Nucleic acid-loaded metal polyphenol nano-drug for targeting macrophages as well as preparation method and application of nucleic acid-loaded metal polyphenol nano-drug

Abstract

The invention discloses a nucleic acid-loaded metal polyphenol nano-drug for targeting macrophages as well as a preparation method and application thereof, the nano-drug takes a metal polyphenol network formed by self-assembly of cerium ions and kaempferol through coordinate bonds as a core carrier, therapeutic nucleic acid is loaded in the nano-drug, and a hyaluronic acid targeting layer is coated on the outer layer of the nano-drug. According to the invention, Ce, Kae and DNA are mixed and self-assembled to form a Ce-Kae-DNA core, and then HA is coated through electrostatic interaction. The nano-drug can deliver nucleic acid to macrophages in a targeted mode, cholesterol excretion is remarkably promoted, meanwhile, reactive oxygen species (ROS) are removed, the inflammation microenvironment is adjusted, and the nano-drug has important application prospects in preparation of drugs for treating inflammation-related diseases such as atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanodelivery technology, specifically relating to a nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages, its preparation method, and its application in the preparation of drugs for the prevention or treatment of cardiovascular inflammatory diseases such as atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is the main pathological basis for serious cardiovascular diseases such as myocardial infarction and stroke, with extremely high mortality and disability rates. Macrophages play a central role in the occurrence and development of AS. Macrophages at the lesion site engulf large amounts of oxidized low-density lipoprotein (ox-LDL). Due to the obstruction of intracellular cholesterol efflux mechanisms, excessive lipid accumulation occurs, eventually transforming into foam cells and forming the lipid core of plaques. Simultaneously, macrophages produce large amounts of reactive oxygen species (ROS) during phagocytosis, triggering severe oxidative stress and secreting large amounts of pro-inflammatory factors (such as IL-6 and TNF-α), creating a persistent chronic inflammatory microenvironment. Therefore, promoting intracellular cholesterol efflux, clearing excess ROS, and alleviating local inflammation are key strategies for treating AS.

[0003] In recent years, gene therapy has shown great potential in the field of cardiovascular disease. For example, studies have found that inhibitors of specific microRNAs (such as miR-33) (Anti-miR-33) can significantly upregulate the expression of proteins related to cholesterol efflux, thereby promoting lipid efflux and reversing foam cell formation. However, free therapeutic nucleic acids face insurmountable physiological barriers when applied in vivo: on the one hand, naked nucleic acids are easily degraded by nucleases in the blood, have a short half-life, and exhibit extremely poor physiological stability; on the other hand, free nucleic acids lack targeting specificity and are difficult to cross the cell membrane barrier to accumulate in macrophages in the lesion area, resulting in low efficacy and potential off-target side effects.

[0004] To overcome the barriers to nucleic acid delivery, researchers have developed various nanocarriers (such as cationic liposomes and polymers). However, existing conventional nanodelivery systems still have many significant drawbacks: First, most traditional nanocarriers are "inert" carriers, possessing only a single delivery function, and are unable to actively regulate the complex "high ROS, high inflammation" microenvironment at atherosclerotic lesions.

[0005] Second, some inorganic or cationic polymer drug delivery systems have problems such as complex preparation processes, low nucleic acid loading rates, easy leakage, and potential cytotoxicity.

[0006] Third, it lacks the ability to specifically recognize macrophages in the lesion area, and the efficiency of targeted delivery needs to be improved.

[0007] Nanomaterials based on metal polyphenol networks (MPNs) have attracted much attention in recent years. However, there is still no mature and excellent technical solution for screening metal-polyphenol combinations with the best antioxidant enzyme activity and simultaneously achieving efficient, stable loading and precise targeted delivery of nucleic acids.

[0008] In summary, existing single nucleic acid drugs or conventional delivery vectors cannot meet the therapeutic needs in complex inflammatory microenvironments. Therefore, there is an urgent need in this field to develop a novel multifunctional nanomedicine system that can precisely target macrophages, efficiently and stably load protective nucleic acids, and simultaneously deliver genes while relying on the vector's own properties to scavenge ROS and reverse the inflammatory microenvironment, in order to achieve a multidimensional synergistic therapeutic effect of "gene-antioxidant-anti-inflammatory". Summary of the Invention

[0009] The present invention aims to solve the technical problems of existing technologies, such as the easy degradation of therapeutic nucleic acids in vivo and the lack of targeting, as well as the single function of traditional nanocarriers and their inability to effectively improve the complex inflammatory microenvironment of atherosclerosis.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A nucleic acid-loaded metallopolyphenol nanomedicine targeting macrophages, comprising, from the inside out: The core carrier is a metal polyphenol network formed by the self-assembly of cerium ions and kaempferol through coordination bonds; Therapeutic nucleic acids are loaded inside a core carrier to form a nucleic acid-containing core carrier; The targeting shell is a hyaluronic acid coating that covers the surface of a nucleic acid-containing core carrier, forming the final nanomedicine.

[0011] In a preferred embodiment, the present invention can be further configured such that therapeutic nucleic acids are physically embedded within the core carrier via electrostatic adsorption.

[0012] In a preferred embodiment, the present invention can be further configured such that the molecular weight of the hyaluronic acid is 30 kDa.

[0013] In a preferred embodiment, the present invention can be further configured such that the nanomedicine has hierarchically assembled particle size and Zeta potential characteristics: the average particle size of the exposed core carrier is 80 nm and the Zeta potential is +18.2 mV; the average particle size of the nucleic acid-containing core carrier is 110 nm and the Zeta potential is +4 mV; after being coated with a targeting shell, the average particle size of the nanomedicine is 140 nm and the Zeta potential reverses to -24.5 mV.

[0014] On the other hand, the present invention provides a method for preparing the aforementioned nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages, comprising the following steps: S1. Mix cerium salt solution, kaempferol solution and therapeutic nucleic acid solution, and after reaction, obtain a core carrier containing nucleic acid; S2. Mix the nucleic acid-containing core carrier obtained in step S1 with a hyaluronic acid solution, and the aforementioned nanomedicine is obtained after the reaction.

[0015] In a preferred embodiment, the present invention can be further configured such that, in step S1, the molar ratio of cerium ions, kaempferol, and therapeutic nucleic acid is 10:4:1.

[0016] In a preferred embodiment, the present invention can be further configured such that, in step S1, the reaction is carried out at room temperature, the stirring speed is 500 rpm, and the reaction time is 30 minutes.

[0017] In a preferred embodiment, the present invention can be further configured such that, in step S2, the reaction is carried out at room temperature, the stirring speed is 500 rpm, and the reaction time is 120 minutes.

[0018] In a preferred embodiment, the present invention may be further configured to include a step of centrifugal washing and / or freeze drying after step S2.

[0019] This invention also provides the application of a nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages in the preparation of drugs for the prevention or treatment of cardiovascular diseases.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. Optimal core carrier with outstanding natural antioxidant capacity: Through extensive screening, this invention has found that the Ce-Kae combination exhibits the best performance in CAT enzyme activity, SOD enzyme activity, and ABTS / ·OH free radical scavenging effect. The carrier itself possesses a strong ability to scavenge reactive oxygen species (ROS), and can actively improve the oxidative stress microenvironment.

[0021] 2. Highly efficient nucleic acid loading and excellent structural stability: This invention demonstrates, through dual verification by fluorescence quantification and gel electrophoresis, that Ce-Kae has the highest loading rate for therapeutic nucleic acids, achieving not only highly efficient loading but also excellent stability through the HA shell coating.

[0022] 3. High biocompatibility and strong cellular stress protection: Cell scratch and MTT assays confirmed that the nanomedicine has good biocompatibility; under the stimulation of H2O2 (oxidative stress) and LPS (inflammatory stress), HA@Ce-Kae-DNA showed the strongest protective ability, which could significantly increase the survival rate of damaged cells to more than 80%.

[0023] 4. Targeted delivery and rescue of mitochondrial function: Cy5 labeling and confocal microscopy confirmed that the nanomaterial can be efficiently taken up by macrophages; at the same time, it can effectively remove excess ROS in the cell (verified by both fluorescence staining and flow cytometry), and maintain the physiological function of macrophages by protecting the mitochondrial membrane potential (verified by JC-1 experiment).

[0024] 5. Effectively reverses foam cells and promotes cholesterol efflux: At the cellular level, this nanomedicine significantly reduces lipid droplet accumulation (verified by Oil Red O staining) and greatly enhances the cholesterol efflux capacity of macrophages by delivering nucleic acids to regulate the expression of related proteins (verified by WB experiment) and fundamentally inhibits the formation of atherosclerotic plaques. Attached Figure Description

[0025] Figure 1 This is a scanning electron microscope (SEM) morphology characterization image of the nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages according to the present invention.

[0026] Figure 2 This is a schematic diagram illustrating the synthesis of the nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages according to the present invention.

[0027] Figure 3 This is a comparative chart showing the antioxidant enzyme activity and free radical scavenging effect of six metal-polyphenol nanomaterials of the present invention (including CAT enzyme activity, SOD enzyme activity and ABTS free radical scavenging effect).

[0028] Figure 4 The diagram shows the hydration size distribution and zeta potential changes of the nanoparticles (Ce-Kae, Ce-Kae-DNA, HA@Ce-Kae-DNA) at each level of the present invention.

[0029] Figure 5 This is a verification diagram of the loading of therapeutic DNA onto the nanoparticles of the present invention (including the fluorescence emission spectrum at 670 nm after Cy5 labeling, and the fluorescence intensity quantification diagram of different metal assembly systems).

[0030] Figure 6 This is an electrophoresis diagram used in this invention to verify the DNA loading efficiency and retardation effect of the nanocarrier using agarose gel electrophoresis.

[0031] Figure 7 The graph shows the evaluation of the ABTS free radical scavenging effect and the hydroxyl free radical (·OH) scavenging effect of different components (Ce, Kae, Ce-Kae, HA@Ce-Kae-DNA) at different concentrations.

[0032] Figure 8This is a diagram showing the in vitro biocompatibility evaluation of the nanomaterials of this invention (including the results of MTT assay cytotoxicity evaluation and cell scratch assay).

[0033] Figure 9 These are confocal fluorescence images of the nanomaterials (Cy5 labeled) of this invention being endocytosed by macrophages at different times.

[0034] Figure 10 This is a graph showing the MTT assay results of the cell-protective ability of the nanomedicine of this invention in a damage model induced by hydrogen peroxide (H2O2) and lipopolysaccharide (LPS).

[0035] Figure 11 This is an evaluation diagram of the effect of the nanomedicine of the present invention on the scavenging effect of reactive oxygen species (ROS) in macrophages (including ROS fluorescent probe staining diagram and flow cytometry quantitative analysis diagram).

[0036] Figure 12 This image shows the evaluation of the protective effect of the nanomedicine of the present invention on the mitochondrial membrane potential of macrophages (JC-1 probe detection diagram) and the regulation of expression of inflammation / lipid metabolism-related proteins (Western Blot diagram).

[0037] Figure 13 This is an evaluation diagram of the effect of the nanomedicine of the present invention on reversing macrophage foaming and promoting cholesterol efflux (including Oil Red O staining diagram and cholesterol efflux measurement diagram by fluorescence labeling method). Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] Example 1: Preparation and physical characterization of nucleic acid-loaded metallopolyphenol nanomedicine (HA@Ce-Kae-DNA) targeting macrophages This embodiment provides a self-assembly preparation method for HA@Ce-Kae-DNA nanomedicine with a core-shell structure, the specific steps of which are as follows: (1) In situ coordination assembly of the core vector (Ce-Kae-DNA) Cerium nitrate hexahydrate aqueous solution was prepared using ultrapure water, kaempferol (Kae) solution was prepared using anhydrous ethanol, and therapeutic DNA was dissolved using ultrapure water. A 20 mM HEPES buffer was prepared and its pH was adjusted to 7.4 using 1 M NaOH solution to maintain the assembly system in a physiologically neutral environment.

[0043] At an ambient temperature of 25°C, 5 mL of pre-prepared cerium nitrate hexahydrate aqueous solution and 1 mL of kaempferol ethanol solution were added sequentially to the reaction vessel, followed by the slow addition of 0.2 mL of therapeutic DNA solution; the molar ratio of cerium ions (Ce), kaempferol molecules and DNA in the system was set to 10:4:1.

[0044] The reaction system was placed on a magnetic stirrer and the stirring speed was set to 500 rpm. Under this speed and temperature conditions, cerium ions and phenolic hydroxyl groups in kaempferol molecules underwent metal-polyphenol network coordination cross-linking. At the same time, negatively charged DNA molecules were embedded in the in-situ formed nanocrystal nuclei through electrostatic adsorption. The mixture was stirred for 30 min to form a Ce-Kae-DNA core carrier mixture.

[0045] (2) Coating and purification of hyaluronic acid (HA) shell After the above core carrier was formed, the stirring parameters were maintained at 25°C and 500 rpm. A hyaluronic acid aqueous solution with a concentration of 5 mg / mL was slowly added dropwise to the system. The hyaluronic acid coated the positively charged Ce-Kae-DNA core surface through electrostatic interaction and hydrogen bond network. After the addition was completed, the reaction was stirred for 120 min.

[0046] After the reaction was completed, the resulting suspension was transferred to a centrifuge and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and an appropriate amount of ultrapure water was added to the bottom precipitate. The precipitate was briefly sonicated to resuspend it, and then centrifuged again to discard the supernatant. This ultrapure water washing step was repeated 3 times to remove unreacted metal ions, polyphenols, and free hyaluronic acid.

[0047] The washed precipitate was pre-frozen at -80°C for 4 hours, and then transferred to a vacuum freeze dryer for 48 hours to obtain powdered HA@Ce-Kae-DNA nanomedicine. The sample was sealed and stored in a 4°C refrigerator in the dark for later use.

[0048] (3) Material characterization and analysis Morphological observation: An appropriate amount of nanoparticles was redispersed in water, drop-coated onto the surface of a silicon wafer, dried, and then sputter-coated with gold. The morphology was then observed using a scanning electron microscope (SEM). Figure 1 As shown, the synthesized HA@Ce-Kae-DNA nanoparticles exhibit a uniform, near-spherical morphology and good dispersibility.

[0049] Particle size and potential analysis: Samples were measured using a dynamic light scattering (DLS) instrument at 25°C. Figure 4 As shown, the hydrated particle size of the Ce-Kae empty vector, Ce-Kae-DNA core, and HA@Ce-Kae-DNA exhibits a gradient increasing trend, confirming the layer-by-layer encapsulation process. Zeta potential testing results show that the nanomedicine possesses hierarchical assembly particle size and Zeta potential characteristics. The average particle size of the bare core carrier is 80 nm, with a Zeta potential of +18.2 mV; the average particle size of the nucleic acid-containing core carrier is 110 nm, with a Zeta potential of +4 mV; after encapsulation with the targeting shell, the average particle size of the nanomedicine is 140 nm, with a Zeta potential of -24.5 mV. That is, the initial Ce-Kae core is positively charged, and after HA encapsulation, the surface potential reverses and stabilizes in the negative potential range, verifying the successful construction of the core-shell structure and the colloidal stability of the system.

[0050] Example 2: Antioxidant Screening and Free Radical Scavenging Evaluation of Six Metal-Polyphenol Nanomaterials To screen for carriers with the best ability to scavenge reactive oxygen species (ROS), this embodiment prepared six groups of metal-polyphenol nanoparticles and quantitatively evaluated their free radical scavenging rate.

[0051] (1) Preparation of comparative materials Cerium nitrate hexahydrate (Ce source) and tungsten chloride (WCl6, dissolved in anhydrous ethanol, as W source) were selected; kaempferol (Kae), curcumin (Cur), and quercetin (Que) were selected, all dissolved in anhydrous ethanol. Following the same assembly process as in Example 1 (25°C, 500 rpm), the metal sources and polyphenols were cross-paired to prepare six core carriers: Ce-Kae, Ce-Cur, Ce-Que, W-Kae, W-Cur, and W-Que.

[0052] (2) Determination of free radical scavenging ability Prepare nanoparticle solutions of different concentrations (12.5, 25, 50, 100, 200 μg / mL).

[0053] ABTS radical scavenging assay: An equal volume of ABTS solution and potassium persulfate solution were mixed and oxidized at room temperature in the dark for 12-16 hours to generate ABTS radical cations. The working solution was diluted with anhydrous ethanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm. 10 μL of each concentration of nanoparticle solution was added to a 96-well plate, followed by 190 μL of ABTS working solution. After mixing, the plate was incubated at room temperature in the dark for 30 min. The absorbance (Abs) at 734 nm was measured using a microplate reader. The scavenging rate was calculated as follows: Scavenging rate (%) = [1 - (Abs_sample - Abs_blank) / Abs_control] × 100%.

[0054] Hydroxyl radical (·OH) scavenging assay: A Fenton reaction system was used. o-Phenanthroline solution, PBS buffer, sample solution, and ferrous sulfate solution were added sequentially, followed by the addition of 1% H₂O₂ to initiate the reaction. After reacting at 37°C for 60 minutes, the absorbance was measured at 536 nm, and the scavenging rate was calculated.

[0055] Simulated enzyme activity assay: According to the kit instructions, the activity levels of the simulated catalase (CAT) and superoxide dismutase (SOD) in the material were determined by colorimetric method.

[0056] (3) Analysis of experimental results like Figure 3 and Figure 7As shown, quantitative results indicate that the free radical scavenging rate of the cerium (Ce) group was generally higher than that of the tungsten (W) group. Among the three polyphenol ligands, the Ce-Kae combination exhibited the best antioxidant performance due to the coordination effect between the specific molecular structure of kaempferol and cerium ions. At all concentration gradients, Ce-Kae showed significantly higher scavenging rates of ABTS and ·OH free radicals than the other five groups, and possessed the highest CAT and SOD mimicry activities. Based on these results, this invention confirms Ce-Kae as the optimal antioxidant core carrier for carrying nucleic acids.

[0057] Example 3: Verification of DNA loading capacity and electrophoretic retardation of nanocarriers (1) Evaluation of loading rate by fluorescence spectroscopy Therapeutic DNA (Cy5-DNA) was labeled with the Cy5 fluorophore. The Cy5-DNA was loaded into six different vectors according to the method described in Example 1. After reaction and purification, the suspensions were spectrally scanned using a fluorescence spectrophotometer (excitation wavelength set to 640 nm).

[0058] like Figure 5 As shown, all samples exhibited the characteristic emission peak of Cy5 at 670 nm. Quantitative data, obtained by converting fluorescence intensity using a standard curve, revealed that the Ce-Kae system had the highest nucleic acid loading efficiency among the six groups, indicating that this coordination network possesses a strong ability to encapsulate DNA molecules.

[0059] (2) Agarose gel electrophoresis retardation assay Free Cy5-DNA was set up as the control group, and DNA encapsulated with HA@Ce-Kae was set up as the experimental group. The DNA and nanocarrier were mixed at a mass ratio of 1:5 and incubated at room temperature (25℃) for 30 min.

[0060] Prepare a 1.2% (w / w) agarose gel using TAE buffer. Mix the incubated sample with the loading buffer and add the mixture to the gel wells. Perform electrophoresis in the TAE buffer system at a constant voltage of 100V for 30 minutes.

[0061] like Figure 6 Electrophoresis patterns showed that in the control group, the free DNA bands migrated significantly towards the positive electrode; while in the experimental group, the fluorescent bands were stably blocked within the sample wells, and no diffuse DNA fluorescence signal was observed behind the lanes. These results confirm that the metal-polyphenol cross-linking network can tightly encapsulate DNA molecules, effectively blocking their free migration in an electric field.

[0062] Example 4: In vitro biosafety evaluation of nanomedicines In this embodiment, the MTT assay and cell scratch assay were used to evaluate the toxicity of the material to macrophages.

[0063] (1) MTT cytotoxicity detection procedure Logarithmically growing RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well. The plates were then incubated at 37°C with 5% CO₂ for 24 hours until cell attachment.

[0064] A drug administration concentration gradient of 12.5, 25, 50, 100, and 200 μg / mL was set up, and culture medium containing the corresponding concentration of HA@Ce-Kae-DNA nanomedicine was added. Five parallel wells were set up for each group, and a blank control group was also set up. The mixture was continuously incubated in an incubator for 24 h.

[0065] After incubation, add 10 μL of 5 mg / mL MTT solution to each well and incubate in the dark for 4 h. Discard the supernatant and add 150 μL of dimethyl sulfoxide (DMSO) to each well, then shake at room temperature in the dark for 10 minutes to dissolve the crystals. Read the absorbance (OD value) at 490 nm using a microplate reader and calculate the relative cell viability.

[0066] (2) Analysis of evaluation results like Figure 8 As shown in the cell viability bar chart, after treatment for 24 hours within the concentration range of 12.5–200 μg / mL, the relative survival rate of RAW264.7 cells in each treatment group remained above 95%, with no statistically significant difference compared to the control group. The cell scratch healing assay also showed that the material did not inhibit normal cell migration. These results indicate that the HA@Ce-Kae-DNA material possesses good in vitro biocompatibility.

[0067] Example 5: Macrophage surface receptor-mediated endocytosis and dynamic uptake experiment In this embodiment, laser confocal scanning microscopy (CLSM) was used to observe the uptake process of nanomedicine by macrophages.

[0068] (1) Confocal Target Tracking Experimental Procedure RAW264.7 cells were seeded in 35 mm confocal culture dishes and cultured to adhere. HA@Ce-Kae-DNA nanomedicine encapsulated with Cy5-DNA was added, and co-incubation was performed at 1 h, 4 h, and 7 h.

[0069] After the predetermined time is reached, the culture medium is aspirated, and the cells are washed three times with ice-cold PBS. Hoechst 33342 staining solution is added and the cells are stained for live cell nuclei at 37°C in the dark for 20 min (paraformaldehyde fixation was not performed in this step to preserve receptor activity).

[0070] After washing with PBS, the cells were observed using a CLSM with the support of a live cell workstation. A 405nm laser was used to excite the blue fluorescence channel (nucleus), and a 633nm laser was used to excite the red fluorescence channel (Cy5-DNA), resulting in a combined dual-channel image.

[0071] (2) Analysis of dynamic intake results like Figure 9 As shown, after 1 hour of incubation, a weak Cy5 red fluorescence signal appeared in the cytoplasm of macrophages, indicating that the hyaluronic acid (HA) shell recognized the CD44 receptor on the macrophage surface and initiated endocytosis; by 4 hours, the intensity of intracellular red fluorescence increased significantly and accumulated in the perinuclear region; after 7 hours of incubation, the intracellular red fluorescence reached a high-density distribution. This time-dependent uptake result confirms that the carrier can effectively enter macrophages via the receptor-mediated pathway.

[0072] Example 6: Protective effect on macrophages under dual stress of oxidation and inflammation To simulate the microenvironment of atherosclerosis, an in vitro injury model induced by H2O2 and LPS was established to evaluate the protective effect of nanomedicines on cells.

[0073] (1) Procedure for establishing a stress injury and protection model RAW264.7 cells were seeded into 96-well plates and allowed to adhere. The experiment was divided into a blank control group, a pure injury model group, and a drug-protected group (concentration gradient 12.5~200μg / mL).

[0074] For the drug-treated protection group, the culture medium containing the drug was added beforehand and incubated for 1 hour. Two injury models were then established: Oxidative damage model: Hydrogen peroxide (H2O2) was added to the system at a final concentration of 500 μM, and the system was stimulated for 1 h.

[0075] Inflammatory injury model: Lipopolysaccharide (LPS) was added to the system at a final concentration of 1 μg / mL, and the system was stimulated for 1 h.

[0076] After stimulation, cell viability was determined using the MTT assay as described in Example 4.

[0077] (2) Analysis of protective effect results like Figure 10 As shown, in the pure injury model group, cell viability significantly decreased to the 40%-50% range under H2O2 or LPS stimulation. In the treatment group, cell viability recovered in a dose-dependent manner after pre-incubation with HA@Ce-Kae-DNA for 1 h. Pretreatment with a concentration of 200 μg / mL maintained cell viability above 80%, showing a statistically significant difference compared to the injury model group. These results confirm that the material effectively resists exogenous oxidation and inflammatory stimulation, providing significant protection for macrophages.

[0078] Example 7: Evaluation of Intracellular Reactive Oxygen Species (ROS) Scavenging and Mitochondrial Function (1) Assessment of intracellular ROS clearance (DCFH-DA probe method) After washing the cells from each group that had undergone H2O2 damage and drug intervention, add DCFH-DA probe working solution diluted 1:1000 with serum-free culture medium and incubate at 37°C in the dark for 20 min.

[0079] After washing to remove the extracellular probe, the cells were imaged using a fluorescence microscope with the FITC channel (green fluorescence). Simultaneously, cells were collected by trypsin digestion, and the average green fluorescence intensity of 10,000 cells per group was detected by flow cytometry for quantitative analysis.

[0080] like Figure 11 As shown, strong green fluorescence signals appeared in the cytoplasm of the damaged model group, indicating high levels of oxidative stress. In the group treated with HA@Ce-Kae-DNA, the intensity of green fluorescence decreased significantly with increasing drug dosage, and flow cytometry data confirmed a significant decrease in ROS levels.

[0081] (2) Detection of mitochondrial membrane potential (ΔΨm) (JC-1 probe method) JC-1 staining working solution was added to each group of cells and incubated at 37°C for 20 min. After washing twice with JC-1 buffer, the distribution of red / green fluorescence was observed under a fluorescence microscope.

[0082] like Figure 12 As shown in the foregoing section, in the damaged model group, the red fluorescence representing healthy mitochondria was weakened, while the green fluorescence increased, and the red / green fluorescence ratio was significantly reduced. After intervention with nanomedicine, macrophages regained their red fluorescence signal. This result indicates that the material effectively maintained the stability of the mitochondrial transmembrane potential by clearing excessively accumulated ROS.

[0083] Example 8: Reversing macrophage foaming and regulating lipid metabolism / inflammatory protein expression This embodiment establishes an in vitro foam cell model to evaluate the regulatory effect of materials on lipid accumulation and inflammatory factor expression.

[0084] (1) Evaluation of anti-lipid accumulation (Oil Red O staining and cholesterol efflux) RAW264.7 cells were treated with oxidized low-density lipoprotein (ox-LDL) at a final concentration of 100 μg / mL for 24 h to establish a foam cell model. The medium containing ox-LDL was then removed, and medium containing nanomedicine was added for intervention.

[0085] After intervention, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. After washing, Oil Red O working solution was added for staining at room temperature for 20 min. After removing the background with 60% isopropanol, lipid droplet distribution was observed under a microscope. Simultaneously, cholesterol efflux rate was determined using a fluorescently labeled cholesterol kit.

[0086] like Figure 13 As shown, a large number of orange-red lipid droplets aggregated in the cytoplasm of the model group cells; after drug treatment, the intracellular lipid droplet density decreased significantly. Quantitative analysis showed a significant increase in cholesterol efflux rate. The results indicate that this drug delivery system can promote cholesterol efflux and reverse the foaming phenotype of macrophages.

[0087] (2) Measurement of inflammation-related protein expression (Western Blot procedure) Cells from each group were collected and lysed for 30 minutes using RIPA lysis buffer containing PMSF. Total protein in the supernatant was extracted by centrifugation at 12000g, quantified, and balanced using a BCA kit.

[0088] After polyacrylamide gel electrophoresis (concentrated at 80V and then separated at 120V), proteins were transferred onto a PVDF membrane under a constant current of 250mA. The membrane was blocked with 5% skim milk for 2 hours, and primary antibodies against IL-6, TNF-α, and IL-10 (Tubulin as an internal control) were added, followed by incubation overnight at 4°C. After washing, the membrane was incubated with HRP-labeled secondary antibody for 1 hour. The bands were recorded using an imaging system after development with ECL chemiluminescence buffer.

[0089] like Figure 12 As shown in the latter part, IL-6 and TNF-α proteins were highly expressed in the ox-LDL-induced model group. After intervention with HA@Ce-Kae-DNA, the expression levels of IL-6 and TNF-α were significantly downregulated, while the expression level of the anti-inflammatory factor IL-10 was significantly upregulated. The results confirm that the drug-loaded nanosystem of this invention can effectively regulate the inflammatory signaling pathway of macrophages, promoting their transformation from a pro-inflammatory phenotype to an anti-inflammatory phenotype.

[0090] Optional implementation methods and variations: Based on the core concept of this invention, those skilled in the art can conceive of the following modified embodiments, all of which fall within the protection scope of this invention: Metal ion substitution: Although the present invention has demonstrated through screening that the combination of Ce ions and Kae is optimal, other metal ions with similar coordination ability and redox activity, such as other lanthanide elements (e.g., La, Gd, etc.), can also form metal polyphenol networks with Kae and possess certain antioxidant and drug-loading capabilities. Such substitutions should be considered equivalent replacements of the present invention.

[0091] Alternatives for polyphenol molecules: Kaempferol (Kae) is the preferred ligand in this invention, but other flavonoids with ortho-phenolic hydroxyl structures, such as quercetin (Que) and curcumin (Cur), although slightly less effective, can also be used to construct similar metal polyphenol networks and may be valuable in certain applications.

[0092] Alternatives to targeting molecules: Hyaluronic acid (HA) is a preferred targeting layer due to its ability to specifically recognize CD44 receptors on the surface of macrophages. Other negatively charged polysaccharides or polymers with macrophage targeting capabilities, such as chondroitin sulfate and alginate, may also achieve similar targeting functions by coating the core surface through electrostatic interactions.

[0093] Expansion of nucleic acid types: The therapeutic nucleic acid exemplified in this invention is Anti-miR-33, but this delivery system is also applicable to other types of therapeutic nucleic acids, including but not limited to other microRNA inhibitors, microRNA mimics, small interfering RNA (siRNA), messenger RNA (mRNA), etc., for the treatment of different diseases.

[0094] Adjustment of preparation parameters: The reaction temperature (25℃), stirring speed (500 rpm), and reaction time (30 min and 120 min) given in the examples are exemplary parameters. Those skilled in the art can adjust them within a certain range according to actual conditions. For example, the target nanomedicine may still be successfully prepared even when the temperature is between 4-37℃ and the stirring speed is between 300-800 rpm.

[0095] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A nucleic acid-loaded metallopolyphenol nanomedicine targeting macrophages, characterized in that, The nanomedicine comprises, from the inside out, the following: The core carrier is a metal polyphenol network formed by the self-assembly of cerium ions and kaempferol through coordination bonds; Therapeutic nucleic acid, wherein the therapeutic nucleic acid is loaded inside the core carrier to form a nucleic acid-containing core carrier; The target shell is a hyaluronic acid coating that covers the surface of the nucleic acid-containing core carrier, forming the final nanomedicine.

2. The nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages according to claim 1, characterized in that: The therapeutic nucleic acid is physically embedded inside the core carrier through electrostatic adsorption.

3. The nucleic acid-loaded metallopolyphenol nanomedicine targeting macrophages according to claim 2, characterized in that: The hyaluronic acid has a molecular weight of 30 kDa.

4. The nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages according to claim 3, characterized in that: The nanomedicine has hierarchical assembly particle size and Zeta potential characteristics, with the exposed core carrier having an average particle size of 80 nm and a Zeta potential of +18.2 mV. The average particle size of the nucleic acid-containing core carrier is 110 nm, and the Zeta potential is +4 mV. After being coated with the target shell, the nanomedicine has an average particle size of 140 nm and a zeta potential of -24.5 mV.

5. A method for preparing the nucleic acid-loaded metallopolyphenol nanomedicine targeting macrophages according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Mix cerium salt solution, kaempferol solution and therapeutic nucleic acid solution, and after reaction, obtain a core carrier containing nucleic acid; S2. Mix the nucleic acid-containing core carrier obtained in step S1 with a hyaluronic acid solution, and the nanomedicine is obtained after the reaction.

6. The preparation method according to claim 5, characterized in that: In step S1, the molar ratio of cerium ions, kaempferol, and therapeutic nucleic acid is 10:4:

1.

7. The preparation method according to claim 6, characterized in that: In step S1, the reaction is carried out at room temperature, with a stirring speed of 500 rpm and a reaction time of 30 minutes.

8. The preparation method according to claim 7, characterized in that: In step S2, the reaction is carried out at room temperature, with a stirring speed of 500 rpm and a reaction time of 120 minutes.

9. The preparation method according to claim 5, characterized in that: The method further includes centrifugal washing and / or freeze drying steps after step S2.

10. The use of the nucleic acid-loaded metal polyphenol nanomedicine targeting macrophages according to any one of claims 1-4 in the preparation of a drug for the prevention or treatment of cardiovascular diseases.

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