A pH-responsive biomimetic nano-preparation based on PC NPs, and a preparation method and application thereof

Abstract

The present application relates to a kind of pH-responsive biomimetic nano-preparation based on PC NPs and its preparation method and application, and the pH-responsive nano-preparation is by procyanidin self-assembly nanoparticles (PC NPs), hydrophobic drug, with the hyaluronic acid of targeting effect Composition, hydrophobic drug is loaded in PC NPs, with the outer layer of the PC NPs of the hydrophobic drug of targeted effect is modified with hyaluronic acid.The pH-responsive nano-preparation can realize drug co-delivery, reduce the influence of carrier, enhance the targeting ability and the controllable release in pathological site, so that the pH-responsive nano-preparation is applied to the preparation of the drug for preventing and / or treating atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pH-responsive biomimetic nanoparticle formulation based on PC NPs, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (As) is the most common cause of CVD, including stroke, myocardial infarction, and coronary artery disease, and its development is the result of multiple cell interactions. The pathogenesis of As involves oxidized low-density lipoprotein (ox-LDL) damaging vascular endothelial cells, leading to a local inflammatory response. At the site of injury, cytokines are secreted, recruiting circulatory system mononuclear cells, which differentiate into macrophages that internalize cholesterol-rich lipoproteins to form foam cells. Foam cells secrete extracellular matrix, further promoting lipoprotein retention and the recruitment of pro-inflammatory factors, monocytes, T cells, and neutrophils. As inflammation and other cytotoxic factors stimulate foam cells, apoptosis occurs, forming a necrotic core. Apoptotic cells release inflammatory contents, creating a positive feedback loop that accelerates the progression of As and the formation of unstable plaques, leading to clinically fatal complications such as myocardial infarction as As progresses.

[0003] To maintain homeostasis, the body eliminates apoptotic cells through "cell burial." Cell burial is the process by which phagocytes, such as macrophages, dendritic cells, epithelial cells, and fibroblasts, clear apoptotic and necrotic cells, protecting surrounding tissues from damage caused by toxic enzymes, oxidants, and other cellular contents within the apoptotic cells. In the early stages of ankylosing spondylitis (AS), cell burial is functioning normally, rapidly clearing apoptotic cells, preventing secondary necrosis, inducing the production of anti-inflammatory factors, and eliminating inflammatory macrophages, preventing their transformation into foam cells. As AS progresses, foam cells accumulate to form a necrotic core. After macrophages engulf apoptotic cells, the rapidly increasing intracellular cholesterol impairs their efflux function, severely damaging cell burial and exacerbating AS progression. Therefore, restoring the cell burial function of macrophages is a practical strategy for alleviating AS.

[0004] Pitavastatin (usually as a calcium salt) is a member of the statin class of lipid-lowering drugs. It has been reported that pitavastatin enhances its cytotoxic effect by activating the ERK5 pathway in macrophages, thereby clearing necrotic cores. However, long-term oral administration results in low blood concentrations and rapid clearance, requiring high doses to achieve therapeutic efficacy. This lack of targeted therapy can easily lead to liver and kidney damage. Therefore, overcoming the shortcomings of traditional administration methods and achieving personalized treatment for patients holds great promise.

[0005] With the rapid development of nanotechnology, nanomedicine has also made significant progress. Nanomedicine refers to the use of nanoscale materials, such as biocompatible nanoparticles (NPs) or nanorobots, for biological diagnosis and drug delivery to achieve precision treatment. NPs are a general term for materials with dimensions less than a few hundred nanometers. These extremely small materials have attracted widespread attention in the biomedical field because they can cross various biological barriers and target and release encapsulated drugs to specific tissues. The core structure of polyphenols (PC) is similar to that of EGCG in tea polyphenols, both being polyphenolic flavonoids. Based on this physicochemical characteristic, PC can polymerize under certain conditions to form linear polyphenol oligomers. As the concentration increases, the linear polyphenol oligomers gradually aggregate to form hollow polyphenol NPs through hydrogen bonding, hydrophobic interactions, and π-π interactions. Subsequently, the NPs are dissolved and reassembled through solvent conversion, and the strong binding between proteins and some drug molecules by the main functional group (phenolic hydroxyl group) of PC is used to construct PC-drug NPs with high drug loading capacity. These NPs have the characteristics of high efficiency, large yield, and flexible control. To address the liver and kidney damage caused by high-dose statin therapy, this study encapsulated pitavastatin (PC@Pita) with hollow linear polyphenol oligomers synthesized from PC to reduce statin toxicity. Studies have shown that EGCG, similar to PC, retains various biological activities after self-assembly into polyphenolic NPs. Therefore, PC, after forming linear polyphenolic NPs, will also retain various biological activities. The polyphenolic NP form can improve the bioavailability of PC and enhance therapeutic efficacy. To further improve the delivery efficiency and release effect of NPs, both NPs and the drug must undergo further functional modification to allow for fine-tuning and drug release at specific target lesion sites, achieving the effect of killing diseased cells. Currently, there are reports of nanocarriers sensitive to various biochemical or physical signals used for drug delivery and sustained release targeting subcellular organelles or lesion sites. Therefore, we linked a layer of HA to the outer layer of PC@Pita to target inflammatory macrophages in plaque areas and achieve drug accumulation. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a pH-responsive nano-formulation based on PC NPs, its preparation method, and its application. This pH-responsive nano-formulation enables co-delivery of drugs, reduces the influence of carriers, enhances targeting capabilities, and allows for controlled release at the lesion site.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a pH-responsive biomimetic nanoparticle based on PC NPs, wherein the pH-responsive nanoparticle includes proanthocyanidin self-assembled nanoparticles (PC NPs), a hydrophobic drug and hyaluronic acid with targeting function, wherein the hydrophobic drug is loaded in the PC NPs and the hyaluronic acid with targeting function is modified on the outer layer of the PC NPs loaded with the hydrophobic drug.

[0008] The PC NPs are formed by the linear polyphenol oligomers generated through phenolic condensation + benzoxazine reaction (phenol + primary amine + aldehyde), and then, as the concentration increases, the polyphenol oligomers gradually aggregate under hydrogen bonding, hydrophobic interaction and π-π interaction to form polyphenol nanoparticles.

[0009] The pH-responsive nanoparticles have a particle size of 140-200 nm.

[0010] Preferably, the hydrophobic drug is pitavastatin.

[0011] This invention also provides a method for preparing pH-responsive biomimetic nanoparticles based on PC NPs, comprising the following steps:

[0012] S1. PC is first ultrasonically stirred with formaldehyde and reacted in the dark for 5 minutes, then glycine is added dropwise and ultrasonically stirred for 2 hours to obtain an emulsion, the emulsion being PC NPs.

[0013] S2. The hydrophobic drug pitavastatin was dissolved and added to the PC NPs for ultrasonic stirring. The resulting solution was then dialyzed and freeze-dried to obtain a nano-formulation loaded with the hydrophobic drug (PC@Pita).

[0014] S3. The nano-formulation loaded with the hydrophobic drug is mixed with hyaluronic acid solution by ultrasonic stirring, and then subjected to dialysis and freeze-drying to obtain pH-responsive nano-formulation (HA@PC@Pita NPs).

[0015] Preferably, in step S1, the concentrations of PC, formaldehyde, and glycine are 2.5 mg / mL, 37%, and 10 mg / mL, respectively.

[0016] Preferably, in step S2, the mass ratio of PC NPs to the hydrophobic drug is 3 mg: 0.2 mg, and in step S3, the mass ratio of PC NPs to the hyaluronic acid is 3 mg: 0.05 mg.

[0017] Preferably, in steps S2 and S3, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa for 6 hours.

[0018] Preferably, the specific preparation process of the PC NPs in step S1 is as follows:

[0019] S101, dissolve 10 mg PC in 4 mL ddH2O to form a reddish-brown solution with a concentration of 2.5 mg / mL, add 10 μL HCHO with a concentration of 37%, and react in the dark at a stirring speed of 800 rpm for 5 min.

[0020] S102: Dissolve 8 mg Gly in 0.8 mL ddH2O to form a colorless and transparent solution with a concentration of 10 mg / mL, and add it dropwise to the solution obtained in S101. Stir at 800 rpm and react in the dark for 2 hours.

[0021] S103: The solution obtained from S102 was centrifuged at 13000 rpm for 10 min, and the precipitate was washed three times with water to obtain PC NPs. The NPs were dispensed in 3 mg vials and stored at -20℃.

[0022] Preferably, in steps S1, S2 and S3, the ultrasonic stirring speed is 800 rpm, the ultrasonic power is 100 W and the ultrasonic time is 2 min.

[0023] Another aspect of the present invention provides the application of pH-responsive biomimetic nanoformulations based on PC NPs in the preparation of drugs for the prevention and / or treatment of atherosclerosis.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This invention designs a green, biodegradable biomimetic nanoparticle formulation. Utilizing the design characteristics of hyaluronic acid and pH responsiveness, it achieves specific aggregation, penetration, and controlled release of drugs at lesion sites. This invention utilizes phenolic condensation and benzoxazine reaction to realize proanthocyanidin self-assembled nanoparticles (PC NPs). Since PC NPs can reassemble in DMSO, solvent conversion enables the dissolution and reassembly of NPs. Furthermore, leveraging the strong binding interaction between the main functional group of PC (phenolic hydroxyl group) and proteins and some drug molecules, PC-drug NPs with high drug loading capacity are constructed. Hyaluronic acid is used to modify the outer layer of PC-drug NPs (HA@PC@Pita NPs) to achieve the targeting capability of the nanoparticle formulation to lesion sites. When this pH-responsive biomimetic nanoparticle formulation reaches the lesion site, the interaction between HA and the activated macrophage surface receptor CD44 successfully enables the pH-responsive nanoparticle formulation to target and enter the activated macrophages. Meanwhile, the slightly acidic environment (pH 6.4-6.8) in the microenvironment promotes the release of hydrophobic drugs from pH-responsive biomimetic nano-formulations, thereby achieving controlled drug release at the lesion site;

[0026] 2. In addition to loading hydrophobic drugs for the treatment and / or prevention of atherosclerosis, the present invention provides a biomimetic nanoparticle formulation with microenvironment responsive properties that can replace different hydrophobic drugs as an alternative platform for the treatment of other diseases, such as tumors and rheumatoid arthritis. Therefore, the stimulation pH-responsive biomimetic nanoparticles provided by the present invention have multiple functions and advantages for the treatment of a variety of diseases.

[0027] 3. The pH-responsive biomimetic nano-formulation based on PC NPs provided by this invention utilizes the anti-atherosclerotic effects of Pita, such as anti-inflammatory properties, promotion of autophagy, and enhanced cell burial, to maximize the prevention and treatment of atherosclerosis.

[0028] 4. This invention solves the problems of irreversible and toxic side effects in the treatment of late-stage atherosclerosis. At the same time, it can improve the target effect of the drug on plaques and activated macrophages, as well as the pH-responsive controllable release ability. It provides new theoretical support for the development of new drugs for the prevention and treatment of atherosclerosis and related clinical prevention and treatment, and has important scientific significance, application value and economic value. Attached Figure Description

[0029] Figure 1 Schematic diagrams of PC self-assembly before and after: 1A, PC before and after synthesis; 1B, UV absorption peaks of PC before and after synthesis; 1C, PCNPs reassemble only in DMSO.

[0030] Figure 2 Infrared spectra of PC NPs;

[0031] Figure 3 Particle size potentials of PC NPs, PC@Pita and HA@PC@Pita NPs: 3A, Particle size of HA@PC@Pita NPs at different ratios; 3B, Particle size of PC NPs, PC@Pita and HA@PC@Pita NPs; 3C, Potentials of PC NPs, PC@Pita and HA@PC@Pita NPs.

[0032] Figure 4 Transmission electron micrographs of PC NPs, PC@Pita NPs and HA@PC@Pita NPs;

[0033] Figure 5 The cumulative release of Pita from HA@PC@Pita NPs at different pH values;

[0034] Figure 6 The effects of different materials on cell burial and the ability to clear apoptotic cells; 6A, the viability of macrophages and apoptotic cells was not affected after co-culturing; 6B, the ability to clear apoptotic cells; 6C, a fluorescence statistical graph of the ability to clear apoptotic cells.

[0035] Figure 7 Targeting of HA@PC@Pita NPs on inflammatory macrophages in vitro; 7A, Fluorescence localization map of inflammatory macrophages; 7B, Statistical graph of fluorescence intensity;

[0036] Figure 8 Targeting of HA@PC@Pita NPs in atherosclerotic plaques in mice; 8A, Fluorescence localization map of mouse blood vessels; 8B, Statistical graph of fluorescence intensity;

[0037] Figure 9 Oil Red O staining images of aortic valves in different treatment groups for atherosclerosis: 9A, Oil Red O staining image; 9B, statistical graph;

[0038] Figure 10 HE staining statistical graphs of aortic valves in different treatment groups for atherosclerosis; 10A, HE staining image; 10B, statistical graph;

[0039] Figure 11 H&E staining images of the heart, liver, spleen, lungs, and kidneys of mice treated with different treatment groups for advanced atherosclerosis. Detailed Implementation

[0040] The following will combine Figure 1-10 The present invention will be described in detail below. The illustrative embodiments and descriptions herein are used to explain the invention, but are not intended to limit the invention.

[0041] In the following embodiments, unless otherwise specified, the raw materials and instruments used are commercially available, and the methods used are conventional methods in the art.

[0042] A pH-responsive biomimetic nano-formulation based on PC NPs includes proanthocyanidin self-assembled nanoparticles (PCNPs), a hydrophobic drug, and hyaluronic acid with targeting function. The hydrophobic drug is loaded inside the PC NPs. Specifically, the hydrophobic drug is pitavastatin, and the hyaluronic acid with targeting function is modified on the outer layer of the PC NPs loaded with the hydrophobic drug.

[0043] PC NPs are formed by the linear formation of polyphenol oligomers through phenolic condensation + benzoxazine reaction (phenol + primary amine + aldehyde). As the concentration increases, the polyphenol oligomers gradually aggregate under hydrogen bonding, hydrophobic interaction and π-π interaction to form polyphenol nanoparticles.

[0044] The particle size of pH-responsive nano-formulations is 140-200 nm.

[0045] A method for preparing pH-responsive biomimetic nanoparticles based on PC NPs includes the following steps:

[0046] S1. PC is first ultrasonically stirred with formaldehyde and reacted in the dark for 5 minutes. Then glycine is added dropwise and ultrasonically stirred for 2 hours to obtain an emulsion. The emulsion is PC NPs, wherein the concentrations of PC, formaldehyde and glycine are 2.5 mg / mL, 37% and 10 mg / mL, respectively.

[0047] The specific preparation process of these PC NPs is as follows:

[0048] S101, dissolve 10 mg PC in 4 mL ddH2O to form a reddish-brown solution with a concentration of 2.5 mg / mL, add 10 μL HCHO with a concentration of 37%, and react in the dark at a stirring speed of 800 rpm for 5 min.

[0049] S102: Dissolve 8 mg Gly in 0.8 mL ddH2O to form a colorless and transparent solution with a concentration of 10 mg / mL, and add it dropwise to the solution obtained in S101. Stir at 800 rpm and react in the dark for 2 hours.

[0050] S103: The solution obtained from S102 was centrifuged at 13000 rpm for 10 min, and the precipitate was washed three times with water to obtain PC NPs. The NPs were dispensed in 3 mg vials and stored at -20℃.

[0051] S2. The hydrophobic drug pitavastatin is dissolved and added to the PC NPs for ultrasonic stirring. The resulting solution is then dialyzed and freeze-dried to obtain a nano-formulation loaded with the hydrophobic drug (PC@Pita), wherein the mass ratio of PC NPs to the hydrophobic drug is 3 mg: 0.2 mg.

[0052] S3. The nano-formulation loaded with the hydrophobic drug is mixed with the hyaluronic acid solution by ultrasonic stirring, and then subjected to dialysis and freeze-drying to obtain a pH-responsive nano-formulation (HA@PC@Pita NPs), wherein the mass ratio of PC NPs to the hyaluronic acid is 3 mg: 0.05 mg.

[0053] Specifically, in steps S1, S2 and S3, the ultrasonic stirring speed is 800 rpm, the ultrasonic power is 100 W and the ultrasonic time is 2 min. In addition, in steps S2 and S3, dialysis is performed for 6 h using a dialysis bag with a molecular weight cutoff of 3.5 kDa.

[0054] Example 1

[0055] (1) Synthesis of HA@PC@Pita NPs nanoformulation

[0056] a. Synthesis of PC NPs

[0057] Weigh out 10 mg PC and 8 mg Gly, and prepare 37% HCHO and ddH2O.

[0058] S101, dissolve 10 mg PC in 4 mL ddH2O to make a reddish-brown solution with a concentration of 2.5 mg / mL, add 10 μL HCHO with a concentration of 37%, and react in the dark at a stirring speed of 800 rpm for 5 min.

[0059] S102: Dissolve 8 mg Gly in 0.8 mL ddH2O to form a colorless and transparent solution with a concentration of 10 mg / mL, and add it dropwise into the solution obtained in step S102. React at 800 rpm in the dark for 2 hours.

[0060] S103: Centrifuge the solution obtained from S102 at a stirring speed of 13000 rpm for 10 min, wash the precipitate three times with water, dispense into 3 mg / vial, and store at -20℃.

[0061] b. Synthesis of PC@Pita NPs

[0062] 1. Prepare 3 mg PC NPs, 20 mg / mL Pita (pitavastatin), and 1% PVA solution; 2. Dissolve 3 mg PC NPs in 100 μL DMSO (dimethyl sulfoxide), and sonicate at 40% for 2 min using a small probe; 3. Add 10 μL of Pita (20 mg / mL) to the solution obtained in step 2, and sonicate at 40% for 2 min using a small probe; 4. Add the solution obtained in step 3 dropwise to 1 mL of 1% PVA solution, stir at 800 rpm for 2 min, sonicate at 40% for 5 min using a small probe, and then stir for 24 h; 5. Dialyze the PC@Pita NPs obtained in step 4 in a 3500 kDa dialysis bag for 6 h, aliquot the internal solution into 600 μg / vial, freeze-dry, and store at -20℃.

[0063] c. Synthesis of HA@PC@Pita NPs

[0064] 1. Prepare 3 mg PC NPs, 20 mg / mL Pita, 1% PVA solution, and 10 mg / mL HA (hyaluronic acid); 2. Dissolve 3 mg PC NPs in 100 μL DMSO and sonicate at 40% for 2 min using a small probe; 3. Add 10 μL of Pita (20 mg / mL) to the solution obtained in step 2 and sonicate at 40% for 2 min using a small probe; 4. Add the solution obtained in step 3 dropwise to 1 mL of 1% PVA solution, stir at 800 rpm for 2 min, sonicate at 40% for 5 min using a small probe, and then stir for 12 h; 5. Add 5 μL of HA (10 mg / mL) to the solution obtained in step 4 and stir for 12 h; 6. Dialyze the HA@PC@Pita NPs obtained in step 5 in a 3500 kDa dialysis bag for 6 h, aliquot the internal solution into 600 μg / vial, freeze-dry, and store at -20℃.

[0065] (2) Characterization of HA@PC@Pita NPs nanoformulation

[0066] like Figure 1 and Figure 2 As shown, UV and IR analyses were performed on the PC before and after self-assembly, and the results showed that PC NPs were successfully prepared.

[0067] like Figure 3 and Figure 4 As shown, transmission electron microscopy (TEM) images and DLS particle size analysis were performed on the PC NPs, PC@Pita NPs, and HA@PC@Pita NPs biomimetic nanoparticles prepared in this embodiment. The results show that uniformly dispersed spherical PC NPs materials were successfully prepared with a particle size of approximately 100 nm; the Pita-loaded nanoparticles (PC@Pita NPs) are spherical with a particle size of approximately 129 nm; the HA-modified biomimetic hybrid film coats the outer layer of PC@Pita NPs, exhibiting a distinct "shell-core" structure, and has a particle size of approximately 146 nm.

[0068] like Figure 5 As shown, the cumulative release of Pita gradually increases as the pH value decreases.

[0069] Example 2

[0070] A pH-responsive nano-formulation based on PC NPs is characterized by its synthesis via phenolic condensation and benzoxazine reaction. Unlike traditional nano-formulations, PC NPs are self-assembled from the drug, reducing the impact of the carrier on the body.

[0071] Example 3

[0072] A pH-responsive biomimetic nano-formulation based on PC NPs is characterized by the ability of PC NPs to reassemble in DMSO and to dissolve and reassemble NPs through solvent conversion. It also combines the strong binding effect of the main functional group (phenolic hydroxyl group) of PC on proteins and some drug molecules to construct PC-drug NPs (PC@Pita NPs) with high drug loading capacity.

[0073] Example 4

[0074] A pH-responsive biomimetic nanoformulation based on PC NPs differs from Example 1 in that the amount of Pita solution added during the synthesis of PC@Pita NPs is 10 μl.

[0075] Example 5

[0076] A pH-responsive biomimetic nano-formulation based on PC NPs differs from Example 1 in that 10 μl of Pita solution is added dropwise to the PC NPs solution and stirred at 800 rpm for 24 h at room temperature.

[0077] Example 6

[0078] The application of the pH-responsive nano-formulation based on PC NPs provided by this invention in the prevention and treatment of atherosclerosis is illustrated by taking the HA@PC@Pita NPs nano-formulation prepared in Example 1 as an example.

[0079] The ability of the HA@PC@Pita NPs nanoparticles prepared in Example 1 to restore macrophage cytotoxicity and the effect of apoptotic cells on macrophage viability after treatment with different materials were determined using fluorescence imaging and the MTT assay. The Pita concentration was 0.2 μM. The experimental results are as follows: Figure 6 As shown in AC. The results showed that, compared with the Pita alone and the PC+Pita group, the HA@PC@PitaNPs biomimetic nanoparticle formulation could effectively restore the cytotoxic function of macrophages and their viability was not affected after the apoptotic cells were cleared.

[0080] The above results demonstrate that the HA@PC@Pita NPs nanoparticles prepared in Example 1 can effectively restore macrophage burial function.

[0081] Example 7

[0082] Using the HA@PC@Pita NPs nanoformulation prepared in Example 1, the in vitro targeting of the HA@PC@Pita NPs nanoformulation was determined by a semi-quantitative method of detecting fluorescence intensity.

[0083] RAW264.7 macrophages were stimulated with LPS for 24 h, and then 500 μL of PC@Ce6 and HA@PC@Ce6 at a concentration of 5 μg / mL were added. Fluorescence imaging was performed 4 h later.

[0084] The results are as follows Figure 7 As shown, compared with the PC@Ce6 group, the HA@PC@Ce6 group accumulated in inflammatory macrophages, indicating that the HA@PC@Pita NPs nanoformulation prepared in Example 1 can effectively target the inflammatory site.

[0085] Example 8

[0086] Using the HA@PC@Pita NPs nanoformulation prepared in Example 1, the in vivo targeting of the HA@PC@Pita NPs nanoformulation was determined by a semi-quantitative method of detecting fluorescence intensity.

[0087] ApoE - / - Mice were fed a high-fat diet for one month, and then injected with 200 μL of Ce6, PC@Ce6 and HA@PC@Ce6 at a concentration of 5 mg / kg via the tail vein. Twelve hours later, the mouse aorta was harvested for fluorescence imaging.

[0088] The results are as follows Figure 8 As shown, compared with the Ce6 and PC@Ce6 groups, the HA@PC@Ce6 group showed significant aggregation in the aortic arch and abdominal aorta, indicating that the HA@PC@Pita NPs nanoformulation prepared in Example 1 can effectively target atherosclerotic plaque sites.

[0089] Example 9

[0090] The HA@PC@Pita NPs nanoformulation prepared in Example 1 was used for the treatment of advanced atherosclerosis.

[0091] After two months of high-fat diet treatment, ApoE- / - mice were subjected to the following treatment modalities: normal diet group; high-fat diet group; single-material group (treated with single-material treatment while being fed a high-fat diet); two-drug combination group (treated with two-drug combination treatment while being fed a high-fat diet); and nanomedicine group (treated with nanomedicine treatment while being fed a high-fat diet). All treatments were administered via tail vein injection. The treatment groups were further divided into PC NPs-only group, PC+Pita group, and HA@PC@Pita NPs group. Three days after the end of treatment, the mouse aorta was dissected and stained with Oil Red O to assess the therapeutic effects of different treatment groups.

[0092] The results are as follows Figure 9 and Figure 10As shown, the HA@PC@Pita NPs treatment group significantly reduced the formation of atherosclerotic plaques in advanced atherosclerosis, indicating that the nano-formulation constructed in this invention can effectively treat advanced atherosclerosis.

[0093] Example 10

[0094] The HA@PC@Pita NPs nanoformulation prepared in Example 1 was used for in vivo biosafety testing.

[0095] Using the treatment regimen adopted in Example 9, after the ApoE- / - mice were treated, the major organs (heart, liver, spleen, lungs and kidneys) of mice with different treatment components under different treatment regimens were collected for H&E staining to examine the biosafety of different treatment groups.

[0096] The results are as follows Figure 11 As shown, no obvious organic lesions were observed in the organs of mice in different treatment groups, indicating that the biomimetic nano-formulation constructed in this invention has good biosafety.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for preparing pH-responsive biomimetic nanoparticles based on proanthocyanidin self-assembled nanoparticles (PC NPs), characterized in that, Includes the following steps: S1. Proanthocyanidins (PC) are first ultrasonically stirred with formaldehyde and reacted in the dark for 5 minutes. Then, glycine (Gly) is added dropwise, ultrasonically stirred, and reacted in the dark for 2 hours to obtain an emulsion. The emulsion is proanthocyanidin self-assembled nanoparticles (PC NPs). The specific preparation process of the proanthocyanidin self-assembled nanoparticles (PC NPs) is as follows: S101, dissolve 10 mg of proanthocyanidins (PC) in 4 mL of ddH2O to form a reddish-brown solution with a concentration of 2.5 mg / mL, add 10 μL of HCHO with a concentration of 37%, and react in the dark at a stirring speed of 800 rpm for 5 min. S102: Dissolve 8 mg of glycine (Gly) in 0.8 mL of ddH2O to form a colorless and transparent solution with a concentration of 10 mg / mL. Add the solution dropwise to the solution obtained in S101. Stir at 800 rpm and react in the dark for 2 hours. S103: The solution obtained from S102 was centrifuged at 13000 rpm for 10 min, and the precipitate was washed three times with water to obtain proanthocyanidin self-assembled nanoparticles (PC NPs). The NPs were dispensed in 3 mg vials and stored at -20℃. S2. The hydrophobic drug pitavastatin was dissolved and added to the proanthocyanidin self-assembled nanoparticles (PC NPs) and ultrasonically stirred. The resulting solution was then dialyzed and freeze-dried to obtain a nano-formulation loaded with the hydrophobic drug (PC@Pita). S3. The nano-formulation loaded with the hydrophobic drug is mixed with hyaluronic acid solution by ultrasonic stirring, and then subjected to dialysis and freeze-drying to obtain pH-responsive nano-formulation (HA@PC@Pita NPs).

2. The method for preparing pH-responsive biomimetic nanoparticles based on PC NPs according to claim 1, characterized in that: In step S1, the concentrations of proanthocyanidins (PC), formaldehyde, and glycine (Gly) are 2.5 mg / mL, 37%, and 10 mg / mL, respectively.

3. The method for preparing pH-responsive biomimetic nanoparticles based on PC NPs according to claim 1, characterized in that: In step S2, the mass ratio of proanthocyanidin self-assembled nanoparticles (PC NPs) to the hydrophobic drug is 3 mg: 0.2 mg, and in step S3, the mass ratio of proanthocyanidin self-assembled nanoparticles (PC NPs) to the hyaluronic acid is 3 mg: 0.05 mg.

4. The method for preparing pH-responsive biomimetic nanoparticles based on PC NPs according to claim 1, characterized in that: In both steps S2 and S3, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa for 6 hours.

5. The method for preparing pH-responsive biomimetic nanoparticles based on PC NPs according to claim 1, characterized in that: In steps S1, S2 and S3, the ultrasonic stirring speed is 800 rpm, the ultrasonic power is 100 W and the ultrasonic time is 2 min.

6. A pH-responsive biomimetic nanoparticle formulation based on proanthocyanidin self-assembled nanoparticles (PC NPs), characterized in that: The pH-responsive biomimetic nanoparticles based on PC NPs are prepared using the preparation method described in any one of claims 1 to 5. The pH-responsive nanoparticles include proanthocyanidin self-assembled nanoparticles (PC NPs), a hydrophobic drug, and hyaluronic acid with targeting function. The hydrophobic drug is loaded within the proanthocyanidin self-assembled nanoparticles (PC NPs), and the hyaluronic acid with targeting function is modified on the outer layer of the proanthocyanidin self-assembled nanoparticles (PC NPs) loaded with the hydrophobic drug. The proanthocyanidin self-assembled nanoparticles (PC NPs) are formed by the generation of linear polyphenol oligomers through phenolic condensation and benzoxazine reaction. As the concentration increases, the polyphenol oligomers gradually aggregate under hydrogen bonding, hydrophobic interaction and π-π interaction to form polyphenol nanoparticles. The pH-responsive nanoparticles have a particle size of 140-200 nm.

7. The pH-responsive biomimetic nano-formulation based on PC NPs according to claim 6, characterized in that: The hydrophobic drug is pitavastatin.

8. The use of a pH-responsive biomimetic nanoparticle preparation obtained by the method for preparing a pH-responsive biomimetic nanoparticle based on PC NPs according to any one of claims 1 to 5, or the use of a pH-responsive biomimetic nanoparticle based on PC NPs according to any one of claims 6 to 7, in the preparation of a drug for the prevention and / or treatment of atherosclerosis.

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