Targeting vesicle based on M1 type macrophage metabolism design and preparation and application thereof

Abstract

The invention discloses a targeting vesicle based on M1 type macrophage metabolism design as well as preparation and application of the targeting vesicle. The targeting vesicles are glucose modified macrophage membrane vesicles, the protein concentration is 2 mg / mL, and the mass ratio of a macrophage membrane to glucose is (10-20): 1. Glucose is a glucose derivative molecule capable of being recognized by GLUT1 or a homologous transporter of GLUT1, and is modified to the surface of a macrophage membrane through membrane insertion, physical adsorption, click chemical reaction, amido bond formation or Michael addition, so that the binding capacity of the vesicles and M1 type macrophages highly expressing GLUT1 and other glucose transporters can be improved. The targeting vesicles are used for loading anti-inflammatory metabolites, anti-inflammatory small molecule drugs or siRNA, targeting intervention drugs under different disease models can be prepared according to different biological membrane sources, pathological microenvironments and drug loading, and the targeting vesicles are suitable for various clinical scenes such as inflammatory diseases, tumor metabolism regulation and control, tissue repair and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical configurations, relates to nano-drugs for targeted delivery, and particularly relates to a targeted vesicle designed based on the metabolism of M1 macrophages, and its preparation and application. Background Art

[0002] Macrophages are key effector cells in the innate immune system and are widely involved in immune surveillance, inflammatory responses, tissue homeostasis maintenance, and injury repair of the body. Under various pathological conditions, such as pathogen infection, tumor immune activation, tissue trauma, and chronic inflammation, the concentration of pro-inflammatory factors (such as IFN-γ, LPS) in the microenvironment increases, inducing macrophages to polarize into M1 macrophages. M1 macrophages achieve functions such as pathogen clearance, antigen presentation, and immune activation by secreting tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS). However, the continuous and excessive activation of M1 macrophages may lead to tissue damage and chronic inflammatory responses, and then induce various immune-related diseases, such as rheumatoid arthritis, atherosclerosis, and lupus erythematosus, etc.

[0003] Therefore, precisely regulating the activation state and function of M1 macrophages is of great significance for the treatment of various inflammatory and autoimmune diseases. On the one hand, tissue damage can be reduced by inhibiting the inflammatory response of M1 macrophages. On the other hand, inflammation resolution and tissue repair can also be promoted by inducing their transformation into M2 macrophages. For this reason, researchers have tried various intervention strategies, including specific drug delivery systems, signal pathway regulation, gene editing, and cell engineering, etc. Common methods include: (1) Constructing nano-carriers modified with antibodies or targeting peptides on the surface, and achieving targeted delivery by recognizing surface markers of M1 macrophages (such as CD86, TREM1). However, such systems usually have problems such as short half-life, poor permeability, complex production, and limited targeting efficiency, and are easily cleared by the mononuclear phagocyte system, resulting in reduced in vivo drug efficacy.

[0004] (2) Using small molecule inhibitors of signal pathways such as NF-κB, JAK-STAT to regulate the polarization process, but due to the lack of cell specificity, systemic immunosuppressive side effects are often caused.

[0005] (3) Using gene editing technologies such as CRISPR / Cas9 to target and intervene in polarization-related genes (such as IRF5, STAT1), but there are off-target effects and delivery problems in vivo, limiting the feasibility of clinical translation.

[0006] Based on this, there is an urgent need to develop a new targeted system that has the ability to specifically recognize cell subtypes while ensuring delivery efficiency and adapts to the high plasticity of macrophages.

[0007] In recent years, nanovesicles such as exosomes and synthetic liposomes have emerged in the delivery system due to their good biocompatibility, low immunogenicity, and high potential for engineering. After surface functionalization by targeting peptides, biotin-avidin, antibody conjugation, etc., their affinity for specific cells can be improved. However, chemical modification methods often damage the vesicle structure or reduce the drug-loading stability, and face the challenges of dynamic changes in marker expression and M1 / M2 phenotype overlap, severely restricting their targeting efficiency.

[0008] In summary, developing a nanovesicle with strong stability, high drug-loading efficiency, and M1-selective targeting function has become the key to improving the precision and safety of immunotherapy. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the present invention proposes a targeted vesicle designed based on M1 macrophage metabolism, its preparation and application. For the cell membrane material derived from macrophages, surface functionalization modification is carried out through DSPE-PEG-Glucose, so as to achieve efficient recognition and targeted delivery of M1 macrophages. This method is simple to operate, has a wide range of raw material sources, has good biocompatibility and delivery efficiency, and is applicable to various immunotherapy scenarios.

[0010] The targeted vesicle designed based on M1 macrophage metabolism, the targeted vesicle is a macrophage cell membrane vesicle modified with glucose, used for targeting M1 macrophages, and the protein concentration is 2 mg / mL.

[0011] Preferably, the glucose is a glucose-derived molecule that can be recognized by GLUT1 or its homologous transporter.

[0012] Preferably, the glucose is SPE-PEG-Glucose, cholesterol-glucose (Cholesterol-Glucose), ceramide-glucose (Ceramide-Glucose), glycosphingolipids, a polymer carrier conjugated with glucose, chitosan-Glucose, a dextran derivative modification layer, phenylboronic acid (PBA), a phenylboronic acid derivative modification structure, or a glycopolymer modification layer.

[0013] A preparation method of a targeted vesicle designed based on M1 macrophage metabolism, comprising the following steps: Step 1: Obtaining the cell membrane material Take a mammalian-derived macrophage cell line, culture it in RPMI-1640 complete medium containing 10% fetal bovine serum, and grow under the conditions of 37 °C and 5% CO2. After the cells grow to 70% - 80% confluence, collect the macrophages and wash them with PBS.

[0014] Step 2: Extraction of cell membrane Suspend the induced macrophages in a hypotonic buffer, break the cells by repeated freezing and thawing 3 times or by ultrasonic lysis, centrifuge the lysate to remove organelles such as the nucleus and mitochondria, and collect the membrane structure of macrophages.

[0015] Step 3: Assembly and modification of vesicles Prepare macrophage cell membrane vesicles by membrane extrusion, and modify glucose on the surface of macrophage cell membrane vesicles by transmembrane insertion, physical adsorption, click chemical reaction, amide bond formation (EDC / NHS coupling) or Michael addition to complete the surface functionalization of nanovesicles. The mass ratio of the macrophage cell membrane to glucose is (10-20):1.

[0016] Preferably, incubate the extracted macrophage cell membrane and glucose gently with shaking in a 37°C water bath for 30 minutes, and then obtain glucose-modified macrophage cell membrane vesicles by membrane extrusion.

[0017] Preferably, use an MWCO 100 kDa ultrafiltration tube to concentrate and purify the vesicles to remove free unmodified materials and free drugs.

[0018] Application method of targeted vesicles designed based on the metabolism of M1 macrophages: Co-incubate small molecule anti-inflammatory drugs, macrophage cell membranes and DSPE-PEG-Glucose, and then obtain DSPE-PEG-Glucose-modified M1 macrophage cell membrane vesicles loaded with drugs by membrane extrusion.

[0019] Preferably, the small molecule anti-inflammatory drug is an anti-inflammatory metabolite, a small molecule anti-inflammatory drug or siRNA.

[0020] Preferably, passively encapsulate drugs into DSPE-PEG-Glucose-modified macrophage cell membrane vesicles by pH gradient encapsulation or by ultrasonic or electroporation methods.

[0021] The present invention has the following beneficial effects: 1. Specific recognition of M1 macrophages: Design glucose as a targeting ligand according to the metabolic characteristics of M1 macrophages, and improve the binding ability to M1 cells with high expression of glucose transporters such as GLUT1 through glucose modification; 2. High biocompatibility and low immunogenicity: The vesicles are derived from natural cell membranes, and the structure and function are naturally biomimetic.

[0022] 3. Simple process and scalable preparation: No complex equipment or highly toxic reagents are required, and it has the potential for industrial transformation.

[0023] 4. Adapt to macrophage phenotypic plasticity: With adjustable structure and expandable function, it can be flexibly selected according to different biofilm sources, pathological microenvironments, and drug loading requirements, and be used to prepare targeted intervention drugs under different disease models, suitable for various clinical scenarios such as inflammatory diseases, tumor metabolic regulation, and tissue repair. Description of the Drawings

[0024] Figure 1 Western blot of the macrophage membrane prepared in Example 1.

[0025] Figure 2 Fluorescence co-localization results of the targeted vesicles prepared in Example 1.

[0026] Figure 3 Measurement results of the particle size and Zeta potential of the targeted vesicles prepared in Example 1.

[0027] Figure 4 Transmission electron microscope image of the targeted vesicles prepared in Example 1.

[0028] Figure 5 Uptake results of the targeted vesicles by macrophages in Example 1.

[0029] Figure 6 Loading efficiency of itaconic acid by the targeted vesicles in Example 5.

[0030] Figure 7 Characterization of the anti-inflammatory effect of the targeted vesicles loaded with itaconic acid in Example 5. Detailed Embodiments

[0031] The present invention will be further explained below with reference to the accompanying drawings.

[0032] Example 1

[0033] This example provides a preparation method of targeted vesicles designed based on the metabolism of M1 macrophages, using DSPE-PEG-Glucose to modify the macrophage membrane. The specific steps are as follows: Step 1. Collection of macrophages RAW264.7 mouse macrophages were inoculated into a T175 culture flask and routinely cultured using RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. They were cultured at 37 °C and 5% CO2 until the cell confluence reached 70%. The macrophages were collected and washed 2 times with PBS.

[0034] Step 2. Extraction of macrophage membrane The collected macrophages were centrifuged at 1500 rpm for 5 min, and the precipitate was collected and resuspended in ice-cold hypotonic lysis buffer and placed on ice for 30 min to promote cell swelling. The hypotonic lysis buffer had a pH of 7.4 and contained 10 mM Tris-HCl and 1 mM EDTA.

[0035] Subsequently, ultrasonic lysis was performed using an ultrasonic disruptor under ice bath conditions. The power of the ultrasonic disruptor was set at 20%, with ultrasonic treatment for 5 s and an interval of 10 s, and this was repeated 6 times.

[0036] The lysate was first centrifuged at 3000 ×g for 10 min to remove cell nuclei and debris, then centrifuged at 12000 ×g for 20 min to remove organelles such as mitochondria, and finally ultracentrifuged at 20000 ×g for 30 min to collect the macrophage membrane fraction, which was resuspended in PBS. Western blotting was performed as Figure 1 shown, and the extracted macrophage membrane retained macrophage cell surface markers.

[0037] Step 3: Construction and modification of nanovesicles The macrophage membrane collected in Step 2 was mixed with DSPE-PEG-Glucose at a mass ratio of 15:1 and slowly oscillated and incubated in a 37°C water bath for 30 min to allow DSPE-PEG-Glucose to insert into the cell membrane structure. Subsequently, a liposome extruder, Avanti Mini-Extruder, was used to extrude the mixture successively through polycarbonate membranes with pore sizes of 500 nm, 200 nm, and 100 nm, repeating 10 times for each pore size to obtain uniformly sized glucose-modified macrophage membrane vesicles. The insertion result of the lipid was shown by fluorescence colocalization as Figure 2 shown.

[0038] Step 4: Purification of nanovesicles The extruded vesicle solution was centrifuged at 4000 ×g for 15 min using an Amicon ultrafiltration centrifugal tube with an MWCO of 100 kDa to remove free DSPE-PEG-Glucose, obtaining purified targeted vesicles with a protein concentration of 2 mg / mL.

[0039] The targeted vesicles were measured using a Zetasizer Nano ZS particle size analyzer as Figure 3 shown. Their particle size was approximately 120 nm, and the Zeta potential was approximately -15.5 mV. Transmission electron microscopy (TEM) was used to observe the targeted vesicles as Figure 4 shown, and the targeted vesicles were intact bilayer membrane spherical structures.

[0040] Target vesicles were labeled with Cy5, and macrophage membrane vesicles modified with glucose (NV-Glc) at a concentration of 10 μg / mL were added to M1-type and M0-type RAW264.7 mouse macrophages and co-incubated for 2 hours respectively. As a control group, macrophage membrane vesicles without glucose modification (NV) at a concentration of 10 μg / mL were added to M1-type and M0-type RAW264.7 mouse macrophages and co-incubated for 2 hours respectively. Confocal fluorescence microscopy was used to take pictures to detect the cell uptake amount. As Figure 5 shown, the cumulative amount of the glucose-modified macrophage membrane vesicles (NV-Glc) in M1-type RAW264.7 mouse macrophages was significantly higher than that in M0-type, while the cumulative amounts of the macrophage membrane vesicles without glucose modification (NV) in M1 and M0 macrophages were close, indicating that the glucose-modified macrophage membrane vesicles had good M1-type macrophage targeting ability.

[0041] Example 2

[0042] This example provides a method for preparing target vesicles designed based on M1-type macrophage metabolism. On the basis of Example 1, cholesterol-glucose was used to modify the surface of macrophage membranes. The cholesterol-glucose was embedded in the macrophage membrane through hydrophobic interaction.

[0043] Example 3

[0044] This example provides a method for preparing target vesicles designed based on M1-type macrophage metabolism. On the basis of Example 1, chitosan-Glucose was used to modify the surface of macrophage membranes. The chitosan-Glucose was modified onto the surface of macrophage membranes through electrostatic adsorption.

[0045] Example 4

[0046] This example provides a method for preparing target vesicles designed based on M1-type macrophage metabolism. On the basis of Example 1, phenylboronic acid was used to modify the surface of macrophage membranes. The phenylboronic acid formed reversible dynamic covalent bonds with the sugar groups on the surface of macrophage membranes.

[0047] Example 5

[0048] This example is about the application of target vesicles designed based on M1-type macrophage metabolism. The specific steps are as follows: Step 1: Take 1 mg of the macrophage membrane modified with DSPE-PEG-Glucose prepared in Example 1 and mix it with 1 mg of itaconic acid (Sigma-Aldrich), and incubate at 4°C in the dark for 30 minutes to promote adsorption and internalization.

[0049] Step 2: Freeze at -80 °C for 15 minutes, then thaw at room temperature for 15 minutes, and repeat 3 times to enhance the passive encapsulation of itaconic acid into the interior of the targeted vesicles.

[0050] Step 3: Extrude the frozen-thawed mixture 10 times through an extrusion membrane with a pore size of 100 nm to form targeted vesicles (IA-NV-Glc) loaded with anti-inflammatory molecules with a stable size distribution.

[0051] Step 4: Use an ultrafiltration centrifugal tube with a MWCO of 100 kDa to centrifuge at 4000 ×g for 15 minutes to separate free itaconic acid. Collect the filtrate and measure its ultraviolet absorption at λ = 210 nm. Calculate the encapsulation efficiency by fitting the standard curve. As Figure 6 shown, the drug loading of itaconic acid is approximately 284 μg / mg (protein), and the loading efficiency is approximately 30.5%.

[0052] Incubate the targeted vesicles loaded with anti-inflammatory molecules at a final concentration of 10 μg / mL with M1-polarized RAW264.7 mouse macrophages for 6 hours. Further collect the cell culture supernatant after treatment and use an ELISA kit to detect the concentrations of TNF-α and IL-6. The results are as Figure 7 shown. Compared with the control PBS group, in the IA-NV-Glc group with the addition of targeted vesicles loaded with anti-inflammatory molecules, the content of pro-inflammatory factors was significantly reduced, indicating that the targeted vesicles loaded with itaconic acid can exert an anti-inflammatory effect by regulating metabolic and inflammatory pathways.

Claims

1. A targeted vesicle designed based on the metabolism of M1 macrophages, characterized in that: The targeted vesicles are macrophage membrane vesicles modified with glucose, which are used to target M1 macrophages, and the mass ratio of the macrophage membrane to glucose is (10-20):

1.

2. The targeted vesicle designed based on the metabolism of M1 macrophages as described in claim 1, characterized in that: The glucose is a glucose-derived molecule that can be recognized by GLUT1 or its homologous transporter.

3. The targeted vesicle designed based on the metabolism of M1 macrophages as claimed in claim 1 or 2, characterized in that: The glucose is SPE-PEG-Glucose, cholesterol-glucose, sphingolipid-glucose, glycosphingolipids, polymer carrier conjugated glucose, chitosan-Glucose, dextran derivative modification layer, phenylboronic acid, phenylboronic acid derivative modification structure or glycosyl polymer modification layer.

4. The targeted vesicle designed based on the metabolism of M1 macrophages as claimed in claim 1 or 2, characterized in that: The glucose is modified onto the surface of macrophage membrane vesicles by means of transmembrane insertion, physical adsorption, click chemical reaction, amide bond formation or Michael addition.

5. The preparation method of the targeted vesicle designed based on the metabolism of M1 macrophages according to claim 1 or 2, characterized in that: Macrophages derived from mammals are taken, the cell membranes of macrophages are collected, macrophage membrane vesicles are prepared by means of membrane extrusion, and the surfaces of the vesicles are functionally modified with glucose.

6. The preparation method of the targeted vesicle designed based on the metabolism of M1 macrophages according to claim 5, wherein: The extracted macrophage cell membrane and glucose are gently shaken and incubated in a water bath; then glucose-modified macrophage membrane vesicles are obtained by means of membrane extrusion.

7. The preparation method of the targeted vesicle designed based on the metabolism of M1 macrophages according to claim 6, characterized in that: The vesicles are concentrated and purified to remove free unmodified materials.

8. The application of the targeted vesicle designed based on the metabolism of M1 macrophages according to claim 1 or 2, characterized in that: The targeted vesicles are used to load small molecule anti-inflammatory drugs and serve as drugs targeting M1 macrophages.

9. The application of the targeted vesicle designed based on the metabolism of M1 macrophages according to claim 8, wherein: The small molecule anti-inflammatory drugs are anti-inflammatory metabolites, small molecule anti-inflammatory drugs or siRNA.

10. The application of the targeted vesicle designed based on the metabolism of M1 macrophages as claimed in claim 8, wherein: The vesicles actively encapsulate small molecule anti-inflammatory drugs by means of co-incubation, or passively load drugs by means of pH gradient encapsulation, ultrasound or electroporation.

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