CN108542880A - A method of structure sequence grade targets ischemic myocardial cells mitochondria medicament-carried nano micelle - Google Patents

Abstract

The invention discloses a drug-loaded drug (model drugs, such as puerarin, baicalin, etc., which can act on the mitochondria of ischemic cardiomyocytes to treat myocardial ischemia-reperfusion injury) targeting ischemic cardiomyocyte mitochondria. Micellar approach. The method comprises the synthesis of TPP-PEG-PE block copolymer and the preparation of sequence-level targeted drug-loaded nano-micelles. The invention uses sequence-level targeted nano-micelles to accurately deliver the puerarin drug to the mitochondria of ischemic cardiomyocytes to exert pharmacological effects. First, the EPR effect of PEG-PE nanomicelles is used to target the drug carrier to accumulate in the ischemic myocardium in the early stage of infarction; then, the charge effect of TPP cations is used to mediate the drug carrier into the negatively charged mitochondria of cardiomyocytes, and the puerarin and other drugs are gradually targeted to the mitochondria of ischemic cardiomyocytes to achieve precise drug delivery.

Description

A method for constructing sequence-level targeted mitochondrial drug-loaded nanomicelles of ischemic cardiomyocytes

technical field

The invention belongs to the field of pharmaceutical preparations, and in particular relates to a method for constructing sequence-level drug-loaded nanomicelles targeting ischemic cardiomyocyte mitochondria.

Background technique

Acute myocardial infarction (AMI) is a common clinical emergency that can lead to sudden death of patients. Coronary intervention to restore coronary blood flow is a common clinical treatment for AMI patients, but the resulting myocardial ischemia/reperfusion (I/R) injury can increase the size of ischemic myocardial infarction. Increase 20%-50%, there is no good solution at present. The mechanism of myocardial I/R injury is mainly manifested as mitochondrial dysfunction, and the induction of myocardial cell apoptosis and autophagy. Restoring the normal function of mitochondria is undoubtedly of great scientific research value for alleviating myocardial I/R injury. At present, most of the drugs used clinically to treat myocardial I/R injury lack tissue specificity, have low selectivity for cardiomyocytes, and reach the mitochondrial effect target at a very low concentration, resulting in unsatisfactory expected curative effects of the drugs. Therefore, it is of great clinical significance to realize the drug targeting mitochondria in ischemic cardiomyocytes.

(1) Myocardial I/R injury is closely related to mitochondrial mPTP opening

Mitochondrial dysfunction is closely related to various aspects of myocardial I/R injury, especially cardiomyocyte apoptosis and autophagy. Under physiological conditions, the permeability transition pore (Mitochondrial permeability transition pore, mPTP) on the mitochondrial membrane is closed, and in the early stage of myocardial I/R, the mitochondria deviate from the physiological equilibrium point, producing a large amount of superoxide anion and reactive oxygen species, resulting in mitochondrial calcium Overload and oxidative stress increase, oxidative damage occurs to mitochondrial membranes, enzymes, and electron transport chains, and the production of ATP decreases, which promotes the opening of mPTP on the mitochondrial membrane, causing a sharp increase in the permeability of the mitochondrial inner membrane, making cytochrome C (Cytochrome C, Cyt C) released from the mitochondria into the cytoplasm, triggering the cascade effect of Caspases, resulting in a decrease in the expression of the apoptosis-related protein Bcl-2 (anti-apoptosis) and an increase in the expression of Bax (pro-apoptosis), thereby inducing cardiomyocyte apoptosis; It can also activate Beclin1-mediated autophagy, manifested by the appearance of a large number of autophagosomes and autolysosomes in cells, and eventually the cells are degraded by their own lysosomes, triggering autophagic cell death. Therefore, effectively inhibiting the opening of mitochondrial mPTP and reducing cardiomyocyte apoptosis and autophagy in the early stage of reperfusion can be used as a new therapeutic strategy for myocardial I/R injury.

(2) The model drug - puerarin can inhibit the opening of mitochondrial mPTP

Puerarin is an active ingredient extracted from Pueraria root, a traditional Chinese medicine. It has various pharmacological effects such as dilating coronary arteries, improving coronary circulation, reducing myocardial I/R injury, and inhibiting platelet aggregation. Puerarin injection is often used clinically to treat coronary heart disease, diseases such as angina pectoris and myocardial infarction. At present, through the research on the mechanism of puerarin reducing myocardial I/R injury, it is found that puerarin can inhibit the opening of mitochondrial mPTP and regulate the physiological function of mitochondria. Gao Q and Yao H found that the protective effect of puerarin on I/R injured myocardium is related to promoting the opening of mitoK _ATP channel and mitoK _Ca channel, thereby stabilizing the mitochondrial membrane potential and inhibiting the opening of mPTP. The above literature reports show that puerarin maintains mitochondrial homeostasis by inhibiting the opening of mPTP, inhibits cardiomyocyte apoptosis and autophagy, and increases mitochondrial tolerance to ischemia, thereby reducing myocardial I/R injury. Delivery to the effect target located in the mitochondria to achieve precise drug delivery will likely greatly improve the therapeutic effect of the drug and reduce the dosage of the drug. The delivery of puerarin drug to mitochondria needs to overcome multiple biological barriers: first, the puerarin drug carrier must be enriched in the ischemic myocardium; secondly, the drug carrier must overcome the trapping of the reticuloendothelial system (RES) in the liver and spleen to ensure that the drug The effective concentration can be maintained when it reaches the target organ; finally, the cell fluid is in a state of high viscosity, which hinders the uptake of the drug by the mitochondria, and at the same time, the intracellular lysosome can also degrade the drug, further reducing the concentration of the drug reaching the effector organelle. All these issues are closely related to the construction of an efficient mitochondrial-targeted drug delivery system, and are worthy of in-depth exploration and systematic research.

(3) PEG-PE nanomicelles can be passively permeated and enriched in the ischemic myocardium, and can also reduce the capture of drugs by RES

Mitochondrial-targeted drug delivery systems can greatly improve the therapeutic effect of drugs, and research reports in this area are mainly found in tumor drugs. For example, Zhang C’s research group has constructed an original innovative mitochondrial targeting system, which provides a safe and efficient carrier platform for mitochondrial-targeted anti-tumor drugs, and constructs a mitochondrial-targeted drug delivery system for ischemic cardiomyocytes, which may be possible. It is a new and challenging research field. Mitochondria are distributed in tissue cells in the body, how to gather drugs in the ischemic myocardium is the prerequisite for the realization of mitochondrial targeted drug delivery in cardiomyocytes. According to literature reports, the intima permeability of the ischemic myocardium is abnormally increased, and PEG polymers have better high permeability and retention effect (Enhanced permeability and retention effect, EPR) in the ischemic myocardium. The blood vessel wall cells in normal tissue are closely arranged and have a complete structure, so the PEG polymer drug-carrying system hardly leaks out during the blood circulation, but in the myocardial ischemia area, the PEG polymer can penetrate the vascular intima with increased permeability , collected in the early myocardial tissue of infarction. Our previous research results found that: the AUC of PEGylated puerarin in myocardial tissue of rats with acute myocardial ischemia was 1.7 times that of normal rats, and it had obvious targeting of ischemic myocardium; puerarin PEG-PE nanomicelle The AUC in the heart of acute ischemia model mice is 1.9 times that in the heart of normal mice, indicating that puerarin PEG-PE nanomicelles have better cardiac targeting in the early myocardial infarction area, and can accumulate drugs in the heart blood myocardium. Lukyanov AN et al. also found that polyethylene glycol-derivatized phosphatidylethanolamine (PEG-PE) nanomicelles mainly aggregated in early infarcted myocardial tissue, and its content was 8 times that of non-infarcted myocardium. Previous studies and literature reports support that PEG polymers and PEG-PE nanomicelles have a better EPR effect in the ischemic myocardium, which can passively accumulate drugs in the lesion of the heart and improve drug selectivity. In addition, the PEG macromolecules around the PEG-PE nanomicelles have good biocompatibility, and the particle size of the nanomicelles is small, which can reduce the trapping of the reticuloendothelial system (RES) in the liver and spleen.

(4) TPP cations can mediate the targeting of drug carriers into mitochondria and reduce the degradation of drugs by lysosomes

After the drug-loaded micelles stay in the ischemic myocardium, how to overcome the blockage of the cell fluid and the destruction of the lysosome is a key issue in realizing the mitochondrial targeting of the drug. At present, a large number of studies have confirmed that Triphenylphosphonium (TPP) cations have mitochondrial targeting, because the mitochondrial inner membrane is negatively charged at 150-180mv, and the cell membrane is negatively charged at 30-60mv, while TPP is positively charged for triphenylphosphonium cations, positive and negative charges Attract each other, promote the ability of TPP cations to gather in mitochondria by 100-500 times, and can overcome the obstacles of high-viscosity cell fluid. It has been reported in the literature that TPP cations can mediate the targeting of tumor drugs, antioxidant drugs and G(5)-PAMAM dendritic block copolymers into mitochondria, and by analogy, TPP cations can also be delivered to cardiomyocyte mitochondria as drug-loaded nanomicelles Active targeting molecules; in addition, TPP cations have a "proton sponge" effect in lysosomes, which can reduce the damage of intracellular lysosomes and endosomes to nanomicelles, play a "lysosome escape" effect, and further improve Drug concentration in effector organelles.

After retrieving domestic and foreign literature, we found reports on hyaluronic acid-modified puerarin PEG-PLGA nanoparticles, puerarin PEG-PE nanomicelles, and PEGylated puerarin. , protective effect on ischemia-reperfusion myocardium, anti-apoptosis, and tissue distribution in animals with acute myocardial ischemia, etc., to construct sequence-level drug-loaded nanomicelles targeting ischemic cardiomyocyte mitochondria needs to overcome many technical bottlenecks, In view of the EPR effect of the PEG-PE macromolecule on the ischemic myocardium, coupled with the charge effect of the TPP cation, the present invention gradually delivers puerarin and other drugs to the mitochondrial effect site of the ischemic cardiomyocyte, and inhibits the permeability on the mitochondrial membrane The conversion hole is opened, thereby reducing the massive apoptosis and autophagy of cardiomyocytes during ischemia-reperfusion period. Compared with the previous puerarin nano-drugs, the precise delivery of drugs to the target of effector organelles not only greatly improves the effect of drugs It can also reduce adverse drug reactions.

Contents of the invention

The present invention aims to overcome the deficiencies of the prior art, and provides a method for constructing drug-loaded nanomicelles targeted to the mitochondria of ischemic cardiomyocytes.

In order to achieve the above object, the technical solution provided by the invention is:

The method for constructing sequence-level targeted mitochondrial drug-loaded nanomicelles of ischemic cardiomyocytes comprises the following steps:

(1) Synthesis of TPP-PEG-PE block copolymer: Dissolve CTPP in chloroform, then add triethylamine, NHS and EDC, stir at room temperature (5—30°C), and then add DSPE-PEG-NH ₂ in chloroform Solution, under the protection of nitrogen, stirred at room temperature (5-30 ℃) overnight, the crude reactant was washed with glacial ether several times and then centrifuged, the lower precipitate was removed, and then the lower precipitate was freeze-dried to obtain TPP-PEG-PE embedded segment copolymer; the structural formula of the TPP-PEG-PE block copolymer is shown in formula I:

(2) Preparation of sequence-level targeted drug-loaded nanomicelles: PEG-PE, TPP-PEG-PE and puerarin are co-dissolved in organic solvents such as methanol, acetonitrile or dichloromethane, and can be prepared by thin film hydration method Sequential targeted nanomicelles loaded with puerarin; the drug is a drug that acts on the mitochondria of ischemic cardiomyocytes to treat myocardial ischemia-reperfusion injury, preferably puerarin or baicalin.

Preferably, the puerarin-loaded ordered nanomicelles have an average particle size of 10-100 nm, preferably about 17 nm, and a good normal distribution, and a zeta potential of -20-+20 mV.

Preferably, in step (1), the mass volume ratio of the CTPP and chloroform is (90-110mg):(9-11mL), preferably 100mg:10mL, and the mass volume ratio of the CTPP and triethylamine is ( 90-110mg): (180-220μL), preferably 100mg:200μL, the mass ratio of CTPP to NHS is 100:(70-80), preferably 100:74, the mass ratio of CTPP to EDC is 100:( 120-125), preferably 100:124, the mass volume ratio of the CTPP and DSPE-PEG- _NH chloroform solution is (90-110mg):(18-28mL), preferably 100mg:25mL.

Preferably, in step (1), the concentration of the CTPP is 0.23mM, the concentration of the NHS is 0.50-0.70mM, preferably 0.64mM, and the DSPE-PEG-NH in _the chloroform solution of the DSPE-PEG- The concentration of NH ₂ is 20-30 mg/mL, preferably 25 mg/mL.

Preferably, in step (2), the molar ratio of the PEG-PE to the drug is (97-80):(15-25), preferably 90:20, the molar ratio of the TPP-PEG-PE to the drug It is (3-20):(15-25), preferably 10:20. The present invention will be further described below:

Synthesis of TPP-PEG-PE block copolymer:

Accurately weigh 100mg (0.23mM) CTPP dissolved in 10mL chloroform, add 200μL triethylamine, 124mg (0.64mM) EDC and 74mg (0.64mM) NHS, stir at room temperature for 2h, then add 25mL concentration of 25mg/mL DSPE- The chloroform solution of PEG-NH2 (molecular weight: 2800, 0.22mM) was stirred overnight at room temperature under the protection of nitrogen, and the crude reactant was washed with glacial ether several times, centrifuged to remove the lower precipitate, and then freeze-dried to obtain TPP-PEG -PE block copolymer, the specific synthetic route is as follows:

Preparation of drug-loaded targeted nanomicelles:

Since PEG-PE has both hydrophilic and lipophilic groups, the drugs that act on the mitochondria of ischemic cardiomyocytes to treat myocardial ischemia-reperfusion injury, such as puerarin and baicalin, are just hydrophobic drugs, which are encapsulated in PEG-PE nanogel bundle of hydrophobic cores. PEG-PE, TPP-PEG-PE and drug (the molar ratio of PEG-PE and drug is 97:20, the molar ratio of TPP-PEG-PE and drug is 3:20) were dissolved in methanol, acetonitrile or di Sequence-level targeted drug-loaded nanomicelles were prepared by thin film hydration method in methyl chloride (see Figure 1 for the preparation route). After testing, it was found that the average particle size of sequence-level targeted drug-loaded nanomicelles was about 17nm, and it was in a good normal distribution. The zeta potential was -1mV, and the stability was good (results shown in Figure 2).

The process of constructing sequence-level drug-loaded nanomicelles targeting ischemic cardiomyocyte mitochondria is as follows: PEG-PE, TPP-PEG-PE and drugs are co-dissolved in methanol, acetonitrile or dichloromethane, and film hydration is used. Sequence-level targeted drug-loaded nanomicelles can be prepared by using the same method: first, the drug-loaded nanomicelles are accumulated in the myocardial tissue of the ischemic site by the EPR effect of PEG-PE nanomicelles, and then the nanomicelles are wrapped around the nanomicelles. The "stealth" function of the coated PEG polymer shell reduces the phagocytosis of the reticuloendothelial system (RES) in the liver and spleen, and transports most drugs to the site of action; with the help of the charge effect of TPP cations, the drug carrier is mediated into ischemia Mitochondria in cardiomyocytes gradually target drugs to effector organelles, improving the specificity of drug action in vivo.

In conclusion, the present invention uses sequence-level targeted nanomicelles to target the drug for treating myocardial ischemia-reperfusion injury to the mitochondria of ischemic cardiomyocytes to exert pharmacological effects. First, use the EPR effect of PEG-PE nanomicelles to target and accumulate the drug carrier in the ischemic myocardium in the early stage of infarction; then use the charge effect of TPP cations to mediate the drug carrier into the negatively charged mitochondria of myocardial cells, which will act on Mitochondria of ischemic cardiomyocytes Drugs for the treatment of myocardial ischemia-reperfusion injury are gradually targeted to the mitochondria of ischemic cardiomyocytes to achieve precise drug delivery.

Description of drawings

Figure 1 is a process diagram of the preparation of sequence-level targeted drug-loaded nanomicelles by film hydration method; TPP-PEG-PE is a TPP-modified PEG-PE amphiphilic polymer material, and PEG-PE is a PEG-PE amphiphilic polymer , Puerarin is puerarin, Self-assembly is the process of self-assembly to form micelles, and solution is the reaction solvent; finally, the puerarin drug self-assembles with the above two polymer amphiphilic polymer materials to form a nano-micelle solution, and the puerarin drug is wrapped in The lipophilic inner layer of the nanomicelle is surrounded by hydrophilic PEG polymers;

Figure 2 is the particle size distribution and zeta potential diagram of the sequence-targeted nanomicelles loaded with puerarin;

Figure 3 shows the sequence targeting nanomicelles loaded with puerarin (PUE@TPP-PEG-PE), PEG-PE nanomicelles loaded with puerarin (PUE@PEG-PE) and the effect of puerarin (PUE) on isopropyl The protective effect of epinephrine (ISO) on apoptosis of H9c2 cardiomyocytes; among them, Figure 3-1 is Hoechst staining, Figure 3-2 is the percentage of Hoechst positive cells ( ^# p<0.05, ^## p<0.01), Figure 3 -3 is Caspase 3 activity ( ^# p<0.05, ^## p<0.01), Figure 3-4 is the expression of apoptosis-related proteins Bcl-2 and Bax ( ^# p<0.05, ^## p<0.01);

Figure 4 shows the ordered targeting nanomicelles loaded with coumarin (with green fluorescent components) (C6@TPP-PEG-PE), PEG-PE nanomicelles loaded with coumarin (C6@PEG-PE) and fragrance Cellular uptake test of beanin (C6).

The specific embodiment (raw materials and reagents not specified in the examples are all commercially available)

Taking the method of constructing puerarin-loaded order-level targeted nanomicelles as an example, it includes the following steps:

(1) Synthesis of TPP-PEG-PE block copolymer:

Accurately weigh 100mg (0.23mM) CTPP dissolved in 10mL chloroform, add 200μL triethylamine, 124mg (0.64mM) EDC and 74mg (0.64mM) NHS, stir at room temperature for 2h, then add 25mL concentration of 25mg/mL DSPE- PEG-NH ₂ (molecular weight: 2790, 0.22mM) in chloroform, under the protection of nitrogen, stirred at room temperature overnight, the crude reaction product was washed with glacial ether several times, centrifuged to remove the lower layer of precipitate, and then freeze-dried to obtain TPP- PEG-PE block copolymer, the specific synthetic route is as follows:

(2) Preparation of sequence-targeted nanomicelles loaded with puerarin:

Due to the presence of both hydrophilic and lipophilic groups in PEG-PE, puerarin happens to be a hydrophobic drug and is entrapped in the hydrophobic core of PEG-PE nanomicelles. PEG-PE, TPP-PEG-PE and puerarin were co-dissolved in methanol, In acetonitrile or dichloromethane, the membrane hydration method was used to prepare sequence-level targeted drug-loaded nanomicelles (see Figure 1 for the preparation route). After testing, it was found that the average particle size of the sequence-targeted nanomicelles loaded with puerarin was about 17nm, and it was in a good normal distribution. The zeta was -1mV, and the stability was good (results shown in Figure 2).

(3) Protective effect of puerarin-loaded sequential targeting nanomicelles on apoptosis of H9c2 cardiomyocytes induced by isoproterenol (ISO):

Con: blank control group; ISO: model group, 10 μM isoproterenol (ISO) was used to induce apoptosis of H9c2 cardiomyocytes; ISO+PUE: H9c2 cardiomyocytes were first incubated with 20 μM puerarin (PUE) for 0.5 h, Then add 10 μM isoproterenol (ISO) to induce apoptosis of cardiomyocytes, and continue co-incubating for 24 h; ISO+PUE@PEG-PE: H9c2 cardiomyocytes were first mixed with 20 μM puerarin-loaded PEG-PE nanomicelles (PUE@PEG- PE, containing an equal dose of PUE) were incubated together for 0.5h, then 10μM isoproterenol (ISO) was added to induce cardiomyocyte apoptosis, and the incubation was continued for 24h; ISO+PUE@TPP-PEG-PE: H9c2 cardiomyocytes were first mixed with 20μM Puerarin-targeted nanomicelles (PUE@TPP-PEG-PE, containing equal doses of PUE) were co-incubated for 0.5 h, then 10 μM isoproterenol (ISO) was added to induce cardiomyocyte apoptosis, and co-incubation was continued for 24 h. Finally, each group was tested as follows.

① Hoechst positive cell percentage The cells in each group were stained with Hoechst 33258, and the apoptosis of the cells in each group was observed under a fluorescent microscope. The results showed that the cells in the ISO-induced apoptosis group of H9c2 cardiomyocytes showed obvious atrophy and cell debris (arrows) , indicating that the model group drug ISO induced apoptosis of H9c2 cardiomyocytes successfully, while PUE, PUE@PEG-PE, PUE@TPP-PEG-PE pretreatment groups can significantly reduce the number of cardiomyocytes, and PUE@TPP-PEG-PE pretreatment The anti-apoptotic effect of the treatment group was significantly better than that of the PUE and PUE@PEG-PE groups, and there were significant differences (see Figure 3-1, 3-2).

②Caspase 3 activity Apoptosis-related Caspase 3 activity was detected in cells in each group. The results showed that the Caspase 3 activity in the ISO-induced H9c2 cardiomyocyte apoptosis group was the highest, indicating that the model group was successful in inducing H9c2 cardiomyocyte apoptosis, while PUE and PUE@PEG-PE , PUE@TPP-PEG-PE pretreatment group can significantly reduce the activity of Caspase 3, and the PUE@TPP-PEG-PE pretreatment group can reduce the activity of Caspase 3 significantly better than PUE and PUE@PEG-PE groups, there is a significant difference ( See Figure 3-3).

③ Expression of apoptosis-related proteins Bcl-2 and Bax The expression of apoptosis-related proteins Bcl-2 (anti-apoptosis) and Bax (pro-apoptosis) was detected in each group of cells, and the results showed that ISO induced the apoptosis of H9c2 cardiomyocytes by histone Bcl -2 expression was the lowest and Bax was the highest, indicating that the model group was successful in inducing the apoptosis of H9c2 cardiomyocytes, while the PUE, PUE@PEG-PE, and PUE@TPP-PEG-PE pretreatment groups could significantly increase the expression of Bcl-2 and reduce the expression of Bax Among them, the increase of Bcl-2 expression and the decrease of Bax expression in the PUE@TPP-PEG-PE pretreatment group were significantly better than those of the PUE and PUE@PEG-PE groups, and there were significant differences (see Figure 3-4).

The above results indicate that TPP-PEG-PE nanomicelles can significantly enhance the anti-apoptotic effect of puerarin, and the activity of Caspase 3 and the expression of apoptosis-related proteins Bcl-2 and Bax are closely related to puerarin's inhibition of mitochondrial mPTP opening. , thus indirectly indicating that TPP-PEG-PE nanomicelles can target the drug sequence to the mitochondria of ischemic cardiomyocytes, and exert pharmacological effects at the mitochondrial targeting site.

(4) Cellular uptake assay of sequence-targeted nanomicelles loaded with coumarin

Coumarin-6 (Coumarin-6, C6) fluorescent substance (green) was loaded into PEG-PE and TPP-PEG-PE nanomicelles by thin film hydration method to prepare coumarin-loaded PEG-PE nanoparticles Micelles (C6@PEG-PE) and sequential targeting nanomicelles loaded with coumarin (C6@TPP-PEG-PE). Then, coumarin-6 (C6), coumarin-loaded PEG-PE nanomicelles (C6@PEG-PE) and coumarin-loaded sequential targeting nanomicelles (C6@TPP-PEG- PE) were co-incubated with H9c2 cardiomyocytes, and the uptake of coumarin-6 fluorescent substance in the cells was observed by fluorescence microscope at 2h and 4h. It was found that the uptake of coumarin-6 in the cells was very little, while the uptake of C6@PEG-PE and C6@TPP-PEG-PE by the cells was time-dependent and basically gathered around the nucleus (DAPI staining: blue fluorescence), C6@TPP-PEG-PE is particularly obvious. It can be seen that TPP cations can accelerate the transmembrane transport of PEG-PE nanomicelles, so the amount of drug uptake by cells will also increase accordingly. It is speculated that the drug-loaded The system can enhance the pharmacological effect of the drug.

Claims (5)

1. A method for constructing sequence-level targeted ischemic cardiomyocyte mitochondria drug-loaded nanomicelles, said method comprising the steps of: (1) Synthesis of TPP-PEG-PE block copolymer: Dissolve CTPP in chloroform, then add triethylamine, NHS and EDC, stir at 5-30°C and then add DSPE-PEG-NH ₂ in chloroform solution, in Under the protection of inert gas such as nitrogen or argon, stir overnight at 5-30°C, wash the crude reactant several times with ice ether and centrifuge (or dialysis bag to remove small molecular substances), remove the lower layer of sediment, and then freeze the lower layer of sediment Dried to obtain TPP-PEG-PE block copolymer; described TPP-PEG-PE block copolymer structural formula is as shown in formula I: (2) Preparation of sequence-level targeted drug-loaded nanomicelles: PEG-PE, TPP-PEG-PE and drugs are co-dissolved in an organic solvent, and the sequence-level targeted drug-loaded nanomicelles can be prepared by thin film hydration method ; The medicament is a medicament for treating myocardial ischemia-reperfusion injury by regulating the mitochondrial function of ischemic cardiomyocytes. 2. The method according to claim 1, characterized in that, the average particle diameter of the sequence-level targeted nanomicelles loaded with puerarin is 10-100nm, which is in a good normal distribution, and the zeta potential is-20- +20mV. 3. the method for claim 1, is characterized in that, in described step (1), the mass volume ratio of described CTPP and chloroform is (90-110mg): (9-11mL), described CTPP and three The mass volume ratio of ethylamine is (90-110mg):(180-220 μ L), the mass ratio of the CTPP and NHS is 100:(70-80), and the mass ratio of CTPP and EDC is 100:(120-125) , the CTPP and DSPE-PEG- _NH The mass volume ratio of the chloroform solution is (90-110mg): (18-27mL). 4. The method according to claim 3, characterized in that, in the step (1), the concentration of the CTPP is 0.20-0.25mM, the concentration of the NHS is 0.60-0.70mM, and the DSPE-PEG The concentration of DSPE-PEG-NH ₂ in the chloroform solution of -NH ₂ is 20-30mg/mL. 5. the method for claim 1, is characterized in that, in described step (2), the mol ratio of described PEG-PE and medicine is (97-80): (15-25), and described TPP- The molar ratio of PEG-PE to drug is (3-20):(15-25).