A method for preparing PSC-coated Prussian blue nanoparticles and its application

Abstract

This invention discloses a method for preparing Prussian blue nanoparticles coated with polydextrose sorbitol carboxymethyl ether (PSC) and its application. The preparation includes: dissolving PSC with precursors 1 and 2 in deionized water to form two mixtures; mechanically stirring the two mixtures to obtain a mixed reaction solution; reacting the mixed reaction solution under water bath heating to generate a product; and dialyzing, ultrafiltration, concentrating, and freeze-drying the product to obtain PSC-coated Prussian blue nanoparticles. The particles prepared by this invention have uniform and stable size, low polydispersity index, and exhibit catalase, peroxidase, and superoxide dismutase-like activities. They also possess good biocompatibility and safety, effectively scavenging ROS in vivo and combating inflammation. After intravenous injection, they can passively target damaged myocardium due to myocardial ischemia-reperfusion injury through penetration and retention in damaged blood vessels, thus being used for the treatment of myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of bionanomaterials, specifically relating to a method for preparing PSC-coated Prussian blue nanoparticles (PBNPs@PSC) and their biomedical applications. Background Technology

[0002] Prussian blue is a metal-organic framework composed of two octahedral coordinated iron centers (ferric and ferrous ions). The ferric ion is attached to the nitrogen atom terminal of the cyanide, while the ferrous ion is linked by carbon atoms of the cyanide. First synthesized by Disbach in Berlin in 1706, Prussian blue has been widely used since its synthesis, including as a dye and for staining iron in biological samples. Prussian blue exhibits good biocompatibility and safety. As early as 2003, it was approved by the US Food and Drug Administration (FDA) as an antidote for radioactive elements cesium and thallium. Currently, it is approved as a safe material for human use and is included in the World Health Organization's List of Essential Medicines. Prussian blue nanoparticles possess multi-enzyme activity, including catalase-like (CAT), peroxidase-like (POD), and superoxide dismutase-like (SOD) activities, effectively scavenging reactive oxygen species (ROS) in the body and showing great potential in treating ROS-induced diseases. Over time, Prussian blue nanoparticles have exhibited excellent properties, such as imaging capabilities, strong photothermal conversion ability, multi-enzyme activity, high specific surface area, surface modifiability, controllable drug release, biocompatibility and biodegradability, and good stability. Therefore, they are often developed for various biomedical applications, including bioimaging, tumor treatment, inflammation treatment, and cardiovascular disease treatment.

[0003] Surface coatings are crucial for maintaining the structural stability and properties of Prussian blue nanoparticles, including reducing their surface energy, inhibiting aggregation, and improving solubility, thus enhancing their applications in the biomedical field. Commonly used coatings include the organic compound polyvinylpyrrolidone (PVP) and the inorganic compound citric acid. Other options include chitosan (CS), hyaluronic acid (HA), polyethyleneimine (PEI), oxalic acid, and polydienedimethylammonium chloride (PDDA). However, these coatings cannot effectively maintain the stability and safety of Prussian blue or impart new activities to the nanoparticles.

[0004] Using non-toxic polysaccharides as coatings offers several advantages, such as reducing the potential toxicity of Prussian blue nanoparticles, improving stability and biocompatibility, and enabling the nanoparticles to acquire new functions through binding with other bioactive substances. Polydextrose sorbitol carboxymethyl ether (PSC) is a dextran used clinically as a surface coating for Ferumoxytol, an intravenous iron supplement for treating iron deficiency anemia. Compared to other intravenous iron supplements, Ferumoxytol is the most effective iron supplement with the fewest adverse reactions, exhibiting excellent biocompatibility and safety. This is attributed to the carboxyl groups abundant in the PSC structure of Ferumoxytol, which can coordinate with iron ions in superparamagnetic iron oxide nanoparticles, thereby improving the stability and biocompatibility of the inorganic nanoparticles. Furthermore, the hemiacetal hydroxyl groups in the PSC structure have antioxidant effects, scavenging reactive oxygen species (ROS). Summary of the Invention

[0005] Objective of the Invention: To address the problems existing in the prior art, this invention provides a novel method for preparing PSC-coated Prussian blue nanoparticles using PSC as a protective agent, and applies this method to the treatment of myocardial ischemia-reperfusion injury associated with ROS generation and inflammatory infiltration. The PSC-coated Prussian blue nanoparticles prepared by this invention effectively solve the safety and stability problems of current Prussian blue nanoparticles, and improve their bioactivity.

[0006] Technical Solution: To achieve the above objective, the present invention provides a method for preparing PSC-coated Prussian blue nanoparticles (PBNPs@PSC). This method utilizes a dual-precursor preparation method and includes the following steps:

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

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

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

[0010] (4) The mixed reaction solution is heated in a water bath under stirring to generate the product;

[0011] (5) The product was subjected to dialysis, ultrafiltration, centrifugation and ultrafiltration, concentration and freeze drying to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC);

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

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

[0014] In the steps (1) and (2), the solution is deionized water, the amount of precursor 1 is 0.01-1 mmol, and the amount of 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] In step (3), the dropping acceleration rate 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] In step (4), the water bath heating temperature is 40-100℃, the reaction time is 0.5-10h, and the mechanical stirring rate is 100-600r / min.

[0019] In step (5), the dialysis bag used for dialysis has a molecular weight cutoff of 5-100KD, the ultrafiltration tube used for ultrafiltration has a molecular weight cutoff of 10-50KD, the centrifugation rate during ultrafiltration is 1000-6000r / min, and the centrifugation temperature is 3-10℃.

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

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

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

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

[0024] (4) The mixed reaction solution is heated in a water bath and mechanically stirred to produce products;

[0025] (5) The product is subjected to dialysis, ultrafiltration tube centrifugation and ultrafiltration, concentration and freeze drying to obtain the final product.

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

[0027] In step 2, the precursor 2 is a water-soluble ferrocyanide, including one of potassium ferrocyanide, ammonium ferrocyanide, or sodium ferrocyanide; the amount of precursor 2 is 0.01-1 mmol, preferably 0.2-0.5 mmol; the molar ratio of PSC to precursor 1 is 1:1-1:10, preferably 1:2-1:6; and the volume of the mixed solution A is 5-50 mL, preferably 10-30 mL.

[0028] In step 3, mixed solutions A and B must be completely dissolved and the solution must be clear; the injection pump drip rate is 0.1-10 mL / min, preferably 1-5 mL / min; the mechanical stirring rate is 100-600 r / min, preferably 200-500 r / min; and the volume of the mixed reaction solution is 10-100 mL, preferably 20-60 mL.

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

[0030] In step 5, the dialysis bag used for dialysis has a molecular weight cutoff of 10-100 KD, preferably 100 KD, and the dialysis time is 72 hours; the ultrafiltration tube used for ultrafiltration has a molecular weight cutoff of 10-50 KD, preferably 20-40 KD; the centrifugation rate during ultrafiltration is 1000-6000 r / min, preferably 3000-5000 r / min, the centrifugation temperature is 3-10℃, preferably 4℃, and the product is freeze-dried for 48 hours.

[0031] The preparation method described in this invention successfully prepared uniform and stable PSC-coated Prussian blue nanoparticles (PBNPs@PSC).

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

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

[0034] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) described in this invention exhibit higher superoxide dismutase (SOD)-like activity.

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

[0036] The PSC-coated Prussian blue nanoparticles (PBNPs@PSC) described in this invention can be passively targeted to damaged myocardium via intravenous injection.

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

[0038] This invention employs a dual-precursor synthesis method to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC). This method is simple to operate, easy to scale up for industrial production, and can obtain high-quality Prussian blue nanoparticles with easily functionalizable surfaces in a short time. The PSC-coated Prussian blue nanoparticles prepared by this invention have uniform and stable size, low polydispersity index, and exhibit catalase-like, peroxidase-like, and superoxide dismutase-like activities. The superoxide dismutase-like enzyme activity is higher than that of existing PVP-coated high-performance Prussian blue nanoparticles with extremely small sizes. The Prussian blue nanoparticles prepared by this invention have good biocompatibility and safety, can effectively scavenge ROS in vivo and exert anti-inflammatory effects, and can be passively targeted to damaged myocardium in myocardial ischemia-reperfusion injury via intravenous injection, making them well-suited for the treatment of myocardial ischemia-reperfusion injury.

[0039] This invention provides a method for preparing PSC-coated Prussian blue nanoparticles. The numerous hydroxyl groups exposed on the PSC surface can coordinate with ferric ions, providing an intrinsic driving force for the formation of Prussian blue nanoparticles and laying the foundation for further functionalization. Furthermore, as a polysaccharide, the terminal hemiacetal hydroxyl groups of PSC possess excellent redox capabilities, playing a role in resisting oxidative stress. Therefore, this invention utilizes PSC as a coating material to develop a simple, reproducible, stable, uniform, easily functionalized Prussian blue nanoparticle with good biocompatibility and safety. The prepared Prussian blue nanoparticles are also easily applicable to biomedical applications. The method of this invention is a novel technique that utilizes the coordination of carboxyl groups on the PSC surface with ferric ions, followed by coordination polymerization with ferrous cyanide ions to form Prussian blue nanoparticles. This method is called the dual precursor method (e.g., Figure 1 (As shown). This invention employs a dual-precursor synthesis method to obtain PSC-coated Prussian blue nanoparticles (PBNPs@PSC). This method is simple to operate, easy to scale up for industrial production, and can obtain high-quality Prussian blue nanoparticles with easily functionalizable surfaces in a short time. This method can successfully prepare uniform, stable Prussian blue nanoparticles with very high SOD-like enzyme activity. The prepared Prussian blue nanoparticles exhibit excellent ROS scavenging and anti-inflammatory effects in vivo, and possess good biocompatibility and safety. When administered intravenously, they can passively target the lesion site through the EPR effect of myocardial ischemia-reperfusion injury, making them well-suited for the treatment of myocardial ischemia-reperfusion injury. This provides great potential for the application of Prussian blue nanoparticles in the biomedical field.

[0040] Existing techniques for preparing Prussian blue nanoparticles primarily use PVP as a protective agent, but the enzyme-like activity of the synthesized Prussian blue nanoparticles needs improvement. This invention proposes for the first time the use of PSC as a protective agent in the preparation of Prussian blue nanoparticles, which improves SOD-like enzyme activity and exhibits good biocompatibility, making it suitable for the treatment of diseases related to ROS generation, such as myocardial ischemia-reperfusion injury.

[0041] This invention provides a novel method for synthesizing Prussian blue nanoparticles, specifically using PSC as a protective agent to synthesize PSC-coated Prussian blue nanoparticles, thereby improving the stability, safety, and activity of Prussian blue nanoparticles. This invention compares PSC-coated high-performance Prussian blue nanoparticles with an extremely small size (3.4 nm) with those using PVP. While the PVP-coated high-performance Prussian blue nanoparticles with an extremely small size (3.4 nm) also exhibit enzyme-like activity, the smaller the size, the greater the toxicity. In contrast, the method of this invention, using PSC as a protective agent in the preparation of Prussian blue nanoparticles, produces PSC-coated Prussian blue nanoparticles with a larger size of 44 nm. However, their SOD enzyme activity—the ability to scavenge superoxide ions and hydroxyl radicals—is increased by 1.6 times and 0.6 times, respectively, compared to the PVP-coated high-performance Prussian blue nanoparticles with an extremely small size (3.4 nm). Furthermore, it exhibits excellent biocompatibility and can be used for intravenous administration. This invention not only has high activity and high biosafety, but also has great therapeutic potential for diseases related to ROS production.

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

[0043] This invention utilizes PSC as a protective agent to prepare PSC-coated Prussian blue nanoparticles via a dual precursor method. The Prussian blue nanoparticles prepared using this invention exhibit uniform and stable size distribution (44 nm), good batch-to-batch stability, and significantly enhanced SOD-like enzyme activity compared to PVP-coated ultra-small (3.4 nm) high-performance Prussian blue nanoparticles (USPBNPs). Specifically, their superoxide ion scavenging ability is increased by 1.6 times, and their hydroxyl radical scavenging ability is increased by 0.6 times. The PSC-coated Prussian blue nanoparticles prepared by this invention demonstrate good biocompatibility, are less prone to hemolysis, and exhibit good ROS scavenging and anti-inflammatory effects. The PSC-coated Prussian blue nanoparticles prepared by this invention can passively target damaged myocardium through the EPR effect. After intravenous injection into a mouse model of myocardial ischemia-reperfusion injury, the retention rate in damaged myocardium was more than 5 times higher than in the sham-operated group. The PSC-coated Prussian blue nanoparticles prepared by this invention can effectively treat myocardial ischemia-reperfusion injury. Attached Figure Description

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

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

[0046] Figure 3These are the hydration size distribution diagram and transmission electron microscope image of PSC-coated Prussian blue nanoparticles.

[0047] Figure 4 This is an electron spin resonance diagram of the peroxidase-like activity measurement of PSC-coated Prussian blue nanoparticles.

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

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

[0050] Figure 7 The images show liver and kidney function, as well as H&E diagrams of the heart, liver, spleen, lungs, and kidneys in mice after intravenous injection of PSC-coated Prussian blue nanoparticles.

[0051] Figure 8 This is a graph showing the results of PSC-coated Prussian blue nanoparticles in clearing ROS from cardiomyocytes.

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

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

[0054] Figure 11 The PSC-coated Prussian blue nanoparticles exhibit ROS clearance and anti-inflammatory effects on damaged myocardium in a mouse model of myocardial ischemia-reperfusion injury.

[0055] Figure 12 This study investigated the effects of PSC-coated Prussian blue nanoparticles on myocardial remodeling and improved cardiac function in a mouse model of myocardial ischemia-reperfusion injury. Detailed Implementation

[0056] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0057] Polydextrose sorbitol carboxymethyl ether (PSC), with a relative molecular weight of 10,000, reference: Ferumoxytol of ultrahigh magnetization produced by hydrocooling and magnetically internalheating co-precipitation. Nanoscale 2018, 10(16), 7369-7376.

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

[0059] Example 1

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

[0061] Weigh 0.5 g of PSC and 67.5 mg of ferric chloride into an Erlenmeyer flask, add 20 mL of deionized water, and stir until clear to obtain mixed solution A. Weigh 0.5 g of PSC and 105.6 mg of potassium ferrocyanide into a 50 mL centrifuge tube, add 20 mL of deionized water, and stir until clear to obtain mixed solution B. Then transfer mixed solution B to a 20 mL syringe and add it dropwise to mixed solution A at a rate of 1 mL / min using a syringe pump, stirring continuously while adjusting the mechanical stirrer speed to 400 rpm. After the addition of mixed solution B is complete, react for 4 hours in a 90°C water bath with a stirring speed of 400 rpm. After the reaction is complete, turn off the water bath and allow the reactants to cool naturally to room temperature. Then transfer them to a dialysis bag with a molecular weight cutoff of 100 KD, dialyze against ultrapure water for 72 hours, and then concentrate using a 30 KD ultrafiltration tube at 4°C and a centrifugation speed of 4000 rpm. The concentrate was 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 determination of PBNPs@PSC

[0064] Take a small amount (50 mg) of PBNPs@PSC powder obtained in Example 1 and add it to the center of the groove in the sample holder. Gently press a glass slide onto the sample surface to smooth the powder sample surface to align with the frame plane. Place the sample holder into the sample chamber of the X-ray measuring instrument for measurement to obtain the X-ray diffraction results of the product. Alternatively, take a small amount (50 mg) of PBNPs@PSC powder obtained in Example 1 and place it in the sample loading port of an infrared spectrometer. Measure the results using an infrared spectrometer to obtain the infrared spectrometer data of the obtained product. The results are as follows... Figure 2 As shown, the X-ray diffraction pattern shows that the PBNPs@PSC powder prepared in this invention has a Prussian blue crystal structure, and the infrared spectrum shows that the surface of the PBNPs@PSC prepared in this invention has a PSC structure. Figure 2 Together, these demonstrate that the present invention successfully prepared PSC-coated Prussian blue nanoparticles.

[0065] Example 3

[0066] PBNPs@PSC hydration particle size, polydispersity index and transmission electron microscopy determination

[0067] The PBNPs@PSC obtained in Example 1 were diluted with water to a concentration of 10 μg / mL, and 1 mL was pipetted into the sample cell. The particle size and polydispersity were determined using a Malvern Nano ZS90 particle size potentiometric analyzer. The PBNPs@PSC obtained in Example 1 were diluted with water to a concentration of 500 μg / mL, and 20 μL was dropped onto a copper grid. After air drying, the samples were imaged using a transmission electron microscope. The results are as follows: Figure 3 As shown in the figure, the results indicate that the PBNPs@PSC prepared by this 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 comparative product (USPBNPs@PVP) (prepared according to the literature "Zhiguo Qin, et al. Achieving Ultrasmall Prussian Blue Nanoparticles as HighPerformance Biomedical Agents with Multifunctions ACS Appl. Mater. Interfaces 2020, 12, 57382-57390") were diluted with water to a concentration of 75 mg / mL. 200 μL of 5 mg / mL FeSO4 solution was added to 20 μL of DMPO and 160 μL of the product obtained in Example 1 or the comparative product. Then, 20 μL of 30% hydrogen peroxide was added, and the mixture was stirred and reacted for 5 min. Samples were taken and tested using an electron spin resonance analyzer to obtain the electron spin resonance data of the obtained product and the comparative product for scavenging hydroxyl radicals.

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

[0072] The results are as follows Figure 4 As shown, the results indicate that the PBNPs@PSC prepared in this invention exhibits a 1.6-fold increased superoxide ion scavenging ability and a 0.6-fold increased hydroxyl radical scavenging ability compared to the high-performance, ultra-small (3.4 nm) PVP-coated Prussian blue nanoparticles prepared in the references. This demonstrates that the PBNPs@PSC prepared in this invention possesses very high SOD-like enzyme activity, enabling better ROS scavenging and providing significant therapeutic potential for ROS-related diseases.

[0073] Example 5

[0074] Cytotoxicity study of PBNPs@PSC

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

[0076] The results are as follows Figure 5 As shown, at a dosage of 100 μg / mL, the PBNPs@PSCs prepared in this invention did not significantly affect cell viability, indicating that they have good cell safety.

[0077] Example 6

[0078] Hemolysis experiment of PBNPs@PSC

[0079] Fresh blood from anticoagulated C57BL / 6 mice was centrifuged at 1200 rpm for 8 minutes, the supernatant was discarded, and the lower precipitate was washed three times with PBS to obtain red blood cells. The red blood cells were then diluted 10-fold with PBS. 100 μL of different concentrations of PBNPs@PSC obtained in Example 1 were added to 1 mL of the diluted red blood cells to achieve stop concentrations of 30, 100, and 300 μg / mL, respectively. The cells were incubated at 37°C for 4 hours, followed by centrifugation at 10000 rpm for 3 minutes. 100 μL of the supernatant was then transferred to a 96-well plate, and the absorbance at 540 nm was measured using a microplate reader. Red blood cells in PBS served as a negative control, and red blood cells in water served as a positive control. Hemolysis rate (%) = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%. A hemolysis rate exceeding 5% was considered hemolysis.

[0080] The results are as follows Figure 6 As shown, at a concentration of 300 μg / mL, the PBNPs@PSC prepared in this invention do not cause hemolysis, indicating their good biocompatibility and safety, and that they can be used for intravenous administration.

[0081] Example 7

[0082] In vivo safety study of PBNPs@PSC

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

[0084] The results are as follows Figure 7 As shown, intravenous injection of PBNPs@PSC prepared in this invention into mice did not affect liver and kidney function, nor did it have any harmful effects on vital organs such as the heart, liver, spleen, lungs, and kidneys, indicating that PBNPs@PSC has good biosafety.

[0085] Example 8

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

[0087] The effect of PBNPs@PSC obtained in Example 1 on cellular ROS scavenging was detected using a ROS detection kit. 200,000 RAW264.7 cells and HL-1 cells were seeded in confocal microplates and cultured for 24 hours. Then, 1 μg / mL and 10 μg / mL PBNPs@PSC were added and incubated for 24 hours, respectively. After discarding the incubation medium, the cells were washed three times with PBS, and then stimulated with 10 mM H2O2 for 30 min to generate ROS. The culture medium was discarded, and the cells were washed three times with PBS. Then, the ROS fluorescent probe DCFH-DA (1:1000, diluted in serum-free culture medium) was added and incubated for 20 minutes. After discarding the incubation medium, the cells were washed three times with serum-free culture medium and observed directly under a fluorescence microscope. Quantitative analysis of ROS scavenging was performed using flow cytometry. Cell processing was the same as above, except that cells were seeded in 12-well plates (100,000 cells per well), and after adding DCFH-DA-labeled ROS, cells were collected for flow cytometry analysis.

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

[0089] Example 9

[0090] Anti-inflammatory effects of PBNPs@PSC at the cellular level

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

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

[0093] Example 10

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

[0095] Prussian blue nanoparticles labeled with the fluorescent dye FITC were synthesized using PBNPs@PSC obtained in Example 1. A classic myocardial ischemia-reperfusion injury model was established using C57BL / 6 mice. After surgery, these mice were intravenously injected with 200 μL of FITC-labeled PBNPs@PSC at a dose of 0.5 mg / kg body weight. Twelve hours later, 200 μL of 1% Evans blue solution was injected intravenously again. The mice were then anesthetized, mechanically inserted, and an observation window was opened in the thoracic cavity to expose the heart. The myocardium was observed using a small animal in vivo in situ cell dynamic analysis imaging system. A sham-operated group of mice was set up as a control. The results showed ( Figure 10 The content of PBNPs@PSC in the myocardial tissue of model mice was 5 times higher than that in the sham-operated group, indicating that PBNPs@PSC can passively target damaged myocardium and be taken up by cardiomyocytes.

[0096] Example 11

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

[0098] Immediately after establishing a myocardial ischemia-reperfusion injury model in C57BL / 6 mice, PBNPs@PSCs were administered intravenously at a dose of 0.5 mg / kg body weight, with repeated administration on days 3 and 7 post-surgery. On day 3, heart tissue from the treated mice was collected to measure ROS and inflammatory factors (IL-6, IL-1β, and TNF-α), examining the ROS clearance and anti-inflammatory effects of PBNPs@PSCs. Results showed ( Figure 11 PBNPs@PSC can effectively remove ROS from the myocardial tissue of model mice, and has a good anti-inflammatory effect.

[0099] Echocardiography was performed on mouse models on days 3, 7, and 14 after drug treatment. Heart tissue was harvested on day 14 for WGA and Masson staining to assess the effect on myocardial remodeling. Results are as follows: Figure 12 As shown, after 14 days of treatment, PBNPs@PSC effectively promoted myocardial remodeling and improved cardiac function in mice with myocardial ischemia-reperfusion injury, 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 0.25 g of PSC and 67.5 mg of ferric chloride into an Erlenmeyer flask, add 20 mL of deionized water, and stir until clear to obtain mixed solution A. Weigh 0.25 g of PSC and 105.6 mg of potassium ferrocyanide into a 50 mL centrifuge tube, add 20 mL of deionized water, and stir until clear to obtain mixed solution B. Then transfer mixed solution B to a 20 mL syringe and add it dropwise to mixed solution A at a rate of 1 mL / min using a syringe pump, stirring continuously while adjusting the mechanical stirrer speed to 400 rpm. After the addition of mixed solution B is complete, react for 4 hours in a 90°C water bath with a stirring speed of 400 rpm. After the reaction is complete, turn off the water bath and allow the reactants to cool naturally to room temperature. Then transfer them to a dialysis bag with a molecular weight cutoff of 100 KD, dialyze against ultrapure water for 72 hours, and then concentrate by ultrafiltration using a 30 KD ultrafiltration tube at a centrifugal speed of 4000 rpm. The concentrate was freeze-dried for 48 hours to obtain PSC-coated Prussian blue nanoparticle powder (PBNPs@PSC).

[0103] Example 13

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

[0105] Weigh 1g of PSC and 67.5mg of ferric chloride into an Erlenmeyer flask, add 20mL of deionized water, and stir until clear to obtain mixed solution A. Weigh 1g of PSC and 105.6mg of potassium ferrocyanide into a 50mL centrifuge tube, add 20mL of deionized water, and stir until clear to obtain mixed solution B. Then transfer mixed solution B to a 20mL syringe and add it dropwise to mixed solution A at a rate of 1mL / min using a syringe pump, stirring continuously while adjusting the mechanical stirrer speed to 400r / min. After the addition of mixed solution B is complete, react for 4 hours in a 90℃ water bath with a stirring speed of 400r / min. After the reaction is complete, turn off the water bath and allow the reactants to cool naturally to room temperature. Then transfer them to a dialysis bag with a molecular weight cutoff of 100KD, dialyze with ultrapure water for 72 hours, and then concentrate by ultrafiltration using a 30KD ultrafiltration tube at a centrifugal speed of 4000r / min. The concentrate was 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, This method uses a dual-precursor preparation method, including the following steps: (1) Polydextrose sorbitol carboxymethyl ether PSC and precursor 1 are dissolved in solution to form mixture A; (2) Dissolve PSC and precursor 2 in a solution to form a mixture B; (3) Add the mixture B dropwise to the mixture A under mechanical stirring to obtain a mixed reaction solution; (4) The mixed reaction solution is heated in a water bath under stirring to generate the product; (5) The product was subjected to dialysis, ultrafiltration, centrifugation and ultrafiltration, concentration 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 mentioned in step (1) is one of ferric chloride, ferric sulfate or ferric nitrate; the precursor 2 mentioned 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 PSC to precursor 1 is 10:1 to 1:10, the molar ratio of PSC to precursor 2 is 10:1 to 1:10, and the molar ratio of precursor 1 to precursor 2 is 10:1 to 1:

10.

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

5. The preparation method according to claim 1, characterized in that, In step (3), the drop rate 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, The water bath heating temperature in step (4) is 40-100℃, the reaction time is 0.5-10h, and the mechanical stirring rate is 100-600 r / min.

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

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

9. The use of PSC-coated Prussian blue nanoparticles (PBNPs@PSC) prepared by the method of claim 1 in the preparation of reagents or drugs for scavenging ROS in organisms and for anti-inflammation.

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

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