Active oxygen response oxygen self-production nanometer material for promoting vascular regeneration and preparation method thereof

Abstract

The invention discloses an active oxygen response oxygen self-production nanometer material for promoting vascular regeneration and a preparation method thereof, and relates to the technical field of biomedical materials. The nano-material comprises self-oxygen-producing PLGA nano-particles and a macrophage membrane, wherein the self-oxygen-producing PLGA nano-particles simultaneously load a mesenchymal stem cell conditioned culture medium which is pretreated under a preset low-oxygen condition and catalase with an active oxygen response characteristic, and the surface of the self-oxygen-producing PLGA nano-particles is coated with the macrophage membrane. The invention provides a synergistic treatment strategy for promoting revascularization and relieving oxygen deficit of a focus part, and solves the problem of slow release speed of a targeted nano-material loaded drug for promoting revascularization.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, in particular to an active oxygen-responsive self-generating nano material for promoting angiogenesis and a preparation method thereof. Background Art

[0002] Blood vessels are the channels for biological transport of blood, oxygen and nutrients needed by the human body, and are one of the important organs of the human body. Vascular system diseases can lead to vascular stenosis or blockage, insufficient blood supply to tissues and organs, and thus cause a series of ischemic diseases. Lower limb ischemia (HI) is a common ischemic disease with high morbidity and high disability rates. At present, the clinical treatment of lower limb ischemia mainly consists of surgical treatment and drug therapy. However, surgical treatment has high requirements on the patient's own physical condition, some patients are not suitable for surgical treatment, and secondary blockage is prone to occur after surgery; while the effect of drug treatment is not satisfactory, and the effect on patients with severe lesions is minimal. Due to arterial stenosis or occlusion, the lower limb extremities are insufficiently supplied with blood, which causes hypoxia in the extremities and produces a large amount of ROS. The excessive production of ROS will further aggravate cell death and loss of tissue function, and hinder angiogenesis. The top priority for the treatment of such diseases is to relieve the hypoxic microenvironment of the ischemic site, remove excessive ROS, and reconstruct the necrotic blood vessels in the ischemic site to restore blood supply.

[0003] Mesenchymal stem cells (MSCs) are widely used in the treatment of ischemic diseases because of their strong paracrine ability. Studies have found that many of the factors paracrine secreted by MSCs are related to angiogenesis, such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1). In addition, the reduction of oxygen in ischemic tissues will lead to the activation of hypoxia-inducible factor-1α (Hif-1α), which can upregulate the expression of angiogenesis-related genes, thereby further promoting vascular regeneration, inhibiting fibrosis and reducing cell apoptosis. Studies have shown that once MSCs migrate to the hypoxic area, the amount of their paracrine factors will increase significantly. Therefore, screening the optimal hypoxic pretreatment conditions for MSCs will maximize the paracrine effect of MSCs, further promote angiogenesis, and achieve the best therapeutic effect. Catalase (CAT) is an antioxidant enzyme that not only removes reactive oxygen species in the lesion site, but also releases oxygen to improve the hypoxic microenvironment in the tissue. Therefore, catalase-based therapy has become a promising treatment method. Catalase has been shown to be effective in a variety of pathological conditions, including vascular dysfunction, hypoxic-ischemic encephalopathy, and myocardial infarction. However, after in vivo administration, catalase cannot reach the lesion site to remove reactive oxygen species and release oxygen due to its short half-life and non-targeting properties.

[0004] In recent years, research on the use of nano drug delivery systems to treat ischemic diseases has gradually emerged, which makes up for the shortcomings of MSC transplantation therapy. Polylactic-co-glycolic acid (PLGA) is a polymer compound approved by the US FDA and the European Medicines Agency for drug delivery. It is safe, non-toxic and highly biocompatible, making it an ideal candidate for loading MSC paracrine factors and catalase. At present, most treatments based on nano drug delivery systems are injected into the body through intravenous injection. However, nanoparticles may be cleared by immune response in the blood and thus fail to achieve the desired therapeutic effect. Studies have shown that macrophage membranes (MM) can be coated on the surface of PLGA nanoparticles (PLGA-NPs) to enhance the biocompatibility of PLGA-NPs and target PLGA-NPs to the site of injury. However, PLGA-NPs degrade slowly, which also leads to a decrease in the release rate of the loaded drugs. Summary of the invention

[0005] The purpose of the present invention is to propose a synergistic treatment strategy for promoting angiogenesis and alleviating hypoxia in the lesion site, thereby solving the problem of slow release of targeted nanomaterial-loaded drugs that promote angiogenesis.

[0006] In a first aspect, an embodiment of the present invention provides an active oxygen-responsive self-generating nanomaterial that promotes angiogenesis, comprising: self-generating PLGA nanoparticles that simultaneously load mesenchymal stem cell conditioned medium pretreated with preset hypoxic conditions and catalase with active oxygen-responsive properties, and a macrophage membrane coated on the surface of the self-generating PLGA nanoparticles.

[0007] The phase change material of the self-oxygenating PLGA nanoparticles includes DPPC.

[0008] In a second aspect, an embodiment of the present invention further provides a method for preparing an angiogenesis-promoting reactive oxygen species-responsive self-oxygen-producing nanomaterial, comprising: pre-treating a mesenchymal stem cell conditioned medium under preset hypoxic conditions to obtain a treated mesenchymal stem cell conditioned medium; dissolving PLGA and DPPC in a dichloromethane solution, adding catalase and the treated mesenchymal stem cell conditioned medium, performing cell disruption and ultrasonic treatment to obtain a first emulsion; preparing self-oxygen-producing PLGA nanoparticles based on the first emulsion; and coating the surface of the self-oxygen-producing PLGA nanoparticles with macrophage membranes to obtain the angiogenesis-promoting reactive oxygen species-responsive self-oxygen-producing nanomaterial.

[0009] The method also includes determining the preset hypoxic condition based on an experimental screening method; including: culturing mesenchymal stem cells in a complete culture medium for three generations and then replacing it with a serum-free culture medium, and culturing them under multiple hypoxic conditions with different oxygen contents; collecting multiple conditioned culture media containing mesenchymal stem cell paracrine factors under multiple hypoxic conditions with different oxygen contents at multiple different culture time points; centrifuging and filtering the multiple conditioned culture media respectively, and storing them in sterile centrifuge tubes to obtain multiple culture medium samples to be tested; using CCK-8 reagent and Calcein / PI cell activity and cytotoxicity detection kit to analyze the mesenchymal stem cell proliferation ability of the multiple culture medium samples to be tested; and determining the oxygen content and culture time point corresponding to the culture medium sample to be tested with the strongest mesenchymal stem cell proliferation ability as the preset hypoxic condition.

[0010] The method of preparing self-oxygenating PLGA nanoparticles based on the first emulsion comprises: adding the first emulsion to a sodium cholate solution and performing ultrasonic treatment to obtain a second emulsion; adding the second emulsion to the sodium cholate solution and stirring to volatilize the organic solvent to obtain self-oxygenating PLGA nanoparticles.

[0011] The macrophage membrane is coated on the surface of the self-oxygenating PLGA nanoparticles, comprising: adding a macrophage membrane solution to the suspension of the self-oxygenating PLGA nanoparticles, mixing evenly, and extruding with a liposome extruder to coat the macrophage membrane on the surface of the self-oxygenating PLGA nanoparticles.

[0012] The present invention provides an active oxygen-responsive self-generating nanomaterial for promoting angiogenesis and a preparation method thereof, wherein the mesenchymal stem cell conditioned medium pretreated under optimal hypoxia conditions and catalase are co-loaded into self-generating PLGA nanoparticles, and macrophage membranes are coated on the surface thereof, thereby targeted delivery to the injured site. At the same time, the active oxygen-responsive characteristics of CAT are utilized to increase the release rate of angiogenesis-related factors and CAT itself, promote angiogenesis, remove active oxygen in the lesion site and release oxygen, thereby achieving the purpose of treating lower limb ischemia, and solving the technical problem of slow release rate of targeted nanomaterial drugs for promoting angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present application.

[0014] Figure 1 A flow chart of a method for preparing an angiogenesis-promoting reactive oxygen-responsive self-generating nanomaterial provided in an embodiment of the present invention;

[0015] Figure 2 A schematic diagram of the CCK-8 method provided in an embodiment of the present invention for verifying the effect of different hypoxic conditions on the proliferation capacity of MSCs;

[0016] Figure 3 A schematic diagram of the effect of 5% hypoxia on MSC proliferation capacity verified by the live-dead staining method provided in an embodiment of the present invention;

[0017] Figure 4 A schematic diagram showing the comparison of Hif-1α expression and fluorescence intensity in MSCs cultured in 5% hypoxia for 1 day and 3 days according to an embodiment of the present invention;

[0018] Figure 5 A schematic diagram showing the comparison of VEGF expression and fluorescence intensity in MSCs cultured in 5% hypoxia for 1 day and 3 days according to an embodiment of the present invention;

[0019] Figure 6 A schematic diagram of a vascular endothelial cell migration experiment and quantification performed using conditioned medium of mesenchymal stem cells collected after 5 days of 5% hypoxia culture provided in an embodiment of the present invention;

[0020] Figure 7 A schematic diagram of a vascular endothelial cell tube formation experiment and quantification using a mesenchymal stem cell conditioned medium collected after 5 days of 5% hypoxia culture for 3 days provided in an embodiment of the present invention;

[0021] Figure 8 TEM images of an active oxygen-responsive self-generating nanomaterial before and after encapsulation provided by an embodiment of the present invention;

[0022] Fig. 9 An electrophoretic image of an active oxygen-responsive self-generating nanomaterial before and after encapsulation provided by an embodiment of the present invention;

[0023] Fig.10 A schematic diagram of the particle size, potential and stability of an active oxygen responsive nanoparticle provided in an embodiment of the present invention;

[0024] Fig.11 A schematic diagram of the release behavior of an active oxygen responsive nanoparticle provided in an embodiment of the present invention;

[0025] Fig.12 A schematic diagram of the uptake of a self-generated oxygen nanomaterial by endothelial cells in response to reactive oxygen species provided in an embodiment of the present invention;

[0026] Fig.13 A schematic diagram of the oxygen production capacity of different nanoparticles provided in an embodiment of the present invention after incubation in H2O2 for 12 hours;

[0027] Fig.14A schematic diagram of the active oxygen scavenging ability of different nanoparticles provided in an embodiment of the present invention after incubation in H2O2 for 12 hours;

[0028] Fig.15 A schematic diagram of the effect of an active oxygen-responsive self-generating nanomaterial on the viability of vascular endothelial cells provided by an embodiment of the present invention;

[0029] Fig.16 A schematic diagram of the effect of an active oxygen-responsive self-generating nanomaterial on the viability of cardiomyocytes provided by an embodiment of the present invention;

[0030] Fig.17 This is an image of the distribution of a reactive oxygen species-responsive self-generating nanomaterial provided in an embodiment of the present invention in the main organs after it is injected into the tail vein of a mouse suffering from lower limb ischemia;

[0031] Fig.18 Representative images of laser Doppler blood perfusion of ischemic lower limbs of mice before and after treatment with each group of materials at different time points provided in the embodiments of the present invention;

[0032] Fig.19 A schematic diagram of the percentage of the blood perfusion of lower limbs to the blood perfusion before limb ischemia provided by the embodiment of the present invention;

[0033] Fig. 20 A schematic diagram showing the statistical results of limb retention in HI mice 21 days after treatment with different nanomaterials provided in an embodiment of the present invention;

[0034] Fig.21 A schematic diagram of the confocal image and quantitative statistical results of CD31 immunofluorescence staining of new blood vessels marked in ischemic lower limb muscle tissue at 21 days of treatment provided by an embodiment of the present invention;

[0035] Fig. 22 The confocal images and quantitative analysis diagram of TUNEL staining of each group at 21 days of treatment provided by the embodiment of the present invention;

[0036] Fig.23 HE staining images of the main organs of mice provided in the embodiments of the present invention;

[0037] Fig.24 HE staining image of ischemic lower limb muscle of mice provided in an embodiment of the present invention;

[0038] Fig.25 This is a Masson staining image of ischemic lower limb muscle of mice provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0040] Embodiment 1

[0041] The present invention provides an active oxygen responsive self-generating nanomaterial for promoting angiogenesis, comprising: self-generating PLGA nanoparticles simultaneously loaded with mesenchymal stem cell conditioned medium pretreated under preset hypoxic conditions and catalase with active oxygen responsive characteristics, and macrophage membranes coated on the surface of the self-generating PLGA nanoparticles.

[0042] In an optional implementation provided by an embodiment of the present invention, the phase change material of the self-oxygenating PLGA nanoparticles includes DPPC.

[0043] The embodiment of the present invention provides an active oxygen-responsive self-generating nanomaterial that promotes angiogenesis, which is used to load the production factors and catalase parasecreted by mesenchymal stem cells, thereby protecting the growth factors and catalase from degradation and improving the drug release efficiency of the nanoparticles, thereby achieving the effect of eliminating active oxygen, releasing oxygen and promoting angiogenesis.

[0044] Embodiment 2

[0045] Figure 1 1 is a flow chart of a method for preparing angiogenesis-promoting reactive oxygen-responsive self-generating nanomaterial according to an embodiment of the present invention. Figure 1 As shown, the method specifically comprises the following steps:

[0046] Step S102, pre-treating the mesenchymal stem cell conditioned medium under a preset hypoxic condition to obtain a treated mesenchymal stem cell conditioned medium.

[0047] Step S104, dissolving PLGA and DPPC in a dichloromethane solution, adding catalase and the treated mesenchymal stem cell conditioned medium, performing cell disruption and ultrasonic treatment, and obtaining a first emulsion.

[0048] Step S106, preparing self-oxygenating PLGA nanoparticles based on the first emulsion.

[0049] Step S108, coating the macrophage membrane onto the surface of the self-oxygenating PLGA nanoparticles to obtain angiogenesis-promoting active oxygen-responsive self-oxygenating nanomaterials.

[0050] The method provided in the embodiment of the present invention further includes: determining the preset hypoxic condition based on an experimental screening method. Specifically, the method includes the following steps:

[0051] After culturing mesenchymal stem cells in complete medium for three generations, they were switched to serum-free medium and cultured under hypoxic conditions with different oxygen contents;

[0052] At multiple different culture time points, multiple conditioned culture media containing mesenchymal stem cell paracrine factors under multiple hypoxic conditions with different oxygen contents are collected respectively;

[0053] Centrifuging and filtering multiple conditioned culture media respectively, and storing them in sterile centrifuge tubes to obtain multiple culture media samples to be tested;

[0054] The proliferation capacity of mesenchymal stem cells in multiple culture medium samples was analyzed using CCK-8 reagent and Calcein / PI cell activity and cytotoxicity detection kit;

[0055] The oxygen content and culture time point corresponding to the culture medium sample to be tested with the strongest mesenchymal stem cell proliferation ability are determined as the preset hypoxic conditions.

[0056] The present invention takes 5% hypoxia condition as an example to illustrate the specific screening process of the preset hypoxia condition provided by the present invention.

[0057] Specifically, in an optional embodiment provided in an embodiment of the present invention, mouse adipose-derived mesenchymal stem cells can be used for experiments after being cultured in complete medium for 3 generations. When the MSC grows to 60% confluence after attachment, the DMEM / F-12 complete medium is replaced with DMEM / F-12 serum-free medium and cultured under 21% O2 and 5% O2 conditions, respectively. At a specified time point (e.g., 1 day, 3 days, and 5 days), the conditional culture containing mesenchymal stem cell paracrine factors cultured at different times in normoxic and hypoxic conditions is collected and placed in a 15 mL sterile centrifuge tube, centrifuged for 3 minutes, and the supernatant is passed through a 0.45 μm filter to remove cell debris and placed in a sterile centrifuge tube at -80°C.

[0058] CCK-8 reagent and Calcein / PI cell activity and cytotoxicity detection kit were used to explore the effect of 5% O2 hypoxia on MSC proliferation. First, Hif-1α and VEGF were selected to perform immunofluorescence analysis on the conditioned medium of mesenchymal stem cells treated under different hypoxic conditions and for different time periods, and the optimal hypoxic conditions for MSC were preliminarily screened; finally, the conditioned medium of mesenchymal stem cells under different hypoxic treatment conditions was used to perform vascular endothelial cell migration experiments and endothelial cell tube formation experiments, and finally the hypoxic conditions that can maximize the paracrine effect of MSC were screened out.

[0059] Figure 2 is a schematic diagram of verifying the effect of different hypoxic conditions on MSC proliferation ability according to the CCK-8 method provided in an embodiment of the present invention, Figure 3 2 is a schematic diagram of verifying the effect of 5% hypoxia on MSC proliferation ability according to the live-dead staining method provided in an embodiment of the present invention, wherein the 21% oxygen concentration condition is the control group; Figure 4 : is a schematic diagram comparing the expression of Hif-1α and the fluorescence intensity in MSCs cultured in 5% hypoxia for 1 day and 3 days according to an embodiment of the present invention; Figure 5 : is a schematic diagram comparing the expression of VEGF and the fluorescence intensity in MSCs cultured in 5% hypoxia for 1 day and 3 days according to an embodiment of the present invention; Figure 6 It is a schematic diagram of a vascular endothelial cell migration experiment and quantification performed using 5% hypoxia culture for 3 days and mesenchymal stem cell conditioned medium collected after 5 days according to an embodiment of the present invention; Figure 7 It is a schematic diagram of a vascular endothelial cell tube formation experiment and a quantitative diagram according to an embodiment of the present invention using 5% hypoxia culture for 3 days and mesenchymal stem cell conditioned medium collected after 5 days.

[0060] like Figure 2-Figure 7 As shown, according to the experimental screening results, it can be determined that the preset hypoxic condition includes a condition of 5% hypoxia pretreatment for 5 days.

[0061] Specifically, step S106 also includes the following steps:

[0062] Step S1061, adding the first emulsion to the sodium cholate solution and performing ultrasonic treatment to obtain a second emulsion;

[0063] Step S1062, adding the second emulsion to the sodium cholate solution and stirring to volatilize the organic solvent to obtain self-oxygenating PLGA nanoparticles.

[0064] Specifically, step S108 further includes the following steps: adding the macrophage membrane solution to the suspension of self-oxygenating PLGA nanoparticles to mix evenly, and extruding with a liposome extruder to coat the macrophage membrane on the surface of the self-oxygenating PLGA nanoparticles.

[0065] Specifically, DPPC (dipalmitoylphosphatidylcholine) is a neutral phospholipid, and its modification helps to improve the biocompatibility of nanomaterials and can prolong the retention time of nanomaterials in the body. Therefore, the present invention selects PLGA to prepare nanoparticles, and selects DPPC as the phase change material of the nanoparticles, and the above-screened 5% hypoxia pretreated 5-day conditioned medium and catalase are encapsulated inside. The surface of the nanoparticles is wrapped with macrophage membranes, so that the nanoparticles can be targeted to the ischemic site while escaping immune clearance, and the contents are released more quickly at the ischemic site, thereby more effectively utilizing the secretory components and catalase of mesenchymal stem cells to promote vascular regeneration, remove reactive oxygen and release oxygen.

[0066] In an optional embodiment provided in an embodiment of the present invention, the preparation method is as follows: 40 mg PLGA and 40 mg DPPC are dissolved in 5 mL of dichloromethane, 10 to 100 μL of conditioned medium dissolved in 5% hypoxia pretreatment for 5 days with catalase is added, the cell disruptor power is adjusted to 40%, ultrasonic for 3 minutes, the ultrasonic emulsion is poured into 4 mL of 1% sodium cholate solution, and the ultrasonic emulsion is poured into 8 mL of 0.5% sodium cholate solution for the second time, and stirred overnight to volatilize the organic solvent. Collect the prepared self-oxygenating PLGA nanoparticles, adjust the centrifuge speed to 10000g, centrifuge for 5 minutes to remove the supernatant, and wash three times with PBS. Take 5 to 50 μL of macrophage membrane with a protein concentration of 1 mg / mL, add it to 1 mL of self-oxygenating PLGA nanoparticle suspension and mix evenly, extrude it 20 times with a liposome extruder to coat the membrane on the surface of the self-oxygenating PLGA nanoparticles to prepare active oxygen responsive self-oxygenating nanomaterials.

[0067] Embodiment 3

[0068] The present invention also provides a method for characterizing a self-generated oxygen nanomaterial in response to active oxygen. Specifically, the method includes the following aspects:

[0069] Morphology: The morphology of self-generated oxygen-producing PLGA nanoparticles and their coated nanoparticles was observed by transmission electron microscopy (TEM). The steps for preparing TEM samples are as follows: 8 μL of sample was dropped onto a copper grid, air-dried at room temperature, dropped into a neutral 2% phosphotungstic acid negative staining solution, negatively stained for 20 seconds, and then excess liquid was removed with filter paper. The sample was observed under TEM. Figure 8 shown.

[0070] Polyacrylamide gel electrophoresis experiment: 1. Uncoated nanoparticles (hySF+CAT-NP); 2. Nanoparticles coated with macrophage membranes (hySF+CAT-NP@MM); 3. Macrophage membranes (MM). The quality of macrophage membranes used in each group is the same. After the electrophoresis, the protein gel was stained with Coomassie Brilliant Blue staining solution, and finally decolorized with decolorizing solution. After decolorization, a photo was taken with an automatic image analysis system. Fig. 9 shown.

[0071] Particle size, potential and stability: The particle size and potential of the nanoparticles were measured by the Malvern Zetasizer Nano ZS (DLS) dynamic light scattering particle size analyzer. To investigate the stability of the nanoparticles, the nanoparticles were resuspended in PBS and the particle size was measured by the dynamic light scattering particle size analyzer at different time points every day for 7 consecutive days. The particle size, potential and stability results are as follows: Fig.10 shown.

[0072] Release behavior: Prepare 10mL of self-oxygenating PLGA nanoparticles encapsulating Cy5, take the supernatant after centrifugation and place it in a UV spectrophotometer to calculate the Cy5 encapsulation rate in PLGA-NP. Place the 10mL PLGA-NPs resuspended after centrifugation in a dialysis bag with a cutoff molecular weight of 1000, and immerse the dialysis bag in 10mL PBS containing 400μM H2O2, so that the H2O2 concentration in the entire drug release system is 200μM. Place the drug release system in a shaker at a constant temperature of 37°C and shake gently at 150rpm. Take samples at specific continuous time points to detect the release of the encapsulated factors. The specific method is as follows: Take 100μL of supernatant at each time point, determine the Cy5 concentration with a UV spectrophotometer, and at the same time add 100μL PBS containing 200μM H2O2 to the system. The test results are as follows: Fig.11 shown.

[0073] Embodiment 4

[0074] Uptake of self-generating oxygen nanomaterials by cells in response to reactive oxygen species:

[0075] Fluorescent dye Cy5 was encapsulated into the nanoparticles as the inner aqueous phase. The preparation process was referred to Example 2. 1 μL Dio was added to 1 mL macrophage membrane for staining. Endothelial cells were cultured at 2.0×10 4The cells / well were inoculated in a 48-well plate. After culturing for 24 hours, the original culture medium was aspirated, and the nanoparticles were added to 1640 complete culture medium. The concentration of the culture medium containing the nanoparticles was adjusted to 20 μg / mL. The well plate was placed in a 37°C incubator for 2 hours. After washing the well plate three times with PBS, 300 μL of DAPI staining solution was added to each well to stain the cell nucleus for 5-10 minutes. After washing with PBS, the well plate was placed under a laser confocal microscope to observe the phagocytosis of nanoparticles by endothelial cells and take pictures.

[0076] Fig.12 A schematic diagram of the uptake of self-generated oxygen nanomaterials by endothelial cells in response to reactive oxygen species provided in an embodiment of the present invention.

[0077] Embodiment 5

[0078] ROS scavenging and oxygen production capabilities of reactive oxygen species-responsive self-generating nanomaterials:

[0079] The nanoparticles were divided into groups according to 1.hySF+CAT-NP: prepared with mesenchymal stem cell culture medium and catalase cultured under 5% hypoxia for 5 days; 2.CAT-NP: prepared with catalase; 3.hySF-NP: prepared with nanoparticles containing mesenchymal stem cell culture medium cultured under 5% hypoxia for 5 days. The concentrations of nanoparticles in each group were adjusted to 4 mg / mL, 10 mg / mL, 20 mg / mL and 50 mg / mL, respectively, and incubated in PBS containing 200 μM H2O2 for 12 hours, and then the residual H2O2 was determined using a hydrogen peroxide assay kit. At the same time, in order to evaluate the oxygen production capacity, the oxygen produced in the mixture after 12 hours of incubation was photographed.

[0080] The results of oxygen generation capacity and active oxygen scavenging capacity are as follows Fig.13 and Fig.14 As shown, Fig.13 A schematic diagram of the oxygen production capacity of different nanoparticles provided in an embodiment of the present invention after incubation in H2O2 for 12 hours. Fig.14 Schematic diagram of the active oxygen scavenging ability of different nanoparticles provided in an embodiment of the present invention after incubation in H2O2 for 12 hours.

[0081] Embodiment 6

[0082] Cytotoxicity of Reactive Oxygen Response Self-generating Nanomaterials:

[0083] In order to determine whether the synthesized reactive oxygen responsive self-generating nanomaterials can be used for in vivo experiments, the present invention used the nanoparticles before and after encapsulation to act on vascular endothelial cells and cardiomyocytes, respectively, and used the CCK-8 method to determine the cytotoxicity of the nanoparticles after treatment. The results are as follows: Fig.15 and Fig.16As shown, Fig.15 is a schematic diagram of the effect of an active oxygen-responsive self-generating nanomaterial on the viability of vascular endothelial cells provided by an embodiment of the present invention, Fig.16 This is a schematic diagram of the effect of an active oxygen responsive self-generating nanomaterial on the viability of myocardial cells provided by an embodiment of the present invention.

[0084] like Fig.15 and Fig.16 As shown, after the vascular endothelial cells and cardiomyocytes were treated with nanoparticles of different concentrations, the cell viability did not change significantly, which indicates that the nanoparticles prepared by the present invention will not have toxic effects on cells and have good biosafety.

[0085] Embodiment 7

[0086] Reactive oxygen species-responsive self-generating oxygen nanomaterials targeting lower limb ischemia:

[0087] In order to verify the targeting of the reactive oxygen responsive self-generated oxygen nanomaterials provided in the embodiment of the present invention to the ischemic part of the lower limbs, the embodiment of the present invention uses a small animal in vivo imaging device for evaluation. Male Balb / c mice aged 8 weeks (mass 20-25g) were selected for lower limb ischemia (HI) modeling, and the mice were strictly fasted one day in advance. The mice were anesthetized by inhalation of isoflurane gas. After the mice were anesthetized, the amount of isoflurane was adjusted to 2% to keep the mice in an anesthetized state. The mouse was placed with its abdomen facing upwards, and its limbs were fixed on a clean foam board with medical tape. The hair on the lower limbs was removed with a depilatory cream and wiped with iodine for disinfection. The outer skin was cut downward along the root of the left lower limb of the mouse with surgical scissors, and the connective tissue was peeled off with blunt forceps to expose the femoral artery, femoral vein and nerve. The femoral artery was carefully separated from the femoral vein and nerve, and the upper and lower edges of the femoral artery were ligated with 6-0 sutures, and the blood vessels were cut in the middle of the two ligature points. The wound was then sutured, the wound was wiped with normal saline, and the wound and surrounding skin were disinfected with iodine.

[0088] In addition, Cy5 at a concentration of 0.02 mg / mL was encapsulated into self-oxygenating PLGA nanoparticles, and then a part of the self-oxygenating PLGA nanoparticles were coated with macrophage membranes to prepare targeted reactive oxygen responsive self-oxygenating nanomaterials. hySF+CAT-NP and hySF+CAT-NP@MM were injected into mice with hind limb ischemia through the tail vein, respectively. After 12 hours, the distribution of different nanoparticles in various organs in the body was observed by the small animal in vivo imaging system.

[0089] Fig.17 This is an image of the distribution of a reactive oxygen-responsive self-generating nanomaterial in the main organs of a mouse with lower limb ischemia after the tail vein injection of the nanomaterial. Fig.17It can be concluded that nanomaterials coated with macrophage membranes can be targeted to ischemic sites.

[0090] Embodiment 8

[0091] The therapeutic effect of reactive oxygen species-responsive self-generated oxygen nanomaterials on ischemic lower limbs:

[0092] First, the mice were modeled for lower limb ischemia with reference to Example 7, and the model mice were randomly divided into 4 groups (5 mice in each group), and given different treatments. The experimental groups were:

[0093] 1. PBS group: injected with 100 μL sterile PBS;

[0094] 2. CAT-NP@MM: Preparation of nanoparticles containing catalase and encapsulation with macrophage membranes;

[0095] 3. hySF-NP@MM: Nanoparticles containing MSC culture medium cultured under 5% hypoxia for 5 days were prepared and coated with macrophage membranes;

[0096] 4. hySF+CAT-NP@MM: Nanoparticles containing catalase and mesenchymal stem cell culture medium cultured under 5% hypoxic conditions for 5 days were prepared and coated with macrophage membranes.

[0097] Doppler blood flowmeter was used to collect blood perfusion images and data of the lower limbs of mice before modeling and 2 hours, 7 days and 21 days after modeling, and the limb retention of mice was counted. After the collection, the mice were killed, and the viscera and lower limbs of the mice were sampled and made into frozen sections and paraffin sections respectively. The frozen sections of the lower limbs were immunofluorescently stained with endothelial cell marker CD31 to explore the effects of different nanomaterials on angiogenesis in damaged tissues; TUNEL staining was performed on frozen sections of the lower limbs to explore the effects of different nanomaterials on cell apoptosis in damaged tissues; HE staining was performed on paraffin sections of the viscera to explore the biosafety of different nanomaterials on mice; HE staining was performed on paraffin sections of the lower limbs to explore the ability of different nanomaterials to inhibit inflammatory response in ischemic lower limbs; Masson staining was performed on paraffin sections of the lower limbs to explore the ability of different nanomaterials to inhibit fibrosis in ischemic lower limbs.

[0098] Fig.18 Representative images of laser Doppler blood perfusion of ischemic lower limbs of mice before and after treatment with various materials at different time points provided in the embodiments of the present invention. Fig.19 A schematic diagram of the percentage of lower limb blood perfusion volume to limb blood perfusion volume before ischemia provided by the embodiment of the present invention, Fig.18 and Fig.19 It can be seen that tail vein injection of hySF+CAT-NP@MM can increase the perfusion volume of ischemic lower limbs and is beneficial to the blood flow recovery of mice.

[0099] Fig. 20 This is a schematic diagram of the statistical results of the limb retention of HI mice after 21 days of treatment with different nanomaterials provided in the embodiments of the present invention. Fig. 20 It can be seen that hySF+CAT-NP@MM can effectively inhibit the necrosis of ischemic lower limb muscles and promote the repair of ischemic tissues in the lower limbs.

[0100] Fig.21 The confocal image and quantitative statistical result of CD31 immunofluorescence staining of ischemic lower limb muscle tissue labeled new blood vessels at 21 days of treatment provided by the embodiment of the present invention are shown in FIG. Fig.21 It can be seen that hySF+CAT-NP@MM can significantly increase the neovascularization of ischemic lower limbs, reduce muscle cell apoptosis in damaged tissues by enhancing the reconstruction of vascular networks, thereby accelerating the repair of damaged tissues and restoring the normal function of the lower limbs of mice.

[0101] Fig. 22 The confocal images and quantitative analysis diagram of TUNEL staining in each group at 21 days of treatment provided by the embodiment of the present invention are as follows: Fig. 22 It can be seen that hySF+CAT-NP@MM can significantly inhibit the apoptosis of cells in ischemic lower limb muscles.

[0102] Fig.23 HE staining images of the main organs of mice provided in the embodiments of the present invention, such as Fig.23 As shown, there is no obvious inflammatory cell infiltration in each organ, indicating that the active oxygen responsive self-generating nanomaterial prepared in the embodiment of the present invention has good biosafety.

[0103] Fig.24 HE staining images of ischemic lower limb muscles of mice provided in the embodiments of the present invention, such as Fig.24 As shown, hySF+CAT-NP@MM has the strongest ability to inhibit inflammatory infiltration.

[0104] Fig.25 The Masson staining image of the ischemic lower limb muscle of mice provided in the embodiment of the present invention is as follows: Fig.25 As shown, the hySF+CAT-NP@MM group inhibited the degree of muscle fibrosis and the state of muscle tissue was restored best.

[0105] As can be seen from the above description, the embodiment of the present invention provides an active oxygen-responsive self-generating nanomaterial that promotes angiogenesis and a preparation method thereof, wherein the mesenchymal stem cell conditioned medium pretreated under optimal hypoxic conditions and catalase are co-loaded into self-generating PLGA nanoparticles, and macrophage membranes are coated on their surfaces for targeted delivery to the site of injury. At the same time, the active oxygen response characteristics of catalase are utilized to increase the release rate of angiogenesis-related factors and catalase itself, promote angiogenesis, remove active oxygen in the lesion site and release oxygen, thereby achieving the purpose of treating lower limb ischemia, alleviating the technical problem of slow release rate of drugs loaded on targeted nanomaterials that promote angiogenesis in the prior art.

[0106] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0107] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A reactive oxygen species-responsive self-generating nanomaterial that promotes angiogenesis, characterized in that: include: The method also loads mesenchymal stem cell conditioned medium pretreated with preset hypoxic conditions, self-oxygenating PLGA nanoparticles with active oxygen response characteristics, and macrophage membranes coated on the surfaces of the self-oxygenating PLGA nanoparticles.

2. The active oxygen responsive self-generating nanomaterial for promoting angiogenesis according to claim 1, characterized in that: The phase change material of the self-oxygenating PLGA nanoparticles includes DPPC.

3. A method for preparing the active oxygen-responsive self-generating nanomaterial for promoting angiogenesis according to claim 1, characterized in that: include: pre-treating the mesenchymal stem cell conditioned medium under a preset hypoxic condition to obtain a treated mesenchymal stem cell conditioned medium; Dissolving PLGA and DPPC in a dichloromethane solution, adding catalase and the mesenchymal stem cell conditioned medium after the treatment, performing cell disruption and ultrasonic treatment to obtain a first emulsion; preparing self-oxygenating PLGA nanoparticles based on the first emulsion; The macrophage membrane is coated on the surface of the self-oxygenating PLGA nanoparticles to obtain the active oxygen responsive self-oxygenating nanomaterial that promotes angiogenesis.

4. The method according to claim 3, characterized in that: The method further includes determining the preset hypoxic condition based on an experimental screening method; comprising: After culturing mesenchymal stem cells in complete medium for three generations, they were switched to serum-free medium and cultured under hypoxic conditions with different oxygen contents; At multiple different culture time points, multiple conditioned culture media containing mesenchymal stem cell paracrine factors under multiple hypoxic conditions with different oxygen contents are collected respectively; Centrifuging and filtering the multiple conditioned culture media respectively, and storing them in sterile centrifuge tubes to obtain multiple culture medium samples to be tested; Analyzing the proliferation capacity of the mesenchymal stem cells of the multiple culture medium samples to be tested using CCK-8 reagent and Calcein / PI cell activity and cytotoxicity detection kit; The oxygen content and culture time point corresponding to the culture medium sample to be tested with the strongest mesenchymal stem cell proliferation ability are determined as the preset hypoxic condition.

5. The method according to claim 3, characterized in that: The method comprises preparing self-oxygenating PLGA nanoparticles based on the first emulsion, comprising: adding the first emulsion to a sodium cholate solution and performing ultrasonic treatment to obtain a second emulsion; The second emulsion is added to the sodium cholate solution and stirred to volatilize the organic solvent to obtain self-oxygenating PLGA nanoparticles.

6. The method according to claim 3, characterized in that: The macrophage membrane is coated on the surface of the self-oxygenating PLGA nanoparticles, comprising: adding a macrophage membrane solution to the suspension of the self-oxygenating PLGA nanoparticles, mixing evenly, and extruding with a liposome extruder to coat the macrophage membrane on the surface of the self-oxygenating PLGA nanoparticles.

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