Procyanidine-loaded NO release type prussian blue bionic composite material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a procyanidine-loaded NO release type prussian blue bionic composite material and a preparation method and application thereof, the composite material comprises NO release type prussian blue, procyanidine, a bionic membrane and hyaluronic acid, and the particle size of the composite material is 200-250 nm. The preparation method comprises the following steps: loading proanthocyanidins into NO release type Prussian blue by adopting pi-pi stacking and hydrophobic interaction to prepare NO release type PB (at) PC NPs loaded with the proanthocyanidins, mixing the NO release type PB (at) PC NPs loaded with the proanthocyanidins with a biomimetic membrane according to a ratio, and repeatedly extruding to carry out biomembrane camouflage, and finally, modifying the surface of the biomimetic membrane with hyaluronic acid to obtain the final product HMP-PC NPs. The nano-composite material disclosed by the invention has a good anti-silicosis effect, can relieve silicosis and can be used for developing potential medicines for treating silicosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a proanthocyanidin-loaded NO-releasing Prussian blue bionic composite material and a preparation method and application thereof. Background Art

[0002] Silicosis is the most common, fastest-progressing, and most serious type of occupational pneumoconiosis. It refers to a systemic disease characterized by diffuse pulmonary fibrosis due to long-term and repeated inhalation of free silica crystal particles. The pathological feature is interstitial pulmonary fibrosis. In the process of pulmonary fibrosis, changes in the local microenvironment of lung tissue determine the response of the tissue to various injuries. The nature and intensity of these reactions determine the occurrence, development, and outcome of tissue inflammation, wound healing, and fibrosis. The main effector cells in the development of silicotic fibrosis are macrophages, fibroblasts, epithelial cells, endothelial cells, and lymphocytes. When macrophages are dysfunctional, their repair and homeostasis functions are subverted and they secrete a large number of inflammatory cytokines and platelet-derived growth factor, which indirectly activate and regulate the activation and migration of endothelial cells, epithelial cells, and fibroblasts in lung tissue and induce extracellular matrix deposition, affecting the process of fibrosis. Macrophages and fibroblasts regulate each other in structure and function. In the pathological process, macrophages mainly show polarization, regulate immunity and fluid balance, while fibroblasts are activated and differentiated into myofibroblasts and secrete collagen. The interaction between the two leads to silicosis fibrosis.

[0003] Therefore, regulating macrophage polarization is a very promising means of treating silicotic fibrosis. Although proanthocyanidins can inhibit the activation of NLRP3 inflammasomes in RAW264.7 macrophages and the recruitment of monocyte-derived macrophages to the lungs, thereby exerting an anti-silicotic fibrosis effect, the systemic administration of proanthocyanidins alone has defects such as non-specific distribution in the body, low bioavailability, short retention time, and large effective dose, which limits the therapeutic effect. How to improve the bioavailability and targeted distribution of proanthocyanidins is a key issue in the treatment of silicotic fibrosis with proanthocyanidins. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a NO-releasing Prussian blue bionic composite material loaded with proanthocyanidins and a preparation method and application thereof. The nanocomposite material of the present invention has good anti-silicotic fibrosis effect, can alleviate silicotic fibrosis, and can be used to develop potential drugs for the treatment of silicotic fibrosis.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A NO-releasing Prussian blue bionic composite material loaded with proanthocyanidins, abbreviated as HMP NPs, comprises NO-releasing Prussian blue, proanthocyanidins, a bionic membrane and hyaluronic acid, wherein the proanthocyanidins are physically encapsulated in NO-releasing Prussian blue nanoparticles, the bionic membrane is camouflaged in the outermost layer of the NO-releasing Prussian blue, and the hyaluronic acid is modified on the surface of the bionic membrane.

[0007] In a preferred embodiment of the present invention, the particle size of the composite material is 200 nm to 250 nm.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned proanthocyanidin-loaded NO-releasing Prussian blue biomimetic composite material, comprising the following steps:

[0009] The collected macrophages were ultrasonically disrupted and repeatedly frozen and thawed, and then centrifuged to obtain a biomimetic membrane, which was then dissolved in PBS to obtain a biomimetic membrane solution;

[0010] After hyaluronic acid is activated by stirring under the action of an activator, amino polyethylene glycol 2000 is added to obtain a mixed solution, the mixed solution is dialyzed to remove free N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and then freeze-dried to obtain activated hyaluronic acid, and the activated hyaluronic acid is dissolved in a PBS solution to obtain a hyaluronic acid solution;

[0011] Adding proanthocyanidins and NO-releasing Prussian blue into water and stirring to obtain NO-releasing Prussian blue loaded with proanthocyanidins;

[0012] The NO-releasing Prussian blue loaded with proanthocyanidins and the PBS solution of the biomimetic membrane are mixed and squeezed repeatedly, and then a hyaluronic acid solution is added and incubated to obtain a Prussian blue biomimetic composite material loaded with proanthocyanidins.

[0013] In a preferred embodiment of the present invention, the mass ratio of hyaluronic acid to DSPE-PEG2000-NH2 is 3 to 5:1.

[0014] In a preferred embodiment of the present invention, the mass ratio of the proanthocyanidins to the NO-releasing Prussian blue is 1-2:1.

[0015] In a preferred embodiment of the present invention, the mass ratio of the bionic membrane, hyaluronic acid, and proanthocyanidin-loaded NO-releasing Prussian blue is 5 to 8:5 to 8:4.

[0016] In a preferred embodiment of the present invention, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 0.5 to 1:2, and the mass ratio of hyaluronic acid to N-hydroxysuccinimide is 1:10.

[0017] In a preferred embodiment of the present invention, the activation reaction stirring speed is 600-800 rpm / min, the activation reaction time is 18-24 h, the ultrasonic power is 80-100 W, the ultrasonic time is 8-10 min, the repeated freezing and thawing temperature is -80°C and 25-37°C, the repeated freezing and thawing times is 3-5 times, and the centrifugal speed is 13000-18000 rpm / min.

[0018] In a preferred embodiment of the present invention, the stirring speed of adding proanthocyanidins and NO-releasing Prussian blue into water is 600-800 rpm, and the reaction temperature is 25-30°C.

[0019] The third object of the present invention is to provide an application of the above-mentioned proanthocyanidin-loaded Prussian blue bionic composite material in the targeted treatment of silicosis fibrosis.

[0020] To avoid the body's immune system from clearing, the present invention uses a macrophage membrane biomimetic membrane system to disguise NO-releasing Prussian blue nanoparticles to reduce systemic immunogenicity and achieve immune escape. However, although biomimetic composite materials have many advantages, they also have some defects in the preparation process and application process, such as high cost and low targeting. Therefore, the present invention utilizes the characteristics of hyaluronic acid that can specifically identify the CD44 receptor of inflammatory macrophages in the pulmonary fibrosis area, and finally modifies the macrophage membrane biomimetic membrane system to disguise NO-releasing Prussian blue nanoparticles to achieve the purpose of targeted drug delivery to treat silicosis fibrosis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention designs a biomimetic nanocomposite with high safety and biodegradability, and utilizes the design features of biomimetic membrane and hyaluronic acid modification to achieve specific aggregation and penetration of drugs in the lesion site and improve the bioavailability of drugs to target cells. The composite nanomaterial of the present invention is physically encapsulated by NO-releasing Prussian blue for proanthocyanidins, the macrophage biomimetic membrane is camouflaged in the outermost layer of the nanocomposite material, and hyaluronic acid is modified to the surface of the biomimetic membrane. In the pathogenesis of silicosis fibrosis, macrophages participate in the body's inherent immunity as key regulatory factors for balancing the tissue microenvironment, and monocyte-derived macrophages are recruited to the lungs. The outermost biomimetic membrane realizes the recruitment of nanocomposite materials to the lesion site through the inherent immune escape and recruitment characteristics of its source cells (macrophages). Utilizing the characteristics of hyaluronic acid and the surface receptor CD44 of inflammatory macrophages, the effective utilization of nanocomposite materials by target cells is realized, and the effect of preventing and treating silicosis fibrosis is improved. Based on the above results, a bionic HMP@PC NPs composite material loaded with proanthocyanidins was designed and prepared, which utilized the anti-silicotic fibrosis effect of proanthocyanidins to achieve targeted treatment of silicotic fibrosis.

[0023] 2. The present invention uses π-π stacking and hydrophobic interaction to load proanthocyanidins into NO-releasing-PB NPs to prepare NO-releasing-PB@PC NPs loaded with proanthocyanidins, mix the NO-releasing-PB@PC NPs loaded with proanthocyanidins with a bionic membrane and repeatedly extrude them for biological camouflage, and finally modify hyaluronic acid onto the surface of the bionic membrane to obtain the final product HMP@PCNPs. The nanocomposite material of the present invention has a good anti-silicosis fibrosis effect, can alleviate silicosis fibrosis, and can be used to develop potential drugs for the treatment of silicosis fibrosis.

[0024] 3. In addition to loading drugs for treating and / or preventing silicosis fibrosis, the drug-loaded bionic nanocomposite provided by the present invention can be used as an alternative platform for treating other diseases, such as tumors, liver fibrosis, etc., by replacing different bionic membrane types and drugs and utilizing the "homing effect" of the bionic membrane and the mechanism of action of different drugs. Therefore, the present invention combines bionic membrane coatings and targeted modified nanocomposites, which can provide multiple functions and advantages to treat a variety of diseases.

[0025] 4. The present invention can improve the immunogenicity of the drug and achieve the targeting effect on the silicosis fibrosis site and target cells. It provides new theoretical support for the development of anti-silicosis fibrosis drugs and related clinical detection and treatment, and has important scientific significance, practical value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A and B are transmission electron microscopy images of HMP@PCNPs prepared in the present invention;

[0027] Figure 2 The particle size (A) and potential (B) diagram of HMP@PCNPs prepared in the present invention;

[0028] Figure 3 The infrared spectrum of HMP@PCNPs prepared in the present invention;

[0029] Figure 4 The ability of HMP@PCNPs prepared in the present invention to inhibit fibroblast activation (A) and macrophage polarization (B);

[0030] Figure 5 The staining pictures of HMP@PCNPs prepared by the present invention in treating silicosis fibrosis in mice; A: days of administration; B: HE staining lung fibrosis results; C: Masson staining lung fibrosis results; D: Sirius red staining lung fibrosis results;

[0031] Figure 6 These are H&E staining images of the heart, liver, spleen and kidney of mice with silicosis fibrosis treated with different treatment groups of the present invention. DETAILED DESCRIPTION

[0032] The following is a detailed description of the technical solutions in the embodiments of the present invention in combination with the preferred embodiments and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] It should be noted that all professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0034] In recent years, natural compounds modified with nanoscale materials as carriers are being widely used in the prevention, diagnosis and treatment of diseases including cancer, cardiovascular disease, diabetes and various inflammatory diseases. Nanomaterials have shown great advantages in drug delivery in the form of nanoformulations, which can effectively improve the targeting, compatibility and immune escape ability of drugs, while improving the bioavailability of drugs to target cells, providing a potential alternative for the prevention and treatment of silicosis fibrosis. Prussian blue nanoparticles are clinical drug carriers approved by the US Food and Drug Administration, with good drug loading, surface modification, biocompatibility and antioxidant capabilities. Biomimetic membrane nanosystems generally refer to the encapsulation of cell membranes with special functions onto the surface of nanomaterials, so that nanomaterials have cell-like properties. However, although biomimetic composite materials have many advantages, there are also some defects in the preparation and application process, such as high cost and low targeting.

[0035] Therefore, the present invention provides a NO-releasing Prussian blue bionic composite material loaded with proanthocyanidins and its preparation method and application, wherein the composite material is mainly composed of NO-releasing Prussian blue, proanthocyanidins, bionic membranes and hyaluronic acid, wherein the proanthocyanidins are physically encapsulated in NO-releasing Prussian blue nanoparticles, the bionic membranes are camouflaged in the outermost layer of NO-releasing Prussian blue, and the hyaluronic acid is modified to the surface of the bionic membranes. The composite nanomaterial of the present invention physically encapsulates proanthocyanidins by NO-releasing Prussian blue, the macrophage bionic membranes are camouflaged in the outermost layer of the nanocomposite material, and the hyaluronic acid is modified to the surface of the bionic membranes. In the pathogenesis of silicosis fibrosis, macrophages participate in the body's inherent immunity as key regulatory factors for balancing the tissue microenvironment, monocyte-derived macrophages are recruited to the lungs, and the outermost bionic membranes realize the recruitment of nanocomposite materials to the lesion site through the inherent immune escape and recruitment characteristics of its source cells (macrophages). By utilizing the properties of hyaluronic acid binding to the surface receptor CD44 of inflammatory macrophages, the target cells can effectively utilize the nanocomposite material and improve its effect in preventing and treating silicosis fibrosis. Based on the above results, a biomimetic HMP@PC NPs composite material loaded with proanthocyanidins was designed and prepared, which utilized the anti-silicosis fibrosis effect of proanthocyanidins to achieve targeted treatment of silicosis fibrosis.

[0036] Example 1

[0037] The synthesis of a proanthocyanidin-loaded NO-releasing Prussian blue biomimetic composite material comprises the following steps:

[0038] (1) Preparation of biomimetic membrane

[0039] The collected macrophages were resuspended in 1 mL of pre-cooled PMSF with a concentration of 1 mM, and lysed at 4°C for 30 min. The mixed cell suspension was ultrasonically disrupted for 10 min at an ultrasonic power of 80 W. Then, the mixture was repeatedly frozen and thawed at -80°C and 37°C for 4 times, centrifuged at 800 rpm / min for 10 min, and the supernatant was collected. The supernatant was centrifuged at 13000 rpm / min for 30 min. The bottom sediment was the macrophage membrane, recorded as: M; the macrophage membrane was dissolved with PBS to obtain a biomimetic membrane solution.

[0040] (2) Preparation of hyaluronic acid

[0041] 25 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 50 mg N-hydroxysuccinimide (NHS) and 5 mg hyaluronic acid (HA) were added to 1 mL PBS in sequence and stirred at 25°C for 30 min. Since DSPE can be inserted into the phospholipid bilayer to promote HA modification to the cell membrane surface, 25 mg aminopolyethylene glycol 2000 (DSPE-PEG2000-NH2) was added to the activated HA solution and stirred at 37°C for 30 min; then, stirred at 25°C for 24 h at a stirring speed of 800 rpm. In order to remove free NHS and EDC, the resulting solution was placed in a dialysis membrane and freeze-dried to obtain activated hyaluronic acid, which was recorded as: HA.

[0042] (3) Preparation of NO-releasing Prussian blue

[0043] 3 g of polyvinyl pyrrolidone (PVP) and 132 mg of potassium ferricyanide (K3[FeCN6]) were added to a HCl solution (0.1 M, 40 mL) and stirred with a magnetic stirrer for 30 min until a clear yellow solution was obtained. The mixture was then heated in an 80°C oil bath for 20 h. The resulting blue mixed solution was centrifuged at 13000 rpm / min for 20 min, and the precipitate was collected. After that, it was washed several times with deionized water until the supernatant was neutral. Finally, the blue precipitate was freeze-dried for 12 h to obtain mesoporous NO-releasing Prussian blue, denoted as: PB NPs.

[0044] (4) Preparation of NO-releasing Prussian blue loaded with proanthocyanidins

[0045] 1 mg / mL proanthocyanidin (PC) and 1 mg / mL PB NPs were added to 1 mL ddH2O and magnetically stirred at 800 rpm / min at 25°C for 24 h to finally obtain NO-releasing Prussian blue loaded with proanthocyanidin, which was recorded as: PB@PC NPs.

[0046] (5) Preparation of biomimetic nanocomposites loaded with proanthocyanidins

[0047] The PB@PC NPs dispersion and the biomimetic membrane dispersion (0.5 mL) were mixed and repeatedly extruded at least 10 times through a micro extruder with a pore size of 200 nm to obtain a biomimetic nanocomposite material loaded with proanthocyanidins, which was recorded as: MP@PC NPs.

[0048] (6) Preparation of NO-releasing Prussian blue biomimetic composite materials loaded with proanthocyanidins

[0049] The activated hyaluronic acid was redissolved in PBS solution, added to MP@PC NPs, and magnetically stirred at 37°C and 800 rpm / min for 30 min to obtain a NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins, denoted as HMP@PCNPs.

[0050] Example 2

[0051] The synthesis of a proanthocyanidin-loaded NO-releasing Prussian blue biomimetic composite material comprises the following steps:

[0052] (1) Preparation of biomimetic membrane

[0053] The collected macrophages were resuspended in 1 mL of pre-cooled PMSF with a concentration of 1 mM, and lysed at 4°C for 30 min. The mixed cell suspension was ultrasonically disrupted for 9 min at an ultrasonic power of 90 W. Then, the mixture was repeatedly frozen and thawed at -80°C and 25°C for 3 times, centrifuged at 800 rpm / min for 10 min, and the supernatant was collected. The supernatant was centrifuged at 13000 rpm / min for 30 min, and the bottom sediment was the macrophage membrane, recorded as: M.

[0054] (2) Preparation of hyaluronic acid

[0055] 12.5 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 50 mg N-hydroxysuccinimide (NHS) and 3 mg hyaluronic acid (HA) were added to 1 mL PBS in sequence and stirred at 25°C for 30 min. Since DSPE can be inserted into the phospholipid bilayer to promote HA modification to the cell membrane surface, 25 mg amino polyethylene glycol 2000 (DSPE-PEG2000-NH2) was added to the activated HA solution and stirred at 37°C for 30 min; then, stirred at 25°C for 20 h at a stirring speed of 600 rpm. In order to remove free NHS and EDC, the resulting solution was placed in a dialysis membrane and freeze-dried to obtain hyaluronic acid, which was recorded as HA.

[0056] (3) Preparation of NO-releasing Prussian blue

[0057] 3 g of polyvinyl pyrrolidone (PVP) and 132 mg of potassium ferricyanide (K3[FeCN6]) were added to a HCl solution (0.1 M, 40 mL) and stirred with a magnetic stirrer for 30 min until a clear yellow solution was obtained. The mixture was then heated in an 80°C oil bath for 20 h. The resulting blue mixed solution was centrifuged at 15,000 rpm / min for 20 min, and the precipitate was collected. After that, it was washed several times with deionized water until the supernatant was neutral. Finally, the blue precipitate was freeze-dried for 12 h to obtain mesoporous NO-releasing Prussian blue, denoted as: PB NPs.

[0058] (4) Preparation of NO-releasing Prussian blue loaded with proanthocyanidins

[0059] 1.5 mg / mL PC and 1 mg / mL PB NPs were added to 1 mL ddH2O and magnetically stirred at 600 rpm / min at 28°C for 24 h to finally obtain NO-releasing Prussian blue loaded with proanthocyanidins, which was recorded as: PB@PC NPs.

[0060] (5) Preparation of biomimetic nanocomposites loaded with proanthocyanidins

[0061] The PB@PC NPs dispersion and the biomimetic membrane dispersion (0.5 mL) were mixed and repeatedly extruded at least 10 times through a micro extruder with a pore size of 200 nm to obtain a biomimetic nanocomposite material loaded with proanthocyanidins, which was recorded as: MP@PC NPs.

[0062] (6) Preparation of NO-releasing Prussian blue biomimetic composite materials loaded with proanthocyanidins

[0063] The activated hyaluronic acid was redissolved in PBS solution, added to MP@PC NPs, and magnetically stirred at 37°C and 800 rpm / min for 30 min to obtain a NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins, denoted as HMP@PCNPs.

[0064] Example 3

[0065] The synthesis of a proanthocyanidin-loaded NO-releasing Prussian blue biomimetic composite material comprises the following steps:

[0066] (1) Preparation of biomimetic membrane

[0067] The collected macrophages were resuspended in 1 mL of pre-cooled PMSF with a concentration of 1 mM, and lysed at 4°C for 30 min. The mixed cell suspension was ultrasonically disrupted for 8 min at an ultrasonic power of 100 W. Then, the mixture was repeatedly frozen and thawed at -80°C and 30°C for 5 times, centrifuged at 800 rpm / min for 10 min, and the supernatant was collected. The supernatant was centrifuged at 18000 rpm / min for 30 min, and the bottom sediment was the macrophage membrane, recorded as: M.

[0068] (2) Preparation of hyaluronic acid

[0069] 20 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 50 mg N-hydroxysuccinimide (NHS) and 4 mg hyaluronic acid (HA) were added to 1 mL PBS in sequence and stirred at 25°C for 30 min. Since DSPE can be inserted into the phospholipid bilayer to promote HA modification to the cell membrane surface, 25 mg amino polyethylene glycol 2000 (DSPE-PEG2000-NH2) was added to the activated HA solution and stirred at 37°C for 30 min; then, stirred at 25°C for 18 h at a stirring speed of 700 rpm. In order to remove free NHS and EDC, the resulting solution was placed in a dialysis membrane and freeze-dried to obtain hyaluronic acid, which was recorded as: HA.

[0070] (3) Preparation of NO-releasing Prussian blue

[0071] 3 g of polyvinyl pyrrolidone (PVP) and 132 mg of potassium ferricyanide (K3[FeCN6]) were added to a HCl solution (0.1 M, 40 mL) and stirred with a magnetic stirrer for 30 min until a clear yellow solution was obtained. The mixture was then heated in an 80°C oil bath for 20 h. The resulting blue mixed solution was centrifuged at 13000 rpm / min for 20 min, and the precipitate was collected. After that, it was washed several times with deionized water until the supernatant was neutral. Finally, the blue precipitate was freeze-dried for 12 h to obtain mesoporous NO-releasing Prussian blue, denoted as: PB NPs.

[0072] (4) Preparation of NO-releasing Prussian blue PB@PCNPs loaded with proanthocyanidins

[0073] 2 mg / mL proanthocyanidin (PC) and 1 mg / mL PB NPs were added to 1 mL ddH2O and magnetically stirred at 700 rpm / min at 30°C for 24 h to finally obtain NO-releasing Prussian blue loaded with proanthocyanidin, which was recorded as: PB@PC NPs.

[0074] (5) Preparation of biomimetic nanocomposites loaded with proanthocyanidins

[0075] The PB@PC NPs dispersion and the biomimetic membrane dispersion (0.5 mL) were mixed and repeatedly extruded at least 10 times through a micro extruder with a pore size of 200 nm to obtain a biomimetic nanocomposite material loaded with proanthocyanidins, which was recorded as: MP@PC NPs.

[0076] (6) Preparation of NO-releasing Prussian blue biomimetic composite materials loaded with proanthocyanidins

[0077] The activated hyaluronic acid was redissolved in PBS solution, added to MP@PC NPs, and magnetically stirred at 37°C and 800 rpm / min for 30 min to obtain a NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins, denoted as HMP@PCNPs.

[0078] Application Example 1

[0079] The HMP@PCNPs bionic nanoformulation prepared in Example 1 was used for the treatment of silicosis fibrosis.

[0080] Experiment 1: The mice were randomly divided into control group, SiO2+Saline group, SiO2+PFD group, SiO2+HMP group, and SiO2+HMP@PC group, with 10 C57BL / 6J mice in each group. After isoflurane anesthesia, 0.1mL 200mg / mL SiO2 suspension was instilled intratracheally to establish a silicosis model, and the control group was instilled with an equal volume of saline. 48h after surgery, Saline, PB NPs (5mg / kg), and HMP@PC NPs (5mg / kg) were injected into the tail vein of the SiO2+Saline group, SiO2+HMP group, and SiO2+HMP@PC, respectively, with an interval of 48h between each administration. During the treatment period, pirfenidone (PFD, 200mg / kg·day gavage) was used as a positive control group. The experiment ended on the 21st day, and lung tissue sections were taken for H&E, Masson, and Sirius red staining to observe pulmonary fibrosis.

[0081] Results Analysis

[0082] Characterization of biomimetic HMP@PC NPs nanocomposites loaded with proanthocyanidins

[0083] like Figure 1 As shown in A and B, the NPBs prepared in Example 1 were subjected to transmission electron microscopy image analysis, and the results showed well-dispersed cubic nanoparticles with an average diameter of about 200 nm; the HA-modified bionic membrane was wrapped around the outer layer of the HMP@PCNPs loaded with proanthocyanidins, showing an obvious "core-shell" structure with a particle size of about 250 nm.

[0084] Figure 2The particle size (A) and potential (B) diagram of HMP@PC NPs prepared in the present invention are shown in FIG. Figure 2 As shown, dynamic light scattering (DLS) system showed that the average diameter of HMP@PCNPs was about 250 nm and the Zeta potential was about -7.00 mV.

[0085] like Figure 3 As shown, infrared spectroscopy (FT-IR) detection showed that the presence of Ar-, -COC and -OH indicated that NPBs were successfully loaded with PC.

[0086] Figure 4 The ability of HMP@PCNPs prepared in the present invention to inhibit fibroblast activation (A) and macrophage polarization (B) is shown in FIG. Figure 4 As shown, immunofluorescence, mobility shift and qRT-PCR experiments were used to determine the ability of the HMP@PCNPs biomimetic nanoformulation prepared in Example 1 to inhibit macrophage polarization and fibroblast activation. The experimental results show that the HMP@PC NPs biomimetic nanoformulation can effectively inhibit macrophage polarization and fibroblast activation.

[0087] Figure 5 The HMP@PCNPs prepared in the present invention are used to treat silicosis fibrosis in mice; A: days of administration; B: HE staining pulmonary fibrosis results; C: Masson staining pulmonary fibrosis results; D: Sirius red staining pulmonary fibrosis results, such as Figure 5 As shown, compared with the model group, the HMP@PCNPs group was able to reduce SiO2 damage to lung tissue to form silicotic nodules, the alveolar structure was relatively intact, the inflammatory infiltration was reduced, and the area of ​​fibrosis was reduced, indicating that the HMP@PCNPs bionic nanoformulation prepared in Example 1 can effectively alleviate silicosis fibrosis.

[0088] The HMP@PC NPs biomimetic nanoformulation prepared in Example 1 was used for in vivo biosafety examination. Using the treatment scheme adopted in Example 1, after the treatment of C57BL / 6J mice, the main organs (heart, liver, spleen and kidney) of mice with different treatment components under different treatment schemes were collected for H&E staining to examine the biosafety of different treatment groups.

[0089] The results are as follows Figure 6 As shown, no obvious organic lesions were found in the organs of mice in different treatment groups, indicating that the bionic nano-preparation constructed by the present invention has good biosafety.

[0090] In summary, the present invention designs a biomimetic nanocomposite with high safety and biodegradability, and utilizes the design features of biomimetic membrane and hyaluronic acid modification to achieve specific aggregation and penetration of drugs in lesions and improve the bioavailability of drugs to target cells. The composite nanomaterial of the present invention physically encapsulates proanthocyanidins by PB NPs, the biomimetic membrane is camouflaged in the outermost layer of the nanocomposite material, and hyaluronic acid is modified on the surface of the biomimetic membrane. In the pathogenesis of silicosis fibrosis, macrophages participate in the body's inherent immunity as key regulatory factors for balancing the tissue microenvironment, and monocyte-derived macrophages are recruited to the lungs. The outermost biomimetic membrane realizes the recruitment of nanocomposite materials to lesions through the inherent immune escape and recruitment characteristics of its source cells (macrophages). The characteristics of hyaluronic acid binding to the surface receptor CD44 of inflammatory macrophages are utilized to achieve effective utilization of nanocomposite materials by target cells, thereby improving the effect of preventing and treating silicosis fibrosis. Based on the above results, a bionic HMP@PC NPs composite material loaded with proanthocyanidins was designed and prepared, which utilized the anti-silicotic fibrosis effect of proanthocyanidins to achieve targeted treatment of silicotic fibrosis.

[0091] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0092] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins, characterized in that: The composite material comprises NO-releasing Prussian blue, proanthocyanidin, bionic membrane and hyaluronic acid, wherein the proanthocyanidin is physically encapsulated in the NO-releasing Prussian blue, the bionic membrane is camouflaged in the outermost layer of the NO-releasing Prussian blue, and the hyaluronic acid is modified on the surface of the bionic membrane.

2. The NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 1, characterized in that: The particle size of the composite material is 200nm-250nm.

3. A method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 1 or 2, characterized in that: The following steps are involved: The collected macrophages were ultrasonically disrupted and repeatedly frozen and thawed, and then centrifuged to obtain a biomimetic membrane, which was then dissolved in PBS to obtain a biomimetic membrane solution; After hyaluronic acid is activated by stirring under the action of an activator, amino polyethylene glycol 2000 is added to obtain a mixed solution, the mixed solution is dialyzed and dried to obtain activated hyaluronic acid, and the activated hyaluronic acid is dissolved in a PBS solution to obtain a hyaluronic acid solution; Proanthocyanidins and NO-releasing Prussian blue nanoparticles are added into water and stirred to obtain NO-releasing Prussian blue loaded with proanthocyanidins; The NO-releasing Prussian blue loaded with proanthocyanidins and the bionic membrane solution are mixed and squeezed repeatedly, and then a hyaluronic acid solution is added and incubated to obtain a NO-releasing Prussian blue bionic composite material loaded with proanthocyanidins.

4. The method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 3, characterized in that: The mass ratio of the hyaluronic acid to aminopolyethylene glycol 2000 is 3 to 5:

1.

5. The method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 3, characterized in that: The mass ratio of the proanthocyanidins to the NO-releasing Prussian blue is 1-2:

1.

6. The method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 3, characterized in that: The mass ratio of the bionic membrane, hyaluronic acid and proanthocyanidin-loaded NO-releasing Prussian blue is 5-8:5-8:

4.

7. The method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 3, characterized in that: The activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 0.5-1:2, and the mass ratio of hyaluronic acid to N-hydroxysuccinimide is 1:

10.

8. The method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 3, characterized in that: The activation reaction temperature is 25°C, and the activation reaction time is 18 to 24 hours.

9. The method for preparing the NO-releasing Prussian blue biomimetic composite material loaded with proanthocyanidins according to claim 3, characterized in that: When preparing the NO-releasing Prussian blue loaded with proanthocyanidins, the reaction temperature is 25-30°C.

10. Use of the proanthocyanidin-loaded NO-releasing Prussian blue bionic composite material according to claim 1 or 2 in the preparation of a drug for the targeted treatment of silicosis fibrosis.

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