CN102861322A - Hemoglobin oxygen carrier and preparation method thereof - Google Patents
Hemoglobin oxygen carrier and preparation method thereof Download PDFInfo
- Publication number
- CN102861322A CN102861322A CN2012103556354A CN201210355635A CN102861322A CN 102861322 A CN102861322 A CN 102861322A CN 2012103556354 A CN2012103556354 A CN 2012103556354A CN 201210355635 A CN201210355635 A CN 201210355635A CN 102861322 A CN102861322 A CN 102861322A
- Authority
- CN
- China
- Prior art keywords
- hemoglobin
- oxygen
- carrier
- taken
- hoptoglobin
- 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
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 142
- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 142
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 85
- 239000001301 oxygen Substances 0.000 title claims abstract description 85
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 85
- 238000002360 preparation method Methods 0.000 title claims description 50
- 239000002105 nanoparticle Substances 0.000 claims abstract description 67
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- -1 polyethylene Polymers 0.000 claims abstract description 24
- 239000004698 Polyethylene Substances 0.000 claims abstract description 23
- 229920000573 polyethylene Polymers 0.000 claims abstract description 23
- 229920000728 polyester Polymers 0.000 claims abstract description 9
- 229920000375 Poly(ethylene glycol)-block-poly(ε−caprolactone) methyl ether Polymers 0.000 claims description 44
- 239000008363 phosphate buffer Substances 0.000 claims description 26
- 229920001610 polycaprolactone Polymers 0.000 claims description 21
- 238000000746 purification Methods 0.000 claims description 19
- 239000003963 antioxidant agent Substances 0.000 claims description 18
- 230000003078 antioxidant effect Effects 0.000 claims description 18
- 235000006708 antioxidants Nutrition 0.000 claims description 18
- 125000000524 functional group Chemical group 0.000 claims description 18
- 239000002245 particle Substances 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 16
- 238000001556 precipitation Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims description 9
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 8
- 239000004626 polylactic acid Substances 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 6
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 6
- 238000005227 gel permeation chromatography Methods 0.000 claims description 6
- 102000016938 Catalase Human genes 0.000 claims description 5
- 108010053835 Catalase Proteins 0.000 claims description 5
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 125000003368 amide group Chemical group 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 102000003992 Peroxidases Human genes 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 2
- 229930003427 Vitamin E Natural products 0.000 claims description 2
- QRSFFHRCBYCWBS-UHFFFAOYSA-N [O].[O] Chemical compound [O].[O] QRSFFHRCBYCWBS-UHFFFAOYSA-N 0.000 claims description 2
- 229940105657 catalase Drugs 0.000 claims description 2
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims 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 claims description 2
- 235000003969 glutathione Nutrition 0.000 claims description 2
- 229960003180 glutathione Drugs 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims description 2
- 229940072417 peroxidase Drugs 0.000 claims description 2
- 239000004632 polycaprolactone Substances 0.000 claims description 2
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 claims description 2
- 235000019165 vitamin E Nutrition 0.000 claims description 2
- 229940046009 vitamin E Drugs 0.000 claims description 2
- 239000011709 vitamin E Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 10
- 230000001988 toxicity Effects 0.000 abstract description 10
- 231100000419 toxicity Toxicity 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 8
- 238000001802 infusion Methods 0.000 abstract description 5
- 206010020772 Hypertension Diseases 0.000 abstract description 3
- 210000002381 plasma Anatomy 0.000 abstract description 3
- 102000014702 Haptoglobin Human genes 0.000 abstract 2
- 108050005077 Haptoglobin Proteins 0.000 abstract 2
- 230000007547 defect Effects 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 28
- 239000000243 solution Substances 0.000 description 26
- 239000008187 granular material Substances 0.000 description 25
- 239000000843 powder Substances 0.000 description 25
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- 238000003756 stirring Methods 0.000 description 19
- 229920000642 polymer Polymers 0.000 description 18
- 238000012360 testing method Methods 0.000 description 18
- INGWEZCOABYORO-UHFFFAOYSA-N 2-(furan-2-yl)-7-methyl-1h-1,8-naphthyridin-4-one Chemical compound N=1C2=NC(C)=CC=C2C(O)=CC=1C1=CC=CO1 INGWEZCOABYORO-UHFFFAOYSA-N 0.000 description 16
- 210000004369 blood Anatomy 0.000 description 16
- 239000008280 blood Substances 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 229920001223 polyethylene glycol Polymers 0.000 description 15
- 229910052757 nitrogen Inorganic materials 0.000 description 14
- 239000002202 Polyethylene glycol Substances 0.000 description 13
- 239000000839 emulsion Substances 0.000 description 13
- 229920000151 polyglycol Polymers 0.000 description 13
- 239000010695 polyglycol Substances 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 230000005540 biological transmission Effects 0.000 description 12
- 238000000502 dialysis Methods 0.000 description 12
- 239000006185 dispersion Substances 0.000 description 12
- 210000004700 fetal blood Anatomy 0.000 description 11
- 239000003960 organic solvent Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000003556 assay Methods 0.000 description 10
- 229920001577 copolymer Polymers 0.000 description 10
- 229940079593 drug Drugs 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 230000001804 emulsifying effect Effects 0.000 description 9
- 239000000376 reactant Substances 0.000 description 9
- PAPBSGBWRJIAAV-UHFFFAOYSA-N ε-Caprolactone Chemical compound O=C1CCCCCO1 PAPBSGBWRJIAAV-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 8
- 238000013019 agitation Methods 0.000 description 8
- 239000003054 catalyst Substances 0.000 description 8
- 238000001914 filtration Methods 0.000 description 8
- 239000003517 fume Substances 0.000 description 8
- 125000005474 octanoate group Chemical group 0.000 description 8
- 239000003921 oil Substances 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 238000004062 sedimentation Methods 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 238000001291 vacuum drying Methods 0.000 description 8
- 229920001400 block copolymer Polymers 0.000 description 7
- 210000003022 colostrum Anatomy 0.000 description 7
- 235000021277 colostrum Nutrition 0.000 description 7
- 229920001553 poly(ethylene glycol)-block-polylactide methyl ether Polymers 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 229920005654 Sephadex Polymers 0.000 description 5
- 239000012507 Sephadex™ Substances 0.000 description 5
- 230000036772 blood pressure Effects 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 238000012827 research and development Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 108010030158 HBOC 201 Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 208000034530 PLAA-associated neurodevelopmental disease Diseases 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000009530 blood pressure measurement Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000008176 lyophilized powder Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000001338 self-assembly Methods 0.000 description 3
- 230000002227 vasoactive effect Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- JJTUDXZGHPGLLC-IMJSIDKUSA-N 4511-42-6 Chemical compound C[C@@H]1OC(=O)[C@H](C)OC1=O JJTUDXZGHPGLLC-IMJSIDKUSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 208000035126 Facies Diseases 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 241000594182 Sarcophaga sigma Species 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000001631 hypertensive effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- KSBAEPSJVUENNK-UHFFFAOYSA-L tin(ii) 2-ethylhexanoate Chemical compound [Sn+2].CCCCC(CC)C([O-])=O.CCCCC(CC)C([O-])=O KSBAEPSJVUENNK-UHFFFAOYSA-L 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010074954 MP4 maleimide-polyethylene glycol-modified hemoglobin Proteins 0.000 description 1
- 229920000881 Modified starch Polymers 0.000 description 1
- 239000004368 Modified starch Substances 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 108010013106 O-raffinose cross-linked human hemoglobin Proteins 0.000 description 1
- 108010034539 PolyHeme Proteins 0.000 description 1
- 229920002564 Polyethylene Glycol 3500 Polymers 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 229940126587 biotherapeutics Drugs 0.000 description 1
- 238000012661 block copolymerization Methods 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 239000003633 blood substitute Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 230000007681 cardiovascular toxicity Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000008497 endothelial barrier function Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 231100000682 maximum tolerated dose Toxicity 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019426 modified starch Nutrition 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 108010070577 polymerized bovine hemoglobin Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 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 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical compound Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 239000005526 vasoconstrictor agent Substances 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a hemoglobin-based oxygen carrier, which comprises hemoglobin, haptoglobin and polyethylene glycol-polyester nanoparticles, wherein the hemoglobin is connected to the surfaces of the polyethylene glycol-polyester nanoparticles through the haptoglobin. The hemoglobin oxygen carrier has no obvious toxicity to cells, no obvious free hemoglobin in blood plasma after infusion, no hypertension reaction, and capacity of overcoming the defect of strong side effect of traditional hemoglobin oxygen carrier.
Description
Technical field
The present invention relates to a kind of hemoglobin and take the carrier of oxygen.
Background technology
Important function has been brought into play in blood transfusion in modern medical service, be clinical operation, combat a natural disaster and the indispensable medical procedure of field rescue.But tradition blood transfusion has a lot of drawbacks, as, the blood source is single, and is main by donating blood, and human blood types is complicated, need to carry out strict could using after joining type, limited it to emergency and severe disease patient's treatment and the application under urgent accident environment.More seriously blood is easy to be subject to hepatitis, and the pollution of the virus such as acquired immune deficiency syndrome (AIDS), and the new and old blood problem of more and more paying close attention to now are so that transfusion safety is on the hazard.Along with the development of medical skill, the demand of blood constantly increases, and the blood source is day by day in short supply safely and effectively.Blood of human body is comprised of blood plasma, erythrocyte, leukocyte and platelet, and composition is very complicated, produce a kind of solution of blood that can replace fully very difficult, hardly may in other words; But develop a kind of interim succedaneum, replace the effect of certain component in the blood but to have feasibility in the lower short time of situation in urgent need.
As the important component part of blood, hemoglobin (Hemoglobin, Hb) is positioned at erythrocyte, is the main media of body transportation oxygen.But, simple hemoglobin solutions can not direct infusion, lose 2 because break away from erythrocytic hemoglobin, the 3-diphosphoglyceric acid (2, adjusting 3-DPG), oxygen affinity sharply raises, can not be to organizing effective oxygen supply, and free hemoglobin under the effect of various enzymes also rapidly depolymerization be dimer and monomer, through renal excretion or stop up renal tubules, produce strong nephrotoxicity.In order to overcome aforementioned drawback, hemoglobin is carried out necessary modification or processing, adjust effective molecular radius of hemoglobin, to viscosity with take oxygen oxygen release ability etc. and be optimized, to reduce the untoward reaction of above-mentioned hemoglobin, increase oxygen and carry effect and the retention time in blood plasma, the products similar of generation is referred to as hemoglobin and takes the carrier of oxygen (HBOCs).
Nearly decades, HBOCs is the emphasis of blood substitute research and development always.The R﹠D process of HBOCs mainly is divided into the three generations.The first generation is with small numerator modified hemoglobin or uses the cross-linking agent polymeric hemoglobin.Second is cross-linked to form polymer on behalf of haemoglobin molecule and antioxidase such as superoxide dismutase, catalase etc.Be used for front two generation HBOCs comparatively ripe chemical modification and cross-linking method and comprise, Polyethylene Glycol (PEG) is modified, and O-Raffinose is crosslinked, two aspirin are crosslinked, glutaraldehyde (GDA) is crosslinked etc., and product comprises HemAssist, Polyheme, HemoLink, Hemopure (HBOC-201), Hemospan (MP4), Gelenpol, HemoTech, Hemoximer etc.Front two generation product in clinical practice, a lot of problems: HemAssist all occurred and belonged to the 1st generation product, because of in the III clinical trial phase, find experimenter's mortality rate obviously increase abandon; Polyheme also stops research because serious cardiac toxicity occurs in the clinical trial; The polymerized bovine hemoglobin of the Hemopure(glutaraldehyde cross-linking of U.S. Biopure company research and development) is present unique product of having got permission in clinical practice, only limit to go on the market in South Africa, but because expose the safety issue of cardiovascular aspect in the III phase clinical research, further research and development have also been stopped.2008, analyze Deng the Meta that discloses 1 piece of relevant HBOC clinical trial at medical journal " JAMA ", point out that it has increased patient's mortality rate and the potential risk that causes myocardial infarction is arranged, point out simultaneously before the potential toxic and side effects of clear and definite HBOC goods and mechanism thereof, should not carry out again further clinical trial.The conclusion of this magazine causes the listing request that U.S. FDA had been refused the HBOC of Northfield company product (PolyHeme) in 2009 indirectly, has affected whole HBOC industry.About the toxicity behind the HBOCs goods infusion, generally believe to be it owing to there being free/unpolymerized hemoglobin among the HBOC, it can see through endothelial barrier, consume endothelium relaxing factor nitric oxide (NO), simultaneously, the oxygen-derived free radicals of the redox reaction of hemoglobin generation itself causes the body response to oxidative stress.
In order to solve among the HBOC problem that has free/unpolymerized hemoglobin, third generation HBOCs hemoglobin outside is with film or macromolecular material parcel, makes it more to be called the parcel hemoglobin near erythrocytic practical structures.Canadian TMS Chang has reported the capsulated hemoglobin of use polyethylene glycol-polylactic acid, about capsule grain diameter 100nm.The Tsuchida Eishun of Japan has prepared cellular type hemoglobin (Hemoglobin Vehicle, HbV), about particle diameter 250nm.Also there is similar report in China, application number: 200810051615.1, denomination of invention: the application for a patent for invention of polypeptide vesicle of biodegradable loaded with hemoglobin and preparation method thereof, a kind of amphipathic block polypeptide vesicle of biodegradable loaded with hemoglobin is disclosed, the inside aqueous phase of this vesicle contains the hemoglobin of CO protection, and the wall of vesicle is made of biodegradable polylysine-polyphenylalanine two block copolymerization peptides; Application number: 200810050914.3, denomination of invention: a kind of biodegradable bonding the nanoparticle of haemoglobin molecule and the application for a patent for invention of method for making, the nanoparticle of haemoglobin molecule that discloses a kind of biodegradable bonding, it is the erythrocytic cellularity of simulation, make the carrier of hemoglobin of the lactic acid based block copolymer, construct polymer/hemoglobin nano-micelle or capsule; Application number: 201010246051.4, denomination of invention: a kind of Patent Application Publication of artificial nano red blood cells a kind of artificial nano red blood cells, it is to have amphiphatic modified starch in the hemoglobin outside by the nanoassemble parcel, and forming particle diameter is the artificial nano red blood cells of the microencapsulation of 200 ~ 300nm.But this based article is defectiveness still, and such as packaging material, especially phospholipid is expensive and unstable, and the hemoglobin behind the physically encapsulation may be dashed forward and be released, and still is difficult to avoid the side effect of free/unpolymerized hemoglobin, and potential infusion risk is arranged.
Summary of the invention
In order to address the above problem, to the invention provides a kind of new hemoglobin and take the carrier of oxygen.
Hemoglobin of the present invention is taken the carrier of oxygen, comprises hemoglobin, hoptoglobin and polyethylene glycol-ester nanoparticles, and hemoglobin is connected to polyethylene glycol-ester nanoparticles surface by hoptoglobin.
Its particle diameter is 80 ~ 400nm.Preferably, its particle diameter is 200 ~ 300nm.
Described polyethylene glycol-ester nanoparticles is PEG-PCL nanoparticle or polyethylene glycol-polylactic acid nanoparticle.
Wherein, the content of hemoglobin is 6 ~ 12%(w/w), and the mol ratio of hemoglobin and globin is 1:1.
Described polyethylene glycol-ester nanoparticles is connected by thioether bond or amido link with hoptoglobin.
Described hemoglobin is taken the carrier of oxygen, oxygen affinity P
50Value is 10 ~ 36mmHg, and the Hill coefficient is 1.30 ~ 2.90, and pH value is 7.2 ~ 7.5.
Described hemoglobin is taken the carrier of oxygen and is also comprised antioxidant, and antioxidant is wrapped in the polyethylene glycol-ester nanoparticles.Described antioxidant is one or more the combination in ascorbic acid, superoxide dismutase, catalase, peroxidase, vitamin E, glutathion, sodium dithionite, the N-acetylcystein.
The present invention also provides above-mentioned hemoglobin to take the preparation method of the carrier of oxygen, and it comprises the steps:
A, get the hoptoglobin of hoptoglobin, sulfhydrylation hoptoglobin or activated carboxylic, and be dissolved in the phosphate buffer with the polyethylene glycol-ester nanoparticles of functional group, the weight ratio of hoptoglobin and nanoparticle is 1:5 ~ 10,4 ℃ shook 18 ~ 30 hours, the phosphate buffer washing, 10000 ~ 14000g is centrifugal, gets precipitation;
B, step a gained precipitation is dissolved in the phosphate buffer, adds hemoglobin, the weight ratio of hemoglobin and hoptoglobin is (2 ~ 4): (1 ~ 3), 4 ℃ of lucifuges were reacted 2 ~ 6 hours, and separation and purification is concentrated freeze-dried, namely gets hemoglobin and takes the carrier of oxygen.
The described functional group of step a is single maleimide, carboxyl or amino.
Wherein, the described polyester of step a is polylactic acid or polycaprolactone.
Wherein, wrap up antioxidant in the described nanoparticle of step a.
Wherein, among the described step a, hoptoglobin and nanoparticle weight ratio are 1:5 or 1:10; Shook 24 hours; 12000g is centrifugal.
Wherein, among the described step b, the weight ratio of hemoglobin and hoptoglobin is 3:2; Lucifuge reaction 2 hours; Isolation and purification method is: centrifugal or gel chromatography column separating purification, to supernatant or eluent till 400-600nm is without obvious absorption peaks, collecting precipitation or eluent.
Be by with functional group's polyethylene glycol-ester di-block copolymer self assembly with the polyethylene glycol-ester nanoparticles of functional group, perhaps the mixture self assembly of itself and poly glycol monomethyl ether-polyester biblock copolymer obtains.The method that self assembly is adopted is organic solvent method, dialysis, emulsion dispersion method or multi-emulsion method.A, organic solvent method: will be with polyethylene glycol-ester di-block copolymer and the poly glycol monomethyl ether-polyester biblock copolymer of functional group, mass ratio 1:0-40, (antioxidant: total copolymer mass ratio=0-1:10) is dissolved in ethyl acetate altogether with antioxidant, through revolve boil off except organic solvent after, add in 40～70 ℃ of hot water vibrating dispersion, be cooled to room temperature, lyophilizing namely gets the nanoparticle with functional group and parcel antioxidant.B, dialysis: will be with polyethylene glycol-ester di-block copolymer and the poly glycol monomethyl ether-polyester biblock copolymer of functional group, mass ratio 1:0-40, with antioxidant (antioxidant: total copolymer mass ratio=0-1:10) be dissolved in can with the miscible organic solvent DMSO of water in, then join in the bag filter, and in water, dialyse.In the dialysis procedure, di-block copolymer is self-assembled into nanoparticle and with antioxidant packages section within it, removed simultaneously organic solvent wherein, do not needed to use surfactant and high-speed stirred, with the dialysis after the solution lyophilizing namely get with functional group and the parcel antioxidant nanoparticle.C, the emulsion dispersion method: with the aqueous solution that contains emulsifying agent as water, first emulsifying on the emulsion dispersion machine, then drip the organic facies that contains with polyethylene glycol-ester di-block copolymer and poly glycol monomethyl ether-polyester biblock copolymer and the antioxidant of functional group with certain speed, drip rear continuation emulsifying 10 minutes on the emulsion dispersion machine, Rotary Evaporators is taken out organic solvent and is namely got nanoparticle with functional group.D, multi-emulsion method (W/O/W method): (antioxidant: the total copolymer mass ratio=0-1:10) water-soluble solution forms interior water with antioxidant first, under the condition of ultrasonic emulsification, dropwise join in the organic facies with the polyethylene glycol-ester di-block copolymer of functional group and poly glycol monomethyl ether-polyester biblock copolymer (mass ratio 1:0-40), form first the W/O colostrum, again colostrum is slowly added the outer aqueous phase that contains emulsifying agent under churned mechanically condition, form the emulsion of W/O/W, fling to lyophilizing behind the organic solvent and namely get nanoparticle with functional group.
Hemoglobin of the present invention is taken in the carrier of oxygen, hemoglobin can form stable complex with hoptoglobin, after even nanoparticle decomposes, can not produce free hemoglobin yet, without hyper tensive reactions, and hoptoglobin/hemoglobin complex is removed through reticuloendothelial system, can greatly alleviate the injury of kidney that may cause through renal excretion, overcomes the long-standing defective of HBOCs based article.And the combination of hemoglobin site-directed quantitative can be realized with the controlled active function groups of quantity in HBOCs of the present invention surface, obtains stay-in-grade end-product, has extremely good potential applicability in clinical practice.
Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
The specific embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 is the schematic diagram that the related novel hemoglobin of the present invention is taken the oxygen nanoparticle
Fig. 2 is Mal-PEG-PCL copolymer among the embodiment 3
1The H-NMR collection of illustrative plates
Fig. 3 is MPEG-PCL copolymer among the embodiment 3
1The H-NMR collection of illustrative plates
Fig. 4 is transmission electron microscope (TEM) figure that embodiment 3 prepared hemoglobins are taken the carrier of oxygen
Fig. 5 is that embodiment 3 prepared hemoglobins are taken the detected result of carrier of oxygen employing MALVERN particle instrument
Fig. 6 is that embodiment 3 prepared hemoglobins are taken the cytotoxicity result that the carrier of oxygen adopts mtt assay to measure
Fig. 7 is transmission electron microscope (TEM) figure that embodiment 4 prepared hemoglobins are taken the carrier of oxygen
Fig. 8 is that embodiment 4 prepared hemoglobins are taken the detected result of carrier of oxygen employing MALVERN particle instrument
Fig. 9 is that embodiment 4 prepared hemoglobins are taken the cytotoxicity result that the carrier of oxygen adopts mtt assay to measure
Figure 10 is the lyophilized powder that embodiment 3 and embodiment 4 prepared hemoglobins are taken the carrier of oxygen
Figure 11 is that embodiment 3 and embodiment 4 prepared hemoglobins are taken the carrier of oxygen to the vasoactive testing result of mice
The specific embodiment
Key instrument and reagent:
Nuclear magnetic resonance analyser: Varian 400, Varian company, USA;
Ma Erwen laser diffraction particle size analyzer: Nano-ZS, Malvern Instrument, UK;
Chromatograph of gel permeation: Agilent 110, Agilent company, USA;
Transmission electron microscope: Hitachi H-6009IV, Hitachi company, Japan;
Blood gas analyzer: ABL 800FLEX, Radiometer company; BC-3000PLUS, MINDRAY, China;
HEMOX
TMAnalyser: TCS Scientific, USA;
PH electrode: Mettler Toledo company, USA;
Noninvasive Blood Pressure Measurement System: BP-2000, Visitech Systems company, USA;
Poly glycol monomethyl ether (MPEG) is available from U.S. Aldrich company, analytical pure;
Single maleimide Polyethylene Glycol (Mal-PEG), mono amino Polyethylene Glycol (NH
2-PEG) and mono carboxylic Polyethylene Glycol (COOH-PEG) all available from the triumphant company of Beijing key, analytical pure;
Caprolactone (Caprolactone, CL), lactide (D, L-lactide), stannous octoate (Stannousoctoate, Sn (oct) 2), dimethyl sulfoxide (DMSO) is available from U.S. Sigma company, analytical pure;
Sulfhydrylization reagent Traut ' s(2-IminothiolaneHCl), available from U.S. ThermoScientific-Pierce company; Acetone, dichloromethane, petroleum ether, ethyl acetate, lactic acid, phosphate buffer (PBS, pH7.4) all are purchased from Chengdu Ke Long chemical reagents corporation;
MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) available from U.S. Sigma company;
Cell culture medium DMEM culture medium is available from Gibico BRL company;
HEKC (HEK293) is from Huaxi Hospital Attached to Sichuan Univ Biotherapeutics National Key Laboratory;
1, preparation method
(1) preparation of Mal-PEG-PCL and MPEG-PCL
Respectively with the poly glycol monomethyl ether (MPEG2000) of single maleimide Polyethylene Glycol (Mal-PEG2000) of 5.0g caprolactone and 5.0g molecular weight 2000 or 5.0g molecular weight 2000 in mass ratio 1:1 place there-necked flask, add the inferior stannum of octoate catalyst and an amount of toluene, in 140 ℃ of oil baths, reacting 4 hours under the condition of mechanical agitation and nitrogen protection;
After the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain Mal-PEG-PCL and MPEG-PCL again.
(2) contain the preparation of the PEG-PCL nano-granule freeze-dried powder of maleimide amine functional group
Then, get respectively 1mg Mal-PEG-PCL and 40mg MPEG-PCL is dissolved in the 5ml ethyl acetate, take out ethyl acetate with Rotary Evaporators, then add the 10ml distilled water, stir in 60 degree heating in water bath, namely obtain containing the PEG-PCL nanoparticle aqueous solution of maleimide amine functional group, lyophilizing namely gets the nano-granule freeze-dried powder of the PEG-PCL that contains the maleimide amine functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Hoptoglobin and sulfhydrylization reagent Traut ' s, according to mol ratio 1:40, the lucifuge stirring reaction is 2 hours under the logical High Purity Nitrogen condition, and the sephadex column separation obtains the sulfhydrylation hoptoglobin;
People's hoptoglobin of 5mg sulfhydrylation is dissolved in the 10ml phosphate buffer, adds the PEG-PCL nano-granule freeze-dried powder that 50mg contains the maleimide amine functional group, 4 ℃ of lucifuges were vibrated PBS solution washing and centrifugalize under 12000g 10 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 8mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 2 hours, reactant liquor centrifugalize under 12000g, regional without till absorbing at 400-600nm to the upper strata clear liquid with the phosphate buffer washing, namely get hemoglobin of the present invention and take the carrier of oxygen.
2, physicochemical property detects
Detection method:
The chemical constitution of copolymer and composition adopt infrared spectrum, and NMR (Nuclear Magnetic Resonance) spectrum is identified and calculated;
The molecular weight and molecualr weight distribution of copolymer uses the gel permeation chromatography device to measure;
Nanoparticle Size and the use MALVERN particle instrument that distributes are measured;
The form of nanoparticle adopts transmission electron microscope to observe and record;
The concentration of hemoglobin (carrying drug ratio) is by the ABL 800FLEX blood gas analyzer of Radiometer company and the BC-3000PLUS analysis-e/or determining of MINDRAY;
The P50 value of the nanoparticle of loaded with hemoglobin and Hill coefficient value are by the HEMOXTM analysis-e/or determining of TCS Scientific company;
Hemoglobin carrying drug ratio (%) represents by the percentage ratio that hemoglobin quality in the finished product accounts for the nano-granule freeze-dried powder gross mass;
PH value is measured by the pH electrode of Mettler Toledo company;
Cytotoxicity detection method: with 100 μ L(1 * 10
5Individual/mL) the HEK293 cell is inoculated in 96 orifice plates, culture medium is DMEM, treat that Growth of Cells is when merging state, the normal saline solution (final concentration be respectively 50,100,200,400,600,800 and 1000ug/ml) that adds the nanoparticle that variable concentrations above-described embodiment obtains, each concentration is established 6 multiple holes, 37 ℃, contain 5%CO
2Hatch and cultivate 24h in the incubator.Then, in culture medium, add MTT, making whole mass concentration is 0.5g/L, in 37 ℃, contain hatching of 5%CO2 and cultivate 4h in the incubator, abandon supernatant, add DMSO (100 μ L/ hole), mixing 10min, detect (measuring wavelength 570nm, reference wavelength 630nm) absorbance (A) with microplate reader, with normal saline as the blank group.Computing formula is as follows: cytoactive (Cell Viability)=A
The variable concentrations nanometer The grain processed group/ A
The blank group* 100%.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 80nm, and transmission electron microscope observing nanoparticle form is rule and spherical in shape, hemoglobin carrying drug ratio 8%, oxygen affinity P
50The value 18mmHg, Hill coefficient 1.47, pH value 7.2, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity.
1, preparation method
(1) Mal-PEG-PCL preparation
Respectively with single maleimide Polyethylene Glycol (Mal-PEG3500) of 8.89g caprolactone and 1.11g molecular weight 3500 in mass ratio 8:1 place there-necked flask, add the inferior stannum of octoate catalyst and an amount of toluene, in 137 ℃ of oil baths, reacting 6 hours under the condition of mechanical agitation and nitrogen protection;
After the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain Mal-PEG-PCL again.
(2) contain the preparation of the PEG-PCL nano-granule freeze-dried powder of maleimide amine functional group
Getting 5mg Mal-PEG-PCL is dissolved in the 2ml dichloromethane, join again in the aqueous solution that 5ml contains dodecyl sodium sulfate (SDS) (SDS concentration is 0.2mg/ml), high-speed stirred is 10～15 minutes under the 10000rpm rotating speed, gained nanoparticle aqueous emulsion is revolved steaming remove organic solvent, lyophilizing namely gets the PEG-PCL nano-granule freeze-dried powder that contains the maleimide amine functional group again.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Hoptoglobin and sulfhydrylization reagent Traut ' s, according to mol ratio 1:40, stirring reaction is 2 hours under logical High Purity Nitrogen, the normal temperature and pressure conditions, and the ultrafiltration and concentration separation obtains the sulfhydrylation hoptoglobin;
People's hoptoglobin of 10mg sulfhydrylation is dissolved in the 20ml phosphate buffer, adds the PEG-PCL nano-granule freeze-dried powder that 50mg contains the maleimide amine functional group, 4 ℃ of lucifuges were vibrated PBS solution washing and centrifugalize under 12000g 10 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg bovine hemoglobin, 4 ℃ of lucifuge stirring reactions 2 hours, reactant liquor centrifugalize under 12000g, with the phosphate buffer washing to the upper strata clear liquid in the 400-600nm zone without till absorbing.
2, physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 180nm, and transmission electron microscope observing nanoparticle form is rule and spherical in shape, hemoglobin carrying drug ratio 12%, oxygen affinity P
50The value 21mmHg, Hill coefficient 2.35, pH value 7.5, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity.
1, preparation method
(1) preparation of Mal-PEG-PCL and MPEG-PCL
Respectively with the poly glycol monomethyl ether (MPEG5000) of single maleimide Polyethylene Glycol (Mal-PEG5000) of 8.33g caprolactone and 1.67g molecular weight 5000 or 1.67g molecular weight 5000 in mass ratio 5:1 place there-necked flask, add the inferior stannum of octoate catalyst and an amount of toluene, in 137 ℃ of oil baths, reacting 6 hours under the condition of mechanical agitation and nitrogen protection;
After the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain Mal-PEG-PCL(such as Fig. 2 again) and MPEG-PCL(such as Fig. 3).
(2) contain the preparation of the PEG-PCL nano-granule freeze-dried powder of maleimide amine functional group
With 5mg superoxide dismutase SOD, the 5mg cat catalase, be dissolved in 2ml PBS (pH7.4), speed with per minute 0.5ml splashes into the dichloromethane solution that 5ml contains 5mg Mal-PEG-PCL and 95mg MPEG-PCL under sonic oscillation, after dripping the colostrum that makes is poured into rapidly in the PVA solution of 20ml0.1%, emulsifying is 5 minutes on the emulsion dispersion machine, then adding in the PVA solution of 100ml 0.1% dilution stirred 30 minutes, fling to organic solvent on Rotary Evaporators, lyophilizing namely gets parcel superoxide dismutase and the catalatic PEG-PCL nano-granule freeze-dried powder that contains the maleimide amine functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Hoptoglobin and sulfhydrylization reagent Traut ' s, according to mol ratio 1:40, stirring reaction is 2 hours under the logical High Purity Nitrogen condition, and the sephadex column separation obtains the sulfhydrylation hoptoglobin;
10mg people's hoptoglobin is dissolved in the 10ml phosphate buffer, adds the PEG-PCL nano-granule freeze-dried powder that 100mg contains the maleimide amine functional group, 4 ℃ of lucifuges were vibrated PBS solution washing and centrifugalize under 12000g 24 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 2 hours, reactant liquor is collected in the eluent that there is the hemoglobin characteristic absorption in the 400-600nm zone through the gel chromatography column purification, lyophilizing behind ultrafiltration and concentration.
2, physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 224nm(such as Fig. 4), transmission electron microscope observing nanoparticle form is rule and (such as Fig. 5) spherical in shape, hemoglobin carrying drug ratio 8%, oxygen affinity P
50The value 36mmHg, Hill coefficient 2.90, pH value 7.3, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity (such as Fig. 6).
1, preparation method
(1) preparation of Mal-PEG-PCL and MPEG-PCL
Respectively with the poly glycol monomethyl ether (MPEG5000) of single maleimide Polyethylene Glycol (Mal-PEG5000) of 8g caprolactone and 2g molecular weight 5000 or 2g molecular weight 5000 in mass ratio 4:1 place there-necked flask, add the inferior stannum of octoate catalyst and an amount of toluene, in 137 ℃ of oil baths, reacting 6 hours under the condition of mechanical agitation and nitrogen protection;
After the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain Mal-PEG-PCL and MPEG-PCL again.
(2) contain the preparation of nano-granule freeze-dried powder of the PEG-PCL of maleimide amine functional group
With 5mg superoxide dismutase SOD, the 5mg cat catalase, the 5mg peroxidase is dissolved in 2ml PBS (PH 7.4), speed with per minute 0.5ml splashes into the dichloromethane solution that 20ml contains 20mgMal-PEG-PCL and 180mg MPEG-PCL under sonic oscillation, emulsifying formed colostrum in 5 minutes on the emulsion dispersion machine, then colostrum is added to stir to spend the night in the PVA aqueous solution of 200ml 0.1% and volatilize organic solvent, lyophilizing namely gets the PEG-PCL nano-granule freeze-dried powder that contains the maleimide amine functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Hoptoglobin and sulfhydrylization reagent Traut ' s, according to mol ratio 1:40, the lucifuge stirring reaction is 2 hours under the logical High Purity Nitrogen condition, and the sephadex column separation obtains the sulfhydrylation hoptoglobin;
10mg people's hoptoglobin is dissolved in the 10ml phosphate buffer, adds the PEG-PCL nano-granule freeze-dried powder that 50mg contains the maleimide amine functional group, 4 ℃ vibrated PBS solution washing and centrifugalize under 12000g 24 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 6 hours, reactant liquor is collected in the eluent that there is the hemoglobin characteristic absorption in the 400-600nm zone through the gel chromatography column purification, lyophilizing behind ultrafiltration and concentration.
2, physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 254nm(such as Fig. 5), transmission electron microscope observing nanoparticle form is rule and (such as Fig. 8) spherical in shape, hemoglobin carrying drug ratio 9%, oxygen affinity P
50The value 10mmHg, Hill coefficient 1.30, pH value 7.4, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity (such as Fig. 9).
Embodiment 5 hemoglobins of the present invention are taken the preparation of the carrier of oxygen
1, preparation method
(1) preparation of Mal-PEG-PCL and MPEG-PCL
Respectively with the poly glycol monomethyl ether (mpeg 3 000) of single maleimide Polyethylene Glycol (Mal-PEG3500) of 8.33g caprolactone and 1.67g molecular weight 3500 or 1.67g molecular weight 3000 in mass ratio 5:1 place there-necked flask; add the inferior stannum of octoate catalyst and an amount of toluene, in 90 ℃ of oil baths, reacting 24 hours under the condition of mechanical agitation and nitrogen protection.Then temperature is risen to 180 ℃, and reaction system is evacuated to vacuum.After 20 minutes, cessation reaction, after the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain Mal-PEG-PCL and MPEG-PCL again.
(2) contain the preparation of nano-granule freeze-dried powder of the PEG-PCL of maleimide amine functional group
The 10mg ascorbic acid is dissolved in 2ml PBS (PH 7.4), speed with per minute 0.5ml splashes into the dichloromethane solution that 20ml contains 8mg Mal-PEG-PCL and 192mg MPEG-PCL under sonic oscillation, emulsifying formed colostrum in 5 minutes on the emulsion dispersion machine, then colostrum is added to stir to spend the night in the PVA aqueous solution of 200ml 0.1% and volatilize organic solvent, lyophilizing namely gets the PEG-PCL nano-granule freeze-dried powder that contains the maleimide amine functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Hoptoglobin and sulfhydrylization reagent Traut ' s, according to mol ratio 1:40, the lucifuge stirring reaction is 2 hours under the logical High Purity Nitrogen condition, and the sephadex column separation obtains the sulfhydrylation hoptoglobin;
10mg people's hoptoglobin is dissolved in the 10ml phosphate buffer, adds the PEG-PCL nano-granule freeze-dried powder that 50mg contains the maleimide amine functional group, 4 ℃ vibrated PBS solution washing and centrifugalize under 12000g 24 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 6 hours, reactant liquor is collected in the eluent that there is the hemoglobin characteristic absorption in the 400-600nm zone through the gel chromatography column purification, lyophilizing behind ultrafiltration and concentration.
2, physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 320nm, and transmission electron microscope observing nanoparticle form is rule and spherical in shape, hemoglobin carrying drug ratio 6%, oxygen affinity P
50The value 23mmHg, Hill coefficient 2.41, pH value 7.3, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity.
1, preparation method
(1) preparation of Mal-PEG-PLA and MPEG-PLA
5g lactide (D, L-lactide) with the poly glycol monomethyl ether (MPEG5000) of single maleimide Polyethylene Glycol (Mal-PEG5000) of 5g molecular weight 5000 or 5g molecular weight 5000 in mass ratio 1:1 place there-necked flask, add the inferior stannum of octoate catalyst and an amount of toluene, in 135 ℃ of oil baths, reacting 10 hours under the condition of mechanical agitation and nitrogen protection, temperature is risen to 180 ℃, and reaction system is evacuated to vacuum;
After 30 minutes, cessation reaction after the question response system temperature drops to room temperature, adds the dichloromethane dissolving, again with cold petroleum ether precipitation washing 2 times.Polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain Mal-PEG-PLA and MPEG-PLA again.
(2) contain the preparation of nano-granule freeze-dried powder of the PEG-PLA of maleimide amine functional group
Get respectively 5mg Mal-PEG-PLA and 150mg MPEG-PLA is dissolved in 5ml acetone, take out acetone with Rotary Evaporators, then add the 10ml distilled water, stir in 60 degree heating in water bath, namely obtain containing the PEG-PLA grain aqueous solution of maleimide amine functional group, lyophilizing namely gets the nano-granule freeze-dried powder of the PEG-PLA that contains the maleimide amine functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Hoptoglobin and sulfhydrylization reagent Traut ' s, according to mol ratio 1:40, the lucifuge stirring reaction is 2 hours under the logical High Purity Nitrogen condition, and the sephadex column separation obtains the sulfhydrylation hoptoglobin;
People's hoptoglobin of 10mg sulfhydrylation is dissolved in the 10ml phosphate buffer, adds the PEG-PLA nano-granule freeze-dried powder that 100mg contains the maleimide amine functional group, 4 ℃ of lucifuges were vibrated PBS solution washing and centrifugalize under 12000g 10 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 2 hours, reactant liquor centrifugalize under 12000g, regional without till absorbing at 400-600nm to the upper strata clear liquid with the phosphate buffer washing.
(2) physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 120nm, and transmission electron microscope observing nanoparticle form is rule and spherical in shape, hemoglobin carrying drug ratio 10%, oxygen affinity P
50The value 27mmHg, Hill coefficient 2.58, pH value 7.2, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity.
Embodiment 7 hemoglobins of the present invention are taken the preparation of the carrier of oxygen
1, preparation method
(1) NH
2The preparation of-PEG-PCL and MPEG-PCL
Respectively with the mono amino Polyethylene Glycol (NH of 8.89g caprolactone and 1.11g molecular weight 3500
2-PEG3500) or the poly glycol monomethyl ether of 1.11g molecular weight 3000 (mpeg 3 000) in mass ratio 8:1 place there-necked flask; add the inferior stannum of octoate catalyst and an amount of toluene, in 100 ℃ of oil baths, reacting 12 hours under the condition of mechanical agitation and nitrogen protection.Then temperature is risen to 130 ℃, and reaction system is evacuated to vacuum;
After 30 minutes, cessation reaction is after the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain NH again
2-PEG-PCL and MPEG-PCL.
(2) contain the preparation of nano-granule freeze-dried powder of the PEG-PCL of amido functional group
PVA aqueous solution with 1% is as water, and then first emulsifying 2 minutes on the emulsion dispersion machine drip 5ml with the speed of per minute 0.5ml and contain 10mg NH
2The dichloromethane solution of-PEG-PCL and 90mg MPEG-PCL, emulsifying 10 minutes on the emulsion dispersion machine after dripping, Rotary Evaporators is taken out dichloromethane, and lyophilizing namely gets the nano-granule freeze-dried powder of the PEG-PCL that contains amido functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Get the above-mentioned nano-granule freeze-dried powder 100mg for preparing, be dissolved in the 10ml PBS buffer;
Again 10mg people's hoptoglobin is dissolved in 10ml PBS buffer (PH 7.4), adds EDC and NHS to solution, the carboxyl of activated protein.Behind room temperature reaction 2h, under the condition that stirs, this solution is added drop-wise in the aqueous solution of nanoparticle, 4 ℃ of lucifuges were vibrated PBS solution washing and centrifugalize under 12000g 24 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 4 hours, reactant liquor centrifugalize under 12000g, regional without till absorbing at 400-600nm to the upper strata clear liquid with the phosphate buffer washing.
2, physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 400nm, and transmission electron microscope observing nanoparticle form is rule and spherical in shape, hemoglobin carrying drug ratio 7%, oxygen affinity P
50The value 19mmHg, Hill coefficient 1.52, pH value 7.4, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity.
1, preparation method
(1) preparation of COOH-PEG-PCL and MPEG-PCL
Respectively with the poly glycol monomethyl ether (MPEG2000) of the mono carboxylic Polyethylene Glycol (COOH-PEG2000) of 8.89g caprolactone and 1.11g molecular weight 2000 or 1.11g molecular weight 2000 in mass ratio 8:1 place there-necked flask; add the inferior stannum of octoate catalyst and an amount of toluene, in 100 ℃ of oil baths, reacting 12 hours under the condition of mechanical agitation and nitrogen protection.Then temperature is risen to 130 ℃, and reaction system is evacuated to vacuum;
After 30 minutes, cessation reaction, after the question response system temperature drops to room temperature, polymer poured in the cold petroleum ether precipitate, with sedimentation and filtration, and precipitation is placed on makes solvent evaporates to polymer cure in the fume hood, the product vacuum drying is by the dialysis purification, and lyophilizing can obtain COOH-PEG-PCL and MPEG-PCL again.
(2) contain the preparation of nano-granule freeze-dried powder of the PEG-PCL of carboxyl functional group
Then, PVA aqueous solution with 1% is as water, first emulsifying 2 minutes on the emulsion dispersion machine, then the speed with per minute 0.5ml drips the dichloromethane solution that 5ml contains 10mg COOH-PEG-PCL and 90mgMPEG-PCL, emulsifying 10 minutes on the emulsion dispersion machine after dripping, Rotary Evaporators is taken out dichloromethane, and lyophilizing namely gets the nano-granule freeze-dried powder of the PEG-PCL that contains amido functional group.
(3) hemoglobin is taken the preparation of the carrier of oxygen
Get the above-mentioned nano-granule freeze-dried powder 100mg for preparing, be dissolved in the 10ml PBS buffer, add EDC and NHS to solution, the carboxyl on the postactivated nanoparticle of room temperature reaction 2h surface;
10mg people's hoptoglobin is dissolved in 10ml PBS buffer (PH 7.4) again, is added drop-wise in the reactant liquor of above-mentioned nanoparticle, 4 ℃ of lucifuges were vibrated PBS solution washing and centrifugalize under 12000g 24 hours;
Collect lower floor's nanoparticle, be dissolved in the 5ml phosphate buffer, add the 15mg human cord blood hemoglobin, 4 ℃ of lucifuge stirring reactions 4 hours, reactant liquor centrifugalize under 12000g, regional without till absorbing at 400-600nm to the upper strata clear liquid with the phosphate buffer washing.
2, physicochemical property detects
Detection method is with embodiment 1.
Testing result: the particle diameter that hemoglobin of the present invention is taken the carrier of oxygen is about 320nm, and transmission electron microscope observing nanoparticle form is rule and spherical in shape, hemoglobin carrying drug ratio 8%, oxygen affinity P
50The value 21mmHg, Hill coefficient 2.29, pH value 7.5, the mtt assay testing result show this nanoparticle to the HEK293 cell without overt toxicity.
The vasoactive that experimental example 1 hemoglobin of the present invention is taken the carrier of oxygen detects
1, prepare respectively according to embodiment 3 and embodiment 4 take the carrier of oxygen 1 and 2(such as Figure 10).
2, vasoactive detects: 9 of male mouse of kunming, 22-25g is divided into Purification of Human cord blood hemoglobin group at random, takes 1 group of the carrier of oxygen, takes 2 groups of the carriers of oxygen, 3 every group.After sobering animal is weighed, place the BP-2000 Noninvasive Blood Pressure Measurement System, the afterbody pressure measurement, mensuration obtains basic blood pressure, then according to dividing into groups respectively from tail intravenous injection 16% blood volume (theoretical maximum tolerated dose, about 0.3ml/25g mice) human cord blood hemoglobin solution is taken the carrier of oxygen 1 solution and is taken the carrier of oxygen 2 solution, detects every group of situation of change of injecting blood pressure in rear 60 minutes.Before injecting, respectively with normal saline with human cord blood hemoglobin, take the carrier of oxygen 1 lyophilized powder and take the carrier of oxygen 2 lyophilized powders and be diluted to final concentration 8g/dl.The result represents with mean ± standard deviation,
*P＜0.05,
*P＜0.01vs basic blood pressure.
Testing result is as shown in figure 11: behind the Purification of Human cord blood hemoglobin infusion, can cause that animal blood pressure obviously improves, showing has significant vasoconstrictor activity; And inject after two kinds of hemoglobins take the carrier of oxygen, all do not find the significant change of animal blood pressure, basically identical with base state, show that hemoglobin of the present invention takes the carrier of oxygen without obviously blood vessel side effect.
Experimental result explanation, hemoglobin of the present invention are taken the carrier of oxygen and are injected in vivo afterwards without hyper tensive reactions, prove that it is safe, can not produce free hemoglobin.
To sum up, hemoglobin of the present invention is taken the carrier of oxygen can not produce free hemoglobin in vivo, overcome the intrinsic defective of HBOCs based article, and, its surface can be quantitatively in conjunction with hemoglobin with the controlled active function groups of quantity, and the product quality that obtains is stable, safety and controllability all significantly improve, and have extremely good potential applicability in clinical practice.
Claims (15)
1. a hemoglobin is taken the carrier of oxygen, it is characterized in that: comprise hemoglobin, hoptoglobin and polyethylene glycol-ester nanoparticles, hemoglobin is connected to polyethylene glycol-ester nanoparticles surface by hoptoglobin.
2. hemoglobin according to claim 1 is taken the carrier of oxygen, it is characterized in that: its particle diameter is 80 ~ 400nm.
3. hemoglobin according to claim 2 is taken the carrier of oxygen, it is characterized in that: its particle diameter is 200 ~ 300nm.
4. hemoglobin according to claim 1 is taken the carrier of oxygen, it is characterized in that: the content of hemoglobin is 6 ~ 12%(w/w), and the mol ratio of hemoglobin and hoptoglobin is 1:1.
5. hemoglobin according to claim 1 is taken the carrier of oxygen, it is characterized in that: described polyethylene glycol-ester nanoparticles is PEG-PCL nanoparticle or polyethylene glycol-polylactic acid nanoparticle.
6. hemoglobin according to claim 1 is taken the carrier of oxygen, it is characterized in that: described polyethylene glycol-ester nanoparticles is connected by thioether bond or amido link with hoptoglobin.
7. hemoglobin according to claim 1 is taken the carrier of oxygen, it is characterized in that: described hemoglobin is taken carrier of oxygen oxygen affinity P
50Value is 10 ~ 36mmHg, and the Hill coefficient is 1.30 ~ 2.90, and pH value is 7.2 ~ 7.5.
8. hemoglobin according to claim 1 is taken the carrier of oxygen, it is characterized in that: it also comprises antioxidant, and antioxidant is wrapped in the polyethylene glycol-ester nanoparticles.
9. hemoglobin according to claim 8 is taken the carrier of oxygen, it is characterized in that: described antioxidant is one or more the combination in ascorbic acid, superoxide dismutase, catalase, peroxidase, vitamin E, glutathion, sodium dithionite, the N-acetylcystein.
10. the described hemoglobin of claim 1 is taken the preparation method of the carrier of oxygen, and it is characterized in that: it comprises the steps:
A, get the hoptoglobin of hoptoglobin, sulfhydrylation hoptoglobin or activated carboxylic, and be dissolved in the phosphate buffer with the polyethylene glycol-ester nanoparticles of functional group, the weight ratio of hoptoglobin and nanoparticle is 1:5 ~ 10,4 ℃ shook 18 ~ 30 hours, the phosphate buffer washing, 10000 ~ 14000g is centrifugal, gets precipitation;
B, step a gained precipitation is dissolved in the phosphate buffer, adds hemoglobin, the weight ratio of hemoglobin and hoptoglobin is (2 ~ 4): (1 ~ 3), 4 ℃ of lucifuges were reacted 2 ~ 6 hours, and separation and purification is concentrated freeze-dried, namely gets hemoglobin and takes the carrier of oxygen.
11. preparation method according to claim 10 is characterized in that: the described functional group of step a is single maleimide, carboxyl or amino.
12. preparation method according to claim 10 is characterized in that: the described polyester of step a is polylactic acid or polycaprolactone.
13. preparation method according to claim 10 is characterized in that: wrap up antioxidant in the described nanoparticle of step a.
14. preparation method according to claim 10 is characterized in that: among the described step a, hoptoglobin and nanoparticle weight ratio are 1:5 or 1:10; Shook 24 hours; 12000g is centrifugal.
15. preparation method according to claim 10 is characterized in that: among the described step b, the weight ratio of hemoglobin and hoptoglobin is 3:2; Lucifuge reaction 2 hours; Isolation and purification method is: centrifugal or gel chromatography column separating purification, to supernatant or eluent till 400-600nm is without obvious absorption peaks, collecting precipitation or eluent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103556354A CN102861322A (en) | 2012-08-09 | 2012-09-21 | Hemoglobin oxygen carrier and preparation method thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210281432 | 2012-08-09 | ||
| CN201210281432.5 | 2012-08-09 | ||
| CN2012103556354A CN102861322A (en) | 2012-08-09 | 2012-09-21 | Hemoglobin oxygen carrier and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102861322A true CN102861322A (en) | 2013-01-09 |
Family
ID=47440634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012103556354A Pending CN102861322A (en) | 2012-08-09 | 2012-09-21 | Hemoglobin oxygen carrier and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102861322A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103251936A (en) * | 2013-04-19 | 2013-08-21 | 中国人民解放军成都军区总医院 | Oxygen carrier based on hemoglobin-haptoglobin compound and preparation method of oxygen carrier |
| CN105749257A (en) * | 2016-01-25 | 2016-07-13 | 四川大学华西医院 | Hemoglobin oxygen-carrying nano gel and preparation method and application thereof |
| CN113552348A (en) * | 2021-07-28 | 2021-10-26 | 四川省医学科学院·四川省人民医院 | Nanoparticle solution for detecting SARS-CoV-2 coronavirus S protein, preparation method, kit and application |
| CN115969969A (en) * | 2023-02-13 | 2023-04-18 | 中国科学院长春应用化学研究所 | Virus-like structure nanoparticle vaccine and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1419936A (en) * | 2001-11-16 | 2003-05-28 | 中国科学院过程工程研究所 | Blood substitute of hemoglobin microcapsule and preparing method thereof |
| CN1733298A (en) * | 2005-08-16 | 2006-02-15 | 华东理工大学 | Microcapsule type blood substitute based on hemoglobin and process for preparing the same |
| CN101306196A (en) * | 2008-07-03 | 2008-11-19 | 中国科学院长春应用化学研究所 | Biodegradable nano-particles bonded with hemoglobin and production method thereof |
-
2012
- 2012-09-21 CN CN2012103556354A patent/CN102861322A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1419936A (en) * | 2001-11-16 | 2003-05-28 | 中国科学院过程工程研究所 | Blood substitute of hemoglobin microcapsule and preparing method thereof |
| CN1733298A (en) * | 2005-08-16 | 2006-02-15 | 华东理工大学 | Microcapsule type blood substitute based on hemoglobin and process for preparing the same |
| CN101306196A (en) * | 2008-07-03 | 2008-11-19 | 中国科学院长春应用化学研究所 | Biodegradable nano-particles bonded with hemoglobin and production method thereof |
Non-Patent Citations (6)
| Title |
|---|
| A. GERSON GREENBURG ET AL.: "Civilian Uses of Hemoglobin-based Oxygen Carriers", 《ARTIFICIAL ORGANS》 * |
| ERIC M KETCHAM ET AL.: "Hemoglobin-Based Oxygen Carriers: Development and Clinical Potential", 《ANNALS OF EMERGENCY MEDICINE》 * |
| TOBY A. SILVERMAN ET AL.: "Hemoglobin-based oxygen carriers: current status and future directions", 《TRANSFUSION》 * |
| 王岱娟: "血红蛋白氧载体研究进展及应用现状", 《中国生化药物杂志》 * |
| 联系人：赵颖: "一种新型血红蛋白类携氧纳米粒子", 《四川省技术转移（乐山）中心》 * |
| 路秀玲: "修饰血红蛋白作为红细胞代用品的研究进展和挑战", 《生物工程学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103251936A (en) * | 2013-04-19 | 2013-08-21 | 中国人民解放军成都军区总医院 | Oxygen carrier based on hemoglobin-haptoglobin compound and preparation method of oxygen carrier |
| CN105749257A (en) * | 2016-01-25 | 2016-07-13 | 四川大学华西医院 | Hemoglobin oxygen-carrying nano gel and preparation method and application thereof |
| CN105749257B (en) * | 2016-01-25 | 2019-12-06 | 四川大学华西医院 | Hemoglobin oxygen-carrying nano gel and preparation method and application thereof |
| CN113552348A (en) * | 2021-07-28 | 2021-10-26 | 四川省医学科学院·四川省人民医院 | Nanoparticle solution for detecting SARS-CoV-2 coronavirus S protein, preparation method, kit and application |
| CN115969969A (en) * | 2023-02-13 | 2023-04-18 | 中国科学院长春应用化学研究所 | Virus-like structure nanoparticle vaccine and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tan et al. | A novel MPEG-PDLLA-PLL copolymer for docetaxel delivery in breast cancer therapy | |
| Luk et al. | Safe and immunocompatible nanocarriers cloaked in RBC membranes for drug delivery to treat solid tumors | |
| Rios-Doria et al. | A versatile polymer micelle drug delivery system for encapsulation and in vivo stabilization of hydrophobic anticancer drugs | |
| Ren et al. | pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery | |
| Jia et al. | Construction and evaluation of hemoglobin‐based capsules as blood substitutes | |
| Bhadra et al. | PEGylated peptide dendrimeric carriers for the delivery of antimalarial drug chloroquine phosphate | |
| KR100904931B1 (en) | Nanoparticles and Method for the Preparation Thereof | |
| Gao et al. | Cationic amylose-encapsulated bovine hemoglobin as a nanosized oxygen carrier | |
| Ding et al. | Dual-functional bio-derived nanoparticulates for apoptotic antitumor therapy | |
| Jansman et al. | Hemoglobin-based oxygen carriers incorporating nanozymes for the depletion of reactive oxygen species | |
| He et al. | Hemocompatibility of folic-acid-conjugated amphiphilic PEG-PLGA copolymer nanoparticles for co-delivery of cisplatin and paclitaxel: treatment effects for non-small-cell lung cancer | |
| Weiss et al. | The First Step into the Brain: Uptake of NIO‐PBCA Nanoparticles by Endothelial Cells in vitro and in vivo, and Direct Evidence for their Blood–Brain Barrier Permeation | |
| CN106727307A (en) | A kind of preparation and application for reducing sensitive nano-micelle | |
| Mu et al. | Hyperbranched polyglycerol-modified graphene oxide as an efficient drug carrier with good biocompatibility | |
| CN102861322A (en) | Hemoglobin oxygen carrier and preparation method thereof | |
| Wagh et al. | A short circulating peptide nanofiber as a carrier for tumoral delivery | |
| WO2011031849A2 (en) | Anionic lipids and lipid nanostructures and methods of producing and using same | |
| Shi et al. | Hemoglobin conjugated micelles based on triblock biodegradable polymers as artificial oxygen carriers | |
| Li et al. | Asymmetric copolymer vesicles to serve as a hemoglobin vector for ischemia therapy | |
| Jansman et al. | Hemoglobin-based oxygen carriers camouflaged with membranes extracted from red blood cells: Optimization and assessment of functionality | |
| Hickey et al. | Synthesis of hemoglobin-based oxygen carrier nanoparticles by desolvation precipitation | |
| Shalaby et al. | Nanoparticles based on linear and star-shaped poly (ethylene glycol)-poly (ε-caprolactone) copolymers for the delivery of antitubulin drug | |
| Li et al. | A programmed cell‐mimicking nanoparticle driven by potato alkaloid for targeted cancer chemoimmunotherapy | |
| JP2017526740A (en) | Compositions and methods for inducing nanoparticle-mediated microvascular embolization of tumors | |
| CN106137962A (en) | A kind of glioma target polymer micelle loading carmustine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130109 |