A high-strength double network gel loaded with hemoglobin and a preparation method thereof

Abstract

The application discloses a kind of high-strength double-network gel of hemoglobin load and preparation method thereof, the preparation method includes providing N-fluoromethoxy carbonyl diphenylalanine-containing organic solution, sodium alginate aqueous solution, hemoglobin solution and calcium ion-containing aqueous solution;Hemoglobin solution is dropped into N-fluoromethoxy carbonyl diphenylalanine-containing organic solution, and gently mixed;Add sodium alginate aqueous solution, and mix evenly;Drop calcium ion-containing aqueous solution, to obtain high-strength double-network gel of hemoglobin load.The high-strength double-network gel of hemoglobin load prepared by the above method can load up to 250% hemoglobin, and the gel structure is complete, with high mechanical strength.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a high-strength dual-network gel loaded with hemoglobin and its preparation method. Background Technology

[0002] Hemoglobin-based oxygen carriers (HBOCs) are oxygen carriers prepared from matrix-free hemoglobin through chemical modification and other methods. They possess oxygen-carrying and oxygen-releasing functions, and can replace red blood cells in transporting oxygen to various organs and tissues in the body. They have significant potential to enhance the body's oxygen supply and are a major international research hotspot in areas such as blood substitutes and cancer treatment. The hemoglobin (Hb) mainly comes from human blood or animal blood such as pigs and cattle. Currently, methods such as polymerization, cross-linking, conjugation, enzymatic modification, and chemical modification are used, but these methods suffer from problems such as the sensitive hemoglobin structure being easily oxidized and inactivated, low strength, low biocompatibility, difficult degradation, and complex preparation processes.

[0003] Double-network reinforcement can effectively address the deficiency of low carrier strength. Researchers such as Gong Jianping, Suo Zhigang, etc. (Gong JP, Katsuyama Y, Kurokawa T, et al. Double-network hydrogels with extremely high mechanical strength [J]. Adv Mater, 2003, 15 (14): 1155-1158. Sun JY, Zhao X, Illeperuma WR, et al. Highly stretchable and toughhydrogels [J]. Nature, 2012, 489(7414): 133-136.) proposed a double-network reinforcement strategy from a materials science perspective. This strategy can construct two interconnected network structures with different properties. For example, poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) can be used as the first network and polyacrylamide (PAAM) can be used as the second network. The energy dissipation mechanism between the two can be used to significantly improve the overall strength and toughness of the material. However, when such gel networks are applied to load active biomolecules, it is difficult to achieve a balance between macromolecular bioactivity, strength, and biocompatibility.

[0004] Previous research results (202210535758.X "A High-Concentration Loading Gel for Doxorubicin and Its Preparation Method") show that a high loading capacity for small molecule drugs can be achieved by constructing a double-network gel using 9-fluorenylmethoxycarbonyl-L-phenylalanine small molecule gelling agent and polymer gelling agent calcium alginate. However, when this system is introduced into macromolecule loading, it is difficult to achieve effective loading of hemoglobin, resulting in defects such as extremely low loading capacity, low gel structure strength, and poor stability.

[0005] Constructing carriers with high loading capacity, strength, and stability for hemoglobin loading is the main approach to solving the above problems. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a high-strength dual-network gel loaded with hemoglobin and its preparation method. The present invention provides a method for preparing a high-strength dual-network gel loaded with hemoglobin based on the self-assembly of N-fluoromethoxycarbonyl diphenylalanine combined with ALG-Ca enhancement. The resulting high-strength dual-network gel loaded with hemoglobin can have a hemoglobin loading of up to 250%, with an intact gel structure and high mechanical strength.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: On the one hand, a method for preparing a high-strength dual-network gel loaded with hemoglobin is provided, comprising: Provide organic solutions containing N-fluoromethoxycarbonyl diphenylalanine, sodium alginate aqueous solutions, hemoglobin solutions, and calcium ion-containing aqueous solutions; Add the hemoglobin solution dropwise into an organic solution containing N-fluoromethoxycarbonyl diphenylalanine and mix gently. Add sodium alginate aqueous solution and mix well; A calcium-containing aqueous solution was added dropwise to obtain a high-strength double-network gel loaded with hemoglobin.

[0008] On the other hand, a high-strength dual-network gel loaded with hemoglobin is provided, prepared by the above-mentioned method for preparing a high-strength dual-network gel loaded with hemoglobin.

[0009] Compared with the prior art, the present invention has the following advantages: 1. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to the present invention involves dropping a hemoglobin solution into an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, followed by adding an aqueous solution of sodium alginate and an aqueous solution containing calcium ions to prepare a high-strength dual-network gel loaded with hemoglobin. This method can achieve high loading of large molecular weight hemoglobin, with a hemoglobin loading of up to 250%. The resulting high-strength dual-network gel loaded with hemoglobin has a complete structure, high mechanical strength, and high structural integrity of the loaded hemoglobin. The high-strength dual-network gel loaded with hemoglobin prepared by the method of the present invention can effectively inhibit the conversion of hemoglobin to methemoglobin, ensuring long-term oxygen-carrying function.

[0010] 2. The method for preparing the high-strength dual-network gel loaded with hemoglobin of the present invention adopts physical cross-linking and non-covalent cross-linking throughout the process, without introducing chemical cross-linking agents, and has good biocompatibility.

[0011] 3. The preparation method of the high-strength dual-network gel loaded with hemoglobin of the present invention has mild process conditions, requires no complicated equipment, and is easy to scale up for production.

[0012] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 The results are the gel strain scanning results for each embodiment and comparative example; Figure 2 Comparison of oxygen binding capacity curves for the Hb-loaded dual-network gel in Example 2; Figure 3 This is a schematic diagram of the RAW264.7 cytotoxicity evaluation results of the Hb-loaded dual-network gel in Example 2. Detailed Implementation

[0014] The technical solution will now be clearly and completely described with reference to the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0015] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0016] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0017] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0018] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] The technical principle upon which this invention is based is to avoid the influence of osmotic pressure caused by the diffusion effect of macromolecules on the carrier network structure. This invention provides a method for constructing an interpenetrating double network oxygen carrier. The self-assembly of N-fluoromethoxycarbonyl diphenylalanine in a solvent system provides a flexible buffer and protective microenvironment for hemoglobin. Then, sodium alginate-Ca is used to construct a macroscopic mechanical support system to obtain a high-strength gel network that can effectively encapsulate a large amount of hemoglobin and maintain its biological activity.

[0021] On the one hand, a method for preparing a high-strength dual-network gel loaded with hemoglobin is provided, comprising: The invention provides an organic solution containing N-fluoromethoxycarbonyl diphenylalanine (Fmoc-FF), an aqueous solution of sodium alginate (ALG), a hemoglobin (Hb) solution, and an aqueous solution containing calcium ions; the organic solution containing N-fluoromethoxycarbonyl diphenylalanine is obtained by dissolving N-fluoromethoxycarbonyl diphenylalanine in an organic solvent; the aqueous solution of sodium alginate is obtained by dissolving sodium alginate in deionized water; the hemoglobin solution is obtained by dissolving hemoglobin in deionized water; the aqueous solution containing calcium ions is obtained by dissolving a soluble calcium salt in deionized water; and the organic solvent is DMSO. Add the hemoglobin solution dropwise into an organic solution containing N-fluoromethoxycarbonyl diphenylalanine and mix gently. Add sodium alginate aqueous solution and mix well; by dropping the hemoglobin solution into an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, based on the hydrophobic interaction and π-π stacking of N-fluoromethoxycarbonyl diphenylalanine in the system, the hemoglobin molecule is used as the assembly core, and the N-fluoromethoxycarbonyl diphenylalanine molecules are oriented and cross-linked around it, so that the N-fluoromethoxycarbonyl diphenylalanine molecules first assemble around the hemoglobin molecules to form the first layer of gel network that encapsulates and initially fixes the hemoglobin. This step aims to pre-isolate the hemoglobin in a flexible small molecule network microenvironment to provide protection for subsequent steps; A high-strength double-network gel loaded with hemoglobin was obtained by adding an aqueous solution containing calcium ions dropwise. The slow addition of the calcium-ion-containing aqueous solution initiated the reaction of sodium alginate with Ca... 2+ Specific ionic cross-linking occurs, forming a rigid second-layer gel network that runs through the first network. This construction method, first the first network and then the second, ensures that Ca... 2+ Primarily confined within the cross-linking process of sodium alginate, greatly reducing free Ca2+. 2+ Direct contact with the embedded hemoglobin molecules effectively avoids calcium deficiency. 2+ It may induce negative effects such as hemoglobin oxidation or aggregation.

[0022] In this invention, the small molecule gel raw material is N-fluoromethoxycarbonyl diphenylalanine. Previous research mainly focused on constructing gel loading carriers for small molecule azithromycin-like drugs based on Fmoc-F. During the research process, the inventors discovered that this gel loading system had difficulty achieving a high effective loading of large molecule hemoglobin molecules. This invention introduces Fmoc-FF to construct a first network structure. Fmoc-FF self-assembles with hemoglobin in a solvent, and then combines with a second network structure to effectively improve the mechanical strength of the gel network, achieving a high effective loading of hemoglobin.

[0023] In some embodiments, the concentration of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine is 1-5 wt%; the concentration of the sodium alginate aqueous solution is 0.4-4 wt%; the concentration of the hemoglobin solution is 1-5 wt%; and the concentration of calcium ions in the calcium ion-containing aqueous solution is 1-15 wt%. Preferably, the concentration of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine is 1-4 wt%, and the concentration of calcium ions in the calcium ion-containing aqueous solution is 2-12 wt%. In some further preferred embodiments, the concentration of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine is 1 wt%, the concentration of the sodium alginate aqueous solution is 0.4 wt%, the concentration of the hemoglobin solution is 5 wt%, and the concentration of calcium ions in the calcium ion-containing aqueous solution is 2 wt%. In some embodiments, the volume ratio of the organic solution containing N-fluoromethoxycarbonyldiphenylalanine, the volume of the sodium alginate aqueous solution, the volume of the hemoglobin solution, and the volume of the calcium ion-containing aqueous solution is (0.1~0.3):(0.2~0.6):(0.2~0.5):0.1; in some further preferred embodiments, the volume ratio of the organic solution containing N-fluoromethoxycarbonyldiphenylalanine, the volume of the sodium alginate aqueous solution, the volume of the hemoglobin solution, and the volume of the calcium ion-containing aqueous solution is 0.2:0.5:0.2:0.1.

[0024] In some embodiments, the mass ratio of sodium alginate to calcium ions is 1:1.

[0025] In some embodiments, in a composite system composed of an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, an aqueous solution of sodium alginate, a hemoglobin solution, and an aqueous solution containing calcium ions, the concentration of N-fluoromethoxycarbonyl diphenylalanine is 0.1~1.5 wt%, the concentration of sodium alginate is 0.2~2 wt%, and the concentration of calcium ions is 0.2~2 wt%. In some preferred embodiments, in a composite system composed of an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, an aqueous solution of sodium alginate, a hemoglobin solution, and an aqueous solution containing calcium ions, the concentrations of N-fluoromethoxycarbonyl diphenylalanine, sodium alginate, and calcium ions are all 0.2~1.2 wt%. In some further preferred embodiments, in a composite system composed of an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, an aqueous solution of sodium alginate, a hemoglobin solution, and an aqueous solution containing calcium ions, the concentrations of N-fluoromethoxycarbonyl diphenylalanine, sodium alginate, and calcium ions are all 0.2 wt%.

[0026] During the research, the inventors discovered that when the concentration of the calcium ion aqueous solution is 2wt% and the volume is 0.1mL, the composite system composed of an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, an aqueous solution of sodium alginate, and a hemoglobin solution can effectively achieve high-level effective loading of Hb. The possible reason is that the calcium ions with a concentration of 2wt% and a final concentration of 0.2wt% in the composite system can effectively reduce the damage to the gel structure caused by excessive metal ion concentration in the construction of the second network structure.

[0027] In some embodiments, the ratio of the volume A of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine to the sum of the volumes B of the sodium alginate aqueous solution, C of the hemoglobin solution, and D of the calcium ion-containing aqueous solution is 1:9 to 3:7; A / (B+C+D), where A, B, C, and D are all volumes with consistent units.

[0028] This invention is based on the self-assembly of N-fluoromethoxycarbonyl diphenylalanine in the solvent system and the specific ionic crosslinking of calcium ions and sodium alginate in the solvent system to achieve an interpenetrating first gel network and second gel network, wherein the solvent in the solvent system is derived from the raw material solution itself.

[0029] In some embodiments, the hemoglobin loading in the high-strength dual-network gel loaded with hemoglobin is 50% to 250%.

[0030] The present invention preferably provides a method for preparing a high-strength dual-network gel capable of loading high amounts of hemoglobin. By the above-mentioned preparation method, which includes dropping a hemoglobin solution into an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, followed by adding an aqueous solution of sodium alginate and an aqueous solution containing calcium ions, a high loading of macromolecular hemoglobin can be effectively achieved, with the hemoglobin loading capacity reaching up to 250%.

[0031] In some embodiments, the hemoglobin is bovine hemoglobin.

[0032] The preferred hemoglobin of this invention is bovine hemoglobin, which has lower hemolytic and brittle properties and is easier to load when combined with the above-mentioned dual-network gel.

[0033] On the other hand, a high-strength double-network gel loaded with hemoglobin prepared by the above method is provided, wherein the hemoglobin loading in the high-strength double-network gel loaded with hemoglobin is 50% to 250%, and the hemoglobin loading can reach up to 250%.

[0034] Prior to this application, a series of experiments were conducted. Some of the experimental results are listed below to provide a more detailed description of the invention. The following is a detailed description in conjunction with the embodiments. Example 1

[0035] This embodiment provides a method for preparing a high-strength dual-network gel loaded with hemoglobin, including: (1) Dissolve Fmoc-FF in DMSO and stir until homogeneous to obtain precursor solution A with a concentration of 1 wt%; (2) Dissolve ALG in deionized water and stir until homogeneous to obtain precursor solution B with a concentration of 0.4 wt%. (3) Dissolve Hb in deionized water and stir until homogeneous to obtain an Hb solution with a concentration of 1 wt%; the Hb is bovine hemoglobin, purchased from Solarbio Science & Technology, 9008-02-0; (4) Dissolve CaCl2 in deionized water and stir until homogeneous to obtain Ca 2+ Solution, concentration 2wt%: (5) Take 0.2 mL of precursor solution A and add 0.2 mL of Hb solution dropwise to obtain the first precursor system; (6) Add 0.5 mL of precursor solution B dropwise to the first precursor system obtained above; (7) Add 0.1 mL of Ca to the system obtained in step (6). 2+ Solution; (8) After standing, an ALG / Fmoc-FF / Hb gel was obtained with a gel volume of 1 mL, a DMSO:H2O volume ratio of 2:8, a final Hb concentration of 0.2 wt%, and an Hb loading rate of 50%. The Hb loading rate E was calculated using the formula E=m Hb / (m ALG +m Fmoc-FF )*100%. Example 2

[0036] This embodiment provides a method for preparing a high-strength dual-network gel loaded with hemoglobin, including: (1) Dissolve Fmoc-FF in DMSO and stir until homogeneous to obtain precursor solution A with a concentration of 1 wt%; (2) Dissolve ALG in deionized water and stir until homogeneous to obtain precursor solution B with a concentration of 0.4 wt%. (3) Dissolve Hb in deionized water and stir until homogeneous to obtain an Hb solution with a concentration of 5 wt%; (4) Dissolve CaCl2 in deionized water and stir until homogeneous to obtain Ca 2+ Solution, concentration 2wt%: (5) Take 0.2 mL of precursor solution A and add 0.2 mL of Hb solution dropwise to obtain the first precursor system; (6) Add 0.5 mL of precursor solution B dropwise to the first precursor system obtained above; (7) Add 0.1 mL of Ca to the system obtained in step (6). 2+ Solution; (8) After standing, ALG / Fmoc-FF / Hb gel was obtained with a gel volume of 1 mL, a DMSO:H2O volume ratio of 2:8, a final Hb concentration of 1 wt%, and an Hb loading rate of 250%.

[0037] Comparative Example 1 This comparative study examines the influence of dual-network gels on the structure and properties of the product, and provides a method for preparing a gel network loaded with hemoglobin. This method is essentially the same as the method for preparing hemoglobin-loaded hemoglobin using a first-network gel, and specifically includes: 1) Dissolve Fmoc-FF in DMSO and stir until homogeneous to obtain precursor solution A with a concentration of 1 wt%; dissolve Hb in deionized water and stir until homogeneous to obtain Hb solution with a concentration of 5 wt%. 2) Take 0.2 mL of precursor solution A and add 0.2 mL of Hb solution dropwise to obtain the first precursor system; 3) Add 0.6 mL of deionized water dropwise to the first precursor system obtained above; 4) After standing, Fmoc-FF / Hb gel was obtained with a gel volume of 1 mL and a DMSO:H2O volume ratio of 2:8.

[0038] Comparative Example 2 This comparative study examines the influence of dual-network gels on the structure and properties of the product, and provides a method for preparing a gel network loaded with hemoglobin. This method is essentially the same as Example 2 for preparing a second-network gel loaded with hemoglobin, and specifically includes: 1) Dissolve ALG in deionized water and stir until homogeneous to obtain precursor solution B with a concentration of 0.4 wt%; 2) Dissolve Hb in deionized water and stir until homogeneous to obtain an Hb solution with a concentration of 5 wt%. 3) Dissolve CaCl2 in deionized water and stir until homogeneous to obtain Ca 2+ Solution, concentration 2wt%: 4) Add 0.2 mL of Hb solution dropwise to 0.5 mL of precursor solution B, and add 0.2 mL of deionized water; 5) Add 0.1 mL of Ca 2+ Solution; 6) Allow to stand to obtain ALG / Hb gel with a gel volume of 1 mL. Example 3

[0039] This embodiment provides a method for preparing a high-strength dual-network gel loaded with hemoglobin, including: (1) Dissolve Fmoc-FF in DMSO and stir until homogeneous to obtain precursor solution A with a concentration of 1 wt%; (2) Dissolve ALG in deionized water and stir until homogeneous to obtain precursor solution B with a concentration of 4 wt%; (3) Dissolve Hb in deionized water and stir until homogeneous to obtain an Hb solution with a concentration of 1 wt%; (4) Dissolve CaCl2 in deionized water and stir until homogeneous to obtain Ca 2+ Solution, concentration 8wt%: (5) Take 0.2 mL of precursor solution A and add 0.5 mL of Hb solution dropwise to obtain the first precursor system; (6) Add 0.2 mL of precursor solution B dropwise to the first precursor system obtained above; (7) Add 0.1 mL of Ca to the system obtained in step (6). 2+ Solution; (8) After standing, ALG / Fmoc-FF / Hb gel was obtained with a gel volume of 1 mL, a DMSO:H2O volume ratio of 2:8, a final Hb concentration of 0.5 wt%, and an Hb loading rate of 50%. Example 4

[0040] This embodiment provides a method for preparing a high-strength dual-network gel loaded with hemoglobin, including: (1) Dissolve Fmoc-FF in DMSO and stir until homogeneous to obtain precursor solution A with a concentration of 4 wt%; (2) Dissolve ALG in deionized water and stir until homogeneous to obtain precursor solution B with a concentration of 2 wt%; (3) Dissolve Hb in deionized water and stir until homogeneous to obtain an Hb solution with a concentration of 4 wt%; (4) Dissolve CaCl2 in deionized water and stir until homogeneous to obtain Ca 2+ Solution, concentration 12 wt%; (5) Take 0.1 mL of precursor solution A and add 0.2 mL of Hb solution dropwise to obtain the first precursor system; (6) Add 0.6 mL of precursor solution B dropwise to the first precursor system obtained above; (7) Add 0.1 mL of Ca to the system obtained in step (6). 2+ Solution; (8) After standing, ALG / Fmoc-FF / Hb gel was obtained with a gel volume of 1 mL, a DMSO:H2O volume ratio of 1:9, a final Hb concentration of 0.8 wt%, and an Hb loading rate of 50%. Example 5

[0041] This embodiment provides a method for preparing a high-strength dual-network gel loaded with hemoglobin, including: (1) Dissolve Fmoc-FF in DMSO and stir until homogeneous to obtain precursor solution A with a concentration of 4 wt%; (2) Dissolve ALG in deionized water and stir until homogeneous to obtain precursor solution B with a concentration of 2 wt%; (3) Dissolve Hb in deionized water and stir until homogeneous to obtain an Hb solution with a concentration of 4 wt%; (4) Dissolve CaCl2 in deionized water and stir until homogeneous to obtain Ca 2+ Solution, concentration 6wt%: (5) Take 0.3 mL of precursor solution A and add 0.3 mL of Hb solution dropwise to obtain the first precursor system; (6) Add 0.3 mL of precursor solution B dropwise to the first precursor system obtained above; (7) Add 0.1 mL of Ca to the system obtained in step (6). 2+ Solution; (8) After standing, ALG / Fmoc-FF / Hb gel was obtained with a gel volume of 1 mL, a DMSO:H2O volume ratio of 3:7, a final Hb concentration of 1.2 wt%, and an Hb loading rate of 67%.

[0042] Performance Evaluation The gel strain scanning results for each embodiment and comparative example are as follows: Figure 1 As shown, the left figure is the strain scan rheological characteristic curve of the first network gel loaded with hemoglobin in Comparative Example 1, the middle figure is the strain scan rheological characteristic curve of the second network gel loaded with hemoglobin in Comparative Example 2, and the right figure is the strain scan rheological characteristic curve of the dual-network gel loaded with Hb in Example 2. The test method is as follows: the corresponding sample is placed on an Anton par MCR302 rotational rheometer, strain control mode is adopted, a flat plate clamp is used to place the gel drug, the lower fixed disk diameter is 25 mm, the upper disk rotor is PP25SN27785, the test spacing is 0.96~1.2 mm, and strain scan rheological tests are performed in the strain range of 0.01~1000%. The results show that compared with the single-network hemoglobin loaded gel, the strength of the high-strength dual-network gel loaded with hemoglobin of the present invention is increased by nearly 10 times, and the strength is significantly improved.

[0043] Figure 2 The oxygen binding capacity curve of the Hb-loaded dual-network gel in Example 2 is shown, with hemoglobin serving as a blank control. The test method was as follows: the oxygen dissociation curve was determined using the HemoCue™ Hb 201+ System at a test temperature of 37 °C. Oxygenated and deoxygenated states were obtained by purging with compressed air and nitrogen, respectively. The oxygen partial pressure (p50) and Hill coefficient (Hill-n) at an oxygen saturation of 50% were calculated. The test results showed that the oxygen binding P50 of the Hb-loaded dual-network gel in Example 2 was higher than that of the control group, while the affinity was lower, remaining within the physiological range (25~30 mmHg), which is beneficial for achieving efficient oxygen binding and on-demand oxygen release.

[0044] Figure 3This is a schematic diagram illustrating the cytotoxicity evaluation results of the Hb-loaded dual-network gel in RAW264.7 cells, as shown in Example 2. The test method included co-culturing the gel sample with macrophages (RAW264.7) for 12 hours, followed by determining cell viability. The sample preparation method for different Hb loading rates was the same as in Example 2, differing only in the concentration of the Hb solution. The results showed that with Hb loading ranging from 0.1% to 200%, the cell viability was above 65%, with the highest biocompatibility observed at a Hb loading of 100%, where the cell viability was >80%.

Claims

1. A method for preparing a high-strength dual-network gel loaded with hemoglobin, characterized in that, include: Provide organic solutions containing N-fluoromethoxycarbonyl diphenylalanine, sodium alginate aqueous solutions, hemoglobin solutions, and calcium ion-containing aqueous solutions; Add the hemoglobin solution dropwise into an organic solution containing N-fluoromethoxycarbonyl diphenylalanine and mix gently. Add sodium alginate aqueous solution and mix well; A calcium-containing aqueous solution was added dropwise to obtain a high-strength double-network gel loaded with hemoglobin.

2. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 1, characterized in that, The organic solution containing N-fluoromethoxycarbonyl diphenylalanine is obtained by dissolving N-fluoromethoxycarbonyl diphenylalanine in an organic solvent; the sodium alginate aqueous solution is obtained by dissolving sodium alginate in deionized water; the hemoglobin solution is obtained by dissolving hemoglobin in deionized water; the calcium ion-containing aqueous solution is obtained by dissolving a soluble calcium salt in deionized water; and / or, the hemoglobin is bovine hemoglobin.

3. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 1, characterized in that, The concentration of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine is 1-5 wt%; the concentration of the sodium alginate aqueous solution is 0.4-4 wt%; the concentration of the hemoglobin solution is 1-5 wt%; the concentration of calcium ions in the calcium ion-containing aqueous solution is 1-15 wt%; and / or, the volume ratio of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine, the volume of the sodium alginate aqueous solution, the volume of the hemoglobin solution, and the volume of the calcium ion-containing aqueous solution is (0.1-0.3):(0.2-0.6):(0.2-0.5):0.

1.

4. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 3, characterized in that, The concentration of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine is 1-4 wt%, and the concentration of calcium ions in the aqueous solution containing calcium ions is 2-12 wt%.

5. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 1, characterized in that, The mass ratio of sodium alginate to calcium ions is 1:

1.

6. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 1, characterized in that, In a composite system consisting of an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, an aqueous solution of sodium alginate, a hemoglobin solution, and an aqueous solution containing calcium ions, the concentration of N-fluoromethoxycarbonyl diphenylalanine is 0.1~1.5wt%, the concentration of sodium alginate is 0.2~2wt%, and the concentration of calcium ions is 0.2~2wt%.

7. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 6, characterized in that, In a composite system consisting of an organic solution containing N-fluoromethoxycarbonyl diphenylalanine, an aqueous solution of sodium alginate, a hemoglobin solution, and an aqueous solution containing calcium ions, the concentrations of N-fluoromethoxycarbonyl diphenylalanine, sodium alginate, and calcium ions are all 0.2–1.2 wt%.

8. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 1, characterized in that, The ratio of the volume of the organic solution containing N-fluoromethoxycarbonyl diphenylalanine to the sum of the volumes of the sodium alginate aqueous solution, the hemoglobin solution, and the calcium ion-containing aqueous solution is 1:9 to 3:

7.

9. The method for preparing a high-strength dual-network gel loaded with hemoglobin according to claim 1, characterized in that, The hemoglobin loading in the high-strength double-network gel is 50% to 250%.

10. A high-strength dual-network gel loaded with hemoglobin prepared by the method described in any one of claims 1 to 9.

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