Preparation method for and use of lactoferrin and formulation thereof

Abstract

Provided are a preparation method for and a use of lactoferrin and a formulation thereof, wherein the iron saturation content of the lactoferrin is 10 mg / g or more. The prepared lactoferrin can quickly reduce the iron content in a microenvironment, and thus can better deplete iron to treat iron overload diseases, including but not limited to myocardial ischemia reperfusion injury, renal ischemia reperfusion injury, cerebral infarction injury and other diseases; moreover, the lactoferrin has enhanced antibacterial activity mediated by iron deprivation.

Description

A kind of preparation method and application of lactoferrin and its preparation Technical Field

[0001] The present application belongs to the technical field of functional proteins and relates to a preparation method and application of lactoferrin and its preparation. Background Art

[0002] Lactoferrin is a small molecular weight natural milk-derived glycoprotein with a relative molecular mass of approximately 80KDa. It is widely present in milk, tears and saliva. Lactoferrin has an iron-absorbing capacity 300 times higher than transferrin and can be used as a natural iron chelator. The sequence of lactoferrin consists of more than 700 amino acids. Structurally, lactoferrin consists of two globular lobes and its structure is stabilized by disulfide bonds. Previous theories believed that the center of each of the two globular lobes of lactoferrin can chelate one iron, and the chelation site is supplemented by carbonate provided by the outside world (for example, a certain concentration of carbonate ions in body fluids) to stabilize the chelation site, making it difficult for the iron to dissociate. Lactoferrin will open the iron chelation site and release iron ions in an environment with a pH below 4.0; in a neutral environment, apolactoferrin can spontaneously re-chelate free iron ions.

[0003] The lactoferrin provided in the prior art has only two iron ion binding sites, such as document 1 (Doi: 10.1016 / j.seppur.2020.117522), document 2 (Doi: 10.3168 / jds.S0022-0302(95)76862-X.), document 3 (Doi: 10.3168 / jds.S0022-0302(84)81345-4.) and patent document CN102459328B. In addition, as recorded in document 2, Lf contains 260 μg / g of iron and 18% of iron, so the iron saturation content is about 1.4 mg / gprot.

[0004] The higher the iron saturation content of lactoferrin, the stronger its iron-absorbing ability. This faster and stronger iron-absorbing ability allows lactoferrin to rapidly reduce the iron content in the microenvironment, effectively reducing iron and treating iron overload diseases. It also has a stronger ability to deprive iron and thus fight bacteria. Therefore, it is of great significance to develop a method for preparing lactoferrin with strong iron-absorbing ability to further improve this ability.

[0005] Free hemoglobin, at a concentration of just 1 μM, can cause cell death and is highly toxic. In many pathological scenarios associated with hemolysis, hemoglobin is released when blood cells rupture, causing organ damage. Therefore, proteins are needed to bind hemoglobin to prevent cells from actively taking it up and thus prevent cell / tissue damage.

[0006] Currently, hemopexin (Sigma-Aldrich, H9291) has been approved as a drug for the treatment of sickle cell anemia. Hemopexin is currently recognized as the strongest hemoglobin-binding protein. Other proteins that can serve as heme-binding proteins include Serum Albumin (nearshore protein, Cat. No.: CP01), Alpha1-Microglobulin (MCE, HY-P7489), Alpha-1-Antitrypsin (nearshore protein, Cat. No.: C533), Lipopolysaccharide-Binding Protein (nearshore protein, Cat. No.: C370), Casein (J&K / J&K, 166199-100G), and Transferrin (Thermo Fisher Scientific, T8158); among them, hemopexin has 1 to 2 binding sites, Serum Albumin has 1 binding site, Alpha 1-Microglobulin has 2 binding sites, alpha-1-Antitrypsin has 1 binding site, Lipopolysaccharide-Binding Protein has 1 binding site, Casein has 1 binding site, and transferrin has 4 binding sites. Technical issues

[0007] However, existing heme-binding proteins have a limited number of binding sites, which limits their efficiency in clearing heme and requires more protein to be injected / implanted per unit amount of heme. Therefore, research into the use of lactoferrin for heme binding is crucial to address the limited number of heme-binding sites on existing proteins. Technical Solutions

[0008] The present application provides a lactoferrin with strong iron-absorbing ability.

[0009] The present application provides lactoferrin with strong iron-absorbing ability, wherein the iron saturation content of the lactoferrin with strong iron-absorbing ability is above 10 mg / g.

[0010] Optionally, the iron saturation content of the lactoferrin with strong iron absorption ability is 10 mg / g to 14.43 mg / g.

[0011] Optionally, the lactoferrin with strong iron absorption ability is obtained by methacrylating lactoferrin with low iron content.

[0012] Low-iron lactoferrin exposes the most potent iron-binding structures within the lactoferrin structure, resulting in a strong iron-absorbing capacity. After methacrylation, the surface positive charge density of low-iron lactoferrin decreases, facilitating the entry of iron ions into the protein. Methacrylation also alters the 3D conformation of low-iron lactoferrin (after chemical structural changes, the protein automatically adjusts its 3D conformation based on the principle of minimum energy), creating more binding sites for iron. This increase in the protein's iron-binding sites significantly enhances its iron-absorbing capacity, raising the iron saturation content of lactoferrin to over 10 mg / g.

[0013] Optionally, the lactoferrin with low iron content refers to lactoferrin with an iron content of less than 0.15 mg / g.

[0014] Optionally, the iron content of the low-iron lactoferrin is 0.0139 mg / g to 0.15 mg / g.

[0015] Optionally, the iron content of the low-iron lactoferrin is 0.010 mg / g to 0.015 mg / g.

[0016] The present application also provides a lactoferrin with strong iron-absorbing ability, wherein the lactoferrin with strong iron-absorbing ability has 14 to 20 iron ion binding sites.

[0017] The present application also provides lactoferrin, wherein the lactoferrin is apolactoferrin that has been methacrylated, and the number of heme binding sites on the lactoferrin is 7 to 13.

[0018] The lactoferrin may be the lactoferrin with strong iron absorption ability described in this application.

[0019] Optionally, the iron content of the apolactoferrin is lower than 0.15 mg / g.

[0020] Optionally, methacrylic anhydride is used for methacrylation treatment, wherein the grafting rate of methacrylic anhydride is 30% to 100%.

[0021] The present application also provides lactoferrin, wherein the KD value of the lactoferrin binding to heme is 0.015 nM to 2.02 nM.

[0022] The present application also provides an application of lactoferrin in binding hemoglobin, that is, the lactoferrin described in the present application is used to bind hemoglobin.

[0023] Heme forms hydrogen bonds, salt bridges, π-π conjugation, and other interactions with the amino acids on lactoferrin. Methacryl-treated lactoferrin increases the exposure range of the protein's hydrophobic domain (methacryloyl is a hydrophobic group, so the hydrophobic domain of methacryloyl-treated lactoferrin increases), and its electrostatic properties change (methacryloyl grafted onto lysine modifies the positively charged group into a neutral group, thus changing the electrostatic properties). The area of ​​the hydrophobic domain on the protein surface increases, making it easier to bind to the hydrophobic heme. Acylation of the amino acid's amino group reduces the positive charge on the protein surface, reducing the repulsion with the central positive iron of the heme, making it easier for heme to enter the protein, significantly increasing both heme affinity and binding capacity (i.e., the number of binding sites).

[0024] Optionally, the lactoferrin is formulated into a solution or prepared into a hydrogel and then injected for binding to free hemoglobin.

[0025] For example, it can be configured into a solution for intravenous / peritoneal / in situ tissue injection, or prepared into a hydrogel for injection filling, to bind to free hemoglobin, which is the free hemoglobin produced by hemolysis in the blood and the free hemoglobin produced by hemolysis in tissue bleeding.

[0026] Optionally, configuring the lactoferrin into a solution comprises:

[0027] The lactoferrin is dissolved in physiological saline to prepare a solution with a concentration of 5 to 10 mg / mL.

[0028] Optionally, preparing the lactoferrin into a hydrogel comprises:

[0029] The lyophilized powder obtained by freeze-drying the lactoferrin is dissolved in a PBS solution to prepare a mother solution, and then an initiator and lithium phenyl 2,4,6-trimethylbenzoylphosphinate are added, and the mother solution is solidified under light to form a hydrogel.

[0030] Optionally, the initiator is a free radical initiator, a redox initiator, a vitamin C-hydrogen peroxide system, or a glutathione-peroxide system.

[0031] Optionally, the free radical initiator is selected from at least one of Irgacure 2959, Irgacure 819, and camphorquinone.

[0032] Optionally, the redox initiator includes:

[0033] Oxidant: at least one selected from ammonium persulfate, potassium persulfate, and sodium persulfate;

[0034] Reducing agent: at least one selected from ascorbic acid, TEMED, sodium sulfite (Na2SO3), and sodium bisulfite (NaHSO3).

[0035] Optionally, the vitamin C-hydrogen peroxide system includes:

[0036] Oxidant: H2O2;

[0037] Reducing agent: ascorbic acid (C5H8O6).

[0038] Optionally, the glutathione-peroxide system includes:

[0039] Oxidant: carbamide peroxide (CO(NH2)2·H2O2);

[0040] Reducing agent: glutathione (GSH, containing sulfhydryl groups).

[0041] Optionally, the initiator is a photoinitiator, lithium phenyl 2,4,6-trimethylbenzoylphosphinate, and is cured under light to form a hydrogel.

[0042] Optionally, the lactoferrin concentration in the mother liquor is 0.12 g / ml, and the amount of lithium phenyl 2,4,6-trimethylbenzoylphosphinate is 0.4 wt %.

[0043] Optionally, the injection method is selected from at least one of intravenous injection, intraperitoneal injection, and in situ tissue injection.

[0044] The present application also provides a method for preparing lactoferrin (i.e., lactoferrin with strong iron-absorbing ability), which comprises performing methacrylation treatment on lactoferrin with low iron content to obtain the lactoferrin with strong iron-absorbing ability.

[0045] Optionally, the method further comprises de-ironizing the lactoferrin to obtain the lactoferrin with low iron content.

[0046] Optionally, the low iron content lactoferrin refers to lactoferrin with an iron content of less than 0.15 mg / g;

[0047] Optionally, the ratio of the iron content of the low-iron lactoferrin to the iron content of the lactoferrin before apo-ironization is 3.97% to 9.8%; the ratio of the iron content of the lactoferrin with strong iron absorption ability to the iron content of the lactoferrin before apo-ironization is 2.86% to 8.5%.

[0048] Optionally, the ratio of the iron content of the lactoferrin with low iron content to the iron content of the lactoferrin before apo-ironization is 9.8%; the ratio of the iron content of the lactoferrin with strong iron absorption ability to the iron content of the lactoferrin before apo-ironization is 8.5%.

[0049] Optionally, the iron saturation content of the lactoferrin with strong iron absorption ability is above 10 mg / g.

[0050] Optionally, the lactoferrin with low iron content is apolactoferrin.

[0051] Optionally, when the lactoferrin is de-ironized, a lactoferrin solution is first prepared, and the retentate is lyophilized after dialysis to obtain lactoferrin with a low iron content.

[0052] Optionally, dialyzing the lactoferrin solution comprises placing the lactoferrin solution (to-be-apoferrin lactoferrin solution) into a dialysis bag, and placing the dialysis bag in a PBS solution for dialysis for 48 to 72 hours.

[0053] Optionally, the retentate after dialysis is freeze-dried to obtain the apolactoferrin.

[0054] Optionally, during the dialysis process, the concentration of the PBS solution in the dialysis bag is gradually reduced. For example, the dialysate PBS solution can be continuously replaced.

[0055] The iron removal efficiency can be increased by gradiently adjusting the concentration of PBS solution in the dialysis step of the de-ironification process. In addition, methacrylation of low-iron lactoferrin can enhance its iron absorption capacity, with the iron saturation content increased to a maximum of 14.43 mg / g and the maximum number of binding sites increased to 20, meaning that the iron ions that can be bound by a unit of lactoferrin are increased by 10 times.

[0056] Optionally, the concentration of the PBS solution is gradually reduced by:

[0057] The concentration of PBS solution was gradually decreased from 0.1 M to 0.05 M during the 0th to 24th hour (excluding 24 hours);

[0058] From 24 to 48 hours (excluding 48 hours), the concentration of PBS solution was gradually reduced from 0.05M to 0.01M.

[0059] The concentration of PBS solution was gradually decreased from 0.01M to 0M during the 48th to 72th hour.

[0060] By gradually reducing the phosphate concentration in the dialysis environment, that is, by continuously bringing out the iron ions released by lactoferrin in an acidic environment through osmotic pressure, the iron removal efficiency can be increased.

[0061] Optionally, the lactoferrin solution is prepared by dissolving lactoferrin powder (lactoferrin powder to be de-ferrified) in a mixed solution at room temperature.

[0062] Optionally, the lactoferrin is lactoferrin from colostrum, milk and whey of other mammals such as cows, humans and camels, as well as recombinant lactoferrin obtained by fermentation of genetically engineered bacteria and plants;

[0063] Optionally, the mixed solution is prepared by adding acetic acid, sodium acetate (the molar ratio of acetic acid to sodium acetate is 1:1) and EDTA (sodium ethylenediaminetetraacetic acid) to a PBS solution (phosphate buffer) with a pH of 2.0 to 4.0. For example, the concentration of PBS in the mixed solution is 0.01M, the total concentration of acetic acid and sodium acetate is 0.1M, and the concentration of EDTA is 40 to 500mM. The pH of the PBS solution used in the preparation of the mixed solution is adjusted to 2.0 to 4.0 by an acid selected from any one of the following: acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid,

[0064] Optionally, the concentration of the lactoferrin solution is 0.5-10 g / 100 ml.

[0065] The method for preparing lactoferrin with strong iron absorption ability as described above includes the following steps of methacrylation treatment of lactoferrin with low iron content:

[0066] Optionally, the low-iron-content lactoferrin is methacrylated with methacrylic anhydride.

[0067] Optionally, the process for methacrylation of low iron lactoferrin includes:

[0068] Step (1) dissolving the low iron content lactoferrin in PBS solution to prepare a lactoferrin stock solution;

[0069] Step (2) adding methacrylic anhydride to the lactoferrin stock solution to react, and obtaining lactoferrin with strong iron absorption ability after the reaction.

[0070] Optionally, the low-iron-content lactoferrin in step (1) is dissolved in a PBS solution at room temperature to prepare a lactoferrin stock solution with a concentration of 1 to 10 g / 100 ml.

[0071] Optionally, the pH value of the PBS solution in step (1) is 2.0 to 4.0 (for example, adjusted to 2.0 to 4.0 by acid, the acid being selected from any one of the following: acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid).

[0072] Optionally, in step (2), the ratio of methacrylic anhydride to low-iron lactoferrin is 0.01-2 mL / 1 g.

[0073] Optionally, in step (2), the ratio of methacrylic anhydride to low-iron lactoferrin is 0.03 mL / 1 g.

[0074] Optionally, during the methacrylation treatment in step (2), the grafting rate of methacrylic anhydride is 10% to 100%.

[0075] Optionally, the addition of methacrylic anhydride to the lactoferrin stock solution in step (2) is performed under high-speed stirring at 400 to 1000 rpm.

[0076] Optionally, in step (2), methacrylic anhydride is added to the lactoferrin stock solution and the pH value is adjusted to 6.5-7.5 for reaction.

[0077] Optionally, after adjusting the pH value to 6.5-7.5 in step (2), the reaction is continued with stirring at 10-25° C. for 0.5-24 hours, and lactoferrin with strong iron absorption ability is obtained through post-treatment after the reaction.

[0078] Optionally, the post-treatment is dialysis for 24 to 72 hours, ultrafiltration concentration followed by two filtrations using a filter membrane, and freeze-drying the filtrate to obtain lactoferrin with strong iron absorption capacity.

[0079] Optionally, the filtration membrane used in step (2) has a pore size of 0.1 μm or 0.22 μm.

[0080] The present application also provides a preparation for clearing hemoglobin, wherein the preparation comprises the aforementioned lactoferrin.

[0081] Optionally, the preparation is a microsphere having a porous structure, and the porosity of the preparation is greater than 80%.

[0082] The microspheres with the porous structure have a larger specific surface area and a better hemoglobin-clearing effect.

[0083] Optionally, the preparation has a specific surface area greater than 35 m2 / g.

[0084] Optionally, the preparation has 7 to 13 binding sites with lactoferrin.

[0085] The present application also provides a method for preparing the preparation for removing hemoglobin, wherein an aqueous phase and an oily phase are mixed to form an emulsion containing a photoinitiator, and the emulsion is photocrosslinked to obtain a preparation in the form of microspheres, wherein the aqueous phase uses water as a solvent and contains a photoinitiator, the lactoferrin with strong iron absorption ability, and methacrylated gelatin, and the oily phase uses liquid paraffin as a solvent and contains an emulsifier.

[0086] Optionally, the content of lactoferrin in the aqueous phase is 5-30% w / v.

[0087] Optionally, the content of methacrylated gelatin in the aqueous phase is 1 to 20% w / v.

[0088] Optionally, the content of the photoinitiator in the aqueous phase is 0.1-1% w / v.

[0089] Optionally, in the aqueous phase, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0090] Optionally, the content of the emulsifier in the oil phase is 1 to 20% w / v.

[0091] Optionally, in the oil phase, the emulsifier is Span 80.

[0092] Optionally, the water phase and the oil phase are driven by injection pumps respectively, and the oil phase and the water phase merge and emulsify through the pipeline at a flow rate ratio of 10,000 to 50,000:1 to form droplets.

[0093] Optionally, the flow rate of the oil phase is 1 to 5 mL / min.

[0094] Optionally, the flow rate of the aqueous phase is 10 to 1000 μL / min.

[0095] Optionally, the emulsion is frozen and then photocrosslinked under 405 nm UV light.

[0096] Alternatively, the emulsion is frozen before photocrosslinking, the emulsion being frozen at -100°C to -30°C and then frozen in liquid nitrogen.

[0097] Alternatively, the morphology can be maintained by placing the plate at 4°C for 5-10 minutes before photocrosslinking.

[0098] Optionally, after the photocrosslinking is completed, the obtained microspheres are washed with petroleum ether, -80°C acetone and deionized water to obtain a preparation.

[0099] The present application also provides a use of a preparation in the treatment of iron overload diseases, wherein the preparation is the preparation for clearing hemoglobin as described in any one of claims 52 to 55.

[0100] Optionally, the iron overload disease is myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury or cerebral infarction-reperfusion injury.

[0101] Reperfusion therapy is a method of reopening occluded blood vessels and reperfusing ischemic tissues through the use of thrombolytic drugs, interventional therapy, or surgical treatment. During reperfusion therapy, blood flow is restored to tissue cells that have suffered a certain period of ischemia, which may aggravate their structural damage and cause injury. One of the reasons for this is related to iron overload.

[0102] The present application also provides a method for treating iron overload diseases, comprising injecting the preparation described in the present application into the lesion site.

[0103] Optionally, the iron overload disease includes myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury or cerebral infarction-reperfusion injury.

[0104] Alternatively, the lesion is injected using an interventional device including a needle catheter.

[0105] The present application also provides a reperfusion treatment method, comprising:

[0106] injecting the preparation described in the present application into the ischemic tissue;

[0107] Reperfusion therapy is performed on the ischemic tissue.

[0108] The preparation described in the present application contains lactoferrin with strong iron absorption ability, which can reduce cell damage during reperfusion therapy. The timing of injection of the preparation can be different stages of reperfusion therapy, especially before reperfusion therapy. Based on this, it can be understood that the present application also provides a method for preventing reperfusion injury.

[0109] Optionally, the preparation is pre-injected into the ischemic tissue and then subjected to reperfusion therapy.

[0110] The present application also provides a reperfusion treatment method for myocardial ischemia, comprising pre-injecting the preparation described in the present application into the left ventricular wall, and then performing reperfusion treatment on the left ventricle.

[0111] Optionally, the preparation is injected into the left ventricular wall using an interventional device comprising a needle catheter, which is passed sequentially through the femoral artery and the aortic valve to the left ventricular endocardium, and the preparation is injected into the left ventricular wall through the needle catheter.

[0112] Optionally, the injection points of the needle catheter are distributed at predetermined locations on the left ventricular wall, and the number of injection points is at least 3.

[0113] Optionally, the injection volume at each injection point is not less than 0.1 mL.

[0114] Optionally, the injection volume at each injection point is 0.1 to 0.3 mL.

[0115] The lactoferrin of the present application with strong iron absorption ability can quickly reduce the iron content in the microenvironment, and can better reduce iron to treat iron overload diseases, including but not limited to myocardial ischemia reperfusion, renal ischemia reperfusion, cerebral infarction and other diseases; at the same time, it has better iron deprivation and antibacterial ability. Beneficial effects

[0116] The present application modifies lactoferrin with a reasonable degree of methacrylic anhydride grafting, which can enhance the hemoglobin absorption ability of lactoferrin. The hemoglobin-binding pockets of methacrylylated lactoferrin are increased, and the affinity and binding capacity for hemoglobin are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIG1 is an ICP test graph of the lactoferrin raw material, apo-lactoferrin (Apo-Lf) and lactoferrin with strong iron absorption ability (Apo-LfMA) of Example 1;

[0118] FIG2 is a nuclear magnetic resonance spectrum of apo-lactoferrin (Apo-Lf) and lactoferrin with strong iron-absorbing ability (Apo-LfMA) of Example 1;

[0119] FIG3 is a MALDI-TOF image of apo-lactoferrin (Apo-Lf) and lactoferrin with strong iron absorption ability (Apo-LfMA) of Example 1;

[0120] FIG4 is a diagram showing the 3D structural changes before and after modification of apo-lactoferrin (Apo-Lf) with methacrylic anhydride to obtain lactoferrin (Apo-LfMA) with strong iron-absorbing ability in Example 1;

[0121] FIG5 is a schematic diagram of specific amino acid sites modified by methacrylic anhydride on Apo-Lf;

[0122] Figure 6 is a molecular docking diagram of lactoferrin (Apo-LfMA) with strong iron absorption ability binding to iron ions, wherein the spheres represent iron ions, indicating that the protein chelates 20 iron ions;

[0123] Figures 7 to 16 are the detailed 20-site analysis of the iron chelation of Apo-LfMA after methacrylic anhydride grafting, i.e., the amino acid composition and the analysis of the chelation ability with iron;

[0124] FIG17 is a comparison of the iron saturation content of apo-lactoferrin (Apo-Lf) of Example 1 and lactoferrin with strong iron absorption ability (Apo-LfMA);

[0125] FIG18 is an ICP test graph of apo-lactoferrin (Apo-Lf) at pH = 4.0 in Example 2 and pH = 2.0 in Example 1;

[0126] FIG19 is a MALDI-TOF diagram of Example 3 (left side in the figure) with a methacrylic anhydride grafting degree of 10% (10% Apo-LfMA) and Example 4 (right side in the figure) with a methacrylic anhydride grafting degree of 100% (100% Apo-LfMA);

[0127] FIG20 shows the iron saturation content of Example 3 with a methacrylic anhydride grafting degree of 10% (10% Apo-LfMA) and Example 4 with a methacrylic anhydride grafting degree of 100% (100% Apo-LfMA);

[0128] FIG21 is an ICP test graph of the lactoferrin raw material, apo-lactoferrin (Apo-Lf), and methacrylylated lactoferrin (Apo-LfMA) of Example 5, showing the iron content of the raw materials;

[0129] FIG22 is a diagram showing the 3D structural changes of apo-lactoferrin (Apo-Lf) and methacrylylated lactoferrin (Apo-LfMA) before and after methacrylic anhydride modification in Example 5;

[0130] FIG23 is a schematic diagram of the specific amino acid sites modified by methacrylic anhydride on Apo-Lf;

[0131] FIG24 shows the changes in surface electrostatic properties of apo-lactoferrin (Apo-Lf) and methacrylylated lactoferrin (Apo-LfMA) before and after modification with methacrylic anhydride in Example 5;

[0132] Figure 25 shows the changes in surface hydrophobicity of apo-lactoferrin (Apo-Lf) and methacrylylated lactoferrin (Apo-LfMA) before and after modification with methacrylic anhydride in Example 5, where a and b represent the hydrophobicity of the two sides of the protein, respectively;

[0133] FIG26 is a molecular dynamics simulation image of the binding of apo-lactoferrin (Apo-Lf) and methacrylylated lactoferrin (Apo-LfMA) to heme in Example 5, wherein Site 1, Site 2, ... represent the heme binding sites on the protein;

[0134] Figure 27 is a surface plasmon resonance (SPR) spectrum of apo-lactoferrin (Apo-Lf) and methacrylated lactoferrin (Apo-LfMA) bound to heme in Example 5, where "Raw" is the original curve and "Fitting" is the fitted curve;

[0135] FIG28 is a comparison of the number of heme-bound apo-lactoferrin (Apo-Lf) and methacrylylated lactoferrin (Apo-LfMA) in Example 5;

[0136] FIG29 is an ICP test graph of apo-lactoferrin (Apo-Lf) at pH = 4.0 of Example 6 and pH = 2.0 of Example 5;

[0137] FIG30 is a surface plasmon resonance (SPR) spectrum of methacrylylated lactoferrin (Apo-LfMA) obtained from apo-lactoferrin (Apo-Lf) obtained from Example 6 at pH = 4.0 and Example 5 at pH = 2.0, and binding to heme, indicating the binding constant between the protein and heme, where “Raw” is the original curve and “Fitting” is the fitted curve;

[0138] FIG31 shows the number of heme-bound lactoferrin (Apo-LfMA) obtained from apo-lactoferrin (Apo-Lf) at pH 4.0 in Example 6 and pH 2.0 in Example 5;

[0139] FIG32 is a surface plasmon resonance (SPR) spectrum of the methacrylated apolactoferrin (100% Apo-LfMA) bound to heme in Example 7, where “Raw” is the original curve and “Fitting” is the fitted curve.

[0140] FIG33 shows the number of heme-bound apo-lactoferrin (100% Apo-LfMA) treated with methacrylation in Example 7;

[0141] FIG34 is a bar graph showing the specific surface areas of ALMG and ALMS;

[0142] Figure 35 is a histogram of the porosity of ALMG and ALMS;

[0143] FIG36 is a bar graph showing the saturated heme binding number of ALMG and ALMS;

[0144] FIG37 shows live / dead staining and intracellular ferrous ion staining of cardiomyocytes treated with heme for 24 hours;

[0145] FIG38 is a graph showing cell viability of cardiomyocytes treated with heme for 24 hours in the presence of GMG, ALMG, or ALMS (the five bars in the figure correspond from left to right to Control, Haem, Haem+GMG, Haem+ALMG, and Haem+ALMS);

[0146] FIG39 shows the intracellular ferrous ions (Fe) in myocardial cells after 24 hours of heme treatment in the presence of GMG, ALMG or ALMS. 2+ ) Mean fluorescence intensity graph (5 bars in the figure, from left to right: Control, Haem, Haem+GMG, Haem+ALMG, Haem+ALMS);

[0147] FIG40 is a TEM image of an I / R rat heart with an IMH phenotype;

[0148] FIG41 is a graph showing hemoglobin concentrations after injection of different substances (the four bars in the figure correspond from left to right to Sham, I / R, GMG, and ALMS);

[0149] FIG42 is a graph showing the iron content in the myocardium after injection of different substances (the four bars in the graph correspond from left to right to Sham, I / R, GMG, and ALMS);

[0150] FIG43 is an immunofluorescence staining image of HBB and Hmox1;

[0151] FIG44 is an immunofluorescence staining image of HBB and TUNEL, scale bar = 20 μm (three lines in each of the upper and lower figures, each set of three lines from top to bottom represents HBB, TUNEL, and DAPI);

[0152] FIG45 is a DAB-Perls' blue and 4-HNE (4-hydroxynonenal) staining image of rat heart sections;

[0153] FIG46 shows the levels of myocardial injury markers in the ALMS group (the figure includes four images, a, b, c, and d, each of which has four bars, corresponding from left to right to Sham, I / R, GMG, and ALMS);

[0154] Figure 47 shows ultrasound images of infarct size and left ventricular wall thickness under different treatment conditions;

[0155] Figure 48 shows the values ​​of LVEF (left ventricular ejection fraction) and LVFS (left ventricular fractional shortening) under different treatment conditions (4 bars in each figure, from left to right corresponding to Sham, I / R, GMG, and ALMS);

[0156] Figure 49 shows the masson staining images under different treatment conditions;

[0157] Figure 50 shows the size of the infarct area and the thickness of the left ventricular wall under different treatment conditions (4 bars in each figure, from left to right corresponding to Sham, I / R, GMG, and ALMS);

[0158] FIG51 is a schematic diagram of the route for injecting ALMS into the inner wall of the left ventricle using a transcatheter system;

[0159] Figure 52 is a schematic diagram of TTC staining of left ventricular tissue;

[0160] Figure 53 is a comparison of the volume of necrotic myocardium under different treatment conditions (from left to right in the figure corresponds to Sham, I / R, and ALMS);

[0161] FIG54 is a comparison of creatine kinase isoenzymes (CK-MB) under different treatment conditions (from left to right in the figure correspond to Sham, I / R, and ALMS);

[0162] FIG55 is a comparison of creatine kinase (CK) under different treatment conditions (from left to right in the figure corresponds to Sham, I / R, and ALMS);

[0163] FIG56 is a comparison of lactate dehydrogenase (LDH) under different treatment conditions (from left to right in the figure corresponds to Sham, I / R, and ALMS);

[0164] FIG57 is a comparison of aspartate aminotransferase (AST) levels under different treatment conditions (from left to right in the figure correspond to Sham, I / R, and ALMS);

[0165] FIG58 is a comparison of cardiac troponin I (cTnI) under different treatment conditions (from left to right in the figure corresponds to Sham, I / R, and ALMS);

[0166] FIG59 is a comparison of cardiac hemoglobin under different treatment conditions (from left to right in the figure corresponds to Sham, I / R, and ALMS);

[0167] Figure 60 is a comparison of heme oxygenase 1 (HMOX1) under different treatment conditions (from left to right in the figure corresponds to Sham, I / R, and ALMS);

[0168] FIG61 shows hematoxylin-eosin (H&E)-stained images of tissue sections of pig liver, spleen, lung, and kidney (n=3). Modes for Carrying Out the Invention

[0169] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0170] The testing method involved in the present invention is as follows:

[0171] Iron content: Add 1 ml of concentrated nitric acid to 5 mg of protein powder and digest it at 70°C for 1 hour. After cooling to room temperature, dilute it with deionized water to 5 ml. The diluted solution is sent for inductively coupled plasma (ICP) testing of iron ion concentration. The obtained concentration value a ug / mL is equal to the iron content a mg / g in the protein.

[0172] Iron saturation content: Dissolve 100 mg of protein in 50 mL of a 50 μg / mL FeCl3 / NaHCO3 (molar ratio 1:1) solution at a pH of 7.2-7.4 and stir for 30 minutes. Centrifuge the mixture in a 50 kDa ultrafiltration tube to remove excess unbound iron ions. Wash the protein three times with deionized water until all unbound iron ions are removed, then lyophilize the protein to a powder. Add 1 mL of concentrated nitric acid to 5 mg of lyophilized protein powder and digest at 70°C for 1 hour. After cooling to room temperature, dilute to 5 mL with deionized water. The diluted solution is tested for iron ion concentration by inductively coupled plasma (ICP). The resulting concentration value, a μg / mL, is equal to the iron saturation content of a mg / g in the protein.

[0173] Binding constant KD value: The technology used is the surface plasmon resonance technology (SPR) of the Biacore instrument. The SPR principle is applied to detect the interaction between the ligand and the analyte on the biosensor chip. To study the interaction between two molecules, one molecule is fixed to the chip surface, while the other molecule flows continuously over the chip surface in the form of a solution. The detector can detect the binding and dissociation process of the molecule in the solution and the molecule on the chip surface in real time, and display it as the SPR response value. The calculation formula is: KD = kd / ka, where ka is the affinity rate of binding between the molecule and the protein, and kd is the dissociation rate.

[0174] Example 1

[0175] A method for preparing lactoferrin with strong iron-absorbing ability, comprising the following steps:

[0176] (1) Acetic acid, sodium acetate (molar ratio of 1:1) and EDTA were added to a PBS solution with a pH of 2.0 to prepare a mixture, and lactoferrin powder (Bega, Australia) was dissolved in the mixture at room temperature to prepare a lactoferrin solution with a concentration of 0.5 g / 100 ml;

[0177] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM.

[0178] (2) the lactoferrin solution obtained in step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution and dialyzed for 72 hours, the retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain apolactoferrin with an iron content of 0.0139 mg / g; the PBS solution is continuously replaced during the dialysis process, and the concentration of the PBS solution is gradually reduced, and the concentration of the PBS solution is gradually reduced from 0.1M to 0.05M within the 0th to 24th hour, the concentration of the PBS solution is gradually reduced from 0.05M to 0.01M within the 24th to 48th hour, and the concentration of the PBS solution is gradually reduced from 0.01M to 0M within the 48th to 72th hour;

[0179] Among them, the molecular weight cut-off of the dialysis bag is 3500Da;

[0180] (3) performing methacrylation on the apolactoferrin obtained in step (2) to obtain lactoferrin with strong iron absorption ability;

[0181] (3.1) dissolving apolactoferrin in a PBS solution with a pH of 2 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0182] (3.2) methacrylic anhydride was added to the apolactoferrin stock solution under high-speed stirring at 600 rpm, and the pH value was then adjusted to 7. The reaction was stirred at 25°C for 2 h, followed by dialysis for 48 h. The solution was then concentrated by ultrafiltration and filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was pre-frozen at -50°C and then lyophilized under a vacuum of 1 Pa to obtain lactoferrin with a saturated ferric iron content of 14.43 mg / g and a strong iron-absorbing capacity;

[0183] The ratio of methacrylic anhydride to lactoferrin is 0.03 mL / 1 g, and the grafting degree of methacrylic anhydride on lactoferrin with strong iron absorption ability is 30%.

[0184] As shown in Figure 1 , Apo-Lf and Apo-LfMA were obtained by de-ironification, with iron contents of 9.8% and 8.5% of Lf, respectively;

[0185] As shown in Figures 2 and 3, methacrylic anhydride successfully grafted reactive double bonds onto Apo-Lf, and thus the molecular weight of Apo-LfMA increased after methacrylation.

[0186] As shown in Figure 4, the changes in the 3D structure of Apo-Lf and Apo-LfMA before and after methacrylic anhydride grafting (protein morphology was constructed by AlphaFold).

[0187] Figure 5 shows a schematic diagram of the specific amino acid (lysine) sites modified by methacrylic anhydride on Apo-Lf. The specific 14 sites are listed in Table 1 below; the lowercase "k" represents the specific lysine modification position. The new Apo-LfMA protein structure was obtained for subsequent molecular docking analysis of iron ions.

[0188] 4 and 6 , it can be seen that after methacrylation, the 3D conformation of low-iron lactoferrin changes (after the chemical structure changes, the protein automatically adjusts its 3D conformation based on the principle of minimum energy), generating more binding sites for iron.

[0189] Through molecular docking of Apo-LfMA and iron ions, it was found that there are 20 iron chelating sites of Apo-LfMA after methacrylic anhydride grafting. The specific analysis diagrams of the 20 sites are shown in Figures 7 to 16.

[0190] Table 1

[0191] As shown in FIG17 , the saturation content of lactoferrin bound to ferric iron increased significantly after methacrylation treatment.

[0192] Example 2

[0193] A method for preparing lactoferrin with strong iron-absorbing ability, comprising the following steps:

[0194] (1) Acetic acid, sodium acetate (molar ratio of 1:1) and EDTA were added to a PBS solution with a pH of 4.0 to prepare a mixed solution, and lactoferrin powder (Bega, Australia) was dissolved in the mixed solution at room temperature to prepare a lactoferrin solution with a concentration of 10 g / 100 ml;

[0195] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 40 mM;

[0196] (2) the lactoferrin solution obtained in step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution and dialyzed for 72 hours, the retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain apolactoferrin with an iron content of 0.149 mg / g; the PBS solution is continuously replaced during the dialysis process, and the concentration of the PBS solution is gradually reduced, and the concentration of the PBS solution is gradually reduced from 0.1M to 0.05M within the 0th to 24th hour, the concentration of the PBS solution is gradually reduced from 0.05M to 0.01M within the 24th to 48th hour, and the concentration of the PBS solution is gradually reduced from 0.01M to 0M within the 48th to 72th hour;

[0197] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0198] As shown in FIG18 , increasing the deferrification pH to 4.0 in Example 2 will reduce the deferrification efficiency. The iron content of Apo-lf obtained by deferrification at pH = 4.0 is 0.149 mg / g, which is 10 times higher than that of Example 1 at pH = 2.0.

[0199] (3) performing methacrylation on the apolactoferrin obtained in step (2) to obtain lactoferrin with strong iron absorption ability;

[0200] (3.1) dissolving apolactoferrin in a PBS solution at pH 4 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0201] (3.2) methacrylic anhydride was added to the apolactoferrin stock solution under high-speed stirring at 600 rpm, and the pH was adjusted to 7.2. The solution was stirred at 25° C. for 2 h, dialyzed for 48 h, and then ultrafiltration and concentration were performed. The solution was filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was pre-frozen at −50° C. and then lyophilized under a vacuum of 1 Pa to obtain lactoferrin with a saturated ferric iron content of 14.43 mg / g and a strong iron-absorbing capacity.

[0202] The ratio of methacrylic anhydride to lactoferrin is 0.03 mL / 1 g, and the grafting degree of methacrylic anhydride on lactoferrin with strong iron absorption ability is 30%.

[0203] Example 3

[0204] A method for preparing lactoferrin with strong iron-absorbing ability, comprising the following steps:

[0205] (1) Acetic acid, sodium acetate (molar ratio of 1:1) and EDTA were added to a PBS solution to prepare a mixture, and lactoferrin powder (Bega, Australia) was dissolved in the mixture at room temperature to prepare a lactoferrin solution with a concentration of 0.5 g / 100 ml;

[0206] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM.

[0207] (2) the lactoferrin solution obtained in step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution and dialyzed for 72 hours, the retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain apolactoferrin with an iron content of 0.0139 mg / g; the PBS solution is continuously replaced during the dialysis process, and the concentration of the PBS solution is gradually reduced. The concentration of the PBS solution is gradually reduced from 0.1M to 0.05M within the 0th to 24th hour, the concentration of the PBS solution is gradually reduced from 0.05M to 0.01M within the 24th to 48th hour, and the concentration of the PBS solution is gradually reduced from 0.01M to 0M within the 48th to 72th hour;

[0208] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0209] (3) performing methacrylation on the apolactoferrin obtained in step (2) to obtain lactoferrin with strong iron absorption ability;

[0210] (3.1) dissolving apolactoferrin in a PBS solution with a pH of 2 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0211] (3.2) Under high-speed stirring at 400 rpm, methacrylic anhydride was added to the apolactoferrin stock solution, and the pH was adjusted to 7.5. The solution was stirred at 20° C. for 5 h, dialyzed for 48 h, and then concentrated by ultrafiltration and filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was pre-frozen at −50° C. and then lyophilized under a vacuum of 1 Pa to obtain lactoferrin with a saturated ferric iron content of 10.95 mg / g as shown in Figures 19 and 20;

[0212] The ratio of methacrylic anhydride to lactoferrin is 0.01 mL / 1 g, and the grafting degree of methacrylic anhydride on lactoferrin with strong iron absorption ability is 10%.

[0213] Example 4

[0214] A method for preparing lactoferrin with strong iron-absorbing ability, comprising the following steps:

[0215] (1) Acetic acid, sodium acetate (molar ratio of 1:1) and EDTA were added to a PBS solution to prepare a mixed solution, and lactoferrin powder (Bega, Australia) was dissolved in the mixed solution at room temperature to prepare a lactoferrin solution with a concentration of 10 g / 100 ml;

[0216] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM.

[0217] (2) the lactoferrin solution obtained in step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution and dialyzed for 72 hours, the retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain apolactoferrin with an iron content of 0.0139 mg / g; the PBS solution is continuously replaced during the dialysis process, and the concentration of the PBS solution is gradually reduced, the concentration of the PBS solution is 0.1 M within the 0th to 24th hour, the concentration of the PBS solution is 0.05 M within the 24th to 48th hour, and the concentration of the PBS solution is 0 M within the 48th to 72th hour;

[0218] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0219] (3) performing methacrylation on the apolactoferrin obtained in step (2) to obtain lactoferrin with strong iron absorption ability;

[0220] (3.1) dissolving apolactoferrin in a PBS solution with a pH of 2 at room temperature to prepare an apolactoferrin stock solution with a concentration of 1 g / 100 ml;

[0221] (3.2) Under high-speed stirring at 1000 rpm, methacrylic anhydride was added to the apolactoferrin stock solution, and then the pH value was adjusted to 6.5. The reaction was stirred at 10°C for 24 hours, and the reaction was dialyzed for 72 hours. After ultrafiltration and concentration, the solution was filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was pre-frozen at a temperature of -50°C and then lyophilized under a vacuum condition of 1 Pa to obtain lactoferrin with a saturated ferric iron content of 12.46 mg / g as shown in Figures 19 and 20;

[0222] The ratio of methacrylic anhydride to lactoferrin is 2 mL / 1 g, and the grafting degree of methacrylic anhydride on lactoferrin with strong iron absorption ability is 100%.

[0223] Example 5

[0224] The application of lactoferrin in binding hemoglobin is as follows:

[0225] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 2.0 to obtain a mixed solution, and then dissolving apoplactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain a 10 g / 100 ml apoplactoferrin solution;

[0226] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM.

[0227] (2) the apolactoferrin solution obtained in step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution and dialyzed for 72 hours, the retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain apolactoferrin with an iron content of 0.0139 mg / g;

[0228] The molecular weight cut-off of the dialysis bag was 7000 Da. The concentration of the PBS solution used for dialysis was gradually reduced from 0.1 M to 0.05 M during the 0th to 24th hour, from 0.05 M to 0.01 M during the 24th to 48th hour, and from 0.01 M to 0 M during the 48th to 72th hour.

[0229] (3) dissolving the apolactoferrin obtained in step (2) in a PBS solution having a pH of 2.0 at room temperature to prepare an apolactoferrin stock solution having a concentration of 10 g / 100 ml;

[0230] (4) adding methacrylic anhydride to the apolactoferrin stock solution under high-speed stirring at 400 rpm, adjusting the pH to 7, and continuously stirring the reaction at 25° C. for 2 h. After the reaction, dialyzing for 72 h, ultrafiltration and concentration were performed, and the solution was filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was freeze-dried at a temperature of −50° C. and a vacuum degree of 1 Pa to obtain apolactoferrin treated with methacrylation;

[0231] The ratio of methacrylic anhydride to apolactoferrin is 0.03 mL / 1 g; and the grafting degree of methacrylic anhydride on the apolactoferrin treated with methacrylic acid is 30%.

[0232] The prepared methacrylated lactoferrin was dissolved in physiological saline to prepare a solution with a concentration of 5 mg / mL and then injected for binding to free hemoglobin;

[0233] There are 13 heme binding sites on methacryloyl-treated lactoferrin; the K D The value is 0.015nM.

[0234] As shown in FIG21 , by de-ironification, Apo-Lf and Apo-LfMA were obtained, each containing 9.8% and 8.5% of Lf iron, respectively.

[0235] As shown in Figure 22, the changes in the 3D structure of Apo-Lf and Apo-LfMA before and after methacrylic anhydride grafting (protein morphology constructed by AlphaFold).

[0236] FIG23 is a schematic diagram showing the specific amino acid sites modified by methacrylic anhydride on Apo-Lf.

[0237] As shown in FIG24 , the surface positive charge density of the methacrylylated lactoferrin with a grafting degree of 30% decreased, and the resistance to the positively charged iron ions at the hemoglobin center entering the interior of the protein decreased.

[0238] As shown in FIG25 , the exposed area of ​​the surface hydrophobic domains of the methacrylated lactoferrin with a grafting degree of 30% is increased, which is beneficial to the binding of hydrophobic heme.

[0239] As shown in FIG26 , the amount of heme bound to methacrylylated lactoferrin with a grafting degree of 30% was higher than that of ungrafted lactoferrin.

[0240] As shown in FIG27 , the saturation content of heme-binding lactoferrin treated with methacrylation at a grafting degree of 30% increased, and the affinity also increased (expressed as K D value decreases).

[0241] As shown in FIG28 , the amount of heme-bound lactoferrin increased after methacrylation.

[0242] Example 6

[0243] The application of lactoferrin in binding hemoglobin is as follows:

[0244] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 4.0 to obtain a mixed solution, and then dissolving apoplactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain a 10 g / 100 ml apoplactoferrin solution;

[0245] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM.

[0246] (2) Preparation of apolactoferrin;

[0247] The apolactoferrin solution obtained in step (1) is placed in a dialysis bag, which is placed in a PBS solution and dialyzed for 72 hours. The retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain an apolactoferrin with an iron content of 0.149 mg / g;

[0248] The molecular weight cut-off of the dialysis bag was 7000 Da. The concentration of the PBS solution used for dialysis was gradually reduced from 0.1 M to 0.05 M during the 0th to 24th hour, from 0.05 M to 0.01 M during the 24th to 48th hour, and from 0.01 M to 0 M during the 48th to 72th hour.

[0249] As shown in FIG29 , increasing the deferrification pH to 4.0 will reduce the deferrification efficiency. The iron content of Apo-lf obtained by deferrification at pH = 4.0 is 0.149 mg / g, which is 10 times higher than that at pH = 2.0.

[0250] (3) dissolving the apolactoferrin obtained in step (2) in a PBS solution having a pH of 4.0 at room temperature to prepare an apolactoferrin stock solution having a concentration of 10 g / 100 ml;

[0251] (4) adding methacrylic anhydride to the apolactoferrin stock solution under high-speed stirring at 40 rpm, adjusting the pH to 7, and continuously stirring the reaction at 25° C. for 2 h. After the reaction, dialyzing was performed for 72 h. After ultrafiltration and concentration, the solution was filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was freeze-dried at a temperature of −50° C. and a vacuum degree of 1 Pa to obtain apolactoferrin treated with methacrylation;

[0252] The ratio of methacrylic anhydride to apolactoferrin is 0.03 mL / 1 g; and the grafting degree of methacrylic anhydride on the apolactoferrin treated with methacrylic acid is 30%.

[0253] The lyophilized powder of the methacrylated apolactoferrin was dissolved in 0.2 M PBS to prepare a 0.12 g / ml stock solution, followed by the addition of 0.4 wt% lithium phenyl 2,4,6-trimethylbenzoylphosphinate and stirring to dissolve the solution. The aqueous solution was sterile filtered and solidified under 405 nm light to form a hydrogel, which was then injected to bind to free hemoglobin.

[0254] There are 11 heme binding sites on methacryloyl-treated lactoferrin; the K D The value is 0.38nM;

[0255] As shown in Figures 30 and 31, the binding constant K of the methacrylated lactoferrin and Heme obtained at pH = 2.0 D The binding amount and the amount of lactoferrin were higher than those obtained by methacryloylation at pH 4.0.

[0256] Example 7

[0257] The application of lactoferrin in binding hemoglobin is as follows:

[0258] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 2.0 to obtain a mixed solution, and then dissolving apoplactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain a 10 g / 100 ml apoplactoferrin solution;

[0259] The concentration of PBS in the mixture was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM.

[0260] (2) Preparation of apolactoferrin;

[0261] The apolactoferrin solution obtained in step (1) is placed in a dialysis bag, which is placed in a PBS solution and dialyzed for 72 hours. The retentate after dialysis is pre-frozen at a temperature of -50°C, and then lyophilized under a vacuum condition of 1 Pa to obtain an apolactoferrin with an iron content of 0.0139 mg / g;

[0262] The molecular weight cut-off of the dialysis bag was 7000 Da. The concentration of the PBS solution used for dialysis was gradually reduced from 0.1 M to 0.05 M during the 0th to 24th hour, from 0.05 M to 0.01 M during the 24th to 48th hour, and from 0.01 M to 0 M during the 48th to 72th hour.

[0263] (3) dissolving the apolactoferrin obtained in step (2) in a PBS solution having a pH of 2.0 at room temperature to prepare an apolactoferrin stock solution having a concentration of 10 g / 100 ml;

[0264] (4) adding methacrylic anhydride to the apolactoferrin stock solution under high-speed stirring at 40 rpm, adjusting the pH to 7, and continuously stirring the reaction at 25° C. for 2 h. After the reaction, dialyzing was performed for 72 h. After ultrafiltration and concentration, the solution was filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was freeze-dried at a temperature of −50° C. and a vacuum degree of 1 Pa to obtain apolactoferrin treated with methacrylation;

[0265] The ratio of methacrylic anhydride to apolactoferrin is 2 mL / 1 g; and the grafting degree of methacrylic anhydride on the apolactoferrin treated with methacrylic acid is 100%.

[0266] The prepared methacrylated lactoferrin was dissolved in physiological saline to prepare a solution with a concentration of 10 mg / mL and then injected for binding to free hemoglobin;

[0267] There are 10 heme binding sites on methacryloyl-treated lactoferrin; the K D The value is 2.02nM;

[0268] As shown in Figures 32 and 33, the binding constant of Apo-LfMA with a methacrylic anhydride modification degree of 100% with Heme is higher than that of Apo-LfMA with a grafting degree of 30%, which means that the affinity of Apo-LfMA with a modification degree of 100% is lower than that of Apo-LfMA with a grafting degree of 30%, and the number of heme binding sites of Apo-LfMA with a methacrylic anhydride modification degree of 100% is lower than that of Apo-LfMA with a grafting degree of 30%.

[0269] Example 8 Preparation of ALMS Preparation

[0270] The aqueous phase contains 12% w / v methacryloylated apolactoferrin (Apo-LfMA) and 3% w / v methacryloylated gelatin (GelMA, for the synthesis of GelMA in this application, see the literature: Zhang, L. et al. Multileveled hierarchical hydrogel with continuous biophysical and biochemical gradients for enhanced repair of full-thickness osteochondral defect. Adv. Mater. 35, 2209565 (2023).), 0.3% w / v LAP photoinitiator; the oil phase is liquid paraffin containing 5% w / v Span 80 emulsifier.

[0271] The aqueous phase and oil phase were driven by syringe pumps respectively. The oil phase passed through the pipeline at a flow rate of 3 mL / min and the aqueous phase at a flow rate of 100 μL / min, and were emulsified to form droplets. The collected droplets were first frozen at -80°C for 1 minute, then frozen in liquid nitrogen, and then photocrosslinked under 405 nm ultraviolet light for 3 minutes. The obtained microspheres were washed three times with petroleum ether, -80°C acetone and deionized water, respectively, freeze-dried, and stored at 4°C.

[0272] Example 9 Preparation of ALMG Preparation

[0273] The aqueous phase contained 12% w / v methacryloylated apolactoferrin (Apo-LfMA), 3% w / v methacryloylated gelatin (GelMA), and 0.3% w / v LAP photoinitiator; the oil phase was liquid paraffin containing 5% w / v Span 80 emulsifier.

[0274] The aqueous phase and oil phase were driven by syringe pumps respectively. The oil phase passed through the pipeline at a flow rate of 3 mL / min and the aqueous phase at a flow rate of 100 μL / min, and were emulsified to form droplets. The emulsion droplets were collected in a 10-cm culture dish and placed at 4°C for 10 minutes to maintain their morphology. They were then photocrosslinked under 405 nm ultraviolet light for 3 minutes. The obtained microspheres were washed three times with petroleum ether, -80°C acetone and deionized water, respectively, freeze-dried, and stored at 4°C.

[0275] Preparation of Comparative Example GMG

[0276] The aqueous phase contained 12% w / v methacryloyl gelatin (GelMA) and 0.3% w / v LAP photoinitiator; the oil phase was liquid paraffin containing 5% w / v Span 80 emulsifier.

[0277] The aqueous phase and oil phase were driven by syringe pumps respectively. The oil phase passed through the pipeline at a flow rate of 3 mL / min and the aqueous phase at a flow rate of 100 μL / min, and were emulsified to form droplets. The emulsion droplets were collected in a 10-cm culture dish and placed at 4°C for 10 minutes to maintain their morphology. They were then photocrosslinked under 405 nm ultraviolet light for 3 minutes. The obtained microspheres were washed three times with petroleum ether, -80°C acetone and deionized water, respectively, freeze-dried, and stored at 4°C.

[0278] Characterization of ALMS and ALMG

[0279] After freeze-drying, the porosity and specific surface area of ​​AMLS and ALMG were measured using a mercury intrusion porosimeter (Micromeritic Auto Pore IV 9510, Micromeritics Instruments, USA). Figure 34 shows the specific surface area of ​​ALMG and ALMS, and Figure 35 shows the porosity of ALMG and ALMS.

[0280] ALMG and ALMS were incubated in a 250 μM / L heme solution at pH 7.2. Aqueous samples were collected at 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours, and the heme content was assessed by the oxalic acid method and inductively coupled plasma spectrometry (ICP) to determine their heme adsorption capacity. Figure 36 shows the saturated heme binding number (Heme / protein ratio) of ALMG and ALMS detected by extracting bound heme.

[0281] In vitro experimental data of ALMS, ALMG, and GMG

[0282] As shown in FIG37 , in the presence of GMG, ALMG or ALMS, the live and dead staining of cardiomyocytes treated with heme for 24 hours and the intracellular ferrous ions (Fe 2+ , stained with Ferro Orange), green represents living cells and red represents dead cells.

[0283] FIG38 shows cell viability of cardiomyocytes (H9C2) treated with heme for 24 hours in the presence of GMG, ALMG or ALMS (n=6). FIG39 shows intracellular ferrous ions (Fe) of cardiomyocytes treated with heme for 24 hours in the presence of GMG, ALMG or ALMS. 2+ ) Mean fluorescence intensity (n=6).

[0284] In Figures 38 and 39 , the control group (control) is a culture medium without hemoglobin, and the blank group (Haem) is a culture medium containing only hemoglobin.

[0285] In vivo experimental data of ALMS, ALMG, and GMG

[0286] In a rat ischemia / reperfusion (I / R) model, ALMS inhibited myocardial ferroptosis induced by intramyocardial hemorrhage (IMH).

[0287] As shown in Figure 40 , the hearts of I / R rats with an IMH phenotype were analyzed by pathological and transmission electron microscopy (TEM) observations. As shown in Figures 41 and 42 , in the rat I / R model, IMH caused free hemoglobin in the left ventricular myocardium to rapidly accumulate to 34.74±5.70 nmol / mgprot within the first 24 hours, resulting in an 89% increase in myocardial iron content. Local implantation of ALMS reduced the amount of free hemoglobin in the myocardium to 16.69±1.87 nmol / mgprot, which was not significantly different from the baseline level of the sham-operated group. The reduction in hemoglobin concentration resulted in an 81% reduction in iron content.

[0288] As shown in Figure 41, the GMG group failed to reduce hemoglobin concentration and cardiomyocyte loss because the hemoglobin was not drained into the implanted microspheres. As shown in Figures 43 and 44, in the GMG and I / R groups, hemoglobin β chain (HBB)-positive erythrocytes were spatially associated with terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) signals, while in ALMS-treated rats, TUNEL signals in HBB-positive areas were sparse and weak, spatially demonstrating the effects of IMH on I / R injury and the salvage effect of ALMS.

[0289] As shown in Figure 45, both GMG and ALMS microspheres accumulated at the injection site, but only ALMS served as a site for draining exogenous heme from the IMH. Iron signals were concentrated at the ALMS injection site, not in the surrounding tissue, whereas severe iron deposition was evident in the surrounding tissue after GMG implantation.

[0290] As shown in Figure 46, the serum levels of myocardial injury markers (including creatine kinase isoenzyme MB (CK-MB), creatine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)) in the ALMS group were maintained at the levels of the sham operation group, which confirmed that ALMS reduced I / R injury by alleviating heme-induced ferroptosis.

[0291] As shown in Figures 47 and 48 , long-term echocardiographic assessment 35 days after surgery demonstrated that ALMS reduced left ventricular remodeling in terms of cardiac function (left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)) and cardiac geometry (left ventricular end-systolic diameter (LVIDS) and left ventricular end-systolic volume (LVESV)) compared with the GMG and I / R groups. Similarly, as shown in Figures 49 and 50 , ALMS reduced the area of ​​fibrosis in the infarct zone by 17.3% and maintained left ventricular wall thickness compared with the I / R group.

[0292] In a porcine cardiac ischemia / reperfusion model, the safety and efficacy of ALMS in the treatment of iron overload diseases (such as myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury, or cerebral infarction-reperfusion injury) were further demonstrated. ALMS is compatible with a minimally invasive transcatheter injection system (TEIS01, Dinova Medtech), which has been used for transendocardial hydrogel injection in patients with heart failure. As shown in Figure 51, under the guidance of digital subtraction angiography (DSA), a steerable double-lumen needle catheter was inserted through the femoral artery and passed through the aortic valve to reach the left ventricular endocardium. The injection site was determined by transthoracic echocardiography and digital subtraction angiography (see Figures 51 (ii-iv). ALMS was injected into the left ventricular wall without leakage, bleeding, or ventricular wall perforation (see Figure 51 (v)). Aggregated ALMS deposits were found in the myocardium, and all pigs survived the operation.

[0293] In the efficacy study, ALMS was injected into nine evenly distributed points in the infarcted left ventricle, followed by immediate reperfusion. Three days after surgery, as shown in Figure 52, TTC staining revealed significant necrotic myocardium in the anterior wall and ventricular septum of the left ventricle. Notably, the volume of necrotic myocardium in the ALMS group was significantly smaller than in the ischemia / reperfusion control group, and the color of the myocardium surrounding the ALMS injection was closer to that of healthy myocardium.

[0294] Compared to the ischemia / reperfusion group, as shown in Figure 53 , the ALMS group showed a 70.4% reduction in necrotic myocardial volume. As shown in Figures 54 to 58 , ALMS implantation also modulated the elevation of serum cardiac enzyme markers (creatine kinase isoenzyme (CK-MB), creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST)) and cardiac troponin I (cTnI) following ischemia / reperfusion injury. As shown in Figure 59 , free hemoglobin levels in the ischemia / reperfusion group increased to 2.02 times that of healthy myocardium. By capturing exogenous hemoglobin, the ALMS reduced hemoglobin levels to healthy levels. As shown in Figure 60 , heme oxygenase 1 (HMOX1) mRNA levels were significantly upregulated in the ischemia / reperfusion (I / R) group, indicating that heme was internalized into intracellular iron. By capturing exogenous hemoglobin, the ALMS significantly reduced hemoglobin 1 levels.

[0295] In a clinically relevant large animal model, ALMS inhibited ferroptosis and ischemia / reperfusion injury induced by intramyocardial hemorrhage. As shown in Figure 61, hematoxylin-eosin analysis of major organs confirmed the biosafety of ALMS in vivo.

Claims

1. A lactoferrin with strong iron-absorbing ability, characterized in that: The iron saturation content of the lactoferrin with strong iron absorption ability is above 10 mg / g.

2. The lactoferrin with strong iron absorption ability according to claim 1, characterized in that The iron saturation content of the lactoferrin with strong iron absorption ability is 10 mg / g to 14.43 mg / g.

3. The lactoferrin with strong iron absorption ability according to claim 1, characterized in that The lactoferrin with strong iron absorption ability is prepared by subjecting lactoferrin with low iron content to methacrylation.

4. The lactoferrin with strong iron absorption ability according to claim 3, characterized in that The low-iron lactoferrin refers to lactoferrin with an iron content lower than 0.15 mg / g.

5. The lactoferrin with strong iron absorption ability according to claim 3, characterized in that The iron content of the low-iron lactoferrin is 0.0139 mg / g to 0.15 mg / g.

6. A lactoferrin with strong iron absorption ability, characterized in that The lactoferrin with strong iron absorption ability has 14 to 20 iron ion binding sites.

7. The use of lactoferrin in the preparation of an iron chelating agent, characterized in that: The lactoferrin is the lactoferrin with strong iron absorption ability according to any one of claims 1 to 6.

8. The use of lactoferrin in reducing the iron content in the microenvironment, characterized in that: The lactoferrin is the lactoferrin with strong iron absorption ability according to any one of claims 1 to 6.

9. Use of lactoferrin in preparing a therapeutic agent for iron overload disease, characterized in that: The lactoferrin is the lactoferrin with strong iron absorption ability according to any one of claims 1 to 6.

10. The use according to claim 9, characterized in that The iron overload disease is myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury or cerebral infarction-reperfusion injury.

11. Lactoferrin, characterized in that The lactoferrin is apolactoferrin that has been subjected to methacrylation treatment, and the number of heme binding sites on the lactoferrin is 7 to 13.

12. The lactoferrin according to claim 11, characterized in that The iron content of the apolactoferrin is lower than 0.15 mg / g.

13. The lactoferrin according to claim 11, characterized in that Methacrylic acid anhydride is used for methacrylation treatment, wherein the grafting rate of methacrylic acid anhydride is 30% to 100%.

14. Lactoferrin, characterized in that The KD value of the lactoferrin binding to hemoglobin is 0.015nM to 2.02nM.

15. The use of lactoferrin according to any one of claims 11 to 14, characterized in that The lactoferrin is used to bind heme.

16. The use of lactoferrin according to claim 15, characterized in that The lactoferrin is configured into a solution or prepared into a hydrogel and then injected for binding with free hemoglobin.

17. The use of lactoferrin according to claim 16, characterized in that Configuring the lactoferrin into a solution comprises: The lactoferrin is dissolved in physiological saline to prepare a solution with a concentration of 5 to 10 mg / mL.

18. The use of lactoferrin according to claim 16, characterized in that The lactoferrin is prepared into a hydrogel comprising: The lyophilized powder obtained by freeze-drying the lactoferrin is dissolved in a PBS solution to prepare a mother solution, and an initiator is added thereto, and the mother solution is solidified to form a hydrogel; The initiator is a free radical initiator, a redox initiator, a vitamin C-hydrogen peroxide system, or a glutathione-peroxide system.

19. The use of lactoferrin according to claim 18, characterized in that The free radical initiator is selected from at least one of Irgacure 2959, Irgacure 819, and camphorquinone.

20. The use of lactoferrin according to claim 18, characterized in that The redox initiator includes: Oxidant: at least one selected from ammonium persulfate, potassium persulfate, and sodium persulfate; Reducing agent: at least one selected from ascorbic acid, TEMED, sodium sulfite, and sodium bisulfite.

21. The use of lactoferrin according to claim 18, characterized in that The vitamin C-hydrogen peroxide system comprises: Oxidant: H2O2; Reducing agent: ascorbic acid.

22. The use of lactoferrin according to claim 18, characterized in that The glutathione-peroxide system comprises: Oxidant: carbamide peroxide; Reducing agent: glutathione.

23. The use of lactoferrin according to claim 18, characterized in that The initiator is a photoinitiator, lithium phenyl 2,4,6-trimethylbenzoylphosphinate, and is cured under light to form a hydrogel.

24. The use of lactoferrin according to claim 23, characterized in that The lactoferrin concentration in the mother liquor is 0.12 g / ml, and the amount of lithium phenyl 2,4,6-trimethylbenzoylphosphinate is 0.4 wt %.

25. The use of lactoferrin according to claim 16, characterized in that The injection method is selected from at least one of intravenous injection, intraperitoneal injection, and in situ tissue injection.

26. A method for preparing lactoferrin with strong iron absorption ability, characterized in that: The method comprises the steps of performing methacrylation treatment on lactoferrin with low iron content to prepare the lactoferrin with strong iron absorption ability.

27. The preparation method according to claim 26, characterized in that The low-iron-content lactoferrin is subjected to methacrylation treatment by using methacrylic anhydride.

28. The preparation method according to claim 27, characterized in that The usage ratio of methacrylic anhydride to low-iron lactoferrin is 0.01-2 mL / 1 g.

29. The preparation method according to claim 28, characterized in that The dosage ratio of methacrylic anhydride to low iron content lactoferrin is 0.03 mL / 1 g.

30. The preparation method according to claim 27, characterized in that During the methacrylation treatment, the grafting rate of methacrylic anhydride is 10% to 100%.

31. The preparation method according to claim 30, characterized in that During the methacrylation treatment, the grafting rate of methacrylic anhydride is 30% to 100%.

32. The preparation method according to claim 26, characterized in that The preparation method further comprises removing iron from the lactoferrin to obtain the lactoferrin with low iron content.

33. The preparation method according to claim 32, characterized in that The low-iron lactoferrin refers to lactoferrin with an iron content of less than 0.15 mg / g; the iron saturation content of the lactoferrin with strong iron absorption ability is above 10 mg / g.

34. The preparation method according to claim 33, characterized in that The iron content of the low-iron lactoferrin is 0.010 mg / g to 0.015 mg / g.

35. The preparation method according to claim 33, characterized in that The iron content of the low-iron lactoferrin is 0.0139 mg / g.

36. The preparation method according to claim 32, characterized in that The ratio of the iron content of the low-iron lactoferrin to the iron content of the lactoferrin before apoirrization is 3.97% to 9.8%; the ratio of the iron content of the lactoferrin with strong iron absorption ability to the iron content of the lactoferrin before apoirrization is 2.86% to 8.5%.

37. The preparation method according to claim 32, characterized in that The ratio of the iron content of the lactoferrin with low iron content to the iron content of the lactoferrin before apoirrigation is 9.8%; the ratio of the iron content of the lactoferrin with strong iron absorption ability to the iron content of the lactoferrin before apoirrigation is 8.5%.

38. The preparation method according to claim 32, characterized in that When the lactoferrin is de-ironized, a lactoferrin solution is first prepared, and the retentate is lyophilized after dialysis to obtain lactoferrin with a low iron content.

39. The preparation method according to claim 38, characterized in that The lactoferrin solution is prepared by dissolving lactoferrin powder in a mixed solution at room temperature.

40. The preparation method according to claim 39, characterized in that The mixed solution is prepared by adding acetic acid, sodium acetate and EDTA into a PBS solution with a pH value of 2.0 to 4.

0.

41. The preparation method according to claim 40, characterized in that The molar ratio of acetic acid to sodium acetate is 1:

1.

42. The preparation method according to claim 39, characterized in that The concentration of PBS in the mixed solution is 0.01 M, the total concentration of acetic acid and sodium acetate is 0.1 M, and the concentration of EDTA is 40-500 mM.

43. The preparation method according to claim 38, characterized in that The concentration of the lactoferrin solution is 0.5-10 g / 100 ml.

44. The preparation method according to claim 38, characterized in that The lactoferrin solution is dialyzed by placing the lactoferrin solution into a dialysis bag, and then placing the dialysis bag in a PBS solution for 48 to 72 hours.

45. The preparation method according to claim 44, characterized in that During the dialysis process, the concentration of the PBS solution in the dialysis bag gradually decreases.

46. ​​The preparation method according to claim 45, characterized in that The concentration of the PBS solution is gradually reduced in the following manner: The concentration of PBS solution was gradually decreased from 0.1 M to 0.05 M during the 0th to 24th hour (excluding 24 hours); From 24 to 48 hours (excluding 48 hours), the concentration of PBS solution was gradually reduced from 0.05M to 0.01M. The concentration of PBS solution was gradually decreased from 0.01M to 0M during the 48th to 72th hour.

47. The preparation method according to claim 26, characterized in that The process for methacrylation of low iron lactoferrin involves: Step (1), dissolving low iron content lactoferrin in PBS solution at room temperature to prepare a lactoferrin stock solution; Step (2), adding methacrylic anhydride to the lactoferrin stock solution to react, and obtaining lactoferrin with strong iron absorption ability after the reaction.

48. The preparation method according to claim 47, characterized in that The low-iron-content lactoferrin in step (1) is dissolved in a PBS solution at room temperature to prepare a lactoferrin stock solution with a concentration of 1 to 10 g / 100 ml.

49. The preparation method according to claim 48, characterized in that The pH value of the PBS solution in step (1) is 2.0-4.

0.

50. The preparation method according to claim 47, characterized in that The usage ratio of methacrylic anhydride to low-iron lactoferrin in step (2) is 0.01-2 mL / 1 g.

51. The preparation method according to claim 50, characterized in that The usage ratio of methacrylic anhydride to low-iron lactoferrin in step (2) is 0.03 mL / 1 g.

52. The preparation method according to claim 47, characterized in that When the methacrylation treatment is performed in step (2), the grafting rate of methacrylic anhydride is 10% to 100%.

53. The preparation method according to claim 47, characterized in that In step (2), the addition of methacrylic anhydride to the lactoferrin stock solution is completed under high-speed stirring at 400 to 1000 rpm.

54. The preparation method according to claim 47, characterized in that In step (2), methacrylic anhydride is added to the lactoferrin stock solution and the pH value is adjusted to 6.5-7.5 for reaction.

55. The preparation method according to claim 54, characterized in that After adjusting the pH value to 6.5-7.5 in step (2), the reaction is continued with stirring at 10-25° C. for 0.5-24 hours. After the reaction, lactoferrin with strong iron absorption ability is obtained through post-treatment.

56. The preparation method according to claim 55, characterized in that The post-treatment comprises dialysis for 24 to 72 hours, ultrafiltration concentration, two filtrations with a filter membrane, and freeze-drying the filtrate to obtain lactoferrin with strong iron absorption capacity.

57. The preparation method according to claim 56, characterized in that The pore size of the filter membrane used for filtration is 0.1 μm or 0.22 μm.

58. A preparation for removing hemoglobin, characterized in that The preparation comprises the lactoferrin according to any one of claims 1 to 5 and 11 to 14.

59. The preparation for removing hemoglobin according to claim 58, characterized in that The preparation is a microsphere with a porous structure, and the porosity of the preparation is greater than 80%.

60. The preparation for removing hemoglobin according to claim 58, characterized in that The specific surface area of ​​the preparation is greater than 35 m2 / g.

61. The preparation for removing hemoglobin according to claim 58, characterized in that The number of binding sites between the preparation and lactoferrin is 7 to 13.

62. A method for preparing a preparation for removing hemoglobin, characterized in that: The preparation is a preparation for removing hemoglobin as described in any one of claims 58 to 61, and the preparation method comprises mixing an aqueous phase and an oily phase to form an emulsion containing a photoinitiator, and photocrosslinking the emulsion to obtain a preparation in the form of microspheres, wherein the aqueous phase uses water as a solvent and contains a photoinitiator, the lactoferrin and methacrylated gelatin, and the oily phase uses liquid paraffin as a solvent and contains an emulsifier.

63. The preparation method according to claim 62, characterized in that In the aqueous phase, the content of lactoferrin is 5-30% w / v.

64. The preparation method according to claim 62, characterized in that In the aqueous phase, the content of methacrylated gelatin is 1-20% w / v.

65. The preparation method according to claim 62, characterized in that In the aqueous phase, the content of the photoinitiator is 0.1-1% w / v.

66. The preparation method according to claim 62, characterized in that In the aqueous phase, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

67. The preparation method according to claim 62, characterized in that In the oil phase, the content of the emulsifier is 1 to 20% w / v.

68. The preparation method according to claim 62, characterized in that In the oil phase, the emulsifier is Span 80.

69. The preparation method according to claim 62, characterized in that The water phase and the oil phase are driven by injection pumps respectively, and the oil phase and the water phase merge and emulsify through the pipeline at a flow rate ratio of 10,000 to 50,000:1 to form droplets.

70. The preparation method according to claim 69, characterized in that The flow rate of the oil phase is 1-5 mL / min.

71. The preparation method according to claim 69, characterized in that The flow rate of the aqueous phase is 10 to 1000 μL / min.

72. The preparation method according to claim 62, characterized in that After the emulsion was frozen, it was photocrosslinked under 405 nm UV light.

73. The preparation method according to claim 62, characterized in that The emulsion was frozen before photocrosslinking. The emulsion was frozen at -100°C to -30°C and then frozen in liquid nitrogen.

74. The preparation method according to claim 62, characterized in that Before photocrosslinking, the cells were placed at 4°C for 5-10 minutes to maintain their morphology.

75. The preparation method according to claim 62, characterized in that After the photocrosslinking was completed, the obtained microspheres were washed with petroleum ether, -80°C acetone and deionized water to obtain a preparation.

76. Use of a preparation in the treatment of iron overload disease, characterized in that: The preparation is the preparation for removing hemoglobin according to any one of claims 58 to 61.

77. The use according to claim 76, characterized in that The iron overload disease is myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury or cerebral infarction-reperfusion injury.

78. A method for treating iron overload diseases, characterized in that: The method comprises injecting the preparation according to any one of claims 58 to 61 into the lesion site.

79. The method of claim 78, wherein the iron overload disease comprises myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury, or cerebral infarction-reperfusion injury.

80. The method of claim 78, wherein the preparation is injected into the lesion site using an interventional device comprising a needle catheter.

81. A reperfusion therapy method, characterized in that include: injecting the preparation according to any one of claims 58 to 61 into ischemic tissue; Reperfusion therapy is performed on the ischemic tissue.

82. The reperfusion therapy method according to claim 81, characterized in that The preparation is injected into the ischemic tissue in advance, and then reperfusion therapy is performed.

83. A method for treating myocardial ischemia by reperfusion, characterized in that: The method comprises pre-injecting the preparation according to any one of claims 58 to 61 into the left ventricular wall, and then performing reperfusion therapy on the left ventricle.

84. The reperfusion therapy method according to claim 83, characterized in that The preparation is injected into the left ventricular wall using an interventional device comprising a needle catheter, which is passed sequentially through the femoral artery and the aortic valve to reach the left ventricular endocardium. The preparation is injected into the left ventricular wall through the needle catheter.

85. The reperfusion therapy method according to claim 84, characterized in that The injection points of the needle catheter are distributed at predetermined locations on the left ventricular wall, and there are at least three injection points.

86. The reperfusion therapy method according to claim 85, characterized in that The injection volume at each injection point should be no less than 0.1 mL.

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