ROS responsive bionic nano drug delivery system, preparation method and application

Abstract

The invention relates to the field of traditional Chinese medicine active ingredient new dosage form pharmaceutical preparations, and discloses an ROS responsive bionic nano drug delivery system, a preparation method and application. The preparation method comprises the following steps: taking lipid components, namely soybean phospholipid (SPC), hydrogenated soybean phospholipid (HSPC), dipalmitoyl phosphatidylcholine (DPPC), pegylated distearoyl phosphatidyl ethanolamine (DSPE-PEG), dioleoyl lecithin (DOPC), (2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate) and a surfactant, as well as an emulsifier, as well as a solvent; the ROS-responsive leonurine lipidosome is prepared by taking 1, 3-dioleoyl-propyl)-trimethylamine (DOTAP), cholesterol and the like as raw materials to prepare nano-lipidosome, taking the nano-lipidosome as an inner core and coating the leonurine serving as a traditional Chinese medicine effective component and catalase capable of catalyzing active oxygen to generate oxygen, so as to form the ROS-responsive leonurine lipidosome. And then realizing bionic fusion with an activated neutrophile granulocyte membrane on the outer layer through a pneumatic nano extruder co-extrusion technology, so as to prepare the activated neutrophile granulocyte membrane coated ROS responsive bionic nano-liposome drug delivery system. The invention can realize targeted therapy of rheumatoid arthritis and the like, and has the advantages of biological safety, slow release and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a ROS-responsive bionic nano drug delivery system coated with a neutrophil membrane targeting rheumatoid arthritis, a preparation method and an application thereof. Background Art

[0002] Rheumatoid arthritis is an autoimmune, chronic inflammatory disease that causes synovial inflammation and progressive destruction of bone and cartilage. Patients with rheumatoid arthritis often experience persistent joint pain, swelling and stiffness, leading to irreversible joint damage and disability. Rheumatoid arthritis affects approximately 1% of adults worldwide, seriously affecting the quality of life. Fibroblast-like synoviocytes in rheumatoid arthritis exhibit tumor-like characteristics such as proliferation, migration and invasion after being activated by proinflammatory factors. They migrate to cartilage / bone and invade the extracellular matrix, causing joint destruction. Uncontrolled rheumatoid arthritis can even lead to multi-organ damage such as cardiovascular complications. Current clinical treatments for rheumatoid arthritis are often accompanied by drug resistance and side effects, and the treatment effect is not ideal. Recent studies have shown that leonurine has the potential to inhibit the dysfunction of fibroblast-like synoviocytes.

[0003] Motherwort was first recorded in Shennong's Herbal Classic. It is the above-ground part of the plant Leonurus of the Lamiaceae family, which has the effect of clearing away heat and detoxifying. The Chinese Pharmacopoeia records that motherwort is a gynecological drug that promotes blood circulation and regulates menstruation. In recent years, more than 200 compounds have been isolated from motherwort, among which leonurine is the most important bioactive compound. Leonurine has a wide range of biological activities, including anti-inflammatory and antioxidant effects. Leonurine can also exert anti-inflammatory effects by inhibiting the secretion of pro-inflammatory factors, but its low solubility, low oral bioavailability and large dosage limit the clinical application of leonurine.

[0004] Liposomes are tiny vesicles with a phospholipid bilayer structure that can encapsulate many water-soluble and fat-soluble drug ingredients. They are biodegradable, have low immunogenicity, are safe and non-toxic, and release drugs slowly after encapsulation. Liposomes deliver drugs into cells through endocytosis and fusion, so liposomes are ideal drug delivery vehicles. Due to the difference in the solubility of leonurine at different pH values, liposomes can effectively encapsulate leonurine using the pH gradient method. Unmodified liposomes only have passive delivery capabilities, and their ability to deliver drugs to arthritis sites is limited. They are not targeted enough, and liposomes are easily phagocytosed by the endothelial system after entering the body. The release of inflammatory factors in the lesions of rheumatoid arthritis attracts neutrophils and other cells to rapidly aggregate, and through the interaction of adhesion molecules on the surface of the cell membrane, they are recruited to the inflamed joints. The delivery of motherwort alkaloids by neutrophil membrane-coated ROS-responsive liposomes can not only reduce the recognition and clearance of liposomes by the endothelial system, but also actively target the synovial membrane of arthritis hyperplasia through adhesion molecules and chemokine receptors on the surface of neutrophil membranes, exert the efficacy of motherwort alkaloids, inhibit the migration and invasion of synovial fibroblasts, and exert anti-inflammatory effects. After entering the arthritis site, the catalase in the neutrophil membrane-coated ROS-responsive motherwort alkaloid liposomes catalyzes the high level of ROS in synovial fibroblasts to produce oxygen, destroy the liposome structure, accelerate the release of motherwort alkaloids, enhance its efficacy in treating rheumatoid arthritis, and further reduce the migration and invasion of synovial fibroblasts and bone destruction in rheumatoid arthritis. At the same time, the inflammatory factor receptors and chemokine receptors on the surface of neutrophil membranes, such as TNF-αR, IL-1R, CXCR2, CCR2, and CCR5, can neutralize the inflammatory factors TNF-α, IL-1β, IL-8 and chemokines CCL2 and CCL5 caused by rheumatoid arthritis, and reduce the level of inflammation. Therefore, the neutrophil membrane-coated ROS-responsive liposome drug delivery system has a wide range of clinical application value. Summary of the invention

[0005] In view of this, it is necessary to provide a neutrophil membrane-coated ROS-responsive bionic nano drug delivery system, preparation method and application targeting rheumatoid arthritis. The purpose of the present invention is to construct a neutrophil membrane-coated ROS-responsive bionic nano drug delivery system targeting rheumatoid arthritis and its preparation method, increase the targeting of traditional Chinese medicine preparations in rheumatoid arthritis lesions, and enhance its therapeutic effect.

[0006] A ROS-responsive bionic nano drug delivery system comprises a liposome nanocarrier encapsulating leonurine and catalase and an activated neutrophil membrane, wherein the liposome is prepared by a thin film hydration method, the catalase is loaded by an ultrasound method, the leonurine is loaded by a pH gradient method, and the catalase liposome and the neutrophil membrane are co-extruded by a membrane extruder to achieve outer membrane coating.

[0007] Preferably, the activated neutrophil membrane is obtained by DMSO-induced differentiation of the HL-60 cell line, and after further inflammatory stimulation, the cell membrane surface overexpresses relevant adhesion molecules, inflammatory factor receptors and chemokine receptors; the activated neutrophil membrane is obtained by mechanical grinding and differential centrifugation extraction in sequence, and cell membrane vesicles are obtained by ultrasonic treatment; the relevant adhesion molecules, inflammatory factor receptors and chemokine receptors include LFA-1, TNF-αR, IL-1R, CXCR2, CCR2 and CCR5.

[0008] Preferably, the encapsulated leonurine is leonurine or its salt, which is an effective ingredient of the traditional Chinese medicine motherwort, and the components of the liposome nanocarrier are phospholipids and cholesterol.

[0009] Preferably, the phospholipid is one or a mixture of soybean lecithin, hydrogenated soybean lecithin, dioleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and their derivatives; the organic solvent is one or a mixture of ethanol and chloroform.

[0010] Preferably, the liposome structure is a hollow phospholipid bilayer structure, which can encapsulate leonurine and catalase, with a corresponding encapsulation rate between 30% and 90%, and a drug loading between 0.5% and 20%.

[0011] Preferably, the method for encapsulating catalase CAT is an ultrasonic method, the ultrasonic intensity is 20kHz to 40kHz, and the ultrasonic time is 5 to 30min. The pH gradient method for encapsulating leonurine Leo uses an initial pH range of 4 to 5 and a pH range of 7 to 8 after adjustment.

[0012] Preferably, the encapsulated contents include catalase CAT, which has the ability to catalyze ROS in inflammatory tissue cells to produce oxygen.

[0013] Preferably, the liposomes and the neutrophil membrane are fused with each other by co-extrusion 10 to 20 times.

[0014] Preferably, the oxygen generated by the encapsulated catalase CAT when encountering a high level of ROS can destroy the liposome structure, thereby accelerating the release of the encapsulated drug under the condition of a high level of ROS.

[0015] Preferably, the average particle size of the liposome is 60 to 100 nm, and the particle size of the neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system is 80 to 130 nm.

[0016] A method for preparing a ROS-responsive bionic nano drug delivery system comprises the following steps:

[0017] Step 1: extracting neutrophil membranes, HL-60 cells were induced to differentiate with DMSO and stimulated with TNF-α to upregulate the expression of LFA-1, TNF-αR, IL-1R, CXCR2, CCR2, and CCR5, and the cell membranes extracted by mechanical grinding and differential centrifugation were dispersed in double distilled water and ultrasonically dispersed;

[0018] Step 2, using dioleoyl phosphatidylcholine, polyethylene glycol-distearoyl phosphatidylethanolamine, cholesterol or dipalmitoyl phosphatidylcholine, polyethylene glycol-distearoyl phosphatidylethanolamine, cholesterol as raw materials to prepare liposomes by thin film hydration method, and encapsulating catalase by ultrasonic treatment, and using citrate buffer to encapsulate the leonurine drug by pH gradient method to prepare ROS-responsive leonurine liposomes;

[0019] Step three, the pre-prepared liposomes encapsulating leonurine and catalase are co-incubated with the neutrophil membrane and repeatedly extruded through the membrane using a membrane extruder to obtain a ROS-responsive bionic nano drug delivery system.

[0020] Preferably, in step 1, HL-60 cells are passaged and expanded in a cell culture incubator with IMDM medium + 20% FBS + 1% PS, DMSO is added to the cell culture medium to induce cell differentiation and TNF-α is given to stimulate the cells, the supernatant is removed by centrifugation, and then washed; the cells are resuspended with membrane extraction buffer, mechanically homogenized on ice, centrifuged, the cell nuclear structure is removed, the supernatant is centrifuged again, the mitochondrial structure is removed, the supernatant is centrifuged, the cell membrane is resuspended with distilled water after centrifugation, and the cells are freeze-dried and stored for later use.

[0021] Preferably, the IMDM medium + 20% FBS + 1% PS is passaged and expanded in a cell culture incubator at 37°C and a CO2 concentration of 5%, DMSO at a concentration of 1% is added to the cell culture medium to induce cell differentiation, and 10 ng / mL TNF-α is given to stimulate the cells for 3 days. After centrifugation at 1000 rpm / min for 5 min to remove the supernatant, the cells are washed 3 times with PBS buffer, the temperature of the PBS buffer is 4°C, the speed of the centrifugal washing is 1000 rpm / min, and the washing time is 5 min.

[0022] Preferably, the membrane extraction buffer is prepared from 225 mM mannitol, 75 mM sucrose, 0.5% BSA, 0.5 mM EDTA, 30 mM Tris-HCl and 1% protease inhibitor. After centrifugation, the cell membrane is resuspended in distilled water, freeze-dried and stored at -80°C for later use.

[0023] Preferably, in step 2, according to ① dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol in a molar ratio of 20:1:10, accurately weighed and dissolved in chloroform; ② soybean lecithin and cholesterol in a molar ratio of 19:2, accurately weighed and dissolved in ethanol; ③ hydrogenated soybean lecithin and cholesterol in a molar ratio of 59:40, accurately weighed and dissolved in ethanol; ④ dioleoyl lecithin, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol in a molar ratio of 58:5:38, accurately weighed and dissolved in chloroform; ⑤ (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol in a molar ratio of 20:70:5.4:30, accurately weighed and dissolved in chloroform, blank liposomes are prepared according to one of the above five raw materials by the thin film hydration method.

[0024] Preferably, in step 2, blank liposomes are treated with catalase at 20kHz to 40kHz for 5 to 30 minutes of ultrasound to obtain ROS-responsive liposomes, leonurine is dissolved in citrate at pH 4.0, and ROS-responsive liposomes are added, and the pH value of the extralipidic aqueous phase is adjusted to 7.4 at 50°C, incubated for 5 minutes, and immediately taken out and cooled on ice.

[0025] The present invention uses lipid components such as soybean lecithin SPC, hydrogenated soybean lecithin HSPC, dipalmitoylphosphatidylcholine (DPPC), polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG), dioleoyl phosphatidylcholine (DOPC), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), and cholesterol as raw materials to prepare nanoliposomes as the inner core and encapsulate the traditional Chinese medicine leonurine and catalase to form ROS-responsive liposome leonurine, which is then co-extruded with the outer activated neutrophil membrane by a pneumatic nano-extruder to achieve biomimetic fusion, HL-60 cells are induced to differentiate into neutrophils by DMSO, and then given inflammatory factor stimulation, the cell membrane-coated ROS-responsive liposome leonurine (Leo@CAT@Lipo) is extracted, and a neutrophil membrane-coated ROS-responsive biomimetic nano drug delivery system (Leo@CAT@NM-Lipo) is constructed. This bionic nano-drug delivery system can not only utilize the molecules on the surface of the neutrophil membrane to increase the biocompatibility of the system and reduce phagocytosis of the endothelial system, but also utilize the active tendency of neutrophils to move toward inflammatory arthritis, target the synovium of rheumatoid arthritis, and deliver the effective ingredients of anti-inflammatory Chinese medicine to the affected joint lesions. It can also enable liposomes to slowly release drugs in the blood circulation, prolong the drug action time, and accelerate the release of drugs in cells with high levels of ROS in inflammatory lesions.

[0026] In the present invention, the drug used is leonurine, an effective ingredient of the Chinese herbal medicine motherwort. Studies have shown that leonurine has anti-inflammatory and synovial migration and invasion inhibitory effects. Leonurine is loaded into ROS-responsive liposomes and coated with neutrophil membranes to increase drug aggregation in rheumatoid arthritis lesions and accelerate drug release, thereby increasing the efficacy of treating rheumatoid arthritis. The average particle size of liposomes prepared with dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, cholesterol, etc. as raw materials is 60-100nm, and it is highly stable at 4°C.

[0027] The present invention uses human synovial fibroblasts (MH7A) to simulate the synovial state of rheumatoid arthritis after being treated with inflammatory factors.

[0028] The present invention constructs a rat rheumatoid arthritis model (AIA) by subcutaneously injecting inactivated tuberculosis bacteria dispersed in mineral oil at the base of the tail, and evaluates the in vivo efficacy of the neutrophil membrane-coated ROS-responsive bionic nano-drug delivery system, which is common and recognized in the art.

[0029] The administration mode of the neutrophil membrane-coated ROS-responsive biomimetic nano drug delivery system prepared by the present invention is intravenous injection.

[0030] The advantages of the present invention are:

[0031] The prepared neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system is different from other targeted nano-carriers modified with a single molecule or multiple molecules. The ROS-responsive biomimetic nano-drug delivery system has unique advantages in the treatment of rheumatoid arthritis:

[0032] 1. Using the surface molecules of neutrophils after differentiation and inflammatory stimulation to actively target the synovial tissue of rheumatoid arthritis;

[0033] 2. The ROS-responsive biomimetic liposomes can effectively encapsulate leonurine through the pH gradient method, and

[0034] It has a sustained release effect in normal blood circulation, prolonging the duration of drug action, and accelerates drug release in synovial cells of rheumatoid arthritis with high ROS levels;

[0035] 3. The neutrophil membrane-coated ROS-responsive biomimetic leonurine liposome has an inflammation neutralizing effect and enhances the therapeutic effect on rheumatoid arthritis;

[0036] 4. The ROS-responsive bionic motherwort liposome base increases the biocompatibility of the system and reduces the phagocytosis of the endothelial system through the coating of the neutrophil membrane, and has high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1This is a characterization of the ROS-responsive bionic nano-drug delivery system of blank liposomes and neutrophil membranes in the present invention. In the figure, A, B, and C are the average particle size and Zeta potential of the ROS-responsive bionic nano-drug delivery system of blank liposomes and neutrophil membranes, respectively.

[0038] Figure 2 The particle size stability of the blank liposome and the neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system in the present invention under storage conditions of 4°C.

[0039] Figure 3 The drug loading capacity and encapsulation efficiency of leonurine in the neutrophil membrane-coated ROS-responsive bionic nano drug delivery system of the present invention.

[0040] Figure 4 It is the qualitative detection result of the surface protein expression of the nano drug delivery system in the present invention. In the figure, A is the protein expression spectrum of the neutrophil membrane and the ROS-responsive bionic nano drug delivery system coated with the neutrophil membrane, and B is the verification of its specific protein expression.

[0041] Figure 5 The ROS responsiveness of the neutrophil membrane-coated ROS-responsive bionic nano drug delivery system of the present invention is verified. In the figure, A is an electron microscope image of the ROS-responsive bionic nano drug delivery system before and after H2O2 treatment, B is the ability of the ROS-responsive bionic nano drug delivery system loaded with different amounts of catalase to produce oxygen after H2O2 treatment, and C is the in vitro release of leonurine from ROS-responsive bionic liposomes loaded with different amounts of catalase in H2O2.

[0042] Figure 6 A is a confocal image of the targeting ability of the biomimetic liposomes of the present invention to synovial fibroblasts, and B is the quantitative analysis of the mean fluorescence intensity.

[0043] Figure 7 The safety evaluation of the ROS-responsive biomimetic nano-drug delivery system coated with neutrophil membrane at different concentrations on synovial fibroblasts (A) and macrophages (B) in the present invention.

[0044] Figure 8 The figure shows the effect of the neutrophil membrane-coated ROS-responsive biomimetic liposome leonurine on the migration and invasion of synovial fibroblasts in the present invention. In the figure, A and B are respectively the migration and invasion result diagrams in the transwell experiment, and C and D are respectively the statistical results of the number of migrated and invaded cells.

[0045] Fig. 9 The neutralizing ability of the ROS-responsive biomimetic nano-drug delivery system coated with neutrophil membranes at different concentrations in the present invention on the inflammatory factors TNF-α (A) and IL-1β (B).

[0046] Fig.10 A is an in vitro imaging diagram of the targeted delivery of the bionic liposomes with different cell membrane protein to liposome mass ratios in the present invention to the hind limbs of a rat model of rheumatoid arthritis, and B is a quantitative diagram of its fluorescence intensity.

[0047] Fig.11 A is the targeted distribution diagram of the bionic liposomes with different cell membrane protein to liposome mass ratios in the present invention in the synovial tissue of the rat model of rheumatoid arthritis, and B is the quantitative diagram of the mean fluorescence intensity.

[0048] Fig.12 The figure shows the therapeutic effect of the neutrophil membrane-coated ROS-responsive bionic nanodrug delivery system on rats with rheumatoid arthritis. In the figure, A is a gross picture of the ankle joint of the rat after treatment, B is the arthritis severity score during the treatment, C is the change in foot volume of the arthritis rat during the treatment, and D is the change in body weight of the arthritis rat during the treatment.

[0049] Fig.13 This is an imaging evaluation of the therapeutic effect of the neutrophil membrane-coated ROS-responsive bionic nanodrug delivery system of the present invention on rats with rheumatoid arthritis. Figure A is the CT imaging result of the ankle joint of the rat after treatment, and Figure B is the ankle joint bone density result of the rat after treatment.

[0050] Fig.14 This is a graph showing the inflammation-alleviating effect of the neutrophil membrane-coated ROS-responsive bionic nanodrug delivery system of the present invention on rats with rheumatoid arthritis, in which A, B, C, and D are respectively the serum TNF-α, IL-6, IL-1β, and IL-10 levels of the rats after treatment. DETAILED DESCRIPTION

[0051] In order to make the technical solution of the present invention easier to understand, the technical solution of the present invention is now clearly and completely described in the form of specific embodiments in conjunction with the accompanying drawings.

[0052] Example 1

[0053] Extraction and preparation of neutrophil membranes:

[0054] HL-60 cells were passaged and expanded in IMDM medium + 20% FBS + 1% PS in a cell culture incubator at 37°C, 5% CO2. 1% DMSO was added to the cell culture medium to induce cell differentiation and 10 ng / mL TNF-α was given to stimulate the cells for 3 days. The cells were centrifuged at 1000 rpm / min for 5 min to remove the supernatant and washed three times with PBS (4°C, 1000 rpm / min, 5 min). Resuspend the cells with prepared membrane extraction buffer (225mM mannitol, 75mM sucrose, 0.5% BSA, 0.5mM EDTA, 30mMTris-HCl, 1% protease inhibitor), mechanically homogenize on ice, centrifuge (4°C, 800g, 10min), remove the nucleus and other structures, centrifuge the supernatant again (4°C, 10000g, 20min), remove the mitochondria and other structures, centrifuge the supernatant (4°C, 100000g, 60min), resuspend the cell membrane with distilled water after centrifugation, freeze-dry and store at -80°C for later use.

[0055] Preparation of blank liposomes:

[0056] According to ① the molar ratio of dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol is 20:1:10, accurately weighed and dissolved in chloroform; or, ② the molar ratio of soybean lecithin and cholesterol is 19:2, accurately weighed and dissolved in ethanol; or, ③ the molar ratio of hydrogenated soybean lecithin and cholesterol is 59:40, accurately weighed and dissolved in ethanol; or, ④ the molar ratio of dioleoyl lecithin, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol is 58:5:38, accurately weighed and dissolved in chloroform; or, ⑤ the molar ratio of (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol is 20:70:5.4:30, accurately weighed and dissolved in chloroform;

[0057] Blank liposomes were prepared according to the thin film hydration method: placed in a rotary evaporator, evaporated at 55°C for 30 min until a uniform film was formed, hydrated with pH 4.0 citrate, ultrasonicated in a water bath for 10 min, and extruded through 400nm, 200nm, and 100nm polycarbonate membranes in an extruder at 55°C. The average particle size of the liposomes was about 93nm as measured by a particle size analyzer, and the zeta potential result was about -20mV. The specific results are shown in Figure 1 The particle size change after storage at 4℃ for one week is relatively stable. Figure 2 The preparation methods of the remaining liposomes are the same as above. The particle sizes, potentials and dispersibility indexes thereof are shown in Table 1. Table 1 shows the particle sizes, potentials and dispersibility indexes of the liposomes with different formulations in the present invention.

[0058] Table 1 Particle size, potential and dispersibility index of liposomes with different formulations

[0059]

[0060] Preparation of ROS-responsive liposomal leonurine:

[0061] Soybean lecithin and cholesterol were accurately weighed and dissolved in ethanol at a molar ratio of 19:2. Blank liposomes were prepared according to the above method. The blank liposomes were treated with catalase at 20kHz ultrasound for 5 minutes to obtain ROS-responsive liposomes. Leonurine was dissolved in citrate at pH 4.0 and added to ROS-responsive liposomes (prepared as described above). The pH of the extralipidic aqueous phase was adjusted to 7.4 at 50°C, incubated for 5 minutes, and immediately removed and cooled on ice. After cooling, all the liquid in the centrifuge tube was transferred to an ultrafiltration centrifuge filter tube, centrifuged (5000rpm, 10min), and free leonurine was filtered out. After repeating the centrifugation three times, the upper layer of the ultrafiltration centrifuge tube was ROS-responsive liposome leonurine. The free leonurine after centrifugation was collected and quantified, and the free leonurine was determined by ultraviolet spectrophotometer.

[0062] According to the formula:

[0063] Encapsulation rate = (total leonurine - free leonurine) / total leonurine × 100%

[0064] Drug loading rate = (total leonurine - free leonurine) / ((total leonurine - free leonurine) + liposome mass) × 100%

[0065] Calculate the encapsulation efficiency and drug loading rate of leonurine, and it can be seen that Figure 3 Under the pH gradient method, the encapsulation rate of leonurine in liposomes reached 80%, and the drug loading rate was about 7.5%.

[0066] Preparation of neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system:

[0067] Hydrogenated soybean lecithin and cholesterol were accurately weighed in a molar ratio of 59:40 and dissolved in ethanol. Blank liposomes were prepared according to the above method. The blank liposomes were treated with catalase at 40 kHz for 30 min. The cell membrane was mixed with ROS-responsive liposomes leonurine or blank liposomes (as described above) at 1:1, 1:10, 1:100, and 1:500, and then treated with ultrasound for 5 min and extruded through 200 nm and 100 nm polycarbonate membranes in a membrane extruder to obtain a regular neutrophil membrane-coated ROS-responsive bionic nano-drug delivery system, as shown in FIG. Figure 1The average particle size of the liposomes was about 93nm as measured by a particle size analyzer, and the particle size of the liposomes coated with neutrophils of different proportions increased to about 100-110nm. The original potential of the liposomes, about -20mV, changed significantly after being coated with the negatively charged neutrophil membrane. The potential of the neutrophil membrane-coated liposomes and the neutrophil membrane-coated ROS-responsive biomimetic liposomes with motherwort alkaloids was about -26mV. Figure 2 The particle size of blank liposomes and neutrophil membrane-coated ROS-responsive biomimetic liposomes of leonurine was relatively stable after storage at 4°C for one week. The free liposomes, neutrophil membranes and biomimetic liposomes were subjected to gel electrophoresis and Coomassie brilliant blue staining and Western blot. Figure 4 B. It can be observed that the bionic nano-drug delivery system has related proteins consistent with the neutrophil membrane. The bionic liposomes retain proteins such as LFA-1, TNF-αR, IL-1R, CXCR2, CCR2 and CCR5 of the neutrophil membrane.

[0068] Example 2

[0069] Verification of the ROS responsiveness of the neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system:

[0070] Dioleylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol were accurately weighed in a molar ratio of 58:5:38 and dissolved in chloroform. The neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system (Leo@CAT@NM-Lipo) was prepared according to the above method. Leo@CAT@NM-Lipo was added to PBS containing 50μM H2O2 and incubated for 12h to observe its structural changes. Figure 5 A. Under transmission electron microscopy, it can be observed that the drug delivery system without H2O2 treatment presents a regular spherical structure, and after H2O2 treatment, it presents a fragmented structure, and the nanoparticles aggregate into clusters. This is because the CAT catalyzes H2O2 to produce oxygen, which destroys the structure of the drug delivery system. 1 mg / mL Leo@NM-Lipo and Leo@CAT@NM-Lipo (containing 10, 20, 50, 100, 200 μg / mL CAT) were added to PBS containing 50 μM H2O2 and incubated for 12 h. Figure 5 B. No O2 production was found in the Leo@NM-Lipo group, while the oxygen production in Leo@CAT@NM-Lipo increased with the increase of CAT content. A large number of oxygen bubbles were observed in both the 100μg / mL and 200μg / mL CAT groups. The in vitro release behavior of motherwort alkaloids in 50μM H2O2 at 37°C PBS was investigated by dialysis, as well as the in vitro release behavior of motherwort alkaloid liposomes and neutrophil membrane-coated biomimetic nano-drug delivery systems. Figure 5C, the release of free leonurine was close to 80% in 2h, while the neutrophil membrane-coated biomimetic liposome leonurine and the neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system continued to release until 12h, and the release curve became flat. It can be seen that the biomimetic nano-drug delivery system has a sustained release effect and can achieve the purpose of continuous drug delivery. However, Leo@NM-Lipo has the slowest release rate because CAT is not added. With the increase of CAT in Leo@CAT@NM-Lipo, the release of Leo gradually accelerates. 100μg / mLCAT and 200μg / mL CAT seem to have the same release behavior, so 100μg / mL CAT was used in subsequent studies. Blank liposomes were treated with catalase at 40kHz ultrasound for 30min, and the method for preparing liposomes was the same as above according to the molar ratio of dioleoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol of 58:5:38. ① The molar ratio of dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol is 20:1:10, accurately weighed, and dissolved in chloroform; ② The molar ratio of soybean lecithin and cholesterol is 19:2, accurately weighed, and dissolved in ethanol; ③ The molar ratio of hydrogenated soybean lecithin and cholesterol is 59:40, accurately weighed, and dissolved in ethanol; ④ The molar ratio of dioleoyl lecithin, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol is 58:5:38, accurately weighed, and dissolved in chloroform; ⑤ The molar ratio of (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol is 20:70:5.4:30, accurately weighed, and dissolved in chloroform.

[0071] Example 3

[0072] Targeting ability of neutrophil membrane-coated biomimetic liposomes to synovial fibroblasts:

[0073] (2,3-Dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol were accurately weighed and dissolved in chloroform at a molar ratio of 20:70:5.4:30, and neutrophil membrane-coated biomimetic liposomes (NM-Lipo) were prepared according to the above method. MH7A cells were seeded on a 24-well plate slide and cultured with a culture medium containing 20 ng / mL TNF-α for 6 h. NM-Lipo-DiD with different membrane protein-lipid ratios (0:1, 1:500, 1:100, 1:10, 1:1) was added to MH7A cells and incubated for 4 h. The concentration of nanoparticles in each group was maintained at 30 μg / mL. In addition, MH7A cells pretreated with excess NM (10 μg / mL) for 1 h were incubated with NM-Lipo-DiD (1:10) for 4 h. After DAPI staining, the images were taken under a confocal microscope. Figure 6, the increase in the fluorescence signal of the nanoparticles targeting MH7A is positively correlated with the amount of NM. This is due to the enhanced interaction between LFA-1 expressed by NM and ICAM-1 on MH7A cells. After NM pretreatment of MH7A cells, followed by incubation with DiD-labeled NM-Lipo (1:10), the fluorescence signal was significantly weakened, further indicating that NM protein plays a key role in the targeting process of MH7A cells.

[0074] Example 4

[0075] Safety evaluation of neutrophil membrane-coated ROS-responsive biomimetic nano-drug delivery system:

[0076] The cell culture density of 96-well plates was 1 × 10 4 MH7A cells and RAW 264.7 cells were incubated with different concentrations (1, 10, 20, 50, 100, 200, 500 and 1000 μg / mL) of Leo@CAT@NM-Lipo for 24 h, then gently rinsed with PBS, replaced with 90 μL of culture medium and 10 μL of CCK-8, and incubated at 37°C for 4 h. The absorbance was measured at 450 nm using an ELISA reader, and the cell viability was calculated to evaluate the cytotoxicity of Leo@CAT@NM-Lipo. Figure 7 As shown, the cell viability of both cells was over 90% at different concentrations, confirming that Leo@CAT@NM-Lipo had no obvious toxicity to MH7A cells and RAW 264.7 cells at the predetermined dose.

[0077] Example 5

[0078] Evaluation of the inhibitory effect of neutrophil membrane-coated ROS-responsive biomimetic nanodrug delivery system on the migration and invasion of synovial fibroblasts:

[0079] MH7A cells were activated with 20 ng / mL TNF-α for 6 h and pretreated with PBS, Leo, Leo@NM-Lipo, and Leo@CAT@NM-Lipo (10 μM Leo), respectively. Cells without any treatment were used as the control group. 1×10 4 Cells were inoculated in a serum-free medium without matrix gel coated Transwell chamber, and complete medium containing 10% FBS was added to the lower chamber. After incubation for 24 hours, the remaining cells in the chamber were gently wiped with a cotton swab, and the cells that migrated through the membrane to the back were fixed with 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet for 15 minutes, and the number of migrated cells was counted in three random fields under an optical microscope. The invasion experiment is similar to the migration experiment, but the matrix gel coated Transwell chamber is used in the invasion experiment. Figure 8TNF-α stimulation significantly enhanced the migration and invasion ability of MH7A cells. Leonurine treatment significantly reduced the migration and invasion of cells. Compared with the PBS group, Leo, Leo@NM-Lipo, and Leo@CAT@NM-Lipo all reduced the number of migrating and invasive cells. Leo@CAT@NM-Lipo had a better inhibitory effect, which was due to the targeted and ROS-responsive drug release of NM.

[0080] Example 6

[0081] Neutralization of inflammation in vitro by neutrophil membrane-coated biomimetic liposomes:

[0082] Leo@CAT@NM-Lipo was diluted to a final concentration of 0, 32, 64, 128, 256, 512, 1024, and 2048 μg / mL and mixed with TNF-α and IL-1β, respectively. After incubation at 37°C for 1 h, the nanoparticles were removed by centrifugation at 12,000 rpm at 4°C for 1 h, and the remaining TNF-α and IL-1β contents in the supernatant were detected by ELISA. The binding ability of Leo@CAT@NM-Lipo to TNF-α and IL-1β was determined to evaluate its ability to neutralize inflammation. Fig. 9 As shown, Leo@CAT@NM-Lipo exhibits concentration-dependent binding ability to TNF-α and IL-1β. The half-maximal inhibition concentration of Leo@CAT@NM-Lipo for TNF-α is as low as 132.8μg / mL, and for IL-1β binding is as low as 117.9μg / mL, indicating that NM-Lipo has a strong ability to neutralize inflammation.

[0083] Example 7

[0084] Targeted study of neutrophil membrane-coated biomimetic nano-drug delivery system on AIA model:

[0085] SD rats weighing about 180 g were subcutaneously injected at the base of their tails with 0.2 mg of heat-killed Mycobacterium tuberculosis (ground in an ice bath) dispersed in 100 μL of mineral oil. About 9-13 days later, the ankle joints of the rats became red and swollen, and the foot volume increased, which was considered a successful model construction and was used for subsequent experiments.

[0086] AIA model rats were randomly divided into 5 groups, 3 rats in each group, and injected with DiD, Lipo-DiD, NM-Lipo-DiD 1∶1, NM-Lipo-DiD 1∶10, and NM-Lipo-DiD 1∶100 through the tail vein. Three healthy rats without modeling were injected with NM-Lipo-DiD 1∶10 through the tail vein for comparison. After 24 hours, the hind limbs of the rats were imaged in vitro using an in vivo imaging system (IVIS, USA), and the fluorescence intensity was calculated. The synovial tissue was taken out and embedded in OCT for frozen sectioning, and the fluorescence distribution and intensity in the synovial tissue were observed under a laser confocal microscope. Fig.10 After in vitro treatment, obvious fluorescence signals were observed in the inflamed joints of AIA rats. The fluorescence intensity of NM-Lipo-DiD prepared with a membrane protein to lipid ratio of 1:100, 1:10, and 1:1 in the inflamed joints was 1.2, 2.2, and 2.8 times that of the Lipo-DiD group, respectively. However, the distribution of NM-Lipo-DiD 1:10 in the normal group was significantly less than that in the AIA model group. In the model mice, the targeted fluorescence intensity of NM-Lipo-DiD 1:10 was significantly higher than that of NM-Lipo-DiD 1:10, but there was no statistical difference with NM-Lipo-DiD 1:1.

[0087] In order to further verify the targeting specificity of NM-Lipo-DiD 1:10 on inflammatory FLS, synovial tissue sections were collected to observe the distribution of biomimetic liposomes in the synovium. Fig.11 The fluorescence intensity of NM-Lipo-DiD1:10 and NM-Lipo-DiD 1:1 in the synovium of AIA model mice was higher than that of the Lipo-DiD group and the NM-Lipo-DiD 1:100 group. The fluorescence intensity of NM-Lipo-DiD1:10 in the inflammatory synovium of rheumatoid arthritis was significantly stronger than that in the normal synovium, indicating that NM-Lipo-DiD has a stronger targeting ability to the inflammatory synovium than to the normal synovium.

[0088] Example 8

[0089] The therapeutic effect of neutrophil membrane-coated ROS-responsive biomimetic nanodrug delivery system on AIA model:

[0090] AIA rat modeling was performed as described in Example 10. AIA model rats were randomly divided into 7 groups, 6 rats in each group. Starting from the 9th day of modeling, PBS, NM-Lipo (membrane protein: lipid is 1:10), Leo, Leo@Lipo, Leo@NM-Lipo (membrane protein: lipid is 1:10), Leo@CAT@NM-Lipo L (membrane protein: lipid is 1:100), Leo@CAT@NM-LipoH (membrane protein: lipid is 1:10) were injected into the tail vein at a dose of 10 mg / kg / day of leonurine for 3 weeks. The PBS group was used as the model group, and 6 healthy rats without modeling were selected as the control group. The remaining groups were named NM-Lipo, Leo, Leo@Lipo, Leo@NM-Lipo, Leo@CAT@NM-Lipo L, and Leo@CAT@NM-Lipo H. During the treatment, arthritis scores were performed every 3 days, and the changes in rat foot volume and weight were measured using a foot volume meter. After the administration, all rats were anesthetized, and blood was collected from the abdominal aorta, and inflammatory factors TNF-α, IL-6, IL-1β, and IL-10 were determined using ELISA kits; the corresponding hind feet were removed and observed and bone density analyzed using a Micro-CT imaging system.

[0091] The scoring rules for arthritis limbs are as follows (the total score of the limbs is used as the final score):

[0092] 0 points: no erythema or swelling;

[0093] 1 point: slight erythema and swelling;

[0094] 2 points: diffuse erythema and slight swelling;

[0095] 3 points: moderate erythema and swelling;

[0096] 4 points: severe erythema and swelling, and joint deformity.

[0097] like Fig.12 As shown in A, Leo, Leo@Lipo, Leo@NM-Lipo, Leo@CAT@NM-Lipo L and Leo@CAT@NM-LipoH can significantly reduce the redness and swelling of arthritic feet. The arthritis score was used to comprehensively evaluate the disease progression of different treatments in each group (Figure). Fig.12B. AIA rats in the model group showed natural disease progression, with a final score of 11.2 points, showing the most severe arthritis manifestations. The arthritis scores of AIA rats in the Leo@CAT@NM-Lipo H treatment group showed a slight upward trend, and the final average score was only 5.2 points, indicating that the treatment effect was the best. Although not as good as the Leo@CAT@NM-Lipo H group, the treatment effects of the Leo@NM-Lipo and Leo@CAT@NM-Lipo L groups were still relatively good, with average arthritis scores of 7.3 and 6.3 points, respectively. The difference in efficacy between the Leo@CAT@NM-Lipo H group and the Leo@CAT@NM-Lipo L group may be related to the amount of neutrophil membrane. Fig.12 C, The change trend of the foot volume increase in each group was consistent with the change trend of the arthritis score. The results showed that compared with the model group, the swelling of the RA foot in the Leo-treated rats was significantly reduced. Compared with the Leo group, Leo@Lipo, Leo@NM-Lipo, Leo@CAT@NM-Lipo L and Leo@CAT@NM-Lipo H all significantly reduced the foot volume, and the therapeutic effect of Leo@CAT@NM-Lipo H was significantly improved compared with Leo@CAT@NM-Lipo L. Leo@CAT@NM-Lipo H showed a better therapeutic effect. The progression of rheumatoid arthritis affects the body weight of rats. Fig.12 As shown in D, the weight gain of AIA rats was significantly slower than that of the normal control group. As the treatment time prolonged, the weight of mice in the Leo@CAT@NM-Lipo H group gradually became similar to that of the control group, and the weight gain trend was consistent with the treatment effect.

[0098] Bone erosion is a key pathological feature of rheumatoid arthritis and is often used as an indicator to assess the severity of the disease. Micro-CT was used to detect changes in bone tissue in each group of rats. Fig.13 As shown, compared with the control group, the inflamed ankle bone surface of the model group was rough and severely eroded, and the bone mineral density (BMD) was significantly reduced. Rats in the model group and NM-Lipo group showed obvious severe bone destruction, rough bone surface, and severe bone erosion. In contrast, only slight bone erosion was observed in the ankle joints of the Leo, Leo@Lipo, Leo@NM-Lipo, Leo@CAT@NM-Lipo L, and Leo@CAT@NM-Lipo H groups, and the degree of bone destruction decreased in turn, indicating that Leo@CAT@NM-Lipo H significantly reduced the degree of bone erosion in rheumatoid arthritis.

[0099] The levels of TNF-α, IL-6, IL-1β and IL-10 in rat serum were detected by ELISA. Fig.14The results showed that the levels of serum TNF-α, IL-6 and IL-1β in AIA rats were significantly higher than those in normal rats. Compared with the model group, the levels of cytokines in the NM-Lipo, Leo, Leo@Lipo, Leo@NM-Lipo, Leo@CAT@NM-LipoL and Leo@CAT@NM-Lipo H groups were all reduced, while the change trend of the anti-inflammatory cytokine IL-10 in serum was the opposite. Among them, the TNF-α, IL-6 and IL-1β in the Leo@CAT@NM-Lipo H group decreased most significantly, the IL-10 level was the highest, and the anti-inflammatory effect was the most obvious.

[0100] It should be noted that the embodiments described herein are only partial embodiments of the present invention, rather than all implementations of the present invention, and the embodiments are only exemplary, and their role is only to provide a more intuitive and clear way to understand the content of the present invention, rather than to limit the technical solutions described in the present invention. Without departing from the concept of the present invention, all other implementation methods that can be thought of by ordinary technicians in this field without creative work, and other simple replacements and various changes to the technical solutions of the present invention, all belong to the protection scope of the present invention.

Claims

1. A ROS-responsive bionic nano drug delivery system, characterized in that: The ROS-responsive bionic nano drug delivery system is a neutrophil membrane-coated ROS-responsive bionic nano drug delivery system, comprising a liposome nanocarrier encapsulating leonurine and catalase and an activated neutrophil membrane, the liposome is prepared by a thin film hydration method, the catalase is loaded by an ultrasound method, the leonurine is loaded by a pH gradient method, and the liposome encapsulating leonurine and catalase and the neutrophil membrane are co-extruded by a membrane extruder to achieve outer membrane coating.

2. The ROS-responsive biomimetic nano drug delivery system according to claim 1, characterized in that: The activated neutrophil membrane is obtained by DMSO-induced differentiation of the HL-60 cell line, and after further inflammatory stimulation, the cell membrane surface overexpresses relevant adhesion molecules, inflammatory factor receptors and chemokine receptors; the activated neutrophil membrane is obtained by mechanical grinding and differential centrifugation extraction in sequence, and cell membrane vesicles are obtained by ultrasonic treatment; the relevant adhesion molecules, inflammatory factor receptors and chemokine receptors include LFA-1, TNF-αR, IL-1R, CXCR2, CCR2 and CCR5.

3. The ROS-responsive biomimetic nano drug delivery system according to claim 2, characterized in that: The encapsulated leonurine is leonurine or its salt, which is the effective ingredient of the traditional Chinese medicine motherwort, and the liposome nanocarrier components are phospholipids and cholesterol; The phospholipid is one or a mixture of soybean lecithin, hydrogenated soybean lecithin, dioleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and their derivatives; the organic solvent is one or a mixture of ethanol and chloroform.

4. The ROS-responsive biomimetic nano drug delivery system according to claim 1, characterized in that: The liposome structure is a hollow phospholipid bilayer structure, which can encapsulate leonurine and catalase, with a corresponding encapsulation rate between 30% and 90%, and a drug loading between 0.5% and 20%.

5. The ROS-responsive biomimetic nano drug delivery system according to claim 1, characterized in that: The method for encapsulating catalase CAT is an ultrasonic method with an ultrasonic intensity of 20kHz to 40kHz and an ultrasonic time of 5 to 30min. The pH gradient method for encapsulating leonurine Leo uses an initial pH range of 4 to 5 and a pH range of 7 to 8 after adjustment.

6. The ROS-responsive biomimetic nano drug delivery system according to claim 1, characterized in that: The liposomes and neutrophil membranes were fused with each other by co-extrusion 10 to 20 times.

7. The ROS-responsive biomimetic nano drug delivery system according to claim 1, characterized in that: The average particle size of the liposome is 60-100 nm, and the particle size of the ROS-responsive bionic nano drug delivery system coated with the neutrophil membrane is 80-130 nm.

8. A method for preparing the ROS-responsive bionic nano drug delivery system as claimed in claim 1, comprising the following steps: Step 1: extracting neutrophil membranes, HL-60 cells were induced to differentiate with DMSO and stimulated with TNF-α to upregulate the expression of LFA-1, TNF-αR, IL-1R, CXCR2, CCR2, and CCR5, and the cell membranes extracted by mechanical grinding and differential centrifugation were dispersed in double distilled water and ultrasonically dispersed; Step 2, using phospholipids such as soybean lecithin, hydrogenated soybean lecithin, dioleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and its derivatives as raw materials to prepare liposomes by a thin film hydration method, encapsulate catalase by ultrasonic treatment, and use citrate buffer to encapsulate the leonurine drug by a pH gradient method to prepare ROS-responsive liposome leonurine; Step three, the pre-prepared liposomes encapsulating leonurine and catalase are co-incubated with the neutrophil membrane and repeatedly extruded through the membrane using a membrane extruder to obtain a ROS-responsive bionic nano drug delivery system.

9. The method for preparing the ROS-responsive bionic nano drug delivery system according to claim 8, characterized in that: In step 2, blank liposomes and catalase are ultrasonically treated at 20kHz to 40kHz for 5 to 30min to obtain ROS-responsive liposomes, leonurine is dissolved in citrate at pH 4.0, and ROS-responsive liposomes are added, and the pH value of the extraliposomal aqueous phase is adjusted to 7.4 at 50°C, incubated for 5min, and immediately taken out and cooled on ice.

10. The use of the ROS-responsive bionic nano drug delivery system according to claim 1, characterized in that: Application of ROS-responsive biomimetic nanodrug delivery system in preparations for targeted treatment of rheumatoid arthritis.

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