Nitrosohemoglobin-loaded membrane fusion bionic nano-carrier as well as preparation method and application of nitrosohemoglobin-loaded membrane fusion bionic nano-carrier

Abstract

The invention discloses a nitrosohemoglobin-loaded membrane fusion bionic nano-carrier as well as a preparation method and application of the nitrosohemoglobin-loaded membrane fusion bionic nano-carrier. According to the membrane fusion bionic nano-carrier, a membrane shell is formed by hybridizing and fusing synthetic phospholipid and a platelet membrane, an aqueous solution of nitrosohemoglobin is encapsulated in the membrane shell, and the nitrosohemoglobin is prepared by carrying out deoxidation treatment on hemoglobin and then reacting the treated hemoglobin with nitric oxide molecules; the membrane fusion bionic nano-carrier has good biocompatibility and natural targeting property, and can quickly and efficiently target the focus of cardiovascular and cerebrovascular diseases, and the delivered nitrosohemoglobin not only retains the biological activity of vasodilation and the like of nitric oxide gas molecules, but also has paramagnetism, can effectively enhance the magnetic resonance development effect, and can be used for preparing a biological medicine for treating cardiovascular and cerebrovascular diseases. And a new tool can be provided for effective delivery of nitric oxide molecules in ischemic cardiovascular and cerebrovascular disease lesions and magnetic resonance imaging diagnosis as a carrier.

Description

Technical Field

[0001] The invention relates to a membrane fusion bionic nanocarrier, in particular to a membrane fusion bionic nanocarrier carrying nitrosohemoglobin, and also to a preparation method and application of the nanocarrier. Background Art

[0002] Nitric oxide (NO) is a small signaling molecule with important biological functions. In the occurrence and development of cardiovascular and cerebrovascular diseases, NO is widely involved in regulating vasodilation, immune response, angiogenesis and other physiological and pathological processes. Therefore, the unique role of NO makes it an intervention target for cardiovascular and cerebrovascular diseases including ischemic stroke, acute myocardial infarction, atherosclerosis, thrombosis, etc.

[0003] However, due to the active chemical properties of NO, its delivery and application face many challenges. At present, the in vivo delivery and application of NO mainly rely on prodrug molecules or carrier systems. For example, NO prodrugs such as nitrates and nitro compounds usually decompose to produce NO through nonspecific pathways in the body. These strategies generally have problems such as uncontrollable NO release rate and uneven biodistribution, which may lead to side effects such as decreased blood pressure and cytotoxicity. Therefore, the problem of NO delivery and transport in vivo needs to be solved urgently. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a membrane fusion biomimetic nanocarrier carrying nitrosohemoglobin, and also to provide a method for preparing the above nanocarrier and its application in magnetic resonance imaging contrast enhancement reagents or NO delivery drugs.

[0005] Technical solution: The present invention discloses a membrane fusion bionic nanocarrier carrying nitrosohemoglobin, wherein the membrane fusion bionic nanocarrier has a membrane shell formed by the fusion of synthetic phospholipids and platelet membranes, wherein the synthetic phospholipid molecules are interlaced with the lipid layer of the platelet membrane through hydrophilic-hydrophobic interactions, the hydrophobic tails of the molecules aggregate with each other, and the hydrophilic heads face the aqueous phase, and an aqueous solution of nitrosohemoglobin is encapsulated in the membrane shell, wherein the nitrosohemoglobin is prepared by reacting hemoglobin with nitric oxide molecules after deoxygenation treatment.

[0006] Wherein, the synthetic phospholipids include one or more combinations of DPPC, DSPC, DSPE, DPPE, DPPA, DLPC, DMPC, DPPG or DSPE-MPEG2000.

[0007] Among them, the mass ratio of the platelet membrane, synthetic phospholipid and nitrosohemoglobin is 1-2:1-2:1, and the mass ratio is preferably 1:1:1, 2:1:1, 1:2:1 or 2:2:1; more preferably 1:2:1.

[0008] Among them, the size of the membrane-fusion bionic nanocarrier carrying nitrosohemoglobin is 100 - 500 nm.

[0009] The preparation method of the above-mentioned membrane-fusion bionic nanocarrier carrying nitrosohemoglobin includes the following steps:

[0010] (1) Physically lyse red blood cells to obtain platelet membranes and hemoglobin; extract the platelet membranes and prepare a platelet membrane suspension; prepare a hemoglobin solution and seal it, and after deoxygenation treatment with an inert gas, fill it with an excessive amount of nitric oxide gas to obtain nitrosohemoglobin;

[0011] (2) Dissolve synthetic phospholipids in a chloroform solution, rotate and evaporate to remove the organic solvent to form a phospholipid envelope, add a physiological saline solution, and hydrate it above the phospholipid phase transition temperature to prepare a liposome emulsion;

[0012] (3) Mix the platelet membrane suspension and the liposome suspension evenly and seal it, perform deoxygenation treatment with an inert gas, add nitrosohemoglobin, and reassemble through extrusion and membrane passing to obtain the membrane-fusion bionic nanocarrier carrying nitrosohemoglobin.

[0013] Among them, the inert gas includes one or more of nitrogen, helium, argon, or sulfur hexafluoride.

[0014] Among them, in step (1), the physical lysis of red blood cells includes a combination of one or more methods of hypotonic lysis, repeated freeze-thaw lysis, or mechanical fragmentation.

[0015] Among them, in step (1), the hemoglobin solution is extracted by hypotonic lysis of red blood cells. Take the red blood cell suspension, centrifuge to separate red blood cells and nutrient solution, resuspend and wash, add ultrapure water to resuspend the red blood cell precipitate to fully lyse the red blood cells, and take the supernatant after centrifugation to obtain the hemoglobin solution; the centrifugation speed for separating the red blood cell suspension is 200 - 500 g, and the centrifugation speed for the red blood cell membrane fragments after lysis is 10,000 - 20,000 g. Preferably, the separation speed of red blood cells is 250 g, and the separation speed of red blood cell membrane fragments after lysis is 18,000 g; the concentration of the hemoglobin solution is 20 - 65 mg / mL, preferably 32.5 mg / mL, that is, 0.5 mM.

[0016] Among them, in step (1), the platelet membrane is extracted by the repeated freeze-thaw method of red blood cells. Repeated freeze-thaw extraction is used to break the membrane and release the inclusions, and after multiple washings and purifications, blank platelet membrane vesicles are obtained. The preferred number of washing and separation times is 3 times.

[0017] Among them, in step (2), the phospholipid phase transition temperature is the temperature when the acyl chains of various phospholipids transition from the crystalline state to the liquid state, and the hydration temperature is 0 - 65 °C.

[0018] Among them, step (3) is specifically as follows: Transfer the suspension of the prepared platelet membrane, liposome, and nitrosylhemoglobin to a sealed micro-liposome extrusion device, and extrude the suspension through the filter membrane multiple times. During the process of passing through the membrane, membrane fusion between the platelet membrane and the liposome and encapsulation of nitrosylhemoglobin are achieved, and finally a membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin is formed; the pore size of the filter membrane used in the pneumatic liposome extrusion device is 100-800 nm, specifically 100 nm, 200 nm, 400 nm, or 800 nm, and the number of times of passing through the membrane multiple times is 5-20 times; preferably, the pore size of the filter membrane is 200 nm, and the number of times of passing through the membrane multiple times is 10 times.

[0019] Among them, the above-mentioned membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin can also be applied in the preparation of magnetic resonance imaging enhanced diagnostic reagents or NO delivery drugs for cardiovascular and cerebrovascular diseases.

[0020] The cardiovascular and cerebrovascular diseases include acute ischemic stroke, acute myocardial infarction, atherosclerosis, microcirculation disorder, or thrombotic disease.

[0021] Principle of the invention: The membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin of the present invention aims at the problems of in vivo delivery and transport of NO. Based on the interaction mechanism between NO and hemoglobin, hemoglobin is used in vitro. After deoxygenation, it reacts with NO gas to prepare nitrosylhemoglobin. Then, a membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin is constructed using natural platelet membranes and synthetic phospholipids to achieve targeted delivery of NO to the lesions of cardiovascular and cerebrovascular diseases, retention of biological activity, and controllable release in response to hypoxia, and to achieve effective dilation of blood vessels in ischemic lesions and restoration of blood supply. At the same time, the magnetic resonance imaging enhancement characteristics of nitrosylhemoglobin are utilized to provide more comprehensive and rich imaging information for the clinical diagnosis of cardiovascular and cerebrovascular diseases.

[0022] Specifically, Hb in red blood cells binds to NO to form nitrosyl hemoglobin (HbNO). During subsequent oxygenation, the NO molecule transfers to the highly conserved βCys93 residue on the β-chain of Hb to form SNO-Hb. This reversible binding is oxygen-dependent. When red blood cells sense a decrease in blood oxygen content in tissues and release oxygen, hemoglobin undergoes a conformational change from the relaxed state (R-state) to the tense state (T-state). At this time, the NO bound to βCys93 is also exposed and released, thereby inducing vasodilation, effectively increasing blood supply, and achieving automatic and precise regulation of tissue oxygenation balance. In addition, after NO binds to the ferrous ion in hemoglobin to form nitrosyl hemoglobin, it changes its magnetic properties, making nitrosyl hemoglobin have the ability to enhance magnetic resonance imaging. Therefore, using the interaction mechanism between NO and hemoglobin may provide new solutions for the direct and effective in vivo transport of NO molecules, the enhanced magnetic resonance imaging diagnosis of disease lesions, and the controllable release in response to hypoxia and the treatment of related diseases.

[0023] Therefore, the membrane-fused biomimetic nanocarrier loaded with nitrosyl hemoglobin of the present invention has a membrane shell formed by the fusion of synthetic phospholipids and natural platelet membranes. The nitrosyl hemoglobin it carries is prepared by directly reacting deoxygenated hemoglobin with NO gas molecules, and the nitrosyl hemoglobin is encapsulated inside the nanocarrier in the form of an aqueous solution. When prepared into a magnetic resonance diagnostic reagent for cardiovascular and cerebrovascular diseases and a nitric oxide delivery drug, it can rapidly target the lesions of cardiovascular and cerebrovascular diseases during in vivo delivery, effectively retain the biological activities such as vasodilation of NO, and can respond to the hypoxic microenvironment to achieve the intelligent and controllable release of NO, thereby achieving the purpose of therapeutic intervention. At the same time, the binding of NO to the ferrous ion in hemoglobin can change its magnetic properties, making the prepared membrane-fused biomimetic nanocarrier loaded with nitrosyl hemoglobin have the function of enhancing magnetic resonance imaging, making it possible to dynamically monitor the magnetic resonance imaging of cardiovascular and cerebrovascular disease lesions while effectively delivering NO by nitrosyl hemoglobin, so as to more comprehensively understand the occurrence and development of the disease.

[0024] Advantages: Compared with the prior art, the present invention has the following remarkable advantages: (1) The membrane-fused biomimetic nanocarrier loaded with nitrosyl hemoglobin of the present invention realizes the direct targeted delivery of NO gas molecules by the loaded nitrosyl hemoglobin and can respond to the hypoxic environment to achieve more controllable NO release; (2) The membrane-fused biomimetic nanocarrier loaded with nitrosyl hemoglobin of the present invention also has an enhanced magnetic resonance imaging effect and can dynamically monitor the magnetic resonance imaging of lesions while delivering NO and treating cardiovascular and cerebrovascular diseases; (3) The preparation method of the membrane-fused biomimetic nanocarrier of the present invention is simple and easy to operate, has a high encapsulation rate, and has broad prospects in practical applications. Description of the Drawings

[0025] Figure 1Schematic diagram of the preparation process of the membrane-fused biomimetic nanocarrier carrying nitrosyl hemoglobin of the present invention;

[0026] Figure 2 Physical photo of nitrosyl hemoglobin provided in Example 1;

[0027] Figure 3 Ultraviolet absorption spectrum of nitrosyl hemoglobin provided in Example 1, and comparison with the ultraviolet absorption spectra of hemoglobin and deoxyhemoglobin;

[0028] Figure 4 Electron paramagnetic resonance (EPR) spectrum of the membrane-fused biomimetic nanocarrier carrying nitrosyl hemoglobin provided in Example 1; and comparison with the EPR spectra of hemoglobin and deoxyhemoglobin;

[0029] Figure 5 Transmission electron microscopy structural characterization diagram of the membrane-fused biomimetic nanocarrier carrying nitrosyl hemoglobin provided in Example 1.

[0030] Figure 6 In vitro magnetic resonance imaging enhancement characterization image of the membrane-fused biomimetic nanocarrier carrying nitrosyl hemoglobin in Test Example 1;

[0031] Figure 7 In vitro magnetic resonance imaging enhancement characterization image of the membrane-fused biomimetic nanocarrier carrying nitrosyl hemoglobin in Test Example 2;

[0032] Figure 8 Vascular dilation biological function characterization result of the membrane-fused biomimetic nanocarrier carrying nitrosyl hemoglobin in Test Example 3. Detailed implementation manners

[0033] The technical solutions of the present invention will be further described below in conjunction with the embodiments. The test materials used in the embodiments can be obtained through conventional channels.

[0034] Example 1

[0035] Preparation method of nitrosyl hemoglobin solution:

[0036] (1) Extract by using a hypotonic lysis method. Centrifuge the fresh red blood cell suspension with a concentration of 1×10 12 / mL at 250 g to separate red blood cells and nutrient solution, and resuspend and wash 3 times with physiological saline to completely remove the nutrient solution;

[0037] (2) Resuspend the red blood cell precipitate with an equal volume of ultrapure water and vortex for 30 seconds to fully lyse the red blood cells;

[0038] (3) Centrifuge the suspension obtained in step (2) at a high speed of 18,000 g, take the supernatant, measure it using a hematology analyzer, calculate the hemoglobin concentration of the resulting solution, and then dilute the solution with ultrapure water to adjust the hemoglobin solution concentration to 32.5 mg / mL;

[0039] (4) Transfer 1 mL of the hemoglobin solution to a 3-mL vial and seal it. Use a 50-mL syringe to draw sulfur hexafluoride gas and fill it into the vial to replace the air in the vial. Then gently shake the solution to gradually deoxygenate the hemoglobin solution, and it can be observed that the color of the solution deepens;

[0040] (5) Repeat step 4 three times until the hemoglobin is completely deoxygenated. Then, use a 50-mL syringe to draw NO gas and fill it into the vial to replace the sulfur hexafluoride in the vial. Gently shake the solution to allow the deoxygenated hemoglobin to react with NO molecules, and it can be observed that the color of the solution returns to bright red;

[0041] The process of hemoglobin deoxygenation and the preparation of nitrosylhemoglobin is as Figure 1 shown, and the photo of the obtained physical diagram is as Figure 2 shown.

[0042] The preparation method of the membrane-fused bionic nanocarrier carrying nitrosylhemoglobin of the present invention includes the following steps:

[0043] (1) Physically lyse red blood cells to obtain platelet membranes and hemoglobin; prepare a hemoglobin solution and seal it, and then deoxygenate it with an inert gas and fill it with an excessive amount of nitric oxide gas to obtain nitrosylhemoglobin;

[0044] Extract platelet membranes by the repeated freeze-thaw method. Take 1 mL of a fresh platelet suspension with a concentration of 1×10 9 / mL and freeze-thaw it 3 times at -80 °C. Then centrifuge at a speed of 4000 g to separate the platelet cell membrane and intracellular contents such as organelles, and wash the precipitate 3 times with physiological saline to obtain purified platelet membranes;

[0045] (2) Take 5 mL of a chloroform solution of DPPC phospholipids with a concentration of 25 mg / mL and add it to a 25-mL pear-shaped flask. Then use a rotary evaporator to vacuum dry it at a speed of 120 rpm for 1 h to obtain a phospholipid film; add 5 mL of physiological saline, and rotate and hydrate it in a water bath at 65 °C at a speed of 120 rpm for 2 h to obtain a DPPC liposome emulsion;

[0046] (3) Seal the platelet membrane suspension and liposome suspension in a vial and deoxygenate them with an inert gas. After initially mixing them for 5 min by means of a 100 W, 42 KHz water bath ultrasound, add nitrosylhemoglobin using a syringe such that the mass ratio of the three is 1:2:1. Transfer the suspension to a micro-liposome extrusion device and, in a sealed state, extrude the suspension through a 200 nm pore size filter membrane 10 times. During the membrane passing process, the fusion of the platelet membrane and liposome and the encapsulation of nitrosylhemoglobin are achieved, ultimately forming a membrane fusion biomimetic nanocarrier carrying nitrosylhemoglobin with a fusion structure of platelet membrane and synthetic phospholipid as the membrane shell and a nitrosylhemoglobin solution as the aqueous core.

[0047] The absorption spectrum of the membrane fusion biomimetic nanocarrier carrying nitrosylhemoglobin prepared in Example 1 was characterized by ultraviolet absorption spectroscopy and compared with the absorption spectra of hemoglobin and deoxyhemoglobin. The results are as Figure 3 shown, indicating that the ultraviolet absorption spectrum of nitrosylhemoglobin has changed significantly.

[0048] The EPR spectrum of the membrane fusion biomimetic nanocarrier carrying nitrosylhemoglobin prepared in Example 1 was characterized by electron paramagnetic resonance (EPR) technology and compared with the EPR spectra of hemoglobin and deoxyhemoglobin. As Figure 4 shown, it indicates that the magnetic properties of hemoglobin have changed significantly after binding with NO and it has paramagnetism.

[0049] The microstructure of the membrane fusion biomimetic nanocarrier carrying nitrosylhemoglobin prepared in Example 1 was characterized by transmission electron microscopy. As Figure 5 described, it shows the effective fusion of the platelet membrane and phospholipid and the successful encapsulation of nitrosylhemoglobin.

[0050] Example 2

[0051] For the membrane fusion biomimetic nanocarrier carrying nitrosylhemoglobin of the present invention, compared with Example 1, in step (3), the mass ratio of platelet membrane, liposome, and nitrosylhemoglobin is 1:1:1.

[0052] Example 3

[0053] For the membrane fusion biomimetic nanocarrier carrying nitrosylhemoglobin of the present invention, compared with Example 1, in step (3), the mass ratio of platelet membrane, liposome, and nitrosylhemoglobin is 2:2:1.

[0054] Example 4

[0055] The membrane-fusion biomimetic nanocarrier loaded with nitrosylhemoglobin of the present invention, compared with Example 1, in step (2), the phospholipid types used for preparing liposomes are a combination of DMPC and DSPE-MPEG2000 at a molar ratio of 10:1, and DMPC can fuse better with platelet membranes.

[0056] Example 5

[0057] The membrane-fusion biomimetic nanocarrier loaded with nitrosylhemoglobin of the present invention, compared with Example 1, in step (3), during the preparation process of multiple extrusion through membranes, the pore size of the filter membrane used is 400 nm, and the number of extrusion through membranes is 15 times. The size of the obtained membrane-fusion biomimetic nanocarrier increases, about 200 - 300 nm.

[0058] Regarding the membrane-fusion biomimetic nanocarriers loaded with nitrosylhemoglobin prepared in Example 1 and Example 2, a 7.0T small animal magnetic resonance imaging system was used to characterize their magnetic resonance imaging enhancement effects:

[0059] Hemoglobin, deoxyhemoglobin, methemoglobin with the same concentration as the nitrosylhemoglobin carried, and normal saline were used as control groups. Each sample was sealed in a 3 mL volumetric vial, the sample volume was 2 mL, and the magnetic resonance scanning sequence used was T1-T2 mapping.

[0060] The experimental results of Example 1 are as Figure 6 shown. In terms of T1-weighted imaging enhancement: the T1 relaxation time of the membrane-fusion biomimetic nanocarrier loaded with nitrosylhemoglobin was measured to be 2049.01 ms. In contrast, the T1 relaxation times of normal saline, hemoglobin, deoxyhemoglobin, and methemoglobin were 2730.42 ms, 2729.42 ms, 2877.07 ms, and 1317.31 ms respectively; in terms of T2-weighted imaging enhancement: the T2 relaxation time of the membrane-fusion biomimetic nanocarrier loaded with nitrosylhemoglobin was measured to be 273.71 ms. In contrast, the T2 relaxation times of normal saline, hemoglobin, deoxyhemoglobin, and methemoglobin were 822.58 ms, 351.36 ms, 305.52 ms, and 233.07 ms respectively. The results show that the membrane-fusion biomimetic nanocarrier loaded with nitrosylhemoglobin has T1 and T2 magnetic resonance dual-modal imaging enhancement effects, but its T2-weighted imaging enhancement effect is stronger.

[0061] The experimental results of Example 2 are as Figure 7As shown in the figure, in terms of T1-weighted imaging enhancement: the T1 relaxation time of the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin was measured to be 1574.66 ms. In contrast, the T1 relaxation times of normal saline, hemoglobin, deoxyhemoglobin, and methemoglobin were 3135.14 ms, 2739.25 ms, 2585.77 ms, and 853.06 ms, respectively. In terms of T2-weighted imaging enhancement: the T2 relaxation time of the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin was measured to be 169.74 ms. In contrast, the T2 relaxation times of normal saline, hemoglobin, deoxyhemoglobin, and methemoglobin were 755.88 ms, 244.09 ms, 175.06 ms, and 131.47 ms, respectively. The results also indicate that the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin has a dual-modal magnetic resonance imaging enhancement effect on T1 and T2, but its T2-weighted imaging enhancement effect is stronger.

[0062] For the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin prepared in Example 3, it was diluted with deoxygenated normal saline to a concentration of 2 mg / mL of nitrosylhemoglobin. Using ischemic stroke as an ischemic cardio-cerebrovascular disease model, the vasodilatory biological effect of the prepared membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin was characterized:

[0063] After the model was established, the diluted solution of the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin was injected into the mice through the tail vein. Using normal saline as the control group, a blood flow meter was used to collect the changes in the blood vessel morphology and blood flow in the stroke lesion area of the mice over time. The results are as Figure 8 shown. After the model was established, the blood vessels in the brains of the control group mice gradually blocked over time, and the blood flow in the lesion area decreased significantly. In contrast, the blood vessels in the brains of the mice injected with the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin showed obvious dilation, and the blood flow in the brain was effectively restored. This indicates that the prepared membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin effectively retains the biological activity of NO. It can rapidly target ischemic lesions, respond to the hypoxic environment of ischemic stroke lesions for controlled release, effectively dilate blood vessels, and has the potential for NO molecular delivery and treatment of ischemic cardio-cerebrovascular diseases.

[0064] In summary, the membrane-fused biomimetic nanocarrier loaded with nitrosylhemoglobin prepared in the present invention not only has an enhanced magnetic resonance imaging effect, but also effectively retains the biological activity of NO. It can achieve targeted delivery of NO molecules in vivo and controlled release in response to hypoxia, realizing a beneficial combination of the diagnosis and treatment of cardio-cerebrovascular diseases.

Claims

1. A membrane fusion biomimetic nanocarrier carrying nitrosohemoglobin, characterized in that: The membrane fusion bionic nanocarrier has a membrane shell formed by hybrid fusion of synthetic phospholipids and platelet membranes, wherein the synthetic phospholipid molecules are interlaced with the lipid layer of the platelet membrane through hydrophilic-hydrophobic interactions, and an aqueous solution of nitrosohemoglobin is encapsulated in the membrane shell, wherein the nitrosohemoglobin is prepared by reacting hemoglobin with nitric oxide molecules after deoxygenation treatment.

2. The membrane fusion biomimetic nanocarrier according to claim 1, characterized in that: The synthetic phospholipids include one or more combinations of DPPC, DSPC, DSPE, DPPE, DPPA, DLPC, DMPC, DPPG or DSPE-MPEG2000.

3. The membrane fusion biomimetic nanocarrier according to claim 1, characterized in that: The mass ratio of the platelet membrane, the synthetic phospholipid and the nitrosohemoglobin is 1-2:1-2:1, and the size of the membrane fusion bionic nanocarrier carrying the nitrosohemoglobin is 100-500nm.

4. A method for preparing the membrane fusion biomimetic nanocarrier carrying nitrosohemoglobin according to claim 1, characterized in that: The following steps are involved: (1) Physically lysing red blood cells to obtain platelet membranes and hemoglobin; extracting platelet membranes and obtaining a platelet membrane suspension; preparing a hemoglobin solution and sealing it, deoxygenating it with an inert gas, and then filling it with excess nitric oxide gas to obtain nitrosohemoglobin; (2) dissolving the synthetic phospholipid in a chloroform solution, removing the organic solvent by rotary evaporation to form a phospholipid envelope, adding a physiological saline solution, and hydrating to prepare a liposome emulsion; (3) The platelet membrane suspension and the liposome suspension are evenly mixed and then sealed, deoxygenated using an inert gas, and nitrosohemoglobin is added. The mixture is reassembled by extruding through a membrane to obtain a membrane fusion biomimetic nanocarrier loaded with nitrosohemoglobin.

5. The preparation method according to claim 4, characterized in that: The inert gas includes one or more of nitrogen, helium, argon or sulfur hexafluoride.

6. The preparation method according to claim 4, characterized in that: In step (1), the physical lysis of red blood cells includes a combination of one or more methods of hypotonic lysis, repeated freeze-thaw lysis or mechanical disruption.

7. The preparation method according to claim 4, characterized in that: In step (1), the hemoglobin solution is extracted by hypotonic lysis of red blood cells, the red blood cell suspension is centrifuged to separate the red blood cells and the nutrient solution, the red blood cells are resuspended and washed, ultrapure water is added to resuspend the red blood cell sediment to fully lyse the red blood cells, and the supernatant is taken after centrifugation to obtain the hemoglobin solution; The platelet membrane is extracted by repeated freezing and thawing of red blood cells, and the contents are released by repeated freezing and thawing extraction and broken membrane, and then washed and purified multiple times to obtain blank platelet membrane vesicles.

8. The preparation method according to claim 4, characterized in that: In step (2), the hydration temperature is 0-65°C.

9. The preparation method according to claim 4, characterized in that: In step (3), the extrusion is passed through a membrane, the pore size of the filter membrane used is 100 to 800 nm, and the number of times of passing through the membrane is 5 to 20 times.

10. Use of the membrane fusion biomimetic nanocarrier carrying nitrosohemoglobin according to claim 1 in the preparation of magnetic resonance imaging enhanced diagnostic reagents for cardiovascular and cerebrovascular diseases or NO delivery drugs.

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