Drug carrier, micelle, anti-inflammatory and / or anti-atherosclerosis pharmaceutical preparation, and preparation method and application thereof

Abstract

The invention provides a drug carrier, a micelle, an anti-inflammatory / anti-atherosclerosis drug preparation and a preparation method and application thereof. The drug carrier is an amphiphilic molecule, the micelle is formed by self-assembling the amphiphilic molecule, and the drug carrier and the micelle comprise: a) a hydrophilic part comprising a hydrophilic heparin compound; and b) a hydrophobic moiety, the hydrophobic moiety being lipoic acid. The hydrophilic heparin compound is connected with the lipoic acid in a specific manner. The micelles can interfere with the functions of mononuclear / macrophages in multiple links, and the anti-inflammatory and anti-atherosclerosis effects are achieved. In addition, the micelle is high in stability, simple in preparation method and convenient for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and relates to pharmaceutical carriers, micelles, anti-inflammatory and anti-atherosclerotic preparations, and preparation methods and uses thereof. Background Art

[0002] Atherosclerosis is a chronic vascular inflammatory disease. At present, the clinical treatment of atherosclerosis mainly includes lipid-lowering therapy represented by statins and antiplatelet therapy represented by aspirin. Statins and antiplatelet drugs can effectively inhibit some links in the development of atherosclerosis, but cannot completely prevent the inflammatory process of atherosclerosis. Atherosclerosis originates from vascular endothelial damage. The damaged vascular endothelial cells overexpress adhesion proteins (including intercellular adhesion factors, intervascular adhesion factors, selectins, integrins, etc.) to recruit monocytes in the blood. Monocytes adhere to, roll and invade the subintimal membrane on the damaged vascular endothelium, and polarize into macrophages under the stimulation of the microenvironment. Macrophages internalize oxidized low-density lipoprotein to form foam cells, which produce reactive oxygen species (ROS), secrete inflammatory factors, and aggravate plaque inflammation. Therefore, vascular endothelial cells, monocytes, macrophages, and foam cells are all involved in the plaque inflammation cascade.

[0003] In order to inhibit the inflammatory response of atherosclerosis, researchers have studied many targets of inflammation and designed a variety of drug delivery systems targeting vascular inflammation. The toxic side effects and poor effects of drug delivery systems in actual applications have greatly limited their clinical use. The complex molecular design of carrier materials increases the uncontrollability of the chemical structure, which can lead to poor therapeutic effects. Therefore, it is not easy to select materials with therapeutic activity and high biosafety and design simple and feasible synthetic routes. Summary of the invention

[0004] One of the purposes of the present invention is to overcome the deficiencies of the prior art and provide a drug carrier and micelle with anti-inflammatory and anti-atherosclerotic effects, as well as their preparation methods and uses. The drug carrier and micelle contain a hydrophilic part and a hydrophobic part. The drug carrier can self-assemble to form nanoscale micelles, which can target the plaque endothelium, block the recruitment of early inflammatory cells by P-selectin, inhibit the adhesion of monocytes to vascular endothelial cells, and reduce the level of plaque reactive oxygen, thereby alleviating atherosclerosis. Finally, the purpose of small side effects, high bioavailability, and good efficacy is achieved.

[0005] To achieve the above and other purposes, the present invention provides a drug carrier, which comprises: a) a hydrophilic part, wherein the hydrophilic part comprises a hydrophilic heparin compound, and b) a hydrophobic part, wherein the hydrophobic part is lipoic acid, and the hydrophilic heparin compound is selected from: one or more of unfractionated heparin, low molecular weight heparin, basil polysaccharide, and kelp polysaccharide; the hydrophilic heparin compound and the lipoic acid are connected by the following connection mode, and the connection mode is selected from: one or more of an ester bond, a pH-sensitive hydrazone bond, an imine bond, an alcohol ether bond, an enzyme-sensitive linker, and an active oxygen-sensitive linker.

[0006] Heparin was first discovered in the liver and is named after it. It is a mucopolysaccharide sulfate composed of glucosamine, L-iduronide, N-acetylglucosamine and D-glucuronic acid in alternation. It has an average molecular weight of 15KDa and is strongly acidic. Hydrophilic heparin compounds are derivatives of heparin and are hydrophilic.

[0007] Lipoic acid can act as a coenzyme to participate in the acyl transfer in the metabolism of substances in the body, and can eliminate free radicals that accelerate aging and cause disease. Lipoic acid enters cells after being absorbed through the intestines in the body, and has the characteristics of both fat solubility and water solubility.

[0008] As certain embodiments of the present invention, the hydrophilic heparin compound is low molecular weight heparin.

[0009] As certain embodiments of the present invention, the low molecular weight heparin is selected from one or more of enoxaparin sodium, dalteparin sodium, nadroparin sodium, and parvaparin sodium.

[0010] As certain embodiments of the present invention, the low molecular weight heparin is selected from one or more of enoxaparin sodium, dalteparin sodium, and nadroparin sodium.

[0011] As certain embodiments of the present invention, the low molecular weight heparin is enoxaparin sodium.

[0012] As certain embodiments of the present invention, the hydrophilic heparin compound and the lipoic acid are connected via one or more of an ester bond, a pH-sensitive hydrazone bond, a thioketal, an oxalate, and a disulfide bond.

[0013] As certain embodiments of the present invention, the hydrophilic heparin compound and the lipoic acid are directly connected via an ester bond.

[0014] The present invention also provides a micelle, which comprises the drug carrier and water or a pharmaceutically acceptable aqueous medium.

[0015] Micelles are aggregates (or supramolecular assemblies) of surfactant phospholipid molecules dispersed in a liquid to form a colloidal suspension (also called a related colloidal system). Typical micelles in water form aggregates with their hydrophilic head regions in contact with the surrounding solvent, isolating the hydrophobic single tail region in the center of the micelle. This phase is caused by the stacking behavior of single-tail lipids in the bilayer. It is difficult to fill all the volume inside the bilayer while adjusting the area of ​​each head group by hydration of the lipid head groups, which leads to the formation of micelles. Such micelles are called normal micelles (oil-in-water micelles). Reverse micelles have a head group in the center and the tail extends outward (water-in-oil micelles). Micelles are roughly spherical. They can also be ellipsoids, cylinders, and bilayers.

[0016] As certain embodiments of the present invention, the aqueous medium is an aqueous solution.

[0017] As certain embodiments of the present invention, the aqueous medium is PBS or physiological saline.

[0018] As certain embodiments of the present invention, the particle size of the micelle is micrometer-scale or nanometer-scale.

[0019] As certain embodiments of the present invention, the particle size of the micelle is in the range of 20 nm to 200 nm.

[0020] The particle size range of the micelles is selected from 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm.

[0021] As certain embodiments of the present invention, the particle size of the micelle is 140nm±20nm.

[0022] The present invention also provides a method for preparing the drug carrier, wherein the method comprises: (1) activating lipoic acid with a carboxyl activator to obtain activated lipoic acid, and (2) mixing a) the activated lipoic acid in (1) above and b) a hydrophilic heparin compound in a reaction solvent, so that a) and b) react to form an amphiphilic copolymer connected by the following connecting bonds, wherein the connecting bonds include: one or more of an ester bond, a pH-sensitive hydrazone bond, an imine bond, an alcohol ether bond, an enzyme-sensitive linker, and an active oxygen-sensitive linker.

[0023] As certain embodiments of the present invention, in step (1), the carboxyl activator is selected from: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), NHS (hydroxysuccinimide), EDC-DMAP (4-dimethylaminopyridine), EDC-HOBT (1-hydroxybenzotriazole), DCC (dicyclohexylcarbodiimide)-HOBT, and a combination of DCC-NHS and DCC-DMAP.

[0024] As certain embodiments of the present invention, in step (1), the molar ratio of the feed is: lipoic acid: activator = 1: (1-10); preferably, the molar ratio of the feed in step (1) is: lipoic acid: activator = 1:2.

[0025] As certain embodiments of the present invention, in step (1), the activation reaction is carried out in a reaction solvent, and the reaction solvent is selected from: water, N,N-dimethylformamide, formamide, a mixed solvent of N,N-dimethylformamide and formamide, dimethyl sulfoxide, a mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide, a mixed solvent of dimethyl sulfoxide and formamide, and a mixed solvent of water and formamide.

[0026] As certain embodiments of the present invention, the activation time in step (1) is 30 minutes to 5 hours, preferably 3-4 hours under the protection of nitrogen and away from light.

[0027] The activation time for activating lipoic acid with a carboxyl activator is selected from 30 min, 40 min, 50 min, 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3 h, 3.1 h, 3.2 h, 3.3 h, 3.4 h, 3.5 h, 3.6 h, 3.7 h, 3.8 h, 3.9 h, 4 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h, 4.9 h, 5 h.

[0028] As certain embodiments of the present invention, in step (2), the hydrophilic heparin compound is selected from one or more of unfractionated heparin, low molecular weight heparin, basil polysaccharide, and undaria pinnatifida polysaccharide, preferably low molecular weight heparin.

[0029] As certain embodiments of the present invention, preferably, the low molecular weight heparin is one or more selected from enoxaparin sodium, dalteparin sodium, nadroparin sodium, and parparin sodium.

[0030] As certain embodiments of the present invention, the low molecular weight heparin is one or more selected from enoxaparin sodium, dalteparin sodium, and nadroparin sodium.

[0031] As certain embodiments of the present invention, the low molecular weight heparin is enoxaparin sodium.

[0032] The molar ratio of lipoic acid:activator is selected from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0033] b) a hydrophilic heparin compound is dissolved in a reaction solvent and mixed with a) and the reaction temperature is selected from 25, 26, 27, 28, 29, 30, 31, 32, 33°C.

[0034] b) a hydrophilic heparin compound is dissolved in a reaction solvent and mixed with a) and the reaction time is selected from 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120 hours.

[0035] As certain embodiments of the present invention, in the step (2), the feed ratio of the activated lipoic acid to the hydrophilic heparin compound is 1-10:1-10 by mass; preferably, in the step (2), the feed ratio of the activated lipoic acid to the hydrophilic heparin compound is 1:1 by mass.

[0036] The mass ratio of the activated lipoic acid to the hydrophilic heparin compound is selected from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 2:3, 2:5, 2:7, 2:9, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 4:1, 4:3, 4:5, 4:7, 4:9, 5:1 , 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 6:1, 6:5, 6:7, 7:1, 7:2, 7:3, 7:4, 7:5, 7:8, 7:9, 7:10, 8:1, 8:3, 8:5, 8:7, 8:9, 9:1, 9:2, 9:4, 9:5, 9:8, 9:10, 10:1, 10:3, 10:7, 10:9.

[0037] As certain embodiments of the present invention, in step (2), b) the hydrophilic heparin compound is first dissolved in a reaction solvent and then mixed with a), and the reaction conditions are preferably 25-33° C. and / or protected from light and nitrogen.

[0038] As certain embodiments of the present invention, the reaction time in step (2) is 8-120 h, preferably 72 h.

[0039] As certain embodiments of the present invention, the connecting bond in step (2) is selected from one or more of an ester bond, a pH-sensitive hydrazone bond, a thioketal, an oxalate and a disulfide bond. Preferably, the hydrophilic heparin compound is directly connected to the lipoic acid via an ester bond.

[0040] As certain embodiments of the present invention, the preparation method further comprises step (3): purifying the amphiphilic copolymer obtained in the above step (2), wherein the purification is performed by precipitation with glacial acetone, and the volume ratio of reaction solution to glacial acetone is 1:(1-20).

[0041] Preferably, in step (3), the volume ratio of reaction solution to glacial acetone is 1:10.

[0042] Preferably, the method further comprises dialyzing the obtained drug carrier against pure water.

[0043] More preferably, the method further comprises drying the obtained drug carrier.

[0044] The volume ratio of reaction solution to glacial acetone is selected from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20.

[0045] The present invention also provides a method for preparing micelles, wherein the micelles are prepared by adopting the method for preparing the drug carrier, and the drug carrier is dissolved in water or a pharmaceutically acceptable aqueous medium.

[0046] Preferably, the aqueous medium in step (4) is an aqueous solution, and more preferably, the aqueous medium is PBS or physiological saline.

[0047] The present invention also provides an anti-inflammatory and / or anti-atherosclerotic drug preparation, wherein the anti-inflammatory and / or anti-atherosclerotic drug is loaded physically or chemically in the hydrophobic part and / or hydrophilic part of the drug carrier or the micelle.

[0048] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug is loaded into the hydrophobic part of the drug carrier or micelle, and the drug loading method is selected from one or more of emulsification ultrasonic rotary evaporation, dialysis, solvent injection, direct dissolution, and solvent volatilization.

[0049] As certain embodiments of the present invention, the drug loading method is selected from dialysis and emulsification ultrasonic rotary evaporation.

[0050] As certain embodiments of the present invention, the drug loading method is selected from emulsification ultrasonic rotary evaporation.

[0051] As certain embodiments of the present invention, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane, water, methanol, ethanol, chloroform, tetrahydrofuran, or a mixed solvent thereof.

[0052] As certain embodiments of the present invention, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from chloroform, dichloromethane, or tetrahydrofuran or a mixed solvent thereof.

[0053] As certain embodiments of the present invention, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane.

[0054] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug is loaded in the hydrophilic part of the drug carrier or micelle.

[0055] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug is coupled to the hydrophilic heparin compound via a linker.

[0056] As certain embodiments of the present invention, the connecting bond is selected from one or more of an amide bond, a hydrazone bond, a disulfide bond, an imine bond or an ester bond.

[0057] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug comprises a cholesterol-lowering drug and / or a triglyceride-lowering drug.

[0058] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drugs include statins, niacin drugs, fibrates, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs.

[0059] The choice of lipid-lowering drugs should be based on the composition of blood lipids. If the cholesterol is high, cholesterol-lowering drugs should be used, such as statins, commonly used ones are atorvastatin and rosuvastatin. If the triglycerides are high, triglyceride-lowering drugs should be used, mainly fibrates.

[0060] As certain embodiments of the present invention, the statin is selected from one or more of fluvastatin, pitavastatin, atorvastatin, rosuvastatin, and simvastatin.

[0061] Statins: including fluvastatin, pitavastatin, atorvastatin, rosuvastatin, simvastatin, etc., suitable for patients with hypercholesterolemia, mixed hyperlipidemia with elevated cholesterol as the main feature, atherosclerosis and coronary heart disease.

[0062] As certain embodiments of the present invention, the nicotinic acid drug is selected from one or more of acipimox, inositol nicotinate, vitamin E nicotinate, mannitol nicotinate, and probucol.

[0063] Niacin drugs include acipimox, inositol nicotinate, vitamin E nicotinate, mannitol nicotinate, probucol, etc., which help lower blood cholesterol and triglyceride levels, dilate blood vessels, and maintain normal insulin function.

[0064] As certain embodiments of the present invention, the fibrate drug is selected from one or more of clofibrate, bezafibrate, and fenofibrate.

[0065] Fibrate drugs include clofibrate, bezafibrate, fenofibrate, etc., which are mainly used for patients with elevated triglycerides. They can also lower very low-density lipoprotein and low-density lipoprotein.

[0066] As certain embodiments of the present invention, the steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs are selected from one or more of aspirin, ibuprofen, indomethacin, rapamycin, and curcumin.

[0067] The present invention also provides a method for preparing the pharmaceutical preparation, wherein the method comprises loading the anti-inflammatory and / or anti-atherosclerotic drug into the hydrophobic part and / or the hydrophilic part of the drug carrier or the micelle by physical or chemical methods.

[0068] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug (A) is loaded into the hydrophobic part of the drug carrier or micelle, and the drug loading method is selected from one or more of emulsification ultrasonic rotary evaporation, dialysis, solvent injection, direct dissolution, and solvent volatilization.

[0069] As certain embodiments of the present invention, the drug loading method is selected from dialysis and emulsification ultrasonic rotary evaporation.

[0070] As certain embodiments of the present invention, the drug loading method is selected from emulsification ultrasonic rotary evaporation.

[0071] As certain embodiments of the present invention, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane, water, methanol, ethanol, chloroform, tetrahydrofuran, or a mixed solvent thereof.

[0072] As certain embodiments of the present invention, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from chloroform, dichloromethane, or tetrahydrofuran or a mixed solvent thereof.

[0073] As certain embodiments of the present invention, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane.

[0074] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug (B) is loaded in the hydrophilic part of the drug carrier or micelle.

[0075] As certain embodiments of the present invention, the anti-inflammatory and / or anti-atherosclerotic drug (B) is coupled to the hydrophilic heparin compound via a linker.

[0076] As certain embodiments of the present invention, the connecting bond is selected from one or more of an amide bond, a hydrazone bond, a disulfide bond, an imine bond or an ester bond.

[0077] The present invention also provides the use of the drug carrier or the micelle, the drug preparation in the preparation of anti-inflammatory and / or anti-atherosclerotic drugs, wherein the anti-inflammatory and / or anti-atherosclerotic drugs are loaded on the hydrophobic part and / or the hydrophilic part of the drug carrier or micelle.

[0078] Therefore, the drug carrier, micelle, anti-inflammatory and / or anti-atherosclerotic drug preparation, and preparation method and application thereof of the present invention have achieved the following beneficial effects:

[0079] 1. The micelles can interfere with the inflammatory response of monocytes / macrophages in atherosclerosis, producing a strong anti-atherosclerotic effect.

[0080] 2. The hydrophilic part and the hydrophobic part of the drug carrier are directly connected, the construction steps are simpler, the structure is simpler, the particles are uniform, and the stability is excellent.

[0081] 3. All components in the drug carrier are preferably made of biocompatible and degradable materials. Compared with the prior art, the toxicity and metabolic discharge pressure caused by non-drug-effective ingredients to the body are eliminated to the greatest extent.

[0082] In short, the drug carrier and micelle of the present invention are simple in construction and highly stable, and can effectively inhibit the inflammatory cascade reaction and alleviate atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Shown is the LMWH-LA nuclear magnetic hydrogen spectrum of Example 1 of the present invention;

[0084] Figure 2 Shown is the LMWH-LA infrared spectrum of Example 1 of the present invention;

[0085] Figure 3 The particle size and potential of L-LA / Cur NPs were measured by dynamic light scattering (DLS) in Example 5, showing that the particle size of L-LA / Cur NPs nanoparticles was about 140 nm, the PDI was 0.135, and the particle size was uniform;

[0086] Figure 4 The nanoparticle morphology is confirmed by transmission electron microscopy (TEM) in Example 5 of the present invention, and the nanoparticles are regular spherical nanoparticles;

[0087] Figure 5 The fluorescence microscope photographs showing the ability of Example 6 of the present invention to inhibit the infiltration of monocytes show that the fluorescence intensity of the enoxaparin sodium and L-LA NPs treatment groups is significantly reduced, indicating that enoxaparin sodium and L-LA NPs can inhibit the adhesion of monocytes by competitively binding to P-selectin;

[0088] Figure 6 The fluorescence intensity comparison of Example 6 of the present invention showing the ability to inhibit the infiltration of monocytes;

[0089] Figure 7 The figure shows the uptake of curcumin and L-LA / Cur NPs by RAW264.7 cells observed by fluorescence microscopy in Example 7 of the present invention, which confirms that the uptake of L-LA / Cur NPs by macrophages is time-dependent and is easier to be taken up than free curcumin;

[0090] Figure 8 The results of observing the generation of ROS by fluorescence microscopy in Example 8 of the present invention show that the fluorescence intensity of the curcumin, L-LA NPs, and L-LA / Cur NPs treatment groups decreased, and the decrease in the L-LA / Cur NPs treatment group was more significant, indicating that L-LA / Cur NPs can inhibit the secretion of reactive oxygen species;

[0091] Fig. 9 The image shows the fluorescence produced by L-LA / Cy5.5 NPs loaded with fluorescent dye and free Cy5.5 in AS model mice observed by the imaging system of Example 9 of the present invention. Compared with free Cy5.5, the fluorescence intensity of the abdominal aorta of AS model mice injected with L-LA / Cy5.5 NPs is stronger, indicating that L-LA NPs can specifically deliver fluorescent dyes to arterial plaques and have atherosclerotic plaque targeting.

[0092] Fig.10 The figure shows the macroscopic oil red staining of the aorta of mice in the PBS, curcumin, L-LA NPs and L-LA / Cur NPs groups in Example 10 of the present invention, indicating that L-LA / Cur NPs can significantly reduce the lipid content on the aortic wall;

[0093] Fig.11 The aorta H&E staining of Example 10 of the present invention shows the sectioning of the mouse aortic sinus, which confirms that L-LA / Cur NPs can reduce the area of ​​arterial plaques and reduce the rate of lumen stenosis. DETAILED DESCRIPTION

[0094] The present invention is further described below in conjunction with specific examples, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0095] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated, the singular forms "a", "an" and "the" include plural forms. When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.

[0096] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0097] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0098] Example 1 Preparation of the pharmaceutical preparation of the present invention

[0099] 1. Synthesis of amphiphilic bonds of enoxaparin sodium and lipoic acid:

[0100] Dissolve 200 mg of enoxaparin sodium in 8 mL of formamide, place in a constant temperature water bath, and accelerate dissolution at 50°C and 120 rpm; take another 200 mg of lipoic acid, 372 mg of EDC, 223 mg of NHS, and 59.0 mg of DMAP and dissolve in 19 mL of DMF, and activate at 35°C and 500 rpm for 3 hours; slowly add the formamide solution of enoxaparin sodium to the activated DMF solution, and react at 35°C and 500 rpm for 72 hours; after the reaction is completed, use a dialysis bag with a cutoff of 3500 to dialyze in DMF for 24 hours, and then dialyze in pure water for 48 hours; freeze the dialysate at -40°C for 8 hours, and freeze-dry it in a freeze dryer, and the resulting product is a white solid. The nuclear magnetic hydrogen spectrum was used to characterize LMWH-LA, such as Figure 1 As shown in the figure, the characteristic peak of H on the LA alkyl chain appears in the LMWH-LA spectrum (shown by the dotted circle), indicating that LMWH-LA was successfully synthesized. Infrared spectroscopy was used to characterize LMWH-LA, as shown in the figure. Figure 2 As shown, the infrared characteristic peak of LA appears on the LMWH-LA molecule, indicating that LMWH-LA was successfully synthesized.

[0101] 2. Preparation of Enoxaparin Sodium and Lipoic Acid Bonded Micellar L-LA NPs:

[0102] The white powder of enoxaparin sodium and lipoic acid bond (LMWH-LA) is dispersed in dichloromethane, pure water is added, and after ultrasonic emulsification, the dichloromethane is removed by rotary evaporation to obtain self-assembled L-LA NPs (L-LA nanoparticles). When used in animal experiments, the bond powder is placed in a physiologically acceptable aqueous medium, such as PBS or saline.

[0103] Example 2 Preparation of the pharmaceutical preparation of the present invention

[0104] 1. Synthesis of amphiphilic bond of dalteparin sodium and lipoic acid:

[0105] 200 mg of dalteparin sodium was dissolved in 8 mL of formamide, and placed in a constant temperature water bath, and the solution was accelerated at 55°C and 120 rpm. Another 200 mg of lipoic acid, 372 mg of EDC, 223 mg of NHS, and 59.0 mg of DMAP were dissolved in 19 mL of DMF, and activated at 35°C and 500 rpm for 3 h. The formamide solution of dalteparin sodium was slowly added to the activated DMF solution, and the reaction was carried out at 35°C and 500 rpm for 72 h. After the reaction was completed, a dialysis bag with a cutoff of 3500 was used to dialyze in DMF for 24 h, and then in pure water for 48 h. The dialysate was frozen at -40°C for 8 h, and then freeze-dried using a freeze dryer, and the obtained product was a white solid.

[0106] 2. Preparation of Dalteparin Sodium and Lipoic Acid Bonded Micelles D-LA NPs:

[0107] The yellow powder of dalteparin sodium and lipoic acid bond (D-LA) is dispersed in dichloromethane, pure water is added, and after ultrasonic emulsification, the dichloromethane is removed by rotary evaporation to obtain self-assembled D-LA NPs (D-LA nanoparticles). When used in animal experiments, the bond powder is placed in a physiologically acceptable aqueous medium, such as PBS or saline.

[0108] Example 3 Preparation of the pharmaceutical preparation of the present invention

[0109] 1. Synthesis of amphiphilic bonds of nadroparin sodium and lipoic acid:

[0110] 200 mg of nadroparin sodium was dissolved in 8 mL of formamide, placed in a constant temperature water bath, and accelerated dissolution at 55°C and 120 rpm; 200 mg of lipoic acid, 372 mg of EDC, 223 mg of NHS, and 59.0 mg of DMAP were dissolved in 19 mL of DMF, and activated at 35°C and 500 rpm for 3 hours; the formamide solution of nadroparin sodium was slowly added to the activated DMF solution, and the reaction was carried out at 35°C and 500 rpm for 72 hours; after the reaction was completed, a dialysis bag with a cutoff of 3500 was used to dialyze in DMF for 24 hours, and then placed in pure water for dialyzation for 48 hours; the dialysate was frozen at -40°C for 8 hours, and then freeze-dried using a freeze dryer, and the obtained product was a white solid.

[0111] 2. Preparation of Nadroparin Sodium and Lipoic Acid Bonded Micelles F-LA NPs:

[0112] The yellow powder of the bond of nadroparin sodium and lipoic acid (F-LA) is dispersed in dichloromethane, pure water is added, and after ultrasonic emulsification, the dichloromethane is removed by rotary evaporation to obtain self-assembled F-LA NPs (F-LA nanoparticles). When used in animal experiments, the bond powder is placed in a physiologically acceptable aqueous medium, such as PBS or saline.

[0113] Example 4 Structural Characterization

[0114] 1. The average particle size, PDI and Zeta potential of L-LA NPs, D-LA NPs and F-LA NPs were measured by dynamic light scattering particle size analyzer (DLS). The results are shown in Table 1 below:

[0115] Table 1

[0116] Group Average particle size (nm) PDI Zeta(mV) L-LA NPs 130.2±5.3 0.134±0.025 -42.2±3.6 D-LA NPs 142.6±4.6 0.157±0.024 -39.6±5.2 F-LA NPs 154.6±3.4 0.125±0.016 -46.3±3.3

[0117] Example 5 Preparation and Characterization of Curcumin (Cur)-loaded L-LA Micelle (L-LA / Cur NPs)

[0118] 1) Example of carrying anti-tumor drugs by physical means

[0119] Taking curcumin as an example, L-LA / Cur NPs were prepared by ultrasonic emulsification. Take hydrophobic curcumin and LMWH-LA copolymer powder (LMWH-LA: curcumin = 9:1, w / w) dispersed in CH2Cl2, stirred for 3h under light-proof conditions. Next, the mixture was added to 5 times the volume of deionized water and ultrasonically emulsified (300W, 2s / 2s, 5min). Next, CH2Cl2 was removed by vacuum rotary evaporation at 37°C, and the yellow clear preparation was placed in a dialysis bag with a molecular weight cutoff of 1000 and dialyzed in deionized water for 3h, and then filtered through a 0.22μm microporous filter membrane to obtain L-LA / Cur NPs.

[0120] 2) Characterization of drug-loaded micelles

[0121] Dynamic light scattering (DLS) was used to measure the particle size and potential of L-LA / Cur NPs, and transmission electron microscopy (TEM) was used to confirm the morphology of the nanoparticles. Figure 3 As shown in Figure 2, the particle size of L-LA / Cur NPs nanoparticles is about 140 nm, the PDI is 0.135, and the particle size is uniform. The transmission electron microscopy image shows that the nanoparticles are regular spherical nanoparticles ( Figure 4 ).

[0122] Example 6 Investigation of the ability to inhibit the infiltration of monocytes:

[0123] Human umbilical vein endothelial cells (HUVECs) were seeded into 24-well plates at a density of 40%. When the cells grew to about 80%, H2O2 was added to activate the cells. After 6 hours of stimulation, PBS, enoxaparin sodium, and L-LA NPs were added to the corresponding wells and incubated for 1 hour. THP-1 cells labeled with BCEDF AM probe were added and incubated with the cells at 37°C and 30rpm for 30 minutes. After washing with PBS, the number of adhered human monocytic leukemia cells (THP-1) was observed under a fluorescence microscope. DMSO was then added to lyse the cells. The fluorescence intensity was detected at Ex=480nm and Em=520nm using a fluorescence spectrophotometer.

[0124] The results are as follows Figure 5 and 6As shown, the fluorescence intensity of the enoxaparin sodium and L-LA NPs treatment groups was significantly reduced, indicating that enoxaparin sodium and L-LA NPs can inhibit the adhesion of monocytes by competitively binding to P-selectin.

[0125] Example 7 Uptake of Nanoparticles by Macrophages

[0126] Mouse macrophage RAW264.7 cells were inoculated into 6-well plates. After adherence, curcumin and L-LA / Cur NPs were incubated with RAW264.7 cells for 1 h and 4 h, respectively. The nucleus was stained with DAPI, and the uptake of nanoparticles by RAW264.7 cells was observed under a fluorescence microscope.

[0127] The results are as follows Figure 7 As shown, the uptake of L-LA / Cur NPs by macrophages was time-dependent and was easier to take up than free Cur.

[0128] Example 8: Examination of the ability to inhibit the secretion of reactive oxygen species (ROS):

[0129] Mouse macrophage RAW264.7 cells were seeded into 6-well plates at a density of 40%. When the cells grew to about 80%, lipopolysaccharide (LPS) was added to activate the cells. After 6 hours of stimulation, PBS, curcumin, L-LA NPs, and L-LA / Cur NPs were added to the corresponding wells and incubated for 24 hours. DCFH-DA probe was added for staining and incubated with the cells at 37°C and 30rpm for 0.5 hours. After washing with PBS, the cells were placed under a fluorescence microscope to observe the production of reactive oxygen species.

[0130] The results are as follows Figure 8 As shown, the fluorescence intensity of the curcumin, L-LA NPs, and L-LA / Cur NPs treatment groups decreased, and the decrease in the L-LA / Cur NPs treatment group was more significant, indicating that L-LA / Cur NPs can inhibit the secretion of reactive oxygen species.

[0131] Example 9 Targeting investigation of atherosclerotic plaques:

[0132] LMWH-LA and fluorescent dye Cy5.5 were dispersed in dichloromethane; the mixed solution was then added to PBS and ultrasonically emulsified, and dichloromethane was removed by rotary evaporator to obtain L-LA / Cy5.5 NPs loaded with fluorescent dye; Apoe- / - mice were fed a high-fat diet to establish the AS model; L-LA / Cy5.5 NPs were injected into the AS model mice through the tail vein, and AS model mice injected with free Cy5.5 served as controls; an imaging system (IVIS Lumina SeriesⅢ, PerkinElmer, USA) was used to observe fluorescence, and the fluorescence intensity was quantified.

[0133] The results are as follows Fig. 9 As shown, compared with free Cy5.5, the fluorescence intensity of the abdominal aorta of AS model mice injected with L-LA / Cy5.5 NPs was stronger, indicating that L-LA NPs can specifically deliver fluorescent dyes to arterial plaques and have atherosclerotic plaque targeting.

[0134] Example 10: Examination of therapeutic effects on atherosclerosis in mice:

[0135] 1. High-fat diet for Apoe- / - mice to establish AS model

[0136] 2. Dosage regimen The atherosclerotic mice were randomly divided into 4 groups (n=8). After the model was established, each group was injected with PBS, curcumin, L-LA NPs and L-LA / Cur NPs through the tail vein, twice a week for 2 months. Normal mice were used as controls. After the treatment, the mouse aorta was isolated and gross oil red staining was performed, and the ratio of oil red positive area to aortic wall area was calculated. The mouse aortic sinus was sectioned and H&E stained to analyze the ratio of plaque to lumen and calculate the vascular stenosis rate.

[0137] Oil red staining showed that L-LA / Cur NPs could significantly reduce the lipid content on the aortic wall. H&E staining of the aorta confirmed that L-LA / Cur NPs could reduce the area of ​​arterial plaque and reduce the rate of lumen stenosis ( Fig.10 and Fig.11 ). This indicates that the preparation has a therapeutic effect on atherosclerosis.

[0138] Example 11 Safety Investigation

[0139] After the treatment, blood was collected from each group of mice; the blood was centrifuged (3000rpm, 5min) to obtain plasma samples, and the expression levels of various enzymes in the plasma were detected using a specific plasma enzyme kit on an automatic biochemical analyzer, and a number of biochemical index parameters related to liver and kidney function were measured.

[0140] The indicators of each treatment group were normal, indicating that L-LA / Cur NPs had no liver and kidney toxicity within the indicators studied.

[0141] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A drug carrier, comprising: a) a hydrophilic portion, the hydrophilic portion comprising a hydrophilic heparin-like compound, and b) a hydrophobic portion, wherein the hydrophobic portion is lipoic acid. The hydrophilic heparin compound is selected from one or more of unfractionated heparin, low molecular weight heparin, basil polysaccharide, and wakame polysaccharide; The hydrophilic heparin compound and the lipoic acid are connected via the following connection modes, which are selected from one or more of an ester bond, a pH-sensitive hydrazone bond, an imine bond, an alcohol ether bond, an enzyme-sensitive linker, and an active oxygen-sensitive linker.

2. The drug carrier according to claim 1, wherein Also includes one or more of the following features: A1) the hydrophilic heparin compound is selected from low molecular weight heparin; the low molecular weight heparin is selected from one or more of enoxaparin sodium, dalteparin sodium, nadroparin sodium, and parparin sodium, preferably, the low molecular weight heparin is selected from one or more of enoxaparin sodium, dalteparin sodium, and nadroparin sodium; more preferably, the low molecular weight heparin is enoxaparin sodium; A2) The hydrophilic heparin compound and the lipoic acid are connected by one or more of an ester bond, a pH-sensitive hydrazone bond, a thioketal, an oxalate, and a disulfide bond; preferably, the hydrophilic heparin compound and the lipoic acid are directly connected by an ester bond.

3. A micelle comprising the drug carrier according to any one of claims 1 to 2 and water or a pharmaceutically acceptable aqueous medium.

4. The micelle according to claim 3, wherein Also includes any one or more of the following features: B1) the aqueous medium is an aqueous solution, more preferably PBS or physiological saline; B2) The particle size of the micelle is micrometer or nanometer; Preferably, the particle size of the micelles ranges from 20 nm to 200 nm; More preferably, the particle size of the micelle is 140 nm±20 nm.

5. A method for preparing the drug carrier according to any one of claims 1 to 2, wherein: The method comprises: (1) activating lipoic acid with a carboxyl activator to obtain activated lipoic acid, (2) mixing a) the activated lipoic acid of (1) above and b) a hydrophilic heparin compound in a reaction solvent, and reacting a) and b) to form an amphiphilic copolymer connected by the following connecting bonds, wherein the connecting bonds include one or more of an ester bond, a pH-sensitive hydrazone bond, an imine bond, an alcohol ether bond, an enzyme-sensitive linker, and an active oxygen-sensitive linker.

6. The preparation method according to claim 5, wherein: Also includes any one or more of the following features: C1) In step (1), the carboxyl activating agent is selected from the group consisting of: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), NHS (hydroxysuccinimide), EDC-DMAP (4-dimethylaminopyridine), EDC-HOBT (1-hydroxybenzotriazole), DCC (dicyclohexylcarbodiimide)-HOBT, and a combination of DCC-NHS and DCC-DMAP; C2) In step (1), the molar ratio of the feed is: lipoic acid: activator = 1: (1-10); preferably, the molar ratio of the feed in step (1) is: lipoic acid: activator = 1: 2; C3) in step (1), the activation reaction is carried out in a reaction solvent selected from the group consisting of water, N,N-dimethylformamide, formamide, a mixed solvent of N,N-dimethylformamide and formamide, dimethyl sulfoxide, a mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide, a mixed solvent of dimethyl sulfoxide and formamide, and a mixed solvent of water and formamide; C4) The activation time in step (1) is 30 min to 5 h, preferably 3-4 h under a nitrogen atmosphere in a dark environment; C5) In step (2), the hydrophilic heparin compound is selected from one or more of unfractionated heparin, low molecular weight heparin, basil polysaccharide, and undaria pinnatifida polysaccharide, preferably low molecular weight heparin; Preferably, the low molecular weight heparin is one or more selected from enoxaparin sodium, dalteparin sodium, nadroparin sodium, and parparin sodium. More preferably, the low molecular weight heparin is one or more selected from enoxaparin sodium, dalteparin sodium, and nadroparin sodium; Further preferably, the low molecular weight heparin is enoxaparin sodium; C6) In the step (2), the ratio of the activated lipoic acid to the hydrophilic heparin compound is 1-10:1-10 by mass; preferably, the ratio of the activated lipoic acid to the hydrophilic heparin compound in the step (2) is 1:1 by mass; C7) In step (2), the hydrophilic heparin compound b) is first dissolved in a reaction solvent and then mixed with a), and the reaction conditions are preferably 25-33° C. and / or protected from light and nitrogen; C8) The reaction time in step (2) is 8-120 h, preferably 72 h; C9) the connecting bond in step (2) is selected from one or more of an ester bond, a pH-sensitive hydrazone bond, a thioketal, an oxalate and a disulfide bond, preferably, the hydrophilic heparin compound is directly connected to the lipoic acid via an ester bond; C10) The preparation method further comprises the step (3): purifying the amphiphilic copolymer obtained in the above step (2), wherein the purification is performed by precipitation with glacial acetone, and the volume ratio of the reaction solution to glacial acetone is 1:(1-20); Preferably, in step (3), the volume ratio of reaction solution to glacial acetone is 1:10; Preferably, the method further comprises dialyzing the obtained drug carrier against pure water; More preferably, the method further comprises drying the obtained drug carrier.

7. A method for preparing micelles, wherein: Prepare micelles using the preparation method of the drug carrier according to any one of claims 5 to 6, and dissolve the drug carrier in water or a pharmaceutically acceptable aqueous medium; Preferably, the aqueous medium in step (4) is an aqueous solution, and more preferably, the aqueous medium is PBS or physiological saline.

8. An anti-inflammatory and / or anti-atherosclerotic pharmaceutical preparation, wherein: The anti-inflammatory and / or anti-atherosclerotic drug is loaded physically or chemically in the hydrophobic part and / or the hydrophilic part of the drug carrier according to any one of claims 1 to 2 or the micelle according to any one of claims 3 to 4.

9. The pharmaceutical preparation according to claim 8, wherein The anti-inflammatory and / or anti-atherosclerotic drug is loaded on the hydrophobic part of the drug carrier or micelle, and the drug loading method is selected from one or more of emulsification ultrasonic rotary evaporation method, dialysis method, solvent injection method, direct dissolution method, and solvent volatilization method; Preferably, the drug loading method is selected from one or more of dialysis and emulsification ultrasonic rotary evaporation; More preferably, the drug loading method is emulsification ultrasonic rotary evaporation; Preferably, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane, water, methanol, ethanol, chloroform, tetrahydrofuran, or a mixed solvent thereof; More preferably, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from chloroform, dichloromethane, or tetrahydrofuran or a mixed solvent thereof; More preferably, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane.

10. The pharmaceutical preparation according to claim 8, wherein The anti-inflammatory and / or anti-atherosclerotic drug is loaded in the hydrophilic part of the drug carrier or micelle; Preferably, the anti-inflammatory and / or anti-atherosclerotic drug is coupled to the hydrophilic heparin compound via a linker; More preferably, the connecting bond is selected from one or more of an amide bond, a hydrazone bond, a disulfide bond, an imine bond or an ester bond.

11. The pharmaceutical preparation according to any one of claims 8 to 10, wherein The anti-inflammatory and / or anti-atherosclerotic drugs include cholesterol-lowering drugs and / or triglyceride-lowering drugs; preferably, the anti-inflammatory and / or anti-atherosclerotic drugs include statins, niacin drugs, fibrates, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs; More preferably, the statin is selected from one or more of fluvastatin, pitavastatin, atorvastatin, rosuvastatin, and simvastatin; The nicotinic acid drug is selected from one or more of acipimox, inositol nicotinate, vitamin E nicotinate, mannitol nicotinate, and probucol; The fibrate drug is selected from one or more of clofibrate, bezafibrate, and fenofibrate; The steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs are selected from one or more of aspirin, ibuprofen, indomethacin, rapamycin and curcumin.

12. A method for preparing the pharmaceutical preparation according to any one of claims 8 to 10, wherein: The method comprises loading the anti-inflammatory and / or anti-atherosclerotic drug into the hydrophobic part and / or the hydrophilic part of the drug carrier according to any one of claims 1 to 2 or the micelle according to any one of claims 3 to 4 by physical or chemical methods.

13. The method according to claim 12, wherein: The anti-inflammatory and / or anti-atherosclerotic drug (A) is loaded on the hydrophobic part of the drug carrier or micelle, and the drug loading method is selected from one or more of emulsification ultrasonic rotary evaporation method, dialysis method, solvent injection method, direct dissolution method, and solvent volatilization method; Preferably, the drug loading method is selected from dialysis and emulsification ultrasonic rotary evaporation; More preferably, the drug loading method is selected from emulsification ultrasonic rotary evaporation; Preferably, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane, water, methanol, ethanol, chloroform, tetrahydrofuran, or a mixed solvent thereof; More preferably, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from chloroform, dichloromethane, or tetrahydrofuran or a mixed solvent thereof; More preferably, the solvent used in the emulsification ultrasonic rotary evaporation method is selected from dichloromethane.

14. The method according to claim 12, wherein: The anti-inflammatory and / or anti-atherosclerotic drug (B) is loaded in the hydrophilic part of the drug carrier or micelle; The anti-inflammatory and / or anti-atherosclerotic drug (B) is coupled to the hydrophilic heparin compound via a linker; Preferably, the connecting bond is selected from one or more of an amide bond, a hydrazone bond, a disulfide bond, an imine bond or an ester bond.

15. Use of the drug carrier according to claim 1 or 2, the micelle according to claim 3 or 4, or the pharmaceutical preparation according to any one of claims 8 to 11 in the preparation of anti-inflammatory and / or anti-atherosclerotic drugs.

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