A macrophage membrane-coated quercetin cerium coordination biomimetic nanomaterial, its preparation method and application

Abstract

The present invention discloses a quercetin cerium coordination biomimetic nanomaterial encapsulated with macrophage cell membrane, its preparation method and application, which relates to the technical field of nanomaterials. The preparation method includes the following steps: adding a quercetin solution to a Ce(NO3)4 solution, and then adding a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution. After mixing and reacting, quercetin cerium coordination nanoparticles are obtained; the quercetin cerium coordination nanoparticles and macrophage cell membrane are uniformly mixed in a PBS buffer solution, and are extruded with a liposome extruder to obtain the quercetin cerium coordination biomimetic nanomaterial encapsulated with macrophage cell membrane. The quercetin cerium coordination biomimetic nanomaterial encapsulated with macrophage cell membrane prepared by the present invention has excellent antioxidant activity, cellular reactive oxygen species scavenging ability, cell protection ability, ability to regulate the expression of inflammatory factors, biosafety and in vivo colitis treatment ability.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and particularly to a macrophage cell membrane-coated quercetin cerium coordination bionic nanomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Ulcerative colitis is a chronic inflammatory disease that affects the colon, and its global incidence is on the rise. The main features of this disease are recurrent and remitting inflammation of the colonic mucosa, usually starting from the rectum and gradually extending proximally to the colon. The etiology of ulcerative colitis is complex and involves multiple factors, including autoimmune responses, infections, genetic factors, and neuropsychiatric factors, etc.

[0003] As a lifelong disease, ulcerative colitis cannot be completely cured at present. The main goal of treatment is to control symptoms by suppressing the inflammatory response and regulating immune function. Current clinical therapies mainly include drug treatment and surgery. Although there are various treatment options available, the existing treatment methods still have limitations:

[0004] Drug treatment: Traditional drug treatment usually focuses on a single pathway, with limited efficacy and difficulty in achieving long-term disease remission.

[0005] Monoclonal antibodies: Although these drugs are effective in some cases, they are easily recognized by the body as foreign substances, triggering immune responses and even potentially leading to acquired immunodeficiency.

[0006] Targeted small molecule drugs: These drugs may cause adverse reactions such as diarrhea, nausea, and fatigue during treatment, further affecting the quality of life of patients.

[0007] Therefore, in the face of this complex medical problem of ulcerative colitis, there is an urgent need to develop a new anti-inflammatory treatment strategy to improve the efficacy and reduce side effects, so as to bring more lasting disease remission and better quality of life to patients. Summary of the Invention

[0008] The purpose of the present invention is to provide a macrophage cell membrane-coated quercetin cerium coordination bionic nanomaterial, a preparation method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art. The macrophage cell membrane-coated quercetin cerium coordination bionic nanomaterial prepared by the present invention has excellent antioxidant activity, cellular reactive oxygen species scavenging ability, cell protection ability, ability to regulate the expression of inflammatory factors, biosafety, and ability to treat colitis in vivo.

[0009] To achieve the above purpose, the present invention provides the following scheme:

[0010] The present invention provides a preparation method of a macrophage cell membrane-coated quercetin cerium coordination bionic nanomaterial, comprising the following steps:

[0011] Add the quercetin solution to the Ce(NO3)4 solution, then add 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution. After mixing and reacting, quercetin cerium coordination nanoparticles are obtained.

[0012] Uniformly mix the quercetin cerium coordination nanoparticles with macrophage cell membranes in PBS buffer solution, and extrude them with a liposome extruder to obtain the quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membranes.

[0013] Furthermore, the concentration of the quercetin solution is 0.76 mg / mL, and the concentration of the Ce(NO3)4 solution is 0.78 mg / mL.

[0014] Furthermore, the volume ratio of the quercetin solution, the Ce(NO3)4 solution and the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution is 1:5:1.

[0015] Furthermore, the temperature of the mixing reaction is 25 °C and the time is 75 min.

[0016] Furthermore, the mass ratio of the quercetin cerium coordination nanoparticles to the macrophage cell membranes is 3 mg:0.75 mg.

[0017] Furthermore, the macrophage cell membranes are obtained by the method of ultrasonic disruption.

[0018] The present invention also provides a quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membranes prepared by the above preparation method.

[0019] The present invention also provides the application of the above quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membranes in the preparation of drugs for treating ulcerative colitis.

[0020] The present invention also provides a drug for treating ulcerative colitis, and the active ingredient includes the above quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membranes.

[0021] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0022] The present invention discloses the following technical effects:

[0023] The quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membrane developed in the present invention has simple preparation steps, mild conditions, easy operation, low-cost and non-toxic raw material sources. The quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membrane has good anti-inflammatory activity, can effectively scavenge excessive reactive oxygen species in the inflammatory microenvironment, reduce oxidative stress, and the macrophage cell membrane surface has inflammatory factor recognition receptors, with good targeting to the inflammatory site, enabling the nanomaterial to effectively accumulate at the inflammatory site. It has been confirmed by biological experiments that the quercetin cerium coordination biomimetic nanomaterial encapsulated by macrophage cell membrane can be used for the treatment of ulcerative colitis, and its cell membrane structure can also assist the nanomaterial to escape from the immune system, with good biological safety, and is an ideal carrier for drug delivery and sustained release. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is a schematic diagram of the synthesis process of the quercetin cerium coordination biomimetic nanomaterial (Ce-Que@MΦ);

[0026] Figure 2 It is the scanning electron microscope (SEM) images of Ce-Que and Ce-Que@MΦ. Among them, A and B are the SEM images of Ce-Que, and C and D are the SEM images of Ce-Que@MΦ;

[0027] Figure 3 It is the Fourier transform infrared spectroscopy (FT-IR) images of cerium nitrate, quercetin, Ce-Que and Ce-Que@MΦ;

[0028] Figure 4 It is the Zeta ultraviolet-visible spectroscopy (UV-Vis) images of cerium nitrate, quercetin, Ce-Que and Ce-Que@MΦ;

[0029] Figure 5 It is the Zeta potential images of cerium nitrate, quercetin, Ce-Que and Ce-Que@MΦ;

[0030] Figure 6 It is a comparison chart of the scavenging ability of cerium nitrate, quercetin, Ce-Que and Ce-Que@MΦ with the same concentration on different oxidative free radicals; among them, A is the comparison chart of the scavenging ability of ·OH; B is the comparison chart of the scavenging ability of ABTS· + scavenging ability comparison chart;

[0031] Figure 7 Superoxide dismutase (SOD) activity comparison chart of cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ at the same concentration;

[0032] Figure 8 Immunofluorescence staining map of reactive oxygen species in macrophages treated with cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ;

[0033] Figure 9 Detection result map of the survival of macrophages treated with cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ and then lipopolysaccharide (LPS) was added; where A is the immunofluorescence staining image of AO, and B is the fluorescence quantification map;

[0034] Figure 10 Detection result map of the mitochondrial membrane potential of macrophages treated with cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ and then lipopolysaccharide was added;

[0035] Figure 11 Scratch migration map (A) and quantification result map (B) of scratch migration rate of NIH-3T3 cells treated with cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ;

[0036] Figure 12 Statistical chart of in vitro hemolysis rate of cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ;

[0037] Figure 13 Detection result map of the levels of ten biochemical indicators in the blood of mice under the action of Ce-Que@MΦ nanomaterials; among them, A is the detection results of total serum protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), glucose (GLU), creatinine (Crea), and urea nitrogen (UREA); B is the detection results of urine creatinine ratio (U / C), lactate dehydrogenase (LDH), and creatine kinase (CK);

[0038] Figure 14 Hematoxylin-eosin (H&E) staining images of the heart, liver, spleen, lung, and kidney of mice treated with the prepared Ce-Que@MΦ nanoparticles;

[0039] Figure 15 Weight change of mice during the treatment of colitis mice with normal saline, dextran sulfate sodium, quercetin, Ce-Que, and Ce-Que@MΦ;

[0040] Figure 16Hematoxylin-eosin (H&E) staining images of the mouse colon and immunohistochemical staining diagrams of different inflammatory factors (TNF-α, IL-10) after treatment with normal saline, dextran sulfate sodium, quercetin, Ce-Que, and Ce-Que@MΦ;

[0041] Figure 17 Imaging diagrams of the mouse colon after treatment with Ce-Que (A) and Ce-Que@MΦ (B) encapsulating rhodamine dye for 24 h in mice. Detailed implementation manners

[0042] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0044] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0045] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0046] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0047] Such as Figure 1As shown in the figure, the present invention synthesizes a nanomaterial by self-assembling cerium nitrate and quercetin through a one-pot method. The nanomaterial has superoxide dismutase-like activity and can effectively scavenge excessive reactive oxygen species. Then, the outer layer is modified with macrophage cell membranes, which have inflammatory factor recognition receptors and can also help the nanomaterial escape from the immune system to increase its biocompatibility and targeting property, thereby preparing a macrophage cell membrane-coated quercetin cerium coordination biomimetic nanomaterial for the treatment of ulcerative colitis.

[0048] The macrophages used in the following examples were purchased from the Cell Culture Center of the Chinese Academy of Sciences.

[0049] Example 1

[0050] 1. Preparation of materials

[0051] Preparation of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution: Accurately weigh 2.383 g of HEPES, add distilled water to make up to 1 L, and adjust the pH value to 7.4 with 0.5 M NaOH aqueous solution.

[0052] Weigh 3.9 mg of Ce(NO3)4 and dissolve it in 5 mL of deionized water. Weigh 0.76 mg of quercetin and dissolve it in 1 mL of ethanol solution. Slowly drop 1 mL of quercetin ethanol solution into 5 mL of Ce(NO3)4 solution, and then add 1 mL of HEPES solution with pH = 7.4. After reacting at room temperature (25 °C) for 75 min, centrifuge at 12000 r for 10 min to obtain a precipitate. Wash the precipitate with ethanol to remove the excess unreacted polyphenol solution, and then lyophilize to obtain quercetin cerium coordination nanoparticles (Ce-Que), which are stored at 4 °C for later use.

[0053] Extraction of macrophage cell membranes: Collect the cultured macrophages, centrifuge at 1200 rpm, and wash twice with PBS. Resuspend the cells at a density of 10 7 cells / mL in a solution containing 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 20 mM HEPES, and 10 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF), with a pH of 7.0. Use an ultrasonic cell disruptor to perform 20 ultrasonic treatments on the cells in an ice bath (ultrasonic power: 100 W, single ultrasonic time: 3 s, interval time: 10 s, frequency range: 20 - 25 KHz). After centrifuging the clarified sample at 14800 rpm for 20 minutes, collect the particles containing cell membranes, wash twice with PBS, and obtain the macrophage cell membranes, which are stored in a solution containing 10 mM PMSF at -20 °C.

[0054] Synthesis of Ce-Que@MΦ: 0.75 mg of extracted macrophage cell membranes was uniformly mixed with 3 mg of Ce-Que nanoparticles in 3 mL of PBS buffer. The mixture was extruded 11 times using a liposome extruder through a polycarbonate membrane with a pore size of 400 nm to obtain the quercetin cerium coordination biomimetic nanomaterial (Ce-Que@MΦ). The nanomaterial was centrifuged at 14,000 rpm for 10 min and then stored at 4 °C after freeze-drying for later use.

[0055] The scanning electron microscope (SEM) images of the quercetin cerium coordination biomimetic nanomaterial prepared in this example are shown in Figure 2 , and as can be seen from Figure 2 A and B therein, the particle size of the Ce-Que nanoparticles is approximately 200 - 400 nm. As can be seen from Figure 2 C and D therein, a film-like substance is attached to the surface of the nanomaterial, indicating that the macrophage cell membrane has been successfully modified on the surface of Ce-Que, and the Ce-Que@MΦ nanomaterial has been successfully synthesized.

[0056] The infrared and visible spectral analysis of the quercetin cerium coordination biomimetic nanomaterial prepared in this example and its raw materials is shown in Figure 3 , and the peaks at 550, 1042, and 3371 cm -1 are caused by the stretching vibrations of Ce-O, C-O, and -C-H, respectively. In addition, quercetin has three peaks at 1612 cm -1 , 1436 cm -1 , and 1285 cm -1 , while Ce-Que is weakened due to polyphenol oxidation, cation-π interaction, and coordination between cerium ions and phenolic hydroxyl groups. The spectrum of Ce-Que@MΦ is not much different from that of Ce-Que, indicating that the macrophage cell membrane is modified on the surface of Ce-Que without causing chemical bond changes in Ce-Que.

[0057] The ultraviolet and visible spectral analysis of the quercetin cerium coordination biomimetic nanomaterial prepared in this example and its raw materials is shown in Figure 4 . In the ultraviolet and visible spectrum, it can be seen that cerium nitrate has a strong absorption peak at 220 nm. After the synthesis of Ce-Que from quercetin and cerium nitrate, the peak intensity decreases. After binding to the macrophage cell membrane, a slightly red-shifted peak can be seen from the absorption curve of Ce-Que@MΦ. This phenomenon is affected by the macrophage cell membrane, and its characteristic absorption peak value is around 260 nm. The study by ultraviolet-visible spectrophotometry further proves the successful binding of Ce-Que@MΦ.

[0058] The Zeta potential analysis spectra of the quercetin cerium coordination biomimetic nanomaterial prepared in this example and its raw materials are shown in Figure 5 . FromFigure 5 It can be seen that cerium nitrate is positively charged (+4.07 mV), quercetin is negatively charged (-17.33 mV), and the absolute value of the potential of the Ce-Que nanoparticles self-assembled from the two is smaller than that of quercetin (-10.10 mV), indicating that cerium nitrate and quercetin have successfully self-assembled, resulting in a change in the potential of the nanoparticles. The Zeta potential of the nanoparticles modified with the cell membrane is -24.60 mV, further indicating that the macrophage cell membrane is loaded on the surface of Ce-Que, and the synthesized Ce-Que@MΦ nanomaterial is more stable.

[0059] Example 2

[0060] Detection of the reactive oxygen species scavenging ability and superoxide dismutase (SOD) activity of the Ce-Que@MΦ nanomaterial:

[0061] ·OH radical scavenging: Tetramethylbenzidine (TMB) was selected as the substrate. Weigh 0.25 mg of cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ, and add 200 μL of 2 mM H2O2 solution, 200 μL of ·OH radical scavenging working solution, 200 μL of 1.25 mM TMB solution, and 400 μL of water to each of them. Incubate at 37 °C for 30 min in the dark, and measure the absorbance (OD 650 ) of the solution at 650 nm using a microplate reader. Set up three parallel experiments, and add 875 μL of water to 125 μL of the sample to be tested as a blank control.

[0062] ABTS· + radical scavenging: Weigh 0.625 mg of cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ, and dissolve them in 500 μL of water respectively. Take 100 μL of the sample to be tested and add 900 μL of ABTS· + working solution diluted 5-fold (add 900 μL of H2O to the control group). Incubate at 37 °C for 3 h in the dark, and measure the absorbance (OD 734 ) at 734 nm using a microplate reader.

[0063] Total SOD enzyme activity detection: The experiment was conducted according to the instructions of the Total SOD Enzyme Activity Detection Kit from Beyotime. Weigh 0.125 mg of the nanomaterial, dissolve it with 100 μL of SOD sample preparation solution, take 20 μL of the sample and place it in a 96-well plate. Then add 120 μL of the WST-8 enzyme solution diluted according to the instructions and 20 μL of the reaction initiation solution. Set up three parallel controls and a blank control group without adding the reaction initiation solution (blank control 3). Also set up a blank control using SOD buffer instead of the test sample (blank control 1), and a blank control without adding the reaction initiation solution (blank control 2). Incubate at 37 °C for 30 min in the dark, and measure the absorbance (OD 450 ) of the solution at 450 nm using an enzyme-linked immunosorbent assay reader.

[0064] The antioxidant capacities of cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ were detected, including a comprehensive comparison of the scavenging abilities for ·OH and ABTS· + . The results are as Figure 6 shown. As can be seen from Figure 6 A, quercetin, Ce-Que, and Ce-Que@MΦ all have good scavenging abilities for ·OH. For ABTS· + , Ce-Que and Ce-Que@MΦ have similar scavenging abilities and can remove more than 90% of ABTS· + ( Figure 6 B), and the scavenging ability of Ce-Que@MΦ is slightly better than that of Ce-Que.

[0065] Regarding the superoxide dismutase activity, as can be seen from Figure 7 , Ce-Que@MΦ shows the best performance among the materials, and its superoxide dismutase activity reaches more than 55%, indicating that Ce-Que@MΦ can simulate superoxide dismutase and scavenge excessive free radicals in the body.

[0066] In summary, Ce-Que@MΦ has good scavenging abilities for ·OH and ABTS·+, and also has corresponding superoxide dismutase activity, so further experimental research can be carried out on the use of Ce-Que@MΦ for inflammation treatment.

[0067] Example 3

[0068] Evaluation of cell protection ability:

[0069] 1. Reactive oxygen species scavenging in vitro

[0070] The abilities of cerium ions, quercetin, Ce-Que, and Ce-Que@MΦ nanoparticles to scavenge reactive oxygen species in cells were investigated by loading the DCFH-DA probe. Mouse macrophages in the logarithmic growth phase were seeded at a density of 2 × 10 per well 5Cells were inoculated in six-well plates at a density of [number] and cultured with DMEM medium for 24 h. Then the medium was discarded and replaced with DMEM complete medium containing cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ nanoparticles, with a final concentration of 20 mg·mL -1 , and one well was reserved for adding H2O2 after 1 h. After incubation in a 37 °C constant temperature incubator for 1 h, H2O2 solution was added respectively to make the final concentration of H2O2 400 μM. After culturing for 24 h, the medium was discarded. After washing three times with PBS to remove the drugs that did not enter the cells, Hoechst 33342 was used for staining for 10 min. After washing three times with PBS to remove the excess dye, 1 mL of the culture medium mixed with DCFH-DA probe was added to each well and incubated for 20 min. After washing three times with PBS to remove the probe that did not enter the cells, a small amount of PBS was dropped on the cells, and a fluorescence inverted microscope was used to observe the cells and take pictures to study the scavenging of ROS.

[0071] From the immunofluorescence staining map of intracellular reactive oxygen species ( Figure 8 ), it can be seen that low-concentration cerium nitrate has little ability to scavenge ROS in macrophages, and low-concentration quercetin also has no good effect on scavenging ROS in macrophages. At the same concentration, Ce-Que and Ce-Que@MΦ have good ability to scavenge the reactive oxygen species generated in macrophages, and nuclear staining shows that the cells treated with the samples still have good cell morphology, indicating that Ce-Que and Ce-Que@MΦ nanomaterials not only have good scavenging effects on the reactive oxygen species generated by cells.

[0072] 2. Cell viability and death staining (AO)

[0073] The acridine orange (AO) dye was used to analyze the viability and death of cells. Observed under a fluorescence microscope, acridine orange can penetrate the normal cell membrane and make the nucleus show uniform green or yellow-green fluorescence; in apoptotic cells, due to chromatin condensation or fragmentation into fragments of different sizes, apoptotic bodies are formed. Acridine orange stains it with dense and intense yellow-green fluorescence. Mouse macrophages in the logarithmic growth phase were inoculated in six-well plates at a density of 2×10 5 cells per well and cultured with DMEM medium for 24 h. Then the medium was discarded and replaced with DMEM complete medium containing blank, cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ nanoparticles, with a final concentration of 20 mg·mL -1 , and one well was reserved for adding lipopolysaccharide (LPS) after 1 h. After incubation in a 37 °C constant temperature incubator for 1 h, LPS solution was added respectively to make the final concentration of LPS 1 μg·mL -1, After culturing for 24 h, discard the culture medium. After washing three times with PBS to remove the drugs that did not enter the cells, add 1 mL of AO staining working solution and incubate in the incubator in the dark for 15 min. After the incubation, discard the supernatant, wash three times with PBS to remove the excess dye, and observe and photograph the cells using a fluorescence inverted microscope at an excitation wavelength of 490 nm.

[0074] As Figure 9 shown by the results of the AO staining experiment, the green fluorescence was the strongest in the blank group. In macrophages induced with lipopolysaccharide to cause inflammation, obvious red fluorescence could be seen. The quantitative data showed that only about 40% of the cells survived in the LPS group, and nearly 60% of the cells died, indicating that LPS could successfully induce cell inflammation and cause cell death. The cell survival rates of cells treated with cerium nitrate, quercetin, and Ce-Que were 50%, 60%, and 74% respectively, while the green fluorescence of macrophages treated with Ce-Que@MΦ was significantly enhanced. The quantitative data graph showed that the cell survival rate of cells treated with Ce-Que@MΦ reached more than 80%, indicating that Ce-Que@MΦ had the best cell protection effect.

[0075] 3. Mitochondrial membrane potential changes

[0076] A mitochondrial membrane potential detection kit was used to evaluate the changes in the mitochondrial membrane potential of cells. When the mitochondrial membrane potential is relatively high, the mitochondrial membrane potential probe (JC-1) aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence; when the mitochondrial membrane potential is relatively low, JC-1 cannot aggregate in the mitochondrial matrix and produces green fluorescence. Mouse macrophages in the logarithmic growth phase were seeded in six-well plates at a density of 2×10 5 cells per well, cultured with DMEM culture medium for 24 h, then the culture medium was discarded and replaced with DMEM complete culture medium containing cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ nanoparticles, and their final concentrations were all 20 mg·mL -1 , and one well was reserved for adding LPS after 1 h. After incubating in a 37 °C constant temperature incubator for 1 h, LPS solution was added to each well to make the final concentration of LPS 1 μg·mL -1 , and after culturing for 24 h, discard the culture medium. After washing three times with PBS to remove the drugs that did not enter the cells, add 1 mL of JC-1 staining working solution and incubate in the incubator in the dark for 15 min. After the incubation, discard the supernatant, wash twice with JC-1 staining buffer, and observe and photograph the cells using a fluorescence inverted microscope at an excitation wavelength of 490 nm.

[0077] As Figure 10As can be seen from the JC-1 immunofluorescence staining images shown, most of the macrophages treated with 1 μg / mL LPS showed green fluorescence, indicating a decrease in mitochondrial membrane potential. The orange fluorescence of the cells treated with cerium nitrate and quercetin was slightly enhanced, while the orange fluorescence of the cells treated with Ce-Que and Ce-Que@MΦ was more significantly enhanced, and the intensity of green fluorescence was significantly reduced, indicating that both Ce-Que and Ce-Que@MΦ have the ability to protect cell mitochondria.

[0078] Example 4

[0079] Biosafety evaluation:

[0080] 1. In vitro hemolysis experiment

[0081] Prepare cerium nitrate, quercetin, Ce-Que and Ce-Que@MΦ sample solutions with a concentration range of 1 - 2000 μg / mL, and prepare a sheep red blood cell reaction system according to the method in Table 1, where Triton is used as the positive control and blank physiological saline is used as the negative control.

[0082] Table 1 Sheep red blood cell reaction system

[0083] [[ID=1%]]

[0084] The treated blood cell hemolysis test reaction system is incubated in a shaker at 37 °C and 180 rpm for one hour. After incubation, the samples are centrifuged at 3000 rpm for 5 min, the supernatant is collected, and the absorbance of the samples at 540 nm is measured using a UV-visible spectrophotometer. Each group is repeated three times, and the hemolysis rate is calculated according to the following formula:

[0085] Hemolysis rate (%) = (A 样品组 - A 阴性对照 ) / (A 阳性对照 - A 阴性对照 ) × 100%.

[0086] The results of the hemolysis experiment are shown in Figure 11 - Figure 12 , when the concentration of cerium nitrate is 16 μg / mL, its hemolysis rate reaches more than 5%, and the biosafety is poor. The hemolysis rate of the quercetin group did not show an obvious upward trend with the increase in quercetin concentration. When the concentration reached 128 μg / mL, the hemolysis rate was 4.9%. When the concentration of Ce-Que was 128 μg / mL, the hemolysis rate was 5.8%. Compared with the quercetin group, its biosafety was poor. When the concentration of Ce-Que@MΦ ≤ 256 μg / mL, the hemolysis rate of the nanomaterial < 5%; the blood compatibility is good. Compared with other groups, Ce-Que@MΦ has the best biosafety and is expected to be used for sustained drug delivery.

[0087] 2. In vivo biosafety evaluation in mice

[0088] Kunming mice were randomly divided into 3 groups, with 3 mice in each group. The mice in each group were injected with normal saline, cerium nitrate, quercetin, Ce-Que, and Ce-Que@MΦ via the tail vein, respectively. The drug was administered once a day for a total of 7 times, and the dosage was 2 mg / kg. After the last administration, the mice were deprived of food and water. The next day, the eyeballs of the mice were removed to collect blood samples for centrifugation, and the obtained serum was analyzed for the top ten routine blood tests before surgery.

[0089] After the mice were sacrificed by taking blood from the eyeballs, the main organs (heart, liver, spleen, lung, kidney) were dissected, and histopathological examinations were performed using hematoxylin and eosin (H&E staining) to evaluate the in vivo systemic toxicity of the preparation.

[0090] The results of the in vivo biosafety evaluation of the mice are as Figure 13 and Figure 14 shown. The blood indexes of the mice injected with Ce-Que@MΦ were all within the normal range, and there was no obvious effect on liver and kidney functions. The mice were dissected, and the main organs of the mice were stained with H&E. The results showed that there were no inflammatory cells and lesions in the tissue sections of the mice injected with Ce-Que@MΦ intravenously, which confirmed its good in vivo biosafety. The above indicates that Ce-Que@MΦ has good biosafety and can be used for subsequent in vivo treatment.

[0091] Example 5

[0092] After 2 - 3 days of adaptive feeding of 5-week-old C57BL6 mice, ear tags were attached to the mice for marking. Except for the 5 mice in the blank group, the drinking water of the others was replaced with 3% dextran sulfate sodium solution (DSS) to establish an ulcerative colitis model (IBD). The mice were given 3% DSS to drink for 6 days. During this period, the success of the mouse colitis model was judged by observing the color (blood in the stool condition), morphology of the mice's feces, and the anal bleeding condition of the mice.

[0093] According to the weight and modeling severity of each mouse, the mice were evenly grouped into blank (Control), 3% dextran sulfate sodium (3% DSS), quercetin (Quercetin), Ce-Que, and Ce-Que@MΦ groups, with 5 mice in each group. After the modeling grouping was completed, the mice in each group were administered drugs. The blank and 3% dextran sulfate sodium groups were injected with 200 μL of normal saline by gavage, and the other groups were injected with 200 μL of quercetin, Ce-Que, and Ce-Que@MΦ with a concentration of 500 μg / mL, respectively. The drugs were administered continuously for seven days, and the mice were weighed during this period. The colon of the dissected mice was measured for length, and the colon tissues were used for H&E and immunohistochemical staining, and the colon of Ce-Que and Ce-Que@MΦ was used for imaging.

[0094] During the drug administration period, the weight changes of the mice were as Figure 15As shown, the body weight of the mice in the 3% dextran sulfate sodium group showed a downward trend, while the body weights of the mice in the blank, Ce-Que, and Ce-Que@MΦ groups showed an overall upward trend. Among them, the body weight of the mice in the Ce-Que@MΦ group increased every day, indicating that the mice were in good condition after drug treatment. Figure 16 The H&E staining of the mouse colon tissue and the immunohistochemical staining of inflammatory factors shown also illustrate this point. Near-infrared dyes (rhodamine dyes) were encapsulated in the material for mouse colon imaging. Figure 17 The colon imaging pictures of Ce-Que and Ce-Que@MΦ were taken under the same environment. Figure 17 It can be seen that after 24 hours of drug administration, more Ce-Que@MΦ accumulated in the mouse colon, preliminarily verifying that the Ce-Que nanomaterial modified with macrophage cell membrane has targeting properties.

[0095] The above results indicate that the quercetin cerium coordination biomimetic nanomaterial (i.e., Ce-Que@MΦ) prepared by the present invention with macrophage cell membrane encapsulation has excellent antioxidant activity, cellular reactive oxygen species scavenging ability, cell protection ability, ability to regulate the expression of inflammatory factors, biosafety, and in vivo colitis treatment ability.

[0096] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a macrophage membrane-coated quercetin cerium coordination bionic nanomaterial, characterized in that, It includes the following steps: Add the quercetin solution into the Ce(NO3)4 solution, then add the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution. After mixing and reacting, quercetin cerium coordination nanoparticles are obtained; Uniformly mix the quercetin cerium coordination nanoparticles with the macrophage cell membrane in PBS buffer solution, and extrude with a liposome extruder to obtain the quercetin cerium coordination biomimetic nanomaterial wrapped by the macrophage cell membrane.

2. The preparation method according to claim 1, wherein The concentration of the quercetin solution is 0.76 mg / mL, and the concentration of the Ce(NO3)4 solution is 0.78 mg / mL.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the quercetin solution, the Ce(NO3)4 solution and the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution is 1:5:

1.

4. The preparation method according to claim 1, wherein, The temperature of the mixing reaction is 25 °C, and the time is 75 min.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the quercetin cerium coordination nanoparticles to the macrophage cell membrane is 3 mg:0.75 mg.

6. The preparation method according to claim 1, characterized in that, The macrophage cell membrane is extracted by the ultrasonic fragmentation method.

7. A quercetin cerium coordination biomimetic nanomaterial wrapped by a macrophage cell membrane prepared by the preparation method according to any one of claims 1-6.

8. An application of the quercetin cerium coordination biomimetic nanomaterial wrapped by a macrophage cell membrane as claimed in claim 7 in the preparation of a drug for treating ulcerative colitis.

9. A drug for treating ulcerative colitis, characterized in that, The active ingredient includes the quercetin cerium coordination biomimetic nanomaterial wrapped by the macrophage cell membrane as claimed in claim 7.

10. The drug according to claim 9, characterized in that, The drug further includes pharmaceutically acceptable excipients.

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