Nanoparticles based on flavonoid compounds from hibiscus flowers, preparation method and application

Abstract

The present invention provides nanospheres based on flavonoid compounds of hibiscus flower and a preparation method thereof. PFC is dispersed as particles in a hibiscus flower solution as an internal aqueous phase, mixed with an oil phase containing a polylactic-co-glycolic acid copolymer, and a glycoside component in the traditional Chinese medicine of hibiscus flower is used as an emulsifier to obtain an emulsion. The emulsion is then treated with an external aqueous phase solution to obtain nanospheres TEA / PFC@PLGA. FITC-labeled CHP polypeptide is grafted onto the surface of the nanospheres TEA / PFC@PLGA to obtain CHP-TEA / PFC@PLGA nanospheres with a targeted effect. The nanospheres of the present invention can be directly administered by intravenous injection and targeted to myocardial cells through the FITC-labeled CHP polypeptide. Ultrasound is used to mediate the release of oxygen from PFC. While the PFC releases oxygen under conditions mediated by ultrasonic vaporization, the flavonoid compounds are released. The two drugs are released synergistically and taken up by myocardial cells at the same time, protecting the morphological structure and function of hypoxic myocardial cells, thereby achieving the purpose of protecting and treating myocardial diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to nano-microspheres based on flavonoid compounds of hibiscus flowers, a preparation method and an application thereof. Background Art

[0002] Myocardial disease is a very difficult and stubborn disease to overcome in modern times. Not only is the cause and mechanism of the disease not yet clear for most patients, making it very difficult to treat, but the myocardial damage and lesions caused by myocardial disease are organic and often irreversible, so it is difficult to cure. Treatment can only be used to improve the patient's clinical symptoms, prevent the malignant development of the disease, reduce the possibility of complications, and improve the patient's quality of life.

[0003] In recent years, as human beings gradually improve their understanding of the causes and genetic mechanisms of myocardial diseases, in-depth research on the pathophysiology of heart diseases, myocardial cell metabolomics and immunology, combined with modern technical means such as electron microscopy, cytological examination, polymer biology and immunology research, has made the detection, diagnosis and treatment of myocardial diseases more complete, and can also improve patients' medical effects and prognosis.

[0004] However, the treatment of myocardial diseases remains one of the major challenges facing world health, not only because some of the causes and pathogenesis of myocardial lesions are not yet fully understood, but also because the related complications that may be caused are very difficult to treat, and there is a lack of safe and feasible treatments and preventive measures. Therefore, the current treatment method is still mainly drug therapy.

[0005] Currently, Western medicine lacks a specific treatment method. Treatments typically involve antibiotics, antivirals, hormones, and nutritional supplements, which are long and associated with significant side effects. Traditional Chinese medicine (TCM) addresses both the underlying cause and symptoms, minimizes toxicity and side effects, and prevents drug resistance. Studies have shown that various flavonoids have a protective effect against damage caused by myocardial ischemia, but they remain ineffective in treating heart disease. Furthermore, oral administration of flavonoids via gavage results in low bioavailability, limiting their efficacy. Summary of the Invention

[0006] The present invention aims to address the deficiencies in the prior art and provide nanoparticles based on flavonoids from hibiscus flower and a preparation method thereof. Nanoparticles formed of polylactic acid-glycolic acid copolymer are used to encapsulate flavonoids from hibiscus flower and perfluorocarbons, and FITC-labeled CHP polypeptides are grafted onto the surface of the nanoparticles to obtain a long-acting injectable CHP-TEA / PFC@PLGA nanoparticles that can be directly injected. The flavonoids from hibiscus flower and PFC can be synergistically released to protect the morphology, structure and function of hypoxic cardiomyocytes, thereby achieving the purpose of protecting and treating myocardial diseases.

[0007] According to a first aspect of the present invention, nanospheres based on flavonoid compounds from hibiscus flowers are provided. The flavonoid compounds from hibiscus flowers and perfluorocarbons are encapsulated in nanospheres formed of poly(lactic acid-co-glycolic acid) copolymer to form nanospheres of flavonoid compounds, TEA / PFC@PLGA. FITC polypeptide is grafted onto the surface of the TEA / PFC@PLGA to form CHP-TEA / PFC@PLGA nanospheres.

[0008] As an optional embodiment, the particle size of the CHP-TEA / PFC@PLGA nanospheres is 100 to 300 nm.

[0009] According to a second aspect of the present invention, there is provided a method for preparing the aforementioned nanospheres based on flavonoid compounds of hibiscus flowers, comprising the following steps:

[0010] Poly(lactic-co-glycolic acid) (PLGA-COOH) was dissolved in dichloromethane, and then sorbitan trioleate was added to prepare an oil phase solution;

[0011] A hibiscus flower powder is dissolved in deionized water to obtain a hibiscus flower solution, and perfluorocarbon (PFC) is slowly added dropwise to the hibiscus flower solution under ultrasonic conditions to prepare an inner aqueous phase solution.

[0012] In an ice bath and sonication, the inner aqueous phase solution is added dropwise to the oil phase solution to obtain an emulsion, the emulsion is then added to a first polyvinyl alcohol solution for sonication to prevent microsphere aggregation, and then added to a second polyvinyl alcohol solution for solidification under magnetic stirring to remove the organic solvent, thereby obtaining a first mixed solution;

[0013] The first mixed solution was centrifuged to collect the solid, and the solid was washed with deionized water to obtain the hibiscus flower flavonoids nanoparticles TEA / PFC@PLGA;

[0014] FITC-labeled CHP polypeptide was grafted onto the surface of TEA / PFC@PLGA to obtain CHP-TEA / PFC@PLGA nanospheres.

[0015] As an optional embodiment, in the oil phase solution, the concentration of poly(lactic acid-co-glycolic acid) is 2-3 mg / mL, and the concentration of sorbitan trioleate is 9-10 mg / mL.

[0016] As an optional embodiment, the concentration of the hibiscus flower solution is 1-3 mg / mL, and the concentration of the perfluorocarbon in the inner aqueous phase solution is 8.5-9 mg / mL.

[0017] As an optional embodiment, the inner aqueous phase solution is added dropwise to the oil phase solution to obtain an emulsion, wherein the volume ratio of the inner aqueous phase solution to the oil phase solution is (0.5-0.6):1.

[0018] As an optional embodiment, the specific process of grafting FITC polypeptide onto the surface of PLGA nanospheres is as follows:

[0019] TEA / PFC@PLGA was dissolved in the first MES buffer, and EDC and NHS were added and stirred at room temperature to obtain a second mixed solution;

[0020] After stirring, the residual EDC and NHS were removed by centrifugation. The obtained precipitate was dissolved in a second MES buffer and then a FITC-labeled CHP polypeptide solution was added to obtain a third mixed solution. The solution was reacted in the dark. After the reaction, the free polypeptide was removed by centrifugation to obtain CHP-TEA / PFC@PLGA nanospheres.

[0021] As an optional embodiment, in the second mixed solution, the concentration of TEA / PFC@PLGA is 0.5-1 mg / mL, the concentration of EDC is 1-2 mg / mL, and the concentration of NHS is 0.5-1.5 mg / mL;

[0022] In the third mixed solution, the mass ratio of the precipitate to the FITC-labeled CHP polypeptide is (4-5):1.

[0023] As an optional embodiment, the concentration of the first MES buffer solution is 0.1 mol / L, and the pH is 6; the concentration of the second MES buffer solution is 0.1 mol / L, and the pH is 8.

[0024] According to a third aspect of the present invention, there is provided a use of the aforementioned nanoparticles based on flavonoid compounds from hibiscus flowers in the preparation of a drug for treating myocardial diseases.

[0025] Compared with the prior art, the present invention has the following significant beneficial effects:

[0026] The nanoparticles based on flavonoid compounds of hibiscus flowers of the present invention can be directly administered by intravenous injection, and target myocardial cells through FITC-labeled CHP polypeptide. Ultrasound-mediated PFC releases oxygen. While the PFC releases oxygen under ultrasonic vaporization-mediated conditions, it expands and bursts the microparticles, allowing the flavonoid compounds to be released. The two drugs are released synergistically and taken up by myocardial cells at the same time, protecting the morphological structure and function of hypoxic myocardial cells, thereby achieving the purpose of protecting and treating myocardial diseases.

[0027] The nanoparticles based on flavonoid compounds of hibiscus flowers of the present invention have the advantages of long-acting injections, can effectively reduce the number of administrations, maintain drug activity, have strong efficacy, low incidence of side effects, have excellent biocompatibility and biodegradability, and have certain mechanical strength and plasticity.

[0028] The invention discloses nano-microspheres based on flavonoid compounds of hibiscus flower and a preparation method thereof. PFC is dispersed as particles in a hibiscus flower solution as an internal aqueous phase, and the mixture is mixed with an oil phase containing a polylactic acid-glycolic acid copolymer. A glycoside component in the traditional Chinese medicine of hibiscus flower is used as an emulsifier to obtain an emulsion. The emulsion is then treated with an external aqueous phase solution to obtain TEA / PFC@PLGA nano-microspheres. FITC-labeled CHP polypeptide is grafted onto the surface of the TEA / PFC@PLGA nano-microspheres to obtain CHP-TEA / PFC@PLGA nano-microspheres with a targeting effect.

[0029] The method of the present invention encapsulates hibiscus flower flavonoids and PFC in nanospheres formed of polylactic acid-glycolic acid copolymer, and grafts FITC-labeled CHP polypeptide on the surface of the nanospheres. No additional emulsifier is required in the system, and the obtained nanospheres have higher purity. The problem that PFC liquid is not suitable for injection into the blood and the current problem of poor drug targeting are solved, thereby obtaining a nano preparation with better therapeutic effect on myocardial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a process flow chart for preparing nano-microspheres based on flavonoid compounds of hibiscus flowers.

[0031] Figure 2 This is a DLS test graph of CHP-TEA / PFC@PLGA nanoparticles according to an example of the present invention.

[0032] Figure 3 This is a TEM test image of CHP-TEA / PFC@PLGA nanospheres according to the present invention.

[0033] Figure 4 1 is an HPLC test chart of the CHP-TEA / PFC@PLGA nanospheres and the standard sample (hibiscus flower compound); wherein a is the HPLC chart of the hibiscus flower compound, and b is the HPLC chart of the CHP-TEA / PFC@PLGA nanospheres.

[0034] Figure 5 These are the binding test diagrams of FITC-labeled CHP polypeptide and TEA / PFC@PLGA of CHP-TEA / PFC@PLGA nanospheres according to the present invention; wherein, a is the fluorescence microscopy test of TEA / PFC@PLGA nanospheres, b is the fluorescence microscopy test of FITC-labeled CHP polypeptide, and c is the fluorescence microscopy test of CHP-TEA / PFC@PLGA nanospheres.

[0035] Figure 6: This is a fluorescence microscope observation test diagram of cell uptake of CHP-TEA / PFC@PLGA nanospheres according to an example of the present invention; wherein, a is a fluorescence diagram of cell uptake of TEA / PFC@PLGA, and b is a fluorescence diagram of cell uptake of CHP-TEA / PFC@PLGA.

[0036] Figure 7 This is a flow cytometric test chart of the cellular uptake of CHP-TEA / PFC@PLGA nanospheres according to the present invention.

[0037] Figure 8 Graph showing the activity of H9C2 cells treated with the CHP-TEA / PFC@PLGA nanospheres of the present invention and a control sample. DETAILED DESCRIPTION

[0038] In order to better understand the technical content of the present invention, specific embodiments are given and described below with reference to the accompanying drawings.

[0039] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to be comprehensive. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, can be implemented in any of a number of ways.

[0040] Hibiscus tiliaceus (Abelmoschus altissima) is a plant of the Malvaceae family. It contains flavonoids, reducing sugars, and other compounds. Flavonoids are the primary active ingredients, with hyperoside, quercetin, and isoquercetin being the most abundant. These compounds have been shown to improve renal function, provide anti-inflammatory effects, provide antipyretic and analgesic benefits, protect the myocardium, protect against ischemic damage to the heart and brain, and promote angiogenesis.

[0041] Based on this, the present invention designs a nano-microsphere, which encapsulates hibiscus flower flavonoids and PFC in nano-microspheres formed of polylactic acid-glycolic acid copolymer, and grafts FITC-labeled CHP polypeptide on the surface of the nano-microspheres by chemical bonds. The hibiscus flower flavonoids and PFC can be synergistically released to protect the morphological structure and function of hypoxic myocardial cells, thereby achieving the purpose of protecting and treating myocardial diseases.

[0042] On the other hand, the present invention utilizes glycoside components in the traditional Chinese medicine of hibiscus flower, such as hyperoside and isoquercetin, as emulsifiers. No additional emulsifier is required in the system, thus avoiding the problem of impure microspheres and increased costs caused by adding additional emulsifiers in the prior art.

[0043] In an exemplary embodiment of the present invention, nanospheres based on flavonoid compounds from hibiscus flower are provided, wherein flavonoid compounds from hibiscus flower and perfluorocarbon are encapsulated in nanospheres formed of poly(lactic acid-co-glycolic acid) copolymer to form nanospheres of flavonoid compounds TEA / PFC@PLGA, and the surface of the TEA / PFC@PLGA is grafted with FITC polypeptide to form CHP-TEA / PFC@PLGA nanospheres.

[0044] As an optional embodiment, the particle size of the CHP-TEA / PFC@PLGA nanospheres is 100 to 300 nm.

[0045] Combine Figure 1 As shown, in another exemplary embodiment of the present invention, a method for preparing the aforementioned nanoparticles based on flavonoid compounds of hibiscus flowers is provided, comprising the following steps:

[0046] Poly(lactic-co-glycolic acid) (PLGA-COOH) was dissolved in dichloromethane, and then sorbitan trioleate (Span85) was added to prepare an oil phase solution;

[0047] A hibiscus flower powder is dissolved in deionized water to obtain a hibiscus flower solution, and perfluorocarbon (PFC) is slowly added dropwise to the hibiscus flower solution under ultrasonic conditions to prepare an inner aqueous phase solution.

[0048] In an ice bath and sonication, the inner aqueous phase solution is added dropwise to the oil phase solution to obtain an emulsion, which is then added to a first polyvinyl alcohol (PVA) solution for sonication to prevent microsphere aggregation, and then added to a second polyvinyl alcohol solution for solidification under magnetic stirring to remove the organic solvent, thereby obtaining a first mixed solution;

[0049] The first mixed solution was centrifuged to collect the solid, and the solid was washed with deionized water to obtain the hibiscus flower flavonoid nanoparticles TEA / PFC@PLGA, where TEA represents hibiscus flower extract, i.e., hibiscus flower flavonoids;

[0050] FITC-labeled CHP peptide was coupled to the surface of TEA / PFC@PLGA through an EDC-mediated coupling reaction between the carboxyl groups of PLGA and the amine groups of CHP to obtain CHP-TEA / PFC@PLGA nanospheres.

[0051] As an optional embodiment, in the oil phase solution, the concentration of poly(lactic acid-co-glycolic acid) is 2-3 mg / mL, and the concentration of sorbitan trioleate is 9-10 mg / mL.

[0052] As an optional embodiment, the concentration of the hibiscus flower solution is 1-3 mg / mL, and the concentration of the perfluorocarbon in the inner aqueous phase solution is 8.5-9 mg / mL.

[0053] As an optional embodiment, the inner aqueous phase solution is added dropwise to the oil phase solution to obtain an emulsion, wherein the volume ratio of the inner aqueous phase solution to the oil phase solution is (0.5-0.6):1.

[0054] As an optional embodiment, the first polyvinyl alcohol solution is a 2 wt. % PVA solution, and the second polyvinyl alcohol solution is a 0.6 wt. % PVA solution.

[0055] As an optional embodiment, the specific process of obtaining the first mixed solution is as follows:

[0056] Under ice bath conditions, ultrasonic cell disruptor was used for sonication, and the inner aqueous phase solution was added dropwise to the oil phase to obtain an emulsion. The emulsion was then added to a 2% PVA solution and sonicated for 8 minutes with a vibration on / off cycle of 2s / 1s to prevent overheating. Thereafter, the solution was added to 30mL of 0.6% PVA under magnetic stirring and cured for 5 hours to remove the organic solvent to obtain a first mixed solution.

[0057] As an optional embodiment, the first mixed solution is centrifuged at 10,000 rpm for 10 minutes to collect solids, and the solids are washed 2 to 3 times with deionized water to obtain nanoparticles TEA / PFC@PLGA of flavonoid compounds from hibiscus flowers.

[0058] As an optional embodiment, the specific process of grafting FITC polypeptide onto the surface of PLGA nanospheres is as follows:

[0059] TEA / PFC@PLGA was dissolved in the first MES buffer, and EDC and NHS were added and stirred at room temperature to obtain a second mixed solution;

[0060] After stirring, the residual EDC and NHS were removed by centrifugation. The obtained precipitate was dissolved in a second MES buffer and then a FITC-labeled CHP polypeptide solution was added to obtain a third mixed solution. The solution was reacted in the dark. After the reaction, the free polypeptide was removed by centrifugation to obtain CHP-TEA / PFC@PLGA nanospheres.

[0061] As an optional embodiment, in the second mixed solution, the concentration of TEA / PFC@PLGA is 0.5-1 mg / mL, the concentration of EDC is 1-2 mg / mL, and the concentration of NHS is 0.5-1.5 mg / mL;

[0062] In the third mixed solution, the mass ratio of the precipitate to the FITC-labeled CHP polypeptide is (4-5):1.

[0063] As an optional embodiment, the concentration of the first MES buffer solution is 0.1 mol / L, and the pH is 6; the concentration of the second MES buffer solution is 0.1 mol / L, and the pH is 8.

[0064] In another exemplary embodiment of the present invention, there is provided a use of the aforementioned nanoparticles based on flavonoid compounds from hibiscus flowers in the preparation of a drug for treating myocardial diseases. After drug injection, the FITC-labeled CHP polypeptide on the surface of the microspheres targets myocardial cells. After targeting the myocardial cells, ultrasound-mediated PFC releases oxygen. While releasing oxygen under ultrasonic vaporization-mediated conditions, the PFC expands and bursts the microspheres, allowing the flavonoid compounds to be released. The two drugs are released synergistically and taken up by the myocardial cells at the same time, protecting the morphological structure and function of the hypoxic myocardial cells, thereby achieving the purpose of protecting and treating myocardial diseases.

[0065] The following will be combined with specific examples and experiments to conduct exemplary experiments and comparisons on the preparation of the aforementioned nano-microspheres and their effects. Of course, the embodiments of the present invention are not limited to these.

[0066] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0067] Example 1

[0068] Preparation of TEA / PFC@PLGA nanoparticles containing flavonoids from hibiscus flowers:

[0069] First, 10 mg of PLGA-COOH copolymer (polymerization ratio of 50:50) was taken and dissolved in 4 mL of dichloromethane. 40 μL of Span85 was added to prepare the oil phase. 5 mg of hibiscus flower Chinese medicine powder was dissolved in 1 mL of deionized water. 100 μL of FDC was added and ultrasonicated for 1 min to prepare the internal aqueous phase.

[0070] Secondly, under ice bath conditions, ultrasonic cell disruptor was used for sonication, and the hibiscus flower solution was added dropwise to the oil phase to obtain an emulsion; then the emulsion was added to a 2% PVA solution and sonicated for 8 minutes (vibration on / off cycle was 2s / 1s), and added to 30mL of 0.6% PVA under magnetic stirring conditions and cured for 5 hours to remove the organic solvent.

[0071] Finally, the solution was centrifuged at 10,000 rpm for 10 min to collect the nanospheres, which were washed 2 to 3 times with deionized water to obtain TEA / PFC@PLGA and stored at 4°C.

[0072] Example 2

[0073] Construction of CHP-TEA / PFC@PLGA nanospheres:

[0074] 2 mg of TEA / PFC@PLGA prepared in Example 1 was dissolved in 5 mL of MES buffer (0.1 mol / L, pH = 6), 10 mg of EDC and 5 mg of NHS (mixed coupling agent) were added, and the mixture was stirred at room temperature for 1 h. After centrifugation at 10,000 rpm three times, the residual EDC and NHS were removed to obtain a precipitate.

[0075] The precipitate was redissolved in 0.1 mol / L MES buffer (pH 8.0), and 1 mL of FITC-labeled CHP peptide solution (1 mg / mL) was added dropwise to the solution. The mixture was allowed to react in the dark for 1 h. The free peptide was then removed by centrifugation three times, and the final CHP-TEA / PFC@PLGA nanospheres were harvested and stored at 4°C.

[0076] Example 3

[0077] DLS, TEM

[0078] The CHP-TEA / PFC@PLGA nanospheres obtained in Example 2 were subjected to dynamic light scattering (DLS) and TEM tests. Figure 2 and Figure 3 shown.

[0079] From the results, it can be seen that CHP-TEA / PFC@PLGA nanospheres are spherical with an average particle size of 260 nm.

[0080] Example 4

[0081] Determination of drug content in microspheres

[0082] 100 mg of the Hibiscus hibiscus flower compound was placed in a 25 mL volumetric flask, and 15 mL of methanol solution was added. The mixture was heated and ultrasonicated for 30 minutes, cooled, diluted to the mark with methanol, shaken, and filtered through a 0.45 μm microporous membrane. The mixture was then gradient eluted using acetonitrile-0.05% formic acid aqueous solution as the mobile phase, and analyzed by HPLC. Simultaneously, the CHP-TEA / PFC@PLGA nanospheres obtained in Example 2 were dissolved in methanol, ultrasonicated, filtered through a 0.45 μm microporous membrane, and analyzed by HPLC.

[0083] like Figure 4 As shown in the figure, it can be seen that the peak time of the liquid phase of CHP-TEA / PFC@PLGA nanoparticles ( Figure 4 b) and hibiscus flower compounds ( Figure 4a) is the same, indicating that the flavonoids of hibiscus flower are successfully encapsulated in the nanospheres. At the same time, the prepared W1 / O / W2 double emulsion solvent showed a uniform emulsion with no oily droplets at the bottom, indicating that the PFC was successfully encapsulated in the nanospheres.

[0084] Example 5

[0085] Binding of FITC-labeled CHP peptide and TEA / PFC@PLGA

[0086] The degree of connection between FITC-labeled CHP and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-labeled TEA / PFC@PLGA nanospheres was determined by examining the connection between FITC-labeled CHP and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) under a fluorescence microscope. Figure 5 shown.

[0087] like Figure 5 c Merged orange image shows TEA / PFC@PLGA nanospheres (red, as Figure 5 a) and FITC-labeled CHP polypeptide (green, as Figure 5 b) There is a perfect connection between the two groups, which proves that the FITC-labeled CHP polypeptide in the present invention is successfully grafted on the surface of TEA / PFC@PLGA.

[0088] The above tests can prove that the present invention successfully prepared CHP-TEA / PFC@PLGA nanoparticles.

[0089] Example 6

[0090] Nanoparticle fluorescence microscopy observation of cellular uptake experiments

[0091] H9C2 cells were collected at 1×10 4 The cells were seeded at a density of 1000 nanoparticles / well in a 96-well plate, and the plate was placed in an incubator at 37°C and cultured overnight. When the cells grew to 70%-80%, the culture medium was removed and cultured for 2.5 hours with serum-free culture medium containing polypeptide (sample of Example 2) and without polypeptide (sample of Example 1). After washing with PBS three times to remove excess nanoparticles not taken up by the cells, 4% paraformaldehyde was added for fixation for 30 minutes, and DAPI was added for staining for 10 minutes. After washing with PBS twice, the cells were observed and photographed using a fluorescence microscope. The results are as follows Figure 6 shown.

[0092] As can be seen from the figure, the nanoparticles with peptides can well target the cardiomyocytes stained with DAPI (4',6-diamidino-2-phenylindole) ( Figure 6 b), while the nanoparticles without FITC peptide did not show obvious fluorescence in the figure ( Figure 6 a) Under a fluorescence microscope, DiI probe shows red fluorescence and DAPI shows blue fluorescence.

[0093] This indicates that the CHP-TEA / PFC@PLGA nanoparticles of the present invention can target ischemic myocardial cells.

[0094] Example 7

[0095] Nanoparticle cellular uptake assay using flow cytometry

[0096] H9C2 cells 2×10 5 The cells were seeded in a 6-well plate at a density of 1000 nanoparticles / well, and the well plate was placed in an incubator at 37°C and cultured overnight. When the cells grew to 70%-80%, the culture medium was removed and replaced with serum-free culture medium containing the polypeptide (sample of Example 2) and the sample without the polypeptide (sample of Example 1) for 2.5 hours, respectively. Untreated cells were used as negative controls. The cells were washed three times with PBS to remove excess nanoparticles that were not taken up by the cells, and the digested cells were transferred to a 1.5 mL centrifuge tube and centrifuged for 5 minutes. The supernatant was removed and the cells were resuspended with 0.2 mL PBS and detected by flow cytometry. The results are as follows: Figure 7 shown.

[0097] As can be seen from the figure, the fluorescence intensity of the cells in the FITC-peptide group is higher than that in the non-peptide group, and the cardiomyocytes have a stronger ability to take up the nanoparticles with the myocardial-targeting peptide, which further proves that the CHP-TEA / PFC@PLGA nanoparticles of the present invention have a strong targeting effect on the myocardium.

[0098] Example 8

[0099] In vitro protective effect of CHP-TEA / PFC@PLGA nanoparticles against X-rays

[0100] CHP-TEA@PLGA preparation process:

[0101] First, 10 mg of PLGA-COOH copolymer (polymerization ratio of 50:50) was taken and dissolved in 4 mL of dichloromethane. 40 μL of Span85 was added to prepare the oil phase. 5 mg of Hibiscus tiliaceus flower Chinese medicine powder was dissolved in 1 mL of deionized water as the internal aqueous phase.

[0102] Secondly, under ice bath conditions, ultrasonic cell disruptor was used for sonication, and the hibiscus flower solution was added dropwise to the oil phase to obtain an emulsion; then the emulsion was added to a 2% PVA solution and sonicated for 8 minutes (vibration on / off cycle was 2s / 1s), and added to 30mL of 0.6% PVA under magnetic stirring conditions and cured for 5 hours to remove the organic solvent.

[0103] Finally, the solution was centrifuged at 10,000 rpm for 10 min to collect the nanospheres, which were washed 2 to 3 times with deionized water to obtain TEA@PLGA, which was then stored at 4°C.

[0104] Take 2 mg of TEA@PLGA and dissolve it in 5 mL of MES buffer (0.1 mol / L, pH = 6). Add 10 mg of EDC and 5 mg of NHS (mixed coupling agent) and stir at room temperature for 1 hour. After centrifugation at 10,000 rpm for 3 times, the residual EDC and NHS were removed to obtain a precipitate.

[0105] The precipitate was redissolved in 0.1 mol / L MES buffer (pH 8.0), and 1 mL of FITC-labeled CHP peptide solution (1 mg / mL) was added dropwise to the solution. The mixture was allowed to react in the dark for 1 h. The free peptide was then removed by centrifugation three times, and the final CHP-TEA@PLGA nanospheres were harvested and stored at 4°C.

[0106] Cell assay:

[0107] H9C2 cells 2×10 5 Cells were seeded in a 6-well plate at a density of 100 μL / well and placed in a 37°C incubator for overnight culture. When the cells grew to 70%-80%, the culture medium was removed and CHP-TEA@PLGA and CHP-TEA / PFC@PLGA nanospheres (0.3 mg / mL) were added respectively. Untreated cells served as negative controls and were irradiated using a VARIAN VitalBeam accelerator with a cumulative dose of 16 Gy. The cells were then placed in an incubator and incubated for 48 hours. The old culture medium was discarded and 100 μL of 10% CCK-8 working solution prepared in high-glucose culture medium was added to each well. The cells were incubated in a cell culture incubator for 1 hour. The absorbance was detected by the microplate reader at a wavelength of 450 nm, and the cell viability was expressed as a percentage of the control.

[0108] like Figure 8 As shown, an effective damage model for H9C2 cells after X-ray irradiation was used. After sufficient exposure to X-rays (16 Gy), H9C2 cell viability decreased to 49%-52%. Subsequently, the protective effects of CHP-TEA@PLGA and CHP-TEA / PFC@PLGA on H9C2 cells were tested.

[0109] The results showed that pretreatment of cells with CHP-TEA@PLGA and CHP-TEA / PFC@PLGA for 4-8 hours before irradiation had an effective protective effect on H9C2 cells exposed to X-rays (16 Gy), resulting in an increase in cell viability from 49%-52% to 64%-67%. At the same time, it can be seen that the cell viability of the PFC-loaded nanoparticles group was 72%-74%, which was significantly better than that of the CHP-TEA@PLGA group, proving that CHP-TEA / PFC@PLGA nanoparticles have a good protective effect on cardiomyocytes. In addition, ultrasound-mediated oxygen release from PFC was used. While releasing oxygen under ultrasonic vaporization-mediated conditions, PFC expanded and burst the microspheres, allowing flavonoids to be released. The two drugs were released synergistically and taken up by cardiomyocytes at the same time, protecting the morphological structure and function of hypoxic cardiomyocytes, achieving the purpose of protecting and treating myocardial diseases.

[0110] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A nanoparticle based on flavonoids from hibiscus flowers, characterized in that: Abelmoschus flower flavonoid compound TEA and perfluorocarbon PFC are encapsulated in nanospheres formed of poly(lactic acid-co-glycolic acid) to form nanospheres of Abelmoschus flower flavonoid compound TEA / PFC@PLGA. The surface of the TEA / PFC@PLGA is grafted with FITC-modified CHP polypeptide to form CHP-TEA / PFC@PLGA nanospheres. Among them, PFC was dispersed as particles in a hibiscus flower solution as the inner aqueous phase, mixed with an oil phase containing polylactic acid-glycolic acid copolymer, and the glycoside component in the traditional Chinese medicine hibiscus flower was used as an emulsifier to obtain an emulsion, which was then treated with the outer aqueous phase solution to obtain nano-microspheres TEA / PFC@PLGA.

2. The nanoparticles based on flavonoids from hibiscus flowers according to claim 1, characterized in that: The particle size of the CHP-TEA / PFC@PLGA nanoparticles is 100-300 nm.

3. A method for preparing nanoparticles based on flavonoid compounds from hibiscus flowers according to any one of claims 1 to 2, characterized in that: The following steps are involved: After polylactic acid-co-glycolic acid copolymer PLGA-COOH was dissolved in dichloromethane, sorbitan trioleate was added to prepare an oil phase solution; A hibiscus flower powder is dissolved in deionized water to obtain a hibiscus flower solution, and perfluorocarbon (PFC) is slowly added dropwise to the hibiscus flower solution under ultrasonic conditions to prepare an inner aqueous phase solution. In an ice bath and sonication, the inner aqueous phase solution is added dropwise to the oil phase solution to obtain an emulsion, the emulsion is then added to a first polyvinyl alcohol solution for sonication to prevent microsphere aggregation, and then added to a second polyvinyl alcohol solution for solidification under magnetic stirring to remove the organic solvent, thereby obtaining a first mixed solution; The first mixed solution was centrifuged to collect the solid, and the solid was washed with deionized water to obtain the hibiscus flower flavonoids nanoparticles TEA / PFC@PLGA; FITC-labeled CHP polypeptide was grafted onto the surface of TEA / PFC@PLGA to obtain CHP-TEA / PFC@PLGA nanospheres.

4. The method for preparing nanoparticles based on flavonoid compounds of hibiscus flowers according to claim 3, characterized in that: In the oil phase solution, the concentration of poly(lactic acid-co-glycolic acid) is 2-3 mg / mL, and the concentration of sorbitan trioleate is 9-10 mg / mL.

5. The method for preparing nanoparticles based on flavonoid compounds of hibiscus flowers according to claim 3, characterized in that: The concentration of the hibiscus flower solution is 1-3 mg / mL, and the concentration of the perfluorocarbon in the inner aqueous phase solution is 8.5-9 mg / mL.

6. The method for preparing nanoparticles based on flavonoid compounds of hibiscus flowers according to claim 3, characterized in that: The inner aqueous phase solution is added dropwise to the oil phase solution to obtain an emulsion, wherein the volume ratio of the inner aqueous phase solution to the oil phase solution is (0.5-0.6):

1.

7. The method for preparing nanospheres based on flavonoid compounds from hibiscus flowers according to claim 3, characterized in that: The specific process of grafting FITC-labeled CHP peptide onto the surface of TEA / PFC@PLGA is as follows: TEA / PFC@PLGA was dissolved in the first MES buffer, and EDC and NHS were added and stirred at room temperature to obtain a second mixed solution; After stirring, the residual EDC and NHS were removed by centrifugation. The obtained precipitate was dissolved in a second MES buffer and then a FITC-labeled CHP polypeptide solution was added to obtain a third mixed solution. The solution was reacted in the dark. After the reaction, the free polypeptide was removed by centrifugation to obtain CHP-TEA / PFC@PLGA nanospheres.

8. The method for preparing nanoparticles based on flavonoid compounds of hibiscus flowers according to claim 7, characterized in that: In the second mixed solution, the concentration of TEA / PFC@PLGA was 0.5–1 mg / mL, the concentration of EDC was 1–2 mg / mL, and the concentration of NHS was 0.5–1.5 mg / mL; In the third mixed solution, the mass ratio of the precipitate to the FITC-labeled CHP polypeptide is (4-5):

1.

9. The method for preparing nanoparticles based on flavonoid compounds from hibiscus flowers according to claim 7, characterized in that: The concentration of the first MES buffer solution is 0.1 mol / L, and the pH value is 6; the concentration of the second MES buffer solution is 0.1 mol / L, and the pH value is 8.

10. Use of the nanoparticles based on flavonoid compounds from hibiscus flowers according to any one of claims 1 to 2 in the preparation of drugs for treating myocardial diseases.

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