure to load antitumor chemotherapeutic drugs to prepare the nano drug. Experiments show that the mechanical property of the nanogel has great influence on the anti-tumor effect of the nano-medicament. Experiments show that the soft nanogel has higher enrichment amount at a tumor part and lower enrichment amount at a liver part; the hard nanogel is lower in enrichment amount at a tumor part and higher in enrichment amount at a liver part. The series of nano-gels can interact with anti-tumor chemotherapy drugs in an electrostatic adsorption manner to realize drug carrying, and series of nano-drugs with different hardness can be obtained. The nano-gel controlled within a proper hardness range is used as a carrier to prepare the nano-drug which has better tumor deep penetration, cell uptake and anti-tumor effects.
(2) The nanogel is obtained by initiating the polymerization reaction of monomers in an aqueous phase by an initiator in the presence of a cross-linking agent and a surfactant by using a temperature-responsive monomer N-isopropyl methacrylamide and a pH-responsive monomer methacrylic acid. The temperature response type monomer N-isopropyl methacrylamide endows the nanogel with excellent hydrophilicity, stability and biocompatibility, and has higher volume phase transition temperature compared with the existing temperature response type monomer, so that the nanogel has enough deformation space under physiological conditions; the pH response monomer methacrylic acid endows the nanogel with negative charges, enhances the stability and endows the nanogel with the capability of carrying a positive water-soluble micromolecule drug, and the carboxyl in the structure can enhance the hydrophilicity of the nanogel after phase change; the crosslinking agent N, N ' -bis (acryloyl) cystamine endows a nanogel network structure, the deformation capacity, namely the hardness, of the nanogel is regulated and controlled by changing the molar ratio of the N, N ' -bis (acryloyl) cystamine to the N-isopropyl methacrylamide, and experiments show that the higher the molar ratio of the N, N ' -bis (acryloyl) cystamine to the N-isopropyl methacrylamide is, the higher the hardness of the prepared nanogel is; the surfactant sodium dodecyl sulfate regulates and controls the particle size of the nanogel, so that the nanogel meets the use requirement, and the more the materials are fed, the smaller the particle size of the nanogel is.
(3) The invention provides a preparation method of nanogel with different hardness series. The temperature response monomer N-isopropyl methacrylamide and the pH response monomer methacrylic acid are crosslinked by N, N' -bi (acryloyl) cystamine in ultrapure water, and series nanogels with different hardness are obtained in an emulsion polymerization mode. Wherein potassium persulfate initiates polymerization reaction, and the hydrated grain size of the nanogel with different hardness is regulated and controlled by sodium dodecyl sulfate. Removing unreacted monomers and other impurities from the nanogel obtained by the reaction after ultrafiltration, washing the nanogel by ultrapure water, and concentrating the nanogel for storage at the temperature of 4 ℃. When the nanogel with higher hardness is prepared, in view of the small solubility of the cross-linking agent in a water phase, the cross-linking agent is firstly dissolved in a small amount of ethanol solvent, and then is added into a reaction system for emulsion polymerization reaction, so that the nanogel with higher hardness is prepared, and the preparation problem of the nanogel with high hardness is solved.
(4) The invention provides a series of nano-drugs with different hardness carrying antitumor chemotherapeutic drug doxorubicin hydrochloride. After the micromolecular chemotherapeutic drug doxorubicin hydrochloride is carried by the nanogel, the stability is obviously improved, and the pharmacokinetic behavior is obviously improved. The nano-drug can be enriched at a tumor part by enhancing the permeation and retention effects, wherein compared with the hard nano-gel, the soft nano-gel with smaller Young modulus can overcome the physical barrier formed by extracellular matrix, can penetrate blood vessels more easily and can permeate deep parts of tumors, and has higher cellular uptake efficiency, thereby realizing higher tumor enrichment amount and more excellent anti-tumor effect.
(5) The invention provides a preparation method of series of nano-drugs with different hardness carrying antitumor chemotherapeutic drug doxorubicin hydrochloride. The antitumor chemotherapeutic drug doxorubicin hydrochloride can be carried on the nanogel in an aqueous solution in an electrostatic adsorption mode by stirring, free doxorubicin hydrochloride is removed by ultrafiltration, and the obtained product is concentrated to a proper concentration for storage and application to obtain a series of nano-drugs with different hardness.
(6) The invention provides a treatment strategy for improving nano-drug tumor delivery and deep penetration by using nano-drug hardness. Although the nano-drug can be enriched at the tumor site by enhancing the penetration and retention effects, the physical barrier formed by the tumor extracellular matrix hinders the process of penetrating the nano-drug through the blood vessel and into the deep part of the tumor, which severely limits the anti-tumor efficacy of the nano-drug. The mechanical property of the nano-drug is changed, the hardness of the nano-drug is reduced, the nano-drug can deform to penetrate through the gap of the tumor extracellular matrix, and the barrier of the tumor extracellular matrix to the deep penetration of the nano-drug is obviously overcome, so that higher tumor enrichment is realized; in addition, reducing the hardness of the nano-drug can promote the uptake of tumor cells and further enhance the anti-tumor efficacy.
Drawings
FIG. 1 shows the particle size distribution and surface charge of nanogels of different hardness series prepared in example 1.
FIG. 2 is a transmission electron microscope image of nanogels of different hardness series prepared in example 1.
FIG. 3 is an atomic force microscope image and Young's modulus of nanogels of different hardness series prepared in example 1.
Fig. 4 is a graph of the triple responsiveness of nanogels of different hardness series prepared in example 1.
FIG. 5 shows the property comparison and stability of the nanogel loaded doxorubicin hydrochloride prepared in example 1 in different hardness series.
Fig. 6 is a graph showing the cytotoxicity of nanogels of different hardness series prepared in example 1.
Fig. 7 is a graph of relative cellular uptake for different hardness series of nanogels prepared in example 1.
FIG. 8 is the cytotoxicity of the nano-drugs of different hardness series prepared in example 3.
FIG. 9 shows the permeation capability of rhodamine B labeled nanogels of different hardness series prepared in example 2.
Fig. 10 is the tumor enrichment of different hardness series of nanogels labeled with indocyanine green prepared in example 4.
Fig. 11 is a tissue distribution of different hardness series of nanogels of indocyanine green label prepared in example 4.
FIG. 12 shows the tumor distribution of rhodamine B labeled nanogels of different hardness series prepared in example 2.
FIG. 13 shows the anti-tumor effect of the nano-drugs of different hardness series prepared in example 3.
FIG. 14 shows the tumor inhibition rates of the nano-drugs of different hardness series prepared in example 3.
FIG. 15 shows tumor necrosis and proliferation after treatment with the nano-drugs of different hardness series prepared in example 3.
Fig. 16 shows the deep tumor penetration of the nano-drugs of different hardness series prepared in example 3.
FIG. 17 is a section of organ tissue after treatment with a series of different hardness nano-drugs prepared in example 3.
FIG. 18 shows the blood biochemistry and blood routine indexes after treatment of nano-drugs of different hardness series prepared in example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The nanogel is obtained by initiating a monomer to carry out random copolymerization (emulsion polymerization) reaction in a water phase by an initiator under the condition that a temperature response type monomer and a pH response type monomer exist in a cross-linking agent and a surfactant, wherein experiments show that the hardness of the nanogel can be regulated and controlled by changing the molar ratio of the cross-linking agent to the monomer, and the larger the molar ratio of the cross-linking agent to the monomer is, the higher the hardness of the obtained nanogel is, and correspondingly, the weaker the deformation capability of the nanogel is. The hydrated particle size of the nanogel can be regulated and controlled through the using amount of the surfactant, and the higher the using amount of the surfactant is, the smaller the hydrated particle size of the prepared nanogel is.
In some embodiments, the monomers include one or more of temperature-responsive monomers, pH-responsive monomers, and reduction-responsive monomers; the temperature response type monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid. The cross-linking agent is one or more of N, N ' -bis (acryloyl) cystamine, N ' -methylene bisacrylamide and N, N ' -vinyl bisacrylamide; the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide. The feeding molar ratio of the pH response type monomer to the temperature response type monomer is (3-8) to 100; the charging molar ratio of the cross-linking agent to the temperature-responsive monomer N-isopropyl methacrylamide is (1-20): 100, and the higher the charging molar ratio is, the higher the Young modulus of the prepared nanogel is, and the higher the hardness is. The mass ratio of the initiator to the temperature-responsive monomer is (5-15): 550. The surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (20-35): 550. The reaction temperature of the polymerization reaction is 70-85 ℃, and the reaction time is 4-8 h.
In a preferred embodiment, the pH responsive monomer is methacrylic acid and the temperature responsive monomer is N-isopropyl methacrylamide. In some embodiments of the present invention, a temperature-responsive monomer is used as a main monomer, a pH-responsive monomer is used as a secondary monomer, and the molar ratio of a cross-linking agent to the temperature-responsive monomer is adjusted to control the hardness of the prepared nanogel. The temperature response type monomer is a main body structure which forms the nanogel, and N-isopropyl methacrylamide with higher phase transition temperature is selected to ensure sufficient deformation space of the nanogel-based nano-drug at the body temperature of an organism; the pH response monomer is used for enhancing the hydrophilicity of the nanogel after phase transition and providing negative charges for drug loading.
The average hydrated particle size of the nanogel prepared by the invention in a PBS buffer solution at 37 ℃ is 150-300 nm, preferably 180-240 nm, and the surface charge distribution is-13.5 mV to-23.8 mV. The Young modulus of the nanogel is adjustable within the range of 79.0-439.2 kPa.
The invention provides a preparation method of nanogel with adjustable hardness, which is obtained by initiating a monomer to carry out polymerization reaction in a water phase by an initiator in the presence of a cross-linking agent and a surfactant; the hardness of the nanogel is regulated and controlled by regulating the molar ratio of the cross-linking agent to the monomer, and the hydrated particle size of the nanogel is regulated and controlled by regulating and controlling the using amount of the surfactant.
In some embodiments, the method of preparing a nanogel according to the invention specifically comprises the following steps:
(1) Preparation of nanogel: dissolving the weighed N-isopropyl methacrylamide and sodium dodecyl sulfate in water, dissolving the weighed N, N '-bis (acryloyl) cystamine in ultrapure water, if the cross-linking agent N, N' -bis (acryloyl) cystamine is in a large dosage and cannot be dissolved in the ultrapure water, dissolving the cross-linking agent N, N '-bis (acryloyl) cystamine in a small amount of ethanol, then adding the cross-linking agent N, N' -bis (acryloyl) cystamine into the aqueous solution, and then adding methacrylic acid into the aqueous solution. The solution is subjected to 3 rounds of air exhaust and argon filling to remove oxygen (or oxygen and ethanol) in the system. Heating the deoxygenated solution to 80 ℃, dissolving the weighed potassium persulfate in a small amount of water, and adding the solution into the system through a syringe to initiate polymerization reaction.
(2) And (3) purifying the nanogel: and (3) after the nano gel aqueous solution obtained by the preparation is cooled to room temperature, adding the nano gel aqueous solution into an ultrafiltration tube. Centrifuging to remove unreacted monomers and other impurities, concentrating the nanogel, adding ultrapure water after concentration, centrifuging and washing for 3 times, detecting the solid content of the washed and concentrated nanogel, and storing at 4 ℃.
The invention also provides a nano-drug based on the nano-gel, which comprises the nano-gel and a water-soluble small-molecule anti-tumor chemotherapeutic drug loaded on the nano-gel through electrostatic adsorption. In some embodiments, the water-soluble small molecule antineoplastic drug is selected from the group consisting of doxorubicin hydrochloride, metformin, and indocyanine green. The drug loading of the water-soluble micromolecule anti-tumor chemotherapeutic drug in the nano-drug is 2-10%. The water-soluble micromolecule anti-tumor chemotherapeutic drug has positive charges, and is carried on the nanogel with the negative charges through electrostatic adsorption. The average hydrated particle size of the nano-drug in PBS buffer solution at 37 ℃ is 150-300 nm, preferably 180-240 nm.
In some embodiments, the method for preparing the nano-drug comprises the following steps: mixing and stirring the aqueous solution of the nanogel and the aqueous solution of the water-soluble micromolecule anti-tumor chemotherapeutic for 36-72 hours, and removing the water-soluble micromolecule anti-tumor chemotherapeutic which is not carried by the nanogel by ultrafiltration to obtain the nano-drug.
In a preferred embodiment, when the feeding molar ratio of the cross-linking agent to the temperature-responsive monomer N-isopropyl methacrylamide in the nano-drug is (1-6): 100, more preferably (1-3): 100, the hardness of the corresponding nano-gel is smaller, and the nano-drug obtained by loading the water-soluble small-molecule anti-tumor chemotherapeutic drug by taking the nano-gel with the hardness range as a carrier through electrostatic adsorption has better tumor deep penetration, cell uptake and anti-tumor effects. Correspondingly, the young's modulus of the nanogel is between 20 and 150kPa, and more preferably between 50 and 100kPa.
The cross-linking agent preferably adopts a reduction-responsive cross-linking agent, and the cross-linking agent with reduction responsiveness can endow the prepared nanogel and the nano-drug with reduction responsiveness; or a monomer with a reduction response type can be adopted, so that the prepared nanogel has the reduction response. The nano-drug with reduction responsiveness is beneficial to biodegradation and metabolism of the nanogel.
The nano-gel-based nano-drug provided by the invention can be used for preparing a drug for preventing and/or treating tumors. The nano-drug has been used for anti-tumor research for decades. The physicochemical properties of the nano-drug, such as particle size, charge, morphology, composition, surface modification, etc., are continually optimized to enhance the anti-tumor efficacy of the nano-drug and reduce toxic side effects. However, most of the nano-drugs still face a complex mechanical microenvironment of the tumor after reaching the tumor part, especially the block of a compact tumor extracellular matrix, so that the nano-drugs can only penetrate the wall of the tumor vessel for a limited distance, which severely limits the anti-tumor efficacy of the nano-drugs. The invention selects proper monomers, and regulates the hardness of the nanogel by regulating the molar ratio of the cross-linking agent to the specific monomers; the particle size of the nanogel is regulated and controlled by adjusting the using amount of the surfactant, so that a series of nanogels with different hardness can be prepared, and the hardness requirement of the nanogel under different application scenes can be met according to the requirement.
In the experimental process, different temperature response type monomers are compared to discover that when N-isopropyl propionamide is used as a monomer, the volume phase change temperature of the prepared nano gel is lower than the body temperature or even room temperature, so that the volume phase change of the nano gel is already generated before entering the organism, and the purposes of deep penetration and cell uptake can be achieved by utilizing the deformation after the phase change; therefore, the monomer is replaced by N-isopropyl methacrylamide, and experiments show that the volume phase transition temperature of correspondingly prepared nanogels with different hardness is improved to a certain extent, so that a larger deformation space is provided for promoting deep penetration and cell uptake after in vivo phase transition. In addition, the temperature response type monomer polymer has phase change along with the temperature rise, the hydrophilicity is reduced, the hydrophobicity is enhanced, and the stability of the temperature response type monomer polymer in an organism is not facilitated.
The carboxyl in the methacrylic acid monomer contained in the nanogel enables the nanogel to be electronegative, and a water-soluble micromolecule anti-tumor drug with positive charges can be loaded through electrostatic adsorption, so that the nano-drug with adjustable hardness is obtained. Experiments show that the nano-drug with the crosslinking degree within the range of 2% -15% prepared in some embodiments of the invention shows stronger tumor deep penetration capability and highest cell uptake when the crosslinking degree is smaller, namely the nano-drug with the hardness is smaller, and the anti-tumor curative effect of the nano-drug corresponding to the relatively soft nano-gel is further improved.
The invention endows the nanogel with excellent deformation capability by utilizing the unique network structure of the nanogel, and changes the mechanical property of the nanogel by changing the crosslinking density of the nanogel. Meanwhile, the abundant surface charges enable the nanogel to carry water-soluble chemotherapeutic drugs in an electrostatic adsorption mode. Therefore, the nano drug delivery system designed based on the nanogels with different hardnesses is the best choice for enhancing the anti-tumor efficacy of the nano drugs through mechanical properties.
According to the invention, the water-soluble micromolecule chemotherapy drug doxorubicin hydrochloride is carried on the nanogels with different hardnesses in an electrostatic adsorption manner, the physicochemical properties, especially the hardness, of the nanogels are not obviously influenced, and the stability of the doxorubicin hydrochloride under saline solution and physiological conditions is obviously improved. Meanwhile, under the load of the nanogel, more doxorubicin hydrochloride is delivered to the tumor region, wherein the soft nanogel further delivers the doxorubicin hydrochloride to the deep part of the tumor and is taken up by tumor cells, so that the anti-tumor curative effect is further enhanced.
The raw material components used in the invention can be obtained commercially, and the reagents used in the embodiment of the invention are all chemically pure.
When the nanogel is prepared in the following examples, the feeding molar ratio of the cross-linking agent to the temperature-responsive monomer N-isopropyl methacrylamide is defined as X:100, the cross-linking degree of the corresponding nanogel is X%, and the prepared nanogel is represented as X% NGs.
The following are examples:
example 1
A nanogel of varying hardness comprising 2% NGs, 5% NGs, 10% NGs, 15% NGs prepared by a process comprising the steps of:
(1) Preparation: 550mg of N-isopropyl methacrylamide was weighed, 35/30/25/20mg (corresponding to respective degrees of crosslinking of 2%/5%/10%/15%) of sodium lauryl sulfate were weighed, 80mL of ultrapure water was added, and ultrasonic dissolution was carried out. Correspondingly, 22.5/56.3/112.6/168.9mg (corresponding to a degree of crosslinking of 2%/5%/10%/15%, respectively) of N, N' -bis (acryloyl) cystamine were weighed out separately, 0.5/1/2/3mL (corresponding to a degree of crosslinking of 2%/5%/10%/15%, respectively) of ethanol was added separately, dissolved by sonication, and then added to the above solution. 3.65mL of methacrylic acid was dissolved in 6.35mL of ultrapure water, and 50. Mu.L of the solution was added. Pumping the obtained solution for 10min, introducing argon, circulating for three times, and fully removing oxygen in the solution. The resulting solution was heated to 80 ℃ for 10min. 10mg of potassium persulfate was weighed, added to 0.5mL of ultrapure water, dissolved by ultrasound, and then added to the above solution by means of a syringe to initiate polymerization for 6 hours.
(2) And (3) purification: after the reaction is finished, the solution obtained in the step (1) is returned to room temperature, the solution is transferred to an ultrafiltration tube, the cut-off value is 10kDa, unreacted monomers and other impurities are removed by centrifugation at 2000rpm, excessive water is removed, after concentration, ultrapure water is repeatedly added for cleaning for 3 times, and finally, the solution is concentrated to 8mL.
(3) Quantification: and (3) taking 300 mu L of the concentrated solution obtained in the step (2), drying and weighing, and calculating the solid content in the concentrated solution.
(4) And (3) storage: based on the calculation results obtained in the above step (3), the concentrated solution was diluted to 20mg/mL to obtain 2% of NGs, 5% of NGs, 10% of NGs, and 15% of NGs, respectively, and the nanogels were stored at 4 ℃.
Example 2
A series of nano-gels of different hardness marked by rhodamine B, rhB @ 3% NGs, including RhB @2% NGs, rhB @5% NGs, rhB @10% NGs, rhB @15% NGs, the preparation method comprises the following steps:
(1) Preparation of RhB-HEMA: 0.5g of rhodamine B,75mg of 4-dimethylaminopyridine and 2.6g of N, N' -dicyclohexylcarbodiimide are weighed, 52.5mL of anhydrous dichloromethane is added, ultrasonic dissolution is carried out, the obtained solution is vacuumized to remove dissolved oxygen, stirring is carried out for 30min under the protection of argon, and then 1.55mL of hydroxyethyl methacrylate is added. The mixed solution is stirred for 25 hours at the temperature of 20 ℃ under the protection of argon. Purifying the reaction product by a silica gel column chromatography, wherein the eluent is a mixed solution of dichloromethane and methanol with the ratio of 90.
(2) Preparation of rhodamine B labeled series of nanogels RhB @ NGs with different hardness: 550mg of N-isopropyl methacrylamide was weighed, 35/30/25/20mg (corresponding to a degree of crosslinking of 2%/5%/10%/15%) of sodium lauryl sulfate were weighed, 80mL of ultrapure water was added, and the mixture was dissolved by sonication. 22.5/56.3/112.6/168.9mg (corresponding to a degree of crosslinking of 2%/5%/10%/15%) of N, N' -bis (acryloyl) cystamine was weighed, added with 0.5/1/2/3mL (corresponding to a degree of crosslinking of 2%/5%/10%/15%) of ethanol, respectively, dissolved by sonication, and then added to the above solution. 255 μ g of RhB-HEMA was added to the above solution, and dissolved with stirring. 3.65mL of methacrylic acid was dissolved in 6.35mL of ultrapure water, and 50. Mu.L of the solution was added. The obtained solution is pumped for 10min, then argon is introduced, and the circulation is carried out for three times, so as to fully remove the oxygen in the solution. The resulting solution was heated to 80 ℃ for 10min. 10mg of potassium persulfate was weighed, added to 0.5mL of ultrapure water, dissolved by ultrasound, and then added to the above solution by means of a syringe to initiate polymerization for 6 hours.
(3) And (3) purification: after the reaction is finished, the solution obtained in the step (2) is returned to room temperature, the solution is transferred to an ultrafiltration tube, the cut-off value is 10kDa, unreacted monomers and other impurities are removed by centrifugation at 2000rpm, meanwhile, redundant water is removed, after the concentration, ultrapure water is repeatedly added for cleaning for 3 times, and finally, the concentration is carried out to 8mL.
(4) Quantification: and (4) taking 300 mu L of the concentrated solution obtained in the step (3), drying and weighing, and calculating the solid content in the concentrated solution.
(5) And (3) storage: according to the calculation result obtained in the step (4), the concentrated solution is diluted to 20mg/mL, so as to obtain RhB @2% NGs, rhB @5% NGs, rhB @10% NGs and RhB @15% NGs respectively, and the concentrated solution is stored at 4 ℃.
Example 3
A series of nanometer medicines with different hardness comprises DOX @2% NGs, DOX @5% NGs, DOX @10% NGs and DOX @15% NGs, and the preparation method comprises the following steps:
(1) Carrying out medicine loading: after 10mg of doxorubicin hydrochloride was weighed, 5mL of ultrapure water was added thereto, and the mixture was ultrasonically dissolved, the solution was added to 5mL of the nanogel solution obtained in example 1 (2% NGs, 5% NGs, 10% NGs, 15% NGs), and the mixture was stirred for 48 hours.
(2) And (3) purification: and (2) transferring the solutions obtained in the step (1) into ultrafiltration tubes respectively, wherein the cut-off value is 10kDa, centrifuging at 2000rpm to remove the non-loaded doxorubicin hydrochloride, and simultaneously concentrating the nano-drugs.
(3) Quantification: and (3) adding 50 mu L of the nano-drug solution obtained in the step (2) into 2.95mL of dimethyl sulfoxide, uniformly mixing, detecting by using an ultraviolet spectrophotometer, wherein the detection wavelength is 483nm, and calculating the concentration of the doxorubicin hydrochloride in the nano-drug solution obtained in the step (2) according to a standard curve of the doxorubicin hydrochloride in the dimethyl sulfoxide under the wavelength of 483 nm.
(4) And (3) storage: according to the calculation result obtained in the step (3), the nano-drug concentrated solution is diluted until the doxorubicin hydrochloride concentration is 1mg/mL, so that DOX @2% NGs, DOX @5% NGs, DOX @10% NGs and DOX @15% NGs are respectively obtained, and the obtained product is stored at 4 ℃.
Example 4
A series of nano-drugs with different hardness marked by indocyanine green comprises ICG @2% NGs, ICG @5% NGs, ICG @10% NGs and ICG @15% NGs, and the preparation method comprises the following steps:
(1) Carrying out medicine loading: 5mg of indocyanine green was weighed, 5mL of ultrapure water was added, ultrasonic dissolution was performed, and then the resulting solution was added to 5mL of the nano-drug solution obtained in example 3 (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs, DOX @15% NGs), followed by mixing and stirring for 48 hours.
(2) And (3) purification: transferring the solution obtained in the step (1) into an ultrafiltration tube, wherein the cut-off value is 10kDa, centrifuging at 2000rpm to remove the non-loaded indocyanine green, and simultaneously concentrating the nano-drug labeled by the indocyanine green.
(3) Quantification: and (3) adding 50 mu L of the nano-drug solution obtained in the step (2) into 2.95mL of dimethyl sulfoxide, uniformly mixing, detecting by using an ultraviolet spectrophotometer, wherein the detection wavelength is 783nm, and calculating the concentration of the indocyanine green in the nano-drug solution labeled by the indocyanine green obtained in the step (2) according to a standard curve of the indocyanine green in the dimethyl sulfoxide under the wavelength of 783 nm.
(4) And (3) storage: according to the calculation result obtained in the step (3), the nano-drug concentrated solution is diluted to the indocyanine green concentration of 1.5mg/mL, so as to obtain ICG @2% NGs, ICG @5% NGs, ICG @10% NGs and ICG @15% NGs respectively, and the solutions are stored at 4 ℃.
Example 5
Detection of hydrated particle size of serial nanogels with different hardness
Dispersing 10 μ L of the nanogel (2% NGs, 5% NGs, 10% NGs, 15% NGs) concentrate obtained in example 1 into 1mL of PBS buffer, and detecting by dynamic light scattering at 37 deg.C for an equilibration time of 15min.
Detection of zeta potential of nanogel with different hardness series
Dispersing 10 μ L of the nanogel (2% NGs, 5% NGs, 10% NGs, 15% NGs) concentrate obtained in example 1 into 1mL of ultrapure water, respectively, and detecting by dynamic light scattering method at 37 ℃ for an equilibration time of 15min.
Fig. 1, content a and content B are the particle size distribution and surface charge of nanogels with different hardness series prepared in example 1, respectively. It can be seen that the nanogels produced in this example, 2% of NGs, 5% of NGs, 10% of NGs, 15% of NGs, were uniformly distributed in PBS buffer at 37 ℃ and the average hydrated particle size was about 220nm. The surface charges are all negative and increase slightly with increasing degree of crosslinking.
Example 6
Detection of series nanometer gel shapes with different hardness
Dispersing the nanogel (2% NGs, 5% NGs, 10% NGs, 15% NGs) concentrate obtained in example 1 with ultrapure water to a concentration of 0.01mg/mL, adding 10. Mu.L of the dispersion dropwise to a carbon support membrane, drying naturally, adding 10. Mu.L of a 1% phosphotungstic acid aqueous solution dropwise to the carbon support membrane after drying, dyeing for 2min, sucking off the excess phosphotungstic acid solution along the edge of the carbon support membrane with filter paper after dyeing is completed, washing with 10. Mu.L of ultrapure water dropwise for 1min, sucking off the excess ultrapure water along the edge of the carbon support membrane with filter paper after washing is completed, drying naturally, and observing with a transmission electron microscope.
FIG. 2 is a transmission electron microscope image of nanogels of different hardness series prepared in example 1. It can be seen that the nanogels with different hardness series are all spherical with uniform particle size distribution.
Example 7
Detection of height and Young modulus of series of nanogels with different hardness
The cover glass is soaked in 1% polyethyleneimine water solution for 24h, the surface of the cover glass is modified with positive charges, the concentrated solution of nanogel (2%; ngs, 5%; ngs, 10%; ngs, 15%; ngs) obtained in example 1 is dispersed with ultrapure water to a concentration of 0.01mg/mL, 100. Mu.L of the above dispersion is dropped onto the positively modified cover glass, the dispersion is electrostatically adsorbed for 10min, the excess dispersion is sucked away, then 300. Mu.L of ultrapure water is dropped to wash away the unadsorbed nanogel, and then the detection environment is a liquid phase by using an atomic force microscope, wherein the image acquisition is a contact mode and the Young's modulus is detected as a tapping mode.
Fig. 3, item a and item B are atomic force microscope images and young's modulus, respectively, of nanogels of different hardness series prepared in example 1. It can be seen that the nanogels with different hardness series are all spherical with uniform particle size distribution, the nanogel with low crosslinking degree is easier to collapse and lower in height, and the nanogel with high crosslinking degree is not easy to collapse and higher in height. The hardness of the nanogel, i.e., the young's modulus, gradually increases with the degree of crosslinking.
Example 8
Series of nanogels of different hardnesses have triple responsivity
(1) Temperature responsiveness: dispersing 10. Mu.L of the nanogel (2% NGs, 5% NGs, 10% NGs, 15% NGs) concentrate obtained in example 1 into 1mL of ultrapure water, and detecting by dynamic light scattering at a detection temperature range of 25 to 55 ℃, at temperature intervals of 1 ℃ and an equilibration time of 1min.
(2) pH responsiveness: mu.L of each of the nanogel (2% NGs, 5% NGs, 10% NGs, 15% NGs) concentrates obtained in example 1 was dispersed in 1mL of ultrapure water, pH was adjusted to 3 to 9, and the concentration was measured by a dynamic light scattering method at a detection temperature of 25 ℃ for an equilibration time of 15min.
(3) Reduction responsiveness: dispersing 10. Mu.L of the nanogel (2% NGs, 5% NGs, 10% NGs, 15% NGs) concentrate obtained in example 1 into 1mL of ultrapure water containing or not containing 10mM glutathione, incubating at room temperature for 24 hours, adding 10. Mu.L of the above dispersion dropwise to a carbon support membrane, drying naturally, adding 10. Mu.L of a 1% phosphotungstic acid aqueous solution dropwise to the carbon support membrane after drying, dyeing for 2 minutes, sucking off the excess phosphotungstic acid solution along the edge of the carbon support membrane with a filter paper after dyeing is completed, washing for 1 minute with 10. Mu.L of ultrapure water dropwise, sucking off the excess ultrapure water along the edge of the carbon support membrane with a filter paper after washing is completed, drying naturally, and observing by a transmission electron microscope.
Fig. 4 is a graph of the triple responsiveness of nanogels of different hardness series prepared in example 1. The content A is temperature responsiveness, and it can be seen that the hydrated particle size of the nanogel is gradually reduced with the increase of temperature, and the shrinkage degree is reduced with the increase of the crosslinking degree; content B is pH responsiveness, and it can be seen that the hydrated particle size of the nanogel gradually increases with the increase of pH, and the expansion degree decreases with the increase of the crosslinking degree; content C is reduction responsiveness. It can be seen that the structure of the nanogel is significantly destroyed after 24h incubation with glutathione.
Example 9
Influence of carried adriamycin hydrochloride on properties of series of nanogels with different hardness
(1) Hydrated particle size: 10 μ L of the concentrated solution of the nano-drug (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs, DOX @15% NGs) obtained in example 3 was dispersed in 1mL of PBS buffer, and detected by dynamic light scattering at 37 ℃ for 15min.
(2) Temperature responsiveness: 10 μ L of the concentrated solution of the nano-drug (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs, DOX @15% NGs) obtained in example 3 was dispersed in 1mL of ultrapure water, and detected by dynamic light scattering at a temperature range of 25 to 55 ℃ at a temperature interval of 1 ℃ for an equilibrium time of 1min.
(3) The appearance is as follows: the nano coagulant drug (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs, DOX @15% NGs) concentrate obtained in example 3 was dispersed with ultrapure water to a concentration of 0.01mg/mL, 10. Mu.L of the above dispersion was dropped onto a carbon support membrane, and naturally dried, 10. Mu.L of 1% phosphotungstic acid aqueous solution was dropped onto the carbon support membrane after drying, and dyeing was carried out for 2min, and after dyeing, excess phosphotungstic acid solution was sucked off along the edge of the carbon support membrane with filter paper, and then 10. Mu.L of ultrapure water was dropped and washed for 1min, and after washing, excess ultrapure water was sucked off along the edge of the carbon support membrane with filter paper, and after natural drying, observation was carried out using a transmission electron microscope.
(4) Height and Young's modulus: soaking a cover glass in a 1% polyethyleneimine water solution for 24h, modifying positive charges on the surface of the cover glass, respectively dispersing the concentrated solution of the nano-drugs (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs and DOX @15% NGs) obtained in example 3 with ultrapure water to a concentration of 0.01mg/mL, dropwise adding 100 μ L of the dispersion solution onto a positively-modified cover glass, performing electrostatic adsorption for 10min, sucking away the redundant dispersion solution, dropwise adding 300 μ L of ultrapure water to wash away the unadsorbed nano-drugs, and detecting by using an atomic force microscope to obtain a liquid phase as a detection environment, wherein the image acquisition is in a contact mode, and the Young modulus is detected in a tapping mode.
(5) Stability: mu.L of the concentrated solution of the nano-drug (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs, DOX @15% NGs) obtained in example 3 was dispersed in 1mL of PBS buffer or 10% of FBS solution, and detected by dynamic light scattering at 37 deg.C for 15min and 24h.
FIG. 5 shows the property comparison and stability of the nanogel loaded doxorubicin hydrochloride prepared in example 1 in different hardness series. Wherein the content A is the particle size distribution of the nanogel before and after drug loading at the condition that the hydrated particle size is 37 ℃ and the particle size distribution of a PBS buffer solution, and the particle size distribution of the nanogel before and after drug loading is basically unchanged; the content B is the temperature responsiveness of the nanogel before and after drug loading, and the temperature responsiveness of the nanogel before and after drug loading is basically unchanged; the content C is a transmission electron microscope image of the nanogel before and after drug loading, and the particle size and the appearance of the nanogel before and after drug loading are basically unchanged; the content D is an atomic force microscope image of the nanogel before and after drug loading, and the particle size, the appearance and the height of the nanogel before and after drug loading can be basically unchanged; content E is the Young modulus of the nanogel before and after drug loading, and the hardness of the nanogel before and after drug loading is basically unchanged; content F is the stability of the drug-loaded nanogel in PBS buffer versus 10% fbs solution, and it can be seen that the hydrated particle size of the drug-loaded nanogel does not change substantially with time, and has excellent stability.
Example 10
Effect of hardness on nanogel cytotoxicity
4T1 cells were plated at 5X 10 3 The concentration of each well was added to a 96-well plate and cultured in an incubator for 12 hours to allow 4T1 cells to adhere. After the adherence, the upper medium was aspirated, 100. Mu.L of serum-free medium containing nanogels of different hardness obtained in example 1 (2% ngs, 5% ngs, 10% ngs, 15% ngs) was added, respectively, the nanogel concentration was 0 to 200. Mu.g/mL, incubated in an incubator for 24h, then the upper medium was aspirated, 100. Mu.L of fresh serum-free medium was added, 20. Mu.L of MTT solution with a concentration of 5mg/mL was added, and the incubation in the incubator was continued for 4h. After incubation, absorbing the upper layer culture medium, adding 150 mu L of dimethyl sulfoxide, incubating for 15min to dissolve the formazan crystal, detecting the light absorption value at the wavelength of 570nm by using an enzyme-labeling instrument, and calculating the cell survival rate according to the light absorption value.
FIG. 6 is a graph showing the cytotoxicity of nanogels of different hardness series prepared in example 1. It can be seen that nanogels of different hardness series are substantially non-toxic.
Example 11
Effect of Nanogel stiffness on cellular uptake
4T1 cells were plated at 5X 10 5 The concentration of each well was added to a 6-well plate and cultured in an incubator for 12 hours to allow 4T1 cells to adhere. After the adherence, the upper layer medium was aspirated, 3mL of serum-free medium containing different-hardness nanogels (RhB @2% NGs, rhB @5% NGs, rhB @10% NGs, rhB @15% NGs) labeled with rhodamine B obtained in example 2 was added, the nanogel concentration was 50. Mu.g/mL, the cells were incubated for 4 hours in an incubator, the upper layer medium was aspirated, washed 3 times with PBS buffer, the cells were digested with trypsin, collected by centrifugation, and the fluorescence intensity was measured with a flow cytometer, the detection channel was PE, and the relative cell uptake was calculated from the fluorescence intensity.
Fig. 7 is a graph of relative cellular uptake for different hardness series of nanogels prepared in example 1. It can be seen that the relative cellular uptake of the nanogels increases with decreasing hardness.
Example 12
Effect of hardness on Nanopharmaceutical cytotoxicity
4T1 cells were plated at 5X 10 3 The concentration of each well was added to a 96-well plate and cultured in an incubator for 12h to allow 4T1 cells to adhere. After adhering to the wall, the upper layer culture medium was aspirated, 100. Mu.L of serum-free medium containing the different hardness nano-drugs obtained in example 3 (DOX @2% NGs, DOX @5% NGs, DOX @10% NGs, DOX @15% NGs) was added, the concentration of the nano-drugs was 0-8. Mu.g/mL based on the doxorubicin hydrochloride concentration, incubation was carried out in the incubator for 24h, then the upper layer culture medium was aspirated, 100. Mu.L of fresh serum-free medium was added, 20. Mu.L of MTT solution with a concentration of 5mg/mL was added, and incubation was continued in the incubator for 4h. After hatching, absorb upper culture medium, add 150 mu L dimethyl sulfoxide, hatch 15min and wait that the formazan crystal dissolves, detect the light-absorption value at 570nm wavelength with the ELIASA, calculate the cell survival rate according to the light-absorption value.
FIG. 8 is a graph showing the cytotoxicity of various hardness series of nano-drugs prepared in example 3. It can be seen that the cell killing effect of the nano-drug increases with decreasing hardness.
Example 13
Effect of hardness on Nanogel deep penetration
(1) Permeability in Matrigel: adding 1mL of Matrigel to the bottom of a 1.5mL EP tube, and adding 0.5mL of the rhodamine B labeled nano gel aqueous solution with different hardness obtained in the example 2 above the Matrigel, wherein the selected nano gels are RhB @2% NGs and RhB @15% NGs, and the concentration is 100 mu g/mL. After 6h incubation, the upper nanogel was blotted off and the depth of penetration of rhb @2% NGs and rhb @15% NGs in Matrigel was observed.
(2) Penetration capacity in 3D tumor spheres: NIH/3T3 cells and 4T1 cells were mixed uniformly in a medium containing 0.24% methylcellulose at a ratio of 1 4 The culture medium of each cell is dripped to the inner side of the upper cover of the culture dish, then the upper cover is turned over to cover the lower dish so that the culture medium containing the cells is suspended on the upper cover, and the cells are cultured in an incubator for 72h till 3D tumor balls are formed. Transferring the 3D tumor spheres to a serum-free culture medium containing rhodamine B labeled nanogels with different hardnesses obtained in example 2, wherein the selected nanogels are RhB @2% NGs and RhB @15% NGs, the concentration is 100 mu g/mL, incubating for 4h in an incubator, transferring the 3D tumor spheres to PBS buffer solution for washing for 3 times, observing along the longitudinal axis of the 3D tumor spheres by using a laser confocal microscope, and selecting excitation light with the wavelength of 561nm.
FIG. 9 shows the permeation capability of rhodamine B labeled nanogels of different hardness series prepared in example 2. The content A is the permeability of rhodamine B-labeled nano-gels with different hardness in Matrigel, and the content B is the permeability of rhodamine B-labeled nano-gels with different hardness in 3D tumor spheres, so that the deep permeability of the soft nano-gel is higher.
Example 14
Influence of hardness on tumor enrichment of nano-drugs
(1) Female BALB/C mice were inoculated subcutaneously on the back near the right hind limb at 1X 10 6 4T1 cells with the volume of 100 mu L are used for constructing a mouse 4T1 breast cancer subcutaneous tumor model. When the tumor volume reaches200mm 3 Later, mice were randomized into two groups, including icg @2% NGs and icg @15% NGs. The indocyanine green labeled nano-drug obtained in example 4 was injected via tail vein at an injection dose of 4mg/kg, and then mice were anesthetized at 1h, 2h, 4h, 8h, 12h, and 24h, respectively, for in vivo imaging, and the fluorescence intensity was quantified. At the end of 24h imaging, mice were sacrificed and tumors and major organs (heart, liver, spleen, lung, kidney) were dissected off for in vitro imaging and fluorescence intensity was quantified.
(2) In order to ensure consistency of hardness to enrichment of nanogel and drug-loaded tumor, tumor enrichment of the rhodamine B labeled nanogel obtained in example 2 was further detected in this study. The rhodamine B is marked on the nanogel molecules in a covalent connection mode, detection difference caused by drug release is avoided, and therefore the fluorescence intensity of the rhodamine B represents the enrichment amount of the nanogel. Establishing a mouse 4T1 breast cancer subcutaneous tumor model according to the method in the step (1). When the tumor volume reaches 200mm 3 Thereafter, mice were randomized into two groups, including RhB @2% NGs and RhB @15% NGs. The rhodamine B labeled nanogel obtained in example 2 was injected via tail vein, the injection dose was the same rhodamine B fluorescence intensity, 4h after injection, mice were sacrificed and tumors were detached, in vitro imaging was performed, and the fluorescence intensity was quantified.
Fig. 10, contents a and B show the tumor enrichment of the nano-gels of indocyanine green labels prepared in example 4 with different hardness series, and it can be seen that the enrichment amount of the soft nano-gel at the tumor site is higher.
Fig. 11 is a tissue distribution of the series of nanogels with different hardness marked by indocyanine green prepared in example 4, and it can be seen that the soft nanogel is more enriched at a tumor part and less enriched at a liver part; the hard nanogel has lower enrichment at tumor parts and higher enrichment at liver parts.
FIG. 12 shows the tumor distribution of rhodamine B labeled nanogels of different hardness series prepared in example 2. It can be seen that the soft nanogel was also enriched more at the tumor site.
Example 15
Influence of hardness on deep penetration and anti-tumor effect of nano-drug
(1) Anti-tumor effect: female BALB/C mice were inoculated subcutaneously on the back near the right hind limb at 1X 10 6 4T1 cells with the volume of 100 mu L are used for constructing a mouse 4T1 breast cancer subcutaneous tumor model. When the tumor volume reaches 100mm 3 Then, the mice were randomly divided into five groups including Control, free DOX, DOX @2% NGs, DOX @10% NGs, DOX @15% NGs, and the number of the groups was recorded as day 0. Physiological saline, free doxorubicin hydrochloride and the nano-drugs (DOX @2% NGs, DOX @10% NGs, DOX @15% NGs) obtained in example 3 were injected through tail veins on the 1 st and 4 th days after grouping, respectively, and the injection dose was 4mg/kg of doxorubicin hydrochloride. From day 0 onwards, the long side (a) and the short side (b) of the mouse subcutaneous tumor were measured daily with a vernier caliper according to the calculation formula: tumor volume V = a × b 2 And/2, calculating the tumor volume. After the measurement on day 15, the mice were sacrificed, the tumors were peeled off, weighed and photographed, and the tumor inhibition rate was calculated from the tumor volume and mass.
(2) Apoptosis and proliferation: fixing the in vitro tumor obtained in the step (1) by using 4% paraformaldehyde, slicing, carrying out H & E, TUNEL and Ki67 staining, then quantifying the ratio of apoptosis and proliferation area by using software, and evaluating the necrosis, apoptosis and proliferation degree of tumor cells.
(3) Deep penetration: fixing the isolated tumor obtained in the step (1) by using 4% paraformaldehyde, slicing, marking tumor blood vessels by using a CD31 antibody marked by fluorescein isothiocyanate, detecting by using fluorescence, scanning the slices, wherein the detection excitation wavelength is 483nm and 495nm respectively, and measuring and counting the nearest distance between the adriamycin hydrochloride and the tumor blood vessels according to a scanning image.
(4) And (3) safety evaluation: in the antitumor effect evaluation process in step (1), the body weight of the mouse was weighed every day from day 0, and after the termination of the measurement on day 15, the main organs (heart, liver, spleen, lung, kidney) were peeled off, fixed with 4% paraformaldehyde and sectioned, and the tissue toxicity was evaluated by H & E staining. Meanwhile, blood is taken from the mouse for blood biochemical and blood routine detection, and the toxicity of the nano-drug is evaluated.
FIG. 13 shows the anti-tumor effect of the nano-drugs of different hardness series prepared in example 3. Wherein, the content A is the change relation of the tumor volume with time after the administration, the content B is the weight of the treated tumor, and the content C is a picture of the treated tumor, so that the treatment effect of the nano-drugs with different hardness series on the tumor is enhanced along with the reduction of the hardness of the nano-drugs; the content D is the change relation of the body weight of the mice along with time after the administration, and the body weight of the mice is slightly reduced and recovered to a normal level after the administration, and the nano-drugs with different hardness series have good safety.
FIG. 14 shows the tumor inhibition rates of the nano-drugs of different hardness series prepared in example 3. Wherein the content A is the tumor inhibition rate based on the tumor volume, and the content B is the tumor inhibition rate based on the tumor weight, and the tumor inhibition rate of the nano-drug can be seen to increase along with the reduction of the hardness. In the figure, G1 is Control, G2 is Free Dox, G3 is DOX @2% NGs, G4 is DOX @10% NGs, and G5 is DOX @15% NGs.
FIG. 15 shows tumor necrosis and proliferation after treatment with the nano-drugs of different hardness series prepared in example 3. Wherein, the content A is H & E, tunel and Ki67 staining images of the tumor slices, the content B is the quantification of the necrotic area proportion of the tumor slices, and the content C is the quantification of the proliferative area proportion of the tumor slices. It can be seen that the sparsity of tumor cells increases with the decrease of the hardness of the nano-drug, the proportion of the necrotic area of the tumor slices increases with the decrease of the hardness of the nano-drug, and the proportion of the proliferative area of the tumor slices decreases with the decrease of the hardness of the nano-drug.
Fig. 16 shows the deep tumor penetration of the nano-drugs of different hardness series prepared in example 3. The content A is a fluorescence image of the tumor section marked with the CD31 antibody, and the soft nano-drug can be seen to have stronger permeability from tumor blood vessels to deep parts of tumors; the content B is the distance between the nano-drug and the nearest tumor blood vessel, and the distance between the soft nano-drug and the tumor blood vessel is farther.
Fig. 17 is an H & E stained image of organ tissue slices treated with different hardness series of nano-drugs prepared in example 3, which shows that the nano-drugs treated with different hardness series have no effect on organs and good safety.
Fig. 18 shows the blood biochemistry and blood routine index after treatment with nano-drugs of different hardness series prepared in example 3, wherein the content a is glutamic-pyruvic transaminase, the content B is glutamic-oxaloacetic transaminase, the content C is glutamic-oxaloacetic transaminase/glutamic-pyruvic transaminase, the content D is creatine kinase, the content E is urea nitrogen, the content F is creatinine, the content G is white blood cells, the content H is red blood cells, and the content I is platelets. It can be seen that all indexes of the nano-drugs with different hardness series are within the normal value range after treatment, and the nano-drugs have good safety.
It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.
Claims (10)
1. The nanogel with adjustable hardness is characterized in that the nanogel is obtained by initiating a monomer to carry out polymerization reaction in an aqueous phase through an initiator in the presence of a cross-linking agent and a surfactant; the hardness of the nanogel can be regulated and controlled by changing the molar ratio of the cross-linking agent to the monomer, and the average hydrated particle size of the nanogel can be regulated and controlled by the using amount of the surfactant.
2. The nanogel of claim 1 wherein said monomers comprise one or more of temperature responsive monomers, pH responsive monomers, and reduction responsive monomers; the temperature response type monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propanesulfonic acid.
3. The nanogel of claim 1 wherein said cross-linking agent is one or more of N, N ' -bis (acryloyl) cystamine, N ' -methylenebisacrylamide, and N, N ' -vinylbisacrylamide; the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide.
4. The nanogel of claim 2 wherein said monomers comprise a pH-responsive monomer and a temperature-responsive monomer; the feeding molar ratio of the pH response type monomer to the temperature response type monomer is (3-8) to 100; the charging molar ratio of the cross-linking agent to the temperature response type monomer is (1-20) to 100; the mass ratio of the initiator to the temperature response type monomer is (5-15) to 550; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (20-35): 550.
5. The nanogel of claim 1 wherein the polymerization reaction is carried out at a temperature of 70 to 85 ℃ for a time of 4 to 8 hours.
6. The method for preparing nanogel according to any one of claims 1 to 5, wherein a monomer is polymerized in an aqueous phase by an initiator in the presence of a cross-linking agent and a surfactant, the hardness of the nanogel is controlled by controlling the molar ratio of the cross-linking agent to the monomer, and the average hydrated particle size of the nanogel is controlled by controlling the amount of the surfactant.
7. A nano-drug based on the nanogel according to any one of claims 1 to 5, comprising the nanogel according to any one of claims 1 to 5 and further comprising a water-soluble small-molecule anti-tumor chemotherapeutic drug loaded on the nanogel by electrostatic adsorption.
8. The nano-drug of claim 7, wherein the water-soluble small molecule antitumor drug is selected from the group consisting of doxorubicin hydrochloride, metformin, and indocyanine green; the drug loading rate of the water-soluble micromolecule antitumor drug in the nano-drug is 2-10%.
9. The NanoTab according to claim 7, wherein said nanogel has a Young's modulus of 20 to 150kPa, preferably 50 to 100kPa.
10. Use of the nano-drug as claimed in any one of claims 7 to 9 in the preparation of an anti-tumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211633371.4A CN115960305B (en) | 2022-12-19 | 2022-12-19 | Nanogel, nanogel-based nano medicine, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211633371.4A CN115960305B (en) | 2022-12-19 | 2022-12-19 | Nanogel, nanogel-based nano medicine, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115960305A true CN115960305A (en) | 2023-04-14 |
| CN115960305B CN115960305B (en) | 2024-04-02 |
Family
ID=87357155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211633371.4A Active CN115960305B (en) | 2022-12-19 | 2022-12-19 | Nanogel, nanogel-based nano medicine, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115960305B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150209447A1 (en) * | 2012-09-11 | 2015-07-30 | University Of Massachusetts | Crosslinked polymer nano-assemblies and uses thereof |
| US20160244546A1 (en) * | 2013-10-11 | 2016-08-25 | Agency For Science, Technology And Research | Direct synthesis of ph-responsive polymer particles and application in control release of hydrophobic therapeutic compounds |
| CN109054049A (en) * | 2018-06-19 | 2018-12-21 | 南华大学 | A kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket and its preparation method and application |
| CN111040075A (en) * | 2019-07-10 | 2020-04-21 | 中国科学院宁波材料技术与工程研究所 | Preparation method of nanogel, nanogel prepared by preparation method and application of nanogel |
-
2022
- 2022-12-19 CN CN202211633371.4A patent/CN115960305B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150209447A1 (en) * | 2012-09-11 | 2015-07-30 | University Of Massachusetts | Crosslinked polymer nano-assemblies and uses thereof |
| US20160244546A1 (en) * | 2013-10-11 | 2016-08-25 | Agency For Science, Technology And Research | Direct synthesis of ph-responsive polymer particles and application in control release of hydrophobic therapeutic compounds |
| CN109054049A (en) * | 2018-06-19 | 2018-12-21 | 南华大学 | A kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket and its preparation method and application |
| CN111040075A (en) * | 2019-07-10 | 2020-04-21 | 中国科学院宁波材料技术与工程研究所 | Preparation method of nanogel, nanogel prepared by preparation method and application of nanogel |
Non-Patent Citations (2)
| Title |
|---|
| YUAN ZHAN等: "Thermo/redox/pH-Triple Sensitive Poly(N-isopropylacrylamide-co-acrylic acid) Nanogels for Anticancer Drug Delivery", 《JOURNAL OF MATERIALS CHEMISTRY B》, vol. 20, no. 3, pages 4221 - 4230 * |
| 刘志景: "PNIPAM及其共聚纳米凝胶的制备与表征", 《中国优秀硕士学位论文全文数据库 工程科技I辑》, no. 5, pages 020 - 119 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115960305B (en) | 2024-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| An et al. | ROS-augmented and tumor-microenvironment responsive biodegradable nanoplatform for enhancing chemo-sonodynamic therapy | |
| Zhao et al. | Glucose-sensitive polypeptide micelles for self-regulated insulin release at physiological pH | |
| Liu et al. | ROS-sensitive biomimetic nanocarriers modulate tumor hypoxia for synergistic photodynamic chemotherapy | |
| Mu et al. | Unsaturated nitrogen-rich polymer poly (l-histidine) gated reversibly switchable mesoporous silica nanoparticles using “graft to” strategy for drug controlled release | |
| Ding et al. | Tumor targeted nanostructured lipid carrier co-delivering paclitaxel and indocyanine green for laser triggered synergetic therapy of cancer | |
| Sun et al. | Oral glucose-and pH-sensitive nanocarriers for simulating insulin release in vivo | |
| Zhou et al. | Preparation of biodegradable PEGylated pH/reduction dual-stimuli responsive nanohydrogels for controlled release of an anti-cancer drug | |
| CN107802840B (en) | Peptide-based dendrimer-modified fluorescent carbon dot-based tumor microenvironment response nanoparticle and preparation method thereof | |
| CN105816920A (en) | Preparation method of modified sodium alginate embolization microspheres | |
| CN103705460B (en) | Preparation method of enzymatic cross-linking medicine carrying nano micelle | |
| Li et al. | Magnetic iron oxide nanoparticles/10-hydroxy camptothecin co-loaded nanogel for enhanced photothermal-chemo therapy | |
| CN105968373A (en) | Zwitter-ion-contained multiple acid-sensitive anti-tumor drug-loading micelle and preparation method and application thereof | |
| US20220378712A1 (en) | Hybrid membrane camouflaged nanomedicine loaded with oxidative phosphorylation inhibitor and preparing method thereof | |
| CN104231155A (en) | Cholesterol modified amphiphilic pH response pennicuius copolymer as well as preparation and micelle of copolymer | |
| CN108409908A (en) | A kind of preparation method of molecularly imprinted polymer and products thereof and application | |
| Qu et al. | Stepwise dual pH and redox-responsive cross-linked polypeptide nanoparticles for enhanced cellular uptake and effective cancer therapy | |
| Ding et al. | Antifouling and pH-responsive poly (carboxybetaine)-based nanoparticles for tumor cell targeting | |
| Liu et al. | Microenvironment-activated nanoparticles for oxygen self-supplemented photodynamic cancer therapy | |
| Feng et al. | Hypoxia-responsive polymeric micelles for enhancing cancer treatment | |
| CN108948413A (en) | The method that wave polymerization prepares thymopeptide-5 molecule imprinting hydrogel | |
| CN105859990A (en) | Polymer with side chains containing lipoyl, preparation method of polymer, polymer vesica prepared from polymer and application of polymer vesica | |
| CN111249253A (en) | Preparation method of core-shell structure stimulus response type drug carrier and drug release method | |
| CN108126210A (en) | A kind of application of single targeting reduction response vesica Nano medication in treatment of brain tumor drug is prepared | |
| CN106075460B (en) | Novel ortho-ester cross-linking agent monomer and method for preparing acid-sensitive nano-drug carrier by using same | |
| Chen et al. | Precise fibrin decomposition and tumor mechanics modulation with hydroxyethyl starch-based smart nanomedicine for enhanced antitumor efficacy |
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 |