Preparation method and application of PSC-coated Prussian blue nanoparticles

Abstract

The invention discloses a preparation method and application of Prussian blue nanoparticles coated with polydextrose sorbitol carboxymethyl ether (PSC), and the preparation method comprises the following steps: respectively dissolving PSC and a precursor 1 and a precursor 2 in deionized water to form two mixed solutions; mechanically stirring the two mixed solutions to obtain a mixed reaction solution; reacting the mixed reaction liquid under the condition of water bath heating to generate a product; and carrying out dialysis, ultrafiltration, concentration and freeze drying on the product to obtain the PSC-coated Prussian blue nanoparticles. The prepared particles are uniform and stable in size and small in polydispersity coefficient, have activity similar to catalase, peroxidase and peroxide dismutase, meanwhile have good biocompatibility and safety, can effectively remove ROS in a living body and resist inflammation, and have good application prospects. After being intravenously injected into the body, the compound can be passively targeted to the myocardial ischemia reperfusion damaged myocardium through the permeation and retention effects of damaged blood vessels, and is used for treating the myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the field of biological nanomaterials, and specifically relates to a preparation method of PSC-coated Prussian blue nanoparticles (PBNPs@PSC) and biomedical applications thereof. Background Art

[0002] Prussian blue belongs to a metal organic framework, which consists of two octahedral coordinated iron centers (ferric iron and ferrous iron). The ferric iron ion is connected to the nitrogen atom end of the cyanide, and the ferrous iron ion is connected in series by the carbon atom of the cyanide. Prussian blue was first synthesized by Disbach in Berlin in 1706. Since its synthesis, it has been widely used, including as a dye and iron staining in biological samples. Prussian blue has good biocompatibility and safety. As early as 2003, Prussian blue was approved by the US Food and Drug Administration (FDA) as an antidote for radioactive elements cesium and thallium. It has been approved as a safe material for human use and is included in the World Health Organization's Essential Medicines List. Prussian blue nanoparticles have multi-enzyme activities, including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), etc., which can effectively remove reactive oxygen species (ROS) in organisms and show great potential in treating diseases caused by ROS. Over time, Prussian blue nanoparticles have shown excellent properties such as imaging function, strong photothermal conversion ability, multi-enzyme activity, high specific surface area, surface modifiability, controllable drug release, biocompatibility and biodegradability, and good stability, and are therefore often developed for various biomedical applications, including bioimaging, tumor therapy, inflammation therapy, cardiovascular disease therapy, etc.

[0003] Surface coatings are crucial in maintaining the structural stability and performance of Prussian blue nanoparticles, including reducing the surface energy of Prussian blue nanoparticles, inhibiting agglomeration, and improving solubility, which can further improve their application in the biomedical field. Commonly used coatings include organic polyvinyl pyrrolidone (PVP) and inorganic citric acid. In addition, there are chitosan (CS), hyaluronic acid (HA), polyethyleneimine (PEI), oxalic acid, and polydimethylammonium chloride (PDDA). However, these coatings cannot effectively maintain the stability and safety of Prussian blue and give Prussian blue nanoparticles new activity.

[0004] The use of non-toxic polysaccharides as coatings shows more advantages, such as reducing the potential toxicity of Prussian blue nanoparticles, improving stability and biocompatibility, and giving Prussian blue nanoparticles new functions by combining polysaccharides with other bioactive substances. Polyglucose sorbitan carboxymethyl ether (PSC) is a dextran and is the surface coating of Ferumoxytol, an intravenous iron supplement used clinically for the treatment of iron deficiency anemia. Compared with other intravenous iron supplements, Ferumoxytol is the most effective iron supplement with the least adverse reactions and has very good biocompatibility and safety. This is due to the fact that PSC in the Ferumoxytol structure is rich in carboxyl groups, which can coordinate with iron ions in superparamagnetic iron oxide nanoparticles, thereby improving the stability and biocompatibility of inorganic nanoparticles. In addition, the hemiacetal hydroxyl group in the PSC structure has an antioxidant effect and can scavenge ROS. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a new method for preparing PSC-coated Prussian blue nanoparticles using PSC as a protective agent, and applies the method to the treatment of myocardial ischemia-reperfusion injury associated with ROS production and inflammatory infiltration. The PSC-coated Prussian blue nanoparticles prepared by the present invention effectively solve the safety and stability problems existing in the current Prussian blue nanoparticles, and improve the biological activity of the Prussian blue nanoparticles.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a method for preparing PSC-coated Prussian blue nanoparticles (PBNPs@PSC), which uses a dual precursor preparation method and comprises the following steps:

[0007] (1) dissolving PSC and precursor 1 in a solution to form a mixed solution A;

[0008] (2) dissolving PSC and precursor 2 in a solution to form a mixed solution B;

[0009] (3) adding the mixed solution B dropwise to the mixed solution A under mechanical stirring conditions to obtain a mixed reaction solution;

[0010] (4) heating the mixed reaction solution in a water bath under stirring conditions to react and generate a product;

[0011] (5) dialyzing the product, performing ultrafiltration by ultrafiltration tube centrifugation, concentrating, and freeze-drying to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC);

[0012] Wherein, the precursor 1 in step (1) is one of ferric chloride, ferric sulfate or ferric nitrate; the precursor 2 in step (2) is one of potassium ferrocyanide, ammonium ferrocyanide or sodium ferrocyanide.

[0013] Wherein, the molar ratio of the PSC to the precursor 1 is 10:1-1:10, the molar ratio of the PSC to the precursor 2 is 10:1-1:10, and the molar ratio of the precursor 1 to the precursor 2 is 10:1-1:10.

[0014] Wherein, the solution in steps (1) and (2) is deionized water, the amount of the substance of the precursor 1 is 0.01-1 mmol, and the amount of the substance of the precursor 2 is 0.01-1 mmol.

[0015] Preferably, the volume of the mixed solution A is 5-50 mL, and the volume of the mixed solution B is 5-50 mL.

[0016] Wherein, the dropping acceleration rate in step (3) is 0.1-10 mL / min, and the mechanical stirring rate is 100-600 r / min.

[0017] Preferably, the volume of the mixed reaction solution is 10-100 mL.

[0018] Wherein, the water bath heating temperature in step (4) is 40-100° C., the reaction time is 0.5-10 h, and the mechanical stirring rate is 100-600 r / min.

[0019] Wherein, the molecular weight cutoff of the dialysis bag used for dialysis in step (5) is 5-100KD, the molecular weight cutoff of the ultrafiltration tube used for ultrafiltration is 10-50KD, the centrifugal rate during ultrafiltration is 1000-6000r / min, and the centrifugal temperature is 3-10°C.

[0020] Preferably, the preparation method of the present invention comprises the following steps:

[0021] (1) PSC and precursor 1 are dissolved in deionized water to form a mixed solution A;

[0022] (2) PSC and precursor 2 are dissolved in deionized water to form a mixed solution B;

[0023] (3) Mixed solution B is added dropwise to mixed solution A using a syringe pump, while mechanically stirring the mixture to obtain a mixed reaction solution;

[0024] (4) the mixed reaction solution is reacted under conditions of water bath heating and mechanical stirring to generate a product;

[0025] (5) The product is dialyzed, ultrafiltered by centrifugation, concentrated, and freeze-dried to obtain.

[0026] Wherein, the precursor 1 described in step 1 is one of ferric chloride, ferric sulfate or ferric nitrate, and its amount is 0.01-1mmol, preferably 0.2-0.5mmol; the molar ratio of PSC to precursor 1 is 1:1-1:10, preferably 1:2-1:6; the volume of the mixed solution A is 5-50mL, preferably 10-30ml.

[0027] Among them, the precursor 2 described in step 2 is a ferrocyanide with good water solubility, including one of potassium ferrocyanide, ammonium ferrohydride or sodium ferrocyanide; the amount of the substance of precursor 2 is 0.01-1mmol, preferably 0.2-0.5mmol; the molar ratio of PSC to precursor 1 is 1:1-1:10, preferably 1:2-1:6; the volume of the mixed solution A is 5-50mL, preferably 10-30ml.

[0028] Wherein, the mixed solution A and the mixed solution B described in step 3 need to be completely dissolved and the solution is clear; the injection pump drop acceleration rate is 0.1-10mL / min, preferably 1-5mL / min; the mechanical stirring rate is 100-600r / min, preferably 200-500r / min; the volume of the mixed reaction solution is 10-100mL, preferably 20-60mL.

[0029] Wherein, the water bath heating conditions described in step 4 are 40-100°C, preferably 60-90°C; the mechanical stirring rate is 100-600r / min, preferably 200-500r / min; and the water bath heating reaction time is 0.5-10h, preferably 2-6 hours.

[0030] Wherein, the molecular weight cutoff of the dialysis bag used for dialysis described in step 5 is 10-100KD, preferably 100KD, and the dialysis time is 72 hours; the molecular weight cutoff of the ultrafiltration tube used for ultrafiltration is 10-50KD, preferably 20-40KD; the centrifugal rate during ultrafiltration is 1000-6000r / min, preferably 3000-5000r / min, the centrifugal temperature is 3-10°C, preferably 4°C, and freeze-dried for 48 hours.

[0031] The preparation method of the present invention successfully prepares uniform and stable PSC-coated Prussian blue nanoparticles (PBNPs@PSC).

[0032] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) prepared by the preparation method of the present invention are used in the preparation of reagents or drugs for removing ROS in organisms and for anti-inflammatory purposes.

[0033] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) prepared by the preparation method of the present invention are used in the preparation of drugs and reagents that are passively targeted to damaged myocardium through intravenous injection and are used for the treatment of myocardial ischemia-reperfusion injury through intravenous injection.

[0034] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) of the present invention have higher superoxide dismutase (SOD)-like activity.

[0035] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) of the present invention can effectively remove reactive oxygen species (ROS) in the body and have a good anti-inflammatory effect.

[0036] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) of the present invention can be passively targeted to the damaged myocardium through intravenous injection.

[0037] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) of the present invention can be used for the treatment of myocardial ischemia-reperfusion injury through intravenous injection.

[0038] The present invention adopts a double precursor synthesis method to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC), which is simple to operate, easy to industrially scale up production, and can obtain high-quality Prussian blue nanoparticles with easy surface functional modification in a short time. The PSC-coated Prussian blue nanoparticles prepared by the present invention have uniform and stable size, small polydispersity coefficient, and have catalase-like, peroxidase-like and superoxide dismutase activities, wherein the enzyme activity of the superoxide dismutase-like is higher than that of the existing PVP-coated extremely small-sized high-performance Prussian blue nanoparticles. The Prussian blue nanoparticles prepared by the present invention have good biocompatibility and safety, can effectively remove ROS in a living body and have an anti-inflammatory effect, can be passively targeted to the damaged myocardium of myocardial ischemia-reperfusion injury by intravenous injection, and can be well applied to the treatment of myocardial ischemia-reperfusion injury.

[0039] The present invention provides a method for preparing Prussian blue nanoparticles coated with PSC, wherein a large number of hydroxyl groups exposed on the surface of PSC can coordinate with trivalent iron ions, which provides an intrinsic driving force for the formation of Prussian blue nanoparticles, is beneficial to the formation of Prussian blue nanoparticles, and lays a foundation for further functional modification of Prussian blue nanoparticles. In addition, as a polysaccharide, the hemiacetal hydroxyl group at the end of PSC has a good redox function, which plays a role in resisting oxidative stress. Therefore, it is feasible for the present invention to use PSC as a coating to develop a technology that is simple to operate, has good repeatability, can obtain stable, uniform, easy to functionally modify the surface, and has good biocompatibility and safety. The prepared Prussian blue nanoparticles are also easy to use in biomedical applications. The method of the present invention is a new technology that uses the carboxyl groups on the surface of PSC to coordinate with trivalent iron ions and then coordinate and polymerize with ferrous cyanide ions to form Prussian blue nanoparticles. This method is called a dual precursor method (such as Figure 1 As shown). The present invention adopts a dual precursor synthesis method to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC). The method is simple to operate and easy to industrially scale up production. It can obtain high-quality Prussian blue nanoparticles with easy surface functional modification in a short time. By this method, uniform, stable, and very high SOD-like enzyme activity Prussian blue nanoparticles can be successfully prepared. The prepared Prussian blue nanoparticles have excellent ROS scavenging and anti-inflammatory effects in vivo, and have good biocompatibility and safety. Through intravenous administration, they can be passively targeted to the lesion site through the EPR effect of the damaged myocardium of myocardial ischemia-reperfusion injury, and can be well used for the treatment of myocardial ischemia-reperfusion injury, providing great potential for the application of Prussian blue nanoparticles in the biomedical field.

[0040] The existing technology for preparing Prussian blue nanoparticles mainly uses PVP as a protective agent, and the enzyme activity of the synthesized Prussian blue nanoparticles needs to be improved. The present invention proposes for the first time to use PSC as a protective agent for the preparation of Prussian blue nanoparticles, which improves the SOD-like enzyme activity and has good biosafety, and can be used to treat diseases related to ROS production, such as myocardial ischemia-reperfusion injury.

[0041] The invention provides a new method for synthesizing Prussian blue nanoparticles, i.e., using PSC as a protective agent to synthesize a PSC-coated Prussian blue nanoparticle, which is used to improve the stability, safety and activity of the Prussian blue nanoparticles. The invention compares the high-performance Prussian blue nanoparticles with extremely small size (3.4nm) coated with PVP. Although the high-performance Prussian blue nanoparticles with extremely small size (3.4nm) coated with PVP also have enzyme-like activity, the smaller the size, the greater the toxicity. The method of the invention, i.e., using PSC as a protective agent for the preparation of Prussian blue nanoparticles, the prepared PSC-coated Prussian blue nanoparticles have a larger size of 44nm, but the SOD enzyme activity, i.e., the ability to remove superoxide ions and the ability to remove hydroxyl radicals are respectively improved by 1.6 times and 0.6 times compared with the high-performance Prussian blue nanoparticles with extremely small size (3.4nm) coated with PVP, and shows very good biosafety, and can be used for intravenous administration. The invention not only has high activity and high biosafety, but also has good therapeutic potential for diseases related to ROS generation.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0043] The present invention adopts PSC as a protective agent, and prepares PSC-coated Prussian blue nanoparticles by a dual precursor method. The size distribution of the Prussian blue nanoparticles prepared by the present invention is uniform and stable, the size is 44nm, the batch stability is good, the SOD-like enzyme activity is significantly improved compared with the extremely small size (3.4nm) high-performance Prussian blue nanoparticles (USPBNPs) coated with PVP, wherein the ability to remove superoxide ions is increased by 1.6 times, and the ability to remove hydroxyl radicals is increased by 0.6 times. The PSC-coated Prussian blue nanoparticles prepared by the present invention have good biosafety, are not prone to hemolysis, and have good ROS removal and anti-inflammatory effects. The PSC-coated Prussian blue nanoparticles prepared by the present invention can passively target the damaged myocardium through the EPR effect, and after intravenous injection into the myocardial ischemia-reperfusion injury model mice, the retention in the damaged myocardium is more than 5 times higher than that of the sham operation group mice. The PSC-coated Prussian blue nanoparticles prepared by the present invention can effectively treat myocardial ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of the synthesis of PSC-coated Prussian blue nanoparticles;

[0045] Figure 2 are the X-ray diffraction patterns and infrared spectra of PSC-coated Prussian blue nanoparticles;

[0046] Figure 3is the hydrated particle size distribution and transmission electron microscopy image of PSC-coated Prussian blue nanoparticles;

[0047] Figure 4 is the electron spin resonance image of the peroxidase-like activity measurement of PSC-coated Prussian blue nanoparticles;

[0048] Figure 5 is the toxicity of PSC-coated Prussian blue nanoparticles at different concentrations to RAW264.7 and HL-1 cells;

[0049] Figure 6 is the hemolysis rate of Prussian blue nanoparticles coated with PSC at different concentrations;

[0050] Figure 7 It is the H&E image of liver and kidney function and heart, liver, spleen, lung and kidney of mice after intravenous injection of PSC-coated Prussian blue nanoparticles;

[0051] Figure 8 This is the result of PSC-coated Prussian blue nanoparticles clearing ROS in cardiomyocytes;

[0052] Fig. 9 It is the anti-inflammatory effect of PSC-coated Prussian blue nanoparticles on LPS-induced macrophages;

[0053] Fig.10 It is the targeting effect of PSC-coated Prussian blue nanoparticles on the damaged myocardium in the myocardial ischemia-reperfusion injury model mice;

[0054] Fig.11 The PSC-coated Prussian blue nanoparticles have ROS-scavenging and anti-inflammatory effects on damaged myocardium in mice with myocardial ischemia-reperfusion injury model;

[0055] Fig.12 The effect of PSC-coated Prussian blue nanoparticles on myocardial remodeling and improvement of cardiac function in mice with myocardial ischemia-reperfusion injury model. DETAILED DESCRIPTION

[0056] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0057] Polyglucose sorbitol carboxymethyl ether (PSC), relative molecular weight of 10000, reference: Ferumoxytol of ultrahigh magnetization produced by hydrocooling and magnetically internalheating co-precipitation. Nanoscale 2018, 10(16), 7369-7376.

[0058] Potassium ferrocyanide is potassium ferrocyanide trihydrate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: P299247, CAS number: 14459-95-1.

[0059] Example 1

[0060] Preparation of PSC-coated Prussian blue nanoparticles (PBNPs@PSC)

[0061] Weigh PSC (0.5g) and ferric chloride (67.5mg) into a conical flask, add 20mL of deionized water, stir and dissolve until clarified to obtain a mixed solution A. Weigh PSC (0.5g) and potassium ferrocyanide (105.6mg) into a 50mL centrifuge tube, add 20mL of deionized water, stir and dissolve until clarified to obtain a mixed solution B. Then transfer the mixed solution B to a 20mL syringe, use a syringe pump to drop it into the mixed solution A at a speed of 1mL / min, stir while injecting, and adjust the speed of the mechanical stirrer to 400r / min. After the mixed solution B is added, react for 4 hours in a 90℃ water bath at a stirring speed of 400r / min. After the reaction is completed, turn off the water bath, wait for the reactants to cool naturally at room temperature, and then transfer them to a dialysis bag with a molecular weight cutoff of 100KD. After dialysis with ultrapure water for 72 hours, use a 30KD ultrafiltration tube to perform ultrafiltration concentration at 4℃ and a centrifugal speed of 4000r / min. The concentrated solution was finally freeze-dried for 48 hours to obtain PSC-coated Prussian blue nanoparticle powder (PBNPs@PSC).

[0062] Example 2

[0063] X-ray diffraction and infrared spectroscopy of PBNPs@PSC

[0064] Take a small amount of 50 mg of the PBNPs@PSC powder obtained in Example 1 and add it to the middle of the groove of the sample holder. Take a glass slide and gently press the sample surface to make the powder sample surface flat and consistent with the frame plane. Put the sample holder into the sample chamber of the X-ray measuring instrument for measurement to obtain the product X-ray diffraction result. Take a small amount of 50 mg of the PBNPs@PSC powder obtained in Example 1 and add it to the sample hole of the infrared spectrometer. Use the infrared spectrometer to measure and obtain the infrared spectrometer data of the obtained product. The results are as follows: Figure 2 As shown, the X-ray diffraction diagram shows that the structure of the PBNPs@PSC powder prepared by the present invention is the crystal structure of Prussian blue, and the infrared spectrum diagram shows that the surface of the PBNPs@PSC prepared by the present invention has the structure of PSC. Figure 2 Together, it is demonstrated that the present invention successfully prepared PSC-coated Prussian blue nanoparticles.

[0065] Example 3

[0066] Hydrated particle size, polydispersity coefficient and transmission electron microscopy of PBNPs@PSC

[0067] The PBNPs@PSC obtained in Example 1 was diluted with water to a concentration of 10 μg / mL, 1 mL was drawn into the sample pool, and the particle size and polydispersity were measured using a MalvernNano ZS90 particle size potential analyzer. The PBNPs@PSC obtained in Example 1 was diluted with water to a concentration of 500 μg / mL, 20 μL was drawn and dropped onto a copper grid, and then naturally dried and photographed using a transmission electron microscope. The results are shown in Figure 2. Figure 3 As shown, the results show that the PBNPs@PSC prepared by the present invention has a small polydispersity coefficient, uniform size and high stability.

[0068] Example 4

[0069] Determination of the ability of PBNPs@PSC to scavenge hydroxyl radicals and superoxide ions

[0070] The PBNPs@PSC obtained in Example 1 and the comparison product (USPBNPs@PVP) (prepared according to the document "Zhiguo Qin, et.al. Achieving Ultrasmall Prussian Blue Nanoparticles as High Performance Biomedical Agents with Multifunctions ACS Appl. Mater. Interfaces 2020, 12, 57382-57390") were diluted with water to a concentration of 75 mg / mL, 200 microliters of 5 mg / ml FeSO4 solution were added to 20 microliters of DMPO and 160 μL of the product or comparison product obtained in Example 1, and then 20 microliters of 30% concentration of hydrogen peroxide were added. The mixture was mixed and reacted for 5 minutes. The samples were taken and tested using an electron spin resonance instrument to obtain the electron spin resonance data of the obtained product and the comparison product for scavenging hydroxyl radicals.

[0071] The PBNPs@PSC obtained in Example 1 and the comparative product obtained above were diluted with water to a concentration of 75 mg / mL, respectively. PBS buffer was used as a solvent to prepare a xanthine solution with a concentration of 10 mM and a xanthine oxidase solution with a concentration of 1 U / ml. 100 μL of the xanthine solution and 100 μL of the xanthine oxidase solution were taken, and then 20 μL of DMPO and 180 μL of the obtained product or the comparative product were added. The reaction was incubated for 10 minutes, and samples were taken for testing using an electron spin resonance instrument to obtain the electron spin resonance data of the obtained product and the comparative product for scavenging superoxide ions.

[0072] The results are as follows Figure 4 As shown, the results show that the PBNPs@PSC prepared by the present invention has a 1.6-fold improvement in the ability to scavenge superoxide ions and a 0.6-fold improvement in the ability to scavenge hydroxyl radicals compared to the high-performance ultra-small size (3.4 nm) PVP-coated Prussian blue nanoparticles prepared in the reference. This indicates that the PBNPs@PSC prepared by the present invention has a very high SOD-like enzyme activity, can better scavenge ROS, and provides great therapeutic potential for diseases related to ROS generation.

[0073] Example 5

[0074] Cytotoxicity investigation of PBNPs@PSC

[0075] The PBNPs@PSC obtained in Example 1 with different concentrations (0.1, 1, 10, 50, 100 μg / mL) were added to a 96-well plate (cell density was grown to 80-90% confluence) with cardiomyocytes (HL-1) or macrophages (RAW 264.7) and incubated for 24 hours, and then 10 μl CCK-8 was added to each well. After incubation at 37°C in the dark for 1 hour, the absorbance at 450 nm was measured using an ELISA reader. Cell survival rate (%) = [(absorbance of experimental well - absorbance of blank well) / (absorbance of control group - absorbance of blank well)] × 100%.

[0076] The results are as follows Figure 5 As shown, at a dosage of 100 μg / mL, the PBNPs@PSC prepared by the present invention did not have much effect on the survival rate of cells, indicating that it has good cell safety.

[0077] Example 6

[0078] Hemolysis experiment of PBNPs@PSC

[0079] Take the fresh blood of anticoagulated C57BL / 6 mice and centrifuge at 1200r / min for 8 minutes, discard the supernatant, wash the lower sediment with PBS 3 times to obtain red blood cells, and dilute the red blood cells 10 times with PBS. 100μL of PBNPs@PSC obtained in Example 1 with different concentrations was added to 1mL of the above diluted red blood cells, so that the termination concentrations were 30, 100 and 300μg / mL, respectively, incubated at 37℃ for 4h, then centrifuged at 10000r / min for 3 minutes, take 100μL of supernatant in a 96-well plate, and measure the absorbance at 540nm using an enzyme reader. The red blood cells in PBS are negative controls, and the red blood cells in water are positive controls. Hemolysis rate (%) = (sample absorbance-negative control absorbance) / (positive control absorbance-negative control absorbance) × 100%, and hemolysis is considered to be hemolysis when the hemolysis rate exceeds 5%.

[0080] The results are as follows Figure 6 As shown, at a concentration of 300 μg / mL, the PBNPs@PSC prepared by the present invention does not cause hemolytic reaction, indicating that it has good biocompatibility and safety and can be used for intravenous administration.

[0081] Example 7

[0082] In vivo safety investigation of PBNPs@PSC

[0083] C57BL / 6 mice were injected with different concentrations of PBNPs@PSC (1, 5 and 10 μg / mL) obtained in Example 1 through the tail vein. Blood was collected 24 hours later to measure liver and kidney function indicators, and important organ tissues such as the heart, liver, spleen, lung and kidney were fixed, dehydrated, paraffin-embedded, and paraffin-sectioned, and then morphologically analyzed by H&E staining.

[0084] The results are as follows Figure 7 As shown, after intravenous injection of the PBNPs@PSC prepared by the present invention into mice, there was no effect on liver and kidney function, and no harmful effects on important organs such as the heart, liver, spleen, lungs and kidneys, indicating that PBNPs@PSC has good biosafety.

[0085] Example 8

[0086] Investigation of ROS scavenging effect of PBNPs@PSC at the cellular level

[0087] The PBNPs@PSC obtained in Example 1 was tested by a ROS detection kit for the scavenging effect of cell ROS. 200,000 RAW264.7 cells and HL-1 cells were planted in confocal dishes for 24 hours, and 1 μg / mL and 10 μg / mL PBNPs@PSC were added for 24 hours, the incubation solution was discarded, PBS was used to wash 3 times, and 10mM H2O2 was added to stimulate the cells for 30min to produce ROS, the culture solution was discarded, PBS was washed 3 times, and then ROS fluorescent probe DCFH-DA (1:1000, serum-free culture solution dilution) was added and incubated for 20 minutes, the incubation solution was discarded, and serum-free culture medium was used to wash three times and then directly observed using a fluorescence microscope. The quantitative analysis of ROS removal was performed by cell flow cytometry, and the cell treatment was as above, except that the cells were planted in 12-well plates (100,000 cells per well), and after adding DCFH-DA to mark ROS, the cells were collected for cell flow analysis.

[0088] The results are as follows Figure 8 As shown, the PBNPs@PSC prepared by the present invention can effectively remove ROS in cells and has a good anti-oxidative stress effect.

[0089] Example 9

[0090] Investigation of the anti-inflammatory effect of PBNPs@PSC at the cellular level

[0091] The anti-inflammatory effect was evaluated by measuring the mRNA levels of IL-6, IL-1β and TNF-α in RAW 264.7 cells by RT-qPCR. RAW 264.7 cells were seeded into 6-well plates (200,000 cells per well) and cultured for 24 hours, and LPS (1 μg / mL) and different concentrations of PBNPs@PSC obtained in Example 1 were added for co-incubation. After 24 hours, the cells were collected, and RNA was extracted according to the operating instructions of the RNA extraction kit, and the RNA concentration was measured. The reverse transcription kit was used to reverse transcribe the IL-6, IL-1β and TNF-α genes, and the transcribed genes were amplified by RT-qPCR and fluorescence quantification was performed.

[0092] The results are as follows Fig. 9 As shown, the PBNPs@PSC prepared by the present invention can effectively reduce the mRNA levels of IL-6, IL-1β and TNF-α in RAW 264.7 cells, and has a good anti-inflammatory effect.

[0093] Example 10

[0094] Investigation of the targeting effect of PBNPs@PSC on damaged myocardium in mice with ischemia-reperfusion injury model

[0095] The PBNPs@PSC obtained in Example 1 was used to synthesize Prussian blue nanoparticles labeled with fluorescent dye FITC. C57BL / 6 mice were used to construct a classic myocardial ischemia-reperfusion injury model mouse. After surgery, 200 μL of FITC-labeled PBNPs@PSC was intravenously injected into the myocardial ischemia-reperfusion injury model mouse. The dosage was 0.5 mg / kg body weight. 12 hours later, 200 μL of 1% Evans blue solution was intravenously injected again to mark the blood vessels. Then, the mouse was anesthetized, a ventilator was inserted, an observation window was opened in the chest cavity, the heart was exposed, and the myocardium was observed using a small animal in situ cell dynamic analysis imaging system. The sham-operated mice were set as controls. The results showed that ( Fig.10 ), the content of PBNPs@PSC in the myocardial tissue of model mice was 5 times higher than that of sham-operated mice, indicating that PBNPs@PSC can passively target the damaged myocardium and be taken up by myocardial cells.

[0096] Embodiment 11

[0097] The therapeutic effect of PBNPs@PSC obtained in Example 1 on ischemia-reperfusion injury model mice was evaluated, and the specific implementation plan is as follows:

[0098] After the myocardial ischemia-reperfusion injury model was established in C57BL / 6 mice, PBNPs@PSC was injected intravenously immediately for treatment at a dose of 0.5 mg / kg body weight, and the administration was repeated on the 3rd and 7th days after surgery. On the 3rd day, the heart tissues of the treated mice were taken to measure ROS and inflammatory factors (IL-6, IL-1β and TNF-α) to investigate the ROS scavenging and anti-inflammatory effects of PBNPs@PSC. The results showed that ( Fig.11 )PBNPs@PSC can effectively remove ROS in the myocardial tissue of model mice and have a good anti-inflammatory effect.

[0099] The model mice underwent cardiac ultrasound examination on the 3rd, 7th and 14th days after drug treatment, and heart tissue was taken on the 14th day for WGA staining and Masson staining to examine the effect of myocardial remodeling. Fig.12 As shown in the figure, after 14 days of treatment, PBNPs@PSC can effectively promote myocardial remodeling and improve cardiac function in mice with myocardial ischemia-reperfusion injury model, indicating that PBNPs@PSC has a good therapeutic effect on myocardial ischemia-reperfusion injury.

[0100] Example 12

[0101] Preparation of PSC-coated Prussian blue nanoparticles (PBNPs@PSC)

[0102] Weigh PSC (0.25g) and ferric chloride (67.5mg) into a conical flask, add 20mL of deionized water, stir and dissolve until clarified to obtain a mixed solution A. Weigh PSC (0.25g) and potassium ferrocyanide (105.6mg) into a 50mL centrifuge tube, add 20mL of deionized water, stir and dissolve until clarified to obtain a mixed solution B. Then transfer the mixed solution B to a 20mL syringe, use a syringe pump to drop it into the mixed solution A at a speed of 1mL / min, stir while injecting, and adjust the speed of the mechanical stirrer to 400r / min. After the mixed solution B is added, react for 4 hours in a 90℃ water bath at a stirring speed of 400r / min. After the reaction is completed, turn off the water bath, wait for the reactants to cool naturally at room temperature, and then transfer them to a dialysis bag with a molecular weight cutoff of 100KD. After dialysis with ultrapure water for 72 hours, use a 30KD ultrafiltration tube to perform ultrafiltration concentration at a centrifugal speed of 4000r / min. The concentrated solution was finally freeze-dried for 48 hours to obtain PSC-coated Prussian blue nanoparticle powder (PBNPs@PSC).

[0103] Embodiment 13

[0104] Preparation of PSC-coated Prussian blue nanoparticles (PBNPs@PSC)

[0105] Weigh PSC (1g) and ferric chloride (67.5mg) into a conical flask, add 20mL of deionized water, stir and dissolve until clarified to obtain a mixed solution A. Weigh PSC (1g) and potassium ferrocyanide (105.6mg) into a 50mL centrifuge tube, add 20mL of deionized water, stir and dissolve until clarified to obtain a mixed solution B. Then transfer the mixed solution B to a 20mL syringe, use a syringe pump to drop it into the mixed solution A at a speed of 1mL / min, stir while injecting, and adjust the speed of the mechanical stirrer to 400r / min. After the mixed solution B is added, react for 4 hours in a 90℃ water bath at a stirring speed of 400r / min. After the reaction is completed, turn off the water bath, wait for the reactants to cool naturally at room temperature, and then transfer them to a dialysis bag with a molecular weight cutoff of 100KD. After dialysis with ultrapure water for 72 hours, use a 30KD ultrafiltration tube to perform ultrafiltration concentration at a centrifugal speed of 4000r / min. The concentrated solution was finally freeze-dried for 48 hours to obtain PSC-coated Prussian blue nanoparticle powder (PBNPs@PSC).

Claims

1. A method for preparing PSC-coated Prussian blue nanoparticles (PBNPs@PSC), characterized in that: The method uses a dual precursor preparation method, comprising the following steps: (1) dissolving PSC and precursor 1 in a solution to form a mixed solution A; (2) dissolving PSC and precursor 2 in a solution to form a mixed solution B; (3) adding the mixed solution B dropwise to the mixed solution A under mechanical stirring conditions to obtain a mixed reaction solution; (4) heating the mixed reaction solution in a water bath under stirring conditions to react and generate a product; (5) dialyzing the product, performing ultrafiltration by ultrafiltration tube centrifugation, concentrating, and freeze-drying to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC); Wherein: the precursor 1 is an iron salt, and the precursor 2 is a ferrous cyanide salt.

2. The preparation method according to claim 1, characterized in that The precursor 1 in step (1) is one of ferric chloride, ferric sulfate or ferric nitrate; the precursor 2 in step (2) is one of potassium ferrocyanide, ammonium ferrocyanide or sodium ferrocyanide.

3. The preparation method according to claim 1, characterized in that The molar ratio of the PSC to the precursor 1 is 10:1-1:10, the molar ratio of the PSC to the precursor 2 is 10:1-1:10, and the molar ratio of the precursor 1 to the precursor 2 is 10:1-1:

10.

4. The preparation method according to claim 1, characterized in that The solution in steps (1) and (2) is deionized water. Preferably, the amount of the substance of the precursor 1 is 0.01-1 mmol, and the amount of the substance of the precursor 2 is 0.01-1 mmol.

5. The preparation method according to claim 1, characterized in that The dropping acceleration rate in step (3) is 0.1-10 mL / min, and the mechanical stirring rate is 100-600 r / min.

6. The preparation method according to claim 1, characterized in that In step (4), the water bath heating temperature is 40-100° C., the reaction time is 0.5-10 h, and the mechanical stirring rate is 100-600 r / min.

7. The preparation method according to claim 1, characterized in that The molecular weight cutoff of the dialysis bag used for dialysis in step (5) is 5-100KD, the molecular weight cutoff of the ultrafiltration tube used for ultrafiltration is 10-50KD, the centrifugal rate during ultrafiltration is 1000-6000r / min, and the centrifugal temperature is 3-10°C.

8. PSC-coated Prussian blue nanoparticles (PBNPs@PSC) prepared by the preparation method according to claim 1.

9. Use of PSC-coated Prussian blue nanoparticles (PBNPs@PSC) prepared by the preparation method of claim 1 in preparing an agent or drug for removing ROS in a living body and for anti-inflammation.

10. Use of PSC-coated Prussian blue nanoparticles (PBNPs@PSC) prepared by the preparation method of claim 1 in the preparation of drugs and reagents for passively targeting damaged myocardium through intravenous injection and for treating myocardial ischemia-reperfusion injury through intravenous injection.

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