Artificial blood substitute

Abstract

Artificial blood substitutes and methods of using the same are provided. In particular, artificial blood replaces a new enzyme (synthetase) instead of a natural enzyme.

Description

Technical Field

[0001] The presently disclosed subject matter relates generally to artificial blood substitutes and methods of using the same, and more particularly, to artificial blood substitutes comprising novel enzymes (synthetases) that replace natural enzymes. Background Art

[0002] Red blood cells (RBCs) have three main functions: (1) oxygen transport, (2) carbon dioxide transport, and (3) antioxidant properties. Dr. Thomas Chang and other researchers have prepared different types of blood substitutes based on nanobiotechnology (A professional book on "Nanobiotherapy Basis of Blood Substitutes", edited by Dr. Chang, co-edited by Bulow, Jahr, Sakai & Yang (all past or current chairs of the International Workshop on Blood Substitutes) World Scientific Publishing / Imperial College London (2021)). Open access is available at: https: / / www.worldscientific.com / doi / epdf / 10.1142 / 12054. One of them is based on cross-linking polyhemoglobin using diacids or glutaraldehyde (TMSChang, Science, 1964, 146: 524-525), (TMSChang, Biochem Biophys Res Common 1971, 44(6): 1531-153).

[0003] It wasn't until 1987, when HIV infection in donor blood sparked interest in developing these blood substitutes, leading to a "catch-up" development of blood substitutes. However, the urgency of the need for this technology focused solely on the oxygen-carrying function of red blood cells (RBCs), accelerating the development of hemoglobin-based oxygen carriers (HBOCs). These products only carried oxygen, lacking the other two key functions of RBCs: carbon dioxide transport and antioxidant activity. One of the more successful first-generation HBOCs was based on Dr. Zhang Mingrui's glutaraldehyde cross-linking technology. Biopure, a US company, has produced a glutaraldehyde-cross-linked polymerized hemoglobin (PolyHb) and has conducted extensive clinical trials. South Africa and Russia have approved this polyHb for routine clinical use and have been using it for many years (Jahr JS, et al. (2008) J Trauma 64:1484–97). However, it only performs one of the three functions of RBCs. The risk / benefit ratio of polyHb has been shown to be favorable in some parts of the world where the risk of HIV in donor blood is high. However, in other parts of the world, where HIV in donor blood is no longer a major problem, the risk / benefit is different.

[0004] In conditions of severe acute and persistent ischemia, such as ischemia-reperfusion (common in cases of myocardial infarction, stroke, or organ transplantation), nanoscale PolyHb, unlike red blood cells, offers a solution that more easily reperfuses blocked blood vessels. However, this process triggers the generation of oxygen free radicals, which in turn damage tissues. To address this issue, Dr. Zhang Mingrui and his team developed a nanobiotechnology-based polymerized hemoglobin-superoxide dismutase-catalase (a hemoglobin oxygen carrier with antioxidant properties, abbreviated as PolyHb-SOD-CAT). Unlike pure PolyHb, PolyHb-SOD-CAT did not induce ischemia-reperfusion injury in a rat intestinal ischemia model. PolyHb solutions can be used in certain specific situations, such as myocardial infarction and stroke, because in these situations, they are more likely to reperfuse partially blocked blood vessels. In a rat hemorrhagic shock model, Dr. Zhang Mingrui's team found that reperfusion with PolyHb after 60 minutes of ischemia significantly exacerbated blood-brain barrier rupture and the development of cerebral edema. However, PolyHb-SOD-CAT did not induce these adverse reactions.

[0005] The main concern associated with PolyHb is the slight increase in nonfatal myocardial infarction observed in clinical trials during hemorrhagic shock. Although red blood cells transport both carbon dioxide and oxygen, worldwide research on blood substitutes has focused on oxygen transport and the removal of oxygen free radicals. A study conducted in a Norwegian animal model showed that the degree of increase in tissue carbon dioxide partial pressure (PCO2) correlated with the degree of myocardial ischemia.

[0006] Therefore, Dr. Zhang Mingrui's team developed a nanobiotechnology formulation that can simultaneously transport carbon dioxide and oxygen and remove oxygen free radicals. It consists of polyhemoglobin-catalase-superoxide dismutase-carbonic anhydrase (PolyHb-SOD-CAT-CA). This nanobiotechnology-constructed formulation achieves an enzyme concentration six times that found in red blood cells (RBCs). In a rat model of hemorrhagic shock, they found that PolyHb-SOD-CAT-CA was more effective than RBCs in reducing tissue PCO2 levels and protecting the heart. This third-generation blood substitute, containing naturally occurring enzymes (superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA)), now possesses all three key functions of RBCs. However, naturally occurring enzymes are difficult to obtain in large quantities and have low stability in vivo or at room temperature. Furthermore, the preparation of this complex (PolyHb-SOD-CAT-CA) requires more sophisticated processing to avoid immunological issues.

[0007] There is also a need for an artificial blood substitute containing new enzymes (synthetases) to replace the natural enzymes and increase the stability of the complex. Summary of the Invention

[0008] According to an embodiment, an artificial blood substitute is provided that contains novel enzymes (synthetases) rather than natural enzymes. This greatly increases the stability of the complex and reduces the cost and complexity of complex preparation as well as potential immunogenicity, because the novel enzymes (neoSOD, neoCAT, and neoCA) used are cheaper and more stable than their natural counterparts.

[0009] According to another embodiment, a synthetase activity solution is provided, comprising at least one compound selected from the following group:

[0010] Neo-carbonic anhydrase (neoCA) compounds with carbonic anhydrase activity selected from ZnHisGly (zinc-histidine-glycerol complex);

[0011] New superoxide dismutase (neoSOD) compounds with superoxide dismutase activity selected from MnTmPyP (manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin);

[0012] A novel catalase (neoCAT) compound with catalase activity selected from EUK-134 (2,2′-[1,2-ethanediylbis(nitrosomethylimino)]bis[6-methoxyphenol]manganese complex) or chloro[2,2′-[1,2-ethanediylbis(nitrosomethylimino)]bis[6-methoxyphenol]-N2,N2′,O1,O1′]-manganese;

[0013] Combinations of the above compounds;

[0014] Wherein, at least one compound is present in phosphate buffered saline (PBS, pH 7.4), physiological saline, lactated Ringer's solution (RL), cell culture medium, organ preservation solution, distilled water (dH2O) or other pharmaceutically acceptable buffer, and the compound is suitable for transporting carbon dioxide and / or scavenging oxygen free radicals.

[0015] According to one embodiment, there is provided an artificial blood substitute solution comprising:

[0016] The synthetase activity solution according to the first aspect of the present invention, which has a combination of up to 3 compounds; and

[0017] An oxygen carrier selected from the group consisting of: PolyHb, other modified hemoglobin (examples include but are not limited to other types of polymerized hemoglobin prepared with other cross-linking agents, conjugated hemoglobin including polyethylene glycol-hemoglobin (PEG-Hb), intramolecularly cross-linked hemoglobin, recombinant hemoglobin, bioengineered hemoglobin, nanoencapsulated hemoglobin in polyethylene glycol-lipid membrane or polyethylene glycol-polylactic acid membrane or other membranes), perfluorinated compounds, synthetic heme, oxygen-saturated solution; liquid for organ and cell preservation, the liquid is used for the preservation of transplanted organs and cells, or for the biotechnological production of stem cells and other cells;

[0018] The artificial blood substitute solution is suitable for simultaneously transporting carbon dioxide and oxygen and removing oxygen free radicals.

[0019] In the artificial blood substitute solution, the ratio of oxygen carrier to neoCA, neoSOD and neoCAT is approximately 1 g:18 mg:18 mg:18 mg.

[0020] According to one embodiment, there is provided a use of the artificial blood substitute solution of the present invention in the preparation of a medicament for treating blood loss, carbon dioxide accumulation disorder, ischemia-reperfusion injury or diver's disease.

[0021] The ischemia-reperfusion injury includes severe hemorrhagic shock, stroke, myocardial infarction or a combination thereof.

[0022] According to one embodiment, a solution of synthetase activity of the present invention is provided for use in treating a fluid (eg, blood, which may be in an artificial lung or dialysis) to remove carbon dioxide.

[0023] While the blood is in the artificial lung, it may be used to transport oxygen within the patient's body, but it may also be needed to remove accumulated carbon dioxide (such as in severe coronavirus patients).

[0024] According to one embodiment, there is provided a use of the artificial blood substitute solution of the present invention for preserving organs, tissues or cells for transplantation.

[0025] Features and advantages of the present subject matter will become more apparent from the following detailed description of selected embodiments, taken in conjunction with the accompanying drawings. The subject matter disclosed and claimed is capable of modification in various respects without departing from the scope of the claims. The drawings and description are, therefore, to be regarded as illustrative in nature and not restrictive, the full scope of the inventive subject matter being set forth by the claims.

[0026] The technical solution of the present invention has the following main advantages:

[0027] The present invention provides an artificial blood substitute containing novel enzymes (synthetases) rather than natural enzymes. This greatly increases the stability of the complex and reduces the cost and complexity of complex preparation, as well as potential immunogenicity, because the novel enzymes (neoSOD, neoCAT, and neoCA) used are cheaper and more stable than their natural counterparts. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Preliminary viability assay results of human hepatocytes treated with different solutions in an in vitro model of ischemia-reperfusion (I / R) shock are presented, as well as a feasibility study for the preservation and recovery of ischemic cells and organs for transplantation.

[0029] Figure 2 The results of viability assays of human hepatocytes treated with different solutions in a 180-min in vitro ischemia-reperfusion (I / R) shock model are presented, as well as a feasibility study on their use in the preservation and recovery of ischemic cells and organs for transplantation.

[0030] Figure 3 This figure presents preliminary results of viability measurements of human cardiomyocytes treated with different solutions in an 180-minute in vitro ischemia-reperfusion (I / R) shock model, as part of a feasibility study for the preservation and recovery of ischemic cells and organs for transplantation. Furthermore, since polymerized hemoglobin (PolyHb) alone has been shown to cause minor cardiac side effects in patients with hemorrhagic shock, this figure also serves as an example of its ability to protect the heart from damage under hemorrhagic shock conditions.

[0031] Figure 4 This figure shows the viability of human cardiomyocytes treated with different solutions in an 180-minute in vitro ischemia-reperfusion (I / R) shock model, and also serves as a feasibility study for the preservation and recovery of ischemic cells and organs for transplantation. Furthermore, given that the use of polymerized hemoglobin (PolyHb) alone has produced minor cardiac side effects in patients with hemorrhagic shock, this figure also serves as an example of its ability to protect the heart from damage under hemorrhagic shock conditions. DETAILED DESCRIPTION

[0032] In the Examples, it is disclosed that a small chemical substance having CAT, SOD and / or CA enzyme activity is used to replace a natural protease solution for creating an enzyme activity solution.

[0033] The enzyme activity solution can be added to any oxygen carrier without cross-linking, including but not limited to PolyHb, other modified Hb (examples include but are not limited to other types of polymerized hemoglobin prepared with other cross-linking agents, conjugated hemoglobin including PEG-Hb, intramolecular cross-linked hemoglobin, recombinant hemoglobin, bioengineered hemoglobin, nano-encapsulated hemoglobin in polyethylene glycol-lipid membrane or polyethylene glycol-polylactic acid membrane or other membranes), perfluorinated compounds, synthetic heme, oxygen-saturated solutions and other oxygen carriers to transport carbon dioxide and oxygen and scavenge oxygen free radicals; and liquids for organ and cell preservation, which are used for the preservation of transplanted organs and cells, or for the biotechnology production of stem cells and other cells.

[0034] The small molecule chemical with enzymatic activity does not cause an immune response and can therefore be used as a free solution. The results showed that replacing the natural enzyme with the chemical with enzymatic activity significantly improved the stability of the entire product, as evidenced by the results. Even after two months of storage at room temperature and 37 degrees Celsius, the enzymatic activity of the synthesized enzyme was the same as on the first day (almost 100% activity).

[0035] Preparation of CAT, SOD, and CA enzyme active chemicals

[0036] Neocarbonic anhydrase (NeoCA):

[0037] Neocarbonic anhydrase (NeoCA) was synthesized according to an earlier established protocol (Zhibo Zhang et al., Separation and Purification Technology. Volume 276.2021.119446. ISSN 1383-5866). Briefly,

[0038] 1. Place 0.51g zinc chloride and 1.72g L-histidine in a beaker.

[0039] 2. Add 4.8 ml of glycerol.

[0040] 3. Then stir at 70°C until a homogeneous liquid phase is formed (approximately 30 minutes). The mixture turns light yellow.

[0041] 4. Subsequently, the mixture was placed in a vacuum oven at 60° C. and dried for 2 h, thereby preparing a new carbonic anhydrase (also known as ZnHisGly or zinc-histidine-glycerol complex); a zinc-based deep eutectic solvent; and a carbonic anhydrase mimetic.

[0042] Neo-superoxide dismutase (NeoSOD):

[0043] SOD catalyzes the decomposition of superoxide radicals into molecular oxygen and hydrogen peroxide, providing protection for cells against reactive oxygen species. The neoSOD model used in this study is MnTmPyP or Mn(III) tetra(1-methyl-4-pyridinyl)porphyrin (purchased from Sigma Aldrich Canada (reference: Faulkner, KM et al. (1994) The Journal of biological chemistry, 269(38), 23471-23476)). It is a cell-permeable SOD mimetic that has been used in many studies, but it has never been reported to be used as an antioxidant in combination with a modified hemoglobin blood substitute.

[0044] Neocatalase (NeoCAT):

[0045] Catalase catalyzes the decomposition of hydrogen peroxide into water and oxygen. It protects cells from oxidative damage caused by reactive oxygen species. EUK-134 (2,2′-[1,2-ethanediylbis(nitromethylene)]bis[6-methoxyphenol manganese complex; chloro[[2,2′-[1,2-ethanediylbis(nitromethylene)]bis[6-methoxyphenoxy]](2-)-N2,N2′,O1,O1′]-manganese) is a commercially available catalase (CAT) mimetic with potent antioxidant activity (purchased from Sigma Aldrich Canada (reference: Ri, MH et al, J Mol Model 28, 168(2022) https: / / doiorg / 101007 / s00894-022-05129-4)). It has been used in many studies, but its use as an antioxidant in combination with modified hemoglobin blood substitutes has never been reported.

[0046] The present invention will be more readily understood by reference to the following examples, which are given to illustrate the present invention, not to limit its scope.

[0047] Example 1

[0048] Protective effects of PolyHb and novel enzyme solution on human hepatocytes in vitro ischemia / reperfusion (I / R) model

[0049] This study also investigates the feasibility of using this fluid to preserve and recover ischemic cells and organs for transplantation. Organs and cells destined for transplantation are often in a state of ischemia during retrieval from donor patients. The fluid can provide needed oxygen, remove accumulated carbon dioxide and oxygen free radicals, and aid in organ and cell recovery prior to transplantation. The following preliminary study demonstrating improved liver cell viability demonstrates this feasibility.

[0050] method:

[0051] Human hepatocyte culture:

[0052] DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) was mixed with the PBS (purchased from Thermofisher) in a water bath. Thaw the frozen hepatocytes in a 37°C water bath for less than 2 minutes. Then, wipe the cryopreserved tubes with 70% alcohol in a clean bench and transfer the contents of the tubes to a warm Then, centrifuge at 100×g for 10 min. Pour the supernatant into the waste bottle. 6 Add about 1 ml of plating medium (DMEM medium with the above ingredients added) to each cell, i.e. 8×10 6 Add 8 ml of the solution to each cell and mix gently. Then count the cells and calculate the cell number as 2.4×10 5 Cells were seeded onto collagen-coated 6-well plates at a density of 10 cells / ml. The plates were labeled according to the different research groups, as follows:

[0053] 1. Control group liver cells

[0054] 2. Hepatocytes treated with 20ul RL after ischemic shock

[0055] 3. After ischemic shock, hepatocytes were treated with 20ul RL+0.626mmol / L PolyHb

[0056] 4. After ischemic shock, hepatocytes were treated with 20ul RL+0.626mmol / L PolyHb+0.05% (w / v) vitamin C

[0057] 5. After ischemic shock, hepatocytes were treated with 20ul RL+55.6mmol / L neoenzyme solution (neoSOD+neoCAT+neoCA)

[0058] 6. After ischemic shock, hepatocytes treated with 0.626mmol / L PolyHb + 55.6mmol / L new enzyme solution

[0059] The culture dish was incubated at 37°C for 6 h. After incubation, the culture dish was gently shaken to loosen the debris, the medium was aspirated, and replaced with warm DMEM medium supplemented with the supplement and kept in the incubator for 24 h.

[0060] I / R in vitro model

[0061] It is also being tested for ischemic cell and organ preservation during transplantation. Cells destined for transplantation are often ischemic during their removal from the donor patient. These fluids provide needed oxygen and remove accumulated CO2 and oxygen free radicals, helping organs and cells recover prior to transplantation. This feasibility has been demonstrated in our preliminary hepatocyte viability studies.

[0062] Remove the plate from the incubator and place it in a shaker at 37°C with nitrogen continuously flowing through the shaker to establish the ischemic model. Maintain the cells in a hypoxic environment for 1 hour. Then, remove the cells from the shaker and replace the culture medium with fresh supplemented DMEM. Treat the cells in the previously labeled wells as follows:

[0063] 1. The liver cells in the control group were only fed with fresh DMEM medium.

[0064] 2. Treat hepatocytes with 20ul RL and fresh DMEM medium

[0065] 3. Hepatocytes treated with 20ul RL+0.626mmol / L PolyHb and fresh DMEM medium

[0066] 4. Hepatocytes treated with 20ul RL+0.626mmol / L PolyHb+0.05% (w / v) vitamin C and fresh DMEM medium

[0067] 5. Hepatocytes treated with 20ul RL+55.6mmol / L new enzyme solution (neoSOD+neoCAT+neoCA) and fresh DMEM medium

[0068] 6. Treat hepatocytes with 0.626mmol / L PolyHb + 55.6mmol / L new enzyme solution and fresh DMEM medium

[0069] The culture dish was then returned to the incubator for 24 h.

[0070] Preliminary feasibility analysis:

[0071] Use a pipette to aspirate the liquid culture medium. Then wash the cells twice with 2ml PBS. Add 1ml of collagenase working solution at a concentration of 4mg / ml to each well. Gently rotate the culture plate and place it at room temperature for 10min. Add 1.5ml (the ratio of culture medium to collagenase is 3:1) DMEM culture medium supplemented with fetal bovine serum to terminate the action of collagenase. Then transfer these liquids to centrifuge tubes respectively. Add 1ml of culture medium to the culture plate, wash and transfer the liquid to the corresponding centrifuge tube. Repeat this operation twice more. Then centrifuge at 1500rpm for 5min, discard the supernatant, and then resuspend the pellet with 5ml of culture medium. The cells are then counted using the trypan blue method, and the calculation formula is: Cell viability (%) = (total number of cells - total number of cells stained blue) / total number of cells × 100%.

[0072] result:

[0073] The cell survival data from this preliminary study showed that the combination of PolyHb in solution and the new enzyme had a more significant positive effect on cells after I / R shock compared to the control group and other groups, such as Figure 1 When PolyHb was used alone, cell survival was modestly improved, suggesting that the generated oxygen free radicals were not scavenged from the culture medium during the 24-hour culture. While the new enzyme alone improved cell survival compared to PolyHb, it also had limitations. Combining the oxygen carrier PolyHb with the three new enzymes resulted in even higher cell survival.

[0074] Example 2

[0075] Protective effects of PolyHb+neoCAT+neoSOD+neoCA solution and PolyHb+neoCAT+neoSOD solution on human hepatocytes in vitro ischemia / reperfusion (I / R) model

[0076] This study also investigated the feasibility of using PolyHb-CAT-SOD-CA for the preservation and recovery of ischemic cells and organs for transplantation. Organs and cells destined for transplantation are often in a state of ischemia during retrieval from donor patients. This fluid can provide essential oxygen, scavenge accumulated carbon dioxide and oxygen free radicals, and aid in organ and cell recovery prior to transplantation. This feasibility was demonstrated in subsequent studies of hepatocyte survival.

[0077] method:

[0078] Human hepatocyte culture:

[0079] DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) was mixed with the PBS purchased from Thermofisher in a water bath. Thaw the frozen hepatocytes in a 37°C water bath for less than 2 minutes. Then, wipe the cryopreserved tubes with 70% alcohol in a clean bench and transfer the contents of the tubes to a warm Then, centrifuge at 100×g for 10 min. Pour the supernatant into the waste bottle. 6 Add about 1 ml of plating medium (DMEM medium with the above ingredients added) to each cell, i.e. 8×10 6 Add 8 ml of the solution to each cell and mix gently. Then count the cells and calculate the cell number as 2.4×10 5 Cells were seeded onto collagen-coated 6-well plates at a density of 10 cells / ml. The plates were labeled according to the different research groups, as follows:

[0080] Hepatocytes not undergoing ischemic shock (negative control)

[0081] Hepatocytes that underwent ischemic shock but received no treatment (untreated control).

[0082] Hepatocytes treated with 20ul RL after ischemic shock

[0083] Hepatocytes treated with 0.626mmol / L PolyHb after ischemic shock

[0084] Hepatocytes treated with a 55.6 mmol / L solution containing three new enzymes (neoSOD+neoCAT+neoCA) after ischemic shock

[0085] Hepatocytes treated with 0.626mmol / L PolyHb + 55.6mmol / L solution containing three new enzymes (neoSOD + neoCAT + neoCA) after ischemic shock

[0086] Hepatocytes treated with 0.626mmol / L PolyHb + 55.6mmol / L solution containing two new enzymes (neoSOD + neoCAT) after ischemic shock

[0087] Incubate the plate at 37°C for 6 h. After incubation, gently shake the plate to loosen debris, aspirate the medium, replace it with warm DMEM supplemented with the supplement, and place in the incubator for 24 h.

[0088] I / R in vitro model

[0089] It is also being tested for preserving ischemic cells and organs for transplantation. Organs and cells destined for transplantation are often ischemic during retrieval from the donor patient. These fluids can provide needed oxygen and remove accumulated CO2 and oxygen free radicals, helping the organs and cells recover prior to transplantation. This feasibility has been demonstrated in our hepatocyte viability studies.

[0090] Remove the plate from the incubator and place it in a shaker at 37°C with nitrogen gas continuously flowing through the shaker to establish the ischemic model. Maintain the cells in the hypoxic environment for 180 minutes. Then, remove the cells from the shaker, replace the culture medium with fresh supplemented DMEM, and treat the cells in the previously labeled wells as follows:

[0091] Control hepatocytes were only fed with fresh DMEM medium

[0092] Hepatocytes treated with 20ul RL

[0093] Hepatocytes treated with 0.626mmol / L PolyHb

[0094] Hepatocytes were treated with a solution containing 55.6 mmol / L of the three new enzymes (neoSOD+neoCAT+neoCA)

[0095] Hepatocytes treated with 0.626mmol / L PolyHb + 55.6mmol / L solution containing three new enzymes (neoSOD+meoCAT+neoCA)

[0096] Hepatocytes treated with 0.626mmol / L PolyHb + 55.6mmol / L solution containing two new enzymes (neoSOD + neoCAT)

[0097] The culture dish was then returned to the incubator for 24 h.

[0098] Cell viability assay:

[0099] Use a pipette to aspirate the liquid culture medium. Subsequently, wash the cells twice with 2 ml of PBS. Add 1 ml of collagenase working solution at a concentration of 4 mg / ml to each well. Gently rotate the culture plate and place it at room temperature for 10 minutes. Add 1.5 ml of DMEM culture medium (the ratio of culture medium to collagenase is 3:1) supplemented with fetal bovine serum to terminate the action of collagenase. Then, transfer the above liquids to individual centrifuge tubes. Add 1 ml of culture medium to the culture plate, wash and transfer to the corresponding centrifuge tube. Repeat this operation twice more. Then, centrifuge the cells at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the pellet with 5 ml of culture medium. Subsequently, the cells were counted using the trypan blue method, and the calculation formula is: Percent viability = (total number of cells - total number of cells stained blue) / total number of cells × 100%.

[0100] result:

[0101] The cell viability percentage results of this preliminary study showed that the combination of PolyHb plus at least two new enzymes had a higher cell viability percentage compared with the control group and other groups, and the group of PolyHb plus three new enzyme solutions had a more significant positive effect on cells after the cells experienced I / R shock, such as Figure 2 As shown in the figure, PolyHb alone was unable to achieve a high percentage of cell viability, indicating that oxygen free radicals generated in the culture medium were not scavenged during the 24-hour culture. Using a solution containing only the three novel enzymes improved cell viability compared to using PolyHb alone. However, this approach also has limitations, as the combination of the oxygen carrier PolyHb and all three novel enzymes showed even better cell viability. The combination of PolyHb plus all three novel enzymes was the most effective, as it can both provide oxygen and scavenge carbon dioxide and oxygen free radicals.

[0102] Example 3

[0103] Cardioprotective effects of PolyHb+neoCAT+neoSOD+neoCA solution and PolyHb+neoCAT+neoSOD solution on human cardiomyocytes in vitro ischemia / reperfusion (I / R) model

[0104] This is also a feasibility study for the use of PolyHb-CAT-SOD-CA for the preservation and recovery of ischemic cells and organs for transplantation. Organs and cells for transplantation are often in an ischemic state during the process of being removed from the donor patient. This study also aims to explore whether PolyHb treatment alone can protect the heart from damage caused by severe hemorrhagic shock. The above-mentioned fluid (PolyHb+neoCAT+neoSOD+neoCA solution) can provide the necessary oxygen, remove accumulated CO2 and oxygen free radicals, and help organs and cells recover before transplantation. Our subsequent study on the improvement of cardiomyocyte viability demonstrated this feasibility.

[0105] method:

[0106] Human cardiomyocyte culture:

[0107] PromoCell Cardiomyocyte Growth Medium (Cat. No. C-22070, Sigma Aldrich, Canada, supplemented with 5% fetal bovine serum and 1% penicillin / streptomycin) was preheated to 37°C in a water bath. The cryopreserved cardiomyocytes were thawed in a 37°C water bath for less than 2 minutes. The cryovials were then wiped with 70% alcohol in a clean bench and the contents were transferred to preheated PromoCell Cardiomyocyte Growth Medium. The cells were then centrifuged at 100 × g for 10 minutes. The supernatant was poured into a waste bottle and 1 × 10 6 Add about 1 ml of cardiomyocyte culture medium, i.e. 8×10 6 Add 8 ml of culture medium to each cell and mix gently. Then count the cells and calculate the number of cells as 2.4×10 5 The cells were seeded into 6-well plates at a density of 10 cells / ml. The plates were labeled according to the different research groups, as follows:

[0108] Cardiomyocytes without ischemic shock (negative control)

[0109] Cardiomyocytes after ischemic shock (untreated control group).

[0110] Cardiomyocytes treated with 20ul RL after ischemic shock

[0111] Cardiomyocytes treated with 0.626mmol / L PolyHb after ischemic shock

[0112] After ischemic shock, myocardial cells were treated with a 55.6 mmol / L solution containing three new enzymes (neoSOD+neoCAT+neoCA)

[0113] After ischemic shock, myocardial cells were treated with 0.626mmol / L PolyHb + 55.6mmol / L solution containing three new enzymes (neoSOD + neoCAT + neoCA)

[0114] After ischemic shock, myocardial cells were treated with 0.626mmol / L PolyHb and 55.6mmol / L solution containing two new enzymes (neoSOD+neoCAT)

[0115] Incubate the culture dish at 37°C for 6 hours. After incubation, gently shake the dish to loosen debris, aspirate the culture medium, replace it with warmed cardiomyocyte culture medium supplemented with the appropriate ingredients, and return it to the incubator for another 24 hours.

[0116] I / R in vitro model

[0117] It is also used to test the preservation of ischemic cells and organs for transplantation. Organs and cells intended for transplantation are often in a state of ischemia during the process of removal from the donor patient. This study also aimed to investigate whether treatment with polymerized hemoglobin (PolyHb) could protect the heart from the damage of severe hemorrhagic shock. This fluid provides needed oxygen, removes accumulated carbon dioxide and oxygen free radicals, and helps organs and cells recover before transplantation. Our studies demonstrating improved cardiomyocyte viability demonstrate this feasibility.

[0118] Remove the culture plate from the incubator and place it in a shaker at 37°C while continuously aerating nitrogen to establish the ischemic model. Allow the cells to remain in this hypoxic environment for 180 minutes. Then, remove the cells from the shaker and replace the original cell culture medium with fresh cardiomyocyte culture medium supplemented with the appropriate ingredients. Treat the cells in the previously labeled wells as follows:

[0119] The control group of cardiomyocytes only had fresh cardiomyocyte culture medium

[0120] Cardiomyocytes treated with 20ul RL

[0121] Cardiomyocytes treated with 0.626mmol / L PolyHb

[0122] Cardiomyocytes treated with a 55.6 mmol / L solution containing three new enzymes (neoSOD+neoCAT+neoCA)

[0123] Cardiomyocytes treated with 0.626mmol / L PolyHb + 55.6mmol / L solution containing three new enzymes (neoSOD + neoCAT + neoCA)

[0124] Cardiomyocytes treated with 0.626mmol / L PolyHb and 55.6mmol / L solution containing two new enzymes (neoSOD+neoCAT)

[0125] The culture dish was then returned to the incubator for 24 h.

[0126] Cell viability assay:

[0127] Use a pipette to remove the liquid culture medium. Then wash the cells twice with 2 ml of PBS. Add 1 ml of trypsin working solution at a concentration of 4 mg / ml to each well. Gently rotate the culture plate and keep it at room temperature for 10 minutes. Then add 1.5 ml (the ratio of culture medium to trypsin is 3:1) of cardiomyocyte culture medium containing fetal bovine serum to terminate the action of collagenase. Then transfer the cells to their respective centrifuge tubes. Add 1 ml of culture medium to the culture plate for washing, and transfer the washed cells to the corresponding centrifuge tubes. Repeat this operation twice. Then, centrifuge the cells at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet with 5 ml of culture medium. Finally, count the cells using the trypan blue method, and calculate the percentage of cell activity as follows: Percentage of cell activity = (total number of cells - number of cells stained with trypan blue) / total number of cells × 100%.

[0128] result:

[0129] The cell viability percentages of this preliminary study showed that the combination of PolyHb and at least two novel enzymes had a high cell viability percentage, and the solution of PolyHb combined with three novel enzymes had a better effect on cells than the control group and other groups, especially after the cells were subjected to I / R shock, such as Figure 3 and Figure 4 As shown, PolyHb alone was unable to achieve a higher percentage of cell viability, indicating that oxygen free radicals generated in the culture medium were not scavenged during the 24-hour incubation period. Combinations using only the novel enzymes resulted in better cell viability than PolyHb alone, but this combination also has limitations, as only novel enzyme combinations combined with the oxygen carrier PolyHb showed improved cell viability. The best effect was achieved with the combination of PolyHb and all three novel enzymes, as this combination can both provide oxygen and scavenge carbon dioxide and oxygen free radicals.

[0130] Although preferred embodiments have been described above and illustrated in the accompanying drawings, it will be apparent to those skilled in the art that modifications may be made without departing from the present disclosure. These modifications are considered possible variations within the scope of this disclosure.

Claims

1. A synthetic enzyme activity solution, characterized in that The invention relates to a novel carbonic anhydrase (neoCA) compound having carbonic anhydrase activity selected from ZnHisGly (histidine zinc glycerol) and at least one compound selected from the following groups: A novel superoxide dismutase (neoSOD) compound having superoxide dismutase activity selected from the group consisting of MnTmPyP, a Mn(III) tetramer (1-methyl-4-pyridyl) porphyrin; A novel catalase (neoCAT) compound having catalase activity selected from the group consisting of EUK-134, or 2,2′-[1,2-ethanediylbis(nitrosomethylimino)]bis[6-methoxyphenol] manganese complex; chloro[[2,2′-[1,2-ethanediylbis(nitrosomethylimino)]bis[6-methoxyphenol]](2-)-N2,N2′,O1,O1′]-manganese; Wherein, the at least one compound and neocarbonic anhydrase (neoCA) are in phosphate buffered saline (PBS solution, pH-7.4), physiological saline, lactated Ringer's solution, cell culture medium, organ preservation medium, distilled water (dH2O) or other pharmaceutically acceptable buffer, suitable for transporting carbon dioxide and / or removing oxygen free radicals.

2. An artificial blood substitute solution, characterized in that: include: The synthetase activity solution of claim 1, comprising a combination of up to three compounds; as well as An oxygen carrier comprising PolyHb, other modified hemoglobin (examples include but are not limited to other types of polyhemoglobin, conjugated hemoglobin including PEG-Hb, intramolecularly cross-linked hemoglobin, recombinant hemoglobin, bioengineered hemoglobin, nanoencapsulated hemoglobin, whose encapsulation materials include polyethylene glycol-lipid membrane or polyethylene glycol-polylactic acid membrane or other membranes), perfluorinated compounds, synthetic heme, and oxygen-saturated solution; Cell and organ preservation fluids for the preservation of organs and cells for transplantation or for the biotechnological production of stem cells and other cells; The artificial blood substitute solution is capable of transporting carbon dioxide and oxygen and removing oxygen free radicals.

3. The artificial blood substitute solution according to claim 2, wherein the ratio of the oxygen carrier to neoCA, neoSOD and neoCAT is about 0-10 g of oxygen carrier and 1 mg-180 mg of the novel enzyme (neoCA / neoSOD / neoCAT).

4. The synthetic enzyme activity solution of claim 1 or the artificial blood substitute solution of claim 2 or 3, wherein the synthetic enzyme activity solution consists of a neo carbonic anhydrase (neoCA) compound having carbonic anhydrase activity, a neo superoxide dismutase (neoSOD) compound having superoxide dismutase activity, and a neo catalase (neoCAT) compound having catalase activity.

5. Use of the artificial blood substitute solution according to any one of claims 2 to 4 for the preparation of a medicament for treating blood loss, conditions caused by carbon dioxide accumulation, ischemia-reperfusion injury or diver's disease.

6. The method according to claim 5, wherein the ischemia-reperfusion injury comprises severe hemorrhagic shock, stroke, myocardial infarction or a combination thereof.

7. Use of the synthetic enzyme active solution according to claim 1 or claim 4 for treating liquid to remove carbon dioxide.

8. The use according to claim 7, wherein the liquid is selected from the group consisting of blood and air.

9. Use according to claim 8, wherein the liquid is blood in an artificial lung.

10. The use according to claim 8, wherein the liquid is dialyzed blood.

11. The method according to claim 9, wherein the blood in the artificial lung is used to transport oxygen and remove carbon dioxide in patients (e.g., patients with severe coronavirus).

12. Use of the artificial blood substitute solution according to any one of claims 2 to 4 for preserving organs or cells for transplantation, or for preserving cells such as stem cells for industrial production and storage.

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