Nanoparticle capable of releasing EGCG (epigallocatechin gallate) in ROS (reactive oxygen species) response as well as preparation method and application of nanoparticle

Abstract

The invention provides a nanoparticle capable of releasing EGCG (epigallocatechin gallate) in response to ROS (reactive oxygen species) as well as a preparation method and application of the nanoparticle. The nanoparticles comprise a carrier and EGCG (epigallocatechin gallate) loaded on the carrier; the carrier is Prussian blue nanoparticles of which the surfaces are modified with polyethyleneimine and phenylboronic acid compounds. The preparation method of the nanoparticles is simple and easy to operate, the nanoparticles are good in dispersity, and through research, the nanoparticles have CAT-like, SOD-like and other multi-enzyme activities, can remove. OH, have a high EGCG drug loading rate and release EGCG in the presence of ROS. Besides, the nanoparticles show a good effect of improving myocardial ischemia reperfusion injury in cell level and rat in-vivo experiments, through comparison of EGCG and PB NPs single-drug treatment, synergistic treatment in the composite material achieves an effect of '1 + 1 > 2', and the composite material has good biological safety.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a nanoparticle capable of releasing EGCG in response to ROS, and a preparation method and application thereof. Background Art

[0002] According to statistics from the World Health Organization (WHO), ischemic heart disease (IHD) is the leading cause of death worldwide, with approximately 9 million people dying from IHD each year. The most common manifestation of ischemic heart disease is acute myocardial infarction (AMI). In the case of AMI, early revascularization through percutaneous coronary intervention (PCI) or surgical coronary artery bypass grafting (CABG) can effectively prevent the loss of contractile myocardial mass, reduce infarct size, and improve clinical outcomes. However, revascularization itself can lead to cardiomyocyte death and aggravate myocardial damage after ischemia, which is called myocardial ischemia / reperfusion injury (I / RI). Its specific mechanism is mainly related to excessive ROS production, inflammation, Ca 2+ Overload is related to the opening of the mitochondrial permeability transition pore (mPTP) channel. Treatment of myocardial ischemia-reperfusion injury can significantly reduce the size of myocardial infarction and improve patient prognosis. However, the current treatment methods for I / RI are limited and the efficacy is poor. The development of new therapeutic drugs based on its pathogenesis is a major need in today's society. Reactive oxygen free radicals (ROS) and inflammation play a core role in promoting myocardial ischemia-reperfusion injury. ROS are natural byproducts of aerobic metabolism initially produced by oxygen reduction, mainly including superoxide anions (O2·-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-). ROS accumulate in myocardial cells during the process of blood flow restoration. Excessive production of ROS can induce oxidative stress, which is a harmful process that can cause severe oxidative damage to cell structures (including lipids, biological membranes, proteins, and DNA), ultimately leading to cell necrosis and apoptosis. At the same time, excessive production of ROS also plays a central role in the initiation and maintenance of the inflammatory phase of myocardial I / RI. Damaged cardiac parenchymal cells, extracellular matrix, and released substances act as danger signals, binding to pattern recognition receptors (PRRs) on parenchymal cells (including cardiac fibroblasts and resident macrophages) and infiltrating leukocytes, activating a series of inflammatory mediators through the innate immune system. Excessive inflammation, in turn, induces the production of ROS, thus forming a positive feedback between inflammation and ROS. Therefore, antioxidant and anti-inflammatory treatments are currently considered to be effective methods for improving myocardial reperfusion injury.

[0003] Epigallocatechin-3-gallate (EGCG) is the most bioactive polyphenol in green tea and has attracted much attention for its potential benefits to human health. The gallic acid part and the extra phenolic ring on EGCG can capture electrons and act as a scavenger of free radicals to reduce oxidative stress. It has antioxidant, antibacterial, anti-obesity, anti-inflammatory, cardiovascular protection and neuroprotective effects. EGCG also showed good therapeutic prospects in the myocardial ischemia-reperfusion injury model. However, pharmacological studies on EGCG have found that EGCG has low bioavailability, poor structural stability and short plasma half-life. In order to obtain a therapeutic effect, a high dose of EGCG is required, and the effective high dose of EGCG is not far from its toxic dose level.

[0004] Therefore, in order to overcome the application barriers of EGCG, carriers can be used to load EGCG, but most of the current solutions for preparing carriers loaded with EGCG have problems such as complex technology, high cost, and unstable yield. In addition, the existing research on carriers loaded with EGCG mainly focuses on cancer treatment, and more research is needed to explore its potential application in myocardial ischemia-reperfusion injury. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a nanoparticle capable of releasing EGCG in response to ROS, and a preparation method and application thereof. The preparation method of the nanoparticle is simple and easy, the raw materials are easy to obtain, the cost is low, it is safe and efficient, and it has a high EGCG loading rate.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a nanoparticle capable of releasing EGCG in response to ROS, comprising a carrier and EGCG loaded on the carrier.

[0008] Preferably, the carrier is Prussian blue nanoparticles whose surface is modified with polyethyleneimine and phenylboronic acid compounds.

[0009] Preferably, the phenylboronic acid compound is selected from any one or more of 3-aminophenylboronic acid, 3-acrylamidophenylboronic acid (abbreviated as: APBA) or 4-formylphenylboronic acid (abbreviated as: FPBA), preferably FPBA.

[0010] Preferably, the loading rate of EGCG is above 34%.

[0011] Preferably, the Prussian blue nanoparticles and the nanoparticles are both cubic structures.

[0012] Preferably, the particle size of the Prussian blue nanoparticles is 100-130 nm.

[0013] Preferably, the particle size of the nanoparticles is 120-150 nm.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned nanoparticles, comprising the following steps:

[0015] Prussian blue nanoparticles whose surfaces are modified with polyethyleneimine and phenylboronic acid compounds are self-assembled with EGCG in the presence of a solvent to obtain nanoparticles.

[0016] Preferably, the solvent is a PBS buffer solution; the pH value of the PBS buffer solution is 7.4.

[0017] Preferably, the self-assembly is carried out under stirring conditions, and the self-assembly time is 2 to 3 hours.

[0018] Preferably, the mass ratio of the Prussian blue nanoparticles whose surfaces are modified with polyethyleneimine and phenylboronic acid compounds to EGCG is (1-3):(5-10), more preferably 3:10.

[0019] Preferably, the Prussian blue nanoparticles modified with polyethyleneimine and phenylboronic acid compounds on the surface are prepared according to the following method:

[0020] The Prussian blue nanoparticles are self-assembled with polyethyleneimine to obtain the Prussian blue nanoparticles with polyethyleneimine modified on the surface, and then the Prussian blue nanoparticles are mixed with an alcohol solution of a phenylboronic acid compound to react.

[0021] Preferably, the mass ratio of the polyethyleneimine, phenylboronic acid compound and Prussian blue nanoparticles is (1-2):(5-10):(1-2).

[0022] Preferably, the self-assembly time of the Prussian blue nanoparticles and polyethyleneimine is 10 to 15 hours, preferably 12 hours.

[0023] Preferably, the reaction is carried out under stirring, and the reaction time is 15 to 30 hours, preferably 24 hours.

[0024] In a third aspect, the present invention provides a use of the above-mentioned nanoparticles in the preparation of a drug for treating myocardial ischemia-reperfusion injury.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a nanoparticle capable of releasing EGCG in response to ROS, comprising a carrier and EGCG loaded on the carrier. The carrier is a Prussian blue nanoparticle whose surface is modified with polyethyleneimine and phenylboronic acid compounds. In the present invention, the Prussian blue nanoparticle (PB NPs for short) has a variety of excellent properties such as high specific surface area, imaging characteristics, surface modification, controlled drug release, biocompatibility and biodegradability, good stability, etc. Therefore, the present invention preferably uses it as a carrier. PB NPs are negatively charged, and PB@PEI is obtained by encapsulating PB NPs with polyethyleneimine (PEI for short), which not only improves the dispersibility and biocompatibility of PB NPs in aqueous media, but also converts PB NPs from negative charge to positive charge, so that PB NPs adsorb negatively charged EGCG through electrostatic adsorption. In addition, since EGCG is unstable, the present invention further adsorbs negatively charged phenylboronic acid compounds on the surface of PB@PEI through electrostatic adsorption, and phenylboronic acid compounds can interact with EGCG under physiological conditions through phenylboronic acid-catechol to form a highly stable borate ester bond, thereby protecting EGCG from oxidation in the blood circulation. At the same time, it is beneficial that the final nanoparticles (i.e., PB@PEI / phenylboronic acid compounds-EGCG) can be cleaved and released in the presence of ROS to exert the effect of EGCG, thereby increasing the concentration of EGCG in the diseased tissue.

[0027] After characterization, the nanoparticles obtained using FPBA as the phenylboronic acid compound can be recorded as: PB@PEI / FPBA-EGCG. The cubic structure of PB NPs is maintained in morphology, the structure is stable, the size is about 140nm, and the dispersion is good, which can be used for intravenous injection.

[0028] It has been verified that the PB@PEI / FPBA-EGCG prepared by the present invention can achieve an EGCG loading rate of 34%, remove ROS by simulating the activity of multiple enzymes such as CAT and SOD, and release EGCG in the presence of ROS. At the same time, PB@PEI / FPBA-EGCG achieves efficient enrichment in the site of myocardial ischemia-reperfusion injury through the "permeation and retention" (EPR) effect of nanoparticles, and shows good improvement of myocardial ischemia-reperfusion injury in both cellular level and rat in vivo experiments, and the synergistic treatment of EGCG and PB NPs achieves the effect of "1+1>2". Furthermore, the PB@PEI / FPBA-EGCG has good biosafety, and the results of the hemolysis test show that the hemolysis rate of the material with a concentration of up to 6 mg / mL is still less than 5%, and the hemolysis test is negative. Below a concentration of 50ug / mL, the inhibition rate of H9c2 cell viability is less than 20%.

[0029] In addition, the preparation method of the nanoparticles capable of releasing EGCG in response to ROS provided by the present invention is simple, the raw materials are easily available, the cost is low, and it is safe and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 SEM images, DLS spectra, and XRD spectra of PB NPs;

[0031] in, Figure 1 A is the SEM image of PB NPs; Figure 1 B is the DLS spectrum of PB NPs; Figure 1 C is the XRD spectrum of PB NPs;

[0032] Figure 2 SEM images, DLS spectra, and XRD spectra of PPFE NPs;

[0033] in, Figure 2 A is the SEM image of PPFE NPs; Figure 2 B is the DLS spectrum of PPFE NPs; Figure 2 C is the XRD spectrum of PPFE NPs;

[0034] Figure 3 Fourier transform infrared spectra of PB NPs and PPFE NPs;

[0035] Figure 4 Zeta potential images, UV absorption curves, and standard curves of different NPs;

[0036] in, Figure 4 A is the zeta potential image of PBNPs, PB@PEI, PB@PEI / FPBA, and PPFE NPs; Figure 4 B is the UV absorption curve of EGCG; Figure 4 C is the UV absorption curve of PB NPs;

[0037] Figure 4 D is the EGCG standard curve calculated by measuring the absorbance of EGCG PBS solution with set concentration gradient; Figure 4 E is the PB NPs standard curve calculated by measuring the absorbance of PB NPs solution with set concentration gradient; Figure 4 F is the UV absorption curves of PB NPs, PB@PEI, PB@PEI / FPBA, and PPFE NPs;

[0038] Figure 5 This is a comparison of the O2 produced by PPFE NPs with different concentrations in H2O2 PBS solution over the reaction time;

[0039] Figure 6 The figure is a comparison of the formazan inhibition rates of PPFE NPs with different concentrations;

[0040] Figure 7 Comparison of the ability of PPFE NPs with different concentrations to eliminate ·OH;

[0041] Figure 8 This is a comparison of the release of EGCG over time from PPFE NPs in H2O2 environments with different concentrations;

[0042] Fig. 9 Comparison of the effects of different concentrations of PPFE NPs on the viability of cardiomyocytes and the cell viability of different treatment groups;

[0043] in, Fig. 9 A is a comparison of the effects of different concentrations of PPFE NPs on the viability of cardiomyocytes; Fig. 9 B is a comparison of cell viability in different treatment groups;

[0044] Fig.10 The ROS fluorescence images of each group and the analysis result images were taken using a laser scanning confocal microscope;

[0045] in, Fig.10 A is the ROS fluorescence image of each group taken using a laser scanning confocal microscope; Fig.10 B is the image of the fluorescence intensity statistical analysis results of each group of pictures using Image J;

[0046] Fig.11 Gross images of TTC staining and calculation results of hearts in different groups;

[0047] in, Fig.11 A is the gross image of TTC staining of the heart in different groups (where the yellow dotted line is used to mark the infarct area); Fig.11 B is the statistical graph of the data obtained after calculating the infarct area of ​​each group using image J;

[0048] Fig.12 The fluorescence intensity images of various internal organs at different times after I / R model rats were injected with ICG-labeled PPFE NPs through the tail vein, the fluorescence intensity images of the hearts of Sham group rats and I / R model rats at different times after intravenous injection of ICG-labeled PPFE NPs, and the statistical graphs of the data;

[0049] in, Fig.12 A is the fluorescence intensity image of each internal organ at 2h, 6h, and 24h after the tail vein injection of ICG-labeled PPFE NPs in I / R model rats; Fig.12B is the cardiac fluorescence intensity images of the sham group rats and I / R model rats at 2h, 6h, and 24h after intravenous injection of ICG-labeled PPFENPs; Fig.12 C is for Fig.12 Statistical diagram of fluorescence intensity of each organ in A; Fig.12 D is for Fig.12 B is the statistical diagram of the fluorescence intensity data of each heart;

[0050] Fig.13 This is the performance test result image of PPFE NPs;

[0051] in, Fig.13 A is the hemolysis test results of PPFE NPs with different concentrations; Fig.13 B shows the body weight changes of I / R model rats 14 days after intravenous injection of PPFE NPs or PBS; Fig.13 C is the statistical graph of liver function (ALT, AST) and renal function (BUN, Cr) in I / R model rats 14 days after intravenous injection of PPFE NPs or PBS; Fig.13 D is the HE staining image of various organs in I / R model rats 1 day and 14 days after intravenous injection of PPFE NPs or PBS. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The gallic acid part and the extra phenolic ring on EGCG can capture electrons, act as a scavenger of free radicals to reduce oxidative stress, and have antioxidant, antibacterial, anti-obesity, anti-inflammatory, cardiovascular protection and neuroprotective effects. In addition, considering that the nanomaterials encapsulating EGCG are more stable than conventional molecules in the circulatory system and have an "enhanced permeation and retention" (EPR) effect, they have a longer half-life, allowing for a reduction in the dosage and reducing side effects while maintaining efficacy. Therapy based on nanocarriers can protect therapeutic drugs from degradation after injection, thereby changing the pharmacokinetic properties of the drug and improving bioavailability and therapeutic efficacy.

[0054] Therefore, the present invention aims to provide a nanoparticle capable of releasing EGCG in response to ROS, and to prepare corresponding drugs.

[0055] Based on the above purpose, the present invention first considers drug carriers. Since Prussian blue nanoparticles (PB NPs) have multi-enzyme activity, they can treat oxidative stress-related diseases by scavenging ROS. PB NPs also have many excellent properties such as high specific surface area, imaging properties, surface modification, controlled drug release, biocompatibility and biodegradability, and good stability. Therefore, we choose PB NPs as a carrier to load EGCG, so that EGCG and PBNZs can synergistically play the role of scavenging ROS and anti-inflammatory.

[0056] Since EGCG is hydrophilic, the active hydroxyl groups in polyphenols are easily oxidized and unstable at physiological pH. The present invention has found that adding phenylboronic acid compounds, such as any one or more selected from 3-aminophenylboronic acid, 3-acrylamidophenylboronic acid or 4-formylphenylboronic acid, is preferred to 4-formylphenylboronic acid.

[0057] In some embodiments of the present invention, 4-formylphenylboronic acid (FPBA for short) is preferably used, which can form a highly stable borate ester bond with EGCG through the interaction of phenylboronic acid-catechol under physiological conditions, thereby protecting EGCG from oxidation in the blood circulation. At the same time, EGCG can be cleaved and released in the presence of ROS to exert its effect, thereby increasing the concentration of EGCG in the diseased tissue.

[0058] EGCG is hydrophilic and PB NPs are hydrophobic, and both are negatively charged. It is difficult to load EGCG in PB NPs through hydrophobic interaction and maintain the stability of EGCG. Therefore, this study introduced polyethyleneimine (PEI) into the material design and used PEI to encapsulate PB NPs to obtain PB@PEI, which not only improves the dispersibility and biocompatibility of PB NPs in aqueous media, but also can convert PB NPs from negative charge to positive charge, thereby allowing PB NPs to adsorb negatively charged phenylboronic acid compounds and EGCG through electrostatic adsorption.

[0059] In some embodiments of the present invention, when adding the materials, the mass ratio of the polyethyleneimine, phenylboric acid compounds and Prussian blue nanoparticles is (1-2):(5-10):(1-2). In the nanoparticles finally prepared, the mass ratio of the polyethyleneimine, phenylboric acid compounds and Prussian blue nanoparticles is (1-2):(1-2):(2-3), preferably 1:1:2.

[0060] Based on the above experimental exploration, the present invention uses FPBA as a phenylboronic acid compound to successfully prepare and provide a nanoparticle with ROS response and release of EGCG (denoted as: PB@PEI / FPBA), which includes a carrier and EGCG loaded on the carrier; the carrier is Prussian blue nanoparticles modified with polyethyleneimine and FPBA on the surface (denoted as: PB@PEI / FPBA-EGCG).

[0061] The present invention also provides a method for preparing the above PB@PEI / FPBA-EGCG nanoparticles, comprising the following steps:

[0062] Prussian blue nanoparticles modified with polyethyleneimine and FPBA on the surface are self-assembled with EGCG in the presence of a solvent to obtain nanoparticles.

[0063] According to the present invention, firstly, Prussian blue nanoparticles, namely, PB NPs, are provided. The present invention has no particular limitation on the preparation of PB NPs, and those skilled in the art can prepare PB NPs according to conventional means in the prior art.

[0064] Exemplarily, the preparation of PB NPs comprises the following steps:

[0065] In a 150 mL beaker, 6 g of PVP-K30 was stirred and dissolved in 70 mL of 0.01 M hydrochloric acid. Then 440 mg of K3[Fe(CN)6] was dissolved in 10 mL of 0.01 M hydrochloric acid and added to the above PVP-K30 solution. The mixture was stirred for 1 to 2 h. The mixture was placed in an 80 ° C water bath to react for 12 h. Finally, the solution was centrifuged at 11000 rpm for 20 min, washed with ultrapure water and ethanol three times, and then freeze-dried for storage to prepare PB NPs.

[0066] The above specific parameters, substances and their dosages can be adjusted according to actual conditions.

[0067] In the present invention, after the PBNPs and PEI are self-assembled, Prussian blue nanoparticles with polyethyleneimine modified on the surface (denoted as: PB@PEI) can be obtained. The self-assembly is preferably carried out under stirring conditions, and the self-assembly time is 10 to 24 hours, preferably 12 to 18 hours.

[0068] Exemplarily, the preparation of PB@PEI includes the following steps:

[0069] 40 mg of PB NPs was dissolved in 20 mL of ultrapure water and dispersed by ultrasound. 80 mg of PEI was dissolved in 20 mL of ultrapure water. Na(OH) was added to adjust the pH to about 5. Under ultrasound conditions, the PB NPs aqueous dispersion was slowly added dropwise to the PEI aqueous solution. The mixture was stirred for 12 h using a magnetic stirrer and washed with ethanol and water for at least 3 times to prepare a PB@PEI precipitate.

[0070] The above specific parameters, substances and their dosages can be adjusted according to actual conditions.

[0071] After obtaining PB@PEI, according to the present invention, PB@PEI is mixed with an ethanol solution of FPBA and reacted to obtain PB@PEI / FPBA. The reaction is carried out under stirring conditions, and the reaction time is 15 to 30 hours, preferably 18 to 24 hours.

[0072] Exemplarily, the preparation of PB@PEI / FPBA includes the following steps:

[0073] The PB@PEI precipitate was dispersed in 20 mL of ultrapure water, 200 mg of FPBA was dissolved in 20 mL of ethanol, and the PB@PEI dispersion was preferably slowly added dropwise to the FPBA ethanol solution under stirring. After fully stirring for 24 h, it was washed three times with ethanol and water to prepare a PB@PEI / FPBA precipitate.

[0074] The above specific parameters, substances and their dosages can be adjusted according to actual conditions.

[0075] Finally, according to the present invention, PB@PEI / FPBA and EGCG are self-assembled in the presence of a solvent to obtain nanoparticles. The solvent is a PBS buffer solution; the pH value of the PBS buffer solution is 7.4. The self-assembly is carried out under stirring conditions, and the self-assembly time is 2 to 3 hours, preferably 2.5 hours. The mass ratio of PB@PEI / FPBA to EGCG is (1 to 3): (5 to 10), preferably 3:10.

[0076] Exemplarily, the preparation of PB@PEI / FPBA-EGCG includes the following steps:

[0077] The precipitate from the previous step was dispersed again in 20 mL 1×PBS (pH=7.4), 200 mg EGCG was dissolved in 20 mL 1×PBS (pH=7.4), and the PB@PEI / FPBA dispersion was slowly added dropwise to the stirred EGCG aqueous solution. After stirring for 2 hours, the mixture was washed three times, and the precipitate was freeze-dried to prepare PB@PEI / FPBA-EGCG.

[0078] The above specific parameters and dosage of substances can be adjusted according to actual conditions.

[0079] After characterization, the PB@PEI / FPBA-EGCG prepared by the present invention has the following characteristics:

[0080] (2) The size of PB@PEI / FPBA-EGCG is about 140 nm, with good dispersion. The cubic structure of PB NPs is maintained in morphology, and the structure is stable and can be used for intravenous injection.

[0081] (2) PB@PEI / FPBA-EGCG has an EGCG loading rate of 34%, which can scavenge ROS by simulating the activities of multiple enzymes such as CAT and SOD, and release EGCG in the presence of ROS.

[0082] It has been verified that the PB@PEI / FPBA-EGCG prepared by the present invention has the following technical effects:

[0083] (1) PB@PEI / FPBA-EGCG achieves efficient enrichment in the myocardial ischemia-reperfusion injury site through the “permeation and retention” (EPR) effect of nanoparticles;

[0084] (2) PB@PEI / FPBA-EGCG showed good effects in improving myocardial ischemia-reperfusion injury both at the cell level and in vivo in rats, and the synergistic treatment of EGCG and PB NPs achieved the effect of "1+1>2";

[0085] (3) The PB@PEI / FPBA-EGCG prepared by the present invention has good biosafety. The hemolysis test results show that the hemolysis rate of the material with a concentration of up to 6 mg / mL is still less than 5%, and the hemolysis test is negative. Below a concentration of 50 ug / mL, the inhibition rate of H9c2 cell viability is less than 20%.

[0086] Based on the above technical effects, the present invention also provides an application of the above nanoparticles in the preparation of a drug for treating myocardial ischemia-reperfusion injury.

[0087] In summary, the present invention synthesized Prussian blue nanoparticles (PB NPs) by a hydrothermal method, and further successfully prepared PB@PEI / FPBA-EGCG nanoparticles with a cubic structure through surface modification, electrostatic adsorption, etc. The preparation method is simple and easy to operate, the raw materials are easy to obtain, the cost is low, and it is safe and efficient.

[0088] In order to further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention are all common commercially available products.

[0089] Example 1

[0090] This embodiment provides a method for preparing nanoparticles having the function of releasing EGCG in response to ROS, and the steps are as follows:

[0091] S1. Preparation of Prussian blue nanoparticles (PB NPs)

[0092] In a 150 mL beaker, 6 g of PVP-K30 was stirred and dissolved in 70 mL of 0.01 M hydrochloric acid. Then 440 mg of K3[Fe(CN)6] was dissolved in 10 mL of 0.01 M hydrochloric acid and added to the above PVP-K30 solution. The mixture was stirred for 2 h. The mixture was placed in an 80 °C water bath for 12 h. Finally, the solution was centrifuged at 11,000 rpm for 20 min, washed with ultrapure water and ethanol three times, and then freeze-dried to prepare PBNPs, which were stored at 4 °C away from light.

[0093] The morphology and size of the prepared PB NPs aqueous dispersion were analyzed by scanning electron microscopy (SEM) and dynamic light scattering (DLS). Figure 1 ,Depend on Figure 1 A shows that the prepared PB NPs have a monodisperse cubic structure with a particle size of about 100 nm. Figure 1 The DLS test results of B also showed this result.

[0094] The crystal structure of PB NPs was analyzed by X-ray powder diffractometer (XRD). Figure 1 C, it can be seen that the prepared PB NPs are consistent with the standard card (PDF#97-002-3102), and the structure can be indexed to the face-centered cubic lattice of Fe4[Fe(CN)6]3 nanocubes, indicating that the PB NPs nanomaterials were successfully prepared.

[0095] S2. Preparation of PB@PEI / FPBA-EGCG nanoparticles (abbreviated as: PPFE NPs)

[0096] 40 mg of PB NPs powder was dissolved in 20 mL of ultrapure water and dispersed by ultrasound, 80 mg of PEI was dissolved in 20 mL of ultrapure water, and Na(OH) was added to adjust the pH to 5. The PB NPs aqueous dispersion was added to the PEI aqueous solution at a rate of 2 drops / min under ultrasound (power of 300 W), and after magnetic stirring (stirring rate of 600 r / min) for 12 h, it was centrifuged at 11000 rpm for 20 min to obtain a blue solid, which was washed with deionized water several times, freeze-dried, and weighed to 52.5 mg.

[0097] Take the above 52.5 mg solid sample and add it to 20 mL ultrapure water for dispersion, and add it to the ethanol solution of FPBA (10 mg / mL), stir it magnetically (stirring rate is 600 r / min) for 24 hours, and centrifuge it at 11000 rpm for 20 minutes to obtain a blue solid, wash it with ethanol and deionized water several times, freeze-dry it, and weigh 60.5 mg. Then add it to 20 mL PBS (pH = 7.4) and mix and disperse it. Take 200 mg of EGCG and dissolve it in 20 mL 1×PBS (pH = 7.4), stir it at 500 rpm for 2 hours, centrifuge it at 11000 rpm for 20 minutes, and obtain a blue solid. Wash it with deionized water several times, freeze-dry it to obtain a blue powder, weighing 76.2 mg.

[0098] The prepared PPFE NPs were analyzed by SEM and DLS, and the results are shown in Figure 2 ,Depend on Figure 2 A shows that the prepared PPFE NPs are monodispersed with rough surface and cubic structure, with a diameter of about 140nm. Figure 2 As can be seen from Part B, the DLS test results are basically consistent with the SEM test results.

[0099] The crystal structure of PPFE NPs was analyzed by XRD. Figure 2 C, the structure of PPFE NPs can still be indexed to the standard card (PDF#97-002-3102), but its peak intensity is lower than that of PB NPs, indicating that PPFE NPs still maintain the Fe4[Fe(CN)6]3 nanocube crystal structure, which is consistent with the morphological structure shown by SEM.

[0100] The surface structure of nanomaterials at different synthesis stages was analyzed by Fourier transform infrared spectroscopy. The results are shown in Figure 3 ,Depend on Figure 3 It can be seen that: 2089cm -1 The strong stretching vibration at 1068 cm shows the characteristic peak of the carbon-nitrogen triple bond (C≡N) of PB NPs. -1 The peak corresponds to the -–C-–N–-stretching of PEI, 1512 cm -1 The peaks at 3640 cm-1 and 3660 cm-2 correspond to the in-plane bending vibration of -NH of PEI. -1 、1211cm -1 There is a stretching vibration of the phenolic hydroxyl group nearby at 1630 cm -1 The characteristic peak at 1355cm is due to the stretching vibration of the -C=O group. -1 The characteristic peak at 1456cm is caused by the asymmetric vibration of the BO bond of the phenylboronic acid group.-1 The characteristic peak corresponding to -CH3 in 4-methylphenylboronic acid. The above results show that PEI, FBPA and EGCG were successfully introduced after synthesis.

[0101] The changes in the potential and ultraviolet absorption curves of nanomaterials at different synthesis stages are analyzed. Figure 4 A. According to relevant data, PB NPs, FPBA, and EGCG are all negatively charged, and PEI is positively charged. It can be seen that the Zeta potential of PB NPs is -11mV, the Zeta potential after PEI encapsulation is 20.9mV, the Zeta potential after FPBA mixing is 0.722mV, and the Zeta potential after EGCG is added is -44mV. The change in potential proves that the material synthesis is successful. Figure 4 B shows that the UV absorption peak of EGCG is around 273nm. Figure 4 C shows that the UV absorption peak of PB NPs is around 707 nm. By calculating the UV-vis calibration curve of EGCG and PB NPs (see Figure 4 D and Figure 4 E), the loading capacity of PB@PEI / PBA-EGCG for EGCG was measured to be 34%. Figure 4 Part F shows that every time a component is added, the ultraviolet absorption curve of the material changes accordingly, proving that the material is successfully synthesized.

[0102] Example 2

[0103] In this example, the effect of the prepared PB@PEI / FPBA-EGCG nanoparticles (PPFE NPs) was verified.

[0104] Effect Example 1 - CAT-like enzyme activity: The oxygen production of PPFE NPs was measured using a portable dissolved oxygen meter (LEICI JPB-607A). PPFE NPs (final concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL) and H2O2 solution (final concentration of 1 M) were added to 3.0 mL PBS solution. The solubility of O2 produced after different reaction times was measured (unit: mg / L). The test results are shown in Figure 1. Figure 5 As shown in the figure, with the increase of material concentration, the solubility of O2 gradually increases, and the number of bubbles in the test tube gradually increases. The higher the material concentration, the faster the oxygen production rate. The results show that PPFE NPs have excellent CAT-like enzyme activity, and the higher the concentration, the higher the enzyme activity.

[0105] Effect Example 2 - SOD-like enzyme activity: The WST-1 method was used to detect the SOD-like enzyme ability of PPFE NPs. Different concentrations of materials were prepared and tested according to the instructions of the superoxide dismutase (T-SOD) activity assay kit (WST-1 method, Elabscience). WST-1 can react with superoxide anions (O 2- ) reaction produces water-soluble formazan dye. Since SOD can catalyze the disproportionation reaction of superoxide anions, this reaction step can be inhibited by SOD. Figure 6 As shown in the figure, PPFE NPs can inhibit the generation of formazan. The higher the concentration of the material, the higher the inhibition rate of formazan, that is, the higher the activity of the SOD-like enzyme. The results show that PPFE NPs have excellent SOD-like enzyme activity. The higher the concentration, the higher the enzyme activity.

[0106] Effect Example 3 - Evaluation of the ability of PB@PEI@PBA-EGCG to eliminate ·OH by electron spin resonance (ESR, Bruker A300, Germany) spectroscopy. ·OH is produced by mixing ferrous sulfate and hydrogen peroxide and captured by the spin trapping agent 5,5-dimethyl-1-pyrrolidine n-oxide (DMPO). Prepare a 5mg / mL FeSO4 solution, take 100μL of the solution, add 10μL DMPO (pure DMPO) and 80μL deionized water, then add 10μL 30% hydrogen peroxide, mix and react for 5min, and take samples for testing as the control group data. For the PPFE NPs test group, replace 80μL deionized water with 80μL PPFE NPs solution of different concentrations (6.25, 12.5, 25μg / mL). The ESR spectrum was recorded at an X-ray microwave power of 19.71 mW, a modulation frequency of 100 kHz, a scanning range of 3460-3560 G, and a modulation amplitude of 1 G. The test results are as follows Figure 7 As shown in the figure, the peak value of ·OH signal of PPFE NPs is lower than that of the control group, and the higher the concentration, the lower the ·OH signal peak. The results show that PPFE NPs can remove ·OH, and the higher the material concentration, the better the effect of removing ·OH.

[0107] Effect Example 4-The drug release rate of PPFENPs was evaluated by measuring the content of EGCG released by PPFE NPs in H2O2 solution at different time points. PPFE NPs were dispersed in 2mL PBS solution (pH=7.4) containing different H2O2 concentrations (50μM, 100μM, 150μM, 200μM), and the H2O2 concentration covered the common pathological concentrations. It was placed in a shaker at 37°C, and the PPFE NPs precipitate was collected by centrifugation at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, and 72h. 2mL of supernatant was taken for detection, and 2mL of fresh PBS solution containing the corresponding H2O2 was added. The EGCG content in the supernatant at different time points was detected using a UV spectrophotometer. The test results are as follows Figure 8 As shown, as the concentration of H2O2 gradually increased, the release amount of EGCG gradually increased, and the release rate was relatively slow, and it was still not completely released at 96 hours. The results showed that the material can slowly release EGCG, and the release amount increased with the increase of H2O2 concentration.

[0108] Effect Example 5 - Investigating the effect of PPFE NPs prepared in Example 1 on improving myocardial ischemia-reperfusion injury at the cellular level, the method is as follows: First, the biosafety of PPFE NPs on rat H9c2 cells was evaluated by CCK8 method. Fig. 9 As shown in A, the higher the concentration of PPFENPs, the greater the effect on the viability of cardiomyocytes. Below 50 μg / mL, the inhibition of cell viability is less than 80%. Therefore, 50 μg / mL and below were used as safe doses for subsequent experiments.

[0109] The H9c2 cells cultured for 1 day were replaced with sugar-free and serum-free DMEM medium, and treated with hypoxia and reoxygenation (H / R) (oxygen concentration < 0.1%) in a three-gas incubator. The cells were hypoxic for 10 h and reoxygenated for 6 h. The CCK8 method was used to detect the cell viability of the Control group, H / R+PBS group, H / R+EGCG group, H / R+PB NPs group, and H / R+PPFE NPs group. The results are shown in Fig. 9 As shown in B, compared with the control group, the cell viability in the H / R+PBS group decreased significantly, indicating that the hypoxia-reoxygenation model was successfully established. When PPFE NPs were used for treatment, the improvement effect on the viability of H9c2 cells injured by H / R was significantly better than that of single therapy. The survival rate of cells after treatment with PPFE NPs reached 77%, which showed the ability to effectively improve myocardial cell hypoxia-reoxygenation injury compared with EGCG and PB NPs monotherapy.

[0110] Effect Example 6 - Investigate the effect of PPFE NPs on the level of ROS in myocardial cells after hypoxia and reoxygenation. The method is as follows: rat H9c2 cells were treated with hypoxia and reoxygenation. The method is the same as that of Effect Example 5, and the fluorescence intensity of ROS in different groups is detected using a ROS kit. The results are as follows Fig.10 A (bar = 50 μm) is the fluorescence image of each group taken using a laser scanning confocal microscope. Fig.10 B is the fluorescence intensity analysis of each group image using image J. The results show that compared with the control group, the ROS fluorescence intensity in the H / R+PBS group was significantly enhanced, indicating that a large amount of ROS was produced in the cells through hypoxia-reoxygenation treatment. After treatment with PPFENPs, the ROS fluorescence intensity decreased significantly, and compared with EGCG and PB NPs monotherapy, its fluorescence intensity was the lowest. The results show that PPFE NPs can effectively remove ROS produced in myocardial cells treated with hypoxia-reoxygenation.

[0111] Effect Example 7-Experimental verification of the effect of PPFE NPs in improving myocardial ischemia-reperfusion injury in vivo, the method is as follows: A myocardial ischemia-reperfusion injury (I / RI) model was constructed using SD male 8-week-old rats. The rats were anesthetized and fixed in a supine position on the operating table, and the four limbs were connected to the electrocardiogram leads. Oral tracheal intubation was given and the animal ventilator was connected. The left chest cavity of the rat was opened to expose the whole picture of the heart. The epicardium was carefully torn open with tweezers, and the needle was inserted 4 to 5 mm below the lower edge of the left atrial appendage. The needle insertion depth was about 1.0 to 1.5 mm, and the needle was removed obliquely in the direction of the cone of the upper right pulmonary artery. The distance between the needle insertion and the needle removal was about 3 to 4 mm. The blood vessel was tied with a slipknot with 6-0 silk thread. It can be seen that the electrocardiogram indicated ST segment elevation, and the myocardium at the lower edge of the ligation was white, which proved that the ligation of the blood vessel was successful. After ligation for 30 minutes, the slipknot was opened, and the electrocardiogram ST segment fell back and Q waves were formed, which proved that reperfusion was successful. PBS or nanomedicine was injected through the tail vein 10 minutes before opening the blood vessel.

[0112] Rats were randomly divided into 5 groups. The sham group (sham operation group) only had the needle inserted without ligation. The I / R+PBS group, I / R+EGCG group (6 mg / kg), I / R+PB NPs group (6 mg / kg), and I / R+PPFE NPs (20 mg / kg) group were injected with PBS or corresponding drugs through the tail vein 10 minutes before opening the blood vessels. After 24 hours of reperfusion, the hearts of the rats were taken for TTC staining to check the myocardial infarction area. The results are shown in Fig.11 A (Bar=2 mm) shows the macroscopic images of TTC staining of hearts in different groups and marks the infarct area with a yellow dotted line. Fig.11 B. Statistical analysis of infarct size using image J. Fig.11 A. Fig.11As shown in the results of B, compared with the Sham control group, obvious myocardial infarction was observed in the I / R+PBS group, proving the success of the model. The myocardial infarction area in the PPFE NPs group was the smallest compared with the I / R+PBS group, I / R+EGCG group, and I / R+PB NPs group, indicating that PPFE NPs can significantly reduce the myocardial infarction area in rats with myocardial ischemia-reperfusion model.

[0113] Effect Example 8 - Evaluated PPFE NPs were enriched and retained in the myocardial ischemic area. Methods: The experimental group carried out myocardial ischemia-reperfusion modeling in rats, and the control group was injected with needles at the coronary artery ligation site without ligation (same as the sham group rats). The same dose of ICG-labeled PPFE NPs was injected into the tail vein of the rats, and the heart, liver, spleen, lung, kidney and other organs of the rats were collected at 2h, 6h, and 24h, and the fluorescence intensity of each organ was collected using a small animal in vivo imaging device. The results are as follows Fig.12 A. Fig.12 As shown in C, 2 hours after intravenous injection, the nanoparticles were mainly enriched and metabolized in the liver. At 6 hours and 24 hours, the fluorescence intensity in the liver gradually weakened, indicating that the nanoparticles were gradually metabolized by the liver over time, and the content of nanomaterials in organs such as the spleen, lungs, and kidneys was relatively low. Fig.12 B. Fig.12 D shows that the fluorescence intensity of the nanoparticles in the heart is strongest at 2 hours. Comparing the fluorescence intensity of the heart blood vessel ligation site at different time periods, the I / R group is stronger than the Sham group, and the fluorescence intensity of the Sham group is evenly distributed throughout the heart. The fluorescence intensity decreases rapidly after 2 hours, while the fluorescence of the I / R group is mainly concentrated at the blood vessel ligation site, and the fluorescence intensity decreases slower than that of the Sham group. Fig.12 Part E shows the fluorescence imaging of the heart slices of the I / R group and the Sham group 6 hours later. It can be seen that the fluorescence signal of the I / R group is mainly concentrated in the myocardial ischemia-reperfusion area, while there is no obvious signal aggregation in the Sham group. The results show that after the tail vein injection of PPFE NPs, it is mainly metabolized by the liver. Compared with the Sham group, the material can be enriched and retained in the myocardial ischemia-reperfusion injury area, which improves the utilization rate of the drug and achieves the purpose of improving the efficacy.

[0114] Effect Example 9-Evaluation of the biosafety of PPFE NPs. First, fresh SD rat blood was collected and then centrifuged at 1200rpm for 10min. The deposited red blood cells (RBC) were washed with saline and dispersed in 0.9% saline to form a 2% RBC suspension. Next, the RBC suspension was treated with gradient concentrations of PPFE NPs at 37°C for 1h, and the positive control group was treated with a 1% Triton aqueous solution. Then, the sample was centrifuged at 1500rpm for 10min, and the absorbance of the supernatant at 600nm was detected using an enzyme reader.

[0115] Rats with myocardial ischemia-reperfusion model were injected with 2 mL PPEF NPs (20 mg / kg) and PBS through the tail vein, respectively, and fed for 14 days. The body weight of the rats was recorded, the cardiac function was tested, and the blood was collected from the abdominal aorta before being killed. The blood routine of the rats was tested, and the aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA) and blood urea nitrogen (BUN) in the serum of the rats were tested by an automatic biochemical analyzer. The liver, spleen, lung, kidney and other major organs were collected and fixed and stained with H&E.

[0116] Fig.13 A shows that the hemolysis rate of PPEF NPs materials with a concentration of 6 mg / mL and below is less than 5%. Fig.13 B shows that the weight gain trend of rats in the PPFENPs group was better than that of rats in the PBS group. Fig.13 C shows that the liver and kidney functions of rats in the PPFE NPs group and PBS group were within the normal range at 14 days. Fig.13 D (HE staining 10×20) showed that the liver, spleen, lung and kidney of rats in the material group and PBS group were normal at 1 day and 14 days, with no significant difference. The results showed that PPFE NPs had good biosafety.

[0117] In summary, the present invention provides a kind of nanoparticles that release EGCG in response to ROS, and its preparation method is simple and easy to operate, and has good dispersibility. After research, it was found that it has multi-enzyme activities such as CAT and SOD, can remove OH, has a high EGCG loading rate, and releases EGCG responsively in the presence of ROS. At the same time, PPFE NPs showed good effects in improving myocardial ischemia-reperfusion injury at the cellular level and in vivo experiments in rats. By comparing the monotherapy of EGCG and PB NPs, the synergistic treatment in the composite material achieved the effect of "1+1>2" and had good biosafety.

[0118] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nanoparticle capable of releasing EGCG in response to ROS, characterized in that: It comprises a carrier and EGCG loaded on the carrier; The carrier is Prussian blue nanoparticles with polyethyleneimine and phenylboronic acid compounds modified on the surface.

2. The nanoparticle according to claim 1, characterized in that The phenylboronic acid compound is selected from any one or more of 3-aminophenylboronic acid, 3-acrylamidophenylboronic acid or 4-formylphenylboronic acid.

3. The nanoparticle according to claim 1 or 2, characterized in that The loading rate of EGCG is above 34%.

4. The nanoparticle according to any one of claims 1 to 3, characterized in that The Prussian blue nanoparticles and the nanoparticles both have a cubic structure; The particle size of the Prussian blue nanoparticles is 100 to 130 nm; The particle size of the nanoparticles is 120-150 nm.

5. A method for preparing nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: Prussian blue nanoparticles whose surfaces are modified with polyethyleneimine and phenylboronic acid compounds are self-assembled with EGCG in the presence of a solvent to obtain nanoparticles.

6. The preparation method according to claim 5, characterized in that: The solvent is a PBS buffer solution; the pH value of the PBS buffer solution is 7 to 7.4; The self-assembly is carried out under stirring conditions, and the self-assembly time is 2 to 3 hours.

7. The preparation method according to claim 5 or 6, characterized in that: The mass ratio of the Prussian blue nanoparticles whose surfaces are modified with polyethyleneimine and phenylboronic acid compounds to EGCG is (1-3):(5-10).

8. The preparation method according to any one of claims 5 to 7, characterized in that The Prussian blue nanoparticles with polyethyleneimine and phenylboronic acid compounds modified on the surface are prepared according to the following method: The Prussian blue nanoparticles are self-assembled with polyethyleneimine to obtain the Prussian blue nanoparticles with polyethyleneimine modified on the surface, and then the Prussian blue nanoparticles are mixed with an alcohol solution of a phenylboronic acid compound to react.

9. The preparation method according to claim 8, characterized in that: The self-assembly time of the Prussian blue nanoparticles and polyethyleneimine is 10 to 15 hours; The reaction is carried out under stirring conditions, and the reaction time is 15 to 30 hours; The mass ratio of the polyethyleneimine, phenylboronic acid compounds and Prussian blue nanoparticles is (1-2):(5-10):(1-2).

10. Use of the nanoparticles according to any one of claims 1 to 4 or the nanoparticles prepared according to the preparation method according to any one of claims 5 to 9 in preparing a drug for treating myocardial ischemia-reperfusion injury.

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