Curcumin-loaded engineered cell membrane nano-vesicle NVs-CUR and preparation method thereof

Abstract

The invention relates to the technical field of medicines, in particular to a curcumin-loaded engineered cell membrane nano-vesicle NVs-CUR and a preparation method thereof, and the preparation method comprises the following steps: S1, extruding a macrophage suspension through an extruder to obtain an NVs resuspension; and S2, adding the CUR mixed solution into the NVs resuspension solution, and carrying out ultrasonic treatment to obtain the nano vesicle NVs-CUR. As a novel drug delivery system, the NVs-CUR shows excellent performance in the aspects of physical stability, drug release and stability, cellular uptake ability, cytotoxicity and the like. The characteristics provide powerful support for application in treatment of diseases such as nerve injury and the like, and indicate the wide prospect in the field of biomedicine.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR and a preparation method thereof. Background Art

[0002] Restoration of neurological function following traumatic injury, neurodegeneration, or neuroinflammation is currently hampered by the lack of effective and clinically feasible biotechnologies for precise, cell-targeted therapeutics or diagnostics. Therefore, breakthroughs in biotechnology are needed to enhance and sustain the delivery of therapeutics and receptor foreign body responses. One promising avenue is to utilize nanotechnology inspired by naturally occurring cells. The composition and biological functions of cells in vivo as carriers avoid potential side effects produced by potential therapeutics. Therefore, understanding approaches that may be safer and easier to handle provides opportunities. For example, cell-derived exosomes are promising drug delivery systems because they are a mechanism of natural extracellular communication. However, new approaches are needed, given that the complexity and variability of cell-derived biomimetic tools adapted for the nervous system reduce their potential for scalable precision medicine.

[0003] Engineered cell membrane nanovesicles (NVs) form nanoscale vesicle structures by specific engineering modifications to cell membranes. Its characteristics include good biocompatibility and the ability to mimic the functions and characteristics of natural cell membranes; at the same time, its nanoscale size gives it unique physical and chemical properties. As an innovative drug delivery system, NVs have shown great potential in the field of drug delivery. It can achieve precise drug delivery, improve the therapeutic effect of drugs and reduce side effects; by adjusting its surface membrane proteins, NVs can achieve targeted drug delivery, thereby improving the targeting and bioavailability of drugs, and can effectively target specific tissues or cells, enhance the targeting of treatment, and thus enhance the efficacy of drugs; in addition, it also shows great potential in the fields of bioimaging and disease diagnosis, bringing new breakthroughs and opportunities to medical and biological research.

[0004] CUR is a natural bioactive substance with multiple beneficial biological effects such as antioxidant and anti-inflammatory effects. It has been widely studied and confirmed to have the potential to resist neuronal cell damage. CUR has the ability to affect multiple biological targets and has shown activity against a variety of diseases (including cancer, cardiovascular disease, neurological diseases, and autoimmune diseases). CUR can regulate a variety of biological targets (including transcription factors, growth factors, inflammatory mediators, cytokines, cell cycle proteins, enzymes, protein kinases, and apoptotic proteins) and cellular pathways. However, curcumin itself has some limitations. For example, barriers to the therapeutic application of CUR include low bioavailability, poor pharmacokinetic characteristics, and a short half-life in the gastrointestinal tract. Another challenge as a potential therapeutic agent is its poor stability under physiological conditions. Therefore, it is of great significance to explore how to use NVs to carry curcumin to achieve effective protection and repair of neurons for the treatment of hypothalamic nerve damage caused by heat stroke. Summary of the invention

[0005] The object of the present invention is to provide a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR and a preparation method thereof.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing curcumin-loaded engineered cell membrane nanovesicles NVs-CUR, comprising the following steps:

[0008] S1, extruding the macrophage suspension through an extruder to obtain a NVs resuspension;

[0009] S2. Add the CUR mixture into the NVs resuspension and perform ultrasonic treatment to obtain nanovesicles NVs-CUR.

[0010] The combination of CUR and NVs carrier can further enhance its pharmacological effects and reduce adverse reactions. This new drug delivery system can not only protect the drug from degradation in the body, but also increase the enrichment of the drug in the target tissue, thereby maximizing the therapeutic effect of curcumin.

[0011] As a new type of nanocarrier, NVs encapsulated curcumin has excellent drug stability, bioavailability, targeting, biocompatibility and cell permeability, and can cross the BBB to reach the central nervous system, providing a new way to treat nerve-related diseases. Therefore, the application prospects of NVs as a carrier of curcumin delivery system in the treatment of nerve injury will be studied.

[0012] Therefore, in order to develop a new drug delivery system with neuron targeting capability, this study prepared macrophage membrane nanocapsules loaded with curcumin (CUR-loaded NVs, NVs-CUR). The preparation technology of nanoparticles has brought new development opportunities to fields such as materials science and medicine. The preparation of engineered cell membrane nanovesicles NVs-CUR loaded with curcumin has expanded the application space of curcumin, and is expected to play an important role in disease treatment and health product development. With the continuous deepening of research, it is believed that more efficient and safe nanoparticles and preparation methods of nanoparticles loaded with curcumin will emerge, making greater contributions to human health and scientific and technological progress.

[0013] In some embodiments of the present invention, the following steps are included:

[0014] S1. Preparation of NVs resuspension

[0015] S11, resuspending the macrophages in PBS buffer to obtain a macrophage suspension;

[0016] S12, extruding the macrophage suspension through a PC membrane with a gradually decreasing pore size for multiple times, the number of extrusions being 2 to 10 times;

[0017] S13, centrifuging the mixed solution after extrusion, washing, and adding 1-10% trehalose PBS buffer solution to obtain a nanovesicle NVs resuspension;

[0018] S2. Preparation of NVs-CUR

[0019] S21, adding the CUR mixed solution into the NVs resuspension solution, the volume ratio of the NVs resuspension solution to the CUR mixed solution is (1-5):1;

[0020] S22, subjecting the mixed solution of S21 to a first ultrasonic treatment, cooling, and then subjecting the mixed solution to a second ultrasonic treatment to obtain nanovesicles NVs-CUR.

[0021] In some embodiments of the present invention, the following steps are included:

[0022] S1. Preparation of NVs resuspension

[0023] S11, resuspending the macrophages in PBS buffer to obtain a macrophage suspension;

[0024] S12, extruding the macrophage suspension through a PC membrane with a gradually decreasing pore size for multiple times, the number of extrusions being 3 to 8 times;

[0025] S13, centrifuging the mixed solution after extrusion for multiple times, washing in PBS solution to obtain crude NVs, and adding 3-8% trehalose PBS buffer solution after washing to obtain 0.2-0.5 μg / μL NVs resuspension;

[0026] S2. Preparation of NVs-CUR

[0027] S21, adding the CUR mixture to the NVs resuspension, the volume ratio of the NVs resuspension to the CUR mixture is (1-3):1, and the concentration of the CUR mixture is 0.01-1 mg / mL;

[0028] S22, subjecting the mixed solution of S21 to a first ultrasonic treatment, cooling, and then subjecting the mixed solution to a second ultrasonic treatment to obtain nanovesicles NVs-CUR.

[0029] In some embodiments of the present invention, the pore sizes of the PC membrane are 8-12 μm, 4-6 μm, 0.8-1.2 μm, 0.35-0.45 μm, and 0.08-0.12 μm, respectively.

[0030] In some embodiments of the present invention, the pore sizes of the PC membrane are 10 μm, 5 μm, 1 μm, 0.4 μm, and 0.1 μm, respectively.

[0031] In some embodiments of the present invention, the preparation process of the crude NVs product in step S13 is: centrifuge the extruded mixed solution at 1500-2500g for 10-20min, and continue to centrifuge at 15000-25000g for 10-15min; remove the supernatant and add it to PBS solution, centrifuge at 180000-220000g for 1.5-2.5h to obtain the crude NVs product.

[0032] In some embodiments of the present invention, the concentration of the NVs resuspension of S13 is 0.25-0.30 μg / μL, and the concentration of the CUR mixed solution of S21 is 0.1-0.7 mg / mL.

[0033] In some embodiments of the present invention, the volume ratio of the NVs resuspension to the CUR mixed solution is (1.8-2.2):1.

[0034] In some embodiments of the present invention, the first ultrasonic treatment in S22 is performed for 10 to 20 minutes, cooled at -25 to -15°C for 3 to 8 minutes, and then the second ultrasonic treatment is performed for 10 to 20 minutes.

[0035] The second aspect of the present invention provides a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR, wherein the nanovesicle NVs-CUR is prepared by the above-mentioned preparation method.

[0036] The present invention includes the following beneficial effects: NVs-CUR, as a new drug delivery system, exhibits excellent performance in physical stability, drug release and stability, cell uptake ability, cytotoxicity, etc. These characteristics provide strong support for its application in the treatment of diseases such as nerve damage, and indicate its broad prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the preparation process of NVs-CUR.

[0038] Figure 2 Western blot was used to detect the protein expression of macrophage markers.

[0039] Figure 3 Bright field images of RAW264.7 cells before and after rupture in hypotonic solution, Scale bar = 100 μm.

[0040] Figure 4 UV-visible spectrum of curcumin.

[0041] Figure 5 UV-visible spectrum of NVs.

[0042] Figure 6 UV-visible spectra of NVs-CUR prepared by freeze-thaw cycles, ultrasonic treatment, and room temperature incubation (the amount of added curcumin was 1.5 mg).

[0043] Figure 7 Comparison of encapsulation efficiency (EE) and drug loading efficiency (LE) among three preparation methods: freeze-thaw cycle, ultrasonic treatment and room temperature incubation.

[0044] Figure 8 Selection of the optimal drug loading amount for NVs-CUR.

[0045] Fig. 9 Western blot was used to detect the protein expression of extracellular vesicle markers.

[0046] Fig.10 Representative TEM images of NVs and NVs-CUR (scale bars are 100 nm and 50 nm from top to bottom, respectively).

[0047] Fig.11 The average particle size of NVs and NVs-CUR was detected by NTA.

[0048] Fig.12 Zeta potential of NVs and NVs-CUR was measured for NTA.

[0049] Fig.13 The concentrations of NVs and NVs-CUR in PBS were varied over 30 days.

[0050] Fig.14 Particle size changes of NVs and NVs-CUR in PBS for 30 days

[0051] Fig.15 Curcumin release from NVs-CUR within 48 hours

[0052] Fig.16 This is the plasma concentration curve of curcumin released by mice in the NVs group and NVs-CUR group.

[0053] Fig.17 The uptake of NVs-CUR by PC12 cells.

[0054] Fig.18 NVs-CUR cytotoxicity assay.

[0055] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0056] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. DETAILED DESCRIPTION

[0057] The present invention specification describes the specific implementation scheme in detail. Those skilled in the art should recognize that the following implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The following examples specifically illustrate the beneficial effects of the present invention.

[0058] Preparation of curcumin-loaded engineered cell membrane nanovesicles NVs-CUR

[0059] 1.1 Research subjects

[0060] 1.1.1 Experimental instruments

[0061] -80℃ ultra-low temperature freezer (Thermo, USA), -20℃ low temperature freezer (Midea, China), 4℃ refrigerator (Midea, China), ultrapure water system (Millipore, USA), ice maker (Sanyo, Japan), paraffin slicer (Leica, Germany), optical microscope (Olympus, Germany), full-wavelength multifunctional microplate reader (Thermo, USA), protein immunoblotting electrophoresis equipment (Bio-Rad, USA), electronic balance (Shanghai Tianping Instrument Factory), low-temperature high-speed centrifuge (Sigma, USA), ultrasonic pulverizer (KQ-600KDE, Kunshan Ultrasonic Instrument Co., Ltd., China), inverted phase contrast microscope (Leica, Germany); transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan); Side-mounted Camera-Megaview III (Soft Imaging System, Münster, Germany); UV-Vis spectrophotometer (U-3900, Hitachi, Japan); confocal laser scanning microscope (Synergy-2, BioTek Instruments, Winooski, Vermont, USA); Nikon ECLIPSE80i (Nikon, Japan).

[0062] 1.1.2 Cell lines and sources used in the experiment

[0063] RAW264.7 is the full name of mouse mononuclear macrophage leukemia cells, which are derived from tumors induced by male Abelson murine leukemia virus. It is one of the commonly used inflammatory cell models and is commonly used in the study of cell phagocytosis, cellular immunity, and molecular immunology. The mouse RAW264.7 cells (SNL-112) used in this experiment were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0064] 1.1.3 Cell culture reagents and consumables

[0065] Dulbecco's Modified Eagle Medium (D5030) was purchased from Sigma-Aldrich, USA.

[0066] Fetal bovine serum (FBS, 10100147) was purchased from Invitrogen, USA.

[0067] 1% penicillin / streptomycin (10378016) was purchased from Invitrogen, USA.

[0068] 0.25% trypsin (T1300) was purchased from Solebao Company, China.

[0069] T25 cell culture flasks and 6-well / 96-well cell culture plates were purchased from Corning, USA.

[0070] Cell cryopreservation tubes, 1.5ml / 15ml / 50ml centrifuge tubes, etc. were purchased from Wuxi Nice Biotechnology Co., Ltd., China.

[0071] Cell Counting Kit-8 (CCK-8, AR1160) was purchased from Boster Biotechnology Co., Ltd., Wuhan, China.

[0072] BeyoAOF TM Serum-free cell freezing medium (C0210B-50ml) was purchased from Biyuntian Company, Shanghai, China.

[0073] The sealing film (PM-996) was purchased from Parafilm Company, USA.

[0074] 1.1.4 Preparation of Macrophage Membrane NVs-CUR Reagents and Consumables

[0075] Polycarbonate (PC) membrane filters with pore sizes of 10 μm, 5 μm, 1 μm, 400 nm and 100 nm were purchased from Whatman Company in the UK.

[0076] 5% trehalose PBS buffer solution (C1049) was purchased from Beijing Baosen Biotechnology Co., Ltd., China.

[0077] CUR (C7727) was purchased from Sigma-Aldrich, USA.

[0078] Dimethyl sulfoxide (DMSO) (CAS: 67-68-5) was purchased from Qingdao Dexin Chemical Co., Ltd., China.

[0079] 1.1.5 Characterization and stability analysis of NVs-CUR Reagents and consumables

[0080] Domestic wide-edge round hole copper mesh (AZH200HH) was purchased from Beijing Zhongjing Keyi Company, China.

[0081] Phosphotungstic acid solution (pH 6.8) (79690) was purchased from Merck, USA.

[0082] PBS, sterile syringes, filter paper and other reagents and consumables were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0083] 1.1.6 Western Blot Reagents

[0084] Cell RIPA lysis buffer (RIPALysis Buffer, P0013C), BCA protein concentration determination kit (enhanced) (Enhanced BCA Protein Assay Kit, P0010S), SDS-PAGE protein loading buffer (5X) (SDS-PAGE Sample Loading Buffer, 5X) Protease inhibitor cocktail (universal, 100X) (P1005), BeyoGel TM TBE-Urea PAGE precast gel (10%, 10 wells) (BeyoGel TM TBE-Urea Precast PAGE Gel, SDS-PAGE) (R0235 / R0236), Tween-20 (Tween-20, ST825-500ml), colored prestained protein molecular weight standard (10-180kD) (P0068), bovine serum albumin (BSA (Australasia origin, protease free, fatty acid free, low endotoxin, pH 7) (≥98%, BioPremium) (ST2254-100 g), and ultrasensitive ECL chemiluminescence kit (BeyoECL Plus) (P0018S) were all purchased from Shanghai Beyotime Co., Ltd., China.

[0085] Tris-HCl (Trimethylolamine hydrochloride) (60102ES80) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., China.

[0086] Glycine (G8200-500 g) and sodium dodecyl sulfate (SDS) (S8010-100 g) were purchased from Changsha Chengrui Biological Co., Ltd. (China).

[0087] 1.1.7 Antibodies related to WB experiments

[0088] NVs markers CD9 (K004635P, dilution ratio 1:1000), CD81 (K000385P, dilution ratio 1:1000), CD63 (K007602P, dilution ratio 1:1000), Alix (K009138P, dilution ratio 1:1000) antibodies and endoplasmic reticulum membrane marker Calnexin (K000499P, dilution ratio 1:1000) antibodies were purchased from Solarbio, Beijing.

[0089] HRP GoatAnti-Rabbit IgG (A21030, dilution ratio 1:10000) was purchased from Abbkine.

[0090] 1.1.8 Drug loading efficiency reagents and consumables

[0091] PC12 cells (SNL-124) were purchased from the Cell Bank of the Chinese Academy of Sciences (China).

[0092] 1,10-Dioctadecyl-3,3,30,30-tetramethylpolycarbonate perchlorate (Dil) (465498, Sigma-Aldrich, USA) was purchased from Sigma-Aldrich Company, USA.

[0093] Anti-fluorescence quenching sealing solution (containing DAPI) (P0131-5ml) was purchased from Shanghai Biyuntian Company, China.

[0094] Confocal laser culture dish (150682, Thermo Scientific TM , USA) was purchased from ThermoScientific TM company.

[0095] 1.1.9 Nucleic acid vesicle cytotoxic reagent consumables

[0096] Fetal bovine serum (FBS, 10100147) and penicillin / streptomycin (10378016) were purchased from Invitrogen, USA.

[0097] 0.25% trypsin (T1300) was purchased from Solebao Company, China.

[0098] DMEM (G4524-500ML) was purchased from Wuhan Saiweier Company, China.

[0099] MTT cell proliferation and cytotoxicity detection kit (MDL number: MFCD00080731) was purchased from Sigma-Aldrich, USA.

[0100] Pipette (CLS7017), pipette tips (10 μL, 200 μL, 1 mL), and 96-well cell plates (CLS7369, Corning, USA) were purchased from Corning Incorporated, USA.

[0101] DMSO (ST038-100ml) was purchased from Biyuntian Company, Shanghai, China.

[0102] H2O2 (HX0640, Supelco, USA) was purchased from Supelco, USA.

[0103] 1.2 Content and Methods

[0104] 1.2.1 Storage and preparation of experimental reagents

[0105] 1.2.1.1 Preparation and storage of cell culture related reagents

[0106] Preparation and storage of Dulbecco's Modified Eagle complete medium: Thaw FBS in a water bath at 37°C, and perform the following operations in a clean biosafety cabinet: add 50 ml of fetal bovine serum to a 500 ml Dulbecco's Modified Eagle culture bottle, and continue to add 5 ml of penicillin / streptomycin to the Dulbecco's Modified Eagle culture bottle. Mix thoroughly to obtain Dulbecco's Modified Eagle complete medium; label it and mark the date and name, seal the mouth of the culture medium bottle with a sealing film, and store it in a 4°C refrigerator.

[0107] Preparation of pH 7.4 PBS buffer solution: weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, dissolve in 800mL UP water, adjust the pH value of the solution to 7.4 with HCl, and finally add UP water to make up to 1L. Sterilize in a high pressure steam sterilizer at 121℃ for 20min. After sterilization, seal the mouth of the culture medium bottle with a sealing film and store it in a 4℃ refrigerator.

[0108] 1.2.1.2 Preparation and storage of WB experiment related reagents

[0109] Preparation of 10× electrophoresis buffer: weigh 30.2g Tris Base, 144.0g glycine, 10.0g SDS and other powders and put them into a large beaker, add 800mL UP water to the beaker, stir to dissolve, dilute to 1000mL with UP water, and store at room temperature for later use. Take 10× mother solution and dilute it to 1× electrophoresis buffer with UP water before use.

[0110] Preparation of 10× transfer buffer: weigh 30.2g Tris Base, 144.0g glycine and other reagents and add them into a large beaker. Add 800mL UP water to the beaker and stir to dissolve. Then make up to 1000mL with UP water and store at room temperature for later use. Mix 100mL of 10× transfer buffer, 200mL of methanol and 700mL of ultrapure water to prepare 1× transfer buffer and store in a brown glass bottle away from light.

[0111] Preparation of TBST: Dissolve 48.4g Tris Base and 160g NaCl powder in 2L ultrapure water to prepare 10×TBS and store at room temperature. Add 100mL of 10×TBS to 900mL of ultrapure water to dilute to 1×TBST.

[0112] Preparation of 5% milk blocking solution: Add 2.5 g skim milk powder into 50 mL 1× TBST buffer and mix thoroughly. Prepare and use immediately.

[0113] Preparation of 10% ammonium persulfate: weigh 0.1 g of ammonium persulfate powder into a 2 mL EP tube, dissolve in 1 ml UP water, vortex to mix, wrap in tin foil and store in a 4 °C refrigerator to avoid light.

[0114] Preparation of 10% SDS solution: Weigh 10 g of SDS powder and add 80 mL of UP water. Incubate in a 52°C water bath until clear, then make up to 100 mL and store at room temperature for later use.

[0115] Preparation of 10% Tween 20: Measure 15 mL of Tween 20 stock solution with a measuring cylinder, make up to 150 mL with UP water, stir the magnetic beads until clear, and place in a 4°C refrigerator.

[0116] 1.2.2 Cell culture and cell membrane isolation

[0117] Mouse RAW264.7 cells (SNL-112) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in Dulbecco's modified Eagle's medium (D5030, Sigma-Aldrich, USA) with 10% fetal bovine serum (10100147, Invitrogen, USA) and 1% penicillin / streptomycin (10378016, Invitrogen, USA) at 37°C in a 5% CO2 cell culture incubator. When the cells were cultured to 90% confluence, adherent cells were separated with a cell scraper. About 1×107 cells were collected and centrifuged, and the cells were resuspended in PBS buffer to obtain a single cell suspension for subsequent nanovesicle (NVs) preparation.

[0118] The above single cell suspension macrophages were placed in a hypotonic solution (universal hypotonic solution) to destroy the cell membrane, and the morphological changes before and after the rupture were observed under a bright field microscope. Subsequently, a Dounce homogenizer was used to fully destroy the cell structure. The membranes were collected by density gradient centrifugation and stored at -80°C for further experiments.

[0119] 1.2.3 Separation and preparation of NVs by continuous extrusion

[0120] NVs were isolated from macrophages (RAW264.7) using a continuous extrusion method. Briefly, after 3 days of cell culture, single-cell suspensions were extruded using a mini-extruder (Avanti Polar Lipids, USA) equipped with a polycarbonate (PC) membrane filter (Whatman, UK) with a pore size of 100 nm. The cell suspension was extruded five times, passing through PC membranes with pore sizes of 10 μm, 5 μm, 1 μm, 400 nm, and 100 nm, respectively. Subsequently, the cells were centrifuged at 2000 g and 20,000 g for 15 min, respectively. After washing in PBS solution (200,000 g, 2 h), NVs were resuspended in 5% trehalose PBS buffer solution (C1049, Beijing Baosen Biotechnology Co., Ltd.).

[0121] 1.2.4 Preparation of curcumin (CUR) loaded macrophage membrane nanovesicles (NVs-CUR)

[0122] 1.2.4.1 Load CUR into NVs by ultrasound as follows:

[0123] First, a 30 mg / ml stock solution of CUR (C7727, Sigma-Aldrich, USA) was prepared in dimethyl sulfoxide (DMSO).

[0124] Different concentrations of CUR (0.14, 0.42, 0.56, and 0.69 mg / ml) were added to 0.28 μg / μL NVs, and the mixture ratio of PBS to DMSO was 2:1 (v / v).

[0125] 1.2.4.2 Three methods for encapsulating CUR into NVs were evaluated, including freeze-thaw cycles, sonication, and room temperature incubation.

[0126] 1.2.4.2.1 In the freeze-thaw cycle, the CUR solution was mixed with NVs, incubated for 30 min, then quickly frozen at -80 °C for 8 min and thawed at room temperature. The freeze-thaw cycle was repeated 3 times. Next, NVs-CUR was obtained by ultrasonic treatment for 15 min (frequency of 2 kHz and power of 70%).

[0127] 1.2.4.2.2 During the ultrasonic treatment, the drug and NV mixture was ultrasonically treated for 15 min (frequency of 2 kHz, power of 70%) (KQ-600KDE, Kunshan Ultrasonic Instrument Co., Ltd., China), cooled at -20 °C for 5 min, and then ultrasonically treated again for 15 min (frequency of 2 kHz, power of 70%) to obtain NVs-CUR.

[0128] 1.2.4.2.3 In the case of incubation at room temperature, the drug and NV mixture was incubated at room temperature for 2 h. NVs-CUR was purified by two-step gradient centrifugation. First, centrifugation was performed at 8000 g for 15 min at 4 °C to separate the unloaded CUR, and then centrifugation was performed at 20000 g for 15 min to obtain a yellow-brown precipitate, i.e., NVs-CUR, which was resuspended in PBS.

[0129] 1.2.5 Characterization and stability analysis of NVs-CUR

[0130] 1.2.5.1 Transmission Electron Microscopy (TEM)

[0131] 20 μL of NVs were dropped onto a copper mesh plate and allowed to stand for 3 min. The liquid was then gently absorbed from the side with filter paper, and 30 μL of phosphotungstic acid solution (pH 6.8) (79690, Merck, USA) was added. The samples were counterstained at room temperature for 5 min and dried under an incandescent lamp. Subsequently, the samples were observed using a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV, and images were captured using a side-mounted Camera-Megaview III (Soft Imaging System, Münster, Germany).

[0132] 1.2.5.2 Nanoparticle Tracking Analysis (NTA)

[0133] The NVs sample was resuspended in PBS and diluted 500 times with Milli-Q water. The diluted NVs were then injected into the sample chamber of the NanoSight LM10 (Malvern, UK) instrument using a sterile syringe to ensure that there were no bubbles and the sample chamber was completely filled. The video was analyzed using NanoSight version 2.3 (Malvern, UK) software, with a gain of 6.0 and a threshold of 11 for all samples. The movement trajectory of the particles was recorded, the concentration and particle size distribution of the diluted sample were output, and the NVs concentration of the original solution was calculated based on the dilution factor.

[0134] 1.2.5.3 Protein quantification and sample preparation

[0135] The protein concentration was detected by BCA method. The detailed operation steps were carried out according to the instructions of the BCA kit: BCA working solution was prepared at a ratio of 50:1 between solution A and solution B, and stored in the dark. 2 mg / ml standard protein was added to a 96-well plate at a volume of 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, and 12 μL, and the volume was made up to 20 μL with PBS buffer. 2 μL of the protein sample to be tested was added to the 96-well plate, and each sample was repeated 3 times, and then 18 μL of PBS buffer was added to each well to a total volume of 20 μL. Finally, 200 μL of BCA working solution was added to each well. The 96-well plate was placed in an incubator and incubated for 30 minutes. The absorbance of each well was detected by an enzyme reader, and the corresponding protein concentration was calculated according to the sample absorbance value. The sample concentration was diluted to the same concentration and stored in a -80 °C refrigerator for later use. Take 80 μL of the above protein sample, add 20 μL of 5× loading buffer, shake evenly on an oscillator, and use a 98 °C metal bath for 5 minutes for protein denaturation. After returning to room temperature, store in a -20℃ refrigerator.

[0136] 1.2.5.4 Identification of NV surface markers using Western blot

[0137] NVs were resuspended in RIPA lysis buffer (R0010, Solarbio, Beijing, China) and subjected to Western blot analysis to detect surface markers including CD9, CD81, CD63 and Alix, and the endoplasmic reticulum marker Calnexin. Each experiment was repeated three times.

[0138] 1.2.6 Determination of drug loading efficiency

[0139] In order to quantitatively determine the amount of incorporated CUR, the purified NVs-CUR was collected and the absorbance of CUR at 436 nm was measured using a UV-Vis ultraviolet-visible spectrometer (U-3900, Hitachi, Japan). Subsequently, the respective CUR concentrations in each NVs-CUR sample were calculated based on the calibration curve of CUR. Secondly, the drug encapsulation efficiency (EE) and drug loading (LC) of NVs were calculated as follows: EE = CNVs-CUR / Cinitial; LC = CNVs-CUR / (Cinitial+NNVs). Among them, CNVs-CUR is the amount of CUR in NVs-CUR, Cinitial is the amount of CUR initially added to the preparation, and NNVs is the amount of NVs.

[0140] 1.2.7 Uptake of drug-loaded cells

[0141] To investigate the internalization and intracellular distribution of NVs-CUR, NVs-CUR labeled with 1,10-dioctadecyl-3,3,30,30-tetramethylpolycarbocyanine perchlorate (Dil) (465498, Sigma-Aldrich, USA) was used to observe the uptake of NVs-CUR by PC12 cells (SNL-124, Cell Bank of the Chinese Academy of Sciences). Dil was used as a fluorescent dye to label NVs-CUR. Dil red fluorescent cell membrane probe (10 mM) was added to the NVs-CUR suspension and incubated for 20 min to prepare Dil-labeled NVs-CUR. PC12 cells were incubated overnight in a laser confocal culture dish. When the cell density reached 70%-80%, Dil-labeled NVs-CUR was added to the culture medium and incubated with PC12 cells. DAPI was used to label the cell nucleus. After incubation for 1, 3, and 6 h, the distribution of Dil-labeled NVs-CUR in PC12 cells was observed using a confocal laser scanning microscope (Synergy-2, BioTek Instruments, Winooski, Vermont, USA).

[0142] 1.2.8 Nucleic acid vesicles (NVs) cell viability and toxicity assay

[0143] PC12 cells were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) and then cultured at 37°C in a humidified environment with 5% CO2. Initially, the MTT assay was used to evaluate the cytotoxicity of blank NVs to PC12 cells at a concentration of 5-25 mg / mL. PC12 cells were seeded in 96-well plates at a density of 104 cells per well. After cell adhesion, NVs-CUR was diluted in culture medium to concentrations of 5, 10, 15, 20, and 25 mg / mL. The culture medium served as a blank control. After 48 h of cell treatment, 20 μL of Sigma-Aldrich's MTT solution (5 mg / mL, 0.5% MTT) was added to each well and incubated at 37°C in a humidified environment with 5% CO2 for 4 h. Subsequently, the liquid in the well was aspirated and replaced with 150 μL DMSO. After low-speed shaking for 10 min, the optical density (OD) was measured at a wavelength of 490 nm using a microplate reader to calculate the cell viability.

[0144] To evaluate the protective effect of NVs-CUR on PC12 cells, the MTT method was used to determine the effect of H2O2 (HX0640, Supelco, USA) on the viability of PC12 cells. After 4 h of H2O2 addition, PC12 cells were treated with PBS, CUR, and NVs-CUR. Subsequently, the viability and proliferation of PC12 cells were evaluated using the MTT method, and the concentrations of CUR and NVs-CUR were both 100 ng / mL. After 5 days, the axonal growth of each well was observed and captured under a Nikon ECLIPSE80i (Nikon, Japan) microscope.

[0145] 1.3 Statistical analysis

[0146] All experiments were performed independently at least three times, and the data are expressed as mean ± standard deviation (SD). Independent sample t-test or one-way analysis of variance (ANOVA) was used to evaluate the differences between groups. If the results of the ANOVA showed significant differences, Tukey's honest significant difference (HSD) post hoc test was further performed to compare the differences between groups. For data with non-normal distribution or uneven variance, the Mann-Whitney U test or Kruskal-Wallis H test was used. GraphPad Prism8.0 software was used for statistical analysis. P<0.05 was considered significant.

[0147] 2 Results

[0148] 2.1 Preparation process of NVs and NVs-CUR

[0149] We prepared NVs from macrophages (RAW264.7) using a continuous extrusion method and then loaded CUR into NVs using freeze-thaw cycles, sonication, and incubation at room temperature to prepare NVs-CUR and NVs.

[0150] Figure 1 Figure 1 is a schematic diagram of the preparation process of NVs-CUR. The general process is as follows: first, NVs were separated from macrophages RAW264.7 using a continuous extrusion method. Using a small extruder combined with a 100nm pore size PC membrane filter, the cell suspension was extruded 5 times, passing through 10μm, 5μm and 1μm PC membranes in turn. Centrifuge for 15min each at 2000g and 20,000g. After washing with PBS, NVs were resuspended in 5% trehalose PBS buffer. CUR at a concentration of 1.5mg was added to 0.28μg / μL NVs, where the ratio of PBS and DMSO mixture was 2:1 (volume ratio). NVs-CUR was prepared using freeze-thaw cycles, ultrasonic treatment, and incubation at room temperature.

[0151] 2.2 Identification of macrophage cell membrane

[0152] Western blot results Figure 2 As shown, Figure 2 Western blot was used to detect the protein expression of macrophage markers. Figure 3 Bright field images of RAW264.7 cells before and after rupture in hypotonic solution, Scale bar = 100 μm.

[0153] from Figure 2 It can be seen that the macrophage characteristic marker proteins F4 / 80 and CD11b are less expressed in the RAW264.7 cell group, but more expressed in the RAW264.7 cell membrane, NVs and NVs-CUR groups. This shows that the RAW264.7 cell membrane has been successfully extracted and the properties of macrophage surface proteins have not changed during the preparation process. The images of RAW264.7 cells before and after bursting in the hypotonic solution also indirectly prove the feasibility of the membrane extraction process. The morphological changes before and after the cell rupture can be clearly seen in the wide field of view of the microscope. We found that compared with normal cells, the cells in the hypotonic solution became significantly larger and ruptured ( Figure 3 ). Due to the abundance of proteins and RNA on the surface of macrophage membranes, NVs derived from them play an important role in targeted therapy of inflammation. Therefore, it has homology with macrophages in inflammatory sites, which indicates that it is suitable as a drug carrier for targeting inflammation.

[0154] 2.3 Selection of the optimal NVs-CUR preparation method

[0155] Figure 4 is the UV-visible spectrum of curcumin, Figure 5 is the UV-visible spectrum of NVs, Figure 6 UV-visible spectra of NVs-CUR prepared by freeze-thaw cycles, ultrasonic treatment and room temperature incubation (the amount of added curcumin was 1.5 mg). Figure 7 Comparison of encapsulation efficiency (EE) and drug loading efficiency (LE) of three preparation methods: freeze-thaw cycle, ultrasonic treatment and room temperature incubation.

[0156] like Figure 4 As shown, the UV-visible spectrum of curcumin shows a main peak centered at 436 nm with no interference, confirming that CUR is not destroyed in the UV-visible spectral range.

[0157] like Figure 5 As shown, the UV-visible spectrum shows that the UV-visible spectrum of NVs has no peak at 436 nm and no interference.

[0158] like Figure 6 and 7 As shown in the figure, the UV-visible spectra showed that the NVs-CUR prepared by loading CUR into NVs using freeze-thaw cycles, ultrasonic treatment and incubation at room temperature showed a main peak centered at 436nm, and the loading efficiency (LE) of NVs-CUR was determined by calculating the UV-visible absorption spectrum. The results showed that the drug loading amounts of the ultrasonic method, incubation method and freeze-thaw method were 125.45, 41.33 and 3.78μg / mg, respectively. Correspondingly, the encapsulation efficiency (EE) values ​​of CUR in NVs were 9.82%, 2.75% and 0.28%, respectively. Figure 6 and 7 ). Therefore, the UV-visible spectrum of the NVs-CUR complex indicated that ultrasound was the optimal drug delivery method. The loading capacity (LC) of the NVs-CUR preparation was closely related to the preparation method. It is speculated that the cavitation or sonochemical effect generated by ultrasound treatment led to the instantaneous repair of the NVs membrane gap, thereby significantly improving the ability of drugs to enter NVs.

[0159] 2.4 Selection of optimal drug loading of NVs-CUR

[0160] Drug loading refers to the total mass of curcumin (CUR) loaded in nanovesicles (NVs). The drug loading choices here (such as 0.5, 1.5, 2.0, and 2.5 mg) correspond to the total amount of CUR added during the preparation process. The stock solution of CUR is 30 mg / ml (in DMSO), and different concentrations of CUR solutions (such as 0.14, 0.42, 0.56, and 0.69 mg / ml) are added to NVs with a concentration of 0.28 μg / μL during the preparation process. The mixing ratio of PBS and DMSO is 2:1 to optimize the dissolution and encapsulation efficiency of CUR.

[0161] Methods to achieve different drug loadings:

[0162] Assuming that the volume of NVs used in the preparation process is a certain amount (such as 1 ml), the amount of CUR added at different concentrations will affect the final CUR drug loading. The following are methods for achieving different drug loadings:

[0163] (1) 0.5 mg CUR:

[0164] When the CUR concentration is 0.14 mg / ml, add 3.57 ml (0.14 mg / ml×3.57 ml≈0.5 mg).

[0165] (2) 1.5 mg CUR:

[0166] When the CUR concentration is 0.42 mg / ml, add 3.57 ml (0.42 mg / ml×3.57 ml≈1.5 mg).

[0167] (3) 2.0 mg CUR:

[0168] When the CUR concentration is 0.56 mg / ml, add 3.57 ml (0.56 mg / ml×3.57 ml≈2.0 mg).

[0169] (4) 2.5 mg CUR:

[0170] When the CUR concentration is 0.69 mg / ml, add 3.62 ml (0.69 mg / ml×3.62 ml≈2.5 mg).

[0171] By selecting different CUR concentrations and adjusting the added volume, a specified drug loading (such as 0.5, 1.5, 2.0, and 2.5 mg) can be achieved in NVs. In the process of loading CUR into NVs using ultrasound, the concentration ratio of CUR to NVs has an effect on the encapsulation efficiency. The higher the concentration, the higher the encapsulation efficiency will usually be until saturation, after which absorbance measurement will help confirm the optimal drug loading.

[0172] The initial CUR loading was optimized by testing 0.5, 1.5, 2.0, and 2.5 mg CUR. Figure 8 The optimal drug loading amount of NVs-CUR was selected. The results showed that the absorption peak of NVs-CUR reached the maximum when 1.5 mg CUR was input. These results further confirmed the rationality of using ultrasound to load CUR into NVs and determined the optimal drug loading amount of CUR.

[0173] 2.5 Identification of NVs-CUR extracellular vesicles

[0174] Fig. 9 Western blot was used to detect the protein expression of extracellular vesicle markers. Fig.10 Representative TEM images of NVs and NVs-CUR (scale bars are 100 nm and 50 nm from top to bottom, respectively).

[0175] Western blot results showed that the extracellular vesicle markers CD9, CD81, CD63 and Alix were positively expressed in NVs and NVs-CUR, and the endoplasmic reticulum membrane marker Calnexin was negatively expressed ( Fig. 9 ). Under transmission electron microscopy (TEM), NVs and NVs-CUR were round and had a lipid bilayer membrane structure (10).

[0176] 2.6 Characterization of membrane biomimetic nanomedicine using NVs and NVs-CUR

[0177] Fig.11The average particle size of NVs and NVs-CUR was detected by NTA. Fig.12 Zeta potential of NVs and NVs-CUR was measured for NTA.

[0178] Nanoparticle tracking analysis (NTA) showed that the average particle sizes of NVs and NVs-CUR were 93.15±4.36nm and 105.39±2.05nm, respectively. Fig.11 ), the corresponding Zeta potentials were -25.23±1.16 and -17.82±2.14 mV ( Fig.12 ). Therefore, both NVs and NVs-CUR exhibit typical features of extracellular vesicles.

[0179] 2.7 Stability analysis of membrane biomimetic nanomedicines using NVs and NVs-CUR

[0180] Fig.13 The concentration changes of NVs and NVs-CUR in PBS for 30 days. Fig.14 The particle size changes of NVs and NVs-CUR in PBS for 30 days. Fig.15 The curcumin release of NVs-CUR within 48 hours. Fig.16 The plasma concentration curves of curcumin released by mice in the NVs group and NVs-CUR group; ns indicates P>0.05, * indicates P<0.05, and the cell experiment was repeated three times.

[0181] After NVs and NVs-CUR were in PBS for 30 days, the concentrations of NVs and NVs-CUR did not change significantly ( Fig.13 ); the particle size of NVs and NVs-CUR did not change significantly after 30 days in PBS ( Fig.14 ); The curcumin release from NVs-CUR was detected within 48 hours, and it was found that the curcumin release from NVs-CUR gradually increased, reaching 80% within 48 hours; the concentration curve of curcumin released in the plasma of mice in the CUR group and the NVs-CUR group was detected within 24 hours, and the stability of curcumin released from the NVs-CUR group was better than that of the CUR group. The above results show that the concentration and size of NVs-CUR did not change significantly, and it was more stable in the blood of mice, indicating that it has the sustained stability required for therapeutic applications.

[0182] 2.8 Uptake of NVs-CUR by PC12 cells

[0183] To further determine the uptake of NVs-CUR by PC12 cells, Dil-labeled NVs-CUR was used to observe the uptake of NVs-CUR by PC12 cells. Fig.17The uptake of NVs-CUR by PC12 cells showed that the red fluorescence signal in PC12 cells increased over time, indicating that NVs-CUR was gradually internalized by PC12 cells in a time-dependent manner.

[0184] 2.9 Evaluation of toxicity of NVs-CUR

[0185] Fig.18 To evaluate the toxicity of NVs-CUR, we performed MTT assay after 24 h of NVs-CUR treatment and found that more than 90% of PC12 cells were still alive, indicating that NVs-CUR is biosafe ( Fig.18 ).

[0186] 3 Discussions

[0187] In this study, we chose to develop an engineered cell membrane nanovesicle as a drug delivery system, which has many advantages. Compared with traditional drug delivery systems, engineered nanovesicles can more effectively cross the blood-brain barrier and achieve accurate targeted delivery of drugs to brain tissue or neurons. In addition, engineered nanovesicles have good stability and biocompatibility, which can improve the bioavailability of drugs and reduce side effects. This innovative drug delivery system has broad application prospects in the treatment of neurological diseases.

[0188] In this study, we successfully developed a new drug delivery system, macrophage membrane nanocapsules loaded with curcumin (NVs-CUR), which provides a new idea for the effective treatment of nerve damage. Through the analysis of the preparation process and its results, we can draw the following important conclusions.

[0189] First, the nanovesicles (NVs) prepared from macrophages (RAW264.7) by continuous extrusion method maintained the integrity of cell membrane surface proteins, which was verified by Western blot results. The high expression of macrophage characteristic marker proteins such as F4 / 80 and CD11b in NVs not only proved the successful extraction of cell membrane, but also showed that the properties of cell membrane proteins did not change significantly during the preparation process. This feature enables NVs to mimic the biological functions of macrophages, especially their inflammation targeting ability, providing a solid foundation for subsequent drug delivery.

[0190] Secondly, in the process of loading CUR into NVs to prepare NVs-CUR, we tried various methods such as freeze-thaw cycles, ultrasonic treatment, and incubation at room temperature, and determined that ultrasonic treatment was the best method through UV-visible spectroscopy analysis. Ultrasonic treatment not only significantly improved the loading efficiency and encapsulation efficiency of CUR in NVs, but also retained the activity of CUR. This efficient loading method may be attributed to the cavitation or sonochemical effects generated by ultrasonic treatment, which promote the transient repair of the NVs membrane gap, thereby enhancing the ability of drug molecules to enter the interior of the nanovesicles.

[0191] In addition, by optimizing the initial loading amount of CUR, we found that when 1.5 mg CUR was input, the absorption peak of NVs-CUR reached the maximum. This result not only verified the rationality of the ultrasound loading method, but also determined the optimal loading amount of CUR in NVs, providing an important reference for future clinical applications.

[0192] In terms of the identification of extracellular vesicles, the results of Western blot and transmission electron microscopy (TEM) showed that NVs and NVs-CUR had typical extracellular vesicle characteristics, including the positive expression of extracellular vesicle markers such as CD9, CD81, CD63 and Alix, and the presence of lipid bilayer membrane structure. These characteristics further confirmed the potential and stability of NVs and NVs-CUR as drug carriers. Nanoparticle tracking analysis (NTA) results showed that the average particle size and Zeta potential of NVs and NVs-CUR were within the appropriate range, which was conducive to their stability and targeting in vivo. In particular, the particle size of NVs-CUR was slightly larger than that of NVs, which may be due to the fact that the loading of CUR increased the volume and surface charge of the nanovesicles, but this change did not affect its overall performance as a drug carrier.

[0193] In the stability analysis part of this study, we evaluated the long-term stability of NVs and NVs-CUR in PBS. The results showed that the concentration and particle size of both NVs and NVs-CUR did not change significantly after 30 days in PBS. This finding shows that these nanodrug carriers have excellent physical stability and can maintain their structural and functional integrity in a physiological environment, which is crucial for the practical application of drug delivery systems. In addition, NVs-CUR can release up to 80% of curcumin within 48 hours, and the released curcumin within 24 hours showed better stability than CUR alone, which further confirmed the potential of NVs-CUR in protecting drug activity and achieving controlled release. These stability data not only support the feasibility of NVs-CUR as a long-acting drug delivery system, but also provide strong support for its application in the treatment of diseases such as nerve damage.

[0194] In the cell uptake experiment, we used Dil-labeled NVs-CUR to observe its uptake by PC12 cells. The results showed that the red fluorescence signal in PC12 cells increased over time, indicating that NVs-CUR was internalized by cells in a time-dependent manner. This finding not only verifies the neuronal targeting ability of NVs-CUR, but also reveals the kinetic process of its cellular uptake, providing important clues for understanding its intracellular drug release and biological effects.

[0195] Finally, we evaluated the toxicity of NVs-CUR to ensure its biosafety as a therapeutic drug. MTT assay results showed that the survival rate of PC12 cells was still over 90% after NVs-CUR treatment for 24 h ( Figure 1-9 ). This high survival rate indicates that NVs-CUR has low toxicity to PC12 cells, thus providing safety assurance for its potential application in the treatment of nerve injury.

[0196] In summary, this study successfully prepared a new drug delivery system NVs-CUR with neuron targeting capability by optimizing the preparation process and methods. This system not only maintains the biological properties of macrophage membranes, but also achieves efficient loading and stable delivery of CUR, providing new strategies and directions for the treatment of nerve damage. Future studies will further explore the pharmacodynamic and pharmacokinetic properties of NVs-CUR in vivo, as well as its therapeutic effects and application prospects in different disease models.

[0197] 4 Summary

[0198] 4.1NVs-CUR has excellent physical stability

[0199] After 30 days in PBS, the concentration and particle size of NVs and NVs-CUR did not change significantly, showing excellent physical stability. This stability is crucial to ensure the integrity and effectiveness of the drug delivery system during circulation in the body, and provides a guarantee for the long-term effectiveness and reliability of NVs-CUR in therapeutic applications.

[0200] 4.2 Efficient drug release and stability

[0201] NVs-CUR can release up to 80% of curcumin within 48 hours, and the curcumin released within 24 hours shows better stability than that of CUR alone. This shows that NVs-CUR can not only effectively load and protect drug molecules, but also achieve controlled release of drugs, thereby improving therapeutic effects and reducing drug side effects.

[0202] 4.3 Good cellular uptake ability

[0203] The uptake experiment of Dil-labeled NVs-CUR by PC12 cells showed that the nano drug carrier was internalized by neurons in a time-dependent manner. This finding verified the neuronal targeting ability of NVs-CUR and revealed the kinetic process of its cell uptake, providing an important basis for understanding its intracellular drug release and biological effects.

[0204] 4.4 Low cytotoxicity

[0205] MTT test results showed that the survival rate of PC12 cells after NVs-CUR treatment exceeded 90%, indicating that the nano drug carrier has low toxicity to neuronal cells. This finding provides a safety guarantee for the potential application of NVs-CUR in the treatment of nerve injury and proves its biocompatibility and safety as a therapeutic drug.

[0206] In summary, NVs-CUR, as a new drug delivery system, exhibits excellent performance in physical stability, drug release and stability, cell uptake ability, and cytotoxicity, etc. These characteristics provide strong support for its application in the treatment of diseases such as nerve damage, and indicate its broad prospects in the biomedical field.

[0207] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing curcumin-loaded engineered cell membrane nanovesicles NVs-CUR, characterized in that: The steps include: S1, extruding the macrophage suspension through an extruder to obtain a NVs resuspension; S2. Add the CUR mixture into the NVs resuspension and perform ultrasonic treatment to obtain nanovesicles NVs-CUR.

2. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 1, characterized in that: The steps include: S1. Preparation of NVs resuspension S11, resuspending the macrophages in PBS buffer to obtain a macrophage suspension; S12, extruding the macrophage suspension through a PC membrane with a gradually decreasing pore size for multiple times, the number of extrusions being 2 to 10 times; S13, centrifuging the mixed solution after extrusion, washing, and adding 1-10% trehalose PBS buffer solution to obtain a nanovesicle NVs resuspension; S2. Preparation of NVs-CUR S21, adding the CUR mixed solution into the NVs resuspension solution, the volume ratio of the NVs resuspension solution to the CUR mixed solution is (1-5):1; S22, subjecting the mixed solution of S21 to a first ultrasonic treatment, cooling, and then subjecting the mixed solution to a second ultrasonic treatment to obtain nanovesicles NVs-CUR.

3. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 2, characterized in that: The steps include: S1. Preparation of NVs resuspension S11, resuspending the macrophages in PBS buffer to obtain a macrophage suspension; S12, extruding the macrophage suspension through a PC membrane with a gradually decreasing pore size for multiple times, the number of extrusions being 3 to 8 times; S13, centrifuging the mixed solution after extrusion for multiple times, washing in PBS solution to obtain crude NVs, and adding 3-8% trehalose PBS buffer solution after washing to obtain 0.2-0.5 μg / μL NVs resuspension; S2. Preparation of NVs-CUR S21, adding the CUR mixture to the NVs resuspension, the volume ratio of the NVs resuspension to the CUR mixture is (1-3):1, and the concentration of the CUR mixture is 0.01-1 mg / mL; S22, subjecting the mixed solution of S21 to a first ultrasonic treatment, cooling, and then subjecting the mixed solution to a second ultrasonic treatment to obtain nanovesicles NVs-CUR.

4. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 3, characterized in that: The pore sizes of the PC membrane are 8-12 μm, 4-6 μm, 0.8-1.2 μm, 0.35-0.45 μm, and 0.08-0.12 μm, respectively.

5. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 3, characterized in that: The pore sizes of the PC membrane are 10 μm, 5 μm, 1 μm, 0.4 μm and 0.1 μm, respectively.

6. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 3, characterized in that: The preparation process of the crude NVs product in step S13 is as follows: centrifuging the extruded mixed solution at 1500-2500 g for 10-20 min, and continuing to centrifuge at 15000-25000 g for 10-15 min; After removing the supernatant, add it into PBS solution and centrifuge it at 180000-220000g for 1.5-2.5h to obtain crude NVs.

7. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 3, characterized in that: The concentration of the NVs resuspension of S13 is 0.25-0.30 μg / μL, and the concentration of the CUR mixed solution of S21 is 0.1-0.7 mg / mL.

8. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 3, characterized in that: The volume ratio of the NVs resuspension to the CUR mixed solution is (1.8-2.2):

1.

9. The method for preparing a curcumin-loaded engineered cell membrane nanovesicle NVs-CUR according to claim 3, characterized in that: In the S22, the first ultrasonic treatment is performed for 10 to 20 minutes, the mixture is cooled at -25 to -15°C for 3 to 8 minutes, and then the second ultrasonic treatment is performed for 10 to 20 minutes.

10. A curcumin-loaded engineered cell membrane nanovesicle NVs-CUR, characterized in that: The nanovesicle NVs-CUR is prepared by the preparation method according to any one of claims 1 to 9.

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