Mesenchymal stem cell membrane-coated kaempferol bionic nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a mesenchymal stem cell membrane coated kaempferol bionic nano-drug as well as a preparation method and application thereof, and relates to the technical field of targeted drug preparation, the bionic nano-drug is composed of nano-particles containing kaempferol and a mesenchymal stem cell membrane coated outside the nano-particles, the mesenchymal stem cell membrane is derived from umbilical cord mesenchymal stem cells; the particle size of the bionic nano-drug is 167.67 + / -1.84 nm, the PDI is 0.181 + / -0.027, the potential is-5.41 + / -0.74 mV, the encapsulation efficiency is 79.97 + / -0.19%, and the entrapment rate is 7.27 + / -0.02%. According to the invention, the water solubility of the kaempferol is enhanced, the specific enrichment of the kaempferol at the diseased region of the AS can be enhanced, the utilization efficiency of Kae is improved, and the defects of small molecular drugs are overcome; in addition, through nanocrystallization of Kae, direct contact of cells and tissues with Kae can be reduced, and cytotoxicity is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted drug preparation, and in particular to a mesenchymal stem cell membrane-coated kaempferol biomimetic nanomedicine and a preparation method and application thereof. Background Art

[0002] Atherosclerotic cardiovascular disease and its complications are common diseases worldwide. The development of atherosclerosis (AS) weakens the elasticity of the arterial wall, causes intimal hyperplasia, and forms plaques. These plaques may rupture, leading to thrombosis, which in turn causes severe vascular obstruction. In terms of prevention of AS, it is mainly controlled through diet; in terms of clinical treatment, statins and antiplatelet drugs are mainly used for treatment, but there is a first-pass effect and they cannot effectively treat AS.

[0003] Yixintong is a commonly used Chinese patent medicine for the treatment and prevention of AS. It has the effects of promoting blood circulation, removing blood stasis, and promoting the flow of heart meridians. It can be used to treat chest pain caused by blood stasis blocking the meridians. Symptoms include chest tightness and shortness of breath, stabbing pain in the precordial area, palpitations, forgetfulness, dizziness and tinnitus. For patients with the above symptoms such as angina pectoris caused by coronary heart disease, hyperlipidemia, and cerebral artery insufficiency, the prescription is flavonoids extracted from hawthorn leaves. The total flavonoids of hawthorn leaves include kaempferol, rhamnoside, hyperoside and quercetin. The results of clinical and pharmacological experiments show that the flavonoids contained in hawthorn leaves have multiple pharmacological activities on the cardiovascular system, mainly manifested in: ① By activating peroxisome proliferator-activated receptor α, it reduces blood triglyceride and low-density lipoprotein levels, increases high-density lipoprotein levels, regulates inflammatory responses, and at the same time increases the expression of liver X receptor and adenosine triphosphate binding cassette transporter A1, promotes total cholesterol reverse transport, reduces total cholesterol load, and thus prevents the occurrence of atherosclerosis; ② By increasing NO concentration, clearing free radicals, inhibiting lipid peroxidation damage and stabilizing cell membranes, it relieves and alleviates myocardial ischemia and protects myocardial cells damaged by ischemia and hypoxia; ③ By reducing blood viscosity, inhibiting platelet aggregation, accelerating blood flow, reducing thrombosis, and reducing the expression of gelatinase B, it reduces the degradation of vascular basement membrane and extracellular matrix proteins, reduces the permeability of the blood-brain barrier, reduces brain edema, reduces the area of ​​cerebral infarction, further reduces brain tissue damage, and plays a brain protective role; ④ By adjusting the contractility and heart rate of the heart, it maintains blood pressure stability and improves coronary blood circulation.

[0004] In addition, kaempferol (Kae), as an effective monomer of total flavonoids in hawthorn leaves, has anti-inflammatory and antioxidant effects and has also attracted much attention in the clinical treatment of AS. However, as a flavonoid compound, this drug has extremely low water solubility and is difficult to reach an effective concentration at the lesion site, thus limiting its use. Summary of the invention

[0005] The invention discloses a mesenchymal stem cell membrane-coated kaempferol biomimetic nanomedicine and a preparation method thereof. The bioavailability of kaempferol is improved by coating a specific cell membrane on the surface of nanoparticles containing kaempferol, and the specific enrichment of kaempferol in AS lesion sites is enhanced, thereby solving the problem that kaempferol is difficult to reach an effective concentration at the lesion site due to its extremely low water solubility.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A mesenchymal stem cell membrane-coated kaempferol bionic nanomedicine, the bionic nanomedicine consists of nanoparticles containing kaempferol and mesenchymal stem cell membranes coated on the outside thereof, the mesenchymal stem cell membranes are derived from umbilical cord mesenchymal stem cells; the bionic nanomedicine has a particle size of 167.67±1.84nm, a PDI of 0.181±0.027, a potential of -5.41±0.74mV, an encapsulation rate of 79.97±0.19%, and an encapsulation rate of 7.27±0.02%.

[0008] Preferably, the nanoparticles containing kaempferol are prepared by combining kaempferol and polylactic acid-glycolic acid copolymer through coprecipitation and emulsification.

[0009] Furthermore, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 1:1.

[0010] Furthermore, the mass ratio of kaempferol to polylactic acid-glycolic acid copolymer is 1:10.

[0011] The method for preparing the bionic nano drug comprises the following steps:

[0012] (1) preparing nanoparticles KP containing kaempferol by combining kaempferol and poly(lactic acid-co-glycolic acid) copolymer;

[0013] (2) extracting mesenchymal stem cell membranes from umbilical cord mesenchymal stem cells;

[0014] (3) The extracted mesenchymal stem cell membrane and the aqueous solution of nanoparticles containing kaempferol were mixed by ultrasonication for 3 min in an ice bath, and then squeezed back and forth through a polycarbonate porous membrane by a liposome co-extruder for dozens of times to obtain the bionic nanomedicine KPM.

[0015] More preferably, in step (1), the preparation process of the nanoparticles KP containing kaempferol is:

[0016] a. The polyvinyl alcohol powder was prepared into a 1% concentration liquid using double distilled water, and then subjected to a gradient water bath to fully denature it and then heat preservation, and after cooling, stirred at a low speed to obtain a liquid A for standby use;

[0017] b. Weigh polylactic acid-glycolic acid copolymer and dissolve it in DMSO, then add kaempferol, mix well and ultrasonicate for 3 minutes to obtain solution B for later use;

[0018] c. Add solution B dropwise into solution A, stir at a constant speed for 1 hour, transfer to a dialysis bag, and dialyze overnight with pure water in the dark for 12 hours. The collected liquid is KP.

[0019] More preferably, in step (2), the extraction process of mesenchymal stem cell membrane is:

[0020] a. Take P2 umbilical cord mesenchymal stem cells, melt them in a 37℃ water bath, 1500r / min, 5min, discard the supernatant, mix with αMEM containing 10% FBS, place in a T25 bottle, and culture at 37℃, 5% CO2, in a cell culture incubator for 24h. Passage the cells when they fill 80% to 90% of the cell bottle;

[0021] b. When the cells grow to 90%, discard the culture medium, wash three times with pre-cooled PBS, discard the PBS and add trypsin digestion solution. After digestion, add PBS and blow into a 4℃ pre-cooled centrifuge, centrifuge at 1500rpm / min for 5min, take 5 dishes of cell pellets, add 990μL lysis solution and 10μLPMSSF, lyse on ice for 15min, use liquid nitrogen, 37℃ water bath, alternately freeze and thaw 5 times, centrifuge at 700g at 4℃ for 10min, take the supernatant, put the supernatant into a new EP tube, centrifuge at 12000g at 4℃ for 30min, the resulting precipitate is the mesenchymal stem cell membrane.

[0022] More preferably, in the step (3), 1 mg of KP is added based on the membranes extracted from 5 dishes of mesenchymal stem cells.

[0023] Furthermore, in the step (3), the polycarbonate porous membrane is provided with two layers, and the pore sizes of the two layers of polycarbonate porous membrane are 400 nm and 200 nm respectively.

[0024] The application of the bionic nano drug in the preparation of drugs for treating atherosclerosis.

[0025] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] 1. The present invention not only enhances the water solubility by coating the surface of nanoparticles containing kaempferol with a specific cell membrane, but also enhances the specific enrichment of Kae in AS lesions, thereby improving the utilization efficiency of Kae and overcoming the shortcomings of small molecule drugs; and the nano-sizing of Kae can effectively reduce the direct contact between cells and tissues and Kae, thereby reducing cytotoxicity;

[0027] 2. The present invention broadens the application of bionic membranes in AS treatment, applies mesenchymal stem cell membranes to AS research, achieves immune escape, and targets inflammatory endothelial cells; reaches the lesion site in vivo, reduces lipid deposition in the aortic arch, stabilizes plaques, and reduces the oxidative stress level of the aorta, and has high biosafety;

[0028] 3. The present invention, through ingenious scheme design, enables bionic nanomedicine containing Kae to be better used for the treatment of AS, solves the pain points and difficulties in this field, gives full play to the role of Kae in the clinical treatment of AS, and provides more options for the effective treatment of AS. Therefore, the present invention has outstanding substantive characteristics and significant progress, and is very suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the particle size distribution diagram of KP / KPM prepared in Example 1;

[0030] Figure 2 This is the potential distribution diagram of KP / KPM prepared in Example 1;

[0031] Figure 3 It is a standard curve diagram of the concentration of Kae in DMSO (horizontal axis) and the absorbance value (vertical axis);

[0032] Figure 4 This is a transmission electron microscopy image of KP / KPM prepared in Example 1;

[0033] Figure 5 The drug release curve of KP / KPM prepared in Example 1;

[0034] Figure 6 It is a protein SDS-PAGE gel image;

[0035] Figure 7 This is a picture of the detection of characteristic proteins such as CD47, Integrinβ1, Cxcr4, and Anti-Na / KATPase by western blotting;

[0036] Figure 8 This is a graph of relative cell survival of human umbilical vein endothelial cells after treatment with different drugs in Example 2;

[0037] Fig. 9 This is a graph of relative cell survival of Raw 264.7 cells after treatment with different drugs in Example 2;

[0038] Fig.10 This is a graph of relative cell survival of smooth muscle cells after treatment with different drugs in Example 2;

[0039] Fig.11This is a laser confocal image of normal macrophages and activated macrophages taking up nanoparticles in Example 2;

[0040] Fig.12 Figure 2 is a flow cytometric analysis of the uptake of unencapsulated / encapsulated fluorescent nanoparticles by normal macrophages and activated macrophages in Example 2, A is a curve graph, and B is a statistical bar graph;

[0041] Fig.13 This is a laser confocal image of the normal endothelial and inflammatory endothelial cells taking up nanoparticles in Example 2;

[0042] Fig.14 Figure 2 is a flow cytometric analysis of the uptake of unencapsulated / encapsulated fluorescent nanoparticles by normal endothelial cells and inflammatory endothelial cells in Example 2, A is a curve graph, and B is a statistical bar graph;

[0043] Fig.15 : This is the antioxidant evaluation diagram of KPM on endothelial cells in the oxidative stress model in Example 2, wherein A is the statistical diagram of the CCK8 experiment of endothelial cells on H2O2 concentration; B is the statistical diagram of the MDA level of endothelial cells in the oxidative stress model after different treatment groups; C is the statistical diagram of the ROS level of endothelial cells in the oxidative stress model after different treatment groups; D is the fluorescence diagram of the ROS level of endothelial cells in the oxidative stress model after different treatment groups;

[0044] Fig.16 The hemolysis experiment and statistical graph in Example 2;

[0045] Fig.17 is a line graph of relative fluorescence intensity of different nanoparticles circulating in vivo in Example 3;

[0046] Fig.18 The fluorescence imaging and statistical graphs of different nanoparticles in the aortic arch in Example 3, A is the imaging graph, and B is the statistical bar graph;

[0047] Fig.19 The fluorescence imaging images of different nanoparticles in major organs in Example 3;

[0048] Fig. 20 ApoE in different treatment groups in Example 3 - / - Oil red O staining of the aortic arch of mice;

[0049] Fig.21 ApoE in different treatment groups in Example 3 - / - Oil red O staining of aortic arch root section of mouse (the upper and lower scales are 500 μm and 50 μm, respectively);

[0050] Fig. 22 ApoE in different treatment groups in Example 3 - / -Masson staining of the aortic arch root of mice (the upper and lower scales are 500 μm and 50 μm, respectively);

[0051] Fig.23 ApoE in different treatment groups in Example 3 - / - Toluidine blue staining of the aortic arch root of mice (the upper and lower scales are 500 μm and 50 μm, respectively);

[0052] Fig.24 ApoE in different treatment groups in Example 3 - / - CD68 immunohistochemistry of mouse aortic arch (the upper and lower scales are 500 μm and 50 μm, respectively);

[0053] Fig.25 ApoE in different treatment groups in Example 3 - / - MMP9 immunohistochemistry of mice (the upper and lower scales are 500 μm and 50 μm, respectively);

[0054] Fig.26 ApoE in different treatment groups in Example 3 - / - Nrf2 immunofluorescence staining of the aortic arch root of mice (scale bar, 100 μm);

[0055] Fig. 27 ApoE in different treatment groups in Example 3 - / - GPX4 immunofluorescence staining of the aortic arch root of mice (scale bar, 100 μm);

[0056] Fig.28 ApoE in different treatment groups in Example 3 - / - HE staining of the main organs of mice (scale bar is 100 μm);

[0057] Fig.29 ApoE in different treatment groups in Example 3 - / - Body weight change curve of mice. DETAILED DESCRIPTION

[0058] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0059] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0060] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0061] The present application will be described in detail below with reference to embodiments and experimental data.

[0062] Example 1

[0063] 1. Preparation of experimental samples

[0064] (1) Synthesis of KP

[0065] ① Pre-treat PVA, prepare PVA powder into 1% liquid with double distilled water, place in gradient water bath to fully denature it, then keep warm, cool, measure 4 mL, place on a magnetic stirrer with rotor and stir at low speed, and set aside.

[0066] ② Weigh 10 mg PLGA and dissolve it fully in DMSO, add 1 mg Kae, mix well and sonicate for 3 minutes.

[0067] ③ Drop the mixed DMSO solution containing Kae and PLGA into ①, stir at a constant speed for 1 hour, transfer to a dialysis bag, and dialyze with pure water in the dark overnight.

[0068] ④After 12 hours of dialysis, the collected fluid is KP.

[0069] (2) Extraction of mesenchymal stem cell membrane (MSCM)

[0070] Umbilical cord mesenchymal stem cell recovery and culture: Umbilical cord mesenchymal stem cells were kindly provided by Chongqing Honghui Stem Cell Platform. P2 generation was taken, thawed in a 37°C water bath, 1500r / min, 5min, supernatant discarded, mixed with αMEM containing 10% FBS and placed in a T25 bottle, 37°C, 5% CO2, cell culture in a cell culture incubator, stably cultured for 24h, and passaged when the cells filled 80% to 90% of the cell bottle.

[0071] MSCM extraction: When the cells grow to 90%, discard the culture medium, wash three times with pre-cooled PBS, discard the PBS and add trypsin digestion solution. After digestion, add PBS and blow into a 4℃ pre-cooled centrifuge, centrifuge at 1500rpm / min for 5min, take 5 dishes of cell pellets, add 990μL lysis solution, 10μLPMSF, lyse on ice for 15min, use liquid nitrogen, 37℃ water bath, alternately freeze and thaw 5 times, centrifuge at 700g at 4℃ for 10min, take the supernatant, put the supernatant in a new EP tube, centrifuge at 12000g at 4℃ for 30min, the obvious precipitate obtained is MSCM.

[0072] (3) Synthetic KPM

[0073] The extracted MSCM (the membrane extracted from 5 dishes of mesenchymal stem cells is counted as one unit) is mixed with a 1 mg KP nanoparticle aqueous solution using ice bath ultrasound for 3 minutes, and then squeezed back and forth through 400 nm and 200 nm polycarbonate porous membranes for dozens of times using a liposome co-extruder to obtain KPM.

[0074] (4) Preparation of fluorescent nanoparticles

[0075] We used the same nanoparticle preparation process and used DIO reagent purchased from Shanghai Aladdin Reagent instead of Kae to form fluorescent non-encapsulated nanoparticles DP and fluorescent coated nanoparticles DPM. DP was used to simulate KP, and DPM represented KPM.

[0076] 2. Determination of particle size, potential, PDI, TEM and encapsulation loading of nanomedicine

[0077] The particle size, potential, and PDI of KP and KPM nanoparticles in water were measured at room temperature using a nanoparticle size potentiometer, and three samples were repeated for each experiment. The morphology of the nanoparticles was measured by transmission electron microscopy. A clean copper mesh was taken, and appropriate concentrations of KP and KPM were dropped into it, covering the copper mesh, and air-dried naturally. After air-drying, phosphotungstic acid was added for negative staining for 5 minutes, and then filter paper was used to absorb the excess phosphotungstic acid. The sample was placed under TEM for selected area photography. The KP and KPM aqueous solutions were placed at room temperature, and the particle size was measured at different time points to observe the stability of the nanoparticles in the aqueous solution.

[0078] The particle size and potential of KP and KPM in aqueous solution were determined by DLS. Figure 1 In the figure, we can see that the particle size curves of KP and KPM are smooth, with only one peak, and the software results are well evaluated, indicating that the particle size uniformity is good. From the characterization data in Table 1, we can see that the particle size of KP is 147.50±1.28nm, and after encapsulation with MSCM, the particle size increases to 167.67±1.84nm. The increase in particle size indicates the success of encapsulation to a certain extent. Figure 2In the potential representative graph, it can be seen that the curves of KP and KPM are relatively flat, and there are no mixed peaks, which also reflects the uniformity of particle size. The potential of KP is -1.18 ± 0.17mV, and the potential of KPM is -5.07 ± 0.19mV. Compared with KP, the potential of KPM also increases negatively, because the cell membrane is negatively charged when in the aqueous phase. PDI is used to evaluate the dispersibility of nanoparticles. It is generally believed that the range of about 0.1-0.2 is optimal. The PDI of KP prepared in this embodiment is 0.137 ± 0.030, and the PDI of KPM is 0.181 ± 0.027, indicating that the KP and KPM prepared in this embodiment have good dispersibility, are not easy to agglomerate and aggregate, and are convenient for the smooth development of subsequent phagocytosis experiments.

[0079] Table 1 The particle size, potential, PDI, TEM and encapsulation loading of nanodrugs

[0080]

[0081] Kae has a characteristic absorption peak at 365nm in DMSO solution. We used DMSO to dilute Kae to concentrations of 1, 2.5, 5, 10, 20, and 25μg / mL. The absorbance values ​​measured in the UV spectrophotometer were 0.0665, 0.1742, 0.3448, 0.7051, 1.3663, and 1.7135, respectively. The standard curve was fitted with Kae concentration as the horizontal axis and absorbance value as the vertical axis. Figure 3 , the equation is y = 0.0684x + 0.0045, R2 = 0.9998. Kae has very low solubility in water and is usually dissolved using hot ethanol, alkali, and ether, but the above method brings inconvenience to practical applications. After we used PLGA and PVA to nano-size kaempferol, we found that 799.7 μg of 1 mg of Kae was encapsulated in the nanocarrier. The freeze-dried sample was re-dissolved in DMSO using an ultraviolet spectrophotometer, and the encapsulation rate of KP was calculated to be 79.97 ± 0.19%, and the encapsulation rate was 7.27 ± 0.02% (as shown in Table 1). This nano-size method is safer than direct dissolution and administration using organic solvents.

[0082] 3. Morphological characterization of nanomedicine

[0083] In order to more intuitively verify whether MSCM is coated on the outside of KP, we used phosphoric acid counterstaining and then observed it using TEM, such as Figure 4As shown in the figure, we found that the size of KP is about 100nm and is round. KPM has the same shape as KP and has a membrane on the outer layer. It is larger than KP. Since the sample was air-dried and dehydrated during preparation, the particle size measured by TEM will be different from that measured by DLS. In addition, the thickness of MSCM measured by TEM is about 10nm, indicating that MSCM successfully coated KP.

[0084] 4. Stability evaluation of nanomedicine

[0085] In order to determine the storage stability of the nanoparticles, we measured the particle size of the nanoparticles at different time points of 12, 24, 36, 48, 60, and 72 hours when they were placed in water medium, and found that KP and KPM were able to maintain a certain stability within 72 hours, and the particle size did not show a large fluctuation. This shows that the combination of Kae and the carrier is relatively stable, and the modification of KP by MSCM has a certain stability.

[0086] 5. Drug release profile of nanomedicine

[0087] In order to study the drug release behavior of KP and KPM, we mixed the nanoparticles with PBS and conducted release experiments in a constant temperature shaker to simulate the in vivo environment. Samples were collected at 0, 1, 2, 4, 8, 12, 24, 36, and 72 hours, and the drug content was determined and the cumulative drug release rate was calculated. Figure 5 It can be seen that both KP and KPM can slow down the burst release of drugs. And the release rate of KPM is slower than that of KP, which shows that the modification of MSCM can slow down the release of drugs.

[0088] 6. Protein characterization of KPM

[0089] Membrane proteins play a key role in various biological activities of cells. Membrane proteins have multiple functions such as information transmission and target binding. First, we performed Coomassie Brilliant Blue staining through WB experiments to observe the expression of total proteins. The protein expression profile of MSCM is similar to that of KPM, indicating that KPM has inherited the proteins of MSCM well. Nano-extrusion of MSCM by ultrasound and liposome micro-liposome extruder will not significantly cause the loss of major proteins ( Figure 6 ).

[0090] In order to confirm whether KPM inherited the proteins on MSCM, we characterized CD47, which inhibits immune cell phagocytosis and promotes circulation, Integrinβ1, which targets inflammatory endothelium, and membrane protein internal reference Anti-Na / KATPase. In addition, Cxcr4 is expressed in mesenchymal cells and can bind to Cxcl12, which is overexpressed in ischemia and inflammation. In order to explore whether MSCM and KPM retain Cxcr4, Cxcr4 protein was also characterized. The results of characteristic protein characterization are shown in 7. CD47, Integrinβ1, and Cxcr4 of MSCM are all retained to a certain extent on KPM. This lays the foundation for subsequent in vitro immune escape, targeting, in vivo circulation, and targeting effects.

[0091] Example 2

[0092] In this example, the following in vitro evaluation experiments were performed on the KP and KPM prepared in Example 1:

[0093] 1. Cytotoxicity evaluation experiment

[0094] In order to evaluate the safety of nanomaterials, we used untreated Kae as the control group and incubated different concentrations of nanoparticles (KP, KPM) with Raw 264.7 cells, human umbilical vein endothelial cells, and smooth muscle cells for 12 hours. When there is no significant difference in the survival rate of cells between the drug-treated group (KP group, KPM group) and the control group (Kae group), the drug is generally considered to be non-toxic. Judging from the relative survival of the three groups of cells, at a concentration of 20μg / mL, there was a significant difference in survival between the treatment group and the control group, while at 10μg / mL, no cytotoxicity was caused. Therefore, in subsequent experiments, the concentration of the nanoparticles used was 10μg / mL. Figure 8 and Fig. 9 It can be seen that under the same Kae equivalent, the toxicity of KPM is lower than that of KP, while that of KP is lower than that of pure Kae, indicating that the synthetic KP and KPM can reduce the toxicity to human umbilical vein endothelial cells and Raw 264.7 cells, which may be derived from the sustained release effect of KP and KPM. Fig.10 In the concentration range of 1-20μg / mL, at the same Kae equivalent, the toxicity of pure Kae is less than that of KP and KPM. It may be that VSMC is not sensitive enough to the drug, and PLGA and MSCM will have an impact on VSMC. However, as the concentration increases to above 20μg / mL, smooth muscle cells are more sensitive to Kae, and KP and KPM play a protective role. Through the toxicity test of three types of cells, it is confirmed that nanoparticles can reduce drug toxicity, and the nanoparticle concentration used in subsequent experiments is determined.

[0095] 2. Immune escape and inflammatory cell targeting evaluation experiments

[0096] LPS was used to induce macrophage activation. Then DP and DPM were added and incubated for 2 hours. Fig.11 It can be seen that macrophages absorb more DP that is not wrapped with MSCM, while after being wrapped with MSCM, macrophages' uptake of DPM is weakened. When macrophages undergo polarization transformation and their phagocytic ability is enhanced, compared with the phagocytic state of normal unactivated macrophages, the phagocytic amount of DPM by activated macrophages is enhanced, but the enhancement intensity is far lower than the uptake of DP by activated macrophages. Fig.12 The flow cytometry results also proved that the modification of nanoparticles by MSCM can indeed inhibit the capture of macrophages, thereby achieving immune escape.

[0097] After the fluorescent nanoparticles were co-incubated with normal endothelium and inflammatory endothelium, the images were taken using a fluorescence confocal microscope ( Fig.13 ), and analysis using flow cytometry ( Fig.14 ). The results showed that both normal and inflammatory endothelium phagocytosed coated nanoparticles DPM more than uncoated nanoparticles DP, indicating that the biomimetic nature of coated nanoparticles has an impact on phagocytosis. By comparing the phagocytosis of the two nanoparticles by inflammatory endothelial cells and normal endothelial cells, it was found that the inflammatory endothelial cells took up more DP than the normal endothelium, and the inflammatory endothelial cells took up much more DPM than the normal endothelium, that is, the total uptake was ranked as ADPM>ADP>DPM>DP. (ADPM is the group of DPM phagocytized after endothelial cells were activated, and ADP is the group of DP phagocytized after endothelial cells were activated). This shows that the coated nanoparticles can indeed target the inflammatory endothelium.

[0098] 3. Antioxidant stress evaluation experiment

[0099] Oxidative stress in endothelial cells plays an important role in AS. Oxidative stress in endothelial cells can cause cells to produce excessive reactive oxygen species (ROS), which can directly damage cells, leading to impaired endothelial diastolic function. Malondialdehyde (MDA) is an important biomarker in oxidative stress. Lipid metabolism disorders are an important cause in AS, and MDA represents the degree of lipid peroxidation. Therefore, we explored the effects of KPM treatment on MDA and ROS in endothelial cells. We used H2O2 to pretreat endothelial cells to construct an oxidative stress model of endothelial cells. Fig.15 The results of A showed that when the concentration of H2O2 was 0.4mM, it could significantly affect the activity of endothelial cells. Therefore, we used 0.2mM as the subsequent induction concentration to maximize its effect without affecting the activity. Fig.15The results of B to D show that KPM can significantly reduce the levels of MDA and ROS in endothelial cells. In addition, we can see that KPM reduces the levels of MDA and ROS in endothelial cells better than KPM, which may also indirectly reflect that KPM promotes phagocytosis and better alleviates the oxidative damage of endothelial cells.

[0100] 4. Hemolysis test

[0101] Hemolytic test is an important indicator to verify the toxicity of materials.

[0102] The steps of the hemolytic assay are as follows:

[0103] ① After the mice were anesthetized, blood was collected from the eye sockets and anticoagulated with EDTA-2K (10X) at a concentration of 1:9;

[0104] ② Dilute the anticoagulated blood with 0.9% NaCl solution at a ratio of 4:5 and mix well;

[0105] ③ Take a 2 mL clean centrifuge tube and add 2 mL of Kae, mesenchymal stem cell membrane, KP, KPM, PBS, and distilled water, respectively, in triplicate for each group;

[0106] ④ Add 40 μL of diluted blood to each centrifuge tube and incubate at 37°C for 1 hour;

[0107] ⑤ After the warm bath, place in a centrifuge at 2000g for 5 minutes;

[0108] ⑥ Take the supernatant after centrifugation, measure the absorbance at 545nm, and calculate the hemolysis rate.

[0109] In national standards, it is generally considered that a hemolysis rate below 5% is non-toxic. Fig.16 The hemolysis experiment chart shows that the hemolysis rates of KP and KPM are both lower than 5%, indicating that KP and KPM have good blood compatibility, which provides a safe basis for subsequent tail vein injection of drugs.

[0110] Example 3

[0111] In this example, the following in vivo evaluation experiments were conducted on the KP and KPM prepared in Example 1:

[0112] 1. Establishment of animal atherosclerosis disease model

[0113] ApoE - / - Male mice were purchased from Jicui Yaokang and fed with ordinary feed for one week and then with high-fat feed for 10 weeks to establish atherosclerosis model mice.

[0114] 2. Long-term circulation evaluation experiment of KPM in mice

[0115] In order to detect whether MSCM also has immune escape function in vivo, we injected fluorescent nanoparticles (DP, DPM) into the tail vein of mice and measured the fluorescence intensity in the blood at different time points after injection. Fig.17 The measurement results show that at 1 hour, both fluorescent nanoparticles have the highest fluorescence intensity, and the fluorescence intensity of DPM is much higher than that of DP, indicating that DP decays quickly in the body, which may be due to metabolism. As time goes by, the fluorescence intensity of DP and DPM both decreases, and the fluorescence decrease rate of DP is faster than that of DPM. At 12 hours, it is basically close to the value before injection (0 hours), which can be considered that DP is cleared. The fluorescence intensity of DPM decreases more slowly, and it is not until 72 hours that it approaches the normal blood level. This shows that the circulation time of DPM is longer than that of DP, proving that in vivo, MSCM can prolong the blood circulation time of nanoparticles.

[0116] 3. Targeted research experiment of KPM in mice

[0117] To verify that MSCM has the ability to target the aortic arch in vivo, we injected different fluorescent nanoparticles into the tail vein. After a certain period of time, we performed dissection, sampling, and small animal imaging. We found that the aortic arch of the DPM group had obvious fluorescence accumulation, while the DP group had less accumulation ( Fig.18 ). We also conducted a statistical analysis of the overall fluorescence intensity and found that the fluorescence accumulation of DPM in the blood vessels was significantly stronger than that of the DP group. This shows that MSCM nanoparticles can target the aorta in vivo, providing an experimental basis for the subsequent treatment of aortic AS plaques. In addition, we sampled the main organs of ApoE- / - mice injected with fluorescent nanoparticles, performed fluorescence imaging, and observed the whereabouts of the fluorescent nanoparticles. We found that the DP group mainly went to the liver and kidneys, while the DPM group only found a small part in the liver ( Fig.19 ). This indicates that DPM is not easily metabolized by organs compared to DP.

[0118] 4. Lipid deposition of KPM in mouse aorta

[0119] A major feature of AS is lipid deposition in the aorta, and the degree of lipid deposition also represents the degree of plaque inflammation. After the treatment cycle, we performed Oil Red O staining on the sections of the aortic arch and the root of the aortic arch. Fig. 20 The aortic arch Oil Red O staining and quantitative analysis showed that after KPM treatment, aortic lipid deposition decreased. - / -The lipid deposition in the aorta of the group treated with Kae was only slightly reduced, while the lipid deposition in the aortic arch was reduced to a certain extent by KP treatment. This is because KP has an EPR effect, which can achieve a certain degree of retention. KPM had the best treatment effect, with a significant reduction in lipid deposition in the aortic arch, as well as lipids in the abdominal aorta and iliac arteries, indicating that MSCM does make the nanoparticles have a targeting effect, and at the same time, prolongs the circulation time, allowing Kae to be released and absorbed more in the plaque area.

[0120] In such Fig.21 The Oil Red O staining and statistical diagram of the aortic arch root section shown in the figure show that compared with wild-type mice, ApoE - / - After feeding with a high-fat diet, a large amount of lipid deposition was found at the root of the aortic arch. After different treatments, KPM showed a good effect in reducing lipid deposition, which was consistent with the results of Oil Red O staining of the aortic arch.

[0121] 5. Plaque stability in mice after KPM treatment

[0122] The synthesis and distribution of collagen fibers affect the stability and vulnerability of AS plaques. In AS plaques, collagen fibers are the main component of the fibrous cap. Therefore, the observation of collagen fibers is crucial for the stability of AS plaques. Masson staining is a major method for observing collagen fibers. We used Masson staining to observe the collagen thickness of different treatment groups. Fig. 22 Show, in ApoE - / - In the Kae free group, KP group, and KPM group, the thickness of collagen fibers was large. The thickness of collagen fibers decreased after treatment in the Kae free group, KP group, and KPM group. Among them, the KPM group had the best effect in reducing collagen thickness, indicating that KPM can stabilize plaques by inhibiting the proliferation of collagen fibers.

[0123] The necrotic core is a key feature of AS vulnerable plaques. The formation of necrotic cores is caused by the necrosis and apoptosis of foam cells caused by the phagocytosis of lipids by macrophages. It reduces the stability of plaques and also aggravates the inflammatory response. Toluidine blue staining is a method for observing necrotic cores. We stained the aortic arch root sections of mice in different treatment groups with toluidine blue. The results are shown in Figure 23. Wild-type mice have almost no necrotic cores, while ApoE - / - Mice developed large areas of necrotic cores. After different treatments, the area of ​​necrotic cores decreased. In the KPM group, we found that the decrease in collagen thickness was accompanied by a decrease in the necrotic core, indicating that KPM can indeed target the plaque site and contribute to the stability of the plaque.

[0124] In AS plaques, macrophages will phagocytize lipids to form foam cells in the AS environment. The occurrence and development of foam cells have an adverse effect on the stability of the plaque. At the same time, it can also play a role in the fibrous cap of the plaque. CD68 is a marker on the surface of macrophages. We performed CD68 immunohistochemical staining on mice in different treatment groups. The results are as follows Fig.24 As shown, ApoE - / - There were many macrophages accumulated in the mice. Compared with the Kae treatment group and the KP treatment group, the relative number of macrophages was significantly reduced after treatment with KPM, which was beneficial to the stability of the plaque.

[0125] In AS plaques, macrophages secrete a matrix metalloproteinase-9 (MMP9), which is a marker of plaque instability. It can decompose the extracellular matrix components that maintain plaque stability, thereby affecting AS plaques. We performed MMP9 immunohistochemical staining on the mouse sections of each group, and the results are as follows Fig.25 As shown, it was found that KPM significantly decreased the relative expression level of MMP9.

[0126] 6. Antioxidant activity of mice after KPM treatment

[0127] Nrf2 / GPX4 plays an important role in the oxidative stress of AS. Nrf2 is responsible for the antioxidant response element in cells and can enhance the antioxidant properties of cells by activating the expression of multiple antioxidant enzymes. GPX4 is an important antioxidant enzyme that can reduce lipid peroxides, thereby protecting cells from damage by lipid peroxidation. Usually, in AS, the Nrf2 / GPX4 pathway is inhibited. In order to evaluate the antioxidant properties of nanoparticles at the tissue level. We performed Nrf2 / GPX4 immunohistochemical staining on the sections of each group of mice, and the results are shown in Fig.26 , Fig. 27 As shown, in ApoE - / - In the experiment, the expression of Nrf2 / GPX4 decreased, but the Kae drug group did not significantly improve the expression of Nrf2 / GPX4. This is because although the Kae drug can effectively relieve oxidative stress, it cannot reach the target position well to achieve antioxidant effect due to its free effect. After nano-sizing Kae, the expression of Nrf2 / GPX4 in the aortic arch root of the KP blood vessel increased compared with the Kae group. This is because the EPR effect is achieved after nano-sizing, so that the nanoparticles can be partially accumulated in the aortic arch root, thereby achieving a certain therapeutic effect through drug sustained release. The results show that the KPM group can significantly increase the expression of Nrf2 / GPX4. This is because KPM encapsulates MSCM on the basis of Kae nano-sizing, which provides conditions for nanoparticles to efficiently target plaque sites, improves the effective delivery of Kae, and achieves treatment.

[0128] We tested the MDA in the blood of different groups of mice and measured the MDA content in the aortic arch by homogenization. Our results showed that KPM could significantly reduce the MDA content in the blood and aortic arch. In terms of the overall therapeutic effect, KPM>KP>Kae.

[0129] 7. Safety evaluation experiment of KPM-treated mice

[0130] Histological pathological sections can accurately judge the lesions, and HE staining is a commonly used pathological staining method. It can clearly show whether the tissue has lesions. In order to evaluate the safety of nanoparticles in vivo, we sliced ​​and stained the main organs of mice in each treatment group. Fig.28 As shown in the HE staining results, we found that the organs of mice in each group were normal, and no serious lesions such as fibrosis and necrosis were found. This shows that the nanoparticles prepared in this study are friendly to organs and will not cause organ damage.

[0131] After one month of injection treatment, we evaluated the four blood lipids of each mouse, mainly including CHO (serum total cholesterol), TG (triglycerides), LDL (low-density lipoprotein cholesterol), and HDL (high-density lipoprotein cholesterol). The balance of blood lipids in the body plays a role in the homeostasis of the individual. After one month of treatment, we found that there was no significant difference between the mice in each group that were continuously injected for one month and the control group, indicating that nanoparticles do not significantly affect blood lipid homeostasis.

[0132] Routine blood tests can reveal inflammatory factors and body function status. We collected blood from mice after one month of treatment and analyzed the four items of routine blood tests: WBC (white blood cells), RBC (red blood cells), HG (hemoglobin), and PLT (platelets). As shown in Table 2, it was found that the four items of routine blood tests in each group were within the reference range, indicating that the drugs and nanoparticles used in this study do not cause inflammation and have good blood safety. ALT (alanine aminotransferase), AST (aspartate aminotransferase), UREA (urea), and CREA (creatinine) in the blood are common biochemical indicators for evaluating liver and kidney function. In order to further evaluate the safety of the nanoparticles we prepared on the liver and kidneys, these main indicators were measured in this embodiment, and it was found that the indicators of each treatment group were within the reference range, indicating that the nanoparticles used in this study will not cause liver and kidney damage (Table 3).

[0133] Table 4.1 ApoE in different treatment groups - / - Blood routine test of mice (n=3)

[0134]

[0135] Table 4.2 ApoE in different treatment groups - / - Main serum parameters of mice (n=3)

[0136]

[0137] During the drug administration period, we monitored the body weight of mice in different groups, such as Fig.29 As shown, we found that the administration did not cause eating disorders in mice. The obvious weight loss after the first injection may be due to stress response, but this loss is not caused by toxicity, because the weight of all four groups of mice including the PBS control group decreased. After the injection, the weight of the mice increased, which is the result of continuous high-fat feeding during the treatment period, indicating that the mice can continue to thrive after administration, and there will be no biological toxicity that causes weight loss.

[0138] Finally, it should be noted that the terms "comprises," "includes," or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0139] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended 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.

[0140] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A mesenchymal stem cell membrane-coated kaempferol biomimetic nanomedicine, characterized in that: The bionic nano drug consists of nano particles containing kaempferol and mesenchymal stem cell membranes coated on the outside thereof, wherein the mesenchymal stem cell membranes are derived from umbilical cord mesenchymal stem cells; the bionic nano drug has a particle size of 167.67±1.84nm, a PDI of 0.181±0.027, a potential of -5.41±0.74mV, an encapsulation rate of 79.97±0.19%, and an encapsulation rate of 7.27±0.02%.

2. The bionic nano drug according to claim 1, characterized in that: The nanoparticles containing kaempferol are prepared by combining kaempferol and polylactic acid-glycolic acid copolymer through coprecipitation and emulsification.

3. The bionic nano drug according to claim 2, characterized in that: The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 1:

1.

4. The bionic nano drug according to claim 2, characterized in that: The mass ratio of the kaempferol to the polylactic acid-glycolic acid copolymer is 1:

10.

5. The method for preparing the bionic nano drug according to any one of claims 1 to 4, characterized in that: The steps include: (1) preparing nanoparticles KP containing kaempferol by combining kaempferol and poly(lactic acid-co-glycolic acid) copolymer; (2) extracting mesenchymal stem cell membranes from umbilical cord mesenchymal stem cells; (3) The extracted mesenchymal stem cell membrane and the aqueous solution of nanoparticles containing kaempferol were mixed by ultrasonication for 3 min in an ice bath, and then squeezed back and forth through a polycarbonate porous membrane by a liposome co-extruder for dozens of times to obtain the bionic nanomedicine KPM.

6. The preparation method according to claim 5, characterized in that: In the step (1), the preparation process of the nanoparticles KP containing kaempferol is as follows: a. The polyvinyl alcohol powder was prepared into a 1% concentration liquid using double distilled water, and then subjected to a gradient water bath to fully denature it and then heat preservation, and after cooling, stirred at a low speed to obtain a liquid A for standby use; b. Weigh polylactic acid-glycolic acid copolymer and dissolve it in DMSO, then add kaempferol, mix well and ultrasonicate for 3 minutes to obtain solution B for later use; c. Add solution B dropwise into solution A, stir at a constant speed for 1 hour, transfer to a dialysis bag, and dialyze overnight with pure water in the dark for 12 hours. The collected liquid is KP.

7. The preparation method according to claim 5, characterized in that: In the step (2), the extraction process of mesenchymal stem cell membrane is: a. Take P2 umbilical cord mesenchymal stem cells, melt them in a 37℃ water bath, 1500r / min, 5min, discard the supernatant, mix with αMEM containing 10% FBS, place in a T25 bottle, and culture at 37℃, 5% CO2, in a cell culture incubator for 24h. Passage the cells when they fill 80% to 90% of the cell bottle; b. When the cells grow to 90%, discard the culture medium, wash three times with pre-cooled PBS, discard the PBS and add trypsin digestion solution. After digestion, add PBS and blow into a 4℃ pre-cooled centrifuge, centrifuge at 1500rpm / min for 5min, take 5 dishes of cell pellets, add 990μL lysis solution and 10μLPMSSF, lyse on ice for 15min, use liquid nitrogen, 37℃ water bath, alternately freeze and thaw 5 times, centrifuge at 700g at 4℃ for 10min, take the supernatant, put the supernatant into a new EP tube, centrifuge at 12000g at 4℃ for 30min, the resulting precipitate is the mesenchymal stem cell membrane.

8. The preparation method according to claim 5, characterized in that: In the step (3), 1 mg of KP was added based on the membrane extracted from 5 dishes of mesenchymal stem cells.

9. The preparation method according to claim 8, characterized in that: In the step (3), the polycarbonate porous membrane is provided with two layers, and the pore sizes of the two layers of polycarbonate porous membrane are 400 nm and 200 nm respectively.

10. Use of the bionic nano drug according to any one of claims 1 to 4 in the preparation of a drug for treating atherosclerosis.

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