CN115920083A - Curcumin-loaded apoferritin nanocage and application - Google Patents
Curcumin-loaded apoferritin nanocage and application Download PDFInfo
- Publication number
- CN115920083A CN115920083A CN202310018804.3A CN202310018804A CN115920083A CN 115920083 A CN115920083 A CN 115920083A CN 202310018804 A CN202310018804 A CN 202310018804A CN 115920083 A CN115920083 A CN 115920083A
- Authority
- CN
- China
- Prior art keywords
- curcumin
- apoferritin
- nanocage
- loaded
- ferritin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 title claims abstract description 255
- 239000002091 nanocage Substances 0.000 title claims abstract description 175
- 229940109262 curcumin Drugs 0.000 title claims abstract description 125
- 235000012754 curcumin Nutrition 0.000 title claims abstract description 125
- 239000004148 curcumin Substances 0.000 title claims abstract description 125
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 title claims abstract description 125
- 102000000546 Apoferritins Human genes 0.000 title claims abstract description 99
- 108010002084 Apoferritins Proteins 0.000 title claims abstract description 99
- 208000009304 Acute Kidney Injury Diseases 0.000 claims abstract description 29
- 210000003734 kidney Anatomy 0.000 claims abstract description 26
- 230000000694 effects Effects 0.000 claims abstract description 25
- 208000028867 ischemia Diseases 0.000 claims abstract description 25
- 201000011040 acute kidney failure Diseases 0.000 claims abstract description 24
- 208000033626 Renal failure acute Diseases 0.000 claims abstract description 22
- 239000003814 drug Substances 0.000 claims abstract description 20
- 210000004926 tubular epithelial cell Anatomy 0.000 claims abstract description 8
- 230000008685 targeting Effects 0.000 claims abstract description 4
- 230000014759 maintenance of location Effects 0.000 claims abstract description 3
- 238000000465 moulding Methods 0.000 claims abstract 2
- 102000008857 Ferritin Human genes 0.000 claims description 80
- 108050000784 Ferritin Proteins 0.000 claims description 80
- 238000008416 Ferritin Methods 0.000 claims description 80
- 102000004169 proteins and genes Human genes 0.000 claims description 54
- 108090000623 proteins and genes Proteins 0.000 claims description 54
- 241000283073 Equus caballus Species 0.000 claims description 17
- 125000002091 cationic group Chemical group 0.000 claims description 15
- 239000000126 substance Substances 0.000 claims description 9
- 230000003393 splenic effect Effects 0.000 claims description 5
- 230000002440 hepatic effect Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000003389 potentiating effect Effects 0.000 claims 2
- 230000008095 long lasting therapeutic effect Effects 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 29
- 210000000952 spleen Anatomy 0.000 abstract description 19
- 210000004027 cell Anatomy 0.000 abstract description 18
- 210000004185 liver Anatomy 0.000 abstract description 18
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 8
- 210000002216 heart Anatomy 0.000 abstract description 4
- 210000004072 lung Anatomy 0.000 abstract description 4
- 230000000638 stimulation Effects 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 abstract description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 90
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 36
- 229910052742 iron Inorganic materials 0.000 description 35
- 239000000243 solution Substances 0.000 description 31
- 238000000502 dialysis Methods 0.000 description 28
- 239000002245 particle Substances 0.000 description 28
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 26
- 239000007974 sodium acetate buffer Substances 0.000 description 21
- 238000002296 dynamic light scattering Methods 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000005540 biological transmission Effects 0.000 description 14
- 229940079593 drug Drugs 0.000 description 14
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 12
- 239000000872 buffer Substances 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 11
- 230000002328 demineralizing effect Effects 0.000 description 10
- 230000036542 oxidative stress Effects 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 7
- 239000003963 antioxidant agent Substances 0.000 description 7
- 230000003078 antioxidant effect Effects 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 230000009920 chelation Effects 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 238000005115 demineralization Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 239000008213 purified water Substances 0.000 description 7
- 238000006722 reduction reaction Methods 0.000 description 7
- 239000001632 sodium acetate Substances 0.000 description 7
- 235000017281 sodium acetate Nutrition 0.000 description 7
- 206010061218 Inflammation Diseases 0.000 description 6
- 206010065973 Iron Overload Diseases 0.000 description 6
- 235000006708 antioxidants Nutrition 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 230000006798 recombination Effects 0.000 description 6
- 238000005215 recombination Methods 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 101710195641 Splenin Proteins 0.000 description 5
- 102400000159 Thymopoietin Human genes 0.000 description 5
- MLKWIQODPVLVLN-SHVFRELUSA-N ac1q1oa8 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(=O)NCC(=O)NCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CN)C(C)C)[C@@H](C)O)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(C)C)C1=CC=C(O)C=C1 MLKWIQODPVLVLN-SHVFRELUSA-N 0.000 description 5
- 230000003064 anti-oxidating effect Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 239000012460 protein solution Substances 0.000 description 5
- GIANIJCPTPUNBA-QMMMGPOBSA-N (2s)-3-(4-hydroxyphenyl)-2-nitramidopropanoic acid Chemical compound [O-][N+](=O)N[C@H](C(=O)O)CC1=CC=C(O)C=C1 GIANIJCPTPUNBA-QMMMGPOBSA-N 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000002438 mitochondrial effect Effects 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000003907 kidney function Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 210000005084 renal tissue Anatomy 0.000 description 3
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 2
- 102100025136 Macrosialin Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 208000037273 Pathologic Processes Diseases 0.000 description 2
- 206010063897 Renal ischaemia Diseases 0.000 description 2
- 206010038540 Renal tubular necrosis Diseases 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 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 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000010438 iron metabolism Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 230000008965 mitochondrial swelling Effects 0.000 description 2
- 230000031787 nutrient reservoir activity Effects 0.000 description 2
- 230000009054 pathological process Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 230000010410 reperfusion Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- XJOTXKZIRSHZQV-RXHOOSIZSA-N (3S)-3-amino-4-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[(2S)-2-[[(2S,3S)-1-[[(1R,6R,12R,17R,20S,23S,26R,31R,34R,39R,42S,45S,48S,51S,59S)-51-(4-aminobutyl)-31-[[(2S)-6-amino-1-[[(1S,2R)-1-carboxy-2-hydroxypropyl]amino]-1-oxohexan-2-yl]carbamoyl]-20-benzyl-23-[(2S)-butan-2-yl]-45-(3-carbamimidamidopropyl)-48-(hydroxymethyl)-42-(1H-imidazol-4-ylmethyl)-59-(2-methylsulfanylethyl)-7,10,19,22,25,33,40,43,46,49,52,54,57,60,63,64-hexadecaoxo-3,4,14,15,28,29,36,37-octathia-8,11,18,21,24,32,41,44,47,50,53,55,58,61,62,65-hexadecazatetracyclo[32.19.8.26,17.212,39]pentahexacontan-26-yl]amino]-3-methyl-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-4-oxobutanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)[C@@H](C)O)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@@H]4CSSC[C@H](NC(=O)[C@H](Cc5ccccc5)NC(=O)[C@@H](NC1=O)[C@@H](C)CC)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](Cc1cnc[nH]1)NC3=O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N2)C(=O)NCC(=O)N4)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XJOTXKZIRSHZQV-RXHOOSIZSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 101100173542 Caenorhabditis elegans fer-1 gene Proteins 0.000 description 1
- 108010075016 Ceruloplasmin Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241000407170 Curcuma Species 0.000 description 1
- 235000014375 Curcuma Nutrition 0.000 description 1
- 244000163122 Curcuma domestica Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000238710 Dermatophagoides Species 0.000 description 1
- 241000238740 Dermatophagoides pteronyssinus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 241000234299 Zingiberaceae Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008809 cell oxidative stress Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000003373 curcuma longa Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- UJHBVMHOBZBWMX-UHFFFAOYSA-N ferrostatin-1 Chemical compound NC1=CC(C(=O)OCC)=CC=C1NC1CCCCC1 UJHBVMHOBZBWMX-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 102000018511 hepcidin Human genes 0.000 description 1
- 108060003558 hepcidin Proteins 0.000 description 1
- 229940066919 hepcidin Drugs 0.000 description 1
- 210000004276 hyalin Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000008811 mitochondrial respiratory chain Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 230000006911 nucleation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000002254 renal artery Anatomy 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- -1 solubility Chemical compound 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 238000000108 ultra-filtration Methods 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
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention provides a curcumin-loaded apoferritin nanocage and application thereof. The invention shows that the cell uptake of the apoferritin nanocage carrying curcumin is obviously more than that of free curcumin, and the cell uptake can be further increased under the stimulation of hydrogen peroxide, which shows that the apoferritin nanocage carrying curcumin can be rapidly absorbed by renal tubular epithelial cells of ischemia reperfusion-acute renal injury, and has stronger internalization capability under the molding condition. The curcumin-loaded apoferritin nanocage has the advantages that the curcumin-loaded apoferritin nanocage has kidney targeting capability and high kidney retention capability, can not be accumulated in other tissues such as heart, liver, spleen and lung, and can still retain obvious fluorescent signals after 12 hours, so that the curcumin-loaded apoferritin nanocage can be prevented from being rapidly cleared by the kidney to maintain a long-acting effect, and can be used for reducing an extrarenal effect and simultaneously preventing the apoferritin nanocage from being rapidly cleared by the kidney to maintain a strong and durable treatment effect. Can be applied to the preparation of the medicine for treating ischemia-reperfusion-acute kidney injury.
Description
Technical Field
The invention belongs to a pharmaceutical composition, in particular relates to a apoferritin nano cage loaded with curcumin and application thereof, and discloses the apoferritin nano cage loaded with antioxidant curcumin and having an autonomous renal tendency effect and application thereof in preparation of a medicament for treating acute renal injury caused by ischemia-reperfusion.
Background
Acute kidney injury has a globally associated mortality rate far exceeding that of breast cancer, heart failure or diabetes, which is high for the past 50 years. Acute kidney injury is caused by various causes and is mainly characterized by rapid decline of kidney function in a short time and is accompanied by a series of clinical syndromes with serious complications, the acute kidney injury is rapidly developed, and the death rate reaches 23.0 percent. Ischemia reperfusion is one of the main causes of acute kidney injury, and ischemia reperfusion-acute kidney injury can be caused by major surgery, hemorrhage, severe burn and the like, and the early intervention treatment is particularly important. However, the current clinical treatment means mainly comprise support treatment such as kidney replacement treatment and the like, and an effective drug treatment means is not available.
Studies have shown that excessive reactive oxygen species production during the pathological course of ischemia reperfusion-acute kidney injury is the major cause of renal function injury. When acute kidney injury occurs, mitochondrial respiratory chain is inhibited, and electrons leaking out of the respiratory chain are combined with free oxygen taken in by reperfusion to increase the production of active oxygen; in addition, endogenous antioxidant consumption such as decreased antioxidant enzyme activity, decreased glutathione synthesis, etc. results in a significant decrease in the ability to scavenge oxygen radicals; the two kinds of medicine can cause abnormal rise of active oxygen, and induce renal cell oxidative stress, cell apoptosis and renal function injury. Therefore, antioxidant therapy is an effective approach for drug therapy of ischemia reperfusion-acute kidney injury.
Curcumin is a natural antioxidant, is a natural effective component extracted from the root tuber of curcuma longa of curcuma genus of zingiberaceae family, has various pharmacological activities of tumor resistance, inflammation resistance, bacteria resistance, oxidation resistance and the like, has low toxicity and has better clinical application prospect. The anti-oxidation effect of curcumin is related to the molecular structure of curcumin, and active groups in molecules provide protons in the anti-oxidation process to play an anti-oxidation effect. In recent years, researches show that curcumin can be used for treating acute renal injury through ways of removing oxygen free radicals, reducing the generation of inflammatory cytokines and the like, but has a plurality of defects in clinical application: curcumin has low water solubility, poor dissolution rate, easy metabolism in vivo, short half-life and other factors, so that the bioavailability is very low, and the curcumin lacks the selection specificity to kidney tissues, so that the curative effect is unsatisfactory.
In addition, since renal tubular cells are highly sensitive to iron overload, oxidative damage of renal tubular epithelial cells induced by iron overload is also one of the causes of ischemia reperfusion-acute renal injury. Under physiological conditions, the majority of absorbed iron is transferrin bound Fe 3+ Is present in the form of (a), and then reduced to Fe after cellular uptake 2+ . One part of the iron enters the iron bath, and the other part is stored in the iron storage protein. However, under pathological conditions, iron metabolism is disturbed, resulting in intracellular Fe 2+ Overload, which reacts with lipids through fenton's reaction to produce abundant reactive oxygen species, ultimately leading to cellular iron death. One study showed that the use of a selective iron death inhibitor, fer-1, to inhibit iron death in the pathological course of ischemia reperfusion-acute kidney injury, had a significant protective effect. Another study showed that hepcidin mediates the protective effects of renal ischemia-reperfusion injury by dynamically regulating intracellular iron homeostasis. Therefore, it is also a key strategy to treat ischemia reperfusion-acute kidney injury by interfering with iron overload.
Ferritin is one of the major intracellular iron storage proteins, consisting of heavy and light chains, and plays a central role in iron homeostasis. When iron is overloaded, ferritin can have a strong destructive redox activity of the excess Fe 2+ Transported into the cavity and oxidized to form harmless Fe 3+ Then Fe 3+ Migration, nucleation, mineralization and formation of iron nuclei. This ferritin-mediated iron metabolism can protect renal tubular epithelial cells from iron overload-induced oxidative stress injury. Studies have shown that mice with a kidney proximal tubule-specific ferritin heavy chain subunit knockout have a more significant mortality rate and more severe functional kidney damage, further suggesting that ferritin eggs with ferroxidase activityWhite is necessary for iron transport and iron toxicity limitations. In addition, the inner cavity of ferritin may also be desferrized to prepare a desferricin nanocage that may encapsulate various therapeutic agents.
In addition, the size of the protein nanoparticles can affect their biodistribution in vivo. Studies have shown that larger protein nanoparticles typically accumulate more in the liver and spleen, while nanoparticles with a particle size of 10nm and below accumulate more in the kidney. The kidney, which is the main organ for removing foreign substances from the body, filters approximately one-fourth of the cardiac blood output, and small proteins are transported to the kidney for removal after entering the blood circulation. Therefore, the apoferritin nanocages with a particle size of around 8nm are considered to be a natural kidney-targeting nanocarrier.
Disclosure of Invention
The invention aims to provide a curcumin-loaded apoferritin nanocage, which consists of curcumin and the apoferritin nanocage, wherein the mass percentage ranges of the substances are as follows: the apoferritin nanocage is 82.44% -91.40%, and curcumin is 8.60% -17.56%, wherein the types of ferritin comprise Ma Pitie protein, human liver ferritin, and Ma Pi cationic ferritin.
The second purpose of the invention is to provide the application of the apoferritin nanocage carrying curcumin in the preparation of the medicine for treating ischemia-reperfusion-acute kidney injury. The cell uptake experiment result shows that the cell uptake of the apoferritin nanocage group carrying curcumin is obviously more than that of the free curcumin group, and the cell uptake can be further increased under the stimulation of hydrogen peroxide, which indicates that the apoferritin nanocage carrying curcumin can be rapidly absorbed by renal tubular epithelial cells of ischemia reperfusion-acute kidney injury. The in vivo distribution results show that the curcumin-loaded apoferritin nanocages can effectively target the injured kidney tissues without accumulating in other tissues such as heart, liver, spleen, lung and the like, and significant fluorescence signals are still remained in the mouse kidney after 12 hours, which indicates that the curcumin-loaded apoferritin nanocages can be prevented from being rapidly cleared by the kidney and maintain the long-acting effect. The cell efficacy results show that compared with the free curcumin group, the apoferritin nanocage group carrying curcumin shows remarkable treatment effects on various indexes of cell survival rate, oxidative stress level and inflammation level. Histopathology and mitochondrial ultramicro results show that compared with free curcumin, the apoferritin nanocage loaded with curcumin is obviously improved in the renal tubular necrosis, hyaline cast and cell shedding conditions, the mitochondrial swelling conditions and the cristae structure recovery after being used for the treatment of mice subjected to ischemia reperfusion-acute renal injury modeling. Immunohistochemical staining results of nitrotyrosine as a sensitive marker of oxidative stress show that the number of nitrotyrosine positive tubules and the degree of positive reaction are obviously reduced after the curcumin-loaded apoferritin nanocage is used for treating mice subjected to ischemia-reperfusion-acute kidney injury modeling. Immunohistochemical staining results of CD68, the most reliable marker for macrophages, represent the level of inflammation and show that curcumin-loaded apoferritin nanocages have the best anti-inflammatory effect, returning to almost normal levels.
According to the invention, the unique cage-shaped space structure and reversible self-assembly capability of the apoferritin nanocages are utilized to load the antioxidant curcumin, so that the properties of the curcumin such as solubility, stability and hemolysis are improved. Meanwhile, as the kidney is used as a main organ for clearing foreign matters in vitro, the small molecular protein can be transported to the kidney to be cleared after entering blood circulation. The apoferritin nanocage has a kidney passive targeting effect and high kidney retention capacity, and can play a powerful and lasting role in the damaged part. The curcumin and the apoferritin nanocages can respectively reduce active oxygen and absorb overloaded iron to cooperatively play an antioxidation role, so that the condition of iron overload is relieved, and the pathological process of ischemia-reperfusion-acute kidney injury is reversed. The curcumin-loaded apoferritin nanocage represents a potential kidney-targeting nano platform and can be used for the synergistic treatment of ischemia-reperfusion-acute kidney injury.
The invention provides an antioxidant curcumin-loaded apoferritin nanocage. On one hand, the passive targeting property of the apoferritin nanocages to the kidney can realize targeted delivery of curcumin and reduce extrarenal effects. And the hydrophobic core of the apoferritin nanocage can be loaded with curcumin, so that the solubilization of curcumin is realized, and the bioavailability is improved. On the other hand, curcumin and apoferritin nanocages can synergistically play an anti-oxidation role by reducing active oxygen and absorbing overloaded iron respectively, so that the condition of iron overload is relieved, and the pathological process of ischemia-reperfusion-acute kidney injury is reversed. Therefore, the curcumin-loaded apoferritin nanocages may be a kidney-targeting nanoplatform for the synergistic treatment of ischemia reperfusion-acute kidney injury.
Drawings
FIG. 1 is a TEM image of curcumin-loaded desferriferous Ma Pitie protein nanocages (example 1).
FIG. 2 is the results of cellular uptake of curcumin-loaded desferriferous Ma Pitie protein nanocages (example 8).
FIG. 3 is the results of in vivo distribution of curcumin-loaded desferriferous horse spleen ferritin nanocages (example 8).
FIG. 4 is a graph of the effect of curcumin-loaded desferrioxamers Ma Pitie protein nanocages on the expression of inflammatory factors, oxidative stress factors (example 8).
Figure 5 is the histopathological, mitochondrial ultrastructural and immunohistochemical results for curcumin-loaded desferri Ma Pitie protein nanocages (example 8).
Detailed Description
The invention is further explained by the accompanying drawings and examples.
Example 1 preparation of apoferritin nanocages and curcumin-loaded apoferritin Ma Pitie nanocages
1. Preparation of apoferritin nanocage from horse's spleen
The horse spleen ferritin nano cage with iron removed is prepared by demineralizing horse spleen ferritin through reduction reaction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS No.: 9007-73-2, product No.: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the fluid in the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The liquid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the apo-ferritin on particle size, as measured by dynamic light scattering, was minimal, with both ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 4.17 ± 0.15% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of transmission electron microscopy is shown in fig. 1, in which the iron core in ferritin is removed and the ferritin nanocages still maintain a hollow spherical structure. The successful preparation of the apoferritin nanocages is proved by the experiments.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of apoferritin nanocages was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 5min, 60. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization Ma Pitie protein nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded by the dynamic light scattering instrument is increased to about 14nm, the result of the transmission electron microscope is shown in figure 1, the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 17.56 percent.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the ferritin nanocage is 82.44% and curcumin is 17.56%.
Example 2 preparation of Deferralid spleen ferritin nanocages and curcumin-loaded Deferralid Ma Pitie protein nanocages
1. Preparation of apoferritin nanocages
The horse spleen ferritin demineralization nano cage with iron removed is prepared by carrying out reduction reaction and iron chelation on the horse spleen ferritin demineralization nano cage. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS No.: 9007-73-2, product No.: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the fluid in the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The liquid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the apo-ferritin on particle size, as measured by dynamic light scattering, was minimal, with both ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 8.15 ± 2.13% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of the transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage still maintains a hollow spherical structure. The successful preparation of the apoferritin nanocages is proved by the experiments.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of apoferritin nanocages was adjusted to 2.0 by adding an appropriate volume of 1M hydrochloric acid solution, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 5min, 60. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M sodium hydroxide solution, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization Ma Pitie protein nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 12nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 12.05%.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 87.95% and curcumin is 12.05%.
Example 3 preparation of Dermatophagoides pteronyssinus ferritin nanocages and curcumin-loaded Dermatophagoides Ma Pitie protein nanocages
1. Preparation of apoferritin nanocages
The horse spleen ferritin demineralization nano cage with iron removed is prepared by carrying out reduction reaction and iron chelation on the horse spleen ferritin demineralization nano cage. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS number: 9007-73-2, product number: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the deferrization of ferritin on particle size, as measured by dynamic light scattering instruments, was small, with ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 9.79 ± 1.56% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of the transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage still maintains a hollow spherical structure. The successful preparation of the apoferritin nanocages is proved by the experiments.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH value changes. First, the pH of the solution of apoferritin nanocages was adjusted to 10.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 5min, 90. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization Ma Pitie protein nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded by the dynamic light scattering instrument is increased to about 15nm, and the result of a transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 16.79%.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the ferritin nanocage is 83.21% and curcumin is 16.79%.
Example 4 preparation of Desferricin Nanocamp and curcumin-loaded Desferricin Ma Pitie protein Nanocamp
1. Preparation of apoferritin nanocages
The horse spleen ferritin demineralization nano cage with iron removed is prepared by carrying out reduction reaction and iron chelation on the horse spleen ferritin demineralization nano cage. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS No.: 9007-73-2, product No.: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the deferrization of ferritin on particle size, as measured by dynamic light scattering instruments, was small, with ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 14.36 ± 2.68% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of the transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage still maintains a hollow spherical structure. The above experiments demonstrate the successful preparation of apoferritin nanocages.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of apoferritin nanocages was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 10min, 90. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 8.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded de-ferred Ma Pitie protein nano cage. The result of a transmission electron microscope shows that the ferritin nanocage is loaded with the drug, and the diameter of the nanocage is increased. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 10nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 14.54 percent.
The data prove that the curcumin-loaded deferrization Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 85.46% and curcumin is 14.54%.
Example 5 preparation of De-ferrated human liver ferritin nanocages and curcumin-loaded De-ferrated human liver ferritin nanocages
1. Preparation of iron-removed human liver ferritin nanocage
The iron-removed human liver ferritin nanocage is prepared by demineralizing human liver ferritin through reduction reaction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of human liver ferritin solution (CAS number 9007-73-2, product number F6754) was diluted to 50mg/ml with 30ml of sodium acetate buffer solution, and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction process is carried out in a dark deoxygenated state, the buffer solution is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core of the human liver ferritin was removed. The liquid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriferous human liver ferritin nanocage. The iron-removing effect of ferritin measured by dynamic light scattering instrument has little effect on the particle size, and both ferritin and apoferritin nanocages have particle size of about 10nm. The residual iron content in the apoferritin nanocages was 7.27 ± 1.37% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of a transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage of the iron-removed human liver still maintains a hollow spherical structure. The experiments prove the successful preparation of the iron-removed human liver ferritin nanocage.
2. Preparation of curcumin-loaded desferriferous human liver ferritin nanocage
Curcumin was encapsulated into the cavity of the desferriferous human hepatic ferritin nanocages by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of desferriferous human hepatic ferritin nanocages was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of the desferriferous human hepatic ferritin nanocages were considered to have depolymerized. After 5min, 70. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifuging (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization human liver ferritin nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 18nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 8.95 percent.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 91.05% and curcumin is 8.95%.
Example 6 preparation of equine splenic cationic ferritin nanocages and curcumin-loaded equine splenic cationic ferritin nanocages
1. Preparation of Ma Pi cation ferritin nano cage
Ma Pi cationic ferritin nanocages were prepared by demineralization of equine splenic ferritin by reduction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml Ma Pi cationic ferritin solution (CAS number: 9007-73-2, product number: F7879) was diluted to 50mg/ml with 30ml sodium acetate buffer and transferred to dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction process is carried out in a dark deoxygenated state, the buffer solution is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag fluid changed from deep red to colorless, at which time the iron core of Ma Pi cationic ferritin was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to yield Ma Pi cationic ferritin nanocages. The effect of the deferrization of ferritin on particle size, as measured by dynamic light scattering instruments, was small, with ferritin and apoferritin nanocages having particle sizes of about 8nm. The residual iron content in the apoferritin nanocages was 11.24 ± 0.37% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of a transmission electron microscope shows that the iron core in the ferritin is removed, and the deferrization Ma Pi cationic ferritin nano cage still maintains a hollow spherical structure. The above experiments demonstrate the successful preparation of apoferritin nanocages from spleen cations.
2. Preparation of Ma Pi cationic ferritin nanocage carrying curcumin
Curcumin was encapsulated into the cavity of Ma Pitie cationic ferritin nanocages by a subunit disassembly/recombination strategy based on pH change. First, the pH of Ma Pi cationic ferritin nanocage solution was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of Ma Pi cationic ferritin nanocage were considered to have depolymerized. After 5min, 70. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifuging (4000rpm, 5min) to remove free curcumin, and obtaining Ma Pi cationic ferritin nanocages loaded with curcumin. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 10nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 10.65%.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 89.35% and curcumin is 10.65%.
Example 7 preparation of Cy 5-labeled deferral Ma Pitie protein nanocages
The horse spleen ferritin nano cage with iron removed is prepared by demineralizing horse spleen ferritin through reduction reaction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution is diluted to 50mg/ml with 30ml of sodium acetate buffer solution and transferred to a dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the apo-ferritin on particle size, as measured by dynamic light scattering, was minimal, with both ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 4.17 ± 0.15% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of a transmission electron microscope shows that the iron core in the ferritin is removed, and the deferrization Ma Pi cationic ferritin nano cage still maintains a hollow spherical structure. The Cy5 fluorescence labeling protein kit is used for successfully labeling the ferremarginine nano cage with fluorescence through steps of coupling reaction, room-temperature light-shielding reaction, ultrafiltration centrifugation and the like, and the Cy5 labeled ferremarginine nano cage is successfully prepared.
Example 8 application of curcumin-loaded desferriferous equine splenic ferritin nanocages to treatment of ischemia reperfusion-acute renal injury
1. Cell uptake study of curcumin-loaded desferri Ma Pitie protein nanocage
Example 1 was prepared. Tubular epithelial cells of kidney were cultured at 2X 10 4 The density of individual cells/well was placed in a 24-well plate with coverslips placed in advance and incubated overnight. Then, the curcumin-loaded apoferritin nanocages, free curcumin (final concentrations of curcumin were 6.4 μ g/ml each) were added to the corresponding wells. After incubation for 4h, cells were fixed and mounted. Cellular internalization of curcumin-loaded apoferritin nanocages and free curcumin by renal tubular epithelial cells was studied using confocal laser microscopy. The result is shown in fig. 2, the cell internalization of the apoferritin nanocage group loaded with curcumin is obviously better than that of the free curcumin group, and the cell uptake capacity is obviously increased under the stimulation of hydrogen peroxide. The hydrogen peroxide stimulated cell selectivity may be due to strong membrane lipid peroxidation and oxidative stress, resulting in an increase in the selective permeability of the plasma membrane. The experimental result shows that the apoferritin nano cage carrying curcumin can be quickly internalized by renal tubular epithelial cells of ischemia reperfusion-acute kidney injury and enter the cells to play a role.
2. In vivo distribution study of curcumin-loaded deferrization Ma Pitie protein nanocages
Example 1, example 6 was prepared. ICR mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg) and were clamped to the bilateral renal pedicles to cause renal ischemia. Mice were placed on a 37 degree celsius heating table to maintain body temperature during ischemia. After 30min, the two clamps were released and an AKI mouse model was established. Cy 5-labeled ferritin nanoparticles were prepared to assess their distribution in vivo. Immediately after the AKI model was established, mice were injected with cy 5-labeled apoferritin nanocages and healthy mice were used as controls. Mice were sacrificed 4 or 12 hours post injection to obtain vital organs including heart, liver, spleen, lung and kidney, and fluorescence visualized with an in vivo imaging system. In vivo distribution results are shown in fig. 3, the curcumin-loaded apoferritin nanocages can effectively target damaged kidney tissue without accumulating in other tissues such as heart, liver, spleen, lung, etc. And after 12 hours, a significant fluorescence signal still remained in the mouse kidney, which indicates that the curcumin-loaded apoferritin nanocage can avoid being rapidly cleared by the kidney to maintain a long-acting effect.
3. Cell efficacy study of curcumin-loaded deferral Ma Pitie protein nanocage
Example 1 was prepared. The ischemia reperfusion-acute kidney injury model was established 30 minutes after the mice were clamped by bilateral renal arteries, and the mice were sacrificed 2 days after administration and the kidneys were collected and homogenized. The oxidative stress level is detected by using an active oxygen, lipid peroxide malondialdehyde and superoxide dismutase kit, and the inflammation level is detected by using an elisa kit interleukin-6 and a tumor necrosis factor. The results are shown in fig. 4, compared with the free curcumin group, the apoferritin nano-cage group carrying curcumin shows remarkable treatment effect on each index of oxidative stress level and inflammation level.
4. Histopathology, mitochondrial ultrastructure and immunohistochemical research of curcumin-loaded deferral Ma Pitie protein nanocage
Example 1 was prepared. Immunohistochemistry and HE staining are carried out, and part of kidney is taken and placed in glutaraldehyde for sample preparation and transmission electron microscope observation. The results are shown in fig. 5, compared with free curcumin, the apoferritin nanocages loaded with curcumin are used for treating mice modeled after ischemia-reperfusion-acute renal injury, the renal tubular necrosis, the transparent casting mold and the cell shedding of the renal structure are obviously improved, the morphology of the mitochondrial cristae is reduced or disappeared, the mitochondrial swelling is also obviously improved, and the structure of the cristae is recovered. Immunohistochemical staining results of nitrotyrosine as a sensitive marker of oxidative stress show that the number of nitrotyrosine positive tubules and the degree of positive reaction are obviously reduced after the curcumin-loaded apoferritin nanocage is used for treating mice subjected to ischemia-reperfusion-acute kidney injury modeling. Immunohistochemical staining results of CD68, the most reliable marker for macrophages, represent the level of inflammation and show that curcumin-loaded apoferritin nanocages have the best anti-inflammatory effect, returning to almost normal levels.
Claims (5)
1. A curcumin-loaded apoferritin nanocage is characterized by consisting of curcumin and the apoferritin nanocage, wherein the mass percentage ranges of the substances are as follows: the apoferritin nano cage is 82.44% -91.40% and curcumin is 8.60% -17.56%.
2. The apoferritin nanocage loaded with curcumin as claimed in claim 1, wherein apoferritin is selected from Ma Pitie protein, human hepatic ferritin or equine splenic cationic ferritin.
3. A curcumin-loaded apoferritin nanocage according to claim 1 or 2 for use in the manufacture of a medicament for the treatment of ischemia reperfusion-acute kidney injury.
4. The use according to claim 3, wherein the curcumin-loaded apoferritin nanocages are rapidly taken up by tubular epithelial cells and have a greater capacity to internalize under molding conditions.
5. The use of claim 3, wherein the curcumin-loaded apoferritin nanocage has potent kidney targeting and high kidney retention, reducing extrarenal effects while avoiding rapid clearance by the kidney and maintaining potent and long lasting therapeutic effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310018804.3A CN115920083A (en) | 2023-01-06 | 2023-01-06 | Curcumin-loaded apoferritin nanocage and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310018804.3A CN115920083A (en) | 2023-01-06 | 2023-01-06 | Curcumin-loaded apoferritin nanocage and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115920083A true CN115920083A (en) | 2023-04-07 |
Family
ID=86552436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310018804.3A Pending CN115920083A (en) | 2023-01-06 | 2023-01-06 | Curcumin-loaded apoferritin nanocage and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115920083A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104096231A (en) * | 2014-07-15 | 2014-10-15 | 中国农业大学 | Targeting nano sono-sensitizer and preparation method thereof |
| CN111544598A (en) * | 2020-05-12 | 2020-08-18 | 华东理工大学 | Ferritin nanoparticle loaded with Survivin double inhibitors as well as preparation method and application thereof |
| CN113546093A (en) * | 2021-07-20 | 2021-10-26 | 中国人民解放军陆军军医大学 | Application of cerium oxide nanoparticles in preparation of medicines for treating and preventing renal ischemia-reperfusion injury |
| US20210393539A1 (en) * | 2018-10-31 | 2021-12-23 | Inthena S.R.L. | Ferritin nanoparticles comprising a chemotherapeutic agent |
-
2023
- 2023-01-06 CN CN202310018804.3A patent/CN115920083A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104096231A (en) * | 2014-07-15 | 2014-10-15 | 中国农业大学 | Targeting nano sono-sensitizer and preparation method thereof |
| US20210393539A1 (en) * | 2018-10-31 | 2021-12-23 | Inthena S.R.L. | Ferritin nanoparticles comprising a chemotherapeutic agent |
| CN111544598A (en) * | 2020-05-12 | 2020-08-18 | 华东理工大学 | Ferritin nanoparticle loaded with Survivin double inhibitors as well as preparation method and application thereof |
| CN113546093A (en) * | 2021-07-20 | 2021-10-26 | 中国人民解放军陆军军医大学 | Application of cerium oxide nanoparticles in preparation of medicines for treating and preventing renal ischemia-reperfusion injury |
Non-Patent Citations (3)
| Title |
|---|
| 周小园等: "姜黄素对肾缺血再灌注损伤的保护作用机制的研究进展", 《中国医药导报》, vol. 16, no. 16, 28 February 2019 (2019-02-28) * |
| 孙世澜等: "《肾衰竭诊断治疗学》", 30 April 2021, 北京：人民军医出版社 * |
| 张进香等: "姜黄素去铁蛋白的制备及初步评价", 《山东化工》, vol. 51, no. 11, 8 June 2022 (2022-06-08) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kalashnikova et al. | Ceria-based nanotheranostic agent for rheumatoid arthritis | |
| Ali et al. | Anthocyanin-loaded PEG-gold nanoparticles enhanced the neuroprotection of anthocyanins in an Aβ 1–42 mouse model of Alzheimer’s disease | |
| Chen et al. | Reactive oxygen species (ROS)-responsive nanomedicine for solving ischemia-reperfusion injury | |
| Gui et al. | Superoxide dismutase-loaded porous polymersomes as highly efficient antioxidant nanoparticles targeting synovium for osteoarthritis therapy | |
| An et al. | Applications of nanomaterials for scavenging reactive oxygen species in the treatment of central nervous system diseases | |
| Gao et al. | An anti-inflammatory and neuroprotective biomimetic nanoplatform for repairing spinal cord injury | |
| ES2608659T3 (en) | Composition to inhibit the activity of NADPH oxidase | |
| You et al. | Subcellular co-delivery of two different site-oriented payloads based on multistage targeted polymeric nanoparticles for enhanced cancer therapy | |
| Han et al. | A novel targeted nanoparticle for traumatic brain injury treatment: combined effect of ROS depletion and calcium overload inhibition | |
| CN113750244A (en) | Engineered platelet carrying chemotherapeutic drug and containing external back immune checkpoint inhibitor nanogel as well as preparation method and application of engineered platelet | |
| Jiang et al. | Cartilage targeting therapy with reactive oxygen species-responsive nanocarrier for osteoarthritis | |
| Zhang et al. | Mitochondrial-targeted and ROS-responsive nanocarrier via nose-to-brain pathway for ischemic stroke treatment | |
| Sun et al. | Protective effect of lipoic acid modification on brain dysfunctions of mice induced by mesoporous silica nanoparticles | |
| Wang et al. | Silk sericin stabilized proanthocyanidins for synergetic alleviation of ulcerative colitis | |
| CN114042147B (en) | Micro-nano hydrogel microsphere for targeted regulation and control of mitochondrial respiratory chain and preparation and application thereof | |
| Jiang et al. | Bone-targeted ICG/Cyt c@ ZZF-8 nanoparticles based on the zeolitic imidazolate framework-8: a new synergistic photodynamic and protein therapy for bone metastasis | |
| Bai et al. | Engineered urinary-derived extracellular vesicles loaded nanoenzymes as Trojan horses to regulate the inflammatory microenvironment for treatment of Alzheimer's disease | |
| You et al. | Surface-tethered ROS-responsive micelle backpacks for boosting mesenchymal stem cell vitality and modulating inflammation in ischemic stroke treatment | |
| Wang et al. | Biomimetic lung-targeting nanoparticles with antioxidative and Nrf2 activating properties for treating ischemia/reperfusion-induced acute lung injury | |
| CN115920083A (en) | Curcumin-loaded apoferritin nanocage and application | |
| Liu et al. | Near-infrared-IIb fluorescent nanozymes for imaging-guided treatment of traumatic brain injury | |
| CN112402378A (en) | ROS (reactive oxygen species) -responsive dexamethasone liposome containing selenium bond as well as preparation method and application thereof | |
| Tang et al. | Melanin nanoparticles enhance the neuroprotection of mesenchymal stem cells against hypoxic‐ischemic injury by inhibiting apoptosis and upregulating antioxidant defense | |
| Saghir et al. | Ameliorative Effect of Thymoquinone‐Loaded PLGA Nanoparticles on Chronic Lung Injury Induced by Repetitive Intratracheal Instillation of Lipopolysaccharide in Rats | |
| Lou et al. | Self-oriented ferritin nanocages mitigate iron overload-induced oxidative stress for acute kidney injury |
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 |