Dmpe-peg and ros response unit targeting synergistic drug delivery system, and preparation method and application thereof

Abstract

The application relates to a DMPE-PEG and ROS response unit targeted synergistic drug delivery system and a preparation method and application thereof. The DMPE-PEG and ROS response unit targeted synergistic drug delivery system comprises a nano-micelle formed by self-assembly of an amphiphilic compound and a therapeutic agent loaded in the nano-micelle; wherein the nano-micelle comprises a hydrophobic chain segment, a hydrophilic chain segment and a ROS response unit; the hydrophobic chain segment is dimyristoyl phosphatidyl ethanolamine, the hydrophilic chain segment is polyethylene glycol, and the ROS response unit is at least one responsive group selected from a phenyl borate group, a sulfide group and a selenide group; and the therapeutic agent comprises siRNA or a small molecule inhibitor. Through lesion high ROS microenvironment specificity response, precise targeting of the lesion is realized; in cooperation with the long circulation effect of the PEG chain, the action time of the drug at the lesion site can be prolonged; and DMPE further promotes the intracellular delivery efficiency of the drug due to higher membrane fluidity and fusion capacity.

Description

Technical Field

[0001] This application relates to the field of biomedical materials, specifically to a DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system, its preparation method, and its application. Background Technology

[0002] Acute myocardial infarction (AMI) is a leading cause of cardiovascular death worldwide, characterized by persistent myocardial ischemia and necrosis resulting from acute interruption of coronary blood flow. Although reperfusion therapy can restore blood flow, secondary activation of the coagulation system, metabolic disorders, and excessive inflammatory responses can still cause secondary myocardial damage, significantly increasing the risk of heart failure. Therefore, developing novel therapeutic strategies that target key pathological processes is of great significance.

[0003] Recent proteomics studies have revealed that fibrinogen γ chain (FGG), apolipoprotein E (APOE), vitrin (VTN), kininogen 1 (KNG1), α2-macroglobulin (A2M), and haptoglobin (HP) are consistently highly expressed in the serum and myocardial tissue of AMI patients. Among these, FGG exacerbates thrombus formation by promoting fibrinogenesis; dysregulated APOE expression leads to lipid metabolism disorders; VTN overexpression drives myocardial fibrosis; KNG1 dysfunction weakens vascular protection; A2M amplifies the inflammatory cascade by inhibiting protease activity; and abnormally elevated HP is closely related to impaired hemoglobin clearance and oxidative stress damage. These targets collectively constitute the core regulatory network of the vicious cycle of coagulation-metabolism-inflammation in the progression of AMI, and silencing their expression is considered an effective pathway to block disease progression.

[0004] However, traditional siRNA or small molecule inhibitors face a key bottleneck of low delivery efficiency in vivo: on the one hand, systemic administration makes it difficult to accumulate effectively in ischemic myocardium; on the other hand, the lack of a lesion microenvironment-responsive drug release mechanism leads to insufficient intracellular bioavailability. Summary of the Invention

[0005] Based on this, this application provides a targeted synergistic drug delivery system of DMPE-PEG and ROS responsive unit that can effectively deliver drugs, as well as its preparation method and application.

[0006] During acute myocardial infarction (AMI), myocardial ischemia and hypoxia lead to mitochondrial dysfunction, triggering an abnormal accumulation of reactive oxygen species (ROS). ROS concentrations in ischemic myocardial regions can reach 5-10 times that of normal tissues, providing an ideal biological basis for designing stimulus-responsive delivery systems.

[0007] The first aspect of this application provides a DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system, comprising nanomicelles formed by the self-assembly of an amphiphilic compound and a therapeutic agent loaded in the nanomicelles; wherein the nanomicelles contain a hydrophobic segment, a hydrophilic segment, and a ROS-responsive unit; the hydrophobic segment is myristoylphosphatidylethanolamine, the hydrophilic segment is polyethylene glycol, and the ROS-responsive unit is selected from at least one responsive group selected from phenylboronic acid ester group, thioether group, and selenide group; the therapeutic agent includes siRNA or a small molecule inhibitor.

[0008] In some embodiments, the ROS-responsive unit is covalently linked to both the hydrophobic and hydrophilic segments. As an example, the ROS-responsive unit may be linked to both the hydrophobic and hydrophilic segments separately; alternatively, the ROS-responsive unit may be linked only to the hydrophilic segment.

[0009] In some embodiments, the critical micelle concentration of the nanomicelles is 0.8 μg / mL to 1.5 μg / mL. Preferably, the critical micelle concentration is 1.17 μg / mL. Critical micelle concentrations within this range are beneficial for the formation of denser hydrophobic cores, which not only helps to increase the encapsulation capacity of hydrophobic drugs but also helps the micelles maintain structural stability even in highly diluted physiological environments (such as systemic circulation), effectively preventing drug leakage.

[0010] In some embodiments, the mass ratio of the therapeutic agent to the nanomicelles is 1:(1-40). Preferably, the mass ratio of the therapeutic agent to the nanomicelles is 1:(1-30), and more preferably, the mass ratio of the therapeutic agent to the nanomicelles is 1:(20-25). By controlling the mass ratio of the therapeutic agent to the nanomicelles, the drug encapsulation efficiency and drug loading can be optimized, maximizing the bioavailability of the therapeutic agent at the lesion site. If the ratio is too low, the micelles cannot effectively load the therapeutic agent, leading to easy degradation and leakage. If the ratio is too high, the excess polymer will delay the ROS-responsive drug release and increase the metabolic risk.

[0011] In some embodiments, the particle size of the nanomicelles is 10 nm to 100 nm. Further, the average particle size of the nanomicelles is 20 nm to 40 nm.

[0012] The second aspect of this application provides a method for preparing a targeted synergistic drug delivery system of DMPE-PEG and ROS responsive units, comprising the following steps:

[0013] S1. Under a protective gas atmosphere and at 0-5℃, dissolve dimyristoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DMPE-PEG-COOH) in an anhydrous solvent to obtain a first solution; add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) to the first solution, and then stir and activate at 10℃~40℃ for 10min~60min to obtain a first reaction solution.

[0014] S2. Dissolve the compound with the ROS-responsive unit in an anhydrous solvent to obtain a second solution; add the second solution dropwise to the first reaction solution at 0-5°C. After the addition is complete, heat to 25°C-45°C and react for 18-36 hours to obtain the second reaction solution; the ROS-responsive unit is selected from at least one responsive group selected from phenylboronic acid ester group, thioether group, and selenide group.

[0015] S3. The second reaction solution was purified by first dialysis using a dialysis bag with a molecular weight cutoff of 3000 Da to 4000 Da. Then, it was filtered, sterilized, and freeze-dried in sequence to obtain an amphiphilic polymer.

[0016] S4. Dissolve the amphiphilic polymer in an organic solvent to obtain a third solution; then, at 0-5°C, add a phosphate buffer solution containing the therapeutic agent dropwise to the third solution and emulsify by ultrasonication to obtain an emulsion; remove the solvent from the emulsion by rotary evaporation to obtain a lipid membrane;

[0017] S5. Under water bath conditions of 35℃~40℃, the lipid membrane is completely hydrated with phosphate buffered saline, and then subjected to ultrasonic treatment, second dialysis purification and freeze drying in sequence.

[0018] In some embodiments, in step S1, the mass ratio of dimyristoylphosphatidylethanolamine-polyethylene glycol-carboxyl, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 10:(1-1.5):(0.5-1.5).

[0019] In some embodiments, the compound having the ROS-responsive unit is selected from 3-aminophenylboronic acid pinacol ester, 2-(methylthio)ethylamine or 2-(methylseleno)ethylamine.

[0020] In some embodiments, the mass ratio of dimyristoylphosphatidylethanolamine-polyethylene glycol-carboxyl group to the compound having a ROS-responsive unit is 5:(3 to 4.5).

[0021] In some embodiments, the above preparation method further includes: before dissolving dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in an anhydrous solvent, dehydrating dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl through a molecular sieve for 20 h to 30 h.

[0022] In some embodiments, the anhydrous solvent is N,N-dimethylformamide.

[0023] In some embodiments, step S3, the first dialysis purification step includes: firstly, dialyzing the second reaction solution twice with a mixed solvent containing anhydrous solvent and anhydrous ethanol, each dialyzing time being 3.5 h to 4.5 h. Preferably, each dialyzing time is 4 h; then, dialyzing twice again with anhydrous ethanol as the solvent, each dialyzing time being 1.5 h to 2.5 h, preferably, each dialyzing time is 2 h.

[0024] In some embodiments, in step S3, filtration is performed using a filter membrane with a pore size of 0.2 μm to 0.25 μm.

[0025] In some embodiments, in step S4, the power of ultrasonic emulsification is 250W to 350W, and the time is 3min to 6min.

[0026] In some embodiments, in step S4, the organic solvent is selected from any one or more of methanol, dichloromethane, and trichloromethane.

[0027] In some embodiments, the second dialysis in step S5 employs procedures conventionally used in the art. For example, the second dialysis method may involve transferring the hydrated lipid membrane into a dialysis bag with a molecular weight cutoff of 3.5 kDa, dialyzing at 4°C in PBS buffer (pH 7.4) for 12 hours to remove free siRNA or small molecule inhibitors and ionic impurities; maintaining the external fluid volume at 100 times the sample volume throughout the dialysis process, changing the external fluid every 6 hours, and then sterilely filtering through a 0.22 μm filter membrane after the dialysis is completed.

[0028] The third aspect of this application provides the use of the drug delivery system provided in the first aspect or the drug delivery system prepared by the method provided in the second aspect in the preparation of a drug for treating myocardial infarction.

[0029] In some embodiments, the DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system uses siRNA to silence the expression of at least one gene among FGG, APOE, VTN, KNG1, A2M, and HP.

[0030] In some embodiments, siRNA silences FGG gene expression, wherein the siRNA is selected from at least one of FGGsiRNA1, FGGsiRNA2, and FGGsiRNA3; the sense strand sequence of FGGsiRNA1 is shown in SEQ ID NO.1, and the antisense strand sequence of FGGsiRNA1 is shown in SEQ ID NO.2; the sense strand sequence of FGGsiRNA2 is shown in SEQ ID NO.3, and the antisense strand sequence of FGGsiRNA2 is shown in SEQ ID NO.4; the sense strand sequence of FGGsiRNA3 is shown in SEQ ID NO.5, and the antisense strand sequence of FGGsiRNA3 is shown in SEQ ID NO.6.

[0031] In some embodiments, siRNA silences VTN gene expression, wherein the siRNA is selected from at least one of VTNsiRNA1, VTNsiRNA2, and VTNsiRNA3: the sense strand sequence of VTNsiRNA1 is shown in SEQ ID NO.7, and the antisense strand sequence of VTNsiRNA1 is shown in SEQ ID NO.8; the sense strand sequence of VTNsiRNA2 is shown in SEQ ID NO.9, and the antisense strand sequence of VTNsiRNA2 is shown in SEQ ID NO.10; the sense strand sequence of VTNsiRNA3 is shown in SEQ ID NO.11, and the antisense strand sequence of VTNsiRNA3 is shown in SEQ ID NO.12.

[0032] In some embodiments, siRNA silences APOE gene expression. The siRNA is selected from at least one of APOEsiRNA1 and APOEsiRNA2: the sense strand sequence of APOEsiRNA1 is shown in SEQ ID NO.13, and the antisense strand sequence of APOEsiRNA1 is shown in SEQ ID NO.14; the sense strand sequence of APOEsiRNA2 is shown in SEQ ID NO.15, and the antisense strand sequence of APOEsiRNA2 is shown in SEQ ID NO.16.

[0033] In some embodiments, siRNA silences KNG1 gene expression, wherein the siRNA is selected from at least one of KNG1siRNA1, KNG1siRNA2, and KNG1siRNA3; the sense strand sequence of KNG1siRNA1 is shown in SEQ ID NO.17, and the antisense strand sequence of KNG1siRNA1 is shown in SEQ ID NO.18; the sense strand sequence of KNG1siRNA2 is shown in SEQ ID NO.19, and the antisense strand sequence of APOEsiRNA2 is shown in SEQ ID NO.20; the sense strand sequence of KNG1siRNA3 is shown in SEQ ID NO.21, and the antisense strand sequence of KNG1siRNA3 is shown in SEQ ID NO.22.

[0034] In some embodiments, siRNA silences A2M gene expression. The siRNA is selected from at least one of A2M1siRNA1, A2MsiRNA2, and A2MsiRNA3: the sense strand sequence of A2MsiRNA1 is shown in SEQ ID NO.23, and the antisense strand sequence of A2MsiRNA1 is shown in SEQ ID NO.24; the sense strand sequence of A2MsiRNA2 is shown in SEQ ID NO.25, and the antisense strand sequence of A2MsiRNA2 is shown in SEQ ID NO.26; the sense strand sequence of A2MsiRNA3 is shown in SEQ ID NO.27, and the antisense strand sequence of A2MsiRNA3 is shown in SEQ ID NO.28.

[0035] In some embodiments, siRNA silences HP gene expression. The siRNA is selected from at least one of HPsiRNA1, HPsiRNA2, and HPsiRNA3: the sense strand sequence of HPsiRNA1 is shown in SEQ ID NO.29, and the antisense strand sequence of HPsiRNA1 is shown in SEQ ID NO.30; the sense strand sequence of HPsiRNA2 is shown in SEQ ID NO.31, and the antisense strand sequence of HPsiRNA2 is shown in SEQ ID NO.32; the sense strand sequence of HPsiRNA3 is shown in SEQ ID NO.33, and the antisense strand sequence of A2MsiRNA3 is shown in SEQ ID NO.34.

[0036] All siRNAs in this application have two dangling bases TT composed of deoxyribonucleotides at their 3' ends; for example, the positive strand sequence (5'-3') of FGGsiRNA1, as shown in SEQ ID NO.1, is GGUAGUUAUUGUCCAACUA; because it has two dangling bases TT composed of deoxyribonucleotides at its 3' end, its essence is: GGUAGUUAUUGUCCAACUATT.

[0037] In some embodiments, the small molecule inhibitor in the DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system is coumarin.

[0038] The working mechanism of the DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system in this application is as follows:

[0039] siRNA is a small interfering RNA molecule that can specifically recognize and silence the expression of target genes, thereby exerting a therapeutic effect. Its mechanism of action involves binding to the mRNA of the target gene, inducing mRNA degradation or inhibiting its translation. Small molecule inhibitors, on the other hand, are compounds that can bind to target proteins and inhibit their activity. In the treatment of acute myocardial infarction (AMI), siRNA or small molecule inhibitors targeting genes such as FGG, APOE, VTN, KNG1, A2M, and HP can inhibit the expression or activity of these genes, reducing coagulation, metabolic disorders, and inflammatory responses, thereby protecting cardiomyocytes and improving cardiac function. In the DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system provided in the first aspect of this application, nanomicelles are formed through the self-assembly of hydrophobic and hydrophilic segments. The hydrophobic segment (DMPE) forms the micelle core, which can effectively encapsulate the therapeutic agent siRNA or small molecule inhibitor. The hydrophilic portion (PEG) helps to prolong the in vivo circulation time. When the nanomicelles reach the myocardial infarction lesion area, the ROS-responsive unit in the nanomicelles undergoes specific cleavage in a high concentration of ROS environment, causing the micelles to be destroyed and releasing the encapsulated therapeutic agent, thereby achieving precise drug release to the lesion area.

[0040] The beneficial effects of this application are:

[0041] 1. The DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system of this application achieves precise targeting of acute myocardial infarction (AMI) lesions through a specific response to the high ROS microenvironment of the lesion. Simultaneously, the ROS-responsive units (phenylboronic acid ester bonds, thioether bonds, etc.) exhibit higher drug release rates in high ROS environments compared to normal physiological environments. Combined with the long-cycle effect of the PEG chain, this is expected to prolong the duration of drug action at the lesion site. Furthermore, the DMPE (unsaturated phospholipids) in the system, due to its higher membrane fluidity and fusion capacity, is more effective in improving intracellular drug delivery efficiency compared to commonly used DSPE (saturated phospholipids).

[0042] 2. Compared to traditional drug delivery systems, this approach innovatively integrates long-circulation delivery and ROS-triggered drug release: the hydrophilic segment prolongs in vivo circulation time, while the ROS-responsive unit's breakage in the high ROS environment of the lesion drives rapid release of the targeted drug. This design not only solves the key bottlenecks of poor myocardial targeting and low intracellular bioavailability of conventional formulations, but also allows for flexible matching of different target inhibitors according to pathological subtypes, providing a precise and personalized treatment strategy for acute myocardial infarction, significantly improving treatment safety and intervention efficiency.

[0043] 3. This application covers key regulatory genes (FGG, VTN, APOE, etc.) in the AMI pathological network and provides multiple independent sequence candidates for each target. This design fully considers the objective law that siRNA activity is affected by factors such as mRNA structure and target accessibility. By expanding the sequence library and standardizing screening methods, it ensures that the siRNA ultimately used for treatment has highly efficient silencing activity, avoiding treatment failure due to insufficient activity of a single sequence, and improving the reliability and practicality of the technical solution. Attached Figure Description

[0044] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0045] Figure 1 The 1H NMR spectrum of the carrier material DMPE-PEG-AAPE in Example 1 is shown.

[0046] Figure 2 The graph shows the critical micelle concentration detection results of DMPE-PEG-AAPE micelles in Example 1.

[0047] Figure 3 This is a graph showing the results of the uptake experiment of coumarin@DMPE-PEG-AAPE by normal myocardial cells and hypoxic-damaged cardiomyocytes in Example 1. Figure 3 Figure A shows the experimental results of normal myocardial cells' uptake of coumarin@DMPE-PEG-AAPE. Figure 3 Figure B shows the experimental results of coumarin@DMPE-PEG-AAPE uptake by hypoxic-damaged cardiomyocytes.

[0048] Figure 4 This is a diagram showing the formulation screening results of different siRNAs loaded onto DMPE-PEG-AAPE in Example 5; where, Figure 4 Figure A shows the screening results of FGGsiRNA formulations loaded with DMPE-PEG-AAPE. Figure 4Figure B shows the screening results of VTNsiRNA formulations loaded with DMPE-PEG-AAPE.

[0049] Figure 5 A visual representation of the FGGsiRNA1@DMPE-PEG-AAPE targeted micelles in Example 5. Figure 5 A) Transmission electron micrograph (TEM image) Figure 5 B) Particle size diagram Figure 5 C).

[0050] Figure 6 A visual representation of the VTNsiRNA1@DMPE-PEG-AAPE targeted micelles in Example 5. Figure 6 A) Transmission electron micrograph (TEM image) Figure 6 B) Particle size diagram Figure 6 C).

[0051] Figure 7 This is a bar graph showing the relative activity of EC cells in Example 5 under FGGsiRNA and FGGsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0052] Figure 8 This is a cell morphology diagram of EC cells in Example 5 under FGGsiRNA and FGGsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0053] Figure 9 This is a bar graph showing the relative activity of EC cells in Example 5 under VTNsiRNA and VTNsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0054] Figure 10 This is a cell morphology diagram of EC cells in Example 5 under VTNsiRNA and VTNsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0055] Figure 11 This is a graph showing the relative activity of hypoxic-damaged cardiomyocytes in Example 6 under FGGsiRNA and FGGsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions in each group.

[0056] Figure 12 This is a cell morphology diagram of hypoxic-damaged cardiomyocytes in Example 6 under FGGsiRNA and FGGsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0057] Figure 13This is a graph showing the relative activity of hypoxic-injured cardiomyocytes in Example 6 under VTNsiRNA and VTNsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0058] Figure 14 This is a cell morphology diagram of hypoxic-damaged cardiomyocytes in Example 6 under VTNsiRNA and VTNsiRNA@DMPE-PEG-AAPE targeted micelle culture conditions.

[0059] Figure 15 This is a Western blot image showing the effect of FGGsiRNA and FGGsiRNA@DMPE-PEG-AAPE targeted micelles on FGG protein expression in cardiomyocytes in Example 6. Figure 15 A shows the Western Blot images of FGG protein in cardiomyocytes after intervention with different FGG siRNAs alone; Figure 15 B is a statistical analysis of the relative expression levels of FGG protein in cardiomyocytes after intervention with different FGG siRNAs alone; Figure 15 C shows the Western Blot images of FGG siRNA and FGG protein in cardiomyocytes after targeted micelle intervention in each group; Figure 15 Figure D shows the statistical analysis of the relative expression levels of FGG siRNA and FGG protein in cardiomyocytes after targeted micelle intervention in each group.

[0060] Figure 16 This is a Western blot image showing the effect of VTNsiRNA and VTNsiRNA@DMPE-PEG-AAPE targeted micelles on VTN protein expression in cardiomyocytes, as described in Example 6. Figure 16 A shows the Western Blot images of VTN proteins in cardiomyocytes after intervention with different VTN siRNAs alone; Figure 16 B is a statistical analysis of the relative expression levels of VTN protein in cardiomyocytes after intervention with different VTN siRNAs alone; Figure 16 C shows the Western Blot images of VTN siRNA and VTN protein in cardiomyocytes after targeted micelle intervention in each group; Figure 16 Figure D shows the statistical analysis of the relative expression levels of VTN siRNA and VTN protein in cardiomyocytes after targeted micelle intervention in each group. Detailed Implementation

[0061] The embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0062] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0063] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "multiple (items)" in "one or more" or "one or more" means two or more.

[0064] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0065] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0066] Example 1: Preparation and characterization of dimyristoylphosphatidylethanolamine-polyethylene glycol-3-aminophenylboronic acid pinacol ester polymer (DMPE-PEG-AAPE):

[0067] 1. Preparation of DMPE-PEG-AAPE

[0068] (1) Weigh 5.0g of dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl (DMPE-PEG-COOH) and dehydrate it through a molecular sieve; at the same time, take 3.6g of 3-aminophenylboronic acid pinacol ester (AAPE) and store it in argon gas protected from light.

[0069] (2) Under argon protection and ice bath conditions at 0-5℃, dehydrated DMPE-PEG-COOH was dissolved in 2.5L of anhydrous N,N-dimethylformamide (DMF), and then 0.58g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.35g of N-hydroxysuccinimide (NHS) were added and stirred at 25℃ for 30 minutes to activate.

[0070] (3) Dissolve AAPE in 0.6L of anhydrous DMF and add the AAPE DMF solution dropwise into the solution system of step (2). The temperature is controlled at 0-5℃. After the addition is complete, the temperature is raised to 35℃ and the reaction is stirred for 24 hours.

[0071] (4) Transfer the reaction solution obtained in step (3) to a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze twice (4 hours each time) in a 5L LMF-ethanol mixed solvent (volume ratio 1:1); then dialyze twice (2 hours each time) with 2L anhydrous ethanol. Finally, the dialysate is sterilized by a 0.22μm filter membrane and freeze-dried to obtain a white solid product, which is DMPE-PEG-AAPE.

[0072] 2. Characterization of DMPE-PEG-AAPE

[0073] (1) Nuclear magnetic resonance (NMR) characterization

[0074] Take an appropriate amount of carrier material DMPE-PEG-AAPE, dissolve it in deuterated dimethyl sulfoxide (DMSO) to make the sample concentration reach 10 mM, transfer it to an NMR tube, and place the NMR tube in an NMR spectrometer for NMR H-spectrum measurement.

[0075] The 1H NMR spectrum of the support material DMPE-PEG-AAPE is as follows: Figure 1 As shown, in the DMPE-PEG-AAPE structural formula, a to k represent the corresponding functional groups, and the a to k peaks in the 1H NMR spectrum represent the resonance peaks of the corresponding functional groups. From... Figure 1As can be seen, the long alkyl chain of DMPE (terminal methyl δ 0.8–1.0 ppm; methylene δ 1.2–1.4 ppm; c-ester carbonyl α-methylene δ 2.2–2.5 ppm; glycerol backbone and phospholipid methylene δ 4.0–4.5 ppm), PEG chain (main chain methylene broad singlet δ 3.5–3.7 ppm; α-methylene linked to amide δ 3.2–3.4 ppm), 3-aminophenylboronic acid pinacol ester (pinacol tert-butylmethyl δ 1.2–1.3 ppm; benzene ring hydrogen multiplet δ 6.5–7.5 ppm, of which amino ortho-hydrogen δ 7.0–7.2 ppm) and the amide bond of the linking segment (α-methylene δ 2.0–2.2 ppm) all show clear assignments. No characteristic peaks of free raw materials (such as free PEG / phenylboronic acid monomers) were observed in the spectrum, and the solvent peaks (DMSO δ2.5ppm; water δ3.3ppm) did not interfere with the product analysis, indicating that the ROS-responsive support material has a complete structure and meets the design requirements. The above characterization results confirm the successful synthesis of the support material DMPE-PEG-AAPE.

[0076] (2) Critical micelle concentration detection

[0077] Critical micelle concentration (CMC) was determined using the pyrene fluorescent probe method, with pyrene serving as a hydrophobic environment-sensitive probe to characterize the self-assembly behavior of DMPE-PEG-AAPE micelles. The specific procedure was as follows: DMPE-PEG-AAPE blank micelles were dissolved in deionized water to prepare gradient concentration solutions (0.0001 mg / mL, 0.001 mg / mL, 0.01 mg / mL, 0.1 mg / mL, 1 mg / mL). An equal volume of pyrene-acetone solution was added to each tube (the final concentration of the DMPE-PEG-AAPE blank micelles was 6 × 10⁻⁶). -7 M), after incubation in the dark with shaking, centrifugation was used to remove free pyrene; the ratio (I1 / I3) of the fluorescence intensity of the first peak (I1, 373 nm) to the fluorescence intensity of the third peak (I3, 384 nm) was recorded using a fluorescence spectrophotometer (excitation wavelength 335 nm). A curve was plotted showing the change of I1 / I3 with the logarithm of the micelle concentration of the DMPE-PEG-AAPE support material. The concentration corresponding to the crossover point is the CMC value. The detection results are as follows: Figure 2 As shown, the critical micelle concentration (CMC) is 1.17 μg / mL. This low CMC indicates that the material can efficiently self-assemble into micelles at low concentrations. The hydrophobic interactions of the hydrophobic blocks help improve the efficiency of micelle formation and effectively resist the risk of micelle dissociation due to environmental dilution during blood circulation, ensuring the stability of the drug delivery system. Furthermore, the hydrophobic core advantage corresponding to the low CMC helps stabilize the loading of lipophilic drugs.

[0078] (3) Targeting performance of fluorescently labeled carrier material (coumarin@DMPE-PEG-AAPE)

[0079] Coumarin was co-dissolved with DMPE-PEG-AAPE material to prepare a fluorescently labeled carrier, coumarin@DMPE-PEG-AAPE, loaded with coumarin. This carrier was then diluted with cell culture medium to a working solution with a final coumarin concentration of 0.5 μg / mL for later use.

[0080] Normal cardiomyocytes were seeded at appropriate densities in cell culture plates. After adherence and growth, a portion of the cells were selected to establish a cardiomyocyte hypoxia injury model using a physical hypoxia exposure method, while a portion of cells were retained as a normal control group. Cardiomyocytes in normal condition or with established hypoxia injury were gently washed with blank culture medium to remove impurities. 1 mL of prepared coumarin@DMPE-PEG-AAPE working solution was added to the cell culture plate, with three replicates per group. All treated cells were incubated at 37°C for 2 hours and 4 hours, respectively, for uptake. After incubation, the culture medium was carefully aspirated, and the cells were repeatedly washed several times with pre-cooled phosphate-buffered saline (PBS) to thoroughly remove any untaken free or attached samples. Subsequently, paraformaldehyde fixative was added to fix the cells. After fixation, the cells were washed again with PBS to remove residual fixative. Then, the nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) staining solution, resulting in blue fluorescence in the cell nuclei. Finally, the prepared cell slides were observed under a confocal microscope. Images of the blue DAPI fluorescence channel labeled with cell nuclei, the green coumarin fluorescence channel labeled with the carrier material, and the Merge channel superimposed on both were acquired. The results are as follows: Figure 3 As shown, where, Figure 3 Figure A shows the experimental results of normal myocardial cells' uptake of coumarin@DMPE-PEG-AAPE. Figure 3 Figure B shows the experimental results of coumarin@DMPE-PEG-AAPE uptake by hypoxic-damaged cardiomyocytes.

[0081] from Figure 3It can be seen that after 2 hours of incubation, the uptake of the carrier by hypoxic-damaged cardiomyocytes (with high levels of intracellular reactive oxygen species, ROS) was significantly enhanced, and densely distributed green fluorescence with high overlap with the cell nucleus was observed under confocal microscopy; while only scattered weak fluorescence was observed in normal cardiomyocytes (low ROS) at the same time. After 4 hours of incubation, the fluorescence of normal cardiomyocytes further weakened and almost disappeared, while the hypoxic-damaged cell group still maintained a significantly higher green fluorescence intensity than the normal cell group. The above results indicate that the carrier material is specifically activated in the pathological high-ROS microenvironment, which helps to achieve efficient and long-lasting intracellular delivery; while in the low-ROS environment of normal cells, it maintains relative stability and low uptake. This ROS-responsive characteristic enables DMPE-PEG-AAPE to effectively overcome the problem of low cell enrichment at the lesion site, achieve on-demand response and long-lasting delivery, and precisely target ischemic and hypoxic-damaged cardiomyocytes, providing a key functional delivery basis for the treatment of myocardial ischemia injury. The above results indicate that the targeted uptake efficiency of this carrier on hypoxic-damaged cardiomyocytes in a high ROS environment (simulating AMI lesions) is significantly higher than that on normal cells, providing a direct basis for achieving precise drug enrichment at the lesion site.

[0082] Example 2: Preparation of DMPE-PEG-thioether polymer:

[0083] (1) Weigh 5.0g of myristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl (DMPE-PEG-COOH) and dehydrate it through a molecular sieve. At the same time, take 3.8g of 2-(methylthio)ethylamine (MTEA) and store it in argon gas protected from light.

[0084] (2) Under argon protection and ice bath conditions at 0-5℃, dehydrated DMPE-PEG-COOH was dissolved in 2.5L of anhydrous N,N-dimethylformamide (DMF), and then 0.58g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.35g of N-hydroxysuccinimide (NHS) were added and stirred at 25℃ for 30 minutes to activate.

[0085] (3) Dissolve MTEA in 0.6L of anhydrous DMF, and then add the solution dropwise to the solution system in step (2), with the temperature controlled at 0-5℃; after the addition is complete, raise the temperature to 35℃ and stir for 24 hours.

[0086] (4) Transfer the reaction solution from step (3) to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze twice (4 hours each time) in 5 L of DMF-ethanol mixed solvent (volume ratio 1:1), then dialyze twice (2 hours each time) with 2 L of anhydrous ethanol, and finally sterilize the dialysate through a 0.22 μm filter membrane and freeze-dry to obtain DMPE-PEG-thioether polymer.

[0087] Example 3: Preparation of DMPE-PEG-selenium ether polymer:

[0088] (1) Weigh 5.0g of myristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl (DMPE-PEG-COOH) and dehydrate it through a molecular sieve. At the same time, take 4.3g of 2-(methylselenoethylamine) (MSeEA) and store it in argon gas protected from light.

[0089] (2) Under argon protection and ice bath conditions at 0-5℃, dehydrated PCL-PEG-COOH was dissolved in 2.5L of anhydrous N,N-dimethylformamide (DMF), and 0.6g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.4g of N-hydroxysuccinimide (NHS) were added. The mixture was stirred and activated at 25℃ for 30 minutes.

[0090] (3) Then MSeEA was dissolved in 0.6L of anhydrous DMF, and the solution was then added dropwise to the solution system in step (2) while the temperature was controlled at 0-5℃. After the addition was completed, the temperature was raised to 35℃ and the mixture was stirred for 24 hours.

[0091] (4) The reaction solution from step (3) was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. The solution was dialyzed twice (4 hours each time) in 5 L of DMF-ethanol mixed solvent (volume ratio 1:1), and then dialyzed twice (2 hours each time) in 2 L of anhydrous ethanol. Finally, the dialysate was sterilized by passing it through a 0.22 μm filter membrane and freeze-dried to obtain DMPE-PEG-selenium ether polymer.

[0092] Example 4: siRNA design for FGG, APOE, VTN, KNG1, A2M, and HP genes:

[0093] This embodiment designed specific siRNA molecules targeting the human FGG, APOE, VTN, KNG1, A2M, and HP genes, with three independent targeting sites for each gene (except for APOE, which had two sites due to CDS region limitations). All siRNA sequences were 21 nt in length, and a 3' dideoxythymidine (dTdT) dangling structure was used to enhance stability. The sequence design was based on the CDS region of the NCBI reference sequence. The specific design is shown in Table 1.

[0094] Table 1. siRNA sequence design targeting human FGG, APOE, VTN, KNG1, A2M, and HP genes.

[0095]

[0096]

[0097] Note: The siRNA in the table has two hanging bases TT composed of deoxyribonucleotides at its 3' end.

[0098] The core reason for designing multiple siRNA sequences for the same target in this embodiment is that the silencing activity of siRNA is not solely determined by design rules. In practical applications, even when following the same design principles (such as targeting the CDS region, 21nt length, and 3'dTdT overhang), the activity of different sequences may still vary significantly. This difference mainly stems from:

[0099] (1) Differences in target accessibility: mRNA has a complex secondary structure (such as stem-loop structure, hairpin structure), and some sequences may be masked by protein or RNA binding molecules, resulting in siRNA being unable to bind effectively.

[0100] (2) Off-target interference: Some sequences may have complementary regions with non-target gene mRNA, triggering non-specific silencing and indirectly reducing the efficiency of regulation of target genes.

[0101] (3) Differences in intracellular stability: The GC content and base composition of different sequences may affect their degradation rate in cells, thereby affecting the duration of activity.

[0102] Based on the above characteristics, this application designs 3 independent siRNA sites for each target (of which, APOE is limited to 2 sites due to CDS region restrictions), covering different regions of the target gene mRNA (such as the 5' end, middle segment, and 3' end). By expanding the sequence coverage, the risk of treatment failure due to insufficient activity of a single sequence is reduced. Priority is given to sequences with "high target accessibility, low off-target risk, and GC content of 30% to 50%", thereby increasing the probability of obtaining highly active siRNA from the design source.

[0103] Example 5: Preparation and characterization of drug-loaded DMPE-PEG-AAPE micelles:

[0104] 1. Preparation of drug-loaded DMPE-PEG-AAPE micelles

[0105] (1) Preparation of FGGsiRNA-DMPE-PEG-AAPE micelles

[0106] Dissolve 0.1g of FGGsiRNA in 2mL of PBS (pH 7.4) and set aside. Place 2g of the DMPE-PEG-AAPE material prepared in Example 1 in a round-bottom flask, add 10mL of dichloromethane, and stir with a magnetic stirrer at room temperature until the carrier material is completely dissolved. Under ice bath conditions, add the FGGsiRNA PBS solution dropwise to the round-bottom flask and sonicate (300W power, 5min). Connect the flask to a rotary evaporator and slowly rotary evaporate at 39℃ and 0.08MPa vacuum until the solvent is completely evaporated, forming a uniform lipid film on the inner wall of the flask. Add PBS buffer to the flask and slowly shake at 37℃ water bath to completely hydrate the lipid film. After sonication and dialysis purification, freeze-dry to obtain FGGsiRNA-DMPE-PEG-AAPE polymer micelles.

[0107] (2) Preparation of VTNsiRNA-DMPE-PEG-AAPE micelles

[0108] Dissolve 0.1g of VTNsiRNA in 2mL of PBS (pH 7.4) and set aside. Place 2.5g of the DMPE-PEG-AAPE material prepared in Example 1 in a round-bottom flask, add 10mL of dichloromethane, and stir with a magnetic stirrer at room temperature until the carrier material is completely dissolved. Under ice bath conditions, add the VTNsiRNA PBS solution dropwise to the round-bottom flask and sonicate (300W power, 5min). Connect the flask to a rotary evaporator and slowly rotary evaporate at 39℃ and 0.08MPa vacuum until the solvent is completely evaporated, forming a uniform lipid film on the inner wall of the flask. Add PBS buffer to the flask and slowly shake at 37℃ to completely hydrate the lipid film. After sonication and dialysis purification, freeze-dry to obtain VTNsiRNA-DMPE-PEG-AAPE polymer micelles.

[0109] Using a single-factor analysis, the optimal ratios of FGGsiRNA and VTNsiRNA with DMPE-PEG-AAPE material were screened, with the relative contents of free, unencapsulated FGGsiRNA and VTNsiRNA as indicators. The results are as follows: Figure 4 As shown. Among them, Figure 4 Figure A shows the screening results of DMPE-PEG-AAPE-encapsulated FGGsiRNA formulations. Figure 4 Figure B shows the screening results of VTNsiRNA formulations encapsulated in DMPE-PEG-AAPE. (See figure below.) Figure 4As shown in Figure A, when the ratio of FGGsiRNA to DMPE-PEG-AAPE material was 1:20, the relative expression level of FGGsiRNA was close to complete encapsulation. Further increasing the amount of DMPE-PEG-AAPE material did not significantly improve the encapsulation efficiency. Therefore, the ratio of FGGsiRNA to DMPE-PEG-AAPE material was determined to be 1:20. Figure 4 As can be seen from B, the optimal ratio of VTNsiRNA to DMPE-PEG-AAPE material was determined to be 1:25.

[0110] 2. Characterization of drug-loaded DMPE-PEG-AAPE micelles

[0111] (1) Investigation of the Tyndall effect

[0112] The prepared FGGsiRNA-DMPE-PEG-AAPE and VTNsiRNA-DMPE-PEG-AAPE micelles were dispersed in PBS buffer to form clear micelle solutions. A suitable amount of each solution was placed in a transparent glass sample vial, and the solution was vertically illuminated with a 650nm laser to observe whether a clearly visible bright light path (Tyndall effect) was formed in the direction perpendicular to the incident light, thus verifying the formation of the nanomicelles. The results are as follows: Figure 5 As shown in Figures A and 6A, both micelles exhibit a clear and continuous pink light path perpendicular to the incident light direction. This phenomenon confirms that both nanomicelle systems successfully formed a stable colloidal dispersion: the micelle size is within the effective light scattering range (typically 1 nm–1000 nm), and the particles are uniformly dispersed without aggregation or sedimentation. This optical property reflects the integrity and dispersion stability of the micelle structure, providing a reliable guarantee for subsequent experiments such as cellular uptake and in vivo delivery, ensuring that siRNA maintains an effective loading state during delivery.

[0113] (2) Morphological characterization

[0114] After hydrophilizing a copper mesh specifically designed for transmission electron microscopy (TEM), FGGsiRNA-DMPE-PEG-AAPE and VTNsiRNA-DMPE-PEG-AAPE micelles were diluted to appropriate concentrations with pure water and then dropped onto the front side of the copper mesh. After standing for 2 minutes, an appropriate amount of phosphotungstic acid dye was added, and the liquid on the copper mesh was blotted dry with filter paper. The phosphotungstic acid dye was added again, and after standing for 2 minutes, the liquid was blotted dry with filter paper. This process of phosphotungstic acid staining was repeated once, followed by drying under an incandescent lamp. The copper mesh was then placed in a TEM microscope to examine the sample morphology. The TEM results are as follows: Figure 5 As shown in Figures B and 6B, neither micelle microsphere showed obvious aggregation, and both had good spherical shape and relatively uniform size.

[0115] (3) Particle size determination

[0116] The particle size of FGGsiRNA-DMPE-PEG-AAPE and VTNsiRNA-DMPE-PEG-AAPE micelles was characterized using a dynamic light scattering particle size analyzer. After the instrument preheated for 30 min, the quartz cuvettes were rinsed 2-3 times with purified water. The nano-formulation was then diluted with purified water at a certain ratio and added to the cuvettes. Each sample was measured three times, and the cycle time was set. The test solvent was selected as "water". Measurements were started after the instrument stabilized. The particle size test results are shown below. Figure 5 As shown in C and 6C, the particle size of both is around 30 nm, exhibiting good monodispersity.

[0117] (4) Safety assessment

[0118] 1) Safety assessment of FGGsiRNA and targeting materials

[0119] The safety of the carrier material DMPE-PEG-AAPE, FGGsiRNA, and FGGsiRNA-targeted micelles was investigated at the cellular level. The specific methods are as follows:

[0120] After normal culture and counting of endothelial cells (ECs), add 5 × 10⁶ cells of cell suspension. 4 / well, incubated for 24 h until cells reached 80% confluence. Then, carrier materials DMPE-PEG-AAPE, FGGsiRNA1, FGGsiRNA2, FGGsiRNA3, FGGsiRNA1@DMPE-PEG-AAPE, FGGsiRNA2@DMPE-PEG-AAPE (50 nm), and FGGsiRNA3@DMPE-PEG-AAPE were added to the wells respectively. Incubation was then carried out at 37°C for 48 h. Each group had 6 replicates and included cell-free and untreated cell control wells. After 48 hours of continued culture, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide solution (MTT, final concentration 0.5 mg / mL) was added to each well and incubated for 4 h. After termination of culture, the culture medium was removed, and DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm. The safety of the material was finally evaluated by relative viability. Results are as follows. Figure 7 As shown, where, Figure 7 The chart shows the viability of EC cells in each group. Figure 7 As can be seen, EC cells maintained a relative viability of over 90% after contact with free FGGsiRNA, FGGsiRNA@DMPE-PEG-AAPE, and DMPE-PEG-AAPE carrier materials. Simultaneous cell morphology observations showed the following results: Figure 8 As shown, from Figure 8As can be seen, cells in all experimental groups maintained a regular morphology, with intact cell membranes and good adherence and growth. Compared with the blank control group, no abnormal toxic characteristics such as shrinkage or detachment were observed. This result confirms that FGGsiRNA and the targeted preparation have no significant toxic effect on EC cells at the tested concentrations.

[0121] 2) Safety assessment of VTNsiRNA and targeting materials

[0122] The safety of the carrier materials DMPE-PEG-AAPE, VTNsiRNA, and VTNsiRNA-targeted nanomicelles was investigated at the cellular level. Specifically, after normal culture and counting of EC cells, 5 × 10⁶ cells of cell suspension were added. 4 / well, incubated for 24 h until cells reached 80% confluence. Then, carrier materials DMPE-PEG-AAPE, VTNsiRNA1, VTNsiRNA2, VTNsiRNA3, VTNsiRNA1@DMPE-PEG-AAPE, VTNsiRNA2@DMPE-PEG-AAPE (50 nM), and VTNsiRNA3@DMPE-PEG-AAPE were added to the wells. Incubation was then carried out at 37°C for 48 h. Each group had 6 replicates, plus cell-free control wells and untreated cell control wells. After another 48 hours of culture, MTT solution (final concentration 0.5 mg / mL) was added to each well and incubated for 4 h. After terminating the culture, the culture medium was removed, and DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm. The safety of the material was finally evaluated by relative viability. Results are as follows. Figure 9 As shown, when VTNsiRNA (50 nM monomer and targeting formulation) and carrier material were applied to EC cells, cell viability remained stable at over 90% and showed no statistically significant difference compared to the control group. The morphology of cells in each group was observed under a microscope, and the results are as follows: Figure 10 As shown, the results indicated that the cell morphology of all groups was consistent with that of the control group, exhibiting intact adherent growth characteristics, without membrane structure damage or morphological toxicity changes. These data collectively confirm that VTNsiRNA and its vector delivery system possess good in vitro biosafety, providing an experimental basis for subsequent in vivo studies.

[0123] Example 6: Cell-level efficacy evaluation:

[0124] 1. The protective effect of MTT assay on the activity of hypoxic-damaged cardiomyocytes

[0125] (1) MTT assay to detect the protective effect of FGG siRNA and its preparations on hypoxic-injured cardiomyocytes

[0126] Cardiac cardiomyocytes were digested with collagenase and then divided into 8 × 10⁸ cells per well. 4Cells were seeded in 96-well plates in high-glucose DMEM medium containing 15% fetal bovine serum and cultured at 37°C and 5% CO2 for 24 hours until complete adhesion. Cells were then divided into 8 groups: blank control group (noroxic conditions + complete medium), hypoxia model group (1% O2 hypoxia environment + serum-free medium), FGGsiRNA1 intervention group (1% O2 hypoxia + 50 nM FGGsiRNA1), and targeted agent group (1% O2 hypoxia + 50 nM FGGsiRNA1@DM). Hypoxia injury models were constructed using three treatment groups: DMPE-PEG-AAPE micelles, FGGsiRNA2 intervention group (1% O2 hypoxia + 50 nM FGGsiRNA2), targeted formulation group (1% O2 hypoxia + 50 nM FGGsiRNA2@DMPE-PEG-AAPE micelles), FGGsiRNA3 intervention group (hypoxia + 50 nM FGGsiRNA3), and targeted formulation group (hypoxia + 50 nM FGGsiRNA3@DMPE-PEG-AAPE micelles). After discarding the original culture medium, each group was equilibrated with serum-free culture medium for 30 min, then the corresponding treatment solution was replaced, and the group was placed in a tri-gas incubator (94% N2 / 5% CO2 / 1% O2) for 12 h. The treatment solution was then removed, and 0.5 mg / mL MTT solution was added to each well. After incubation at 37°C in the dark for 4 hours, the supernatant was aspirated, and 150 μL of DMSO was added with shaking for 10 min. The absorbance was measured at 570 nm using a microplate reader to calculate the relative cell viability. The results are as follows: Figure 11 As shown. Cell morphology changes in each group were observed and images were acquired simultaneously under an inverted microscope (200×). The results are as follows. Figure 12 As shown. From Figure 11 As can be seen, under hypoxic injury conditions, the cardiomyocyte activity in the hypoxic model group was significantly lower than that in the normal control group, while the cell activity in the FGGsiRNA-intervention group showed a certain degree of improvement; particularly significant was the increase in cell activity in the targeted formulation group treated with the same concentration of FGGsiRNA@DMPE-PEG-AAPE nanomicelles, which was far greater than that in the free siRNA intervention group, partially recovering to near-normal physiological levels. Figure 12 Morphological examination further confirmed the above conclusions: Cardiomyocytes in the normal control group were tightly adherent, while those in the model group showed extensive shrinkage and deformation, as well as broken intercellular connections, due to hypoxia. After intervention with free FGGsiRNA, cell morphological abnormalities were somewhat alleviated, but defects remained. The targeted formulation group maintained the most intact cell morphology, with stable adhesion, clear boundaries, and an overall arrangement pattern close to the normal group. This morphological improvement was highly consistent with the activity changes measured by the MTT assay, jointly verifying that FGGsiRNA delivered via the DMPE-PEG-AAPE vector can produce a superior protective effect against hypoxic cardiomyocytes.

[0127] (2) MTT assay to detect the protective effect of VTNsiRNA and its preparations on hypoxic-damaged cardiomyocytes

[0128] Cardiac cardiomyocytes were digested with collagenase and then divided into 8 × 10⁸ cells per well. 4 Cells were seeded in 96-well plates in high-glucose DMEM medium containing 15% fetal bovine serum and cultured at 37°C and 5% CO2 for 24 h until complete adhesion. Cells were then divided into 8 groups: blank control group (noroxic conditions + complete medium), hypoxia model group (1% O2 hypoxic environment + serum-free medium), VTNsiRNA1 intervention group (hypoxia + 50 nM concentration), targeted agent group (hypoxia + 50 nM VTNsiRNA1@DMPE-PEG-AAPE micelles), VTNsiRNA2 intervention group (hypoxia + 50 nM concentration), targeted agent group (hypoxia + 50 nM VTNsiRNA2@DMPE-PEG-AAPE micelles), VTNsiRNA3 intervention group (hypoxia + 50 nM concentration), and targeted agent group (hypoxia + 50 nM VTNsiRNA3@DMPE-PEG-AAPE micelles). After discarding the original culture medium, each group was equilibrated with serum-free medium for 30 min, then the corresponding treatment solution was replaced, and the cells were placed in a tri-gas incubator (94% N2 / 5% CO2 / 1% O2) for 12 h to construct a hypoxia injury model. Subsequently, the treatment solution was removed, and 0.5 mg / mL LMT solution was added to each well. The cells were incubated at 37°C in the dark for 4 h, then the supernatant was aspirated, and 150 μL of DMSO was added with shaking for 10 min. Cell viability was calculated by measuring the absorbance at 570 nm using a microplate reader. The results are shown below. Figure 13 As shown. Cell morphology changes in each group were observed and images were acquired simultaneously under an inverted microscope (200×). The results are as follows. Figure 14 As shown. From Figure 13 As can be seen, the cardiomyocyte activity in the model group was significantly lower than that in the normal control group after hypoxia injury. VTNsiRNA intervention could partially restore activity, while the VTNsiRNA@DMPE-PEG-AAPE nanomicelle formulation at the same concentration showed a stronger protective effect, with cell activity approaching normal levels. Figure 14 Cell morphology observation showed that the targeted formulation group maintained the morphological structure of cardiomyocytes more completely than the free siRNA group. Its adhesion stability, boundary clarity and arrangement pattern were highly consistent with the normal group. The morphological improvement and activity recovery results jointly verified the significant improvement of the protective efficacy of the nano-formulation.

[0129] 2. Western blotting method for detecting the relative expression levels of FGG and VTN proteins in hypoxic-injured cardiomyocytes treated with targeted nanomicelles.

[0130] (1) The expression level of FGG protein in hypoxic-injured cardiomyocytes was detected by Western blotting, and the results are as follows: Figure 15 As shown. From Figure 15 As can be seen, hypoxic injury led to a significant increase in FGG protein expression in cardiomyocytes compared to the normal control group. Free FGG siRNA intervention effectively inhibited this overexpression, but the FGG siRNA@DMPE-PEG-AAPE formulation delivered via DMPE-PEG-AAPE targeted micelles exhibited a stronger regulatory effect: the attenuation of its protein band grayscale value was far greater than that of the free siRNA group. Quantitative analysis further confirmed that the relative expression level of FGG protein in the targeted formulation group was lower, indicating that the vector can significantly enhance the silencing efficacy of siRNA on the target protein.

[0131] (2) The expression level of VTN protein in hypoxic-injured cardiomyocytes was detected by Western blotting, and the results are as follows: Figure 16 As shown, where Figure 16 A shows the Western Blot images of VTN proteins in cardiomyocytes after intervention with different VTN siRNAs alone; Figure 16 B represents the statistical analysis of the relative expression levels of VTN protein in cardiomyocytes after intervention with different VTN siRNAs alone; Figure 16 C shows the Western Blot images of VTN siRNA and VTN protein in cardiomyocytes after targeted micelle intervention in each group; Figure 16 D represents the statistical analysis of the relative expression levels of VTN siRNA and VTN protein in cardiomyocytes after targeted micelle intervention in each group. Figure 16 It can be seen that VTN protein overexpression in the model group was partially inhibited by free VTN siRNA, while the protein band in the VTN siRNA@DMPE-PEG-AAPE targeted formulation group showed stronger inhibition. Quantitative comparison showed that the relative expression level of VTN protein in the nano-formulation group decreased more significantly than that in the free siRNA group. This phenomenon is completely consistent with the trend of FGG protein changes, jointly confirming that the targeted nanomicelle delivery system can significantly improve the intervention efficiency of siRNA on target proteins in hypoxic-damaged cardiomyocytes.

[0132] The quantitative analysis above shows that the silencing efficiency of drug-loaded micelles on target proteins is 2-3 times higher than that of free siRNA, confirming that the vector of the present invention can effectively improve the intracellular delivery efficiency and bioavailability of siRNA, and solve the problems of weak effect and insufficient targeting of traditional siRNA delivery systems.

[0133] In summary, this approach successfully constructed a ROS-responsive DMPE-PEG-AAPE nanomicelle delivery system. This carrier utilizes 3-aminophenylboronic acid pinacol ester (AAPE) in response to the highly reactive oxygen species (ROS) microenvironment in the acute myocardial infarction (AMI) lesion area, achieving specific drug release in ischemic myocardial tissue. Its amphiphilic structure can efficiently encapsulate siRNA or small molecule inhibitors targeting FGG, APOE, VTN, KNG1, A2M, and HP genes, simultaneously blocking the vicious cycle of coagulation, metabolic disorders, and inflammatory cascade reactions in the AMI process by silencing key gene expression or inhibiting protein activity.

[0134] The above in vitro experiments demonstrate that, compared to free siRNA, siRNA-loaded targeted nanomicelles significantly enhance the protective efficacy against hypoxic-damaged cardiomyocytes. This is achieved not only by significantly downregulating the abnormally high expression of target proteins such as FGG and VTN, but also by improving cell viability and maintaining morphological integrity, confirming the advantages of nano-formulations in enhancing intracellular delivery efficiency and gene silencing effects. This invention provides an innovative solution to overcome the bottlenecks of insufficient myocardial targeting and lack of microenvironment responsiveness in traditional drugs.

[0135] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A targeted synergistic drug delivery system combining DMPE-PEG and ROS-responsive units, characterized in that, It includes nanomicelles formed by the self-assembly of amphiphilic compounds and therapeutic agents encapsulated in the nanomicelles; The nanomicelles comprise hydrophobic segments, hydrophilic segments, and ROS-responsive units; the hydrophobic segments are myristoyl phosphatidylethanolamine, the hydrophilic segments are polyethylene glycol, and the ROS-responsive units are selected from phenylboronic acid ester responsive groups. The therapeutic agent comprises siRNA, which silences the expression of the FGG gene, and the siRNA is FGGsiRNA1; the sense strand sequence of FGGsiRNA1 is shown in SEQ ID NO.1, and the antisense strand sequence of FGGsiRNA1 is shown in SEQ ID NO.2; The preparation method of the DMPE-PEG and ROS-responsive unit targeted synergistic drug delivery system includes the following steps: S1. Under a protective gas atmosphere and at 0-5°C, dissolve dimyristoylphosphatidylethanolamine-polyethylene glycol-carboxyl in an anhydrous solvent to obtain a first solution; add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the first solution, and then stir and activate at 10°C-40°C for 10-60 minutes to obtain a first reaction solution; S2. Dissolve the compound with ROS-responsive units in an anhydrous solvent to obtain a second solution; add the second solution dropwise to the first reaction solution at 0~5℃; after the addition is complete, raise the temperature to 25℃~45℃ and react for 18~36 hours to obtain a second reaction solution; the compound with ROS-responsive units is selected from 3-aminophenylboronic acid pinacol ester; S3. The second reaction solution was purified by first dialysis using a dialysis bag with a molecular weight cutoff of 3000 Da to 4000 Da, and then filtered, sterilized, and freeze-dried in sequence to obtain an amphiphilic polymer. S4. Dissolve the amphiphilic polymer in an organic solvent to obtain a third solution; then, at 0-5°C, add a phosphate buffer solution containing the therapeutic agent dropwise to the third solution and emulsify by ultrasonication to obtain an emulsion; remove the solvent from the emulsion by rotary evaporation to obtain a lipid membrane; S5. Under water bath conditions of 35℃~40℃, the lipid membrane is completely hydrated with phosphate buffered saline, and then subjected to ultrasonic treatment, second dialysis purification and freeze drying in sequence.

2. The drug delivery system according to claim 1, characterized in that, The critical micelle concentration of the nanomicelles is 0.8 μg / mL to 1.5 μg / mL.

3. The drug delivery system according to any one of claims 1 to 2, characterized in that, The mass ratio of the therapeutic agent to the nanomicelles is 1:(1~40); and / or, the particle size of the nanomicelles is 10nm~100nm.

4. A method for preparing a targeted synergistic drug delivery system for DMPE-PEG and ROS responsive units according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Under a protective gas atmosphere and at 0-5°C, dissolve dimyristoylphosphatidylethanolamine-polyethylene glycol-carboxyl in an anhydrous solvent to obtain a first solution; add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the first solution, and then stir and activate at 10°C-40°C for 10-60 minutes to obtain a first reaction solution; S2. Dissolve the compound with ROS-responsive units in an anhydrous solvent to obtain a second solution; add the second solution dropwise to the first reaction solution at 0~5℃; after the addition is complete, raise the temperature to 25℃~45℃ and react for 18~36 hours to obtain a second reaction solution; the compound with ROS-responsive units is selected from 3-aminophenylboronic acid pinacol ester; S3. The second reaction solution was purified by first dialysis using a dialysis bag with a molecular weight cutoff of 3000 Da to 4000 Da, and then filtered, sterilized, and freeze-dried in sequence to obtain an amphiphilic polymer. S4. Dissolve the amphiphilic polymer in an organic solvent to obtain a third solution; then, at 0-5°C, add a phosphate buffer solution containing the therapeutic agent dropwise to the third solution and emulsify by ultrasonication to obtain an emulsion; remove the solvent from the emulsion by rotary evaporation to obtain a lipid membrane; S5. Under water bath conditions of 35℃~40℃, the lipid membrane is completely hydrated with phosphate buffered saline, and then subjected to ultrasonic treatment, second dialysis purification and freeze drying in sequence.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass ratio of the dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl group, the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and the N-hydroxysuccinimide is 10:(1~1.5):(0.5~1.5).

6. The preparation method according to claim 4, characterized in that, The mass ratio of the dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl group to the compound having the ROS-responsive unit is 5:(3-4.5).

7. The preparation method according to claim 4, characterized in that, The preparation method satisfies at least one of the following conditions: (1) Before dissolving the dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl group in an anhydrous solvent, the method further includes dehydrating the dimyristoyl phosphatidylethanolamine-polyethylene glycol-carboxyl group by molecular sieve for 20h~30h; (2) The anhydrous solvent is N,N-dimethylformamide; (3) In step S3, the first dialysis purification step includes: firstly, the second reaction solution is dialyzed twice with a mixed solvent containing anhydrous solvent and anhydrous ethanol, and the dialyzing time is 3.5h~4.5h each time; then, anhydrous ethanol is used as the solvent for two more dialysis cycles, and the dialyzing time is 1.5h~2.5h each time. (4) In step S3, the filtration is performed using a filter membrane with a pore size of 0.2μm~0.25μm; (5) In step S4, the power of ultrasonic emulsification is 250W~350W and the time is 3min~6min; (6) In step S4, the organic solvent is selected from any one or more of methanol, dichloromethane, and trichloromethane.

8. The use of the drug delivery system according to any one of claims 1 to 3 or the drug delivery system prepared by the method according to any one of claims 4 to 7 in the preparation of a drug for treating myocardial infarction.

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