Bionic lipid nano composite material, preparation method and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a bionic lipid nano composite material, a preparation method and application. The composite material is composed of lipid nanoparticles, Anxa1mRNA, a biomimetic membrane and phosphatidylated hyaluronic acid, the Anxa1mRNA is physically packaged in the lipid nanoparticles to form Anxa1mRNA-loaded lipid nanoparticles, the biomimetic membrane is biologically camouflaged on the outermost layer of the Anxa1mRNA-loaded lipid nanoparticles, the phosphatidylated hyaluronic acid is inserted into the surface of the biomimetic membrane, and the lipid nanoparticles are loaded with the Anxa1mRNA. The lipid nanoparticles loaded with the Anxa1 mRNA are prepared from the Anxa1 mRNA and the lipid nanoparticles; the particle size of the bionic lipid nano composite material ranges from 100 nm to 150 nm. According to the bionic lipid nano composite material disclosed by the invention, the anti-inflammatory effect of Anxa1 is utilized, so that the targeted therapy of acute pancreatitis is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a bionic lipid nanocomposite material, a preparation method and an application thereof. Background Art

[0002] Acute pancreatitis, or AP, is caused by abnormal activation of trypsin due to multiple factors, leading to acinar cell apoptosis and pancreatic aseptic inflammation, often accompanied by systemic inflammatory response syndrome. Severe acute pancreatitis manifests itself in persistent organ failure and multi-organ dysfunction.

[0003] Annexin A1, or Anxa1, is the first member of the annexin superfamily of calcium-dependent phospholipid-binding proteins discovered. Studies have shown that Anxa1 is involved in anti-inflammatory effects. In addition, studies have confirmed that Anxa1 can enhance the recognition, phagocytosis and clearance of apoptotic cells by phagocytes. In different inflammatory models, the application of Anxa1 and its derivative peptides can also play an anti-inflammatory role. The inflammatory response runs through the entire process of AP occurrence and development. The Anxa1 protein expressed by inflammatory macrophages plays an important role in AP inflammation, promoting macrophages to clear apoptotic cells and inhibiting inflammation.

[0004] Studies have found that Anxa1-derived peptides have a short half-life and lack targeting. Therefore, in the existing technology, the application of Anxa1 in the treatment of AP is limited, and there is a lack of products that can load Anxa1 and deliver it to the area where inflammation occurs. Summary of the invention

[0005] The purpose of the present invention is to provide a bionic lipid nanocomposite material, a preparation method and an application thereof, which solve the shortcomings of Anxa1 being easily metabolized and cleared quickly and lacking active targeting.

[0006] The technical scheme adopted by the present invention is a bionic lipid nanocomposite material loaded with membrane annexin A1 mRNA, wherein the composite material is composed of lipid nanoparticles, Anxa1 mRNA, a bionic membrane and phospholipidated hyaluronic acid, wherein the Anxa1 mRNA is physically encapsulated in the lipid nanoparticles to form lipid nanoparticles loaded with Anxa1 mRNA, wherein the bionic membrane is biologically camouflaged in the outermost layer of the lipid nanoparticles loaded with Anxa1 mRNA, the phospholipidated hyaluronic acid is inserted into the surface of the bionic membrane, and the lipid nanoparticles loaded with Anxa1 mRNA are made of Anxa1 mRNA and lipid nanoparticles; and the particle size of the bionic lipid nanocomposite material is 100nm to 150nm.

[0007] A method for preparing a bionic lipid nanocomposite material loaded with membrane annexin A1 mRNA comprises the following steps: collecting macrophage membranes to prepare a bionic membrane dispersion; preparing a phospholipidated hyaluronic acid solution; preparing a lipid nanoparticle solution; dissolving Anxa1 mRNA in a citrate buffer to prepare an Anxa1 mRNA solution; mixing the lipid nanoparticle solution with the Anxa1 mRNA solution, physically encapsulating Anxa1 mRNA in the lipid nanoparticles to generate lipid nanoparticles loaded with Anxa1 mRNA; mixing the lipid nanoparticles loaded with Anxa1 mRNA and the bionic membrane dispersion, extruding, and finally camouflaging the bionic membrane in the outermost layer of the lipid nanoparticles loaded with Anxa1 mRNA to prepare bionic lipid nanoparticles loaded with Anxa1 mRNA; adding the phospholipidated hyaluronic acid solution to the bionic lipid nanoparticles loaded with Anxa1 mRNA, mixing, and inserting the phospholipidated hyaluronic acid into the surface of the bionic membrane to prepare a bionic lipid nanocomposite material loaded with Anxa1 mRNA.

[0008] Preferably, the macrophages are any one of RAW264.7, THP-1 and primary macrophages. Preferably, when preparing the Anxa1 mRNA solution, the mass volume ratio of the Anxa1 mRNA to the citrate buffer is 0.2-1:1.

[0009] Preferably, when generating lipid nanoparticles loaded with Anxa1 mRNA, the volume ratio of the lipid nanoparticle solution to the Anxa1 mRNA solution is 1 to 2:3.

[0010] Preferably, when preparing the Anxa1 mRNA-loaded biomimetic lipid nanoparticles, the mass volume ratio of the Anxa1 mRNA-loaded lipid nanoparticles to the biomimetic membrane dispersion is 5 to 10:1.

[0011] Preferably, when preparing the Anxa1 mRNA-loaded biomimetic lipid nanocomposite material, the mass ratio of the phospholipidated hyaluronic acid solution to the Anxa1 mRNA-loaded biomimetic lipid nanoparticles is 1:2-4.

[0012] An application of a bionic lipid nanocomposite material loaded with annexin A1 mRNA, wherein the bionic lipid nanocomposite material is used for preparing an anti-pancreatitis preparation.

[0013] Preferably, the pancreatitis is acute pancreatitis.

[0014] The beneficial effects of the present invention are as follows: the present invention provides a biomimetic nanocomposite with high safety and biodegradability, and utilizes the design features of biomimetic membrane and hyaluronic acid modification to achieve specific aggregation and penetration of drugs in lesions and improve the bioavailability of drugs to target cells. The composite nanomaterial of the present invention physically encapsulates membrane annexin A1 by lipid nanoparticles, the biomimetic membrane is camouflaged in the outermost layer of the nanocomposite material, and the phospholipidated hyaluronic acid is inserted into the surface of the biomimetic membrane. When acute pancreatitis occurs, the inflammatory environment of the pancreatic site secretes chemokines to recruit macrophages, and the outermost biomimetic membrane realizes the long blood circulation and lesion site targeting ability of the nanocomposite through its macrophage inherent immune escape and recruitment characteristics. Utilizing the characteristics of hyaluronic acid interacting with the target cell surface receptor CD44, the target cell effectively utilizes the nanocomposite material, and improves its effect in treating acute pancreatitis. Using the biomimetic lipid nanocomposite material loaded with membrane annexin A1 mRNA described in the present invention, the anti-inflammatory effect of membrane annexin A1 is utilized to achieve targeted treatment of acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Material characterization of HA-M@Lip@Anxa1 NPs, A: encapsulation efficiency of HA-M@Lip@Anxa1 NPs; B: mRNA encapsulation concentration of HA-M@Lip@Anxa1 NPs; C: representative transmission electron microscopy image of HA-M@Lip@Anxa1 NPs; D: particle size detection of HA-M@Lip@Anxa1 NPs; E: potential detection of HA-M@Lip@Anxa1 NPs.

[0016] Figure 2 Figure 2 shows the targeting of HA-M@Lip@Anxa1 NPs to inflammatory cells in vitro. A: Cell fluorescence image of inflammatory macrophages. a is the fluorescence image of the Lip@ce6 group when not treated with LPS. b is the DAPI nuclear staining result of a. m is the composite image of a and b. c is the fluorescence image of the M@Lip@ce6 group when not treated with LPS. d is the DAPI nuclear staining result of c. n is the composite image of c and d. e is the fluorescence image of the HA-M@Lip@ce6 group when not treated with LPS. f is the DAPI nuclear staining result of e. o is the composite image of e and f. Composite image, g is the fluorescence image of the M@Lip@ce6 group after LPS treatment, h is the DAPI nuclear staining result of g, p is the composite image of g and h, i is the fluorescence image of the HA-M@Lip@ce6 group after LPS treatment, j is the DAPI nuclear staining result of i, q is the composite image of i and j, k is the fluorescence image of the HA-M@Lip@ce6+HA solution group after LPS treatment, l is the DAPI nuclear staining result of k, and r is the composite image of k and l; B: Fluorescence numerical statistics of inflammatory macrophages.

[0017] Figure 3 The targeting of HA-M@Lip@Anxa1 NPs to the pancreatic inflammation site in mice. A: Luminescence imaging of pancreatic tissue. From left to right, representative images of the control group, Ce6 group, Lip@ce6, and HA-M@Lip@ce6 group; B: Statistics of mouse luminescence imaging; C: Data statistics of pancreatic tissue luminescence imaging; D: Data statistics of luminescence imaging of major organs of mice.

[0018] Figure 4 This is the evaluation of the efficacy of different treatment groups in the treatment of acute pancreatitis. A: Schematic diagram of the treatment of mouse pancreatitis model; B: Detection and evaluation index pancreatic pathology score; C: Detection and evaluation index pancreatic tissue dry-wet weight ratio; D: Detection and evaluation index serum lipase; E: Detection and evaluation index myeloperoxidase; F: Detection and evaluation index interferon-β; G: Detection and evaluation index tumor necrosis factor-α.

[0019] Figure 5 The pictures of pancreatic tissue staining in different treatment groups for acute pancreatitis, A: 5x image of pancreatic tissue of mice in normal control group; B: 20x image of pancreatic tissue of mice in normal control group; C: 5x image of pancreatic tissue of mice in PBS group; D: 20x image of pancreatic tissue of mice in PBS group; E: 5x image of pancreatic tissue of mice in Anxa1mRNA AP group; F: 20x image of pancreatic tissue of mice in Anxa1 mRNA AP group; G: 5x image of pancreatic tissue of mice in Lip@Anxa1 AP group; H: 20x image of pancreatic tissue of mice in Lip@Anxa1AP group; I: 5x image of pancreatic tissue of mice in HA-M@Lip@Anxa1 AP group; J: 20x image of pancreatic tissue of mice in HA-M@Lip@Anxa1 AP group; K: 5x image of pancreatic tissue of mice in Ac2-26AP group; L is 20x image of pancreatic tissue of mice in Ac2-26 AP group.

[0020] Figure 6 Safety evaluation of HA-M@Lip@Anxa1 NPs. A: Body weight changes of mice within 30 days; B: Staining images of major organ tissues of mice; C~F: Bar graphs of changes in serum alanine aminotransferase, aspartate aminotransferase, creatinine and urea nitrogen levels of mice. DETAILED DESCRIPTION

[0021] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0022] The structure of traditional nanomaterials loaded with drugs usually combines drug molecules with the surface of nanomaterials. The drug molecules are connected to the surface of polymer nanoparticles through covalent bonds or the drug molecules are adsorbed on the surface of nanomaterials through physical forces such as van der Waals forces or electrostatic forces. However, these structures have the disadvantages of insufficient targeting, inability to deliver drugs to the lesion area, and easy to be quickly metabolized and cleared, which reduces the treatment efficiency.

[0023] The present invention proposes a novel bionic lipid nanocomposite material loaded with membrane annexin A1 mRNA, the structure of which consists of lipid nanoparticles, Anxa1 mRNA, a bionic membrane and phospholipidated hyaluronic acid. The Anxa1 mRNA is physically encapsulated in the lipid nanoparticles to form lipid nanoparticles loaded with Anxa1 mRNA. The bionic membrane is biologically camouflaged in the outermost layer of the lipid nanoparticles loaded with Anxa1 mRNA, and the phospholipidated hyaluronic acid is inserted into the surface of the bionic membrane, thereby solving the problem of being able to load Anxa1 and deliver it to the area where AP inflammation occurs.

[0024] Based on the same inventive concept, the present invention also provides a method for preparing a biomimetic lipid nanocomposite material, comprising the following steps:

[0025] First, a biomimetic membrane solution, a phospholipidated hyaluronic acid solution and a lipid nanoparticle dispersion loaded with Anxa1 mRNA were prepared respectively; then, the biomimetic membrane solution and the lipid nanoparticle dispersion loaded with Anxa1 mRNA were mixed and extruded to prepare biomimetic lipid nanoparticles loaded with Anxa1 mRNA; and the phospholipidated hyaluronic acid solution was added to the biomimetic lipid nanoparticles loaded with Anxa1 mRNA to obtain a biomimetic lipid nanocomposite material loaded with Anxa1 mRNA.

[0026] The bionic lipid nanocomposite material loaded with Anxa1 mRNA of the present invention physically encapsulates Anxa1 mRNA by lipid nanoparticles, the bionic membrane is camouflaged in the outermost layer of the nanocomposite material, and the phospholipidated hyaluronic acid is inserted into the surface of the bionic membrane. Cells are the most basic unit of the body, and characteristic proteins exist on their membranes to help them adapt to the complex internal environment of the body and move to specific parts. Therefore, by utilizing this characteristic of the cell membrane, bionic nanopreparations are constructed to escape the clearance of the body's immune system, to prolong the circulation half-life, and to increase the aggregation of nanopreparations at the disease site through the ligand-receptor interaction in the inflammatory microenvironment. When acute pancreatitis occurs, the inflammatory environment of the pancreas secretes chemokines to recruit macrophages. The bionic membrane of the outermost layer of the nanocomposite material of the present invention realizes the long blood circulation and lesion site targeting ability of the nanocomposite material through its macrophage inherent immune escape and recruitment characteristics. By utilizing the characteristics of the interaction between hyaluronic acid and the target cell surface receptor CD44, the target cells can effectively utilize the nanocomposite material, thereby improving its effect in treating acute pancreatitis.

[0027] Example 1: A method for preparing a biomimetic lipid nanocomposite material

[0028] 1. Preparation of biomimetic membrane solution

[0029] The membranes of macrophages were collected by using a membrane protein extraction kit purchased from Beyotime. Briefly, 1 mL of pre-cooled membrane protein extraction reagent A containing 1 mM PMSF was added to 1.0 × 10 7 RAW264.7 cells were treated at 4°C for 30 minutes. The cell suspension was then ultrasonically disrupted for 10 minutes, and then repeatedly frozen and thawed at -80°C and 37°C for 4 times. The supernatant was collected by centrifugation at 800 rpm for 10 minutes, and then centrifuged at 13000 rpm for 30 minutes to obtain macrophage membrane precipitation. The macrophage membrane was dissolved with PBS to obtain a biomimetic membrane solution, which was recorded as:

[0030] 2. Preparation of phospholipidated hyaluronic acid

[0031] 8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC, 16 mg of N-hydroxysuccinimide NHS and 1 mg of hyaluronic acid were weighed and dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of hyaluronic acid. Subsequently, 1 mg of phospholipid polyethylene glycol amino DSPE-PEG was added. 2000 -NH2 was added to the above solution and stirred at 800 rpm for 24 hours at room temperature. The above mixed solution was placed in a 3.5 kDa dialysis bag and dialyzed for 24 hours to remove free EDC, NHS and DSPE-PEG 2000 -NH2. Finally, the purified material was freeze-dried by freeze drying to obtain phospholipid hyaluronic acid, HA, and the freeze-dried phospholipid hyaluronic acid was placed in a -20°C refrigerator for later use.

[0032] 3. Preparation of lipid nanoparticle dispersion loaded with annexin A1 mRNA

[0033] Preparation of DMG-PEG containing ionizable lipid, auxiliary lipid, cholesterol and 3-(2-methoxyethoxy)propane-1,2-diyldioctadecanoate 2000 The ionizable lipid is 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate SM-102, and the auxiliary lipid is dioleoylphosphatidylethanolamine DOPE.

[0034] In anhydrous ethanol, SM-102, DOPE, cholesterol and DMG-PEG 2000The molar ratio is 0.35:0.11:0.465:0.02.5.

[0035] An aqueous phase containing annexin A1 mRNA was prepared in 50 mM citrate buffer at pH 4, with a mass volume ratio of Anxa1 mRNA to citrate buffer of 1:1. TM The ethanol phase and the water phase were combined in a L nanomixer at a volume ratio of 1:3. The mixture was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h to obtain lipid nanoparticles loaded with membrane annexin A1 mRNA, denoted as: Lip@Anxa1 NPs.

[0036] 4. Preparation of biomimetic lipid nanoparticle dispersion loaded with annexin A1 mRNA

[0037] The lipid nanoparticles loaded with membrane annexin A1 mRNA and the biomimetic membrane dispersion were mixed, and the mass ratio of the lipid nanoparticles loaded with Anxa1 mRNA and the biomimetic membrane was 5: 1. The dispersion of biomimetic lipid nanoparticles loaded with membrane annexin A1 mRNA was obtained by repeated extrusion 11 times with a micro extruder with a pore size of 200 nm, which was recorded as: M@Lip@Anxa1 NPs.

[0038] 5. Preparation of biomimetic lipid nanocomposites loaded with annexin A1 mRNA

[0039] The phospholipidated hyaluronic acid was redissolved in PBS solution, 500 μg of the phospholipidated hyaluronic acid solution was added to the M@Lip@Anxa1 NPs solution, and stirred in a water bath at 37°C and 800 rpm for 1 hour to obtain HA-M@Lip@Anxa1 NPs.

[0040] Example 2: A method for preparing a biomimetic lipid nanocomposite material

[0041] 1. Preparation of biomimetic membrane solution

[0042] The membranes of macrophages were collected by using a membrane protein extraction kit purchased from Beyotime. Briefly, 1 mL of pre-cooled membrane protein extraction reagent A containing 1 mM PMSF was added to 1.0 × 10 7 RAW264.7 cells were treated at 4°C for 30 minutes. The cell suspension was then ultrasonically disrupted for 10 minutes, and then repeatedly frozen and thawed at -80°C and 37°C for 4 times. The supernatant was collected by centrifugation at 800 rpm for 10 minutes, and then centrifuged at 13000 rpm for 30 minutes to obtain macrophage membrane precipitation. The macrophage membrane was dissolved with PBS to obtain a biomimetic membrane dispersion, which was recorded as:

[0043] 2. Preparation of phospholipidated hyaluronic acid

[0044] 8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC, 16 mg of N-hydroxysuccinimide NHS and 1 mg of hyaluronic acid were weighed and dissolved in PBS and stirred at 800 rpm for 30 min at room temperature to activate the carboxyl groups on the surface of hyaluronic acid. Subsequently, 5 mg of phospholipid polyethylene glycol amino DSPE-PEG 2000 -NH2 was added to the above solution and stirred at 1000 rpm for 24 hours at room temperature. The above mixed solution was dialyzed in a 3.5 kDa dialysis bag for 24 hours to remove free EDC, NHS and DSPE-PEG 2000 -NH2. Finally, the purified material was freeze-dried by freeze drying to obtain phospholipid hyaluronic acid, HA, and the freeze-dried phospholipid hyaluronic acid was placed in a -20°C refrigerator for later use.

[0045] 3. Preparation of lipid nanoparticle dispersion loaded with annexin A1 mRNA

[0046] Preparation of DMG-PEG containing ionizable lipid, auxiliary lipid, cholesterol and 3-(2-methoxyethoxy)propane-1,2-diyldioctadecanoate 2000 The ionizable lipid is 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate SM-102, and the auxiliary lipid is dioleoylphosphatidylethanolamine DOPE.

[0047] In anhydrous ethanol, SM-102, DOPE, cholesterol and DMG-PEG 2000 The molar ratio is 0.5:0.16:0.385:0.015.

[0048] An aqueous phase containing annexin A1 mRNA was prepared in 50 mM citrate buffer at pH 4, with a mass volume ratio of Anxa1 mRNA to citrate buffer of 0.2:1. TM The ethanol phase and the water phase were combined in a L nanomixer at a volume ratio of 2:3. The mixture was placed in a 3.5 kDa dialysis bag and dialyzed with deionized water for 24 h to obtain lipid nanoparticles loaded with membrane annexin A1 mRNA, denoted as: Lip@Anxa1 NPs.

[0049] 4. Preparation of biomimetic lipid nanoparticle dispersion loaded with annexin A1 mRNA

[0050] The lipid nanoparticles loaded with membrane annexin A1 mRNA and the biomimetic membrane dispersion were mixed, and the mass ratio of the lipid nanoparticles loaded with Anxa1 mRNA and the biomimetic membrane was 10:1. The dispersion was repeatedly extruded 12 times by a micro extruder with a pore size of 200 nm to obtain a dispersion of biomimetic lipid nanoparticles loaded with membrane annexin A1 mRNA, which was recorded as: M@Lip@Anxa1 NPs.

[0051] 5. Preparation of biomimetic lipid nanocomposites loaded with annexin A1 mRNA

[0052] The phospholipidated hyaluronic acid was redissolved in PBS solution, 250 μg of the phospholipidated hyaluronic acid solution was added to the M@Lip@Anxa1 NPs solution, and stirred in a water bath at 37°C and 800 rpm for 1 hour to obtain HA-M@Lip@Anxa1 NPs.

[0053] The biomimetic lipid nanocomposites prepared in Example 1 and Example 2 were characterized. Figure 1 This is the biomimetic lipid nanocomposite prepared in Example 1, A: encapsulation efficiency of HA-M@Lip@Anxa1 NPs; B: mRNA encapsulation concentration of HA-M@Lip@Anxa1 NPs; C: representative TEM image of HA-M@Lip@Anxa1 NPs; D: particle size detection of HA-M@Lip@Anxa1 NPs; E: potential detection of HA-M@Lip@Anxa1 NPs.

[0054] Example 3: Application of a biomimetic lipid nanocomposite material

[0055] 1. Using the Lip@Anxa1 NPs prepared in Example 1, the in vitro and in vivo targeting of the HA-M@Lip@Anxa1 NPs biomimetic lipid nanocomposite material was determined by semi-quantitative means of detecting fluorescence intensity.

[0056] Experiment 1: A red fluorescent dye dihydrochlorin e6 was used to label Lip@Anxa1 NPs, M@Lip@Anxa1 NPs and HA-M@Lip@Anxa1 NPs with ce6, which were respectively denoted as Lip@ce6, M@Lip@ce6 and HA-M@Lip@ce6 to track the cellular uptake process. Transwell chambers were used to construct an acute pancreatitis microenvironment, where acinar cells and RAW264.7 cells were inoculated in the upper and lower chambers of a 24-well plate, respectively. The concentration of acinar cells in the upper chamber was 0.3 mM, and the concentration of RAW264.7 in the lower chamber was 1×10 per well. 5 After 24 h of treatment with 100 ng / mL lipopolysaccharide (LPS), the above-mentioned different materials were added to each upper chamber, and after incubation for 4 h, the cell nuclei were stained with DAPI and fluorescence analysis was performed.

[0057] Among them, Lip@ce6 was prepared by the following method: using the Lip@Anxa1NPs prepared in Example 1, 20 mg / mL ce6 prepared with DMSO was added dropwise to the Lip@Anxa1NPs. After thorough mixing, it was stirred at 800 rpm at 37°C for 1 hour, and then the mixture was dialyzed in pure water for 12 hours. The retention molecular weight was 3500Da to obtain Lip@ce6.

[0058] M@Lip@ce6 was prepared by the following method: Lip@ce6 and the bionic membrane treated with water bath ultrasound were mixed in a mass ratio of 1:2, and the mixture was repeatedly extruded 10 times through a micro extruder with a pore size of 200 nm to obtain the M@Lip@ce6 bionic nanoformulation.

[0059] HA-M@Lip@ce6 was prepared by the following method: phospholipidated hyaluronic acid was redissolved in PBS solution, 250 μg of phospholipidated hyaluronic acid solution was added to the M@Lip@ce6 solution, and stirred in a water bath at 37°C and 800 rpm for 1 hour to obtain HA-M@Lip@ce6.

[0060] like Figure 2 As shown in the figure, Lip@ce6 was obviously taken up by RAW264.7 cells. Strong red fluorescence signals were seen in RAW264.7 cells incubated with Lip@ce6 for 4 h, while almost no fluorescence was seen in RAW264.7 cells incubated with M@Lip@ce6 and HA-M@Lip@ce6, which indicated that Hybridization can increase the immune escape ability of the drug delivery system with the assistance of the original cell membrane protein. At the same time, modifying HA on the surface of nanoparticles NPs does not affect the immune escape ability of NPs. In addition, when HA-M@Lip@ce6 was incubated with LPS-treated RAW264.7 cells, the red fluorescence in inflammatory macrophages was significantly higher than that in RAW264.7 cells not treated with LPS. However, when inflammatory macrophages were incubated with M@Lip@ce6 or pretreated with HA solution, only weak fluorescence was observed. The above results indicate that endocytosis of inflammatory macrophages has a significant high uptake efficiency for HA-M@Lip@ce6 NPs.

[0061] Experiment 2: Six-week-old C57BL / 6J mice were injected with caerulein, 50 mg / kg caerulein was injected intraperitoneally 7 times, once every 1 hour, to establish a mouse acute pancreatitis model, and 4 mg / kg ce6, 4 mg / kg Lip@ce6 and 4 mg / kg HA-M@Lip@ce6 were injected through the tail vein. After 12 hours, the pancreas and major organs of the mice, including the heart, liver, spleen, lungs and kidneys, were isolated and imaged under a fluorescence imaging system. The targeting ability of HA-M@Lip@Anxa1 NPs to the pancreas in vivo was studied.

[0062] like Figure 3 As shown in Figure 2, 12 hours after intravenous administration, the fluorescence in the pancreas of mice treated with HA-M@Lip@ce6 NPs was stronger than that in other groups. However, only weak fluorescence was observed in the pancreas after treatment with ce6 and Lip@ce6 NPs. These results indicate that HA modification and The encapsulation technology effectively targeted inflammatory macrophages in the acute pancreatitis model. Fluorescence images of major organs showed that different NPs accumulated in the liver, and the fluorescence signal of the HA-M@Lip@Anxa1 NPs group was lower than that of the Lip@ce6 NPs group. These results indicate that Hybridization is beneficial to avoid the first-pass effect in the liver, thereby increasing the accumulation of HA-M@Lip@Anxa1 NPs in the pancreas.

[0063] 2. The HA-M@Lip@Anxa1 NPs prepared in Example 1 were used to treat acute pancreatitis.

[0064] The AP model of C57BL / 6J mice was established by intraperitoneal injection of 50 mg / kg caerulein 7 times with an interval of 1 hour. After the first injection of caerulein, the mice were randomly divided into 5 groups (n=8), and intravenously injected with PBS, 4 mg / kg Anxa1 mRNA, 4 mg / kg Lip@Anxa1 NPs, 4 mg / kg HA-M@Lip@Anxa1 NPs and 1.5 mg / kg active N-terminal peptide Ac2-26 of annexin A1, respectively, which were recorded as PBS AP group, Anxa1 mRNA AP group, Lip@Anxa1 AP group, HA-M@Lip@Anxa1AP group, and Ac2-26 AP group; the normal mouse group without caerulein injection was recorded as normal group or con group. The mice were killed 12 hours later, and the pancreatic tissue and blood of the mice were taken for detection to evaluate the therapeutic effect of different treatment groups.

[0065] The results are as follows Figure 4 and Figure 5As shown, HA-M@Lip@Anxa1 NPs can significantly reduce pancreatic damage, including pancreatic necrosis, edema and inflammation, and significantly reduce systemic inflammatory response in the treatment of acute pancreatitis, indicating that the bionic nanoformulation constructed by the present invention can effectively treat acute pancreatitis.

[0066] 3. The HA-M@Lip@Anxa1 NPs prepared in Example 1 were used for in vivo biosafety testing.

[0067] The results are as follows Figure 6 As shown, there was no significant weight loss in the mice in the HA-M@Lip@Anxa1NPs group compared with the PBS group and the Anxa1 mRNA group. In addition, no evidence of histological damage to the major organs in the HA-M@Lip@Anxa1NPs group, including the heart, liver, spleen, lung, and kidney, was observed compared with the PBS group. At the same time, the levels of alanine aminotransferase ALT, aspartate aminotransferase AST, creatinine, and urea in the serum of mice in the HA-M@Lip@Anxa1NPs group compared with the PBS group indicated that there was no significant damage to liver / kidney function. The results showed that HA-M@Lip@Anxa1NPs are safe for in vivo treatment.

[0068] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A biomimetic lipid nanocomposite material loaded with annexin A1 mRNA, characterized in that: The composite material is composed of lipid nanoparticles, Anxa1 mRNA, a biomimetic membrane and phospholipidated hyaluronic acid, the Anxa1 mRNA is physically encapsulated in the lipid nanoparticles to form Anxa1 mRNA-loaded lipid nanoparticles, the biomimetic membrane is biologically camouflaged in the outermost layer of the Anxa1 mRNA-loaded lipid nanoparticles, the phospholipidated hyaluronic acid is inserted into the surface of the biomimetic membrane, and the Anxa1 mRNA-loaded lipid nanoparticles are made of Anxa1 mRNA and lipid nanoparticles; The particle size of the bionic lipid nanocomposite material is 100nm-150nm.

2. The method for preparing the biomimetic lipid nanocomposite material according to claim 1, characterized in that: The following steps are involved: collecting macrophage membranes and preparing biomimetic membrane dispersions; preparing a phospholipidated hyaluronic acid solution; preparing lipid nanoparticle solutions; Anxa1 mRNA solution was prepared by dissolving Anxa1 mRNA in citrate buffer; The lipid nanoparticle solution is mixed with the Anxa1 mRNA solution, and the Anxa1 mRNA is physically encapsulated in the lipid nanoparticles to generate Anxa1 mRNA-loaded lipid nanoparticles; The Anxa1 mRNA-loaded lipid nanoparticles and the biomimetic membrane dispersion are mixed and extruded, and finally the biomimetic membrane is biologically camouflaged on the outermost layer of the Anxa1 mRNA-loaded lipid nanoparticles to prepare the Anxa1 mRNA-loaded biomimetic lipid nanoparticles; The phospholipidated hyaluronic acid solution is added to the Anxa1 mRNA-loaded biomimetic lipid nanoparticles, and the mixture is evenly mixed. The phospholipidated hyaluronic acid is inserted into the surface of the biomimetic membrane to prepare the Anxa1 mRNA-loaded biomimetic lipid nanocomposite material.

3. The preparation method according to claim 2, characterized in that: The macrophages are any one of RAW264.7, THP-1 and primary macrophages.

4. The preparation method according to claim 2, characterized in that: When preparing the Anxa1 mRNA solution, the mass volume ratio of the Anxa1 mRNA to the citrate buffer is 0.2-1:

1.

5. The preparation method according to claim 2, characterized in that: When generating lipid nanoparticles loaded with Anxa1 mRNA, the volume ratio of the lipid nanoparticle solution to the Anxa1 mRNA solution is 1 to 2:

3.

6. The preparation method according to claim 2, characterized in that: When preparing the Anxa1 mRNA-loaded bionic lipid nanoparticles, the mass volume ratio of the Anxa1 mRNA-loaded lipid nanoparticles to the bionic membrane dispersion is 5-10:

1.

7. The preparation method according to claim 2, characterized in that: When preparing the Anxa1 mRNA-loaded bionic lipid nanocomposite material, the mass ratio of the phospholipidated hyaluronic acid to the Anxa1 mRNA-loaded bionic lipid nanoparticles is 1:2-4.

8. The use of the biomimetic lipid nanocomposite material according to claim 1, characterized in that: The bionic lipid nanocomposite material is used for preparing an anti-pancreatitis preparation.

9. The use of the biomimetic lipid nanocomposite material according to claim 8, characterized in that: The pancreatitis is acute pancreatitis.

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