CN108542880B - Method for constructing order-level targeted ischemic myocardial cell mitochondrion drug-loaded nano-micelle - Google Patents
Method for constructing order-level targeted ischemic myocardial cell mitochondrion drug-loaded nano-micelle Download PDFInfo
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- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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Abstract
The invention discloses a method for constructing sequence-level targeted ischemic myocardial cell mitochondria drug-loaded (model drugs such as puerarin, baicalin and the like, the commonness of which is that the drug-loaded nanoparticles can act on the ischemic myocardial cell mitochondria to treat myocardial ischemia reperfusion injury) nano-micelles. The method comprises the synthesis of TPP-PEG-PE segmented copolymer and the preparation of the order targeted drug-loaded nano-micelle. The invention adopts the sequence-level targeting nano-micelle to accurately deliver the puerarin medicament to the mitochondria of ischemic myocardial cells, thereby playing pharmacological action. Firstly, the drug carrier is accumulated in ischemic myocardium at the early stage of infarction in a targeted manner by using the EPR effect of the PEG-PE nano micelle; then, the drug carrier is mediated to enter myocardial cell mitochondria with negative charge by the charge effect of TPP cation, and the drugs such as puerarin and the like are gradually targeted to the ischemic myocardial cell mitochondria, thereby realizing precise drug delivery.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a method for constructing a sequence-level targeted ischemic cardiomyocyte mitochondrion drug-loaded nano-micelle.
Background
Acute Myocardial Infarction (AMI) is a clinically common critical condition that can lead to sudden death in humans. Coronary artery interventional therapy for recovering coronary blood flow is a common treatment method for clinically saving AMI patients at present, but the caused myocardial Ischemia/reperfusion (I/R) injury can promote the ischemic myocardial infarction area to increase by 20-50%, and no good solution is available at present. The myocardial I/R injury mechanism is mainly expressed as mitochondrial dysfunction and myocardial cell apoptosis and autophagy induced by the mitochondrial dysfunction, and the recovery of the normal function of mitochondria undoubtedly has great scientific research value for relieving myocardial I/R injury. At present, most of medicines for clinically treating myocardial I/R injury lack tissue specificity, have low selectivity on myocardial cells, and have low concentration reaching mitochondria effect targets, so that the expected curative effect of the medicines is not ideal. Therefore, the realization of the drug targeting to the mitochondria of ischemic myocardial cells has very important clinical significance.
(1) Myocardial I/R injury is closely related to mitochondrial mPTP patency
Mitochondrial dysfunction is closely related to each link of myocardial I/R injury, especially myocardial apoptosis and autophagy. Under physiological conditions, permeability transition pores (mPTP) on a Mitochondrial membrane are in a closed state, at the early stage of cardiac muscle I/R, mitochondria deviate from a physiological equilibrium point, a large amount of superoxide anions and active oxygen are generated, so that Mitochondrial calcium overload and oxidative stress are increased, oxidative damage is generated on the Mitochondrial membrane, enzymes and electron transfer chains, ATP is reduced, the mTP on the Mitochondrial membrane is promoted to be opened, the permeability of the Mitochondrial inner membrane is increased rapidly, Cytochrome C (Cytochrome C, Cyt C) is released into cytoplasm from the mitochondria, the cascade effect of Caspases is triggered, the expression of apoptosis-related protein Bcl-2 (anti-apoptosis) is reduced, the expression of Bax (pro-apoptosis) is increased, and myocardial cell apoptosis is induced; beclin 1-mediated autophagy can also be activated, and is characterized by the presence of a large number of autophagosomes and autophagosomes in the cell, and finally the cell is degraded by the autophagosomes to trigger autophagic cell death. Therefore, the preparation method can effectively inhibit the opening of mitochondrion mPTP and reduce the apoptosis and autophagy of myocardial cells in the early reperfusion stage, and can be used as a new treatment strategy for myocardial I/R injury.
(2) Puerarin as model drug has effect of inhibiting opening of mitochondria mPTP
Puerarin is an effective component extracted from Chinese medicinal material pueraria root, and has several pharmacological actions of dilating coronary artery, improving coronary circulation, reducing myocardial I/R injury and inhibiting platelet aggregation, etc. Puerarin injection is clinically used to treat coronary heart disease, angina pectoris, myocardial infarction, etc. At present, the research on a mechanism of reducing myocardial I/R injury by puerarin discovers that the puerarin can inhibit the opening of mitochondria mTP and regulate the physiological function of mitochondria. Gao Q and Yao H researches and the like find that puerarin protects the effect of I/R on damaging cardiac muscle and promotes mitoKATPChannel and mitoKCaThe opening of the channel further stabilizes the mitochondrial membrane potential and inhibits the mPTP opening. The above literature reports show that: puerarin can maintain steady state balance of mitochondria, inhibit apoptosis and autophagy of myocardial cells and increase tolerance of mitochondria to ischemia by inhibiting mTP opening, so that myocardial I/R injury is reduced, drug delivery to effect target sites of mitochondria is speculated, accurate drug delivery is realized, and drug treatment effect and drug administration dosage are improved to a great extent. Delivery of puerarin drugs to mitochondria requires overcoming multiple biological barriers: firstly, puerarin drug carriers must be enriched in ischemic myocardium; secondly, the drug carrier needs to overcome the capture of liver-spleen reticuloendothelial system (RES) and ensure that the drug can maintain effective concentration when reaching a target organ; finally, the cytosol is in a high viscosity state, which prevents mitochondria from taking in the drug, while intracellular lysosomes are also able to degrade the drug, further reducing the concentration of the drug reaching effector organelles. All the problems are closely related to the construction of a high-efficiency mitochondrial targeting drug delivery system, and are worthy of further discussion and systematic research.
(3) The PEG-PE nano micelle can be passively permeated and enriched in ischemic cardiac muscle parts, and can reduce the capture of the medicine by RES
The mitochondrion targeting drug delivery system can greatly improve the treatment effect of the drug, and the research report in the aspect is mainly found in tumor drugs. For example, Zhang C group establishes a source innovative mitochondrial targeting system, provides a safe and efficient carrier platform for mitochondrial target antitumor drugs, and establishes an ischemic myocardial cell mitochondrial targeting drug delivery system with similar functions, which may be a brand-new and challenging research field. Mitochondria are distributed in vivo histiocytes, and how to gather the medicine at the ischemic myocardial part is the precondition of realizing targeted administration of the mitochondria of the myocardial cells. According to the reports in the literature, the permeability of the vascular intima in the ischemic myocardial region is abnormally increased, and PEG (polyethylene glycol) polymers have better high permeability and retention Effect (EPR) in the ischemic myocardial region. The normal tissue vascular wall cells are closely arranged and have complete structure, so the PEG polymer drug delivery system can hardly seep out along with the blood circulation process, but PEG polymers can penetrate through the vascular intima with increased permeability in the myocardial ischemia area and gather in the myocardial tissue in the early stage of infarction. The results of our earlier studies found that: the AUC of the PEG puerarin in the myocardial tissue of an acute myocardial ischemia model rat is 1.7 times that of a normal rat, and the PEG puerarin has obvious ischemic myocardial targeting property; the AUC of the puerarin PEG-PE nano micelle in the heart of the acute ischemia model mouse is 1.9 times that of the heart of the normal mouse, which shows that the puerarin PEG-PE nano micelle has better heart targeting property in the early region of myocardial infarction and can accumulate the medicament in the ischemic myocardium. Lukyanov AN et al also found that polyethylene glycol-derivatized phosphatidylethanolamine (PEG-PE) nano-micelles mainly aggregated myocardial tissues in the early infarct stage, and the content thereof was 8 times that of the myocardial tissues in the non-infarct region. The previous research and literature report support that the PEG polymer and the PEG-PE nano micelle have better EPR effect in the ischemic myocardial part, can passively accumulate the medicament in the pathological change part of the heart, and improve the selectivity of the medicament. In addition, PEG high molecules at the periphery of the PEG-PE nano micelle have good biocompatibility, and the nano micelle has small particle size, so that the capture of a liver-spleen reticuloendothelial system (RES) can be reduced.
(4) TPP cation can mediate drug carrier to target into mitochondria and reduce degradation of drug by lysosome
After the drug-loaded micelle is retained in an ischemic myocardial part, the key problem of how to overcome the blockage of cell sap and the damage of lysosome is that the drug-loaded micelle is targeted to mitochondria. At present, a large number of researches prove that Triphenyl phosphonium (TPP) cations have mitochondrial targeting, because mitochondrial inner membranes have 150-180mv negative charges, cell membranes have 30-60mv negative charges, and TPP is triphenylphosphine cations with positive charges, the positive charges and the negative charges attract each other, the capacity of the TPP cations for gathering the TPP cations on the mitochondria is improved by 100-500 times, and the obstruction of high-viscosity cell sap can be overcome. The literature reports that TPP cations can mediate tumor drugs, antioxidant drugs and G (5) -PAMAM dendritic block copolymers to enter mitochondria in a targeted manner, and by analogy, the TPP cations can also be used as active targeting molecules of drug-loaded nano-micelles to be delivered to the mitochondria of cardiac muscle cells; in addition, TPP cation has a 'proton sponge' effect in lysosomes, can reduce the damage of intracellular lysosomes and endosomes to nano micelles, has a 'lysosome escape' effect, and further improves the concentration of the drug in effector organelles.
By searching domestic and foreign documents, the reports of hyaluronic acid modified puerarin PEG-PLGA nano particles, puerarin PEG-PE nano micelles, PEG puerarin and the like are found, the contents mainly relate to the slow release of medicaments in vitro of nano materials, the reduction of erythrocyte hemolysis, the protection effect on ischemia reperfusion cardiac muscle, the anti-apoptosis, the tissue distribution in acute myocardial ischemia animals and the like, and the construction of the medicament-carrying nano micelle of the sequence targeted ischemia cardiac muscle cell mitochondria needs to overcome a plurality of technical bottlenecks, in view of the EPR effect of PEG-PE macromolecules at the ischemia cardiac muscle part and the charge effect of TPP cations, the medicament such as puerarin and the like is gradually delivered to the effect part of the ischemia cardiac muscle cell mitochondria, the permeability transition hole opening on the mitochondrial membrane is inhibited, thereby reducing a large amount of apoptosis and autophagy of cardiac muscle cells in the ischemia reperfusion period, compared with the prior puerarin nano medicaments, the drug is accurately delivered to the action target of the effector organelle, so that the action effect of the drug is improved to a great extent, and the adverse reaction of the drug can be reduced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for constructing a sequence-level targeted ischemic myocardial cell mitochondrion drug-loaded nano micelle.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the method for constructing the sequence-level targeted ischemic myocardial cell mitochondrion drug-loaded nano-micelle comprises the following steps:
(1) synthesis of TPP-PEG-PE Block copolymer: dissolving CTPP in chloroform, adding triethylamine, NHS and EDC, stirring at room temperature (5-30 ℃), and adding DSPE-PEG-NH2Stirring the chloroform solution at room temperature (5-30 ℃) overnight under the protection of nitrogen, washing a crude reaction product with ethyl acetate for multiple times, centrifuging, taking a lower-layer precipitate, and freeze-drying the lower-layer precipitate to obtain a TPP-PEG-PE segmented copolymer; the structural formula of the TPP-PEG-PE segmented copolymer is shown as a formula I:
(2) preparing a sequence-level targeting drug-loaded nano micelle: dissolving PEG-PE, TPP-PEG-PE and puerarin in organic solvent such as methanol, acetonitrile or dichloromethane, and preparing puerarin-loaded sequence-level targeting nano micelle by thin film hydration method; the medicine is a medicine acting on ischemic myocardial cell mitochondria to treat myocardial ischemia reperfusion injury, and is preferably puerarin or baicalin and the like.
Preferably, the puerarin-loaded sequence-targeted nano-micelle has an average particle size of 10-100 nm, preferably about 17nm, and is in a good normal distribution, and the zeta potential is-20- +20 mV.
Preferably, in the step (1), the mass-to-volume ratio of the CTPP to the chloroform is (90-110 mg): 9-11 mL, preferably 100mg:10mL, the mass-to-volume ratio of the CTPP to the triethylamine is (90-110 mg): 180-220 μ L, preferably 100mg:200 μ L, the mass-to-NHS ratio is 100 (70-80), preferably 100:74, the mass-to-EDC ratio is 100 (120-125), preferably 100:124, and the mass-to-volume ratio of the CTPP to the DSPE-PEG-NH is 100 (70-80)2The mass-to-volume ratio of the chloroform solution (2) to (90-110 mg) to (18-28 mL), preferably 100mg to 25 mL.
Preference is given toIn step (1), the concentration of CTPP is 0.23mM, the concentration of NHS is 0.50-0.70 mM, preferably 0.64mM, and the concentration of DSPE-PEG-NH is 0.23mM2In chloroform solution of (3) DSPE-PEG-NH2The concentration of (b) is 20 to 30mg/mL, preferably 25 mg/mL.
Preferably, in step (2), the molar ratio of PEG-PE to drug is (97-80): 15-25), preferably 90:20, and the molar ratio of TPP-PEG-PE to drug is (3-20): 15-25, preferably 10: 20. The invention is further illustrated below:
synthesis of TPP-PEG-PE Block copolymer:
100mg (0.23mM) of CTPP was precisely weighed and dissolved in 10mL of chloroform, 200. mu.L of triethylamine, 124mg (0.64mM) of EDC and 74mg (0.64mM) of NHS were added thereto, and the mixture was stirred at room temperature for 2 hours, then 25mL of a chloroform solution of DSPE-PEG-NH2 (molecular weight: 2800, 0.22mM) was added thereto, and the mixture was stirred at room temperature overnight under nitrogen protection, the crude reaction was washed with ethyl acetate several times, the lower layer precipitate was centrifuged, and freeze-dried to obtain a TPP-PEG-PE block copolymer, which was specifically synthesized by the following scheme:
preparing a drug-loading sequence-level targeting nano micelle:
because PEG-PE has both hydrophilic and lipophilic groups, drugs acting on ischemic myocardial cell mitochondria to treat myocardial ischemia reperfusion injury, such as puerarin, baicalin and the like, belong to hydrophobic drugs, and are encapsulated in the hydrophobic core of the PEG-PE nano micelle. PEG-PE and TPP-PEG-PE are respectively co-dissolved with drugs (the molar ratio of the PEG-PE to the drugs is 97:20, and the molar ratio of the TPP-PEG-PE to the drugs is 3:20) in methanol, acetonitrile or dichloromethane, and a film hydration method is adopted to prepare the sequence-level targeting drug-loaded nano-micelle (the preparation route is shown in figure 1). Through detection, the average particle size of the sequence-level targeting drug-loaded nano-micelle is about 17nm and is in good normal distribution, the zeta potential is-1 mV, and the stability is good (the result is shown in figure 2).
The invention constructs the order targeted ischemic myocardial cell mitochondrion drug-loaded nano-micelle with the action process as follows: PEG-PE, TPP-PEG-PE and the medicine are dissolved in methanol, acetonitrile or dichloromethane together, and a film hydration method is adopted to prepare the carrier-order targeted medicine-carrying nano micelle: firstly, the drug-loaded nano-micelle is accumulated in the myocardial tissue of an ischemic part by the EPR effect of the PEG-PE nano-micelle, and the phagocytosis of a liver-spleen reticuloendothelial system (RES) is reduced by means of the invisible function of a PEG high-molecular shell layer coated on the periphery of the nano-micelle, and most of the drugs are transported to an action part; by means of the charge effect of TPP cation, the mediated medicine carrier enters mitochondria in ischemic myocardial cell, and the medicine is gradually targeted to effect organelle, so as to raise the specificity of medicine in vivo action.
In a word, the invention adopts the sequence-level targeting nano micelle to deliver the medicament for treating the myocardial ischemia reperfusion injury to the mitochondria of ischemic myocardial cells in a targeting way, and plays a pharmacological role. Firstly, the drug carrier is accumulated in ischemic myocardium at the early stage of infarction in a targeted manner by using the EPR effect of the PEG-PE nano micelle; then, the drug carrier is mediated to enter myocardial cell mitochondria with negative charge by the charge effect of TPP positive ions, and the drug which acts on the ischemic myocardial cell mitochondria to treat myocardial ischemia reperfusion injury is targeted to the ischemic myocardial cell mitochondria step by step, thereby realizing accurate drug delivery.
Drawings
FIG. 1 is a process diagram of preparing a sequence-level targeting drug-loaded nano-micelle by a thin film hydration method; TPP-PEG-PE is a TPP modified PEG-PE amphiphilic polymer material, PEG-PE is a PEG-PE amphiphilic polymer, Puerarin is Puerarin, Self-assembly is carried out to form micelle, and solubilization is a reaction solvent; finally, puerarin medicine and the 2 kinds of macromolecular amphiphilic macromolecular materials are self-assembled to form nano micelle solution, the puerarin medicine is wrapped in the lipophilic core inner layer of the nano micelle, and hydrophilic PEG macromolecules are arranged on the periphery of the nano micelle;
FIG. 2 is a graph of the particle size distribution and zeta potential of the puerarin-loaded sequence-targeted nanomicelle;
FIG. 3 shows the protective effect of puerarin-loaded sequence-targeted nanomicelles (PUE @ TPP-PEG-PE), puerarin-loaded PEG-PE nanomicelles (PUE @ PEG-PE) and Puerarin (PUE) on Isoproterenol (ISO) induced H9c2 cardiomyocyte apoptosis; wherein, FIG. 3-1 is HHoechst staining, FIG. 3-2. Hoechst Positive cell percentage (#p<0.05,##p<0.01), FIGS. 3-3 are Caspase 3 activities (A)#p<0.05,##p<0.01), FIGS. 3-4 are the expression of apoptosis-related proteins Bcl-2, Bax: (#p<0.05,##p<0.01)；
FIG. 4 shows the cell uptake assay of coumarin-loaded (with green fluorescent component) sequence-targeted nanomicelles (C6@ TPP-PEG-PE), coumarin-loaded PEG-PE nanomicelles (C6@ PEG-PE) and coumarin (C6).
Specific examples (the starting materials and reagents not specifically described in the examples are commercially available)
Taking a method for constructing the puerarin-loaded sequence-level targeting nano micelle as an example, the method comprises the following steps:
(1) synthesis of TPP-PEG-PE Block copolymer:
100mg (0.23mM) of CTPP was precisely weighed, dissolved in 10mL of chloroform, added with 200. mu.L of triethylamine, 124mg (0.64mM) of EDC and 74mg (0.64mM) of NHS, stirred at room temperature for 2 hours, and then added with 25mL of DSPE-PEG-NH having a concentration of 25mg/mL2(molecular weight: 2790, 0.22mM) chloroform solution, under the protection of nitrogen, stirring overnight at room temperature, washing the crude reaction product with diethyl ether for multiple times, centrifuging to remove the lower layer precipitate, and freeze-drying to obtain the TPP-PEG-PE block copolymer, wherein the specific synthetic route is as follows:
(2) preparing a puerarin-loaded sequence-level targeting nano micelle:
because the PEG-PE has both hydrophilic and lipophilic groups, the puerarin just belongs to hydrophobic drugs and is encapsulated in the hydrophobic core of the PEG-PE nano micelle. Dissolving PEG-PE and TPP-PEG-PE and puerarin (the molar ratio of PEG-PE to puerarin is 97:20, and the molar ratio of TPP-PEG-PE to puerarin is 3:20) in methanol, acetonitrile or dichloromethane respectively, and preparing the sequence-level targeting drug-loaded nano-micelle by a thin film hydration method (the preparation route is shown in figure 1). Through detection, the puerarin-loaded sequence-level targeting nano micelle has an average particle size of about 17nm and is in good normal distribution, zeta is-1 mV, and stability is good (see a result in FIG. 2).
(3) The puerarin-loaded sequence-level targeting nano micelle has a protective effect on Isoproterenol (ISO) induced H9c2 myocardial cell apoptosis:
con: blank control group; ISO: model group, using 10 μ M Isoproterenol (ISO) to induce H9c2 myocardial cell apoptosis modeling; ISO + PUE: h9c2 cardiac muscle cells are incubated with 20 mu M Puerarin (PUE) medicine for 0.5H, then 10 mu M Isoproterenol (ISO) is added to induce the cardiac muscle cells to die, and the incubation is continued for 24H; ISO + PUE @ PEG-PE: h9c2 myocardial cells are incubated with 20 mu M puerarin-loaded PEG-PE nano micelle (PUE @ PEG-PE, containing equal dose of PUE) for 0.5H, then 10 mu M Isoproterenol (ISO) is added to induce the myocardial cells to die, and the incubation is continued for 24H; ISO + PUE @ TPP-PEG-PE: h9c2 myocardial cells and 20 mu M puerarin-loaded sequence-level targeting nano-micelle (PUE @ TPP-PEG-PE, containing equal dose of PUE) are incubated for 0.5H, 10 mu M Isoproterenol (ISO) is added to induce the myocardial cells to die, the incubation is continued for 24H, and finally the following detection is carried out on each group.
the percentage of Hoechst positive cells of each group is stained by Hoechst 33258, the apoptosis condition of each group of cells is observed under a fluorescence microscope, and the result shows that the cell morphology of the ISO-induced H9c2 myocardial cell apoptosis group has obvious atrophy and cell debris (indicated by an arrow), which indicates that the experiment of the ISO-induced H9c2 myocardial cell apoptosis of the model group medicament is successful, and the PUE, PUE @ PEG-PE and PUE @ TPP-PEG-PE pretreatment groups can obviously reduce the number of the myocardial cells, wherein the anti-apoptosis effect of the PUE @ TPP-PEG-PE pretreatment groups is obviously superior to that of the PUE and PUE @ PEG-PE groups, and the significant difference exists (see figures 3-1 and 3-2).
secondly, detecting apoptosis-related Caspase 3 activity of cells of each group of Caspase 3 activity, wherein the result shows that the Caspase 3 activity of the H9c2 myocardial cell apoptosis group induced by ISO is highest, which indicates that the H9c2 myocardial cell apoptosis test induced by the model group is successful, and the PUE, PUE @ PEG-PE and PUE @ TPP-PEG-PE pretreatment groups can obviously reduce the Caspase 3 activity, wherein the PUE @ TPP-PE pretreatment groups reduce the Caspase 3 activity and are obviously better than the PUE and PUE @ PEG-PE groups, and significant difference exists (see figure 3-3).
expression of apoptosis-related proteins Bcl-2 and Bax cells of each group are used for detecting expression of apoptosis-related proteins Bcl-2 (anti-apoptosis) and Bax (pro-apoptosis), results show that ISO-induced H9c2 myocardial cell apoptosis histone Bcl-2 expression is lowest, Bax is highest, and the results show that a model group is successful in inducing H9c2 myocardial cell apoptosis tests, and PUE, PUE @ PEG-PE and PUE @ TPP-PEG-PE pretreatment groups can obviously increase Bcl-2 expression and reduce Bax expression, wherein the PUE @ TPP-PEG-PE pretreatment groups are obviously superior to the PUE and PUE @ PEG-PE groups in terms of increasing Bcl-2 expression and reducing Bax expression, and significant differences exist (see fig. 3-4).
The results show that the TPP-PEG-PE nano micelle can obviously enhance the anti-apoptosis effect of puerarin, and the expression of Caspase 3 activity, apoptosis-related proteins Bcl-2 and Bax are closely related to the inhibition of mitochondria mPTP opening by puerarin, so that the TPP-PEG-PE nano micelle indirectly shows that the TPP-PEG-PE nano micelle can target the drug sequence level to the mitochondria of ischemic myocardial cells and play a pharmacological effect at the mitochondria target position.
(4) Coumarin-loaded sequence-targeted nano-micelle cell uptake assay
Loading a Coumarin-6 (Coumarin-6, C6) fluorescent substance (green) into PEG-PE and TPP-PEG-PE nano micelles by adopting a film hydration method to prepare Coumarin-loaded PEG-PE nano micelles (C6@ PEG-PE) and Coumarin-loaded sequence-targeted nano micelles (C6@ TPP-PEG-PE). Then, co-incubating coumarin-6 (C6), coumarin-loaded PEG-PE nano-micelle (C6@ PEG-PE) and coumarin-loaded sequence-targeted nano-micelle (C6@ TPP-PEG-PE) with H9C2 myocardial cells, and observing the intracellular uptake condition of the coumarin-6 fluorescent substance by adopting a fluorescence microscope at 2H and 4H. The result shows that the coumarin-6 group has little intracellular uptake, while the C6@ PEG-PE and the C6@ TPP-PEG-PE have obvious time-dependent resistance to the cell uptake and are basically gathered around the cell nucleus (DAPI staining: blue fluorescence), and the C6@ TPP-PEG-PE has obvious performance, so that TPP cation can accelerate the transmembrane transport of PEG-PE nano micelle, the cell uptake drug amount is correspondingly increased, and the drug loading system is supposed to enhance the pharmacological action of the drug.
Claims (3)
1. A method for constructing a sequence-level targeted ischemic cardiomyocyte mitochondrion drug-loaded nano-micelle is characterized by comprising the following steps:
(1) synthesis of TPP-PEG-PE Block copolymer: dissolving CTPP in chloroform, adding triethylamine, NHS and EDC, stirring at 5-30 ℃, and then adding DSPE-PEG-NH2Stirring the chloroform solution at 5-30 ℃ overnight under the protection of nitrogen or argon, washing the crude reaction product by using glacial ethyl ether for multiple times, centrifuging, taking the sediment at the lower layer, and freeze-drying the sediment at the lower layer to obtain the TPP-PEG-PE segmented copolymer; the structural formula of the TPP-PEG-PE segmented copolymer is shown as a formula I:
(2) preparing a drug-loading sequence-level targeted drug-loading nano micelle: mixing PEG-PE, TPP-PEG-PE and medicine according to the following ratio (97-80): (3-20): (15-25) are dissolved in an organic solvent together, and a thin film hydration method is adopted to prepare the drug-loaded sequence-level targeted drug-loaded nano-micelle; the medicine is a medicine acting on ischemic myocardial cell mitochondria to treat myocardial ischemia reperfusion injury; the medicine is puerarin; the average particle size of the puerarin-loaded sequence-targeted drug-loaded nano micelle is 10-17 nm, the puerarin-loaded sequence-targeted drug-loaded nano micelle is in good normal distribution, and the zeta is-20- +20 mV.
2. The method of claim 1, wherein in step (1), the mass-to-volume ratio of CTPP to chloroform is (90-110 mg): 9-11 mL), the mass-to-volume ratio of CTPP to triethylamine is (90-110 mg): 180-220 μ L), the mass ratio of CTPP to NHS is 100 (70-80), the mass ratio of CTPP to EDC is 100 (120-125), and the mass ratio of CTPP to DSPE-PEG-NH is 100 (120-125)2The mass-to-volume ratio of the chloroform solution (2) to (90-110 mg) to (18-27 mL).
3. The method of claim 2, wherein in the step (1), the concentration of the CTPP is 0.20 to 0.25mM, the concentration of the NHS is 0.60 to 0.70mM, and the DSPE-PEG-NH is2In chloroform solution of (3) DSPE-PEG-NH2In a concentration of 20-30 mgmL。
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