A macrophage-targeting functionalized extracellular vesicle and a preparation method and application thereof

Abstract

The present application relates to a macrophage-targeting functional extracellular vesicle and a preparation method and application thereof, and relates to the technical field of biological medicine. The preparation method comprises the following steps: preparing COFs particles loaded with ATP citrate lyase inhibitors by one-pot method through covalent organic framework material and ATP citrate lyase inhibitors; and preparing the macrophage-targeting functional extracellular vesicle by ultrasonic thin film extrusion method through the COFs particles loaded with ATP citrate lyase inhibitors and apoptotic body membranes. The macrophage-targeting functional extracellular vesicle provided by the present application can target macrophages and accurately release drugs to regulate the polarization state of macrophages, thereby providing an effective means for constructing anti-inflammatory treatment products. Meanwhile, the constructed functional extracellular vesicle can reprogram the polarization state of macrophages, regulate the local immune microenvironment, promote the formation of new blood vessels of diabetic skin wounds, and promote wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a macrophage-targeting functional extracellular vesicle and a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a chronic metabolic disease that is prevalent worldwide, with high incidence and mortality. Delayed wound healing is a common complication of diabetes, which seriously affects people's normal life. Chronic wound infection in diabetes is the main cause of non-traumatic amputation, and macrophages play a key role in all stages of tissue repair. In a hyperglycemic environment, the polarization state of macrophages changes continuously to the pro-inflammatory M1 phenotype, hindering wound healing. Therefore, it is crucial to target macrophages to change their polarization state and accelerate angiogenesis for wound healing.

[0003] Extracellular vesicles (EVs) are a kind of lipid bilayer-enclosed heterogeneous membrane structure secreted by cells. According to its size and source, it can be divided into three subtypes: exosomes (50-200nm), microvesicles (100-1000nm), and apoptotic bodies (50-4000nm). EVs, as a small membrane vesicle, play an important role in intercellular communication. Through its surface proteins, intracellular biological information molecules such as proteins, nucleic acids, and transported lipids and glycan, it affects the function of other cells. Apoptotic bodies are extracellular vesicles released during apoptosis, mainly referring to bubble-like bodies formed by membrane ruffling. Apoptotic bodies are then phagocytosed by macrophages, parenchymal cells, or tumor cells and degraded in phagosomes. In recent years, the research on apoptotic bodies has gradually gained momentum. Carrying targeted or therapeutic molecules as drug carriers and having better safety, apoptotic bodies do not trigger immune responses, and have broad prospects in the fields of biomedical applications, especially immune regulation, vascular protection, and tissue regeneration. SUMMARY

[0004] To solve the above problems, the present application provides a macrophage-targeting functional extracellular vesicle and a preparation method and application thereof.

[0005] In a first aspect, the present application provides a preparation method of a macrophage-targeting functional extracellular vesicle, which comprises the following steps:

[0006] The covalent organic framework material and the ATP citrate lyase inhibitor are prepared into COFs particles loaded with the ATP citrate lyase inhibitor by one-pot method;

[0007] The ATP-citrate lyase inhibitor loaded COFs particles and the apoptotic body membrane are prepared into the macrophage targeted functional extracellular vesicle through ultrasonic film extrusion.

[0008] Further, the covalent organic framework material is prepared from DVA (2,5-divinyl terephthaldehyde) and TPB (1,3,5-tris (4-aminophenyl) benzene); and the ATP-citrate lyase inhibitor includes SB 204990.

[0009] Further, the molar ratio of the DVA and the TPB is (3-9):(2:3).

[0010] Further, the apoptotic body membrane is derived from macrophages, and the macrophages include RAW264.7.

[0011] Further, the preparation method of the apoptotic body membrane includes the following processes:

[0012] The RAW264.7 cells are used, 1 μM staurosporine is added to induce apoptosis, and the apoptotic bodies are obtained through differential centrifugation;

[0013] The apoptotic bodies are subjected to hypotonic treatment and ultrasonic treatment to remove the vesicle contents, and the apoptotic body membrane is obtained.

[0014] Further, the time for the staurosporine to induce apoptosis is 3-5 h.

[0015] Further, the mass-volume ratio of the covalent organic framework material and the ATP-citrate lyase inhibitor is 1:(0.5-1) in mg / ml, and the weight ratio of the ATP-citrate lyase inhibitor loaded COFs particles and the apoptotic body membrane is 1:(2-4).

[0016] Further, the working condition parameters of the one-pot method include: rotating mixing at room temperature for 8-12 h, and drying under high vacuum for 24 h; and the working condition parameters of the ultrasonic film extrusion method include: water bath intermittent ultrasonic treatment for 5 min, then loading the nanoparticles into an extruder, and repeatedly extruding 10-15 times through a polycarbonate filter membrane with a diameter of 1000 nm.

[0017] In the second aspect, the present application provides a macrophage targeted functional extracellular vesicle, which is prepared by the preparation method of the macrophage targeted functional extracellular vesicle according to any one of the first aspect.

[0018] Further, the particle size of the macrophage targeted functional extracellular vesicle is 600-800 nm.

[0019] In a third aspect, the present application provides the macrophage-targeting functionalized extracellular vesicle of any one of the first aspect and the second aspect for use as a drug carrier and in the preparation of a medicament for treating a diabetic wound healing disease.

[0020] Compared with the prior art, the above technical solution provided by the embodiments of the present application has at least the following advantages:

[0021] The embodiments of the present application provide a macrophage-targeting functionalized extracellular vesicle, a preparation method and application thereof. The macrophage-targeting functionalized extracellular vesicle provided by the present application has the functions of targeting macrophages and accurately releasing drugs to regulate the polarization state of macrophages, thereby providing an effective means for constructing an anti-inflammatory treatment product. Meanwhile, the constructed functionalized extracellular vesicle can reprogram the polarization state of macrophages, regulate the local immune microenvironment, promote the formation of new blood vessels of a diabetic skin wound, and promote wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate an embodiment consistent with the present application and, together with the description, serve to explain the principles of the application.

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, for those skilled in the art, other drawings can also be obtained based on these drawings without any creative effort.

[0024] Figure 1 The bar is 100 μm.

[0025] Figure 2 The bar is 50 μm.

[0026] Figure 3 The bar is 500 nm.

[0027] Figure 4 The bar is 1 μm.

[0028] Figure 5 The bar is 500 nm.

[0029] Figure 6 Figure 6 is a graph of the results of ABm@COF cytotoxicity detection at different times in the present application.

[0030] Figure 7 Figure 7 is a graph of the regulation of the polarization state of macrophages by SB 204990 in the present application.

[0031] Figure 8 Figure 8 is the uptake of nanoparticles by macrophages in the present application, bar = 20 μm.

[0032] Figure 9 Figure 9 is the specific engulfment of macrophages in the present application, bar = 100 μm.

[0033] Figure 10 Figure 10 is a graph of the results of diabetic wound healing experiments in the present application. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0035] Unless otherwise specifically indicated, all the various raw materials, reagents, instruments and equipment, etc. used in the present application can be purchased from the market or can be prepared by existing methods.

[0036] In a first aspect, the present application provides a preparation method of macrophage-targeted functionalized extracellular vesicles, comprising the following steps:

[0037] The covalent organic framework material and the ATP citrate lyase inhibitor are prepared into COFs particles loaded with the ATP citrate lyase inhibitor by one-pot method;

[0038] The COFs particles loaded with the ATP citrate lyase inhibitor and the apoptotic body membrane are prepared into the macrophage-targeted functionalized extracellular vesicles by ultrasonic thin film extrusion method.

[0039] The embodiment of the present application provides a macrophage-targeting functional extracellular vesicle, the macrophage-targeting functional extracellular vesicle provided by the present application has the functions of targeting macrophages and accurately releasing drugs to regulate the polarization state of macrophages, and provides an effective means for constructing anti-inflammatory treatment products. Meanwhile, the constructed functional extracellular vesicle can reprogram the polarization state of macrophages, regulate the local immune microenvironment, promote the formation of new blood vessels of a wound of a diabetic rat, and promote wound healing.

[0040] In some specific embodiments, the covalent organic framework material is prepared from DVA and TPB as raw materials; the ATP citrate lyase inhibitor comprises SB 204990.

[0041] In the present application, the Chinese full name of DVA is 2,5-divinyl terephthaldehyde, which is one of the key raw materials for synthesizing COFs. It contains two aldehyde groups, which can react with compounds containing amino groups and other functional groups to form the skeleton structure of COFs.

[0042] In the present application, the Chinese full name of TPB is 1,3,5-tris(4-aminophenyl)benzene, and its CAS registration number is 118727-34-7.

[0043] In the present application, SB 204990 is an effective and specific ATP citrate lyase (ACLY) inhibitor, and its CAS number is 154566-12-8.

[0044] In some specific embodiments, the molar ratio of the DVA to the TPB is (3-9):(2:3).

[0045] In some specific embodiments, the apoptotic body membrane is derived from macrophages, and the macrophages include RAW264.7.

[0046] In the present application, RAW264.7, the full name of mouse monocyte macrophage leukemia cell, is a leukemia cell line derived from mice, and the RAW 264.7 cell has the characteristics of macrophages, such as strong phagocytic ability, antigen presentation and cytokine production. These cells are easy to culture and operate in vitro, and show similar functions to primary macrophages.

[0047] In some specific embodiments, the preparation method of the apoptotic body membrane comprises the following processes:

[0048] RAW264.7 cells are used, 1 mu M star-shaped spore bacteria are added to induce apoptosis, and the apoptotic bodies are obtained by differential centrifugation;

[0049] The apoptotic bodies are subjected to hypotonic and ultrasonic treatment to remove the vesicle contents, and the apoptotic body membrane is obtained.

[0050] The time of apoptosis induction by staurosporine is 3-5h.

[0051] In some embodiments, the mass-volume ratio of the covalent organic framework material and the ATP-citrate lyase inhibitor is 1:(0.5-1) in mg / ml, and the weight ratio of the COFs particle loaded with ATP-citrate lyase inhibitor and the apoptotic body membrane is 1:(2-4).

[0052] In some embodiments, the working condition parameters of the one-pot method include: drying for 24h under high vacuum after rotary mixing at room temperature for 8-12h; and the working condition parameters of the ultrasonic film extrusion method include: loading the above nanoparticles into an extruder, passing through a polycarbonate filter film with a diameter of 1000nm, and repeatedly extruding 10-15 times.

[0053] In a second aspect, the present application provides a macrophage-targeted functionalized extracellular vesicle, which is prepared by the preparation method of the macrophage-targeted functionalized extracellular vesicle according to any one of the first aspect.

[0054] In some embodiments, the particle size of the macrophage-targeted functionalized extracellular vesicle is 600-800nm.

[0055] In a third aspect, the present application provides the use of the macrophage-targeted functionalized extracellular vesicle according to any one of the first aspect and the second aspect as a drug carrier and in the preparation of a drug for treating a diabetic wound healing disease.

[0056] It should be noted that the components involved in the macrophage-targeted functionalized extracellular vesicle and the preparation method thereof provided in the embodiments of the present application can be directly used as commercially available products or self-made by using the existing public preparation method if there is no special limitation or specific description. Meanwhile, the steps and parameters involved can be carried out according to the existing process or directly using the existing equipment if there is no special limitation or specific description, and the present application document will not be described one by one.

[0057] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are generally determined according to the national standards. If there is no corresponding national standard, the general international standards, conventional conditions, or the conditions suggested by the manufacturer are used.

[0058] Example 1

[0059] This example provides the preparation and characterization of the apoptotic body membrane, which specifically includes the following processes:

[0060] Preparation method of apoptotic bodies

[0061] Raw264.7 cells were cultured at 37℃ in 5% CO2, and apoptosis was induced by adding 1 μM staurosporine to the culture medium for 3 h. Cell surface bubbling and spherical protrusions were observed under a light microscope, and gradually shed as shown in Figure 2 The culture medium was gently blown and washed with PBS, and the supernatant was centrifuged at 300 g for 10 min. The supernatant was collected, and the precipitate was removed. Then, the precipitate was collected by centrifugation at 1000 g for 10 min, and the apoptotic bodies were obtained. After washing with PBS three times, the PBS was resuspended, and the corresponding characterization detection was performed. Immunofluorescence staining and flow cytometry were used to detect the apoptosis-related marker Annexin V, and the positive rate was about 88.6%.

[0062] Based on the above experiment, the apoptosis induction time of staurosporin was explored, and the experimental results are shown in Figure 1

[0063] Preparation of apoptotic body membranes

[0064] The apoptotic bodies were resuspended in a hypotonic lysis solution and placed at 4℃ for 1 h. Ultrasonic treatment was performed for 5 s, and the fragments were removed by centrifugation at 100 g for 10 min. The supernatant was collected and centrifuged at 10000 g for 10 min to collect the precipitate, which was the apoptotic body membrane (ABm). ddH2O was used to rinse and remove the cell membrane contents, and the resuspension was stored at 4℃.

[0065] In this embodiment, the cell contents were expelled from the apoptotic body membrane by hypotonic treatment and ultrasonic treatment. The cells were placed in a hypotonic lysis solution for 1 h, and then ultrasonic treatment was performed for 2 min. PBS was used for repeated washing three times, and Hochest was used to label the nucleus, and Annexin V was used to label the apoptotic body membrane. As shown in the figure, the amount of Hochest in the apoptotic body was small, indicating that the contents were expelled, and the diameter of the apoptotic body membrane was about 800-1000 nm.

[0066] Example 2

[0067] In this example, the preparation of macrophage-targeted functional extracellular vesicles (extracellular vesicle-COF complex drug delivery system, abbreviated as ABm@COF) was carried out, which specifically included the following processes:

[0068] Preparation of covalent organic framework COFs

[0069] ​Take 0.12 mM DVA and 0.08 mM TPB into a centrifuge tube. Add 8 ml of ACN (acetonitrile) and ultrasonic for 1 min to make it completely dissolved. Add 0.4 ml of 17M HAc (acetic acid) to the test tube. Shake the mixture in a vortex mixer for 30 s and centrifuge at room temperature for 72 h. Wash 3 times with dry THF (dry tetrahydrofuran) and ethanol respectively. Dry at 60℃ under vacuum for 24 h. The COF transmission electron microscope image is shown in Figure 4 .

[0070] Loading of ACLY inhibitor

[0071] COF has the advantages of large specific surface area and high porosity, and high drug loading. SB 204990 is loaded in the pores of COF by electrostatic adsorption. The COF particles are ultrasonically dispersed in the SB 204990 solution, and after stirring for 8-12 h, the suspension is ultrasonically treated for 5 min, washed 3 times with ddH2O, and dried under high vacuum for 24 h.

[0072] Preparation of extracellular vesicle-COF composite drug-loaded system

[0073] Mix 1 mg of COF-SB with 2 mg of apoptotic body membrane, ultrasonic for 2 min in an ultrasonic instrument, and then centrifuge the suspension at 5000g for 10 min. The precipitate is the extracellular vesicle-COF composite drug-loaded system. Load the above nanoparticles into an extruder, pass through a polycarbonate filter membrane with a diameter of 1000 nm, and repeatedly extrude 10-15 times to obtain ABm@COF. The scanning electron microscope image is shown in Figure 5 .

[0074] Example 3

[0075] This example detects the performance of the macrophage-targeted functionalized extracellular vesicle (extracellular vesicle-COF composite drug-loaded system) obtained in Example 2.

[0076] 1) Cytotoxicity detection

[0077] First, different concentrations of ABm@COF were added to macrophages. CCK-8 was used to detect the cytotoxicity of nanoparticles. The CCK8 results showed that even if the concentration of ABm@COF nanoparticles was increased to 200 μg / mL, there was no obvious cytotoxicity. With the increase of culture time, high concentration of ABm@COF had a partial effect on cell activity after 72 h of culture, proving that ABm@COF has good biocompatibility, as shown in Figure 6 .

[0078] 2) Extracellular vesicle-COF macrophage phagocytosis experiment

[0079] Extract BMDMs cells, inoculate cells in confocal culture dishes, add 50 μg / ml Rh B labeled ABm@COF for 6h, remove the culture medium, PBS dip after washing, add 4% paraformaldehyde for 1h, PBS dip after fixing three times, add goat serum, 37°C dark blocking for 30min. Remove the blocking solution and add diluted primary antibody F4 / 80 4°C overnight. PBS dip the slide 3 times, 5min each time. Add diluted fluorescent secondary antibody, dark wet box 37°C for 1h, PBS dip the slide 3 times, 5min each time. Add DAPI dropwise and incubate in the dark for 5min. Stain the specimen with DAPI, and wash the excess DAPI with PBS. Use absorbent paper to dry the liquid on the slide, and seal the slide with an anti-fluorescence quenching agent containing mounting medium. Observe and collect images under confocal microscope, as shown in Figure 7 .

[0080] 3) Effect of SB 204990 on the polarization state of macrophages

[0081] ATP citrate lyase (ACLY) is an important enzyme that produces acetyl-CoA from citrate, which is an important intermediate metabolite of material metabolism and an intermediate hub of sugar, fat and amino acid metabolism. Excessive nutrient input and inflammation can cause abnormal glycolipid metabolism of cells. SB 204990 is an effective inhibitor of ATP citrate lyase (ACLY) and can effectively reprogram macrophages. Raw264.7 cells were given different treatment conditions (LG, HG, HG+LPS, HG+TLPS+SB204990), and after protein extraction, Western blot was used for detection. The protein expression of ACLY, macrophage M1 marker CD86 and macrophage M2 marker CD206 was detected. The results showed that compared with the HG+LPS group, the addition of SB204990 could reduce the expression of ACLY, and the expression of pro-inflammatory M1 macrophages CD86 was reduced, and the expression of anti-inflammatory M2 macrophages CD206 was increased, changing the polarization state of macrophages from pro-inflammatory to anti-inflammatory, as shown in Figure 8 .

[0082] 4) Macrophage targeting experiment of extracellular vesicle-COF composite nanoparticles

[0083] To explore the targeting of ABm@COF to macrophages, we added 25 μg / ml of RhB-labeled ABm@COF to different cell lines for 6 h. Under fluorescence, it can be observed that Raw264.7 (macrophages) obviously phagocytose, HcASMC (smooth muscle cells), HFB, HCF (fibroblasts) basically do not phagocytose nanoparticles, and only some fluorescent particles adhere to the cell surface, proving that the synthesized ABm@COF has good macrophage targeting, as shown in Figure 9 .

[0084] 5) Construction of skin damage model

[0085] Male C57 mice aged 6-8 weeks were selected, and after adaptive feeding for 1 week, they were fasted for 12 h, and 50 mg / ml STZ was injected intraperitoneally, and the injection was continued for 5 days. If the fasting blood glucose is higher than 11 mmol / L, it is considered to be successfully modeled. The diabetic mice were randomly divided into 4 groups (PBS group, ABm group, COF-SB group, ABm@COF-SB group), and after anesthesia by intraperitoneal injection of sodium pentobarbital, the mice were shaved, and a circular full-thickness skin wound was created using a skin punch. Photographs were taken at 0 day, 3 days, 7 days, 10 days and 14 days after the hole was opened to observe the wound healing, and the effect of ABm@COF-SB on the healing of the wound of the diabetic rats was verified, as shown in Figure 10 . The functional extracellular vesicles have good wound healing effect.

[0086] Various embodiments of the present application can exist in a range of forms; it should be understood that the description in a range form is merely for the convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the described range has specifically disclosed all possible sub-ranges and single values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the described range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated in this document, it refers to any cited number (fraction or integer) within the indicated range.

[0087] The above description is only a specific embodiment of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features applied herein.

Claims

1. A method for preparing macrophage-targeted functionalized extracellular vesicles, characterized in that: The preparation method comprises the following steps: Covalent organic framework materials and ATP citrate lyase inhibitors were prepared through a one-pot method to prepare COFs particles loaded with ATP citrate lyase inhibitors; The COFs particles loaded with ATP citrate lyase inhibitor and apoptotic body membranes are subjected to ultrasound plus film extrusion to prepare the macrophage-targeted functionalized extracellular vesicles; the apoptotic body membranes are derived from macrophages, and the macrophages include RAW264.7; The covalent organic framework material is prepared using 2,5-divinylterephthalaldehyde and 1,3,5-tris(4-aminophenyl)benzene as raw materials; the molar ratio of the 2,5-divinylterephthalaldehyde to the 1,3,5-tris(4-aminophenyl)benzene is (3-9):2:3; The ATP citrate lyase inhibitors include SB 204990.

2. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: Preparation method of the apoptotic body membrane The following processes are included: RAW264.7 cells were used to induce apoptosis by adding 1 μM staurosporine, and apoptotic bodies were obtained by differential centrifugation. The apoptotic bodies are subjected to hypotonic and ultrasonic treatment to remove vesicle contents and obtain apoptotic body membranes; Among them, the time for staurosporine to induce apoptosis is 3-5 hours.

3. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: In mg / ml, the mass-to-volume ratio of the covalent organic framework material and the ATP citrate lyase inhibitor is 1:(0.5-1), and the weight ratio of the COFs particles loaded with the ATP citrate lyase inhibitor and the apoptotic body membrane is 1:(2-4).

4. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: The working condition parameters of the one-pot method include: rotating mixing at room temperature for 8-12 hours, and drying under high vacuum for 24 hours. The working condition parameters of the ultrasound plus thin film extrusion method include: after intermittent ultrasound in a water bath for 5 minutes, the nanoparticles are loaded into an extruder, passed through a polycarbonate filter with a diameter of 1000 nm, and repeatedly extruded 10 to 15 times.

5. A macrophage-targeted functionalized extracellular vesicle, characterized in that: The macrophage-targeted functionalized extracellular vesicles are prepared by the preparation method according to any one of claims 1 to 4.

6. The macrophage-targeting functionalized extracellular vesicle according to claim 5, characterized in that: The particle size of the macrophage-targeted functionalized extracellular capsule is 600-800 nm.

7. Use of the macrophage-targeted functionalized extracellular vesicle according to any one of claims 1 to 6 as a drug carrier and in the preparation of a drug for treating diabetic wound healing diseases.

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