CN102861323A - A novel blood substitute with complete red blood cell functions - Google Patents
A novel blood substitute with complete red blood cell functions Download PDFInfo
- Publication number
- CN102861323A CN102861323A CN2012101796705A CN201210179670A CN102861323A CN 102861323 A CN102861323 A CN 102861323A CN 2012101796705 A CN2012101796705 A CN 2012101796705A CN 201210179670 A CN201210179670 A CN 201210179670A CN 102861323 A CN102861323 A CN 102861323A
- Authority
- CN
- China
- Prior art keywords
- sod
- hemoglobin
- cat
- soluble complex
- sfhb
- 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
- 210000003743 erythrocyte Anatomy 0.000 title description 37
- 239000003633 blood substitute Substances 0.000 title description 4
- 230000003915 cell function Effects 0.000 title 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 50
- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 48
- 102000001554 Hemoglobins Human genes 0.000 claims abstract description 48
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 25
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 74
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 74
- 102000003846 Carbonic anhydrases Human genes 0.000 claims description 70
- 108090000209 Carbonic anhydrases Proteins 0.000 claims description 70
- 102000016938 Catalase Human genes 0.000 claims description 68
- 108010053835 Catalase Proteins 0.000 claims description 68
- 108010001708 stroma free hemoglobin Proteins 0.000 claims description 53
- 239000002473 artificial blood Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 32
- 238000004132 cross linking Methods 0.000 abstract description 6
- 230000003078 antioxidant effect Effects 0.000 abstract description 3
- 230000002708 enhancing effect Effects 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract 2
- 239000003963 antioxidant agent Substances 0.000 abstract 1
- 230000002459 sustained effect Effects 0.000 abstract 1
- 210000004369 blood Anatomy 0.000 description 30
- 239000008280 blood Substances 0.000 description 30
- 239000000203 mixture Substances 0.000 description 22
- 201000010099 disease Diseases 0.000 description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 16
- 239000012530 fluid Substances 0.000 description 15
- 238000000034 method Methods 0.000 description 13
- 206010063837 Reperfusion injury Diseases 0.000 description 12
- 208000024891 symptom Diseases 0.000 description 12
- 208000032456 Hemorrhagic Shock Diseases 0.000 description 11
- 206010049771 Shock haemorrhagic Diseases 0.000 description 11
- 230000032258 transport Effects 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000002255 enzymatic effect Effects 0.000 description 9
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 235000018977 lysine Nutrition 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 239000004472 Lysine Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 208000010125 myocardial infarction Diseases 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 206010008190 Cerebrovascular accident Diseases 0.000 description 6
- 208000006011 Stroke Diseases 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000036770 blood supply Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 102000018832 Cytochromes Human genes 0.000 description 3
- 108010052832 Cytochromes Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000006703 hydration reaction Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 108010050918 polyhemoglobin Proteins 0.000 description 2
- 108010048524 polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase Proteins 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000008156 Ringer's lactate solution Substances 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 239000006096 absorbing agent Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 108700042971 cyanomethemoglobin Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 210000001105 femoral artery Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 150000002669 lysines Chemical class 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 1
- 229960002695 phenobarbital Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108010075164 polyhemoglobin-superoxide dismutase-catalase Proteins 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present document describes a soluble complex which comprises a hemoglobin cross-linked with an antioxidant enzyme, and acid-base and carbon dioxide transport enzyme. Cross-linking is enhancing in vivo circulation time of the soluble complex, and prevents harmful in vivo effects that could be sustained by an individual subjected to the individual free components of the soluble complex.
Description
The related application of quoting
The application requires to enjoy in the U.S. Provisional Patent Application US61/490 that submitted on May 26th, 2011,304 priority.
Technical field
The method that the present invention relates generally to the artificial blood succedaneum and uses and make.Particularly, the artificial blood succedaneum comprises soluble complex, the preparation of this complex is by with stroma-free hemoglobin (SFHb) or hemoglobin (Hb) and antioxidase [superoxide dismutase (SOD) and catalase (CAT)] and enzyme (carbonic anhydrase with Acid-Base balance and carbon dioxide transport function, CA) crosslinked, thus poly--[SFHb-SOD-CAT-CA] soluble complex formed.
Background technology
In first aid and surgical operation, the raising of blood transfusion demand caused the urgent needs to erythrocyte (RBC) succedaneum.We investigate the dissimilar oxygen blood substitutes of taking.A kind of poly-hemoglobin (polyHb) that is based on nanometer biotechnology in this series products.Hemoglobin and bifunctional reagent such as diacid or glutaraldehyde cross-linking can be caused the formation of polyHb.Studies show that these 4-5 hemoglobin (Hb) thus the complex of molecule can be by keeping the Hb level in a safety range and transporting enough oxygen and replenish losing blood in the surgical operation.Wherein two kinds of nanometer biotechnology products have reached III clinical trial phase [Jahr JS et al, J Trauma 64:1484-97 (2008) and Moore EE et al, J Am Coll Surg.208:1-13 (2009)], but because side effect, U.S. FDA is not ratified these products.These side effect are included among the first aid injury patient who gathers hemoglobin, 3% incidence rate of myocardial infarction is arranged, and by contrast, the patient who only accepts saline solution only have 0.6% incidence rate.This is likely because ischemical reperfusion injury causes, especially for patients that ischemic heart desease is arranged before those.In addition, in the case of all hemoglobin shock, also there is the variation [Moore EE et al, J Am Coll Surg.208:1-13 (2009)] with soda acid gathered of carbon dioxide.
Based on the blood substitute of hemoglobin such as polyHb compared to the advantage of blood donor's blood supply be:
(1) they do not contain blood group antigen.Therefore they can there and then be transfused blood and not need that the patient is transferred to hospital and carry out cross matching and bracket for blood grouping, and this needs blood donor's blood supply.This shifts in first aid or in nature or human-made disasters and is even more important in very difficult.
(2) different from RBC, polyHb can carry out sterilization treatment, therefore in epiphytotics situation, there is not the possibility of infective agent (now, infect in the epidemic diseases of cause in other types in the future, the early stage case of blood donor's blood supply that AIDS and the HIV that causes thus pollute just can be avoided).
(3) blood donor's blood supply must be stored in 4 ℃ and can only preserve about 42 days.PolyHb can be stored in room temperature more than a year.This just makes PolyHb be stored in to be placed in the plastic bag any place that may need this product at least 1 year, and if be stored in the lower temperature time can be longer.
Yet polyHb only has the wherein a kind of of three kinds of critical functions of RBC.Three kinds of critical functions of RBC are: (1) transports and for oxygen supply, carbon dioxide is transported and removed in (2), and (3) remove the oxygen-derived free radicals that is harmful to.At present, the RBC succedaneum of the industrialization of polyHb form can only be transported oxygen, and because side effect, they do not obtain the approval of U.S. FDA.In addition, the antioxidase SOD of RBC extract and the activity of CAT are fewer than prevention in serious hemorrhagic shock, apoplexy, the ischemical reperfusion injury in myocardial infarction and other situations.Now SOD and CAT have been combined with polyHb and have formed polyHb-SOD-CAT (D ' United States Patent (USP) 5,606,025 that has of Agnillo and Chang).Yet this complex still lacks a kind of important function of RBC, and namely it can not transport and remove carbon dioxide from blood, therefore can cause side effect.The more perfect RBC succedaneum that therefore, need to have whole three kinds of functions of RBC is treated and is lost blood.
In addition, also need more perfect RBC succedaneum to prevent ischemical reperfusion injury.
Summary of the invention
According to embodiment, a kind of soluble complex is provided, it comprises with the antioxidase that is used for the minimizing reactive oxygen species and is used for transhipment CO
2And O
2Acid-Base balance and the crosslinked hemoglobin of carbon dioxide transport enzyme.
Antioxidase can be selected from superoxide dismutase (SOD), catalase (CAT), or its combination.
Antioxidase can be the combination of superoxide dismutase (SOD) and catalase (CAT).
The ratio of hemoglobin and superoxide dismutase (SOD) can for about 1g: 4000U to about 1g: 25000U.
The ratio of hemoglobin and superoxide dismutase (SOD) can be about 1g: 18000U.
The ratio of hemoglobin and catalase (CAT) can be for about 25, and 000U is to about 1g: 310,000U.
The ratio of hemoglobin and catalase (CAT) can be about 1g: 310000U.
Acid-Base balance and carbon dioxide transport enzyme can comprise carbonic anhydrase (CA).
The ratio of hemoglobin and carbonic anhydrase (CA) can be about 1g: 80000U to 1g: 250000U.
The ratio of hemoglobin and carbonic anhydrase (CA) can be about 1g: 130000U.
Hemoglobin can comprise stroma-free hemoglobin (SFHb) or hemoglobin (Hb).
Soluble complex can have the molecular weight greater than 100kDa.
Soluble complex can have greater than about 100kDa to the molecular weight greater than about 450kDa.
According to another embodiment, a kind of artificial blood succedaneum is provided, it comprises the soluble complex of the present invention that exists with suitable medicinal liquid form.
According to another embodiment, a kind of method for the treatment of disease symptoms is provided, comprise the soluble complex of the present invention of granting the treatment effective dose to the patient of needs, or artificial blood succedaneum of the present invention.
This disease symptoms can be selected from and lose blood, carbon dioxide characteristic of concentration symptom, ischemical reperfusion injury and caisson disease.
Ischemical reperfusion injury can comprise serious hemorrhagic shock, apoplexy, myocardial infarction, or its combination.
According to another embodiment, a kind of method of removing carbon dioxide from fluid is provided, comprise the soluble complex of the present invention with effective dose, or artificial blood succedaneum of the present invention is processed this fluid.
This fluid can be selected from blood and air.This fluid can be the blood of dialysis.This fluid can be the blood in the artificial lung.
This fluid can be with CO in the environment
2Air after the removing.
According to another embodiment, provide the purposes of soluble complex of the present invention, for the preparation of the medicine for the treatment of disease symptoms.
According to another embodiment, the purposes of soluble complex of the present invention is provided, be used for the treatment of disease symptoms.
According to another embodiment, provide the purposes of artificial blood succedaneum of the present invention, for the preparation of the medicine for the treatment of disease symptoms.
According to another embodiment, the purposes of artificial blood succedaneum of the present invention is provided, be used for the treatment of disease symptoms.
This disease symptoms can be selected from and lose blood, carbon dioxide characteristic of concentration symptom, ischemical reperfusion injury, caisson disease.
Ischemical reperfusion injury comprises serious hemorrhagic shock, apoplexy, myocardial infarction, or its combination.
According to another embodiment, the purposes of soluble complex of the present invention or artificial blood succedaneum of the present invention is provided, remove wherein carbon dioxide for the treatment of fluid.
This fluid can be selected from blood and air.This fluid can be the blood in the artificial lung.This fluid can be the blood of dialysis.
By following detailed description, by reference to the accompanying drawings, the more characteristics and advantages of the present invention can be able to clearly:
Fig. 1 illustrates carbonic anhydrase activity (CA): (1) stroma-free hemoglobin (SFHb)-have erythrocyte composition (SFHb) of CA activity; (2) poly-stroma-free hemoglobin (polySFHb)-be cross-linked to form the erythrocyte composition of polySFHb, it is active to have a very low CA; (3) stroma-free hemoglobin+SOD+CAT+CA (SFHb+SOD+CAT+CA)-enzyme is not by in the crosslinked extract that directly adds the erythrocyte composition to, and (4) poly--Poly-[Hb-SOD-CAT-CA that [Hb-SOD-CAT-CA] (Poly-[Hb-SOD-CAT-CA]) expression is cross-linked to form with said components according to embodiments of the invention], it is active to produce thus gratifying CA.SFHb: stroma-free hemoglobin; PolySFHb: crosslinked stroma-free hemoglobin; SFHb+SOD+CAT+CA: every gram SFHb adds 15000U SOD, 300,000UCAT and 15,300U CA; Poly-[SFHb-SOD-CAT-CA]: crosslinked SFHb+SOD+CAT+CA.
Fig. 2 illustrates superoxide dismutase (SOD): stroma-free hemoglobin (SFHb)-the have erythrocyte composition (SFHb) of very low SOD activity; Poly-stroma-free hemoglobin (polySFHb)-be cross-linked to form the erythrocyte composition of polySFHb has very low SOD activity; Stroma-free hemoglobin+SOD+CAT+CA (SFHb+SOD+CAT+CA)-enzyme is not by in the crosslinked extract that directly adds the erythrocyte composition to, and the Poly-[SFHb-SOD-CAT-CA that poly--[SFHb-SOD-CAT-CA] (Poly-[SFHb-SOD-CAT-CA])-expression is cross-linked to form with said components according to embodiments of the invention], consequent SOD activity improves greatly.SFHb: stroma-free hemoglobin; PolySFHb: crosslinked stroma-free hemoglobin; SFHb+SOD+CAT+CA: every gram SFHb adds 15000U SOD, 300,000U CAT and 15,300U CA; Poly-[SFHb-SOD-CAT-CA]: crosslinked SFHb+SOD+CAT+CA.
Fig. 3 illustrates catalatic activity (CAT): stroma-free hemoglobin (SFHb)-the have erythrocyte composition of very low SOD activity; Poly-stroma-free hemoglobin (polySFHb)-be cross-linked to form the erythrocyte composition of polySFHb has very low SOD activity; Stroma-free hemoglobin+SOD+CAT+CA (SFHb+SOD+CAT+CA)-enzyme is not by in the crosslinked extract that directly adds the erythrocyte composition to, and poly--[SFHb-SOD-CAT-CA] (Poly-[SFHb-SOD-CAT-CA]) represent to be cross-linked to form Poly-[SFHb-SOD-CAT-CA according to embodiments of the invention with said components], consequent CAT activity improves greatly.SFHb: stroma-free hemoglobin; PolySFHb: crosslinked stroma-free hemoglobin; SFHb+SOD+CAT+CA: every gram SFHb adds 15000U SOD, 300,000U CAT and 15,300U CA; Poly-[SFHb-SOD-CAT-CA]: crosslinked SFHb+SOD+CAT+CA.
Test in the body of Fig. 4 explanation according to the soluble complex of embodiments of the invention acquisition.RBC: whole blood, PHcsc: the PolyHb that enzymatic activity is low, PHCSC: the PolyHb-PolyHbCAT-SOD-CA that enzymatic activity is high.
The specific embodiment
In an embodiment, disclose soluble complex, it is by with hemoglobin and antioxidase and Acid-Base balance and the carbon dioxide transport enzyme is crosslinked forms.The erythrocyte extract comprises with very low antioxidase (such as SOD and CAT) activity and acid-base balance and the active hemoglobin of carbon dioxide transport (CA).Yet, when direct crosslinked these enzymes, SOD, the enzymatic activity of CAT and CA can greatly be reduced to the optimum level that is significantly less than.And the antioxidase in the erythrocyte extract (such as SOD and CAT) enough is not used for preventing serious hemorrhagic shock, apoplexy, the ischemical reperfusion injury that occurs in myocardial infarction and the other diseases.In addition, crosslinked CA level afterwards is not enough to keep Acid-Base balance and carbon dioxide transport function, especially when serious ischemia.
Therefore, according to embodiments of the invention, soluble complex of the present invention (for example Poly-[SFHb-SOD-CAT-CA]) comprises three kinds of functions of RBC: it is to have antioxidant activity (SOD, CAT) and can promote transhipment and the carrier of oxygen (PolySFHb) of acid-base balance function (CA activity).It also comprises the SOD of enhancing, thereby the activity of CAT and CA has strengthened the function of this new erythrocyte succedaneum.This crosslinked poly-hemoglobin Poly-[SFHb-SOD-CAT-CA] in SOD and the CAT level than much higher in RBC.Therefore, believe that it also can be used as therapeutic agent and treat or prevent ischemical reperfusion injury, and can promote to remove the carbon dioxide that in serious hemorrhagic shock and other diseases, gathers.
According to embodiments of the invention, the hemoglobin composition in the soluble complex is as a kind of carrier of oxygen.The hemoglobin composition of soluble complex can be prepared by any suitable hemoglobin, suitable hemoglobin is by erythrocyte, the organism of human or animal (cattle, pig etc.) or restructuring Hb or other types, even extract in the synthetic hemoglobin.
Soluble complex of the present invention also comprises the antioxidase for the treatment of effective dose.Antioxidase can be superoxide dismutase (SOD), catalase (CAT), or its combination.These enzymes can be from the source that comes from hemoglobin outward.The SOD enzyme can correspondingly be that the 1g hemoglobin is than the SOD of 4000U to 25000U with the ratio of hemoglobin composition.Preferably, the preferred proportion of hemoglobin and superoxide dismutase (SOD) is about 1g: 18000U.The ratio of CAT enzyme and hemoglobin can correspondingly be the 1g hemoglobin than 25000U to 310, the CAT of 000U.Preferably, the ratio of hemoglobin goods and above-mentioned catalase (CAT) is about 1g: 310000U.
Soluble complex of the present invention also comprises acid-base balance and the carbon dioxide transport enzyme for the treatment of effective dose.Preferably, acid-base balance and carbon dioxide transport enzyme can be carbonic anhydrase (CA), but also can use the synzyme (for example having provides the CA synzyme of active necessary avtive spot) with identical activity.These enzymes can be from the source of external source.The CA enzyme can correspondingly be that about 1g hemoglobin is than the CA of 80000U to 250000U with the ratio of hemoglobin composition.Preferably, the ratio of hemoglobin and above-mentioned carbonic anhydrase (CA) is about 1g: 130000U.
The composition of soluble complex is that chemistry connects, and be separated rapidly because understand after hemoglobin and enzyme are with the free form input, and they may also can cause illeffects.Especially sensitivity, anaphylaxis and Immunoreactivity.Therefore, according to embodiments of the invention, soluble complex can be Poly-[SFHb-SOD-CAT-CA], SFHb wherein, SOD, CAT and CA are crosslinked mutually.
The specific activity normal presence of SOD of the present invention and CAT wants high a lot of in RBC, therefore can strengthen antioxidant activity.The activity that strengthens can be prevented the illeffects of oxygen-derived free radicals, and this is in serious hemorrhagic shock, apoplexy, and the ischemia-reperfusion that faces in the myocardial infarction is very important.The CA activity is very important to be because under any hemorrhagic shock or ischemic situation, also has gathering of carbon dioxide, and this also is to need to remove.
According to another embodiment, soluble complex Poly-[SFHb-SOD-CAT-CA] can have at least molecular weight of 100kD.Preferably, Poly-[SFHb-SOD-CAT-CA] soluble complex molecular weight for about 100kDa extremely greater than 450kDa, most preferably, Poly-[SFHb-SOD-CAT-CA] soluble complex has the molecular weight greater than 450kDa.Below table 1 show Poly-[SFHb-SOD-CAT-CA] molecular weight distribution and enzymatic activity.Poly-[SFHb-SOD-CAT-CA] show three kinds of molecular weight constituents: (1) low (＜100kDa), (2) medium (100-450kDa), and (3) high molecular (＞450kDa).Sample comprises the composition that about 86% molecular weight is higher than 100kDa.With low molecular weight part (＜100kDa) removal.Hb, SOD, CAT and CA most of active (being respectively 70%, 82%, 90% and 84%) remaines in molecular weight greater than the Poly-[SFHb-SOD-CAT-CA of 450kDa] in the part.The Hb that the part of molecular weight between 100kDa and 450kDa is contained, SOD, CAT and CA activity are respectively 16%, 11%, and 9% and 13%.Molecular weight is less than the Hb of the part of 100kDa, SOD, and the activity of CAT and CA lower (14%, 5%, 0.5% and 0.05%)---this part comprises uncrosslinked Hb and enzyme and is removed.
Table 1 Poly-[SFHb-SOD-CAT-CA] molecular weight distribution and enzymatic activity
According to another embodiment of the invention, the method for the treatment of disease symptoms by the soluble complex of the present invention for the treatment of effective dose is disclosed.
According to some embodiment, described disease symptoms can be to lose blood, carbon dioxide characteristic of concentration disease symptoms, ischemical reperfusion injury and caisson disease.
Ischemical reperfusion injury can comprise serious hemorrhagic shock, apoplexy, myocardial infarction, or its combination.
According to another embodiment, disclose with the soluble complex of the present invention of effective dose or artificial blood succedaneum of the present invention is removed carbon dioxide from fluid method.
This fluid can be for example blood and air, also can be any fluid of advantageously removing carbon dioxide.For example, this fluid can be the dialysis or artificial lung in blood.According to another embodiment, this fluid can be with CO in the environment
2Air after the removing.
According to another embodiment, a kind of method for preparing soluble complex of the present invention is disclosed.The method comprises the step that mixture is crosslinked, and this mixture comprises stroma-free hemoglobin, antioxidase, acid-base balance and carbon dioxide transport enzyme, and lysine.
The mol ratio of lysine and stroma-free hemoglobin can be about 7: 1 to about 12: 1, or about 8: 1 to about 12: 1, or about 9: 1 to about 12: 1, or about 10: 1 to about 12: 1, or about 11: 1 to about 12: 1, or 7: 1 to about 8: 1, or about 7: 1 to about 9: 1, or about 7: 1 to about 10: 1, or about 7: 1 to about 11: 1, or about 8: 1 to about 9: 1, or about 8: 1 to about 10: 1, or about 8: 1 to about 11: 1, or about 8: 1 to about 12: 1, or about 9: 1 to about 10: 1, or about 9: 1 to about 11: 1, or about 9: 1 to about 12: 1, or about 10: 1 to about 11: 1, or about 11: 1 to about 12: 1.Preferably, the mol ratio of lysine and stroma-free hemoglobin is 7: 1.
According to embodiment, undertaken crosslinked by glutaraldehyde.The mol ratio of glutaraldehyde and stroma-free hemoglobin can be about 8: 1 to about 32: 1, or about 8: 1 to about 12: 1, or about 8: 1 to about 16: 1, or about 8: 1 to about 20: 1, or about 8: 1 to about 24: 1, or about 8: 1 to about 28: 1, or about 12: 1 to about 16: 1, or about 12: 1 to about 20: 1, or about 12: 1 to about 24: 1, or about 12: 1 to about 28: 1, or about 12: 1 to about 32: 1, or about 16: 1 to about 20: 1, or about 16: 1 to about 24: 1, or about 16: 1 to about 28: 1, or about 16: 1 to about 32: 1, or about 20: 1 to about 24: 1, or about 20: 1 to about 28: 1, or about 20: 1 to about 32: 1, or about 24: 1 to about 28: 1, or about 24: 1 to about 32: 1, or about 28: 1 to about 32: 1.The mol ratio of glutaraldehyde and stroma-free hemoglobin can change according to the time length of crosslinked insulation.Preferably, this mol ratio is 16: 1, the insulation cycle can be for example 4 ℃ about 24 hours.Can compare excessive lysine (for example 200: 1) with stroma-free hemoglobin by interpolation and come cessation reaction.This mixture can pass through purification subsequently, dialyses and concentrates to obtain soluble complex.
The present invention can be more readily understood by reference following examples, and these embodiment are for describing the present invention rather than being used for limiting its scope.
Embodiment 1 soluble complex Poly-[SFHb-SOD-CAT-CA] preparation
Raw material
Employed Hb be by erythrocyte extract and come without substrate Hb.SOD, CAT and CA (EC 4.2.1.1, the activity of manufacturer's regulation of 2720 units/milligram solid) are available from Sigma's aldrich (Sigma-Aldrich Ontario, Canada).Other chemical substance of every other AG or reagent are all available from Sigma's aldrich.
Method
With SOD (1050 unit/ml), catalase (21,000 unit/ml) and carbonic anhydrase (1070 unit/ml) add in the solution (7g/dl) of stroma-free hemoglobin that 20mL is dissolved in 50mM sodium phosphate buffer (pH7.4).Before the beginning cross-linking reaction, add the lysine of 1.3M with 7: 1 lysines/Hb mol ratio.With the full reaction vessel of nitrogen, thus the formation of prevention metahemoglobin.With 16: thus 1 glutaraldehyde/Hb mol ratio is added 5% glutaraldehyde beginning cross-linking reaction.Glutaraldehyde is divided into the quartering in 15 minutes, adds, make reaction crosslinked 24 hours by constantly being stirred in 4 ℃ under aerobic conditions.Stop cross-linking reaction by the lysine that adds 2.0M with 200: 1 lysine/Hb mol ratio.Then goods are filtered with 0.45 aseptic μ M filter, use subsequently molecule porous bag filter (MWCO:12000-14000) dialysed overnight in ringer lactate solution.With the micro-concentration tube (amicon, Bei Fuli, MA) of 100kDa at 23 ℃ of crosslinker solutions that concentrated 500 μ L five equilibriums with 2500g in centrifugal 55 minutes.
Embodiment 2 molecular weight distribution
In order to analyze Poly-[SFHb-SOD-CAT-CA] molecular weight distribution, use Sephacryl-300HR post (V with 36 ml/hours flow velocitys
Always=560ml).With 0.1M Tris-HCl and 0.15M NaCl (pH 7.4) eluent balance pillar.Record molecular weight distribution with 280nm UV detector with the recording rate of 1mm/m in.The part of collecting is: (1) molecular weight is higher than 450kDa, and (2) 100-450kDa and (3) are lower than 100kDa.For zoopery, will remove less than the part of 100kDa.Use bag filter (MWCO:12000-14000) and spectrum/gel absorbers to come concentrating sample, and with the concentration of (Drabkin ' s) method Measuring hemoglobin of De Labojin.The sample of five equilibrium is stored in-80 ℃.
The quantitative assay of embodiment 3Hb concentration
The colorimetric determination of the Hb concentration in polySFHb and the polySFHb-CA goods is with sample and De Labo gold reagent (Sigma's aldrich) reaction, then detects the concentration of the cyanomethemoglobin solution that obtains at 540nm by spectrophotometry.
The mensuration of embodiment 4CA activity
The hydrated carbon dioxide effect of CA is by based on Henry (Henry) method (14), and the electricity δ pH detection method of Weir uncle and Anderson (Wilbur andAnderson) method (15) is measured.Weir primary-Anderson active unit of CA is defined as pH value that per minute can cause 0.02M Tris buffer and is down to 6.3 enzyme amount from 8.3.Before use, will comprise that the specimen that contains CA and the reagent solution of 0.02M Tris.HCl buffer (pH8.0) are stored in 0-4 ℃.By with CO
2Bubbling prepares the CO of dissolving in distilled water
2Thereby obtain the substrate of experiment usefulness.Come initial action by adding substrate, and the pH of record reactant mixture is down to for 6.3 time (T) from pH8.3.(CO of CA catalysis PA) measures in Fisher Scientific, Pittsburgh to use the Fisher Accumet Basic pH meter of band MI-407 (P) pH electrode pin (Microelectrodes Inc., Bedford, NH)
2The variation of the pH that hydration reaction causes.Contrast (the T of experiment
0) the same except not containing mixture contained the specimen.According to following formula, can within very short time, convert measured value to W-A unit:
W-A?unit＝[2x(T
0-T)]/T
The unit of drafting is to the figure of Hb/CA concentration (mg/mL) subsequently.
The mensuration of embodiment 5 CAT activity
For catalatic mensuration, measure H with the spectrophotometer of UV240nm
2O
2Rate of disappearance.Reactant mixture comprises the 50mM phosphate buffer of 2mL, the 30mM H of pH7.0 and 1mL
2O
2At 240nm monitoring H
2O
2The reduction of level 15 seconds.With the blood sample of same concentrations or do not contain H
2O
2The phosphoric acid buffer liquid mixture as blank.Catalase activity is expressed as unit corresponding to every gram hemoglobin.
The mensuration of embodiment 6 SOD activity
The mensuration of SOD activity is based on peroxide and reduces cytochrome C.Reagent solution comprises and is dissolved in the 50mL kaliumphosphate buffer, 0.1mM EDTA, the xanthine among the pH7.8 (50mM), cytochrome C (10 μ M) and CAT (500 unit/ml).Reaction system comprises specimen and reagent solution.Come initial action by adding xanthine oxidase.With 0.154M NaCl as blank.With the reduction of spectrophotometer at 550nm place record cytochrome C.
Embodiment 7 SFHb, polySFHb and soluble complex Poly-[SFHb-SOD-CAT-CA] enzymatic activity
Referring now to Fig. 1-3.PolySFHb-SOD-CAT-CA forms by nanometer biotechnology as cross-linking agent with glutaraldehyde.Measure (1) SFHb, (2) PolySFHb, (3) PolySFHb+SOD+CAT+CA is (with SOD, CAT and CA add among the PolySFHb with the solution form) and (4) Poly-[SFHb-SOD-CAT-CA] enzymatic activity of (with SOD, the PolySFHb that CAT and CA are crosslinked).In order to eliminate error, prepared three crowdes of polySFHb and Poly-[SFHb-SOD-CAT-CA] and minute BT(batch testing).Cross-linking reaction has greatly reduced SOD, the enzymatic activity of CAT and CA.Therefore, independent PolySFHb does not contain the enzymatic activity the same just like RBC.The amount that is used for crosslinked SOD and CAT is more a lot of than the height that RBC finds.This is essential for SOD and the CAT level that is used for the treatment of ischemical reperfusion injury.Simultaneously in PolySFHb and CA are crosslinked, must add the CO that CA (1070 unit/ml) catches up with SFHb among the RBC
2Hydration is active.The enrichment of enzyme has improved Poly-[SFHb-SOD-CAT-CA greatly] in SOD, the activity of CAT and CA.Add the preferred Poly-[SFHb-SOD-CAT-CA of following enzyme] required Hb: SOD: CAT: the CA ratio is 1g: 18,000: 310,000: 130,000U.
The patient recovers to organize CO in relatively poor and the hemorrhagic shock
2Raising relevant.Therefore, in rat, new method is reduced the CO that increases in hemorrhagic shock
2Efficient be studied.
Experimental design
The body weight of every rat is 300 grams.Rat is divided into 6 groups, 3 every group.Phenobarbital anesthesia.At left side femoral artery and venous cannulation.Continue record mean blood pressure (mmHg) and organize CO
2Tension force (mmHg).Every animal is emitted the blood of total blood volume 50%.Kept average 30mmHg blood pressure 1 hour.Then every rat is accepted following intravenous a kind of.
The 1st group: the saline solution of 3 times of blood losses;
The 2nd group: erythrocyte (rbc 15gm/dl): pour into again blood loss (half of total blood volume);
The 3rd group: PHcsc:5gm/dl, the poly-hemoglobin-csc for preparing by crosslinked stroma-free hemoglobin;
The 4th group: PHCSC:5gm/dl prepares the C=catalase by crosslinked stroma-free hemoglobin and the more enzyme that adds above-mentioned concentration; S=superoxide dismutase and C=carbonic anhydrase.Particularly, with SOD (1050 unit/ml), catalase (21,000 unit/ml), and carbonic anhydrase (1070 unit/ml) adds in the stroma-free hemoglobin (7g/dl), then polymerization forms PolySFHb-SOD-CAT-CA, and the HB of crosslinked rear generation: the SOD ratio is 1g: 8,000U, Hb: the CAT ratio is 1g: 310,000U, and Hb: the CA ratio is 1g: 130,000U.
The 5th group: PHcsc:10gm/dl prepares by crosslinked stroma-free hemoglobin as the 3rd group.Then goods are concentrated into 10gm/dl.
The 6th group: PHCSC:10gm/dl prepares as the 4th group, but subsequently goods is concentrated into 10gm/dl.
The result
The 1st group: the efficient of the saline solution of 3 times of blood losses is minimum because after inject salt solution CO
2Even increased.
The 4th group: poly-hemoglobin-catalase of 5gm/dl-superoxide dismutase-carbonic anhydrase (PHCSC) is slightly higher than the efficient of the erythrocyte (15gm/dl) of triplication.
The 6th group: the poly-hemoglobin-catalase of the 10gm/dl shown in the lines of bottommost-superoxide dismutase-carbonic anhydrase (PHCSC) is high more a lot of than the erythrocytic efficient of more substantial 15gm/dl.
Sum up
The goods that this new method produces are organized CO what reduction increased
2Horizontal aspect is high more a lot of than erythrocytic efficient.If the CO that increases in the hemorrhagic shock
2Level is not lowered, and then can cause shock to recover relatively poor.
Preferred embodiment is as above described and is aided with accompanying drawing and is illustrated, and under the application it to be made amendment be easily to those skilled in the art not deviating from.This class is revised and should be thought as possible variation and to be contained within the application's the scope.
Claims (14)
1. a soluble complex comprises with the antioxidase that is used for the minimizing reactive oxygen species with for transhipment CO
2And O
2Acid-Base balance and the crosslinked hemoglobin of carbon dioxide transport enzyme.
2. soluble complex as claimed in claim 1, wherein said antioxidase is selected from superoxide dismutase (SOD), catalase (CAT), or its combination.
3. soluble complex as claimed in claim 2, wherein said antioxidase is the combination of superoxide dismutase (SOD) and catalase (CAT).
4. such as each described soluble complex in the claim 2 to 3, wherein the ratio of hemoglobin and described superoxide dismutase (SOD) is that 1g: 4000U is to about 1g: 25000U.
5. such as each described soluble complex in the claim 2 to 3, wherein the ratio of hemoglobin and described superoxide dismutase (SOD) is about 1g: 18000U.
6. such as each described soluble complex in the claim 2 to 3, wherein the ratio of hemoglobin and described catalase (CAT) is that 1g: 25000U is to about 1g: 310000U.
7. such as each described soluble complex in the claim 2 to 3, wherein the ratio of hemoglobin and described catalase (CAT) is about 1g: 310000U.
8. such as each described soluble complex among the claim 1-7, wherein said Acid-Base balance and carbon dioxide transport enzyme comprise carbonic anhydrase (CA).
9. soluble complex as claimed in claim 8, wherein the ratio of hemoglobin and described carbonic anhydrase (CA) is that 1g: 80000U is to about 1g: 250000U.
10. soluble complex as claimed in claim 8, wherein the ratio of hemoglobin and described carbonic anhydrase (CA) is about 1g: 130000U.
11. such as each described soluble complex among the claim 1-9, wherein said hemoglobin comprises stroma-free hemoglobin (SFHb) or hemoglobin (Hb).
12. such as each described soluble complex among the claim 1-11, wherein said soluble complex has the molecular weight greater than about 100kDa.
13. such as each described soluble complex among the claim 1-11, wherein said soluble complex has approximately greater than 100kDa to the molecular weight greater than 450kDa.
14. an artificial blood succedaneum comprises each described soluble complex among the claim 1-13 that exists with suitable medicinal liquid form.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161490304P | 2011-05-26 | 2011-05-26 | |
| US61/490,304 | 2011-05-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102861323A true CN102861323A (en) | 2013-01-09 |
Family
ID=47262863
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101796705A Pending CN102861323A (en) | 2011-05-26 | 2012-05-28 | A novel blood substitute with complete red blood cell functions |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN102861323A (en) |
| CA (1) | CA2778010C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112546206A (en) * | 2020-12-30 | 2021-03-26 | 云锦华彰(北京)生物科技有限公司 | Preparation method of human hemoglobin oxygen carrier, product and application thereof |
| CN112618705A (en) * | 2020-12-30 | 2021-04-09 | 云锦华彰(北京)生物科技有限公司 | Human-derived hemoglobin oxygen carrier and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357940A (en) * | 2008-09-18 | 2009-02-04 | 上海市血液中心 | Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof |
-
2012
- 2012-05-23 CA CA2778010A patent/CA2778010C/en active Active
- 2012-05-28 CN CN2012101796705A patent/CN102861323A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357940A (en) * | 2008-09-18 | 2009-02-04 | 上海市血液中心 | Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof |
Non-Patent Citations (2)
| Title |
|---|
| T.M.S. CHANG: "Modified hemoglobin-based blood substitutes: crosslinked,recombinant and encapsulated hemoglobin", 《VOX SANGUINIS》, vol. 74, 31 December 1998 (1998-12-31), pages 233 - 241 * |
| YUZHU BIAN ET AL.: "Polyhemoglobin-superoxide Dismutase-catalase-carbonic Anhydrase: A Novel Biotechnology-based Blood Substitute that Transports both Oxygen and Carbon Dioxide and also Acts as an Antioxidant", 《ARTIFI CIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY》, vol. 39, 30 June 2011 (2011-06-30), pages 127 - 136 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112546206A (en) * | 2020-12-30 | 2021-03-26 | 云锦华彰(北京)生物科技有限公司 | Preparation method of human hemoglobin oxygen carrier, product and application thereof |
| CN112618705A (en) * | 2020-12-30 | 2021-04-09 | 云锦华彰(北京)生物科技有限公司 | Human-derived hemoglobin oxygen carrier and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2778010A1 (en) | 2012-11-26 |
| CA2778010C (en) | 2022-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| D'Agnillo et al. | Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties | |
| KR910009343B1 (en) | Hemoglobin-based blood substitute | |
| TWI322184B (en) | Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates | |
| US9498537B2 (en) | Method of enhancing efficacy of blood transfusions | |
| JPS6289630A (en) | Hemoglobin-polyoxyalkylene conjugate | |
| EP2682407B1 (en) | Hemoglobin-albumin complex, and artificial plasma expander and artificial oxygen carrier, each containing same | |
| JP2010529198A (en) | Targeted oxygen delivery by intravenous or intraarterial infusion of oxygenated polymerized hemoglobin solution | |
| CN107137699B (en) | Deoxygenation method and preparation process of natural hemoglobin blood substitute | |
| Boccu et al. | Coupling of monomethoxypolyethyleneglycols to proteins via active esters | |
| CN102614843B (en) | Protein A immunoadsorption material and preparation method thereof | |
| RU2337705C2 (en) | Polymerised haemoglobin solutions with lowered tetramer content and method of production thereof | |
| CN102861323A (en) | A novel blood substitute with complete red blood cell functions | |
| CN102026654A (en) | Oxygen-transferring blood substitute and a pharmaceutical composition (variants) | |
| AU650439B2 (en) | Polyhemoglobin stabilized by purine derivatives and glutathione | |
| CN102859364A (en) | Methods for preparing peg-hemoglobin conjugates using reduced reactant ratios | |
| CN112546206A (en) | Preparation method of human hemoglobin oxygen carrier, product and application thereof | |
| CN103251936B (en) | Oxygen carrier based on hemoglobin-haptoglobin compound and preparation method of oxygen carrier | |
| CN102370993A (en) | Preparation method for novel red blood cell substitute-artificial red blood cell fluorescent nanoparticles | |
| CN117693364A (en) | Stable storage of enzymes | |
| Pişkin | Therapeutic potential of immobilized enzymes | |
| CN114303462B (en) | Preparation method of red blood cell substitute | |
| CN118976102B (en) | A method for preparing a high molecular polymer nanozyme for regulating tumor immunosuppressive microenvironment | |
| CN114303461B (en) | Preparation method of EDC cross-linked hemoglobin for red blood cell substitute | |
| Chen et al. | Dual Antioxidant and Pro-Oxidation Effects of Ascorbic Acid on Bovine Hemoglobin | |
| Savla | Scalable Purification of Ultra-Pure Lumbricus terrestris Mega-Hemoglobin and Surface Modification using Bio-compatible Polymers for use as an Oxygen Therapeutic |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1178449 Country of ref document: HK |
|
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: Montreal, Quebec, Canada Applicant after: Zhang Mingrui Applicant after: Bian Yuzhu Address before: Montreal, Quebec, Canada Applicant before: Zhang Mingrui Applicant before: Bian Yuzhu |
|
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Mingrui Inventor after: Bian Yuzhu Inventor before: Zhang Mingrui Inventor before: Bian Yuzhu |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130109 |
|
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1178449 Country of ref document: HK |