Drug delivery system and related use thereof in treating spinal cord injury

Abstract

The invention discloses a drug delivery system and related application thereof in treatment of spinal cord injury, and relates to the field of biological medicine. The invention provides a novel drug delivery system, which comprises microvesicles and ginger-derived exosome-like nanoparticles entrapped in the microvesicles, not only retains the characteristic that the ginger-derived exosome-like nanoparticles load ginger active ingredients, but also has the biological characteristics of CCR2 + cell membranes, can accurately target a spinal cord injury area, and has a good application prospect. Effective treatment of spinal cord injury and / or related diseases or symptoms caused by spinal cord injury is achieved, immunogenicity is low, and large-scale production can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a drug delivery system and related applications in treating spinal cord injuries. Background Art

[0002] Spinal cord injury (SCI) is an irreversible disorder of spinal cord motor and sensory function caused by direct or indirect external factors. It is the "number one killer" of death and disability among young and middle-aged people in modern society, and existing clinical treatment measures have limited efficacy.

[0003] Ginger is a typical traditional Chinese medicine that can be used as both medicine and food. Ginger exosome-like nanoparticles (GELNs) are nanoscale vesicles secreted by ginger cells. They are rich in ginger active ingredients such as gingerol and shogaol. They are widely available, non-toxic, pollution-free, low in immunogenicity, highly biocompatible, and highly adaptable, making them ideal drug delivery vehicles. Studies have shown that GELNs have antioxidant, anti-inflammatory, immunomodulatory, and gut microbiome-regulating properties. Oral administration of GELNs has protective effects on the intestine and liver. However, experimental studies have found that due to their lack of targeting, intravenous administration of GELNs has limited protective effects on distant organs.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug delivery system and related applications in treating spinal cord injury.

[0006] The present invention is achieved in that: In a first aspect, an embodiment of the present invention provides a composition comprising: CCR2 + Cell membrane vesicles and ginger-derived exosome-like nanoparticles.

[0007] In a second aspect, an embodiment of the present invention provides a drug delivery system comprising: microvesicles and ginger-derived exosome-like nanoparticles contained in the microvesicles; a method for preparing the drug delivery system comprises: + The mixture of cell membrane vesicles and ginger-derived exosome-like nanoparticles was physically extruded.

[0008] In a third aspect, an embodiment of the present invention provides a drug, the active ingredient of which includes the composition described in the aforementioned embodiment or the drug delivery system described in the aforementioned embodiment.

[0009] In a fourth aspect, embodiments of the present invention provide uses of the composition described in the preceding embodiments or the drug delivery system described in the preceding embodiments in the preparation of products for treating spinal cord injury and / or related diseases or symptoms caused by spinal cord injury.

[0010] The present invention has the following beneficial effects: The present application successfully prepared a new drug delivery system, which includes microvesicles and ginger-derived exosome-like nanoparticles contained in the microvesicles. The system not only retains the characteristics of ginger-derived exosome-like nanoparticles loaded with ginger active ingredients, but also has CCR2 + Biological properties of cell membranes.

[0011] This drug delivery system has good biocompatibility, can accurately target the area of ​​spinal cord injury, enhance the efficacy of ginger-derived exosome-like nanoparticles, and achieve effective treatment of spinal cord injury and / or related diseases or symptoms caused by spinal cord injury. It has low immunogenicity and can be produced on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 Preparation and characteristics of samples in each group; (A) Schematic diagram of the preparation process of CCR2-MV / GELN; (B) Flow cytometry identification of mouse bone marrow-derived Ly6C ++ CCR2 ++ Results of classic monocyte subsets; (CE) Particle size and zeta potential detection and quantitative analysis of GELN, CCR2-MV, and CCR2-MV / GELN; (F) Transmission electron microscopy images of GELN, CCR2-MV, and CCR2-MV / GELN; (G) Laser confocal microscopy image showing colocalization (yellow fluorescence) of the CCR2-MV "shell" (green fluorescence) and the GELN "core" (red fluorescence) of CCR2-MV / GELN in HeLa cells; (H, I) High-performance liquid chromatography and quantitative analysis of 6-gingerol and 6-shogaol in GELN and CCR2-MV / GELN; Figure 2 Expression analysis of membrane surface chemokine receptors and membrane surface markers of each group of samples; (A, B) Flow cytometry detection of GELN (green curve), CCR2 +Expression and quantitative analysis of chemokine receptors CCR2, CX3CR1, and CCR5 on the membrane surface of monocytes (gray curve), CCR2-MV (blue curve), and CCR2-MV / GELN (red curve); (C, D) Western Blot analysis of GELN, CCR2 + Expression and quantitative analysis of monocyte, CCR2-MV, and CCR2-MV / GELN membrane surface markers Ly6C, CD163, CD62L, CD36, CD64, and CD11b; Figure 3 The effects of each group of nanoparticles on the tissue structure of important organs; Figure 4 Figure 3 shows the chemotaxis of each group of nanoparticles to the SCI area; (A, B) Dynamic distribution and quantitative analysis of the fluorescence signal of each group of nanoparticles (GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high) in the blood cells and plasma of SCI rats over time (6 h, 12 h, 1 d, and 3 d); (C, D) Distribution characteristics and quantitative analysis of each group of nanoparticles in the spinal cord injury area and major organs of rats 3 d after SCI; Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. before injury; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 5 Effects of nanoparticles in each group on neurological function after SCI; (A, B) BBB motor function scores and inclined board test scores of rats in each group at 1, 3, 7, and 14 days after SCI; (CF) Quantitative analysis of hindlimb footprints and step width, step length, and dorsum drag rate of rats in each group at 14 days after SCI; (G1) Waveforms of hindlimb motor evoked potentials and quantitative analysis of their latency and amplitude at 14 days after SCI; (JL) T2-weighted magnetic resonance imaging and gross observation of the spinal cord and quantitative analysis of the injury area and edema area of ​​rats in each group at 14 days after SCI; Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 6 Effects of nanoparticles in each group on the permeability of BSCB after SCI; (AC) Fluorescence signal (red fluorescence) of Evans blue penetrating into the rat spinal cord 14 days after SCI and quantitative analysis of fluorescence intensity and content; (D, E) Quantitative analysis of spinal cord water content and MPO activity in each group of rats 14 days after SCI; Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 7 Figure 3. Effects of nanoparticles in each group on spinal cord histomorphology after SCI. (A) Nissl staining of the anterior horn, intermediate zone, and posterior horn of the spinal cord gray matter in each group of rats 14 days after SCI. Neurons with granular Nissl bodies in the cytoplasm, loose chromatin, and prominent nucleoli were considered normal neurons (indicated by ↑). Neurons with shrunken cell bodies, no Nissl bodies in the cytoplasm, and dark staining were considered injured neurons (indicated by ▲). (B) Analysis of the number of normal neurons in the anterior horn, intermediate zone, and posterior horn of the spinal cord gray matter in each group of rats 14 days after SCI. Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 8 Effects of nanoparticles in each group on myelin sheath in the spinal cord after SCI. (AC) Transmission electron microscopy observation of myelin sheath ultrastructure and myelinated axons in the spinal cord of rats in each group 14 days after SCI, and quantitative analysis of the G-ratio (axon diameter / axon diameter + myelin thickness); Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 9Effects of various nanoparticle groups on spinal cord neuronal apoptosis after SCI; (A, B) TUNEL staining and analysis of apoptotic cell numbers in the anterior horn, intermediate zone, and posterior horn of the spinal cord gray matter of rats in each group 14 days after SCI; (C, D) Representative visual fields and quantitative analysis of colocalization (yellow fluorescence) of activated Caspase-3 (red fluorescence) and neurons (NeuN, green fluorescence) in the anterior horn, intermediate zone, and posterior horn of the spinal cord gray matter of rats in each group 14 days after SCI; Statistical markers: * P <0.05,** P <0.01,*** P <0.001vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 10 Effects of nanoparticles in each group on spinal cord apoptosis-related proteins after SCI; (A, B) Western blot analysis of the expression levels of Bcl-2, Bax, Cyt c, activated Caspase-9, and activated Caspase-3 in the spinal cord of rats in each group 14 days after SCI and quantitative analysis; Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 11 Effects of each group of nanoparticles on the Nrf2 signaling pathway and oxidative stress-related indicators in the spinal cord after SCI; (A, B) Detection and quantitative analysis of Nrf2 protein expression; (C) Analysis of Nrf2 DNA binding activity; (DJ) Analysis of SOD activity, CAT activity, and GSH, GSSG, O2⁻·, H2O2, and MDA content; Statistical symbols: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n = 8 per group); Figure 12Effects of each group of nanoparticles on the NF-κB signaling pathway and inflammatory-related indicators in the spinal cord after SCI; (A, B) Detection and quantitative analysis of phosphorylated NF-κB p65 protein expression; (C) NF-κB DNA binding activity analysis; (D-I) TNF-α, IL-6, IL-1β, TGF-β, IL-10, and IL-4 content analysis; (J, K) Detection and quantitative analysis of MMP2 and MMP3 protein expression; Statistical markers: Statistical markers: * P <0.05,** P <0.01,*** P <0.001 vs. SCI group; # P <0.05, ## P <0.01 vs. GELN-high group (n=8 in each group). DETAILED DESCRIPTION

[0014] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0015] Definition of noun The term "CCR2" herein is the same as "CC motif chemokine receptor 2", which is a chemokine receptor expressed at high levels on the membrane of classical monocytes.

[0016] In this article, the term “CCR2 + "Cells" refers to cells expressing CCR2.

[0017] In this article, the term “CCR2 + Cell membrane vesicles", English is "CCR2 + membrane vesicles", abbreviated as CCR2-MV, composed of CCR2 + Derived from cell membrane preparation.

[0018] The term "ginger exosome-like nanoparticles," abbreviated as GELN, is used in this article. GELNs are rich in miRNAs, polyphenols such as 6-gingerol and 6-shogaol, and possess potent anti-inflammatory, antioxidant, and pro-regenerative properties.

[0019] In this article, the term “CCR2 +The mixture of cell membrane vesicles and ginger-derived exosome-like nanoparticles was physically extruded to obtain "microvesicles" which can be abbreviated as CCR2-MV / GELN.

[0020] As used herein, the term "treating" includes preventing or alleviating a condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0021] The lack of targeting of ginger-derived exosome-like nanoparticles in vivo limits their efficacy, and engineering modification is needed to improve their efficiency in delivering to target organs. + Combination of cell membrane vesicles and ginger-derived exosome-like nanoparticles to obtain CCR2 by physical extrusion + A drug delivery system comprising cell membrane-encapsulated ginger-derived exosome-like nanoparticles has targeted properties and can target the spinal cord injury area to achieve effective treatment of spinal cord injury or related diseases or conditions.

[0022] In one aspect, an embodiment of the present invention provides a composition comprising: CCR2 + Cell membrane vesicles and ginger-derived exosome-like nanoparticles.

[0023] In some embodiments, the CCR2 + Cell membrane vesicles derived from CCR2 + The cell membrane of a cell.

[0024] In some embodiments, the CCR2 + cells including CCR2 + Monocytes.

[0025] In some embodiments, the CCR2 + The origin of monocytes can be human and / or mouse.

[0026] In some embodiments, the CCR2 + Monocytes are isolated from bone marrow cells.

[0027] In some embodiments, the CCR2 + The method for isolating the cell membrane includes: + The cells were lysed by ultrasonication and centrifuged by sucrose density gradient to obtain CCR2 + The cell membrane of a cell.

[0028] In some embodiments, the CCR2 + The cell density was 1×10 6 ~9×106 / ml. The density can be 1×10 6 , 3×10 6 , 5×10 6 , 7×10 6 and 9×10 6 Any one or the range between any two of the above.

[0029] In some embodiments, the ultrasonic lysis conditions include: a frequency of 15-25 kHz, a power of 300-500 W, an ultrasonic time of 5-15 s / time, an interval of 5-15 s, and a number of 30-50 times. The frequency can be any one of 15, 17, 19, 21, 22, 23, 24 and 25 kHz or a range between any two of them; the power can be any one of 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 and 500 w or a range between any two of them; the ultrasonic time can be any one of 5, 7, 9, 11, 13 and 15 s / time or a range between any two of them; the interval time can be any one of 5, 7, 9, 11, 13 and 15 s or a range between any two of them; the number of times can be any one of 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 times or a range between any two of them.

[0030] In some embodiments, the conditions for sucrose density gradient centrifugation include: a sucrose density gradient (m / v) of 20%-30%, 35%-45%, 50%-60%, a centrifugal acceleration of 20,000-30,000 g, and a centrifugation time of 40-60 min. The sucrose density gradient can be any one or a range between any two of 20%, 22%, 24%, 26%, 28%, 30%, 35%, 37%, 39%, 41%, 43%, 45%, 50%, 52%, 54%, 56%, 58%, and 60%; the centrifugal acceleration can be any one or a range between any two of 20,000, 22,000, 24,000, 26,000, 27,000, 28,000, and 30,000 g; and the centrifugation time can be any one or a range between any two of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60 min.

[0031] In some embodiments, the CCR2 + The cell membrane concentration of the cells is 1.0-2.0 g / ml, and the concentration can be any one of 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 g / ml or a range between any two of them.

[0032] In some embodiments, the CCR2 + The preparation method of cell membrane vesicles comprises: using water bath ultrasound and / or physical extrusion method to + The cell membrane is converted into CCR2 + Cell membrane vesicles.

[0033] In some embodiments, the conditions of the water bath ultrasound include: a frequency of 20-30 kHz, a power of 200-400 W, an ultrasound time of 10-20 s / time, an interval of 10-20 s, and a number of 20-100 times. The frequency can be any one of 20, 22, 24, 26, 28 and 30 kHz or a range between any two of them; the power can be any one of 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 and 400 w or a range between any two of them; the ultrasonic time can be any one of 10, 12, 14, 16, 18 and 20 s / time or a range between any two of them; the interval time can be any one of 10, 12, 14, 16, 18 and 20 s or a range between any two of them; the number of times can be any one of 20, 30, 40, 50, 60, 70, 80, 90 and 100 times or a range between any two of them.

[0034] In some embodiments, after water bath sonication and before physical extrusion, the CCR2 + The method for preparing cell membrane vesicles further includes a centrifugation step. In some embodiments, the centrifugation conditions are 10,000-15,000 g for 20-40 min. The centrifugal acceleration can be any one of 10,000, 11,000, 12,000, 13,000, 14,000, and 15,000 g, or a range between any two thereof; and the centrifugation time can be any one of 20, 30, and 40 min, or a range between any two thereof.

[0035] In some embodiments, the conditions of the physical extrusion method include: 0-8°C, a polycarbonate membrane, a membrane pore size of 0.3-0.6 μm, and a number of extrusions of 10-30 times. The temperature can be any one of 0, 2, 4, 6, and 8°C, or a range between any two thereof; the membrane pore size can be any one of 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, and 0.6 μm, or a range between any two thereof; and the number of extrusions can be any one of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 times, or a range between any two thereof.

[0036] In some embodiments, after physical compression, the CCR2 +The method for preparing cell membrane vesicles further comprises centrifugation at 10,000 to 15,000 g for 20 to 40 minutes. The centrifugal acceleration can be any one of 10,000, 11,000, 12,000, 13,000, 14,000, and 15,000 g, or a range between any two thereof; and the centrifugation time can be any one of 20, 30, and 40 minutes, or a range between any two thereof.

[0037] In some embodiments, the method for preparing ginger-derived exosome-like nanoparticles comprises: centrifuging ginger juice for a first time, taking the supernatant after the first centrifugation and performing a second centrifugation; taking the supernatant after the second centrifugation and mixing it with a polyethylene glycol solution to obtain a mixed solution; taking the mixed solution and performing a third centrifugation, and collecting the precipitate to obtain ginger-derived exosome-like nanoparticles.

[0038] In some embodiments, the conditions of the first centrifugation are: 0-8°C, 2000-6000 g, 20-60 min; the temperature can be any one of 0, 2, 4, 6 and 8°C or a range between any two of them, the centrifugal acceleration can be any one of 2000, 3000, 4000, 5000 and 6000 g or a range between any two of them, and the centrifugation time can be any one of 20, 30, 40, 50 and 60 min or a range between any two of them.

[0039] In some embodiments, the conditions for the second centrifugation are: 0-8°C, 8000-12000 g, 80-120 min; the temperature can be any one of 0, 2, 4, 6 and 8°C or a range between any two of them, the centrifugal acceleration can be any one of 8000, 9000, 10000, 11000 and 12000 g or a range between any two of them, and the centrifugation time can be any one of 80, 90, 100, 110 and 120 min or a range between any two of them.

[0040] In some embodiments, the conditions of the third centrifugation are: 0-8°C, 2000-6000 g, 20-60 min; the temperature can be any one of 0, 2, 4, 6 and 8°C or a range between any two of them, the centrifugal acceleration can be any one of 2000, 3000, 4000, 5000 and 6000 g or a range between any two of them, and the centrifugation time can be any one of 20, 30, 40, 50 and 60 min or a range between any two of them.

[0041] In some embodiments, the mass volume percentage concentration of polyethylene glycol in the mixed solution is 5% to 25%, specifically any one of 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23% and 25%, or a range between any two of them.

[0042] In some embodiments, the molecular weight of the polyethylene glycol is 5000-7000, specifically any one of 5000, 6000 or 7000 or a range between any two of them.

[0043] In some embodiments, after the second centrifugation and before mixing with the polyethylene glycol solution, the method for preparing ginger-derived exosome-like nanoparticles further comprises: filtering the supernatant after the second centrifugation.

[0044] In some embodiments, the filtration includes coarse filtration and fine filtration. The coarse filtration uses a filter membrane with a pore size of 0.6 to 1.0 μm, specifically any one of 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and 1.0 μm, or a range between any two thereof; the fine filtration uses a filter membrane with a pore size of 0.2 to 0.6 μm, specifically any one of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, and 0.6 μm, or a range between any two thereof.

[0045] In some embodiments, the CCR2 + The mass ratio of the cell membrane vesicles to the ginger-derived exosome-like nanoparticles is 1:1-3, specifically any one of 1:1, 1:2 and 1:3 or a range between any two of them.

[0046] On the other hand, an embodiment of the present invention provides a drug delivery system comprising: microvesicles and ginger-derived exosome-like nanoparticles contained in the microvesicles; a method for preparing the drug delivery system comprises: + The mixture of cell membrane vesicles and ginger-derived exosome-like nanoparticles was physically extruded.

[0047] This drug delivery system has good biocompatibility and targets the injured spinal cord through the CCR2-CCL2 signaling axis to enhance the protective effect of GELN on the spinal cord. Its mechanism is related to anti-apoptosis, anti-oxidation and anti-inflammation.

[0048] In some embodiments, the microvesicles are bound to the CCR2 + The particle size ratio of the cell membrane vesicles is 1:1.5-3, specifically any one of 1:1.5, 1:2, 1:2.5 and 1:3 or a range between any two of them.

[0049] In some embodiments, the ginger-derived exosome-like nanoparticles and CCR2 + The conditions for physical extrusion of cell membrane vesicles include: a polycarbonate membrane with a pore size of 0.1 to 0.4 μm, and an extrusion cycle of 10 to 30 times. The membrane pore size can be any one of 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, and 0.4 μm, or a range between any two thereof; and the extrusion cycle can be any one of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 times, or a range between any two thereof.

[0050] In some embodiments, the preparation method of the drug delivery system further comprises centrifuging the product after physical extrusion, wherein the centrifugation conditions are 10,000 to 15,000 g for 20 to 40 min. The centrifugal acceleration can be any one of 10,000, 11,000, 12,000, 13,000, 14,000, and 15,000 g, or a range between any two thereof; and the centrifugation time can be any one of 20, 30, and 40 min, or a range between any two thereof.

[0051] On the other hand, an embodiment of the present invention further provides a drug, the active ingredient of which includes the composition described in any of the foregoing embodiments or the drug delivery system described in any of the foregoing embodiments.

[0052] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier, including but not limited to diluents, buffers, suspensions, emulsions, granules, encapsulations, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorption carriers.

[0053] In addition, embodiments of the present invention also provide use of the composition described in any of the foregoing embodiments or the drug delivery system described in any of the foregoing embodiments in the preparation of a product for treating spinal cord injury and / or related diseases or symptoms caused by spinal cord injury.

[0054] In some embodiments, the diseases or symptoms associated with spinal cord injury include direct functional impairment and / or complications.

[0055] In some embodiments, the direct dysfunction includes at least one of a motor disorder, a sensory disorder, and a bladder and rectal dysfunction.

[0056] In some embodiments, the complications include: infections (e.g., pressure sores, urinary / respiratory tract infections), circulatory and metabolic problems (e.g., deep vein thrombosis, orthostatic hypotension, osteoporosis), musculoskeletal abnormalities (e.g., joint stiffness / contracture, heterotopic ossification), autonomic dysfunction (e.g., autonomic hyperreflexia, temperature regulation disorders), pain and spasm (e.g., neuropathic pain, spasticity), and psychological and social functioning (e.g., depression, anxiety, suicidal tendencies).

[0057] In some embodiments, the product comprises a pharmaceutical.

[0058] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0059] Example 1. Preparation and identification of CCR2-MV / GELN 1 Materials and Methods 1.1 Cell lines This example uses a human cervical cancer cell line, HeLa cells. After STR identification, they were cultured in Opti-MEM medium containing 10% fetal bovine serum in a 37°C-5% CO2 environment. They were passaged when the confluence reached 70%-80% to ensure that cells in the logarithmic growth phase were used in this experiment.

[0060] 1.2 Experimental Animals The experimental protocols in all examples of this application were approved by the Ethics Committee of the 900th Hospital of the Joint Logistics Support Force, and the experimental procedures followed the "Guidelines for Ethical Review of Laboratory Animal Welfare." The experimental animals in this example were clean-grade healthy male C57BL / 6 mice, 6 to 8 weeks old, weighing 24 to 30 g, provided by the Animal Experiment Center of the 900th Hospital of the Joint Logistics Support Force. They were allowed to eat and drink freely before the experiment.

[0061] 1.3 Experimental methods 1.3.1 Isolation of GELN Fresh ginger was squeezed into juice and filtered through a gauze screen to remove the residue. The collected ginger juice was aliquoted into centrifuge tubes and centrifuged at 4000 g for 40 minutes at 4°C to remove fibers and large particles. The resulting supernatant was then centrifuged at 10,000 g for 100 minutes at 4°C to remove impurities such as cell debris. A 0.8 μm microporous filter was used to remove some impurities, followed by a 0.4 μm microporous filter to further remove smaller particles. 30% polyethylene glycol 6000 and the filtered ginger juice were thoroughly mixed in an equal volume ratio (the final polyethylene glycol 6000 concentration (m / v) in the mixture was 15%). After standing overnight, the mixture was centrifuged at 4000 g for 40 minutes at 4°C, and the supernatant was discarded to obtain GELN.

[0062] 1.3.2 Preparation of CCR2-MVs (1) Isolation and culture of mouse bone marrow-derived Ly6C ++ CCR2 ++ Classical monocytes Mice were killed by dislocation and disinfected by soaking in 75% alcohol for 2 minutes. Both femurs were completely removed in a sterile laminar flow hood and placed in pre-chilled PBS buffer, with residual muscle tissue removed. The femurs were transferred to another dish, and the femoral heads were excised to expose the bone marrow cavity. PBS was injected into the distal end of the dish using a sterile 2 ml syringe and flushed three times. The bone marrow tissue was then pipetted and mixed to create a single-cell suspension. The suspension was filtered through a sterile 400-mesh nylon sieve into a centrifuge tube and centrifuged at 1000 g for 4 minutes at 4°C. The supernatant was discarded. After adding 3 ml of erythrocyte lysis buffer and incubating on ice for 5 minutes, an equal volume of PBS was added to terminate erythrocyte lysis. The suspension was centrifuged as above, and the supernatant discarded. The suspension was resuspended in 3 ml of DMEM supplemented with 10% fetal bovine serum and plated onto a 10 cm dish. The suspension was incubated at 37°C in a 5% CO2 atmosphere for 6–8 hours. The unattached cell suspension in the dish was aspirated into a centrifuge tube and centrifuged as above. The supernatant was discarded. Resuspend in 3 ml of 10% FBS DMEM culture medium, inoculate in 6-well plates, add 25 ng / ml of granulocyte-macrophage colony stimulating factor, and culture at 37°C-5% CO2 for 1 day to obtain Ly6C ++ CCR2 ++ Classical monocytes.

[0063] (2) Identification of mouse bone marrow-derived Ly6C ++ CCR2 ++ Classical monocytes Ly6C was digested with trypsin. ++ CCR2 ++ For classic monocytes, digestion is terminated when the cells become round. Gently pipette until the cells are completely detached, transfer to a centrifuge tube, centrifuge as above, and discard the supernatant. Add 10 μl of mouse serum to block the cells. Incubate on ice for 10 minutes, then add 40 μl of buffer and resuspend by vortexing. Then add fluorescently conjugated flow cytometric antibodies for staining: PerCP anti-mouse CD45, PE anti-mouse F4 / 80, APC anti-mouse CD11b, FITC anti-mouse Ly6C, and AF647 anti-mouse CCR2. Incubate at 4°C in the dark for 30 minutes. Centrifuge as above to discard excess antibody, and add 500 μl of buffer for flow cytometry analysis.

[0064] (3) Isolation of CCR2 + Monocyte cell membrane Ly6C ++ CCR2 ++Transfer the classic monocytes into a centrifuge tube, centrifuge as above, discard the supernatant, and resuspend in PBS to a cell density of 5×10 6 The centrifuge tube was placed in an ultrasonic cell disruptor (frequency 20 kHz, power 400 W) and the cells were disrupted and lysed using pulse mode (ultrasound time 10 s / time, interval time 10 s, number of times 40) in a 4°C water bath. The cells were centrifuged as above and the supernatant was collected. The supernatant was placed on top of a sucrose solution with increasing concentrations of sucrose in volume from top to bottom (25%, 40%, 55%) and centrifuged at 25,000 g for 50 min. The liquid layers between 25% and 40% and between 40% and 55% were collected to obtain CCR2. + Monocyte cell membrane fragments. (4) Preparation of CCR2-MV ① Water bath ultrasound combined with physical extrusion method The obtained CCR2 + The cell membrane fragments of monocytes were diluted with PBS to a concentration of 1.5 g / ml in a centrifuge tube. The centrifuge tube was placed in a 4°C water bath on an ultrasonic oscillator (frequency 25 kHz, power 300 w) and oscillated using a pulse mode (ultrasound time 15 s / time, interval time 15 s, number of times 60). The tube was centrifuged at 12,000 g for 30 min, and the supernatant was discarded to obtain CCR2-MV. CCR2-MVs prepared by water bath ultrasound were extruded 20 times through a polycarbonate membrane with a pore size of 0.45 μm in an Avanti microextruder at 4°C, centrifuged at 12,000 g for 30 min, and the precipitate was collected to obtain CCR2-MVs of homogenous size.

[0065] ②Physical extrusion method The obtained CCR2 + The cell membrane fragments of monocytes were squeezed 20 times through a polycarbonate membrane with a pore size of 0.45 μm in an Avanti microextruder at 4°C, centrifuged at 12,000 g for 30 min, and the precipitate was collected to obtain CCR2-MVs of uniform size.

[0066] 1.3.3 Preparation of CCR2-MV / GELN CCR2-MV and GELN were thoroughly mixed at a mass ratio of 1:2 to form a suspension. The suspension was extruded 20 times through a polycarbonate membrane with a pore size of 0.25 μm in an Avanti microextruder at 4°C, centrifuged at 12,000 g for 30 min, and the precipitate was collected to obtain CCR2-MV / GELN.

[0067] 1.3.4 Particle Size, Zeta Potential, and Electron Microscopy Observation of GELN, CCR2-MV, and CCR2-MV / GELN (1) Particle size and Zeta potential detection Detection was performed using a nanoparticle tracking analyzer. The instrument was calibrated with standard samples, and the measuring cell was cleaned with ddH2O. GELN, CCR2-MV, and CCR2-MV / GELN suspensions were then injected into the measuring cell, ensuring the absence of bubbles. The particle size and zeta potential values ​​were recorded for each measurement.

[0068] (2) Transmission electron microscopy observation GELN, CCR2-MV, and CCR2-MV / GELN suspensions were dropped onto a copper mesh with a carbon support film. After 10 minutes of stagnation, any remaining water droplets were removed with filter paper. A 3% phosphotungstic acid solution was dripped onto a wax tray. The sample-coated copper mesh was gently inverted and placed on the phosphotungstic acid droplet, ensuring full contact. After 1 minute, the mesh was removed, rinsed with ddH2O, and dried before observation under a transmission electron microscope.

[0069] 1.3.5 Observation of Colocalization of CCR2-MV and GELN in CCR2-MV / GELN (1) DiR lipophilic red fluorescent probe labeled GELN GELN suspension (5 × 10 6 The cells / ml) were mixed with DiR working solution (1 mg / ml, DMSO as solvent) at a volume ratio of 1:1, incubated at 37°C in the dark for 30 min, centrifuged at 1000 g for 5 min, the supernatant was discarded, and the pellet was resuspended in PBS.

[0070] (2) DiO lipophilic green fluorescent probe labeling CCR2-MV CCR2-MV suspension (5 × 10 6 The cells / ml) were mixed with DiO working solution (1 mg / ml, DMSO as solvent) at a volume ratio of 1:1, incubated at 37°C in the dark for 30 min, and centrifuged as above. The supernatant was discarded and the pellet was resuspended in PBS.

[0071] (3) Preparation of DiO-CCR2-MV / DiR-GELN Thoroughly mix DiO-labeled CCR2-MV (DiO-CCR2) and DiR-labeled GELN (DiR-GELN) at a mass ratio of 1:2 to form a suspension. Other steps are the same as in 1.3.3.

[0072] (4) Observation of colocalization of CCR2-MV and GELN in CCR2-MV / GELN HeLa cells were seeded in 6-well plates (5 × 10 4Cells were cultured in Opti-MEM medium (10% FBS) supplemented with 10% FBS at 37°C in 5% CO₂ until confluence reached 70%–80%, and then replaced with serum-free Opti-MEM medium. HeLa cells were transfected with the components prepared according to Table 1 (triplicate wells per sample). Cells were incubated at 37°C in 5% CO₂ for 4 hours, fixed with 4% paraformaldehyde for 15 minutes, and stained with DAPI for 5 minutes. Observations were made under a confocal laser scanning microscope using excitation wavelengths of 488 nm (DiO), 647 nm (DiR), and 405 nm (DAPI).

[0073] Table 1 Components of HeLa cells transfected with each sample

[0074] 1.3.6 Detection of 6-gingerol and 6-shogaol in GELN and CCR2-MV / GELN High performance liquid chromatography was used for detection. 6 The sample was eluted with 10 μl of the standard solution (250 mm × 4.6 mm, 5 μm) through an automatic injector. The column temperature was 30°C, the detection wavelength was 282 nm, the mobile phase was a gradient elution of acetonitrile and formic acid, the flow rate was 1.0 ml / min, and the analysis time was 50 min. Quantification was performed by comparing the peak areas of the sample and the standard.

[0075] 1.3.7 GELN, CCR2 + Detection of chemokine receptors and surface markers on monocytes, CCR-MVs, and CCR2-MV / GELNs (1) Detection of chemokine receptors on membrane surface Flow cytometry was used for detection. Fluorescence-coupled flow cytometry antibodies were added to the suspension for staining: AF647 anti-mouse CCR2, PerCP anti-mouse CX3CR1, and PE anti-mouse CCR5. Other procedures were the same as 1.3.2 (2). The mean fluorescence intensity was used to reflect the expression level of the receptor.

[0076] (2) Detection of membrane surface markers Western blot analysis was performed. Membrane surface proteins from GELN, CCR2+ monocytes, CCR-MVs, and CCR2-MV / GELN were extracted using a membrane protein extraction kit, and protein concentrations were determined by the BCA assay. Twenty μg of protein was mixed with an equal volume of sample buffer, boiled for 5 minutes, and electrophoresed on SDS-PAGE gels. The membranes were then transferred to nitrocellulose membranes, blocked with 5% skim milk powder for 3 hours, washed with 0.1% PBST, and incubated overnight at 4°C with rabbit anti-mouse Ly6C, CD163, CD62L, CD36, CD64, and CD11b primary antibodies (1:1000 dilution). After washing three times with PBST, HRP-conjugated donkey anti-rabbit secondary antibodies (1:2000 dilution) were added and incubated at room temperature for 1 hour. The membranes were washed with PBST, developed with ECL chemiluminescence, and imaged using a gel scanning system. ImagePro Plus software was used for analysis, and the ratio of the gray value of the target protein band to the gray value of the internal reference β-actin band was used to reflect the expression level of the target protein.

[0077] 2 Results 2.1 Preparation and characteristics of each group of samples Figure 1 (A) is a schematic diagram of the preparation process of CCR2-MV / GELN. Figure 1 (B) shows that Ly6C isolated and cultured in this example ++ CCR2 ++ The purity of the classical monocyte subsets was as high as 98.1%, laying the foundation for the next step of preparing CCR2-MV. Figure 1 Emission cytometry (CE) revealed that GELN had an average particle size of 235 nm, a PDI of 0.10, and a Zeta potential of -25.7 mV; CCR2-MV had an average particle size of 455 nm, a PDI of 0.04, and a Zeta potential of +25.1 mV; and CCR2-MV / GELN had an average particle size of 265 nm, a PDI of 0.08, and a Zeta potential of +25.0 mV. These results confirm that CCR2-MV and GELN can be brought together by attraction between positive and negative charges, and further squeezed to form CCR2-MV / GELN. The particle size distributions of GELN, CCR2-MV, and CCR2-MV / GELN are uniform.

[0078] Figure 1 (F) shows that GELN is a small vesicle with a single membrane, CCR2-MV is a large vesicle with a single membrane, and CCR2-MV / GELN is a vesicle with a double membrane. Figure 1(G) shows that in HeLa cells, GELN exhibits red fluorescence, CCR2-MV exhibits green fluorescence, and CCR2-MV / GELN displays colocalization (yellow fluorescence) of CCR2-MV green fluorescence (shell) and GELN red fluorescence (core). These results confirm that CCR2-MV / GELN is formed by CCR2-MV encapsulating GELN.

[0079] Figure 1 Figures (H, I) show that the 6-gingerol content in GELN was (4.80±0.55) μmol / million particles, and the 6-shogaol content was (4.15±0.57) μmol / million particles. The 6-gingerol content in CCR2-MV / GELN was (4.65±0.59) μmol / million particles, and the 6-shogaol content was (4.02±0.63) μmol / million particles. These results confirm that CCR2-MV / GELN retains the GELN property of loading ginger active ingredients.

[0080] 2.2 Expression analysis of membrane surface chemokine receptors and membrane surface markers in each group of samples Figure 2 (A, B) show that GELN, CCR2 + Monocytes, CCR2-MVs and CCR2-MV / GELNs all highly expressed CCR2 and lowly expressed CX3CR1 and CCR5 on their membrane surfaces. Figure 2 (C, D) show that GELN, CCR2 + The membranes of monocytes, CCR2-MVs, and CCR2-MV / GELNs all highly expressed classical monocyte markers, including Ly6C, CD163, CD62L, CD36, CD64, and CD11b. These results confirm that CCR2-MV / GELNs possess the biological properties of classical monocyte membranes.

[0081] Example 2: Effect of CCR2-MV / GELN on Alleviating Spinal Cord Injury 1 Materials and Methods 1.1 Experimental animals The experimental animals used in this example were clean-grade, healthy male Sprague Dawley (SD) rats, 3 months old, weighing 300–350 g, provided by the Animal Experiment Center of the 900th Hospital of the Joint Logistics Support Force. All rats with normal neurological function were housed in a vivarium with appropriate temperature, humidity, and day / night cycle for at least one week before surgery, with free access to food and water.

[0082] 1.2 Experimental methods 1.2.1 Establishment of rat SCI model The modified Allen percussion method was used to remove T10 The spinal cord was exposed through the vertebral lamina, and a 1 mm thick pad was attached to the dura mater. A 30 g Kirschner wire was dropped vertically from a height of 3.0 cm along the cannula to hit the pad, causing SCI. The rat's whole body quickly retracted and shook, indicating that the model was successfully established.

[0083] 1.2.2 Experimental Grouping (1) SCI group: rats with T 10 The spinal cord is exposed through the lamina and SCI is performed; (2) GELN-high group: rats were injected with high-dose GELN suspension (5×10 8 GELN / ml) 5 ml / kg; (3) CCR2-MV / GELN-low group: rats were injected with high-dose CCR2-MV / GELN suspension (5×10 8 CCR2-MV / GELN / ml) 1 ml / kg; (4) CCR2-MV / GELN-high group: rats were injected with low-dose CCR2-MV / GELN suspension (5×10 8 CCR2-MV / GELN / ml) 5 ml / kg.

[0084] 1.2.3 Biocompatibility testing of each group of nanoparticles on important organs Three days after SCI, arterial blood was drawn from the rats in each group to test blood routine (RBC, HB, PLT), myocardial enzymes (CK, CK-MB), blood gas (PaO2, PaCO2, pH, HCO3 - ), liver function (ALT, AST, TBil, DBil, ALB, Glu), renal function (BUN, Cr, UA) and electrolytes (K + 、Na + , Ca 2+ 、Cl - ) indicators; the heart, lung, liver, spleen and kidney of the rats were taken for HE staining to observe the changes in the tissue structure and cell morphology of each organ.

[0085] 1.2.4 Chemotaxis of each group of nanoparticles to the SCI area The lipid membranes of GELN and CCR2-MV / GELN were labeled with a DiR lipophilic red fluorescent probe to form DiR-GELN and DiR-CCR2-MV / GELN. DiR-GELN and DiR-CCR2-MV / GELN were administered to the rats in the same group as described in 1.2.2. Three days after SCI, the rats' backs were shaved and placed in a prone position in the imaging chamber of a small-animal in vivo fluorescence imaging system. The thoracic and lumbar regions of interest were selected, and their mean fluorescence intensity was measured. Venous blood was drawn from the rats at 6, 12, 1, and 3 days after SCI and placed in centrifuge tubes containing anticoagulant. Blood cells and plasma were separated by centrifugation at 2000 g for 15 minutes at 4°C. The blood was then transferred to separate centrifuge tubes and placed in the imaging chamber. The mean fluorescence intensity of the blood cells and plasma was measured. Three days after SCI, the spinal cord, heart, lung, liver, and kidney were collected from the rats and placed in the imaging chamber. The mean fluorescence intensity of each organ was measured.

[0086] 1.2.5 Neurobehavioral Assessment (1) BBB motor function score Hindlimb motor function in rats was assessed using the Basso, Beattie, and Bresnahan (BBB) ​​score at 1, 3, 7, and 14 days after SCI. The score consists of three parts: the first part, ranging from 0 to 7, evaluates hindlimb joint movement; the second part, ranging from 8 to 13, assesses hindlimb gait and coordination; and the third part, ranging from 14 to 21, evaluates fine motor movements during movement. Complete hindlimb paralysis is scored as 0, and complete normal function is scored as 21.

[0087] (2) Inclined plate test Hindlimb muscle strength in rats was assessed using an inclined board test at 1, 3, 7, and 14 days after SCI. The board was padded with a 6 mm thick rubber pad. The rat was placed with its body axis perpendicular to the board's longitudinal axis. The angle between the board and the horizontal plane was gradually increased until the rat could rest on the board for 5 seconds. This angle was recorded and the average of the three measurements was calculated.

[0088] (3) Footprint analysis Fourteen days after SCI, a piece of white paper of the same length and width was placed on a narrow board. The dorsum of the rat's hind limbs was smeared with red ink, and the sole of the hind limb was smeared with blue ink. The rat was immediately placed on one side of the white paper and walked toward a dark box. After the rat entered the dark box, the white paper was folded away, and the step width (distance between the left and right footprints), step length (distance between the two footprints on the same side), and dorsum drag rate (red footprint pixel value / red footprint pixel value + blue footprint pixel value) of the hind limb were measured.

[0089] 1.2.6 Motor evoked potential detection Motor evoked potentials (MEPs) were measured 14 days after SCI in rats. Needle-shaped stimulating electrodes were implanted 2 mm anterior to the coronal suture and 2 mm lateral to the sagittal suture (in the motor area of ​​the cerebral cortex). Stimulation intensity was 40 mA, with a 0.1 ms pulse width, a 1 Hz frequency, 300-500 superpositions, a sweep speed of 5 ms / D, and a sensitivity of 5 mV / D. The latency and amplitude of the response wave were recorded.

[0090] 1.2.7 Nuclear Magnetic Resonance Detection Fourteen days after SCI, rat spinal column tomography was performed using a 7.0T small animal MRI. T2-weighted images were used with a 0.5 mm slice thickness to observe morphological and structural changes at the SCI site and measure edema area.

[0091] 1.2.8 Gross observation of the spinal cord On day 14 after SCI, the rats were deeply anesthetized and perfused with 4% paraformaldehyde through the left ventricle for fixation. 10 ~L2 spinal cord, observe its gross morphology and measure the injury area.

[0092] 1.2.9 Blood-spinal cord barrier permeability test The permeability of the blood-spinal cord barrier (BSCB) was reflected by the fluorescence intensity and content of Evans blue infiltrated into the spinal cord, the level of neutrophils (myeloperoxidase [MPO] activity), and the water content of the spinal cord.

[0093] (1) 14 days after SCI, 45 mg / kg of Evans blue solution was injected into the rats through the tail vein. One hour later, the rats were deeply anesthetized and physiological saline was perfused through the left ventricle. When the outflow fluid from the right atrial appendage became colorless, the T 10 ~L2 spinal cord.

[0094] ①Evans blue fluorescence intensity: T 10 The ~L2 spinal cord was fixed in a light-proof, sealed container with 4% paraformaldehyde for 48 hours and frozen (10 μm thick) sections were cut. Evans blue that had penetrated the spinal cord appeared red under a fluorescence microscope (excitation wavelength: 532 nm, emission wavelength: 588 nm). Fluorescence intensity was calculated using Image Pro Plus analysis software.

[0095] ② Evans blue content: weigh T 10 The weight of the ~L2 spinal cord was measured, 3 ml of formamide was added, the cells were incubated at 45°C for 48 h, and centrifuged at 3000 g for 15 min. The supernatant was collected and the OD value of the supernatant was measured at a wavelength of 623 nm using a microplate reader. The Evans blue content in the spinal cord was calculated with reference to the standard curve.

[0096] (2) On day 14 after SCI, the rats were deeply anesthetized and physiological saline was perfused through the left ventricle. T was removed after the outflow fluid from the right atrial appendage became colorless. 10 ~L2 spinal cord.

[0097] ①MPO activity: T 10 Protease inhibitors and RIPA lysis buffer were added to the ~L2 spinal cord, homogenized at 4°C, and centrifuged at 3000 g for 15 min. The supernatant was collected to obtain total cellular protein, which was then detected according to the MPO activity assay kit operating procedures.

[0098] ② Spinal cord water content: Accurately weigh T 10 The ~L2 spinal cord was weighed (wet weight), placed in an oven for 1 h to lose moisture, and then weighed again (dry weight). The spinal cord water content was calculated as (wet weight − dry weight) × 100% / wet weight.

[0099] 1.2.10 Spinal cord neuron survival assay T 10 The ~L2 spinal cord was fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned (5 μm thick). Three sections were equidistantly sampled from each spinal cord segment for Nissl staining. Five non-overlapping fields of view in the anterior horn, intermediate zone, and posterior horn of the spinal cord were selected under a light microscope. The neurons containing granular Nissl bodies in the cytoplasm and with normal morphology were counted and the average value was calculated.

[0100] 1.2.11 Spinal cord neuron apoptosis detection The number of apoptotic neurons and the expression level of apoptosis-related proteins were used to reflect the apoptosis of spinal cord neurons.

[0101] (1) Detection of spinal cord neuron apoptosis: TUNEL staining was used for detection. Three paraffin sections were obtained in 1.2.10 for each spinal cord segment, dewaxed to water, and operated according to the kit steps. The nuclei were then stained with DAPI. Under a fluorescence microscope, TUNEL-positive cells showed green fluorescence (excitation wavelength: 488 nm, emission wavelength: 507 nm), and DAPI-labeled nuclei showed blue fluorescence (excitation wavelength: 400 nm, emission wavelength: 450 nm). Five non-overlapping fields of view were randomly selected, and the number of TUNEL and DAPI co-localized cells in the anterior horn, intermediate zone, and posterior horn of the spinal cord gray matter was counted using Image Pro Plus analysis software, and the average value was taken.

[0102] (2) Detection of activated Caspase-3 (Cleaved Caspase-3) in spinal cord neurons: Double immunofluorescence staining was used. Three paraffin sections were extracted from each spinal cord segment obtained in 1.2.10, dewaxed and blocked with BSA for 15 min, and rabbit anti-mouse Cleaved Caspase-3 primary antibody (dilution 1:1000) and goat anti-mouse NeuN primary antibody (dilution 1:800) were added dropwise. The sections were incubated overnight at 4°C. Donkey anti-rabbit secondary antibody labeled with Alexa Fluor 594 (dilution 1:200) and donkey anti-goat secondary antibody labeled with Alexa Fluor 488 (dilution 1:200) were added dropwise in a dark environment. Under a fluorescence microscope, Cleaved Caspase-3 showed red fluorescence (excitation wavelength 532 nm, emission wavelength 588 nm), and NeuN showed green fluorescence (excitation wavelength 488 nm, emission wavelength 507 nm). Five non-overlapping fields were randomly selected, and the number of cells co-localized with Cleaved Caspase-3 and NeuN in the anterior horn, intermediate zone, and posterior horn of the spinal cord gray matter was counted using Image Pro Plus analysis software, and the average value was taken.

[0103] (3) Detection of apoptosis-related proteins: Western blot was used for detection. 1.2.9 (2) ① After denaturation, electrophoresis, and membrane transfer, the total spinal cord cell protein was added with rabbit anti-mouse Bcl-2, Bax, cytochrome c (Cyt c), activated Caspase-9 (Cleaved Caspase-9), and activated Caspase-3 (Cleaved Caspase-3) primary antibodies (dilution 1:800), and the rest was the same as 1.3.7 (2).

[0104] 1.2.12 Spinal cord myelin sheath detection T was removed 14 days after SCI. 10 ~L2 spinal cord trimmed to approximately 1 mm 3 The cells were fixed in 2.5% glutaraldehyde for 6 h, embedded in epoxy resin, and ultrathinly sectioned (70 nm thick). The sections were placed on copper grids and stained with uranyl acetate for 30 min and then with lead citrate for 10 min. Five fields of view were randomly selected under a transmission electron microscope, and the number of myelinated axons and the G-ratio (axon diameter / axon diameter + myelin sheath thickness) reflecting the thickness of the myelin sheath were counted using Image Pro Plus analysis software, and the average value was taken.

[0105] 1.2.13 Detection of spinal cord Nrf2 signaling pathway and oxidative stress-related indicators (1) Detection of nuclear factor erythroid 2 related factor (Nrf2): Rats were killed at 1, 3, 7, and 14 days after SCI, and T 10 Protease inhibitors and RIPA lysis buffer were added to the L2 spinal cord, homogenized at 4°C, and centrifuged at 3000 g for 15 min. The supernatant was collected and separated into cytoplasmic and nuclear proteins according to the nuclear-cytoplasmic separation kit. The cytoplasmic proteins were used to detect Nrf2 expression by Western blot, following the procedure in step 1.3.7 (2) of Example 1.

[0106] (2) Nrf2 DNA binding activity detection: 1.2.13 (1) Add the isolated nuclear protein to the Nrf2 DNA binding activity detection kit and measure the OD value at an emission wavelength of 450 nm using a microplate reader.

[0107] (3) Antioxidant enzymes and oxidative stress metabolites detection: ELISA method was used for detection. 1.2.13 (1) The isolated cytoplasmic proteins were added with superoxide dismutase (SOD) activity, catalase (CAT) activity, reduced glutathione (GSH), oxidized glutathione (GSSG), superoxide (O2 - ), hydrogen peroxide (H2O2), and malondialdehyde (MDA) detection kits were used, and the corresponding OD values ​​were measured using a microplate reader.

[0108] 1.2.14 Detection of NF-κB signaling pathway and inflammatory markers in the spinal cord (1) Nuclear factor kappa-B (NF-κB) detection: 1.2.13 (1) The cytoplasmic proteins isolated were used to detect NF-κB expression by Western blot, using the same method as step 1.3.7 (2) in Example 1.

[0109] (2) Nrf2 DNA binding activity detection: 1.2.13 (1) Add the isolated nuclear protein to the NF-κB DNA binding activity detection kit and measure the OD value at an emission wavelength of 450 nm using a microplate reader.

[0110] (3) Detection of pro-inflammatory and anti-inflammatory factors: ELISA was used. 1.2.13 (1) The isolated cytoplasmic proteins were added to the detection kits for tumor necrosis factor-α (superoxide dismutase, TNF-α), interleukin-6 (interleukin-6, IL-6), interleukin-1β (interleukin-1β, IL-1β), transforming growth factor-β (transforming growth factor-β, TGF-β), interleukin-10 (interleukin-4, IL-10), and interleukin-4 (interleukin-4, IL-4), and the corresponding OD values ​​were measured using a microplate reader.

[0111] 1.2.15 Statistical Processing SPSS 27.0 statistical software was used for analysis, and the normally distributed quantitative data were expressed as mean ± standard deviation ( ) indicated that the comparison between and within groups was conducted using one-way analysis of variance, and P < 0.05 was considered statistically significant.

[0112] 2. Results 2.1 Biocompatibility of each group of nanoparticles on important organs Table 2 shows that all test indicators in each group were within the normal range, and there was no statistically significant difference between the groups (P>0.05). Figure 3 The results showed that there were no significant abnormalities in the tissue structure and cell morphology of the heart, lungs, liver, spleen, and kidneys. These results confirmed that the nanoparticles in each group had no significant impact on important organs and had good biocompatibility.

[0113] Table 2 Effects of nanoparticles in each group on blood routine, myocardial enzyme spectrum, arterial blood gas, liver and kidney function and electrolyte indicators in rats

[0114] 2.2 Chemotaxis of each group of nanoparticles to the SCI area Figure 4 (A, B) show that the fluorescence intensity of blood cells and plasma in the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups reached peak values ​​at 6 h after SCI ( P <0.001 vs. before injury), and gradually decreased at 12 h, 1 d, and 3 d ( P <0.01 for 12 h, P <0.05 for 1 d vs. before injury). Figure 4(C, D) show that the fluorescence intensity of the thoracic and lumbar spinal cord in the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups was greater than that in the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high), the fluorescence intensity in the liver and kidney of the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups was lower than that of the GELN-high group ( P The above results confirmed that CCR2-MV / GELN has a better chemotaxis to the spinal cord than GELN.

[0115] 2.3 Effects of each group of nanoparticles on neurological function after SCI Figure 5 (A, B) showed that compared with the SCI group, the BBB scores and tilt plate test scores of the GELN-high group, CCR2-MV / GELN-low group, and CCR2-MV / GELN-high group increased at 1, 3, 7, and 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the CCR2-MV / GELN-low group and the CCR2-MV / GELN-high group were higher than the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). Figure 5 The (CL) in the middle showed that compared with the SCI group, the step width, dragging rate, motor evoked potential latency, spinal cord edema area and injury area of ​​the GELN-high, CCR2-MV / GELN-low and CCR2-MV / GELN-high groups were reduced, and the step length and motor evoked potential amplitude were increased 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P<0.001 for CCR2-MV / GELN-high); the effects of the CCR2-MV / GELN-low group and the CCR2-MV / GELN-high group were better than those of the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). The above results confirmed that CCR2-MV / GELN is more effective than GELN in improving neurological function after SCI.

[0116] 2.4 Effects of each group of nanoparticles on BSCB permeability after SCI Figure 6 Figures (AE) show that compared with the SCI group, the fluorescence intensity and content of Evans blue infiltrated into the spinal cord, the water content of the spinal cord, and the MPO activity were decreased in the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the fluorescence intensity and content of Evans blue infiltrated into the spinal cord, spinal cord water content, and MPO activity in the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups were lower than those in the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P The above results confirmed that CCR2-MV / GELN was more effective than GELN in reducing BSCB permeability after SCI.

[0117] 2.5 Effects of each group of nanoparticles on spinal cord histomorphology after SCI Figure 7 (A) shows that SCI leads to a significant loss of normal neurons in the anterior horn, intermediate zone and posterior horn of the spinal cord gray matter, showing a large number of pyknotic and darkly stained damaged neurons. More normal neurons containing granular Nissl bodies in the cytoplasm can be seen in the spinal cords of the GELN-high group, CCR2-MV / GELN-low group and CCR2-MV / GELN-high group. Figure 7(B) shows that compared with the SCI group, the number of normal neurons in the anterior horn of the spinal cord gray matter and the posterior horn of the intermediate zone nucleus in the GELN-high group, CCR2-MV / GELN-low group, and CCR2-MV / GELN-high group increased 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the number of normal neurons in the anterior horn of the spinal cord gray matter and the posterior horn of the intermediate zone nucleus in the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups was higher than that in the GELN-high group ( P <0.05 forCCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). The above results confirmed that CCR2-MV / GELN was better than GELN in protecting the morphological structure of spinal cord tissue after SCI.

[0118] 2.6 Effects of each group of nanoparticles on the spinal cord myelin sheath after SCI Figure 8 (A) shows that SCI leads to a large loss of spinal cord myelin sheaths and thinning of thickness. More and thicker myelin sheaths can be seen in the spinal cords of the GELN-high group, CCR2-MV / GELN-low group, and CCR2-MV / GELN-high group. Figure 8 (B, C) showed that compared with the SCI group, the number of myelinated axons increased and the G-ratio decreased in the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the effects of the CCR2-MV / GELN-low group and the CCR2-MV / GELN-high group were better than those of the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P The above results confirmed that CCR2-MV / GELN was better than GELN in protecting the myelin structure of the spinal cord after SCI.

[0119] 2.7 Effects of each group of nanoparticles on spinal cord neuron apoptosis after SCI Figure 9 (A, B) show that SCI can lead to a large number of spinal cord cell apoptosis, and the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups can reduce spinal cord cell apoptosis. Compared with the SCI group, the number of TUNEL-positive cells in the anterior horn, intermediate zone, and posterior horn of the spinal cord in the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups was reduced 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the number of TUNEL-positive cells in the anterior horn, intermediate zone and posterior horn of the spinal cord in the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups was less than that in the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 forCCR2-MV / GELN-high). Figure 9 (C, D) showed that compared with the SCI group, the number of activated Caspase-3 / NeuN-positive neurons in the anterior horn, intermediate zone, and posterior horn of the spinal cord in the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups was reduced 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the number of activated Caspase-3 / NeuN-positive neurons in the anterior horn, intermediate zone, and posterior horn of the spinal cord in the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups was less than that in the GELN-high group ( P <0.05 forCCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). The above results confirmed that CCR2-MV / GELN is more effective than GELN in alleviating spinal cord neuron apoptosis after SCI.

[0120] 2.8 Effects of each group of nanoparticles on spinal cord apoptosis-related proteins after SCI Figure 10 (A, B) show that compared with the SCI group, the expression level of Bcl-2 in the spinal cord of the GELN-high group, CCR2-MV / GELN-low group, and CCR2-MV / GELN-high group was increased, and the expression levels of Bax, Cyt c, activated Caspase-9, and activated Caspase-3 were decreased ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the effects of the CCR2-MV / GELN-low group and the CCR2-MV / GELN-high group were better than those of the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). The above results further confirmed that CCR2-MV / GELN is more effective than GELN in alleviating spinal cord neuron apoptosis after SCI.

[0121] 2.9 Effects of each group of nanoparticles on the Nrf2 signaling pathway and oxidative stress-related indicators in the spinal cord after SCI Figure 11 (AJ) showed that compared with the SCI group, Nrf2 expression, DNA binding activity, SOD activity, CAT activity, and GSH content in the spinal cord of the GELN-high, CCR2-MV / GELN-low, and CCR2-MV / GELN-high groups were increased at 1, 3, 7, and 14 days after SCI, while GSSG and O2 - , H2O2, MDA content decreased ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the effects of the CCR2-MV / GELN-low group and the CCR2-MV / GELN-high group were better than those of the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). The above results confirmed that CCR2-MV / GELN is more effective than GELN in alleviating oxidative damage after SCI.

[0122] 2.10 Effects of each group of nanoparticles on the NF-κB signaling pathway and inflammation-related indicators in the spinal cord after SCI Figure 12 The (AI) showed that compared with the SCI group, the expression of phosphorylated NF-κB p65 and its DNA binding activity, the levels of TNF-α, IL-6, and IL-1β in the spinal cord of the GELN-high group, CCR2-MV / GELN-low group, and CCR2-MV / GELN-high group were decreased, and the levels of TGF-β, IL-10, and IL-4 were increased at 1, 3, 7, and 14 days after SCI ( P <0.05 for GELN-high, P <0.01 forCCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the effects of the CCR2-MV / GELN-low group and the CCR2-MV / GELN-high group were better than those of the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 forCCR2-MV / GELN-high). Figure 12 (J, K) showed that compared with the SCI group, the expression levels of MMP2 and MMP3 in the spinal cord of the GELN-high group, CCR2-MV / GELN-low group, and CCR2-MV / GELN-high group were decreased 14 days after SCI ( P <0.05 for GELN-high, P <0.01 for CCR2-MV / GELN-low, P <0.001 for CCR2-MV / GELN-high); the expression levels of MMP2 and MMP3 in the spinal cord of the CCR2-MV / GELN-low and CCR2-MV / GELN-high groups were lower than those of the GELN-high group ( P <0.05 for CCR2-MV / GELN-low, P <0.01 for CCR2-MV / GELN-high). The above results confirmed that CCR2-MV / GELN is more effective than GELN in alleviating the inflammatory response after SCI.

[0123] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A composition, characterized in that It includes: CCR2 + Cell membrane vesicles and ginger-derived exosome-like nanoparticles.

2. The composition according to claim 1, characterized in that CCR2 + Cell membrane vesicles derived from CCR2 + the cell membrane of a cell; Optionally, the CCR2 + cells including CCR2 + Monocytes; Optionally, the CCR2 + The origin of monocytes is human and / or mouse; Optionally, the CCR2 + Monocytes are isolated from bone marrow cells.

3. The composition according to claim 2, characterized in that CCR2 + The method for isolating the cell membrane includes: + The cells were lysed by ultrasonication and centrifuged by sucrose density gradient to obtain CCR2 + the cell membrane of a cell; Optionally, the CCR2 + The cell density was 1×10 6 ~9×10 6 Pieces / ml; Optionally, the ultrasonic lysis conditions include: frequency of 15-25 kHz, power of 300-500 W, ultrasonic time of 5-15 s / time, interval time of 5-15 s, and number of times of 30-50 times; Optionally, the conditions for the sucrose density gradient centrifugation include: a sucrose density (m / v) gradient of 20% to 30%, 35% to 45%, 50% to 60%, a centrifugal acceleration of 20,000 to 30,000 g, and a centrifugation time of 40 to 60 min; Optionally, the CCR2 + The cell membrane concentration of cells is 1.0~2.0 g / ml; Optionally, the CCR2 + The preparation method of cell membrane vesicles comprises: using water bath ultrasound and / or physical extrusion method to + The cell membrane is converted into CCR2 + cell membrane vesicles; Optionally, the conditions of the water bath ultrasound include: a frequency of 20-30 kHz, a power of 200-400 W, an ultrasound time of 10-20 s / time, an interval of 10-20 s, and a number of 20-100 times; Optionally, after water bath sonication and before physical extrusion, the CCR2 + The preparation method of cell membrane vesicles also includes a centrifugation step, wherein the centrifugation conditions are 10,000 to 15,000 g for 20 to 40 minutes; Optionally, the conditions of the physical extrusion method include: 0-8°C, polycarbonate membrane, membrane pore size of 0.3-0.6 μm, and extrusion times of 10-30 times; Optionally, after physical extrusion, the CCR2 + The preparation method of cell membrane vesicles also includes centrifugation, and the centrifugation conditions are 10,000-15,000 g for 20-40 min.

4. The composition according to claim 1, characterized in that The preparation method of the ginger-derived exosome-like nanoparticles comprises: performing a first centrifugation on ginger juice, taking the supernatant after the first centrifugation and performing a second centrifugation; taking the supernatant after the second centrifugation and mixing it with a polyethylene glycol solution to obtain a mixed solution; taking the mixed solution and performing a third centrifugation, and collecting the precipitate to obtain the ginger-derived exosome-like nanoparticles; Optionally, the conditions for the first centrifugation are: 0-8°C, 2000-6000 g, 20-60 min; Optionally, the conditions for the second centrifugation are: 0-8°C, 8000-12000 g, 80-120 min; Optionally, the conditions for the third centrifugation are: 0-8°C, 2000-6000 g, 20-60 min; Optionally, the mass volume percentage concentration of polyethylene glycol in the mixed solution is 5% to 25%; Optionally, the molecular weight of the polyethylene glycol is 5000-7000; Optionally, after the second centrifugation and before mixing with the polyethylene glycol solution, the method for preparing ginger-derived exosome-like nanoparticles further comprises: filtering the supernatant after the second centrifugation; Optionally, the filtration includes coarse filtration and fine filtration; the pore size of the filter membrane used in the coarse filtration is 0.6-1.0 μm, and the pore size of the filter membrane used in the fine filtration is 0.2-0.6 μm.

5. The composition according to any one of claims 1 to 4, characterized in that CCR2 + The mass ratio of the cell membrane vesicles to the ginger-derived exosome-like nanoparticles is 1:1-3.

6. A drug delivery system, characterized in that The drug delivery system comprises: microvesicles and ginger-derived exosome-like nanoparticles contained in the microvesicles; the preparation method of the drug delivery system comprises: combining the ginger-derived exosome-like nanoparticles and CCR2 + The mixture of cell membrane vesicles is physically extruded.

7. The drug delivery system according to claim 6, characterized in that The microvesicles and the CCR2 + The particle size ratio of cell membrane vesicles is 1:1.5~3.

8. The drug delivery system according to claim 6 or 7, characterized in that The physical extrusion conditions include: polycarbonate membrane, membrane pore size of 0.1-0.4 μm, extrusion times of 10-30 times; Optionally, the method for preparing the drug delivery system further comprises centrifuging the product after physical extrusion; Optionally, the product after physical extrusion is centrifuged at 10,000-15,000 g for 20-40 min.

9. A drug, characterized in that The active ingredient comprises the composition according to any one of claims 1 to 5 or the drug delivery system according to any one of claims 6 to 8.

10. Use of the composition according to any one of claims 1 to 5 or the drug delivery system according to any one of claims 6 to 8 in the preparation of a product for treating spinal cord injury and / or related diseases or symptoms caused by spinal cord injury; Optionally, the related diseases or symptoms caused by spinal cord injury include direct functional impairment and / or complications; Optionally, the direct functional disorder includes at least one of a motor disorder, a sensory disorder, and a bladder and rectal dysfunction.

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