A mesenchymal stem cell membrane-coated kaempferol biomimetic nanomedicine, its preparation method and application

Abstract

This invention discloses a biomimetic nanomedicine containing kaempferol (Kaempferol) coated with a mesenchymal stem cell membrane, its preparation method, and its application, relating to the field of targeted drug preparation technology. The biomimetic nanomedicine consists of nanoparticles containing kaempferol and an external mesenchymal stem cell membrane, the mesenchymal stem cell membrane being derived from umbilical cord mesenchymal stem cells. The biomimetic nanomedicine has a particle size of 167.67±1.84 nm, a PDI of 0.181±0.027, a potential of -5.41±0.74 mV, an encapsulation efficiency of 79.97±0.19%, and an encapsulation loading rate of 7.27±0.02%. This invention not only enhances the water solubility of kaempferol but also specifically enriches and enhances it at AS lesion sites, improving the utilization efficiency of Kaempferol (Kae) and overcoming the shortcomings of small molecule drugs. Furthermore, the nano-sizing of Kae reduces direct contact between Kae and cells / tissues, thus reducing cytotoxicity.

Description

Technical Field

[0001] This invention relates to the field of targeted drug preparation technology, specifically to a mesenchymal stem cell membrane-coated kaempferol biomimetic nanomedicine, its preparation method, and its application. Background Technology

[0002] Atherosclerotic cardiovascular disease and its complications are prevalent worldwide. The development of atherosclerosis (AS) weakens the elasticity of the arterial walls, causes intimal hyperplasia, and leads to plaque formation. These plaques can rupture, causing thrombosis and resulting in severe vascular obstruction. Prevention of AS primarily involves dietary control; while clinical treatment mainly uses statins and antiplatelet drugs, but these have a first-pass effect and are not effective in treating AS.

[0003] Yixintong is a commonly used traditional Chinese medicine for the treatment and prevention of AS. It has the effects of promoting blood circulation, removing blood stasis, and clearing the heart vessels. It can be used to treat chest pain caused by blood stasis obstructing the vessels, with symptoms such as chest tightness, shortness of breath, stabbing pain in the precordial region, palpitations, forgetfulness, dizziness, and tinnitus. It can also be used for coronary heart disease angina pectoris, hyperlipidemia, and cerebral artery insufficiency with the above symptoms. Its prescription is made from flavonoids extracted from hawthorn leaves. The total flavonoids in hawthorn leaves include kaempferol, rhamnoside, hyperoside, and quercetin. Clinical and pharmacological experiments have shown that the flavonoids contained in hawthorn leaves have various pharmacological activities on the cardiovascular system, mainly manifested in the following ways: ① By activating peroxidase proliferator-activated receptor α, it reduces blood triglyceride and low-density lipoprotein levels, increases high-density lipoprotein levels, regulates inflammatory responses, and simultaneously increases the expression of liver X receptor and adenosine triphosphate binding cassette transporter A1, promoting reverse transport of total cholesterol, reducing total cholesterol load, thereby preventing the occurrence of atherosclerosis; ② By increasing NO concentration, scavenging free radicals, inhibiting lipid peroxidation damage, and stabilizing cell membranes, it alleviates and reduces myocardial ischemia, protecting ischemic and hypoxic myocardial cells; ③ By reducing blood viscosity, inhibiting platelet aggregation, accelerating blood flow velocity, reducing thrombus formation, and simultaneously reducing gelatinase B expression, it reduces the degradation of vascular basement membrane and extracellular matrix proteins, reduces blood-brain barrier permeability, reduces cerebral edema, reduces cerebral infarction area, further reduces brain tissue damage, and exerts a neuroprotective effect; ④ By adjusting cardiac contractility and heart rate, it maintains stable blood pressure and improves coronary blood circulation.

[0004] In addition, kaempferol (Kae), as an effective monomeric component of total flavonoids from hawthorn leaves, has anti-inflammatory and antioxidant effects and has attracted much attention in the clinical treatment of AS. However, as a flavonoid compound, this drug has extremely low water solubility, making it difficult to achieve an effective concentration at the lesion site, thus limiting its application. Summary of the Invention

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

[0006] The technical solution adopted in this invention is as follows:

[0007] A biomimetic nanomedicine containing kaempferol and coated with a mesenchymal stem cell membrane is disclosed. The biomimetic nanomedicine comprises nanoparticles containing kaempferol and an external mesenchymal stem cell membrane derived from umbilical cord mesenchymal stem cells. The biomimetic nanomedicine has a particle size of 167.67±1.84 nm, a PDI of 0.181±0.027, a potential of -5.41±0.74 mV, an encapsulation efficiency of 79.97±0.19%, and a loading rate of 7.27±0.02%.

[0008] Preferably, the nanoparticles containing kaempferol are prepared by a combination of co-precipitation and emulsification of kaempferol and polylactic acid-glycolic acid copolymer.

[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 biomimetic nanomedicine includes the following steps:

[0012] (1) Kaempferol was combined with polylactic acid-glycolic acid copolymer to prepare nanoparticles KP containing kaempferol;

[0013] (2) Extract the mesenchymal stem cell membrane from umbilical cord mesenchymal stem cells;

[0014] (3) The extracted mesenchymal stem cell membrane and the aqueous solution of nanoparticles containing kaempferol were ultrasonically mixed for 3 minutes under ice bath conditions. Then, the biomimetic nanodrug KPM was obtained by extruding it back and forth dozens of times through a polycarbonate porous membrane using a liposome co-extruder.

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

[0016] a. Prepare a 1% concentration liquid by using double-distilled water to prepare polyvinyl alcohol powder, then perform a gradient water bath to fully denature it, keep it at the temperature, cool it down and stir it at low speed to obtain liquid A for later use.

[0017] b. Weigh out the polylactic acid-glycolic acid copolymer and dissolve it thoroughly with DMSO. Then add kaempferol, mix well, and sonicate for 3 minutes to obtain solution B for later use.

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

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

[0020] a. Take P2 generation umbilical cord mesenchymal stem cells, thaw them in a 37℃ water bath, 1500r / min, 5min, discard the supernatant, mix with αMEM containing 10% FBS, place in a T25 flask, culture at 37℃, 5% CO2 in a cell culture incubator, stabilize for 24h, and passage when the cells have filled 80%-90% of the cell flask.

[0021] b. Once the cells have grown to 90%, discard the culture medium, wash three times with pre-cooled PBS, discard the PBS, add trypsin digestion solution, digest, add PBS, pipette and place in a pre-cooled centrifuge at 4°C, centrifuge at 1500 rpm / min for 5 min, take 5 plates of cell pellet, add 990 μL of lysis buffer and 10 μL of LPMSF, lyse on ice for 15 min, use liquid nitrogen, water bath at 37°C, repeat freeze-thaw cycle 5 times, centrifuge at 700g speed at 4°C for 10 min, take the supernatant, place the supernatant in a new EP tube, centrifuge at 12000g speed at 4°C for 30 min, the resulting pellet is the mesenchymal stem cell membrane.

[0022] More preferably, in step (3), 1 mg of KP is fed into the membrane extracted from 5 dishes of mesenchymal stem cells.

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

[0024] The application of the aforementioned biomimetic nanomedicine in the preparation of drugs for treating atherosclerosis.

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

[0026] 1. This invention enhances the water solubility of kaempferol by coating the surface of nanoparticles containing kaempferol with a specific cell membrane, thereby increasing the specific enrichment and enhancement of kaempferol at the site of AS lesions, improving the utilization efficiency of kaempferol, and overcoming the shortcomings of small molecule drugs. Furthermore, the nano-sizing of kaempferol can effectively reduce the direct contact between cells and tissues, thereby reducing cytotoxicity.

[0027] 2. This invention broadens the application of biomimetic membranes in AS treatment by applying mesenchymal stem cell membranes to AS research, enabling immune escape and targeting of inflammatory endothelial cells; it allows the membrane to reach the lesion site in vivo, reducing lipid deposition in the aortic arch, stabilizing plaques, and lowering oxidative stress levels in the aorta, while exhibiting high biocompatibility.

[0028] 3. Through ingenious design, this invention enables biomimetic nanomedicines containing Kae to be better used in the treatment of AS, solving key challenges in the field and fully leveraging the role of Kae in the clinical treatment of AS, providing more options for effective treatment. Therefore, this invention has outstanding substantive features and significant progress, making it highly suitable for large-scale application. Attached Figure Description

[0029] Figure 1 The particle size distribution of KP / KPM prepared in Example 1;

[0030] Figure 2 The KP / KPM potential distribution map prepared in Example 1;

[0031] Figure 3 This is a standard curve of Kae concentration (horizontal axis) versus absorbance (vertical axis) in DMSO.

[0032] Figure 4 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 This is an SDS-PAGE gel image of the protein.

[0035] Figure 7 Detection graphs for characteristic proteins such as CD47, Integrinβ1, Cxcr4, and Anti-Na / KATPase by Western blotting;

[0036] Figure 8 This is a relative cell survival diagram of human umbilical vein endothelial cells after different drug treatments in Example 2;

[0037] Figure 9 This is a graph showing the relative cell survival of Raw 264.7 cells after different drug treatments in Example 2;

[0038] Figure 10 This is a diagram showing the relative cell survival of smooth muscle cells after different drug treatments in Example 2;

[0039] Figure 11This is a laser confocal image showing the uptake of nanoparticles by normal macrophages and activated macrophages in Example 2;

[0040] Figure 12 The following is a flow cytometry analysis of the uptake of uncoated / coated fluorescent nanoparticles by normal macrophages and activated macrophages in Example 2. A is a curve graph and B is a statistical bar graph.

[0041] Figure 13 This is a laser confocal image of the nanoparticles taken up by normal and inflamed endothelial cells in Example 2;

[0042] Figure 14 The following is a flow cytometry 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] Figure 15 The image shows the antioxidant assessment of endothelial cells in the oxidative stress model using KPM in Example 2. A represents the statistical graph of the effect of H2O2 concentration on the CCK8 assay in endothelial cells; B represents the statistical graph of MDA levels in endothelial cells of the oxidative stress model after different treatment groups; C represents the statistical graph of ROS levels in endothelial cells of the oxidative stress model after different treatment groups; and D represents the fluorescence graph of ROS levels in endothelial cells of the oxidative stress model after different treatment groups.

[0044] Figure 16 The hemolysis experiment and statistical chart in Example 2;

[0045] Figure 17 This is a line graph showing the relative fluorescence intensity of different nanoparticles circulating in vivo in Example 3;

[0046] Figure 18 The following are fluorescence images and statistical graphs of different nanoparticles in the aortic arch in Example 3: A is the imaging image, and B is the statistical bar chart.

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

[0048] Figure 20 ApoE in different treatment groups in Example 3 - / - Oil Red O staining image of the aortic arch in mice;

[0049] Figure 21 ApoE in different treatment groups in Example 3 - / - Oil Red O staining image of a section of the aortic arch root of a mouse (the upper and lower bars are 500 μm and 50 μm, respectively);

[0050] Figure 22 ApoE in different treatment groups in Example 3 - / -Masson staining image of the aortic arch root of a mouse (the upper and lower icons are 500 μm and 50 μm respectively);

[0051] Figure 23 ApoE in different treatment groups in Example 3 - / - Toluidine blue staining image of the aortic arch root of a mouse (the upper and lower icons are 500 μm and 50 μm respectively);

[0052] Figure 24 ApoE in different treatment groups in Example 3 - / - Immunohistochemical image of CD68 in mouse aortic arch (the upper and lower bars are 500μm and 50μm, respectively);

[0053] Figure 25 ApoE in different treatment groups in Example 3 - / - Immunohistochemical images of MMP9 in mice (the top and bottom bars are 500 μm and 50 μm, respectively);

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

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

[0056] Figure 28 ApoE in different treatment groups in Example 3 - / - HE staining images of major organs of a mouse (scale bar 100 μm);

[0057] Figure 29 ApoE in different treatment groups in Example 3 - / - A graph showing the change in body weight of mice. Detailed Implementation

[0058] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0059] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

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

[0061] The present application will now be described in detail 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 by preparing a 1% concentration liquid from PVA powder using double-distilled water, subjecting it to a gradient water bath to fully denature it, then keeping it warm, and after cooling, measuring 4 mL and placing it in a magnetic stirrer with a rotor for low-speed stirring, for later use.

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

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

[0068] ④ The collected fluid after 12 hours of dialysis is KP.

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

[0070] Umbilical cord mesenchymal stem cell resuscitation and culture: Umbilical cord mesenchymal stem cells were provided by Chongqing Honghui Stem Cell Platform. P2 passage cells were used. The cells were thawed in a 37℃ water bath at 1500 rpm for 5 min. The supernatant was discarded, and the cells were mixed with αMEM containing 10% FBS and placed in a T25 flask. The cells were cultured at 37℃ in a 5% CO2 cell culture incubator for 24 h to stabilize. When the cells reached 80%–90% confluence in the cell culture flask, they were passaged.

[0071] MSCM extraction: When the cells reach 90% cell growth, discard the culture medium, wash three times with pre-cooled PBS, discard the PBS, add trypsin digestion solution, digest, add PBS, pipette and place in a pre-cooled centrifuge at 4°C, centrifuge at 1500 rpm / min for 5 min, take 5 plates of cell pellet, add 990 μL of lysis buffer and 10 μL of PMSF, lyse on ice for 15 min, use liquid nitrogen, 37°C water bath, repeat freeze-thaw five times, centrifuge at 700g speed, 4°C for 10 min, take the supernatant, place the supernatant in a new EP tube, centrifuge at 12000g speed, 4°C for 30 min, the resulting clear precipitate is MSCM.

[0072] (3) Synthesis of KPM

[0073] The extracted MSCM (each unit is defined as the membrane extracted from 5 dishes of mesenchymal stem cells) was mixed with an aqueous solution of 1 mg KP nanoparticles in an ice bath and sonicated for 3 minutes. Then, using a liposome co-extruder, it was extruded back and forth dozens of times through 400 nm and 200 nm polycarbonate porous membranes to obtain KPM.

[0074] (4) Preparation of fluorescent nanoparticles

[0075] We used the same nanoparticle preparation process, but replaced Kae with DIO reagent purchased from Aladdin Reagents in Shanghai, to form fluorescent uncoated nanoparticles DP and fluorescent coated nanoparticles DPM. DP was used to simulate KP, while DPM represented KPM.

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

[0077] The particle size, potential, and PDI of KP and KPM nanoparticles in water were measured using a nanoparticle size potentiometer at room temperature, with three replicates for each experiment. The morphology of the nanoparticles was determined using transmission electron microscopy (TEM). A clean copper mesh was prepared, and appropriate concentrations of KP and KPM were added, covering the mesh and allowing it to air dry. After drying, phosphotungstic acid was added for negative staining for 5 minutes, and excess phosphotungstic acid was absorbed using filter paper. The sample was then placed under TEM for selective area imaging. The KP and KPM aqueous solutions were left at room temperature, and 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 As can be seen from the data, the particle size curves of KP and KPM are smooth with only one peak, and the software results indicate good uniformity in particle size. Table 1 shows that the particle size of KP is 147.50±1.28 nm, while after encapsulation with MSCM, the particle size increases to 167.67±1.84 nm. This increase in particle size indicates the success of the encapsulation process. Figure 2In the potential graph, the curves of KP and KPM are relatively flat and free of impurities, reflecting the uniformity of particle size. The potential of KP is -1.18±0.17mV, and that of KPM is -5.07±0.19mV. Compared to KP, the potential of KPM is negatively increased because the cell membrane is negatively charged in the aqueous phase. PDI is used to evaluate the dispersibility of nanoparticles, and it is generally considered to be optimal in the range of 0.1-0.2. In this example, the PDI of KP is 0.137±0.030, and that of KPM is 0.181±0.027, indicating that the KP and KPM prepared in this example have good dispersibility, are not prone to clumping or aggregation, and facilitate the smooth conduct of subsequent phagocytosis experiments.

[0079] Table 1. Results of particle size, potential, PDI, TEM, and encapsulation loading of nanomedicines.

[0080]

[0081] Kae exhibits a characteristic absorption peak at 365 nm in DMSO solution. We diluted Kae to concentrations of 1, 2.5, 5, 10, 20, and 25 μg / mL using DMSO, and measured the absorbance values ​​using a UV spectrophotometer to be 0.0665, 0.1742, 0.3448, 0.7051, 1.3663, and 1.7135, respectively. A standard curve was fitted with Kae concentration on the x-axis and absorbance value on the y-axis, as shown below. Figure 3 The equation is y = 0.0684x + 0.0045, R² = 0.9998. Kalene glycol (KP) has extremely low solubility in water and is usually dissolved using hot ethanol, alkali, or ether, but these methods are inconvenient in practical applications. We nano-sized KP using PLGA and PVA and found that 799.7 μg of 1 mg KP was encapsulated in the nanocarrier. The lyophilized sample was reconstituted with DMSO using a UV spectrophotometer, and the encapsulation efficiency was calculated to be 79.97 ± 0.19%, with a loading rate of 7.27 ± 0.02% (Table 1). This nano-sizing method is safer than direct dissolution with organic solvents for drug delivery.

[0082] 3. Morphological characterization of nanomedicines

[0083] To more intuitively verify whether MSCM is coated on the outside of KP, we used phosphoric acid counterstaining followed by TEM observation. Figure 4As shown, we found that KP particles are approximately 100 nm in size and are spherical. KPM has the same shape as KP but is coated with an outer film and is larger than KP. Because the samples underwent air-drying and dehydration during preparation, the particle size measured by TEM differs from that measured by DLS. Furthermore, the MSCM thickness measured by TEM is approximately 10 nm, indicating that the MSCM successfully coated KP.

[0084] 4. Stability evaluation of nanomedicines

[0085] To determine the storage stability of the nanoparticles, we measured the particle size of the nanoparticles after standing in an aqueous medium for 12, 24, 36, 48, 60, and 72 hours. We found that KP and KPM maintained a certain level of stability within 72 hours, with no significant fluctuations in particle size. This indicates that the binding of Kae to the support is relatively stable, and that MSCM-modified KP exhibits a certain degree of stability.

[0086] 5. Drug release curves of nanomedicines

[0087] To investigate the drug release behavior of KP and KPM, we mixed nanoparticles with PBS and conducted a release experiment 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 for drug content determination and calculation of the cumulative drug release rate. Figure 5 It can be seen that both KP and KPM can slow down the sudden release of the drug. Moreover, the release rate of KPM is slower than that of KP, indicating that the modification of MSCM can slow down the release of the drug.

[0088] 6. Protein characterization of KPM

[0089] Membrane proteins play a crucial role in various cellular biological activities, possessing multiple functions such as information transduction and target binding. First, we used Western blotting (WB) with Coomassie brilliant blue staining to observe the expression of total proteins. The MSCM protein expression profile was similar to that of KPM, indicating that KPM effectively inherited the proteins from MSCM. Ultrasound and the use of microliposome extruders for nano-liposome processing did not significantly cause loss of the main proteins in MSCM. Figure 6 ).

[0090] To confirm whether KPM inherits proteins from MSCM, we characterized CD47, which inhibits phagocytosis and promotes circulation, as well as Integrinβ1, which targets inflammatory endothelium, and the membrane protein internal control Anti-Na / KATPase. In addition, Cxcr4 is expressed in mesenchymal cells and can bind to Cxcl12, which is overexpressed in ischemia and inflammation. To investigate whether MSCM and KPM retain Cxcr4, we also characterized the Cxcr4 protein. The characteristic protein characterization results are shown in Figure 7. KPM retains a certain amount of CD47, Integrinβ1, and Cxcr4 from MSCM. This lays the foundation for subsequent in vitro immune escape and targeting, and in vivo circulation and targeting effects.

[0091] Example 2

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

[0093] 1. Cytotoxicity evaluation experiment

[0094] To evaluate the safety of nanomaterials, we used untreated Kae cells as a control group and co-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 was no significant difference in cell survival between the drug-treated groups (KP and KPM groups) and the control group (Kae group), the drug was generally considered non-toxic. Regarding the relative survival of the three cell groups, a significant difference in survival was observed between the treated and control groups at a concentration of 20 μg / mL, while no cytotoxicity was observed at 10 μg / mL. Therefore, in subsequent experiments, the concentration of nanoparticles used was 10 μg / mL. Figure 8 and Figure 9 It can be seen that, at the same Kae equivalent, KPM is less toxic than KP, while KP is less toxic than pure Kae. This indicates that the synthesized KP and KPM can reduce the toxicity to human umbilical vein endothelial cells and Raw 264.7 cells, which may be due to the sustained-release effect of KP and KPM. Meanwhile... Figure 10 Within the concentration range of 1-20 μg / mL, at the same Kae equivalent, pure Kae showed lower toxicity than KP and KPM, possibly because VSMCs were not sensitive enough to the drug, while PLGA and MSCM could affect VSMCs. However, as the concentration increased to above 20 μg / mL, smooth muscle cells became more sensitive to Kae, and KP and KPM played a protective role. Through toxicity tests on three cell types, it was confirmed that nanoparticles can reduce drug toxicity, and the concentration of nanoparticles used in subsequent experiments was determined.

[0095] 2. Evaluation experiments on immune escape and inflammatory cell targeting

[0096] Macrophage activation was induced using LPS. Then, DP and DPM were added and co-incubated for 2 hours. Figure 11 It can be seen that macrophages absorb more DP without MSCM coating, while the uptake of DPM by macrophages is weakened after being coated with MSCM. When macrophages undergo polarization transformation and their phagocytic capacity is enhanced, although the amount of DPM phagocytosis by activated macrophages is increased compared with that of normal, unactivated macrophages, the intensity of the enhancement is far lower than the amount of DP uptake by activated macrophages. Figure 12 The flow cytometry results also demonstrated that MSCM modification of nanoparticles can indeed inhibit macrophage capture, thereby enabling immune escape.

[0097] Fluorescent nanoparticles were co-incubated with normal and inflamed endothelium, and then photographed and observed using a fluorescence confocal microscope. Figure 13 ), and analysis using flow cytometry ( Figure 14 The results showed that both normal and inflamed endothelial cells phagocytosed more DPM nanoparticles than uncoated DP nanoparticles, indicating that the biomimetic effect of the coated nanoparticles influenced phagocytosis. Comparing the phagocytosis results of the two types of nanoparticles by inflamed and normal endothelial cells revealed that inflamed endothelial cells took up more DP than normal endothelial cells, while inflamed endothelial cells took up significantly more DPM than normal endothelial cells. The overall uptake ranking was ADPM > ADP > DPM > DP. (ADPM represents the group of endothelial cells phagocytosing DPM after activation, and ADP represents the group of endothelial cells phagocytosing DP after activation). This demonstrates that the coated nanoparticles can indeed target inflamed endothelial cells.

[0098] 3. Antioxidant stress evaluation experiment

[0099] Oxidative stress in endothelial cells plays a crucial role in ankylosing spondylitis (AS). Endothelial stress leads to excessive production of reactive oxygen species (ROS), which directly damage cells, resulting in impaired endothelial diastolic function. Malondialdehyde (MDA) is an important biomarker of oxidative stress. Lipid metabolism disorder is a significant cause in AS, and MDA represents the degree of lipid peroxidation. Therefore, we investigated the effects of KPM treatment on MDA and ROS in endothelial cells. We used H2O2 pretreatment of endothelial cells to construct an oxidative stress model of endothelial cells. Figure 15 The results from study A showed that a H₂O₂ concentration of 0.4 mM significantly affected endothelial cell activity. Therefore, we used 0.2 mM as the subsequent induction concentration to maximize its effect without affecting activity. Figure 15The results from B to D indicate that KPM significantly reduced the levels of MDA and ROS in endothelial cells. Furthermore, we observed that KPM reduced the levels of MDA and ROS in endothelial cells more effectively than KPM alone, which may indirectly reflect KPM's ability to promote phagocytosis and thus better alleviate oxidative damage to endothelial cells.

[0100] 4. Hemolysis test

[0101] Hemolysis test is an important indicator for verifying the toxicity of materials.

[0102] The hemolytic test procedure is as follows:

[0103] ① After anesthetizing the mice, blood was collected from the orbital cavity and anticoagulated with EDTA-2K (10X) at a 1:9 ratio;

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

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

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

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

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

[0109] In national standards, a hemolysis rate of less than 5% is generally considered non-toxic. For example... Figure 16 The hemolysis test results showed that the hemolysis rates of KP and KPM were both less than 5%, indicating that KP and KPM have good blood compatibility, which provides a safe basis for subsequent tail vein injection of the drug.

[0110] Example 3

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

[0112] 1. Establishment of an animal model of atherosclerosis

[0113] ApoE - / - Male mice were purchased from Jicui Pharmaceutical Co., Ltd. and fed a normal diet for one week, followed by a high-fat diet for 10 weeks to establish an atherosclerosis model.

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

[0115] To investigate whether MSCM also has immune evasion capabilities in vivo, we injected mice with fluorescent nanoparticles (DP, DPM) via the tail vein and measured the fluorescence intensity in the blood at different time points after injection. Figure 17 Measurement results showed that both fluorescent nanoparticles exhibited peak fluorescence intensity at 1 hour, with DPM showing a significantly higher fluorescence intensity than DP, indicating that DP decayed rapidly in vivo, likely due to metabolism. Over time, the fluorescence intensity of both DP and DPM decreased, with DP decreasing faster than DPM, reaching near-zero levels at 12 hours, suggesting DP was cleared. DPM fluorescence intensity decreased more slowly, only approaching normal blood levels at 72 hours. This indicates that DPM has a longer circulation time than DP, demonstrating that MSCM can prolong the blood circulation time of nanoparticles in vivo.

[0116] 3. Targeting study of KPM in mice

[0117] To verify that MSCM targets the aortic arch in vivo, we injected different fluorescent nanoparticles into the tail vein. After a certain period of time, we dissected, sampled, and photographed the small animals. We found that the aortic arch of the DPM group had significant fluorescence accumulation, while the DP group had less accumulation. Figure 18 We also performed statistical analysis on the overall fluorescence intensity and found that the fluorescence accumulation of DPM in blood vessels was significantly stronger than that of the DP group. This indicates that MSCM nanoparticles can target the aorta in vivo, providing an experimental basis for subsequent treatment of aortic AS plaques. Furthermore, we collected samples from the major organs of ApoE- / - mice injected with fluorescent nanoparticles and performed fluorescence imaging to observe the destination of the fluorescent nanoparticles. We found that the DP group mainly received samples from the liver and kidneys, while the DPM group only had a small amount of samples found in the liver. Figure 19 This indicates that, compared to DP, DPM is less easily metabolized by organs.

[0118] 4. KPM in lipid deposition in the mouse aorta

[0119] A key characteristic of aortic atherosclerosis (AS) is lipid deposition in the aorta, and the degree of lipid deposition reflects the severity of plaque inflammation. After the treatment cycle, we stained sections of the aortic arch and its root with Oil Red O. From... Figure 20 The Oil Red O staining image of the aortic arch and quantitative analysis showed that lipid deposition in the aorta decreased after KPM treatment. This was in contrast to ApoE. - / -In the Kae treatment group, lipid deposition in the aorta was only slightly reduced, while KP treatment resulted in a certain reduction in lipid deposition in the aortic arch. This is because KP has an EPR effect, which can achieve a certain degree of retention. KPM treatment showed the best therapeutic effect, with a significant reduction in lipid deposition in the aortic arch, as well as in the abdominal aorta and iliac arteries. This indicates that MSCM does indeed enable the nanoparticles to have a targeting effect, and at the same time, it prolongs the circulation time, allowing for greater release and absorption of Kae at the plaque site.

[0120] In such Figure 21 The Oil Red O staining image and statistical graph of the aortic arch root section shown reveal that, compared to wild-type mice, ApoE... - / - After being fed a high-fat diet, there was a large amount of lipid deposition at the root of the aortic arch. After different treatments, KPM showed a good effect in reducing lipid deposition. This is 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 ankylosing spondylitis (AS) plaques. Collagen fibers are the main component of the fibrous cap in AS plaques; therefore, the observation of collagen fibers is crucial for AS plaque stability. Masson staining is a primary method for observing collagen fibers. We used Masson staining to observe the collagen thickness in different treatment groups. Figure 22 This shows that in ApoE - / - In the group, the collagen fiber thickness was large. Treatment of the Kae free group, KP group and KPM group showed that the collagen fiber thickness was reduced. Among them, the KPM group had the best effect in reducing collagen thickness, indicating that KPM can effectively stabilize plaques by inhibiting the proliferation of collagen fibers.

[0123] Necrotic cores are a key characteristic of vulnerable plaques in ankylosing spondylitis (AS). The formation of necrotic cores results from macrophages phagocytizing lipids and transforming into foam cells, followed by necrosis and apoptosis of these foam cells. This reduces plaque stability and exacerbates the inflammatory response. Toluidine blue staining is a method for observing necrotic cores. We stained sections of the aortic arch root from mice in different treatment groups with toluidine blue. The results are shown in Figure 23. Wild-type mice showed almost no necrotic cores, while ApoE mice... - / - The mice developed large areas of necrotic cores. After different treatments, the area of ​​the necrotic core decreased. In the KPM group, we found that the decrease in collagen thickness was accompanied by a reduction in the necrotic core, indicating that KPM can indeed target plaque sites and contribute to plaque stability.

[0124] In ankylosing spondylitis (AS) plaques, macrophages engulf lipids and form foam cells within the AS environment. The development and formation of foam cells negatively impact plaque stability, and they 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, and the results are as follows: Figure 24 As shown, ApoE - / - The mice had a high number of macrophages. Compared with the Kae treatment group and the KP treatment group, the relative number of macrophages was significantly reduced after KPM treatment, which is beneficial to the stability of the plaque.

[0125] In AS plaques, macrophages secrete matrix metalloproteinase-9 (MMP9), a marker of plaque instability. MMP9 can break down extracellular matrix components that maintain plaque stability, thus affecting AS plaques. We performed MMP9 immunohistochemical staining on mouse sections from each group, and the results are as follows: Figure 25 As shown, KPM significantly reduced the relative expression level of MMP9.

[0126] 6. Antioxidant performance experiment of mice after KPM treatment

[0127] Nrf2 / GPX4 plays a crucial role in oxidative stress in AS. Nrf2 is responsible for intracellular antioxidant responses and can enhance cellular antioxidant capacity by activating the expression of various antioxidant enzymes. GPX4 is an important antioxidant enzyme that can reduce lipid peroxides, thereby protecting cells from lipid peroxidation damage. Normally, the Nrf2 / GPX4 pathway is inhibited in AS. To assess the antioxidant performance of nanoparticles at the tissue level, we performed Nrf2 / GPX4 immunohistochemical staining on sections of mice from each group. The results are shown below. Figure 26 , Figure 27 As shown, in ApoE - / - In the study, Nrf2 / GPX4 expression decreased, but the Kae drug group did not significantly improve Nrf2 / GPX4 expression. This is because although Kae can effectively alleviate oxidative stress, its free nature prevents it from reaching the target site effectively for antioxidant effects. Nanoparticle-based KPM showed increased Nrf2 / GPX4 expression at the aortic arch root compared to the Kae group. This is because nanoparticle-based KPM achieves the EPR effect, allowing some nanoparticles to accumulate at the aortic arch root, thus achieving a therapeutic effect through sustained drug release. The results also showed that the KPM group significantly improved Nrf2 / GPX4 expression. This is because KPM encapsulates MSCM on top of Kae nanoparticles, providing conditions for efficient targeting of plaque sites by nanoparticles, improving the effective delivery of Kae, and achieving therapeutic effects.

[0128] We tested the MDA levels in the blood of mice from different groups and measured the tissue-level MDA content in the aortic arch homogenate. Our results showed that KPM significantly reduced the MDA content in both the blood and the aortic arch. In terms of overall treatment efficacy, KPM > KP > Kae.

[0129] 7. Safety assessment experiment of KPM treatment in mice after treatment

[0130] Histological pathological sections can accurately determine lesions, and HE staining is a commonly used pathological staining method. It can clearly show whether lesions have occurred in the tissue. To assess the safety of nanoparticles in vivo, we performed sectioning and staining of major organs in mice of each treatment group. Through methods such as... Figure 28 The HE staining results shown indicate that the organs of the mice in all groups were normal, with no severe lesions such as fibrosis or necrosis. This demonstrates that the nanoparticles prepared in this study are organ-friendly and will not cause organ damage.

[0131] After one month of injection treatment in mice, we assessed four lipid parameters, including CHO (total cholesterol), TG (triglycerides), LDL (low-density lipoprotein cholesterol), and HDL (high-density lipoprotein cholesterol). Blood lipid balance plays a role in individual homeostasis. After one month of treatment, we found no significant differences between the groups of mice that received continuous injections for one month and the control group, indicating that nanoparticles do not significantly affect blood lipid homeostasis.

[0132] Complete blood count (CBC) can reveal inflammatory factors and the body's functional status. We collected blood samples from mice one month after treatment and analyzed four CBC parameters: WBC (white blood cells), RBC (red blood cells), HG (hemoglobin), and PLT (platelets). As shown in Table 2, all four CBC parameters 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 used to assess liver and kidney function. To further evaluate the safety of our prepared nanoparticles for the liver and kidneys, this example measured these key indicators. We found that the indicators in all treatment groups were within the reference range, indicating that the nanoparticles used in this study do not cause liver or kidney damage (Table 3).

[0133] Table 4.1 ApoE in different treatment groups - / - Complete blood count in mice (n=3)

[0134]

[0135] Table 4.2 ApoE in different treatment groups - / - Major serum markers in mice (n=3)

[0136]

[0137] During the administration period, we monitored the body weight of mice in different groups, such as Figure 29 As shown, we found that the administration did not cause feeding disorders in mice. The significant weight loss after the first injection was likely due to a stress response, but this loss was not caused by toxicity, as all four groups of mice, including the PBS control group, experienced weight loss. Following the injection, the mice's weight increased, a result of the continuous high-fat diet during treatment, indicating that the mice continued to grow healthily after administration and that it did not cause biotoxicity leading to weight loss.

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

[0139] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0140] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A mesenchymal stem cell membrane-coated kaempferol biomimetic nanodrug, characterized in that, The biomimetic nanomedicine consists of nanoparticles containing kaempferol and an externally coated mesenchymal stem cell membrane, wherein the mesenchymal stem cell membrane is derived from umbilical cord mesenchymal stem cells; the biomimetic nanomedicine has a particle size of 167.67±1.84 nm, a PDI of 0.181±0.027, a potential of -5.41±0.74 mV, an encapsulation efficiency of 79.97±0.19%, and an encapsulation loading of 7.27±0.02%; the nanoparticles containing kaempferol are prepared by a combination of co-precipitation and emulsification of kaempferol and polylactic acid-glycolic acid copolymer, wherein the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 1:1, and the mass ratio of kaempferol to polylactic acid-glycolic acid copolymer is 1:

10.

2. The method for preparing mesenchymal stem cell membrane-coated kaempferol biomimetic nanomedicine as described in claim 1, characterized in that, Includes the following steps: (1) Kaempferol was prepared into nanoparticles KP containing kaempferol by copolymerizing kaempferol with polylactic acid-glycolic acid; (2) Extract the mesenchymal stem cell membrane from umbilical cord mesenchymal stem cells; (3) The extracted mesenchymal stem cell membrane and the aqueous solution of nanoparticles containing kaempferol were ultrasonically mixed for 3 minutes under ice bath conditions. Then, the biomimetic nanodrug KPM was obtained by extruding the membrane back and forth dozens of times through a liposome co-extruder.

3. The preparation method according to claim 2, characterized in that, In step (1), the preparation process of the nanoparticles KP containing kaempferol is as follows: a. Prepare a 1% concentration liquid by mixing polyvinyl alcohol powder with double-distilled water, then perform a gradient water bath to fully denature it, keep it at the temperature, cool it down and stir it at low speed to obtain liquid A for later use. b. Weigh out the polylactic acid-glycolic acid copolymer and dissolve it thoroughly with DMSO, then add kaempferol, mix well and sonicate for 3 minutes to obtain solution B for later use; c. Add solution B to 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.

4. The preparation method according to claim 2, characterized in that, In step (2), the extraction process of the mesenchymal stem cell membrane is as follows: a. Take P2 generation umbilical cord mesenchymal stem cells, thaw them in a 37 ℃ water bath, 1500 r / min, 5 min, discard the supernatant, mix with αMEM containing 10% FBS, place in a T25 flask, culture at 37 ℃, 5% CO2 in a cell culture incubator, stabilize for 24 h, and passage when the cells have filled 80%~90% of the cell flask; b. Once the cells have grown to 90%, discard the culture medium, wash three times with pre-cooled PBS, discard the PBS, add trypsin digestion solution, digest, add PBS, pipette and place in a pre-cooled centrifuge at 4 ℃, centrifuge at 1500 rpm / min for 5 min, take 5 plates of cell pellet, add 990 μL of lysis buffer and 10 μL of PMSF, lyse on ice for 15 min, use liquid nitrogen, water bath at 37 ℃, repeat freeze-thaw five times, centrifuge at 700 g at 4 ℃ for 10 min, take the supernatant, place the supernatant in a new EP tube, centrifuge at 12000 g at 4 ℃ for 30 min, the resulting pellet is the mesenchymal stem cell membrane.

5. The preparation method according to claim 2, characterized in that, In step (3), 1 mg of KP was fed into the membrane extracted from 5 dishes of mesenchymal stem cells.

6. The preparation method according to claim 5, characterized in that, In step (3), the polycarbonate porous membrane has two layers, and the pore sizes of the two polycarbonate porous membranes are 400 nm and 200 nm, respectively.

7. The application of the biomimetic nanomedicine as described in claim 1 in the preparation of drugs for treating atherosclerosis.

Images