A kind of EGCG-PLGA-CHP nano targeted preparation and its preparation and application

Abstract

The present invention relates to an EGCG-PLGA-CHP nano-targeted preparation and its preparation and application, and relates to the technical field of nanoparticles. The EGCG-PLGA-CHP nano-targeted preparation is mainly made of EGCG and PLGA-CHP targeting carriers, and the PLGA-CHP targeting carrier is mainly prepared by grafting a cardiac homing peptide on a polylactic-co-glycolic acid copolymer. The present invention adopts PLGA-CHP as a targeting carrier of EGCG, and the EGCG-PLGA-CHP nano-targeted preparation has good stability and dispersibility, realizes EGCG controlled release, and protects the antioxidant property of EGCG; the targeting carrier PLGA-CHP can accurately target the drug EGCG to the heart, and the nano-targeted preparation can inhibit cell apoptosis and resist myocardial ischemia damage.

Description

Technical Field

[0001] The invention relates to the technical field of nanoparticles, and in particular to an EGCG-PLGA-CHP nano targeted preparation and a preparation and application thereof. Background Art

[0002] The 2021 "Guidelines for the Primary Prevention of Cardiovascular Disease in China" reported that the number of deaths from cardiovascular disease in my country accounts for about 40% of the total number of deaths each year, and cardiovascular disease has become one of the main causes of death among Chinese residents. A large number of studies have shown that a variety of cardiovascular diseases are closely related to oxidative stress damage. Oxidative stress damage refers to the destruction of the homeostasis of the body's intracellular environment, which causes an imbalance in the production and elimination of intracellular oxygen free radicals, leading to cytotoxicity and is an important pathogenesis of myocardial damage. The onset of vascular disease is relatively serious, and early and effective drug intervention can slow the progression of the disease and achieve therapeutic effects.

[0003] Epigallocatechin gallate (EGCG) is the main active and water-soluble component of green tea, accounting for 80% of catechins. Related studies have shown that long-term drinking of green tea can significantly reduce the incidence of cardiovascular and cerebrovascular diseases. EGCG has multiple phenolic hydroxyl groups in its chemical structure, and factors such as pH, temperature, oxygen level, antioxidant level, metal ions and EGCG concentration can affect the stability of EGCG. EGCG is easily oxidized under cell culture conditions, with a half-life of about 0.5-2h, and can generate oxidation products such as EGCG quinone, dimer, and dimer quinone. In addition, the effective dose of EGCG is not far from the toxic dose, the biological efficacy is low and it may cause damage to the body. Therefore, how to maintain the stability of EGCG to maintain its biological activity, improve its bioavailability and reduce its toxicity needs to be solved urgently. In view of the excellent antioxidant activity of EGCG, the construction of an EGCG nano-drug delivery system with a cardiac targeting effect is of great significance for the treatment of cardiovascular diseases.

[0004] The construction of intelligent nano drug delivery system in nanomedicine can protect drugs from damage by external media, realize active or passive targeting and controllable release of drugs, make drugs act precisely on lesions, improve drug bioavailability, and reduce drug toxicity to normal tissues and cells. At present, common drug carriers include liposomes, polymer micelles, and polymer nano hydrogels. Studies have found that liposomes encapsulate EGCG to form nanocapsules, which significantly improve the stability of EGCG; EGCG encapsulated by chitosan has a significantly reduced degree of degradation, improved stability, and improved bioavailability under heat treatment conditions; EGCG encapsulated by polylactic acid / polyethylene glycol (PLA / PEG) forms a nanosystem, which significantly improves bioavailability in inhibiting cell growth, promoting apoptosis, and inhibiting angiogenesis. It can be seen that EGCG nano-encapsulation can improve its stability and bioavailability and reduce its toxicity, and has application prospects in disease treatment.

[0005] At present, most of the application research on EGCG nano drug delivery system focuses on tumor treatment, and no research reports on the treatment of cardiovascular diseases have been found. Therefore, constructing an EGCG nano drug delivery system with cardiac targeting effect is of great significance for the treatment of cardiovascular diseases. The present invention provides an EGCG-PLGA-CHP nano targeted preparation and its preparation and application. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an EGCG-PLGA-CHP nano-targeted preparation and its preparation and application. The purpose is to design a targeted cardiac homing peptide (CHP) grafted on polylactic acid-co-glycolic acid (PLGA) as an EGCG carrier, so that the prepared nano-delivery system actively targets the heart, and EGCG can better exert its efficacy in the target tissue.

[0007] The present invention solves the above technical problems. The first aspect is to provide: a preparation of an EGCG-PLGA-CHP nano-targeted preparation, wherein the EGCG-PLGA-CHP nano-targeted preparation is mainly made of EGCG and a PLGA-CHP targeting carrier, and the PLGA-CHP targeting carrier is mainly prepared by grafting a cardiac homing peptide onto a polylactic acid-glycolic acid copolymer.

[0008] Among them, poly(lactic-co-glycolic acid), PLGA, has good biocompatibility and biodegradability. Its hydrolysis produces metabolites, monomers lactic acid and glycolic acid. Since these two monomers are endogenous and can be easily metabolized by the body through the Krebs cycle, systemic toxicity is minimal when PLGA is used for drug delivery or biomaterial applications. PLGA has been approved by the US FDA and the European Medicines Agency (EMA) for use in various drug delivery systems for the human body. PLGA nanoparticles encapsulated with chemical drugs, peptides, proteins or nucleic acid molecules can be used to treat some difficult diseases, including cardiovascular diseases.

[0009] Cardiac homing peptide (CHP, CSTSMLKAC) is a short peptide composed of 9 amino acids. Studies have shown that CHP can specifically target cardiomyocytes injured by ischemia-reperfusion (IRI). Due to the high sensitivity of CHP to ischemic cardiac hypertrophy, CHP can enhance the specificity and effectiveness of nanoparticles directly targeting hypertrophic myocardium. CHP-XOs formed by the combination of CHP and cardiac stem cell exosomes can improve cardiac function after infarction by promoting cardiomyocyte proliferation and angiogenesis.

[0010] The cardiac homing peptide is grafted onto the poly(lactic acid-co-glycolic acid) copolymer via an amide bond. The EGCG-PLGA-CHP nano-targeted preparation needs to be stored at -20°C.

[0011] The EGCG-PLGA-CHP nano targeted preparation of the present invention has good stability and dispersibility, realizes the controlled release of EGCG, protects the antioxidant property of EGCG, and the targeted carrier PLGA-CHP can accurately target the drug EGCG to the heart, and the nano targeted preparation inhibits apoptosis, and can be used to prevent and treat myocardial ischemia-related diseases.

[0012] The beneficial effects of the present invention are:

[0013] (1) The present invention uses PLGA-CHP as a targeting carrier of EGCG, thereby achieving controlled release of EGCG and protecting the antioxidant properties of EGCG. In addition, the targeting carrier PLGA-CHP can accurately target the drug EGCG to the heart.

[0014] (2) The EGCG-PLGA-CHP nanoparticles of the present invention have good stability and dispersibility.

[0015] (3) The present invention investigates the application value of EGCG-PLGA-CHP nano-targeted preparations in cardiac ischemia and provides a preventive treatment plan.

[0016] Furthermore, the molecular weight of the polylactic acid-glycolic acid copolymer is 15000Mw.

[0017] Furthermore, the weight ratio of the EGCG to the polylactic acid-glycolic acid copolymer is (0.1-12):(3-40); the weight ratio of the total weight of the EGCG and the polylactic acid-glycolic acid copolymer to the cardiac homing peptide is (10-30):(1-3).

[0018] Furthermore, the particle size range of the EGCG-PLGA-CHP nano-targeted preparation is 90 to 120 nm, the average particle size of the EGCG-PLGA-CHP nano-targeted preparation is 95.65±0.227 nm, and the Zeta potential of the EGCG-PLGA-CHP nano-targeted preparation is -1.67±0.01 mV.

[0019] The second aspect of the present invention is to provide a method for preparing an EGCG-PLGA-CHP nano-targeted preparation, comprising the following steps:

[0020] (1) EGCG and poly(lactic acid-co-glycolic acid) copolymer were treated by a double emulsion solvent evaporation method to prepare EGCG-PLGA nanoparticles;

[0021] (2) activating the EGCG-PLGA nanoparticles with a coupling activator (such as EDC, NHS) in a MES buffer to obtain activated EGCG-PLGA nanoparticles, and coupling the activated EGCG-PLGA nanoparticles with a cardiac homing peptide in a MES buffer to obtain an EGCG-PLGA-CHP nano-targeted preparation.

[0022] The concentration of the MES buffer used in the activation was 0.1 mol / L, pH 5.5; the concentration of the MES buffer used in the coupling reaction was 0.1 mol / L, pH 8.0.

[0023] The beneficial effects of adopting the above scheme are: the method of the present invention has a simple preparation process, low toxicity, environmental protection, and safety, and the obtained nanoparticles have good stability and dispersibility.

[0024] Furthermore, the double emulsion solvent evaporation method in step (1) comprises the following specific steps: mixing an EGCG aqueous solution and a PLGA ethyl acetate solution to obtain a mixed solution of EGCG and PLGA, emulsifying the mixed solution of EGCG and PLGA in an ice bath to obtain colostrum; adding a polyether F-127 solution to the colostrum, and demulsifying the solution in an ice bath to obtain a double emulsion; adding a polyether F-127 solution to the double emulsion at room temperature, and then sequentially performing solvent evaporation, ultrafiltration, and freeze-drying to obtain EGCG-PLGA nanoparticles.

[0025] The ultrafiltration is carried out using an ultrafiltration centrifuge tube and centrifuged at 3000-3500 rpm / min. The centrifugation and freeze-drying time are determined according to needs.

[0026] Furthermore, the mass concentration of the EGCG aqueous solution is 10-60 mg / mL; the mass concentration of the PLGA ethyl acetate solution is 3-20 mg / mL; the mass concentration of the polyether F-127 solution added to the colostrum is 10-30 mg / mL; the mass concentration of the polyether F-127 solution added to the double emulsion is 1-5 mg / mL; the volume ratio of the EGCG aqueous solution, the PLGA ethyl acetate solution, the polyether F-127 solution added to the colostrum and the polyether F-127 solution added to the double emulsion is (0.1-0.2):(1-2):(5-10):(15-20).

[0027] Furthermore, the demulsification is specifically performed by using an ultrasonic cell disruptor for 90 to 120 seconds; and the emulsification is specifically performed by using an ultrasonic cell disruptor for 90 to 120 seconds.

[0028] Furthermore, the coupling activator is EDC and NHS, the mass ratio of the EDC to the NHS is (2-4): (1-2); the mass ratio of the EGCG-PLGA nanoparticles to the EDC is (5-15): (6-12); the weight ratio of the EGCG-PLGA nanoparticles to the cardiac homing peptide is (10-30): (1-3).

[0029] The activation principle of the coupling activator EDC and NHS is to use the activator EDC and NHS to activate the carboxyl group of PLGA, and then couple with the amino group of CHP.

[0030] Furthermore, the activation and coupling temperature is 20-28° C., the activation time is 1 to 2 hours, and the coupling reaction time is 8 to 24 hours.

[0031] The third aspect of the present invention is to provide an application of an EGCG-PLGA-CHP nano targeted preparation, wherein the above-mentioned EGCG-PLGA-CHP nano targeted preparation is used in a drug for preventing or treating myocardial ischemic injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the particle size image of the EGCG-PLGA nanoparticles of the present invention;

[0033] Figure 2 This is the potential image of the EGCG-PLGA nanoparticles of the present invention;

[0034] Figure 3 This is the particle size image of the nanoparticles of EGCG-PLGA-CHP of the present invention;

[0035] Figure 4 This is the potential image of the nanoparticles of EGCG-PLGA-CHP of the present invention;

[0036] Figure 5 This is a transmission electron microscopy image of the EGCG-PLGA nanoparticles of the present invention;

[0037] Figure 6 This is a transmission electron microscope image of the EGCG-PLGA-CHP nanoparticles of the present invention;

[0038] Figure 7 Infrared spectroscopic images of PLGA, PLGA-CHP, EGCG-PLGA, EGCG-PLGA-CHP, CHP and EGCG of the present invention;

[0039] Figure 8 This is the drug release diagram of EGCG-PLGA nanoparticles of the present invention;

[0040] Fig. 9 This is the drug release image of EGCG-PLGA-CHP nanoparticles of the present invention;

[0041] Fig.10 The effects of the nanocarrier PLGA-CHP and the nanocomposite EGCG-PLGA-CHP of the present invention on cell survival rate;

[0042] Fig.11 This is the cell uptake diagram of EGCG-PLGA and EGCG-PLGA-CHP of the present invention;

[0043] Fig.12 This is a graph showing the coupling rate of EGCG-PLGA-CHP of the present invention;

[0044] Fig.13 Western blot was used to detect the expression of Bax and Bcl-2 proteins in cardiomyocytes;

[0045] Fig.14 Western blot was used to detect the expression of Bax and Bcl-2 proteins in mouse heart tissue. DETAILED DESCRIPTION

[0046] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0047] Description of the sources of reagents and materials in the following examples:

[0048] EGCG was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; PLGA: Jinan Daigang Bioengineering Co., Ltd.; F-127: TCI (Shanghai) Chemical Industry Development Co., Ltd.; EDC: Shanghai Aladdin Biochemical Technology Co., Ltd.; NHS: Sigma-Aldrich (Shanghai) Trading Co., Ltd.; MES buffer: Beijing Regen Biotechnology Co., Ltd.; CHP peptide: Shanghai Chupide Biotechnology Co., Ltd.; HL-1 cell line: Shanghai Chinese Academy of Sciences Cell Bank; DMEM culture medium: Gibco; MTT reagent: Nanjing Jiancheng Bioengineering Institute Co., Ltd.

[0049] Example 1

[0050] This embodiment relates to a method for preparing an EGCG-PLGA-CHP nano-targeted preparation, comprising the following steps:

[0051] (1) EGCG-PLGA nanoparticles were prepared by the double emulsion solvent evaporation method: 0.2 mL (10 mg / mL) EGCG aqueous solution was mixed with 1 mL (20 mg / mL) PLGA ethyl acetate solution, and emulsified for 90 s in an ultrasonic cell disruptor under ice bath conditions to obtain colostrum; the colostrum was mixed with 5 mL (10 mg / mL) F-127 solution, and emulsified for 90 s in an ultrasonic cell disruptor under ice bath conditions to obtain a double emulsion; the double emulsion was mixed with 15 mL (5 mg / mL) F-127 solution, the solvent was evaporated at room temperature for 12 h, ultrafiltration centrifuged at 3000 rpm for 5 min, repeated three times, freeze-dried for 24 h, and stored at -20°C;

[0052] (2) Preparation of EGCG-PLGA-CHP nanoparticles: First, 24 mg EDC, 12 mg NHS (coupling activator) and 30 mg EGCG-PLGA nanoparticles were dispersed in 10 mL (0.1 mol / L, pH 5.5) MES buffer, shaken and activated for 1 h; the activated EGCG-PLGA mixed solution was ultrafiltered and centrifuged at 3000 rpm, and the precipitate was washed for 10 min, and repeated three times; the precipitate was redispersed in MES buffer (0.1 mol / L, pH 8.0), and 1 mL of 1 mg / mL cardiac homing peptide solution was added dropwise, and the coupling reaction was carried out overnight under continuous shaking conditions; ultrafiltered and centrifuged at 3000 rpm, the precipitate was washed 3 times, the precipitate was collected, freeze-dried, and stored at -20°C.

[0053] Example 2

[0054] This embodiment relates to a method for preparing an EGCG-PLGA-CHP nano-targeted preparation, comprising the following steps:

[0055] (1) EGCG-PLGA nanoparticles were prepared by the double emulsion solvent evaporation method: 0.1 mL (40 mg / mL) EGCG aqueous solution was mixed with 1 mL (10 mg / mL) PLGA ethyl acetate solution, and emulsified for 90 s in an ultrasonic cell disruptor under ice bath conditions to obtain colostrum; the colostrum was mixed with 5 mL (30 mg / mL) F-127 solution, and emulsified for 120 s in an ultrasonic cell disruptor under ice bath conditions to obtain a double emulsion; the double emulsion was mixed with 15 mL (5 mg / mL) F-127 solution, the solvent was evaporated at room temperature for 16 h, ultrafiltration centrifugation was performed at 3000 rpm for 5 min, repeated three times, freeze-dried for 35 h, and stored at -20°C;

[0056] (2) Preparation of EGCG-PLGA-CHP nanoparticles: First, 24 mg EDC, 12 mg NHS (coupling activator) and 20 mg EGCG-PLGA nanoparticles were dispersed in 10 mL (0.1 mol / L, pH 5.5) MES buffer, shaken and activated for 1 h; the activated EGCG-PLGA mixed solution was ultrafiltered and centrifuged at 3000 rpm for 10 min, and the precipitate was washed, and repeated three times; the precipitate was redispersed in MES buffer (0.1 mol / L, pH 8.0), and 1 mL of 1 mg / mL cardiac homing peptide solution was added dropwise, and the coupling reaction was carried out overnight under continuous shaking conditions; ultrafiltered and centrifuged at 3500 rpm, the precipitate was washed 3 times, the precipitate was collected, freeze-dried, and stored at -20°C.

[0057] Example 3

[0058] This embodiment relates to a method for preparing an EGCG-PLGA-CHP nano-targeted preparation, comprising the following steps:

[0059] (1) EGCG-PLGA nanoparticles were prepared by the double emulsion solvent evaporation method: 0.2 mL (30 mg / mL) EGCG aqueous solution was mixed with 1 mL (10 mg / mL) PLGA ethyl acetate solution, and emulsified for 120 s in an ice bath using an ultrasonic cell disruptor to obtain colostrum; the colostrum was mixed with 5 mL (30 mg / mL) F-127 solution, and emulsified for 120 s in an ice bath using an ultrasonic cell disruptor to obtain a double emulsion; the double emulsion was mixed with 15 mL (1 mg / mL) F-127 solution, the solvent was evaporated at room temperature for 20 h, ultrafiltration centrifuged at 3000 rpm for 5 min, repeated three times, freeze-dried for 48 h, and stored at -20°C;

[0060] (2) Preparation of EGCG-PLGA-CHP nanoparticles: First, 12 mg EDC, 6 mg NHS (coupling activator) and 20 mg EGCG-PLGA nanoparticles were dispersed in 10 mL MES buffer (0.1 mol / L, pH 5.0), shaken and activated for 1 h; the activated EGCG-PLGA mixed solution was ultrafiltered and centrifuged at 3000 rpm for 10 min, and the precipitate was washed, and repeated three times; the precipitate was redispersed in MES buffer (0.1 mol / L, pH 8.0), and 1 mL of 1 mg / mL cardiac homing peptide solution was added dropwise, and the coupling reaction was carried out overnight under continuous shaking conditions; ultrafiltered and centrifuged at 3000 rpm and the precipitate was washed 3 times, the precipitate was collected, freeze-dried, and stored at -20°C.

[0061] Example 4

[0062] This embodiment relates to a method for preparing an EGCG-PLGA-CHP nano-targeted preparation, comprising the following steps:

[0063] (1) EGCG-PLGA nanoparticles were prepared by the double emulsion solvent evaporation method: 0.2 mL (60 mg / mL) EGCG aqueous solution was mixed with 1 mL (3 mg / mL) PLGA ethyl acetate solution, and emulsified for 120 s in an ultrasonic cell disruptor under ice bath conditions to obtain colostrum; the colostrum was mixed with 5 mL (10 mg / mL) F-127 solution, and emulsified for 120 s in an ultrasonic cell disruptor under ice bath conditions to obtain a double emulsion; the double emulsion was mixed with 15 mL (1 mg / mL) F-127 solution, the solvent was evaporated for 24 h at room temperature with stirring, and ultrafiltration centrifugation was performed at 4000 rpm for 5 min, repeated three times, freeze-dried for 48 h, and stored at -20°C;

[0064] (2) Preparation of EGCG-PLGA-CHP nanoparticles: First, 24 mg EDC, 12 mg NHS (coupling activator) and 30 mg EGCG-PLGA nanoparticles were dispersed in 10 mL MES buffer (0.1 mol / L, pH 5.0), shaken and activated for 1 h; the activated EGCG-PLGA mixed solution was ultrafiltered and centrifuged at 3000 rpm for 10 min, and the precipitate was washed, and repeated three times; the precipitate was redispersed in MES buffer (0.1 mol / L, pH 8.0), and 1 mL of 1 mg / mL cardiac homing peptide solution was added dropwise, and the coupling reaction was carried out overnight under continuous shaking conditions; ultrafiltered and centrifuged at 3500 rpm, the precipitate was washed 3 times, the precipitate was collected, freeze-dried, and stored at -20°C.

[0065] Experimental example

[0066] (1) Dynamic light scattering (DLS) to measure particle size and potential

[0067] The particle size and potential of the EGCG-PLGA nanoformulation and the EGCG-PLGA-CHP targeted nanoformulation obtained in Example 1 were measured by dynamic light scattering (DLS).

[0068] like Figure 1-2 As shown in Figure 2, the EGCG-PLGA nanoparticle preparation was found to have uniform distribution of the prepared nanoparticles through the particle size and potential curves, with an average particle size of 71.06±2.392nm ( Figure 1 ), good dispersion performance (PDI is 0.252±0.008), potential distribution -29.6±2.19 ( Figure 2 ).

[0069] like Figure 3-4 As shown in Figure 2, the EGCG-PLGA-CHP nano-targeted preparation was found to have uniform distribution of the prepared nanoparticles through the particle size and potential curves, with an average particle size of 95.65±0.227nm ( Figure 3 ), good dispersion performance (PDI is 0.227±0.017), potential distribution -1.67±0.01mV ( Figure 4 ).

[0070] (2) Transmission electron microscopy characterization

[0071] The EGCG-PLGA nanoparticles prepared in Example 1 of the present invention and the targeted EGCG-PLGA-CHP nanoparticles were characterized by transmission electron microscopy.

[0072] like Figure 5-6 As shown, EGCG-PLGA nanoformulation ( Figure 5 ) and EGCG-PLGA-CHP nano-targeted preparations ( Figure 6 ) are spherical with a diameter of 70-100 nm. In addition, the size of EGCG-PLGA-CHP targeting nanoparticles increased significantly to a diameter of 90-120 nm.

[0073] (3) Infrared spectroscopy characterization

[0074] like Figure 7 As shown in FIG. 1 , the functional groups of EGCG-PLGA and EGCG-PLGA-CHP prepared in Example 1 were characterized by Fourier transform infrared spectroscopy. -1 , 1618cm -1 , 3487cm -1 , 3364cm -1 There are larger peaks at the four characteristic peaks at 1000-1500cm. In the EGCG-PLGA complex and single PLGA, the above peaks do not appear. At the same time, some characteristic absorption peaks of EGCG, such as 1000-1500cm -1 and 700-900cm -1The peak in the region did not appear in the infrared spectrum of the composite, indicating that PLGA and EGCG formed a composite through non-covalent bond interaction.

[0075] When using infrared absorption spectroscopy to study protein structure, the characteristic peak of amide bonds can be used as an important marker. Amide bonds usually produce two characteristic peaks, which are located at 1620-1750cm -1 and 1250-1350cm -1 In CHP, it appears at 1620-1750cm -1 This characteristic peak is related to the vibration of the oxygen-containing amino group in the amide bond; the amide bond is located at 1250-1350cm -1 Another characteristic peak is related to the vibration of the oxygen-containing acyl group in the amide bond. This characteristic peak does not appear in EGCG-PLGA and pure PLGA, but exists in EGCG-PLGA-CHP and PLGA-CHP, indicating that EGCG-PLGA and CHP are grafted onto the targeting peptide through amide reaction coupling.

[0076] (4) Detection of drug loading rate and encapsulation efficiency of EGCG-PLGA and EGCG-PLGA-CHP

[0077] The drug loading rate and encapsulation rate of EGCG-PLGA and EGCG-PLGA-CHP prepared in Example 1 were detected, and the drug loading rate and encapsulation rate of the nanoformulation were determined by an extraction method: EGCG-PLGA and EGCG-PLGA-CHP were dissolved in dichloromethane, and EGCG therein was extracted with deionized water; the absorbance of the extract was detected at a wavelength of 274 nm by a UV-visible spectrometer, and the mass of EGCG in the extract was substituted into the EGCG standard curve equation, and the drug loading amount and encapsulation rate of the nanoparticles were calculated according to the following formula:

[0078] Drug loading rate = EGCG mass in nanoparticles / nanoparticle mass*100%;

[0079] Encapsulation efficiency = mass of EGCG in nanoparticles / total mass of added EGCG*100%.

[0080] The results are shown in Table 1. The encapsulation efficiency of EGCG-PLGA was 80.89%±1.10, and the drug loading rate was 1.61%±0.03; the encapsulation efficiency of EGCG-PLGA-CHP was 70.41%±1.57, and the drug loading rate was 1.40%±0.03.

[0081] Table 1 Drug loading rate and encapsulation efficiency of EGCG-PLGA and EGCG-PLGA-CHP (x±s, n=3)

[0082]

[0083]

[0084] (5) Release rate detection

[0085] Accurately weigh 40 mg of the EGCG-PLGA-CHP nano-targeted preparation prepared in Example 1 of the present invention and place it in a dialysis bag with a cutoff molecule of 3500, place the dialysis bag in 20 mL of PBS release solution, shake on a shaker at room temperature (37°C) for 72 h, take 1 mL of release solution in an EP tube at 1, 2, 5, 24, 48, and 72 h of oscillation, and add 1 mL of PBS solution to the original release system. Use an ultraviolet-visible spectrometer to detect the absorbance of the release solution at different time points at a wavelength of 274 nm, substitute it into the EGCG standard curve equation, calculate the concentration of EGCG in the release solution at different time points, repeat three times, and calculate the cumulative release percentage of EGCG.

[0086] like Figure 8-9 As shown, the release rate of EGCG is rapid in the first 24 hours. After 24 hours, as time goes by, the release rate of EGCG begins to slow down and tends to be gentle.

[0087] (6) Cell activity detection

[0088] The cell activity of the nanocarrier PLGA-CHP and EGCG-PLGA-CHP targeted preparation prepared in Example 1 of the present invention was detected by MTT method.

[0089] HL-1 cells were routinely cultured at 5×10 3 Cells were inoculated in 96-well plates and randomly divided into 11 groups, including normal, PLGA-CHP and EGCG-PLGA-CHP (final concentrations were 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL, respectively). Each group had 6 replicate wells. After adding the corresponding drugs and incubating for 48 h, MTT reagent was added. The absorbance was detected at a wavelength of 490 nm according to the instructions of the kit, and the relative cell survival rate was calculated.

[0090] like Fig.10 As shown, the nanocarrier PLGA-CHP has no cytotoxicity at 0.1-1.6 mg / mL and has good biocompatibility to HL-1. The EGCG-PLGA-CHP nano-targeted preparation has low cytotoxicity at 0.1-0.8 mg / mL and has significant cytotoxicity at a concentration above 0.8 mg / mL.

[0091] (7) Cell uptake experiment

[0092] According to the method of Example 1, PKH67 fluorescent dye was added to the EGCG aqueous solution to obtain EGCG-PLGA and EGCG-PLGA-CHP encapsulating PKH67.

[0093] HL-1 cardiomyocytes were diluted with culture medium to a density of 1×10 4 mL -1 The suspension was inoculated into a 6-well cell culture plate and cultured for 12 h. After the cells adhered to the wall, they were divided into two groups and treated with drugs (EGCG-PLGA or EGCG-PLGA-CHP, both with a final concentration of 12.5 μM). The uptake was observed under a fluorescence microscope after 24 h, and the process was repeated three times.

[0094] The results are as follows Fig.11 , EGCG-PLGA and EGCG-PLGA-CHP nano-targeted preparations were taken up by HL-1 cells, and the in vitro targeting ability of the coupled targeting peptide CHP as a carrier was significantly better than that of the non-targeted carrier.

[0095] (8) Coupling rate determination

[0096] According to the method of Example 1, DiI fluorescent dye was added to the EGCG aqueous solution, and CHP-FITC was used as the targeting peptide to obtain EGCG-PLGA-CHP-FITC encapsulated with DiI.

[0097] The binding of FITC-labeled CHP and DiI-labeled EGCG-PLGA nanoparticles was observed by inverted fluorescence microscopy. 15 different batches of EGCG-PLGA-CHP were prepared repeatedly, and the CHP connection rate ≥90% was considered to be successful in the preparation of nanoparticles.

[0098] like Fig.12 As shown, DiI-labeled EGCG-PLGA exhibited red fluorescence, FITC-labeled CHP peptide exhibited green fluorescence, and the fusion of the two was orange with a connection rate of (94.11±3)%, indicating that EGCG-PLGA was well connected with the targeting peptide CHP.

[0099] Table 2 EGCG-PLGA-CHP coupling rate determination results

[0100] name Coupling rate EGCG-PLGA-CHP 94.11±3％

[0101] (9) Western blot detection of Bax and Bcl-2 protein expression

[0102] ① In vitro experiments

[0103] HL-1 cells were randomly divided into normoxia group, hypoxia group, hypoxia+EGCG group and hypoxia+EGCG-PLGA-CHP nanoformulation group.

[0104] HL-1 cells were routinely cultured. 48 h before modeling, the HL-1 cell culture medium was replaced with serum-free sugar-free DMEM, and the corresponding drugs (final concentration 25 μmol / L) were added for pretreatment. The modeling was carried out in an anaerobic glove box. A 3% O2-92% N2-5% CO2 mixed gas was pumped in by a vacuum pump to replace the conventional culture, and the cells were cultured in anoxic state at 37°C for 18 h to simulate myocardial ischemia and hypoxia. The cell culture medium was collected, and the expression of cell proteins in each group was detected using a kit according to the operating instructions.

[0105] ② In vivo experiments

[0106] C57BL / 6 mice were randomly divided into sham operation, AMI, AMI+EGCG and AMI+EGCG-PLGA-CHP nanoformulation groups, and the corresponding drugs were injected into the tail vein 30 minutes before ischemia for pretreatment. The sham operation group was given the same volume of PBS. The model was established as follows:

[0107] Acute myocardial infarction (AMI) model was established: mice were intraperitoneally injected with sodium pentobarbital (60 mg / kg), fixed in the supine position, a midline incision was made in the neck, endotracheal intubation was performed, and the mice were connected to a small animal ventilator (respiratory rate 60 times / min, tidal volume 16 ml / kg, respiratory ratio 1:2.5). The electrocardiogram was continuously monitored by the MS4000 biological signal quantitative recording and analysis system. A longitudinal incision was made about 0.5 cm to the left of the sternum to expose the heart. A 6 / 0 suture was passed under the left anterior descending coronary artery about 2 mm from the origin of the left ventricular branch of the coronary artery, and a silicone tube was placed on it for standby use. After the electrocardiogram stabilized for 15 minutes, the suture was tightened in all groups except the sham operation group. The QRS amplitude was increased, the ST segment was elevated, and the T wave was tall or inverted as the success criteria of ischemia. After 18 hours of ischemia, the mouse myocardial AMI model was established. The sham operation group only passed the suture but did not ligate.

[0108] like Fig.13 As shown in AC and 14A-C, compared with the normal (sham operation) group, the expression of Bax protein in the model group was increased and the expression of Bcl-2 protein was decreased (P < 0.05); both EGCG and targeted nanoparticle pretreatment significantly downregulated the expression of Bax protein and upregulated the expression of Bcl-2 protein, and the effect of the targeted group was stronger than that of the EGCG group (P < 0.05), indicating that EGCG-PLGA-CHP has a significant inhibitory effect on apoptosis.

[0109] In summary, the present invention uses PLGA-CHP as a targeting carrier of EGCG and accurately targets the drug to the heart. The EGCG-PLGA-CHP nano-targeted preparation of the present invention has good stability and dispersibility, realizes controlled release of EGCG, and protects the antioxidant properties of EGCG; the targeting carrier PLGA-CHP can accurately target the drug EGCG to the heart, and the nano-targeted preparation can inhibit cell apoptosis and resist myocardial ischemia damage.

[0110] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An EGCG-PLGA-CHP nano-targeted preparation, characterized in that: The EGCG-PLGA-CHP nano-targeted preparation is made of EGCG and a PLGA-CHP targeting carrier, and the PLGA-CHP targeting carrier is prepared by grafting a cardiac homing peptide onto a polylactic acid-glycolic acid copolymer; The preparation method of the EGCG-PLGA-CHP nano targeted preparation comprises the following steps: (1) EGCG and poly(lactic acid-co-glycolic acid) copolymer were treated by a double emulsion solvent evaporation method to prepare EGCG-PLGA nanoparticles; (2) activating the EGCG-PLGA nanoparticles and a coupling activator in a MES buffer to obtain activated EGCG-PLGA nanoparticles, and subjecting the activated EGCG-PLGA nanoparticles to a coupling reaction with a cardiac homing peptide in a MES buffer to obtain an EGCG-PLGA-CHP nano-targeted preparation; The double emulsion solvent evaporation method in step (1) comprises the following specific steps: mixing an EGCG aqueous solution and a PLGA ethyl acetate solution to obtain a mixed solution of EGCG and PLGA, emulsifying the mixed solution of EGCG and PLGA in an ice bath to obtain colostrum; adding a polyether F-127 solution to the colostrum, demulsifying the solution in an ice bath to obtain a double emulsion, adding a polyether F-127 solution to the double emulsion at room temperature, and then successively performing solvent evaporation, ultrafiltration, and freeze-drying to obtain EGCG-PLGA nanoparticles; the mass concentration of the polyether F-127 solution added to the colostrum is 10 to 30 mg / mL; the mass concentration of the polyether F-127 solution added to the double emulsion is 1 to 5 mg / mL; The particle size range of the EGCG-PLGA-CHP nano-targeted preparation is 90 to 120 nm, the average particle size of the EGCG-PLGA-CHP nano-targeted preparation is 95.65±0.227 nm, and the Zeta potential distribution of the EGCG-PLGA-CHP nano-targeted preparation is -1.67±0.01 mV.

2. An EGCG-PLGA-CHP nano targeted preparation according to claim 1, characterized in that: The weight ratio of the EGCG to the polylactic acid-glycolic acid copolymer is (0.1-12):(3-40); the weight ratio of the total weight of the EGCG and the polylactic acid-glycolic acid copolymer to the cardiac homing peptide is (10-30):(1-3).

3. An EGCG-PLGA-CHP nano targeted preparation according to claim 1, characterized in that: The mass concentration of the EGCG aqueous solution is 10-60 mg / mL; the mass concentration of the PLGA ethyl acetate solution is 3-20 mg / mL; the volume ratio of the EGCG aqueous solution, the PLGA ethyl acetate solution, the polyether F-127 solution added to the colostrum and the polyether F-127 solution added to the double emulsion is (0.1-0.2):(1-2):(5-10):(15-20).

4. An EGCG-PLGA-CHP nano targeted preparation according to claim 1, characterized in that: The emulsification described herein specifically involves emulsification using an ultrasonic cell disruptor for 90 to 120 seconds; the demulsification described herein specifically involves demulsification using an ultrasonic cell disruptor for 90 to 120 seconds.

5. An EGCG-PLGA-CHP nano targeted preparation according to any one of claims 3 to 4, characterized in that: The mass ratio of the EGCG-PLGA nanoparticles to the EDC is (5-15):(6-12); the weight ratio of the EGCG-PLGA nanoparticles to the cardiac homing peptide is (10-30):(1-3).

6. An EGCG-PLGA-CHP nano-targeted preparation according to any one of claims 3 to 4, characterized in that: The activation temperature is 20-28° C., and the activation time is 1-2 hours; the coupling reaction temperature is 20-28° C., and the coupling reaction time is 8-24 hours.

7. An application of an EGCG-PLGA-CHP nano targeted preparation, characterized in that: The EGCG-PLGA-CHP nano targeted preparation according to any one of claims 1 to 3 is used to prepare a drug for preventing or treating myocardial ischemic injury.

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