Icariin-loaded PH response type modified polydopamine nano preparation and application thereof in myocardial ischemia resistance

Abstract

The invention discloses a PH response type modified polydopamine nano preparation loaded with icariin and application of the PH response type modified polydopamine nano preparation in myocardial ischemia resistance, and belongs to the technical field of biological medicine. The nano preparation takes polydopamine (PDA) as a carrier core, the carrier core is modified by methoxy polyethylene glycol (mPEG) and then loaded with icariin (ICA), and the nano preparation is named as mPEG-PDA (at) ICANPs; the preparation process comprises the steps of synthesis of mPEG-OTs, synthesis of mPEG-EDA, synthesis of PDA, preparation of mPEG-PDANPs and preparation of mPEG-PDA (at) ICANPs, under the optimal condition, the particle size of the preparation is 179.6 + / -0.432 nm, PDI is 0.130 + / -0.004, the Zeta potential is 23.4 + / -0.436 mV, the stability is good after the preparation is stored in a dark place at 4 DEG C for 30 days, the ICA drug loading capacity is 14.6 + / -1.08%, the encapsulation efficiency is 64.37 + / -2.31%, and the drug cumulative release rate reaches 73.31 + / -1.64% (pH response drug release) in an environment with the pH value of 6.5 for 72 hours. An in-vitro H9c2 cell OGD model experiment proves that the preparation can remarkably improve the survival rate of ischemic myocardial cells, inhibit apoptosis and reduce LDH release, and the effect of the preparation is superior to that of free ICA. The invention provides an efficient targeting drug delivery system for resisting myocardial ischemia, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pH-responsive modified polydopamine nanoparticle loaded with icariin and its application in anti-myocardial ischemia. Background Technology

[0002] Myocardial ischemia is caused by insufficient oxygen supply to the myocardium due to reduced coronary blood flow. Traditional drug treatment faces the key problem of low delivery efficiency, making it difficult for drugs to target and accumulate in the ischemic area and easily being metabolized and cleared.

[0003] Ischemic myocardium has a unique microenvironment: first, cellular hypoxia leads to anaerobic metabolism, and lactic acid accumulation forms an acidic region with a pH of 6.5–6.8; second, reduced blood flow increases vascular endothelial permeability, producing an enhanced permeation and retention effect (EPR effect) similar to that of tumors, providing conditions for targeted enrichment by nanodelivery systems.

[0004] Icariin (ICA) is a natural flavonoid compound that exerts cardioprotective effects through anti-inflammatory, antioxidant, anti-apoptotic, and angiogenesis-promoting mechanisms. However, it suffers from poor water solubility, significant first-pass effect, short in vivo half-life, and low bioavailability (<12%), limiting its clinical translation. Existing ICA nanoformulations (such as nanoemulsions and liposomes) lack pH-responsive drug release characteristics and have not resolved the issue of carrier stability, resulting in insufficient targeting and drug utilization.

[0005] Polydopamine (PDA) has good biocompatibility and biodegradability, and can depolymerize in acidic environments (possessing pH-responsive potential). It can also help inhibit inflammation and oxidative stress. However, unmodified PDA is prone to aggregation and sedimentation in aqueous solutions, and needs to be modified with polyethylene glycol monomethyl ether (mPEG) to improve its stability.

[0006] Current technologies have not yet constructed nano-formulations that "use mPEG-modified PDA as a carrier, load ICA, and adapt to the ischemic myocardial microenvironment (pH response + EPR effect)," and cannot simultaneously solve the problems of low ICA delivery efficiency, poor carrier stability, and insufficient targeted drug release. Therefore, developing such formulations is of great significance for improving the therapeutic effect of anti-myocardial ischemia. Summary of the Invention

[0007] The purpose of this invention is to provide a pH-responsive modified polydopamine nanoparticle loaded with icariin and its application in anti-myocardial ischemia, so as to solve the defects of the prior art, such as low bioavailability of icariin (ICA), lack of targeted responsive delivery system adapted to the microenvironment of ischemic myocardium, and insufficient stability of polydopamine (PDA) carrier.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A pH-responsive modified polydopamine nanoparticle formulation loaded with icariin, wherein the nanoparticle formulation uses polydopamine (PDA) as the carrier core, and is loaded with icariin (ICA) after modification with polyethylene glycol monomethyl ether (mPEG), and is named mPEG-PDA@ICANPs; Its preparation method includes the following steps: (1) Synthesis of mPEG-OTs: mPEG was dissolved in anhydrous dichloromethane, p-toluenesulfonyl chloride (TsCl) and triethylamine were added, and the reaction was carried out in a nitrogen atmosphere at 25°C in the dark for 24 h. After filtration, concentration, washing with ether, and vacuum drying, white solid mPEG-OTs were obtained. (2) Synthesis of mPEG-EDA: mPEG-OTs were dissolved in anhydrous N,N-dimethylformamide (DMF), and excess ethylenediamine was added. The mixture was stirred at 60°C in the dark for 24 h. The reaction solution was dialyzed through a dialysis bag with a molecular weight cutoff (MWCO) of 500 Da and then freeze-dried to obtain mPEG-EDA. (3) Synthesis of PDA: Pluronic® F-127 was dissolved in deionized water at 40°C, anhydrous ethanol and 3,3',5,5'-tetramethylbenzidine (TMB) were added, and the mixture was stirred for 30 min to form an emulsion. Dopamine hydrochloride was then added, and the pH of the system was adjusted with Tris-HCl buffer (pH>8.5). The reaction was carried out at 35°C in the dark for 24 h. The reaction product was collected by centrifugation at 20,000 rpm for 20 min, resuspended in ethanol solution, and stored at 4°C to obtain PDA. (4) Preparation of mPEG-PDANPs: The PDA obtained in step (3) was dispersed in Tris-HCl buffer, and after sonication, the mPEG-EDA obtained in step (2) was added. The mass ratio of mPEG-EDA to PDA was controlled to be 2:1. The mixture was stirred at 30°C in the dark for 24 hours. The reaction solution was dialyzed through a dialysis bag with a MWCO of 10000 Da and then freeze-dried to obtain mPEG-PDA nanopolymers. (5) Preparation of mPEG-PDA@ICANPs: The mPEG-PDA and ICA obtained in step (4) were ultrasonically dispersed in dimethyl sulfoxide (DMSO). After mixing, the mass ratio of mPEG-PDA to ICA was controlled to be 1:1. The mixture was stirred at 30°C in the dark for 48 hours. The reaction solution was dialyzed through a dialysis bag with a MWCO of 10000 Da and then freeze-dried to obtain the pH-responsive modified polydopamine nanoparticles loaded with icariin.

[0009] In a further embodiment, in step (1), the mass of mPEG is 5.0 g, the volume of anhydrous dichloromethane is 50 mL, the mass of TsCl is 714 mg, and the volume of triethylamine is 1.0 mL. In step (3), the mass of Pluronic® F-127 is 0.2g, the volume of deionized water is 20mL, the volume of anhydrous ethanol is 20mL, the mass of TMB is 0.2g, and the mass of dopamine hydrochloride is 60mg; in step (4), the mass of PDA is 10mg, and the mass of mPEG-EDA is 20mg. In step (5), the mass of mPEG-PDA is 20 mg, the mass of ICA is 2 mg, and the volume of DMSO is 5 mL.

[0010] In a further step, the pH of the Tris-HCl buffer in step (3) is >8.5, which is used to adjust the pH of the PDA synthesis system to ensure the self-polymerization of dopamine; in steps (4) and (5), the dialysis process uses deionized water as the dialysis medium and the dialysis time is not less than 24 hours. During this period, the dialysis medium is replaced regularly to remove unreacted impurities.

[0011] In a further embodiment, the nano-formulation possesses the following physicochemical properties and performance: (1) Physicochemical properties: The particle size was 179.6±0.432nm, the polydispersity index (PDI) was 0.130±0.004, and the zeta potential was -23.4±0.436mV. After being stored at 4℃ in the dark for 30 days, there were no significant changes in particle size, PDI and zeta potential (P>0.05). (2) Drug loading capacity: The drug loading capacity of icariin was (14.6±1.08)%, and the encapsulation efficiency was (64.37±2.31)%. (3) pH-responsive release characteristics: In PBS buffer at pH 6.5 (simulating the acidic microenvironment of ischemic myocardium), the cumulative release rate of icariin within 72 h was (73.31±1.64)%; in PBS buffer at pH 7.4 (simulating the normal physiological environment), the cumulative release rate of icariin within 72 h was (54.24±1.51)%.

[0012] This invention also discloses the application of the above-mentioned pH-responsive modified polydopamine nanoparticles loaded with icariin in the preparation of anti-myocardial ischemia drugs.

[0013] A further embodiment of the application targets myocardial cell damage caused by myocardial ischemia, specifically including: improving the survival rate of ischemic cardiomyocytes (such as H9c2 rat cardiomyocytes), inhibiting apoptosis of ischemic cardiomyocytes, and reducing the release rate of lactate dehydrogenase (LDH) from ischemic cardiomyocytes; wherein, in the myocardial cell oxygen-glucose deprivation (OGD) model, the cell survival rate of the nano-preparation treatment group was significantly higher than that of the free icariin treatment group (P < 0.005), the cell apoptosis rate was significantly lower than that of the free icariin treatment group (P < 0.0005), and the LDH release rate was significantly lower than that of the free icariin treatment group (P < 0.005).

[0014] The present invention has the following beneficial effects: Combining targeting and responsiveness: The passive targeted enrichment of nano-formulations is achieved by utilizing the EPR effect of ischemic myocardium, while pH-responsive drug release is achieved by leveraging the acidic depolymerization properties of PDA, thus avoiding premature drug release in non-target areas and increasing the local concentration of ICA in ischemic myocardium. Excellent carrier stability: The problem of PDA aggregation and sedimentation is solved by mPEG modification. The physicochemical properties of the nano-formulation do not change significantly after 30 days of storage at 4℃ in the dark, which provides a guarantee for the storage of the formulation and its clinical application. Improved drug utilization: Nanoformulation significantly improves the water solubility and bioavailability of ICA, and its cellular uptake efficiency is higher than that of free ICA, thus enhancing the drug's effect on ischemic cardiomyocytes; Significant therapeutic effects: In vitro experiments have confirmed that this preparation can effectively improve the survival rate of ischemic cardiomyocytes, inhibit cell apoptosis, and reduce cell membrane damage. Its comprehensive anti-myocardial ischemia effect is superior to that of free ICA, providing a new strategy and dosage form for the treatment of ischemic heart disease. Attached Figure Description

[0015] Figure 1 Synthesis route diagram of mPEG-PDA nanomaterials, showing the synthesis process of mPEG-OTs→mPEG-EDA→mPEG-PDA; Figure 2 Fourier transform infrared spectra of DA, mPEG-EDA, PDA, and mPEG-PDA, with the characteristic absorption peaks of each material marked; Figure 3 Particle size and zeta potential distribution of PDANPs, mPEG-PDANPs, and mPEG-PDA@ICANPs; Figure 4 Transmission electron microscope image of mPEG-PDA@ICANPs; Figure 5 The UV standard curve of ICA, with the horizontal axis representing ICA concentration (μg / mL) and the vertical axis representing absorbance, has the regression equation y = 0.011x - 0.004 and R² = 0.9999. Figure 6 The 30-day stability curve of mPEG-PDA@ICANPs shows the changes in particle size, PDI, and Zeta potential over time. Figure 7 In vitro release curves of mPEG-PDA@ICANPs and free ICA; Figure 8: An assessment of cellular uptake of FITC-labeled free ICA and mPEG-PDA@ICA NPs after 24 hours of treatment; Figure 9 Bar chart showing cell viability in different groups; Figure 10 Bar charts showing apoptosis rates in different groups and AO / EB staining patterns; Figure 11 : Bar chart showing the apoptosis rate compared to the normal group; Figure 12 Bar chart showing LDH release rates in different groups. Detailed Implementation

[0016] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0017] 1. Materials 1.1 Drugs and Reagents Dopamine hydrochloride, polyethylene glycol monomethyl ether (mPEG), Pluronic® F-127, icariin (ICA), Tris-HCl buffer (pH > 8.5), dimethyl sulfoxide (DMSO), PBS buffer, CCK-8 reagent, AO / EB staining reagent, LDH detection kit, 4% paraformaldehyde, DAPI, FITC, sugar-free culture medium, fetal bovine serum (FBS), penicillin-streptomycin solution.

[0018] 1.2 Cells and Culture Methods Rat cardiomyocytes (H9c2 cells) were purchased from Cybio (Shanghai) Biotechnology Co., Ltd. These cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C and 5% CO2.

[0019] 1.3 Main Instruments Thermostatic magnetic stirrer (DF-101S, Zhengzhou Great Wall Science & Industry Co., Ltd.); Vacuum freeze dryer (FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd.); UV-Vis spectrophotometer (UV-Probe-2450, Shimadzu); Fourier transform infrared spectrometer (Nicolet 360, Thermo Fisher Scientific); Dynamic light scattering instrument (Zetasizer Nano ZS90, Malvern); Transmission electron microscope (Tecnai F20, FEI Company); Carbon dioxide incubator (HF-151, Lixin Instrument Co., Ltd.); Inverted fluorescence microscope (DMi8, Leica Instruments Co., Ltd.); Microplate reader (Multiskan FC, Thermo Fisher Scientific). 2 methods 2.1 Synthesis of mPEG-OTs 5.0 g of mPEG was dissolved in 50 mL of anhydrous dichloromethane, and 714 mg of TsCl and 1.0 mL of triethylamine were added sequentially. The mixture was reacted under a nitrogen atmosphere at 25 °C in the dark for 24 h. After routine post-treatment (filtration, concentration, and washing with ether), the mixture was dried under vacuum to obtain a white solid, mPEG-OTs.

[0020] 2.2 Synthesis of mPEG-EDA 2.0 g of mPEG-OTs was dissolved in 10 mL of anhydrous DMF, and excess ethylenediamine (1.2 mL) was added. The mixture was stirred at 60 °C in the dark for 24 h. The reaction solution was dialyzed (MWCO: 500 Da) and freeze-dried to obtain the product mPEG-EDA.

[0021] 2.3 Synthesis of PDA at Different Temperatures Pluronic® F-127 (0.2 g) was dissolved in 20 mL of deionized water at 40 °C, followed by the addition of anhydrous ethanol (20 mL) and TMB (0.2 g), and the mixture was stirred for 30 minutes to form an emulsion. Dopamine hydrochloride (60 mg) was then added, and the pH was adjusted to >8.5 using Tris-HCl buffer. The reaction system was incubated at 25 °C, 30 °C, and 35 °C in the dark for 24 hours, respectively. The product was collected by centrifugation (20,000 rpm, 20 min), resuspended in ethanol, and stored at 4 °C.

[0022] 2.4 Preparation of mPEG-MPDANPs with different feed ratios PDA (10 mg) was dispersed in Tris-HCl buffer, sonicated, and then mPEG-EDA (20 mg) was added. The mixture was stirred at 30 °C in the dark for 24 hours. The reaction solution was dialyzed (MWCO: 10000 Da) and freeze-dried to obtain mPEG-PDA nanopolymer. To investigate the effect of the feed ratio, parallel experiments were conducted with mPEG-EDA to PDA mass ratios of 12:1, 8:1, and 2:1.

[0023] 2.5 Preparation of mPEG-MPDA@ICANPs with different feed ratios mPEG-MPDA (20 mg) and ICA (2 mg) were ultrasonically dispersed separately in 5 mL DMSO, and the mixture was stirred at 30 °C in the dark for 48 hours. The reaction solution was dialyzed (MWCO: 10000 Da) and freeze-dried to obtain mPEG-MPDA@ICANPs. To investigate the drug loading conditions, parallel experiments were conducted with mPEG-MPDA to ICA mass ratios of 10:1, 5:1, and 1:1.

[0024] 2.6 Structural Characterization Fourier transform infrared spectroscopy was used to characterize the chemical structures of each material (DA, mPEG-EDA, PDA, mPEG-PDANPs); dynamic light scattering was used to determine the particle size, zeta potential, and polydispersity index (PDI) of PDANPs, mPEG-PDANPs, and mPEG-PDANPs with different formulation ratios; and transmission electron microscopy was used to observe the morphology and particle size distribution of mPEG-PDA@ICANPs.

[0025] 2.7 Drug loading and encapsulation efficiency A standard curve for ICA at 270 nm was plotted using UV-Vis spectrophotometry, with a linear range of 1-100 μg / mL. The concentration of ICA in the nanoparticles was calculated based on this equation, and the drug loading (DL) and encapsulation efficiency (EE) were calculated using the following formulas: Stability of 2.8mPEG-MPDA@ICANps The prepared mPEG-MPDA@ICANPs aqueous dispersion was stored at 4℃ in the dark. Samples were taken at fixed time points on days 1, 5, 10, 15, 20, 25, and 30 to determine the particle size, PDI, and Zeta potential, and to evaluate the physical stability of the nanoparticles.

[0026] 2.9 In vitro drug release curve 10 mg mPEG-MPDA@ICANPs were placed in PBS at pH 6.4 and 7.4, respectively, and placed in MWCO3000 dialysis bags, shaken at 37°C and 100 rpm. Samples (2 mL) were taken at 1, 2, 4, 8, 12, 24, 48, and 72 h, and an equal volume of PBS was added. Drug concentration (Cn) was determined by UV-Vis spectrophotometry, and the cumulative release rate (Qn%) was calculated using the following formula: Where V is the volume of the release medium; C n V is the drug concentration measured at the nth time point; s It is the volume of each sample taken; It is the sum of drug concentrations measured from time point 1 to time point n-1; M is the total mass (mg) of the drug in the nanoparticles.

[0027] 2.10 OGD Model Establishment and Grouping Logarithmic growth phase H9c2 cells were seeded into 6-well or 96-well plates. When confluence reached 70-80%, an oxygen / glucose deprivation (OGD) model was established: the medium was replaced with glucose-free medium, and the cells were cultured in an oxygen-deprived chamber at 37°C for 5 hours, followed by replacement with normal complete medium to restore normoxic culture. The experiment was divided into 5 groups: (1) Control group: routine culture; (2) OGD group: Only OGD treatment is performed; (3) ICA group: After OGD treatment, the culture medium was replaced with one containing 40 μM free ICA; (4) mPEG-PDA@ICANPs group: After OGD treatment, the culture medium containing drug-loaded nanoparticles with an equivalent ICA concentration (40 μM) was replaced; (5) mPEG-PDANPs group: After OGD treatment, the culture medium containing an equal amount of blank nanoparticles was replaced.

[0028] 2.11 Cell uptake experiment H9c2 cells in logarithmic growth phase were seeded at 1.8 × 10⁵ cells / well in 6-well plates and cultured for 24 hours to allow them to adhere. Four experimental groups were established: Control + FreeFITC, Control + mPEG-MPDA-FITC, OGD + FreeFITC, and OGD + mPEG-MPDA-FITC. The OGD group underwent 5 hours of glucose- and hypoxic treatment, after which all groups were incubated for another 24 hours in medium containing either FreeFITC or mPEG-MPDA-FITC. After washing with PBS, fixing with 4% paraformaldehyde, and staining the nuclei with DAPI, the uptake of FITC or nanoparticles was observed and analyzed using fluorescence microscopy.

[0029] 2.12 Cell viability assay H9c2 cells in the logarithmic growth phase were fed a dose of 9 × 10⁻⁶. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours. After grouping and culturing for another 24 hours according to method "2.10", 10 μL of LCK-8 solution was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: The OD blank represents the absorbance value of the blank well containing only culture medium and MTT, and without cells.

[0030] 2.13 Apoptosis Experiment After being treated in groups according to "2.10" for 24 hours, cells were double-stained with AO / EB and the nuclear fluorescence was observed under a fluorescence microscope to distinguish live cells (green), apoptotic cells (bright green / aggregated) and necrotic cells (red).

[0031] 2.14 Detection of lactate dehydrogenase (LDH) release levels After cells were treated in groups according to "2.10" for 24 hours, the absorbance (OD value) at 490 nm was measured according to the LDH kit instructions, and the LDH release rate was calculated using the following formula: The OD blank represents the absorbance value of the blank well containing only culture medium and MTT, and without cells.

[0032] 3 Results 3.1 Infrared Spectroscopic Analysis In Fourier transform infrared spectroscopy, compared to DA, PDA shows better performance at 1630 cm⁻¹. -1 A new peak with a C=O (quinone group) structure was observed at 1630 cm⁻¹, which is consistent with the characteristics of PDA polymerization. Simultaneously, mPEG-PDA also showed a peak at 1630 cm⁻¹. -1 The peak was observed at 2886 cm⁻¹, consistent with that of PDA. Compared with mPEG-EDA and PDA, mPEG-PDA showed a peak at 2886 cm⁻¹. -1 1108cm -1 and 1068cm -1 A distinct absorption peak appears at this point, which is consistent with the stretching vibrations of the CH, COC, and CO bonds in mPEG-EDA, indicating that mPEG-EDA is linked. Figure 2 ); Where: Vertical axis: Intensity (arbitrary unit, au) x-axis: wavenumber (cm⁻¹) curve: DA: Dopamine mPDA: Polydopamine mPEG-PDA: Polyethylene glycol monomethyl ether modified polydopamine mPEG-EDA: Polyethylene glycol monomethyl ether-ethylenediamine.

[0033] 3.2 Observation of particle size, potential, and morphology of nanoparticles The physicochemical properties of three nanoparticles were evaluated using a dynamic light scattering system. Figure 3 (See Tables 1, 2, and 3). PDANPs exhibited optimal characteristics at 35°C: smallest particle size (134.5 ± 2.20 nm), highest absolute value of Zeta potential (-34.8 mV), and PDI all below 0.2. Therefore, this temperature was selected for subsequent preparation.

[0034] Figure 3 (A) Particle size distribution of PDA NPs with different feed ratios; (B) Particle size distribution of mPEG-PDA NPs with different feed ratios; (C) Particle size distribution of mPEG-PDA@ICA NPs with different feed ratios; (D) Potential distribution of PDA NPs with different feed ratios; (E) Potential distribution of mPEG-PDA NPs with different feed ratios; (F) Potential distribution of mPEG-PDA@ICA NPs with different feed ratios; Particle size distribution (A) and potential distribution (D) of polydopamine nanoparticles (PDA NPs) prepared at 25℃, 30℃, and 35℃; particle size distribution (B) and potential distribution (E) of polyethylene glycol monomethyl ether modified polydopamine nanoparticles (mPEG-PDA NPs) prepared at a feed ratio of polyethylene glycol monomethyl ether-ethylenediamine (mPEG-EDA) to polydopamine (PDA) of 2:1, 8:1, and 12:1; particle size distribution (C) and potential distribution (F) of icariin-loaded polyethylene glycol monomethyl ether modified polydopamine nanoparticles (mPEG-PDA@ICA NPs) prepared at a feed ratio of polyethylene glycol monomethyl ether modified polydopamine (mPEG-PDA) to icariin (ICA) of 1:1, 5:1, and 10:1. Further investigation into the effect of the mPEG-EDA to MPDA feed ratio revealed that as the mPEG-EDA ratio increased, the nanoparticle size gradually increased, but the PDI remained <0.25, and the Zeta potential, although slightly decreased, remained at a high level. The optimal feed ratio of 2:1 was ultimately determined to be the best condition (particle size 190.5±2.21 nm, Zeta potential -25.6±0.93 mV).

[0035] The effect of the drug loading ratio on the nanoparticle properties was then investigated. As the mPEG-PDA:ICA ratio decreased from 10:1 to 1:1, the nanoparticle size exhibited a regular change. Among them, the mPEG-PDA@ICANPs prepared at a 1:1 ratio showed the best performance: smallest particle size (179.6±0.432 nm), moderate PDI (0.130±0.004), and maintained a high negative Zeta potential (-23.4±0.436 mV). Furthermore, transmission electron microscopy (TEM) revealed... Figure 4 The results showed that the drug had a regular morphology and good dispersion, and the dried size was consistent with the DLS results. Based on the drug loading and encapsulation efficiency data, a 1:1 ratio was ultimately selected as the optimal drug loading condition for subsequent experiments.

[0036] PDA synthesis: PDA was synthesized at 25℃, 30℃ and 35℃ respectively. The physicochemical properties were detected by dynamic light scattering (Table 1): PDA synthesized at 35℃ had the smallest particle size (134.5±2.20nm), the highest absolute value of Zeta potential (-34.8±0.265mV) and PDI<0.2. Therefore, 35℃ was selected as the optimal synthesis temperature. Table 1 Physicochemical parameters of PDANPs prepared at different temperatures; Preparation of mPEG-PDANPs: mPEG-PDANPs were prepared with mPEG-EDA to PDA mass ratios of 12:1, 8:1, and 2:1, respectively, and their physicochemical properties were tested (Table 2). At a mass ratio of 2:1, the particle size was 190.5±2.21 nm, the Zeta potential was -25.6±0.929 mV, and the PDI was 0.183±0.01, showing the best stability and uniformity. Therefore, 2:1 was selected as the optimal feed ratio.

[0037] Table 2 Physicochemical parameters of mPEG-PDANPs prepared with different feed ratios Preparation of mPEG-PDA@ICANPs: Drug-loaded nanoparticles were prepared with mPEG-PDA and ICA at mass ratios of 10:1, 5:1, and 1:1, respectively. The physicochemical properties and drug loading performance were tested (Table 3). At a mass ratio of 1:1, the particle size was the smallest (179.6±0.432 nm), the PDI was moderate (0.130±0.004), and the Zeta potential was -23.4±0.436 mV. Furthermore, the drug loading (14.6±1.08%) and encapsulation efficiency (64.37±2.31%) were optimal. Therefore, 1:1 was selected as the optimal drug loading ratio. Table 3 Physicochemical parameters of mPEG-PDANPs prepared with different feed ratios Drug loading and encapsulation efficiency The standard curve of ICA at 270 nm was determined by UV-Vis spectrophotometer, and the regression equation was y = 0.011x - 0.004 (R²). 2 =0.9999). Based on the standard curve calculation, at the optimal ratio (mPEG-PDANPs:ICA=1:1), the drug loading and encapsulation efficiency of ICA in the drug-loaded nanoparticles were (14.6±1.08)% and (64.37±2.31)%, respectively. Figure 5 ); Stability of mPEG-MPDA@ICANps To evaluate the stability of mPEG-MPDA@ICANPs, they were stored in the dark at 4℃, and their particle size, zeta potential, and polydispersity index (PDI) were monitored periodically. The results showed that during the 30-day observation period, the average particle size, PDI, and PDI of the nanoparticles did not change significantly (P > 0.05). This indicates that mPEG-MPDA@ICANPs possesses good physical stability in the short term. Figure 6 ); In vitro drug release curve The in vitro release behavior of drug-loaded nanoparticles mPEG-PDA@ICANPs in PBS buffer (pH 7.4 and 6.5) was investigated using dialysis. Free ICA was rapidly released within 24 hours under both pH conditions, with cumulative release rates exceeding 80%; while mPEG-PDA@ICANPs exhibited significant sustained-release characteristics. At pH 7.4, the cumulative release rate of ICA within 72 hours was (54.24 ± 1.51)%; at pH 6.5, the release was significantly accelerated, reaching (73.31 ± 1.64)%. These results indicate that mPEG-PDA@ICANPs have a good sustained-release effect and can promote drug release in response to acidic conditions in ischemic environments. Figure 7 ); Figure 7 Comparison of cumulative release rates under pH 6.5 and pH 7.4 conditions; Cell uptake experiment To assess the uptake of nanoparticles by H9c2 cells, we performed qualitative analysis using inverted fluorescence microscopy. The results showed that, compared to the free ICA group, cells treated with mPEG-PDA@ICANPs exhibited significantly enhanced FITC green fluorescence, indicating that the nanoparticles effectively promoted cellular uptake. Furthermore, this fluorescence signal intensity reached its maximum in H9c2 cells treated with OGD, suggesting that OGD damage may further enhance the uptake efficiency of mPEG-PDA@ICANPs by increasing cell membrane permeability. Figure 8 ); Explanation of the diagram: Top channel: DAPI: 4',6-Diamidinyl-2-phenylindole (used for nuclear staining) FITC: Fluorescein isothiocyanate (fluorescent marker) Merge: (overlay image of DAPI and FITC fluorescence channels) Grouping: ICA: Free icariin group mPEG-PDA@ICA: mPEG-PDA@Epimedium nanoparticles OGD / R+ICA: Oxygen-glucose deprivation / reoxygenation + free icariin group OGD / R+mPEG-PDA@ICA: Oxygen-glucose deprivation / reoxygenation + mPEG-PDA@icariin nanoparticles Scale: 200 micrometers; Cell viability assay The protective effect of mPEG-PDA@ICANPs against OGD-induced H9c2 cardiomyocytes was systematically evaluated using the CCK-8 assay. Results showed that compared with the normal group, the cell viability in the OGD model group was significantly reduced (P<0.0001), indicating successful model establishment. The cell viability in the mPEG-PDA@ICANPs group was significantly higher than that in the OGD group (P<0.001) and the free ICA group (P<0.005). This indicates that it can effectively alleviate OGD-induced H9c2 cardiomyocyte damage and improve cell viability. Figure 9 ); Among them: Oxygen-glucose deprivation group (OGD) Oxygen-glucose deprivation + icariin group (OGD+ICA) Oxygen-glycemic deprivation + blank mPEG-PDA nanoparticle assembly (OGD+mPEG-PDA) Oxygen-glycemic deprivation + mPEG-PDA@icariin nanoparticles (OGD+mPEG-PDA@ICA); The survival rates of each group and statistical differences were marked (P < 0.0001, ###P < 0.001, &&P < 0.005). Apoptosis experiment The anti-apoptotic effect of mPEG-PDA@ICANPs on OGD-induced damage in H9c2 cardiomyocytes was evaluated using the AO / EB fluorescence double staining method. The results showed that the apoptosis rate in the OGD / R model group was significantly increased compared to the normal group (P < 0.0001). Treatment with free ICA and blank nanoparticles (mPEG-PDANPs) significantly reduced the apoptosis rate (P < 0.0005, P < 0.005), exhibiting good anti-apoptotic effects; however, the apoptosis rate in the mPEG-PDA@ICANPs group was significantly lower than that in the free ICA group (P < 0.0005), showing the best anti-apoptotic effect. These results indicate that ICA and its nanoparticle formulations have significant anti-apoptotic effects on OGD-damaged H9c2 cardiomyocytes. Figure 10 and 11 ); Figure 10 In the text: apoptosis rates and statistical differences between groups are indicated (####P < 0.0001). P < 0.0005, &&&P < 0.0005) Figure 11 China: Oxygen-glucose deprivation group (OGD) Oxygen-glucose deprivation + icariin group (OGD+ICA) Oxygen-glycemic deprivation + blank mPEG-PDA nanoparticle assembly (OGD+mPEG-PDA) Oxygen-glycemic deprivation + mPEG-PDA@icariin nanoparticles (OGD+mPEG-PDA@ICA); Lactate dehydrogenase (LDH) release level detection The effect of mPEG-PDA@ICANPs on LDH in OGD-damaged H9c2 cardiomyocytes was evaluated. Results showed that the LDH release rate in the OGD model group was significantly increased compared to the normal group (P < 0.0001), indicating that ischemic injury led to severe cell membrane damage. Compared with the OGD model group and the free ICA group, the LDH release rate in the mPEG-PDA@ICANPs group was significantly decreased (P < 0.0005, P < 0.005), indicating that mPEG-PDA@ICANPs can reduce LDH release in OGD-damaged H9c2 cardiomyocytes. Figure 12 ); Among them: control group (control) Oxygen-glucose deprivation group (OGD) Oxygen-glucose deprivation + icariin group (OGD+ICA) Oxygen-glycemic deprivation + blank mPEG-PDA nanoparticle assembly (OGD+mPEG-PDA) Oxygen-glycemic deprivation + mPEG-PDA@icariin nanoparticles (OGD+mPEG-PDA@ICA); in conclusion This invention successfully constructed a pH-responsive polydopamine nanodelivery system (mPEG-PDA@ICANPs) for loading icariin. The system exhibits uniform particle size distribution, good stability, and significant pH-responsive drug release characteristics. In vitro experiments show that mPEG-PDA@ICANPs can be effectively taken up by OGD-damaged H9c2 cardiomyocytes, significantly improving cell viability, inhibiting apoptosis, and reducing LDH release; its overall protective effect is superior to free ICA.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pH-responsive modified polydopamine nanoparticle formulation loaded with icariin, characterized in that, The nano-formulation uses polydopamine PDA as the carrier core, and is loaded with icariin ICA after being modified with polyethylene glycol monomethyl ether mPEG. Its preparation method includes the following steps: Step 1: Synthesize mPEG-OTs; Step 2: Synthesize mPEG-EDA; Step 3: Synthesize the PDA; Step 4: Preparation of mPEG-PDANPs; Step 5: Preparation of mPEG-PDA@ICANPs: The mPEG-PDA and ICA obtained in step four were ultrasonically dispersed in dimethyl sulfoxide (DMSO). After mixing, the mass ratio of mPEG-PDA to ICA was controlled at 1:

1. The mixture was stirred at 30°C in the dark for 48 hours. The reaction solution was dialyzed through a dialysis bag with a MWCO of 10000 Da and then freeze-dried to obtain a pH-responsive modified polydopamine nanoparticle loaded with icariin.

2. The pH-responsive modified polydopamine nanoparticle formulation loaded with icariin according to claim 1, characterized in that: Step 1: Synthesis of mPEG-OTs: mPEG was dissolved in anhydrous dichloromethane, p-toluenesulfonyl chloride TsCl and triethylamine were added, and the reaction was carried out in a nitrogen atmosphere at 25°C in the dark for 24 hours. After filtration, concentration, washing with diethyl ether, and vacuum drying, white solid mPEG-OTs were obtained. Step 2: Synthesis of mPEG-EDA: mPEG-OTs were dissolved in anhydrous N,N-dimethylformamide DMF, and excess ethylenediamine was added. The mixture was stirred at 60°C in the dark for 24 hours. The reaction solution was dialyzed through a dialysis bag with a molecular weight cutoff of 500 Da and then freeze-dried to obtain mPEG-EDA. Step 3: PDA Synthesis: Pluronic® F-127 was dissolved in deionized water at 40°C, anhydrous ethanol and 3,3',5,5'-tetramethylbenzidine were added, and the mixture was stirred for 30 min to form an emulsion. Dopamine hydrochloride was then added, and the pH of the system was adjusted with Tris-HCl buffer. The reaction was carried out at 35°C in the dark for 24 h. The reaction product was collected by centrifugation at 20,000 rpm for 20 min, resuspended in ethanol solution, and stored at 4°C to obtain PDA. Step 4: Preparation of mPEG-PDANPs: The PDA obtained in Step 3 was dispersed in Tris-HCl buffer, sonicated, and then the mPEG-EDA obtained in Step 2 was added. The mass ratio of mPEG-EDA to PDA was controlled at 2:

1. The mixture was stirred at 30°C in the dark for 24 hours. The reaction solution was dialyzed through a dialysis bag with a MWCO of 10000 Da and then freeze-dried to obtain mPEG-PDA nanopolymers.

3. The pH-responsive modified polydopamine nanoparticle formulation loaded with icariin according to claim 2, characterized in that, In step one, the mass of mPEG is 5.0 g, the volume of anhydrous dichloromethane is 50 mL, the mass of TsCl is 714 mg, and the volume of triethylamine is 1.0 mL.

4. The pH-responsive modified polydopamine nanoparticle formulation loaded with icariin according to claim 2, characterized in that, In step three, the mass of Pluronic® F-127 is 0.2g, the volume of deionized water is 20mL, the volume of anhydrous ethanol is 20mL, the mass of TMB is 0.2g, and the mass of dopamine hydrochloride is 60mg; in step four, the mass of PDA is 10mg, and the mass of mPEG-EDA is 20mg.

5. The pH-responsive modified polydopamine nanoparticle formulation loaded with icariin according to claim 2, characterized in that, In step five, the mass of mPEG-PDA is 20 mg, the mass of ICA is 2 mg, and the volume of DMSO is 5 mL.

6. The pH-responsive modified polydopamine nanoparticle formulation loaded with icariin according to claim 5, characterized in that, In step three, the pH of the Tris-HCl buffer is >8.5, which is used to adjust the pH of the PDA synthesis system to ensure the self-polymerization of dopamine. In steps four and five, the dialysis process uses deionized water as the dialysis medium and the dialysis time is not less than 24 hours. During this period, the dialysis medium is replaced regularly to remove unreacted impurities.

7. The pH-responsive modified polydopamine nanoparticle formulation loaded with icariin according to claim 1, characterized in that, The nano-formulation: Physicochemical properties: Particle size was 179.6±0.432 nm, polydispersity index (PDI) was 0.130±0.004, and zeta potential was -23.4±0.436 mV. After storage at 4℃ in the dark for 30 days, there were no significant changes in particle size, PDI, and zeta potential (P>0.05). Drug loading capacity: The drug loading of icariin was (14.6±1.08)%, and the encapsulation efficiency was (64.37±2.31)%. pH-responsive release characteristics: In PBS buffer at pH 6.5, the cumulative release rate of icariin within 72 h was (73.31±1.64)%; in PBS buffer at pH 7.4, the cumulative release rate of icariin within 72 h was (54.24±1.51)%.

8. The use of a pH-responsive modified polydopamine nanoparticle loaded with icariin as described in any one of claims 1-7 in the preparation of anti-myocardial ischemia drugs.

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