CN111529505B

CN111529505B - Functional chimeric apoptotic body and preparation method and application thereof

Info

Publication number: CN111529505B
Application number: CN202010215437.2A
Authority: CN
Inventors: 刘文娜
Original assignee: Xi'an tissue engineering and regenerative medicine research institute
Current assignee: Xi'an tissue engineering and regenerative medicine research institute
Priority date: 2020-03-25
Filing date: 2020-03-25
Publication date: 2023-04-11
Anticipated expiration: 2040-03-25
Also published as: CN111529505A

Abstract

The functional chimeric apoptotic body comprises an apoptotic body membrane and mesoporous silica nanoparticles wrapped in the apoptotic body membrane, wherein the apoptotic body membrane is derived from immune cells, and the mesoporous silica nanoparticles are loaded with curcumin. Provides an effective means for constructing a specific therapeutic product with a targeted delivery function and a precise drug release function.

Description

Functional chimeric apoptotic body and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a functional chimeric apoptotic body, and a preparation method and application thereof.

Background

Extracellular Vesicles (EVs) are carriers for transmitting signal transduction molecules in intercellular communication, and can carry various functional molecules such as nucleic acids, proteins and lipids to play a role. Meanwhile, the natural membrane endows the extracellular vesicles with low immunogenicity, strong homologous targeting, long circulation time, biological barrier accessibility and the like, and is incomparable with artificial materials. Inspired by these remarkable properties, extracellular vesicles are considered as a new generation delivery system. The preparation of engineered extracellular vesicles with modifications of various bioactive molecules or targeting ligands, which produce therapeutic effects at the desired site, is an attractive strategy for disease treatment.

Apoptotic Bodies (ABs) are extracellular vesicles secreted by Apoptotic cells, produced by the outward bubbling of the cell membrane. There is substantial evidence that phagocytosis of apoptotic cells can modulate the anti-inflammatory direction of the immune response. Similar results are also reflected only in apoptotic cell/vesicle membrane phagocytosis, which may be closely related to surface phosphatidylserine.

The preparation of the engineered extracellular vesicles adopts various therapeutic molecule loading methods, which mainly comprise an electroporation method, a pre-complexation method, a parental cell transfection method before extracellular vesicle separation, a saponin infiltration method and an ultrasonic method. However, the existing methods still have considerable limitations in that, for example, residual components not relevant to the therapeutic purpose cannot be removed, which may cause potential threats; in the existing method, various functional molecules are biochemically coupled on the surface, which can destroy the targeting structure and function of the extracellular vesicles; lack of intelligent intracellular control switches, ensure the accurate and effective release of functional therapeutic molecules carried by engineered extracellular vesicles.

Ulcerative colitis is a chronic nonspecific intestinal inflammatory disease with uncertain etiology and pathogenesis, the incidence rate is increased year by year in China, and the pathogenesis is not clear. The pathological change parts of the ulcerative colitis are mainly limited to the colon mucous membrane and the submucosa, the colon mucous membrane is congested and edematous during the onset of diseases, and the ulcerative colitis is difficult to completely eradicate by the existing treatment means. Repeated onset and difficult healing of ulcerative colitis affect on the one hand the quality of life of the patient and on the other hand increase the possibility of canceration, seriously threatening the life health of the patient. The lack of specific approaches to treatment of ulcerative colitis has also become a hotspot in the digestive field of recent years. The currently available treatments are to reduce inflammation, alleviate symptoms, and in the best cases result in long-term remission. However, long-term use of these drugs can cause a variety of side effects, such as infection, kidney or liver damage.

Meanwhile, the traditional and existing medicines for treating skin injury and ulcerative colitis have side effects on other tissues and organs and poor treatment effect, and a medicine with a specific treatment function is required to replace the traditional and existing medicines so as to achieve a better effect and reduce or eliminate the side effects caused by the medicines. Thus, suitable primitive extracellular vesicles with natural biological function and targeting, as well as novel manufacturing strategies are needed to address these issues.

Disclosure of Invention

The invention aims to provide a functional chimeric apoptotic body, a preparation method and application thereof aiming at the problems in the prior art, and provides an effective means for constructing a specific therapeutic product with a targeted delivery function and an accurate drug release function.

The purpose of the invention is realized by the following technical scheme:

in one aspect, a functional chimeric apoptotic body is provided, and the functional chimeric apoptotic body comprises an apoptotic body membrane and mesoporous silica nanoparticles wrapped in the apoptotic body membrane, wherein the apoptotic body membrane is derived from an immune cell, and the mesoporous silica nanoparticles are loaded with curcumin.

Preferably, the immune cells are selected from lymphocytes selected from T cells, B lymphocytes or NK lymphocytes.

Preferably, after the curcumin is loaded in the pores of the mesoporous silica nanoparticles by physical adsorption, the mesoporous silica nanoparticles are encapsulated with the polyglycerol dimethacrylate.

Preferably, when the functional chimeric apoptotic body is targeted to a site of inflammation and phagocytosed by macrophages, the polyglycerol dimethacrylate can be degraded by esterase in the target cells, thereby responsively releasing curcumin in the target cells.

In another aspect, there is provided a method of making a functional chimeric apoptotic body as described above, comprising the steps of:

1) Mixing mesoporous silica nanoparticles, curcumin and glyceryl dimethacrylate in water, ultrasonically mixing uniformly, and vacuumizing under a vacuum condition to generate mesoporous silica nanoparticles co-loaded with the glyceryl dimethacrylate and the curcumin;

2) Respectively dissolving persulfuric acid and tetramethylethylenediamine in water, uniformly mixing 2500 mu l of ammonium persulfate solution and 440 mu l of tetramethylethylenediamine solution with the mesoporous silica nanoparticles co-loaded with the glycerol dimethacrylate and the curcumin under nitrogen to generate a polymerization reaction to generate mesoporous silica nanoparticles co-loaded with the glycerol dimethacrylate and the curcumin, after suspending, stirring for 1h under the conditions of nitrogen and room temperature to generate mesoporous silica nanoparticles co-loaded with the glycerol dimethacrylate and the curcumin, and after cleaning, carrying out vacuum drying for later use;

3) Mixing the mesoporous silica nano particles co-loaded with the polyglycerol dimethacrylate and the curcumin with an apoptotic body membrane, then intermittently carrying out ultrasonic treatment on the mixed solution for 2min in an ultrasonic instrument, centrifuging the treated suspension for 10min under the condition of 5000g, taking the precipitate to obtain the functional chimeric apoptotic body, and suspending the functional chimeric apoptotic body in water for storage at 4 ℃.

Preferably, in the step 3), the mass ratio of the mesoporous silica nanoparticles co-loaded with the polyglycerol dimethacrylate and the curcumin to the apoptotic body membrane is 1.

Preferably, there is provided the use of the functional chimeric apoptotic bodies described above in the manufacture of a product for the treatment of inflammatory diseases.

Preferably, the inflammatory disease comprises skin inflammation, colitis, pneumonia, hepatitis or nephritis.

Further, a medicament for treating inflammatory diseases is provided, comprising the functional chimeric apoptotic body described above and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier is selected from water, PBS buffer or physiological saline.

Interpretation of terms:

apoptotic small body membrane: refers to a vesicle membrane obtained after the contents of apoptotic bodies have been removed.

Mesoporous silica nanoparticles: refers to nano-scale micro-particles which are prepared by taking silicon dioxide as a raw material and have larger specific surface area and adsorption capacity.

T cell: t lymphocytes.

Compared with the prior art, the invention has the beneficial effects that:

the functional chimeric apoptotic body provided by the invention utilizes the potential inflammation targeting effect of T cells and the inflammation regulating characteristic of an apoptotic body membrane derived from T cells to fuse the apoptotic body membrane derived from T cells with a mesoporous nanoparticle delivery system, so as to construct the functional chimeric apoptotic body capable of targeted delivery of curcumin, wherein the functional chimeric apoptotic body removes the inclusion components in the apoptotic body, maintains the targeting property and the inflammation regulating characteristic of the apoptotic body membrane, can accurately release curcumin in target cells, avoids the occurrence of off-target effect or curcumin leakage of the functional chimeric apoptotic body loaded with curcumin, inherits the advantages of a natural apoptotic body membrane, can actively target inflammation parts and macrophages, and solves the problems of poor treatment effect and large side effect on other tissues and organs of the traditional medicine; the problem that the utilization efficiency of curcumin loaded by the functional chimeric apoptotic bodies is greatly reduced by the endocytosis and disposal of the internalized functional chimeric apoptotic bodies by a lysosome system in the receptor cells after downstream signal conduction is started in the receptor cells by the functional chimeric apoptotic bodies constructed by using common apoptotic body membranes is solved, and the transfer efficiency and the utilization rate of the curcumin in the receptor cells are greatly improved.

In addition, before the apoptotic small membrane and the mesoporous nanoparticles are transferred and fused, the mesoporous nanoparticles are preloaded with curcumin through electrostatic interaction and further modified with various stimulus response molecules, and intracellular esterase is used for degrading poly-glyceryl dimethacrylate encapsulating the curcumin so that the curcumin is released in response in cells, and therefore the curcumin can be accurately released in target cells, the macrophage phenotype is effectively regulated and controlled, and inflammation is improved.

Further, an efficient strategy is provided for the manufacture of standardized engineered extracellular vesicles with modular design and specific therapeutic functions. Under the condition of not changing the conception of the invention, according to the basic principle that the mesoporous silica nano particles designed by the invention load active substances and release the active substances in target cells in a responding way, different loading methods and functional chimeric apoptotic bodies with different functions can be designed aiming at different active substances.

The functional chimeric apoptotic body prepared by the preparation method of the functional chimeric apoptotic body provided by the invention removes the inclusion components in the apoptotic body, simultaneously maintains the targeting property and the inflammation regulation characteristic of the apoptotic body membrane, can accurately release curcumin in target cells, avoids the occurrence of off-target effect or curcumin leakage of the functional chimeric apoptotic body loaded with curcumin, and meanwhile, the constructed functional chimeric apoptotic body inherits the advantages of a natural apoptotic body membrane and can actively target to an inflammation part.

Experimental data in the embodiment can prove that the functional chimeric apoptotic body provided by the invention can be used for preparing medicines for treating inflammatory diseases, and particularly can be used for preparing medicines for treating skin inflammation, colitis, pneumonia, hepatitis or nephritis.

Drawings

FIG. 1 shows the core-shell structure of chimeric apoptotic bodies under a transmission electron microscope;

FIG. 2A is a statistical chart of the stability detection results of the chimeric apoptotic bodies and the mesoporous silica nanoparticles in water and PBS, respectively;

figure 2B shows the stability assay of chimeric apoptotic bodies before lyophilization and after lyophilization resuspension;

fig. 2C shows the stability detection results of the chimeric apoptotic bodies and the mesoporous silica nanoparticles in serum;

FIG. 3A shows the staining pattern of the macrophage marker F4/80 in the PBS group (control group) in example 7;

FIG. 3B shows the PKH26/RhB staining pattern of the PBS group in example 7 (control group);

FIG. 3C shows a Hoechst staining pattern of the PBS group (control group) in example 7;

FIG. 3D shows a Merge plot for the PBS group (control group) in example 7;

FIG. 3E shows a staining pattern for macrophage marker F4/80 of apoptotic membrane group in example 7;

FIG. 3F shows a PKH 26-labeled apoptotic-envelope staining profile of the apoptotic-envelope population in example 7;

FIG. 3G shows a Hoechst staining pattern of apoptotic membrane groups in example 7;

FIG. 3H shows Merge plots of apoptotic membrane groups in example 7;

FIG. 3I shows a staining pattern for macrophage marker F4/80 of the mesoporous silica nanoparticle group of example 7;

fig. 3J shows a graph of the staining of RhB-labeled mesoporous silica nanoparticles of the set of mesoporous silica nanoparticles of example 7;

FIG. 3K shows a Hoechst staining pattern of the mesoporous silica nanoparticle group of example 7;

FIG. 3L shows a Merge plot of the mesoporous silica nanoparticle group of example 7;

FIG. 3M shows a staining pattern for macrophage marker F4/80 of the chimeric apoptotic body set in example 7;

FIG. 3N shows a staining pattern of RhB-labeled chimeric apoptotic bodies of the chimeric apoptotic body group in example 7;

FIG. 3O shows a Hoechst staining pattern of the chimeric apoptotic body group of example 7;

FIG. 3P shows Merge plots for the chimeric apoptotic body group of example 7;

FIG. 4A is the result of TNF-. Alpha.assay in example 8;

FIG. 4B shows the results of IL-6 detection in example 8;

FIG. 4C shows the result of TGF-. Beta.assay in example 8;

FIG. 4D shows the results of IL-10 detection in example 8;

FIG. 5 shows the results of detection of release of MicroRNA-21 triggered by glutathione in example 9;

FIG. 6 is the release profile of curcumin initiated by esterase degradation of poly (glycerol dimethacrylate) in example 10;

FIG. 7 is the result of the biodistribution measurement of the damaged part of the skin inflammation damage model in example 12;

FIG. 8 shows the results of wound healing tests in skin inflammation injury models;

FIGS. 9A-9E are normal skin group, PBS group, cABs group, respectively ^MicroRNA-21 Group and cABs ^Cur HE staining results of skin lesion sites in groups;

FIG. 10 is the biodistribution at the lesion site of ulcerative colitis model in different administration groups in example 15;

FIG. 11A shows the results of monitoring the body weight of mice in different administration groups in example 16;

FIG. 11B shows the results of colon length measurements of different administration groups in example 16;

FIGS. 12A-12E are normal, PBS, cABs, and cABs, respectively ^MicroRNA-21 Group and cABs ^Cur HE staining of colon tissue.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.

Example 1 Targeted responsive Release System loaded with MicroRNA-21

The targeted responsive release system comprises a chimeric apoptotic body, wherein the chimeric apoptotic body comprises an apoptotic body membrane and mesoporous silica nanoparticles wrapped in the apoptotic body membrane, the apoptotic body membrane is derived from T cells, the apoptotic body membrane is derived from immune cells, and the mesoporous silica nanoparticles are loaded with MicroRNA-21. In some embodiments, the MicroRNA-21 is supported on the mesoporous nanoparticles by electrostatic interaction or physical adsorption. In some embodiments, disruption of disulfide bonds by glutathione in the cytoplasm of the target cell by MicroRNA-21 results in release of MicroRNA-21 into the target cell. In a preferred embodiment, the MicroRNA-21 is labeled with Cy5.

Example 2 curcumin loaded targeted responsive release systems

The targeted responsive release system comprises a chimeric apoptotic body, wherein the chimeric apoptotic body comprises an apoptotic body membrane and mesoporous silica nanoparticles wrapped in the apoptotic body membrane, the apoptotic body membrane is derived from T cells, the apoptotic body membrane is derived from immune cells, and the mesoporous silica nanoparticles are loaded with curcumin.

In some embodiments, after the curcumin is loaded in the pores of the mesoporous silica nanoparticles by physical adsorption, the mesoporous silica nanoparticles are encapsulated with the polyglycerol dimethacrylate.

In some embodiments, the curcumin is responsively released into the target cell by degrading the polyglycerol dimethacrylate with an enzyme within the target cell.

Example 3: preparation of apoptotic microsomal membranes

T cell isolation, activation and identification

Primary T cells were derived from C57BL/6 mouse splenocytes and cultured in RPMI-1640 medium with 20% FBS and 1% penicillin/streptomycin. First, 2. Mu.g/ml of CD 3. Epsilon. Antibody was inoculated into a 6-well plate and cultured at 37 ℃ for 4 hours. Separating and taking out the spleen of the mouse, fully grinding the spleen by using a sterile syringe handle, filtering to form a cell suspension, adding a sterile erythrocyte lysate, gently and fully blowing and uniformly mixing, cracking on ice for 10min, centrifuging cells after cracking is finished, and then re-suspending the cells by using a culture medium. Then, the cell suspension is filtered by a 100 mu m filter to remove cell masses, the cell suspension is inoculated into a 6-well plate precoated with a CD3 epsilon antibody after 2 mu g/ml of CD28 antibody is added, T cells are activated after the cell suspension is cultured for 24-72 hours under the conditions of 37 ℃ and 5% CO2, and then 1 mu g/ml of lipopolysaccharide is added into a culture system to act for 12 hours to stimulate the T cells. Morphological changes before and after T cell activation were observed under an inverted microscope.

2. Macrophage isolation and identification

Primary macrophages were derived from bone marrow mononuclear cells of C57BL/6 mice cultured in high glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Specifically, a mouse is killed after being decapped and is soaked in a 75% ethanol solution for disinfection, the skin is cut off along the hind limb of the mouse by using sterile scissors and tweezers, the femur and the tibia are separated and taken out, soft tissues attached to the surfaces of the femur and the tibia are stripped off in ice PBS, epiphyseal ends on two sides of the bone are subtracted, bone marrow is washed by PBS, the collected suspension is fully blown to form single cell suspension, red blood cell lysate is added for lysis for 2-3min, the cells are collected by centrifugation for 5min at 800rpm, the cells are inoculated in a 6-well plate after being completely cultured and are subjected to basic suspension, recombinant mouse macrophage colony stimulating factor addition at the concentration of 20ng/ml to induce the cell differentiation, and the cells become mature macrophages after 7 days of induction for standby.

3. Isolation and characterization of apoptotic bodies

Treating T cells for 3-4h with 0.5 mu M staurosporine, inducing T cell apoptosis, collecting cell supernatant, centrifuging 50g for 5min, removing cells and debris, repeating the steps twice, centrifuging 1000g of supernatant for 10min, collecting precipitates, namely apoptotic bodies, washing 2-3 times with PBS, resuspending, measuring the protein concentration of the apoptotic bodies with a BCA protein detection kit, and storing at-80 ℃ for later use.

4. Preparation of apoptotic microsomal membranes

Thawing apoptotic bodies stored at-80 ℃, centrifuging for 10min under the condition of 1000g, taking the precipitate to be suspended in hypotonic lysis solution, carrying out hypotonic treatment for 1h under 4 ℃, then carrying out low-power ultrasonic treatment for 5s, centrifuging for 10min under the condition of 100g to remove debris, centrifuging for 10min under the condition of 10000g again to take the precipitate, then washing for at least 3 times by water until components in the vacuole are removed, suspending the collected apoptotic body membrane in water, and storing for later use at 4 ℃. The hypotonic lysis solution comprises the following components: 10mM Tris, 10mM MgCl ₂ And 1mM phenylmethylsulfonyl fluoride.

Example 4 preparation of mesoporous silica nanoparticles

The mesoporous silica nano particle is prepared by the following method: adjusting the pH value of 1000 mL of deionized water to 11 by using 52.8 mL of ammonia water solution, heating to 50 ℃, adding 1.12 g of hexadecyl trimethyl ammonium bromide, dropwise adding 5.8 mL of ethyl orthosilicate while quickly stirring after the hexadecyl trimethyl ammonium bromide is completely dissolved, standing the mixture at room temperature overnight after 2 hours, then centrifuging, and thoroughly washing by using distilled water and ethanol to collect a product; and (3) extracting by using acidic methanol at 70 ℃ to remove the surfactant, further centrifuging, collecting the precipitate, washing by using ethanol for a plurality of times, and drying for 20 hours in vacuum to obtain the mesoporous silica nanoparticles with uniform particle size distribution.

Example 5: construction of chimeric apoptotic bodies

In order to ensure that the apoptotic body membrane is completely covered on the surface of the mesoporous silica nanoparticle, 1mg of mesoporous silica nanoparticle and 2mg of apoptotic body membrane are mixed, then the mixed solution is subjected to intermittent ultrasonic treatment for 2min in an ultrasonic instrument, the treated suspension is centrifuged for 10min under the condition of 5000g, and a precipitate, namely the chimeric apoptotic body, is taken out and is stored in water at 4 ℃.

The morphology of the chimeric apoptotic bodies was determined by transmission electron microscopy. 4ul of the chimeric apoptotic body solution with the concentration of 1mg/ml is placed on a 400-mesh-square copper grid of a carbon coating for deposition treatment, the copper grid is washed by deionized water after 10min of deposition, then the copper grid is negatively dyed by 1% phosphotungstic acid for 30 s, the copper grid is naturally dried after being washed by the deionized water, and observation is carried out by using a transmission electron microscope. The results are shown in FIG. 1, and FIG. 1 is a transmission electron microscope image directly showing the core-shell structure of chimeric apoptotic bodies.

Example 6: stability assay for chimeric apoptotic bodies

For short-term storage stability, the mesoporous silica nanoparticles and the chimeric apoptotic bodies are respectively resuspended in water and PBS, and the size of the nanoparticles in different media is detected by DLS for 1 week. The results are shown in FIG. 2A, and show that the chimeric apoptotic bodies were uniform in size within 7 days in both solutions. While the mesoporous silica nanoparticles are only stable in water, they aggregate to a size of 1-2 μm in PBS.

To assess long-term storage stability, we examined the size of chimeric apoptotic bodies before lyophilization and after resuspension. The results are shown in fig. 2B, and show that the chimeric apoptotic bodies before and after freeze-drying were almost the same size.

To assess serum stability, we suspended the chimeric apoptotic bodies in 100% serum at a concentration of 0.1 mg/ml for serum stability testing, with water as the control. The absorbance was measured at 560 nm at different time points (1 min, 5min, 10min, 15min, 30min, 60min, 90 min) after resuspension in serum using a spectrophotometer. The change in absorbance is due to the poor stability of the nanoparticles in serum. As shown in FIG. 2C, after resuspension in 100% serum, the absorbance change of the chimeric apoptotic bodies was very small, while the mesoporous silica nanoparticles were significantly increased.

Example 7: in vitro phagocytic ability detection of cABs by macrophages

To verify the specific phagocytic capacity of macrophages for chimeric apoptotic bodies, PKH 26-labeled apoptotic body membranes, rhB-labeled mesoporous silica nanoparticles, and RhB-labeled chimeric apoptotic bodies at a concentration of 50 μ g/ml were added to the macrophages for treatment for 3h, with PBS as control. Cells were then fixed with 4-cent pfa overnight and then blocked with goat serum at 37 ℃ for 30min. Finally, after incubation for 1h at room temperature using F4/80 antibody and FITC-labeled secondary antibody, nuclei were counterstained with Hoechst 33342 and fluorescence imaging was observed by CLSM. The detection result is shown in fig. 3, compared with the control group and the mesoporous silica nanoparticle group, the apoptotic body membrane and the chimeric apoptotic body group both have obvious red fluorescent signals, and the result shows that the specific phagocytosis of the chimeric apoptotic body by the macrophage is the same as that of the apoptotic body membrane.

Example 8: immunomodulation of apoptotic microsomal membranes

To explore the immunomodulatory effects mediated by apoptotic microsomal membranes, macrophages were treated with Lipopolysaccharide (LPS) at a concentration of 1 μ g/ml to induce inflammatory macrophages to mimic inflammation in vitro. Meanwhile, apoptotic body membranes, mesoporous silica nanoparticles and chimeric apoptotic bodies at a concentration of 25 μ g/ml were added to the culture system, while unstimulated macrophages and PBS groups served as controls. After 24h, supernatants from cultured cells were collected and stored at-80 ℃ until cytokine levels were detected. Levels of tumor necrosis factor (TNF-. Alpha.), IL-6, transforming growth factor (TGF-. Beta.), IL-10 were measured using ELISA kits as indicated. Pro-inflammatory cytokines: TNF-alpha, IL-6; anti-inflammatory cytokines: TGF-beta, IL-10. The detection results are shown in fig. 4A to 4D: FIG. 4A shows the results of TNF- α assays; FIG. 4B shows the result of detection of IL-6; FIG. 4C shows the result of TGF-. Beta.assay; FIG. 4D shows the results of IL-10 detection. The results show that LPS stimulation significantly promoted TNF-alpha and IL-6 release at levels much higher than the unstimulated control group; the addition of apoptotic bodies membranes inhibited the inflammatory burst and promoted the secretion of TGF- β and IL-10, the same results were found in the same chimeric apoptotic body group, which means that the treatment of chimeric apoptotic bodies resulted in a remission of inflammation.

Example 9: construction of MicroRNA-21-loaded targeted responsive release system

MicroRNA-21 is loaded on mesoporous silica nanoparticles through electrostatic interaction. Specifically, 50mg of mesoporous silica nanoparticles and 200ul of 3-mercaptopropyltrimethoxysilane are suspended in 20ml of ethanol solution, and complete dispersion is promoted by 100W ultrasound for 10 min. The suspension will be stirred overnight at 60 ℃ under nitrogen. Subsequently, 225mg of 2,2' -dithiodipyridine and 145mg of 2-dimethylaminoethanethiol were added to the above solution to continue the reaction for 12 hours. The reaction product is washed several times with ethanol and centrifuged to obtain a precipitate, which is dried under vacuum to obtain mesoporous silica nanoparticles (abbreviated as MSN +) grafted with dimethylamine. Next, 0.06mg MSN + and 15nmol MicroRNA-21 were mixed well and blown in 60ul of enzyme-free water at 4 ℃ for 1h. Washing the reaction product with non-enzyme water for several times, and centrifuging to obtain MicroRNA-21 loaded mesoporous silica nanoparticles (MSN for short) ^MicroRNA-21 ). The same procedure was followed to synthesize Cy 5-labeled MicroRNA-21-loaded mesoporous silica nanoparticles (abbreviated as MSN) ^{MicroRNA-21-cy5} ）。

In the experiments of glutathione-responsive release systems, a certain amount of MSNs was first administered at room temperature ^MicroRNA ^-21-cy5 Dispersed in 400. Mu.l of enzyme-free water with and without glutathione, respectively. Subsequently, centrifugation was carried out at 10000rpm for 10min to obtain a supernatant. The amount of released MicroRNA-21-cy5 from nanoparticles into solution was determined using a fluorescence microplate reader (HH 3400, perkinElmer) to measure the fluorescence intensity in the supernatant (excitation wavelength = 649 nm/emission wavelength = 670 nm).

Finally, 1mg MSN was added ^{MicroRNA-21-cy5} Mixing with 2mg apoptotic body membrane, and subjecting the mixture to intermittent ultrasound in a sonicator for 2min. Centrifuging the treated suspension for 10min under the condition of 5000g, and taking out the precipitate to obtain a targeted responsive release system (abbreviated as cABs) loaded with MicroRNA-21 ^MicroRNA-21 ) And re-suspended in water for storage at 4 ℃ for subsequent experiments.

Specifically, microRNA-21 binds to positively charged mesoporous silica nanoparticles via electrostatic interaction, wherein the positively charged dimethylamine groups are linked to the mesoporous silica nanoparticles via disulfide bonds. Therefore, after the macrophage internalizes the chimeric apoptotic body, high concentration of glutathione in the cytoplasm of the target cell destroys the disulfide bond, resulting in rapid release of MicroRNA-21.

The detection results are shown in fig. 5: FIG. 5 shows the detection of release of MicroRNA-21 triggered by glutathione, and the results show that the final release rate of MicroRNA-21 is less than 20% after 7 days of the control group. In contrast, in the presence of glutathione, the release of MicroRNA-21 increased significantly over time, reaching 90% after 7 days. The capability of loading MicroRNA-21 on the mesoporous silica nano particles is determined.

Example 10: construction of curcumin-loaded targeted responsive release system

Completely and uniformly mixing 10mg of mesoporous silica nanoparticles, 1mg of curcumin and 10ul of glyceryl dimethacrylate in 10ul of water, and performing ultrasonic treatment at 100W for 20min to fully and uniformly mix. And vacuumizing for 3 times and 10min each time under the vacuum condition to generate the mesoporous silica nano particles loaded with the glyceryl dimethacrylate and the curcumin together. At the same time, 24mg of peroxosulfuric acid was dissolved in 3ml of water under nitrogen for 20min, and then 80ul of tetramethylethylenediamine was dissolved in 800ul of water. Then, 2500 mul of ammonium persulfate solution and 440ul of tetramethylethylenediamine solution are mixed with the mesoporous silica nanoparticles co-loaded with the glycerol dimethacrylate and the curcumin uniformly under nitrogen to generate a polymerization reaction to generate the mesoporous silica nanoparticles (abbreviated as MSN: MSN) co-loaded with the glycerol dimethacrylate and the curcumin ^{-Cur-polyster} ) And leakage of curcumin is prevented. The suspension was stirred under nitrogen at room temperature for 1h to facilitateAnd (4) carrying out polymerization reaction. The resulting nanoparticles were washed 3 times with water and dried overnight at 50 ℃ under vacuum.

The mesoporous silica nanoparticles and polyester-coated mesoporous silica nanoparticles (abbreviated as MSN-polyester) were observed using a transmission electron microscope.

The intracellular release of curcumin can be detected in vitro by a fluorescence spectrophotometer. Specifically, 10mg of MSN was added ^{-Cur-polyster} After dispersing in 10mL of PBS containing esterase and without esterase at 37 ℃ with shaking, the supernatant was collected by centrifugation at various time points, and the fluorescence intensity of the supernatant was measured to obtain in vitro release results.

Finally, 1mg of MSN-Cur-polyster was mixed with 2mg of apoptotic bodies, and the mixture was subjected to intermittent ultrasound for 2min in an ultrasound apparatus. Centrifuging the treated suspension for 10min at 5000g, and collecting precipitate to obtain target-response curcumin-loaded release system (cABs) ^Cur ) Resuspended in water and stored at 4 ℃.

Specifically, curcumin is loaded in the pores of mesoporous silica nanoparticles by physical adsorption, and then encapsulated with polyglycerol dimethacrylate to prevent the nanoparticles from causing leakage of curcumin during cycling. When it is targeted to the site of inflammation and phagocytosed by macrophages, the polyglycerol dimethacrylate can be degraded by esterases in the cells to initiate the release of curcumin.

The detection results are shown in fig. 6: FIG. 6 shows MSN ^Cur The results show that the release performance of the compound is highly dependent on the enzymolysis reaction of esterase.

Example 11: establishment of skin inflammation injury model

Before depilation, pentobarbital sodium 1% is used in an amount of 70mg/kg for intraperitoneal injection anesthesia, hair is cut by a bent knife, hairs on the back and around the animal are removed by sodium sulfide 8%, and the hairs are scraped off slightly after a plurality of minutes and are wiped clean by clear water. Fixing the mouse in the prone position on an operation plate, disinfecting the center of the back with 75% alcohol, disinfecting with iodophor, marking out a trace with the diameter of 1.0cm, and establishing a full-layer circular skin inflammation injury model with the same size.

Example 12: effect of inflammation in skin injury model

The mouse full-layer skin injury model is simple, convenient and quick, has better balance among the wound surfaces, and is one of ideal models for researching the effect of medicaments on promoting the wound surface repair. In order to evaluate the application of the functional chimeric apoptotic body constructed by the inventor in inflammatory diseases, the influence on the development of the inflammatory diseases and the treatment effect, the inventor uses a mouse skin injury model for verification, and provides a brand-new and effective treatment method for the mouse skin injury model.

To investigate the effect of chimeric apoptotic bodies on inflammation in vivo, the inflammatory site-specific ability of chimeric apoptotic bodies was assessed by an in vivo imaging system. Injecting the DiR-marked mesoporous silica nanoparticles, the apoptotic body membrane, the chimeric apoptotic body and PBS into a mouse body through a tail vein, detecting the biodistribution of the DiR-marked mesoporous silica nanoparticles at the damaged part of the mouse after 24 hours, and carrying out quantitative statistics on the DiR-marked mesoporous silica nanoparticles, the apoptotic body membrane, the chimeric apoptotic body and the PBS according to the fluorescence intensity of an imaging system. The detection result is shown in fig. 7, and according to the in vivo imaging result, the chimeric apoptotic bodies or apoptotic body membranes have enhanced targeting ability to inflammatory wound sites compared to the naked mesoporous silica nanoparticles. The results indicate the targeting ability of chimeric apoptotic bodies in vivo, while mesoporous silica nanoparticles accumulate little around the inflammatory region.

Example 13: treatment of skin inflammation injury model

To assess the effect of chimeric apoptotic bodies on wound healing, we treated model mice. Mice, a skin inflammation injury model, were randomly divided into four groups (n = 6 per group), PBS and cABs, respectively ^{null group} Group, cABs ^MicroRNA-21 Group and cABs ^Cur After the operation, 100. Mu.l PBS + 100. Mu.g cABs were injected into the caudal vein ^MicroRNA-21 And 100. Mu.l PBS + 100. Mu.g cABs ^Cur . Wound size was measured at 0, 3, 5, 7, 10 and 12d after treatment and calculated using image J for statistics. At the end point, all mice were euthanized and the skin at the site of injury was collected for histological studies. The detection results are shown in FIGS. 8-9E, and FIG. 8 is the mouse wound healing detection result, nodeFruit display, with cABs ^null Injection of cABs compared to PBS group ^MicroRNA-21 Or cABs ^Cur The wound closure rate of the mouse is obviously improved, and particularly shows that the mouse has obvious therapeutic action; FIGS. 9A-9E are HE staining results of different groups of skin lesions and skin tissue results showing injection of cABs compared to that observed in PBS and chimeric apoptotic bodies ^MicroRNA-21 Can remarkably enhance the healing of the skin, has good organization and layered neuroepithelium at the injured part, and has a large amount of collagen deposition. Furthermore, cABs compared to PBS group ^MicroRNA-21 The groups had increased hair follicle and sebaceous gland formation and decreased inflammatory cell infiltration, similar to normal skin structure. Likewise, cABs ^Cur The healed skin of the group had a layered structure, reduced inflammatory cell infiltration, and concomitant production of skin appendages. In conclusion, the results show that chimeric apoptotic bodies can be released by MicroRNA-21 and curcumin metastasis, promoting the regression of inflammation, collagen deposition and formation of subcutaneous appendages and skin regeneration during wound healing.

Example 14: establishment of ulcerative colitis model

To further investigate the effect of chimeric apoptotic bodies on inflammation and therapeutic effects, we established another acute inflammation model. A2.5% dextran sodium sulfate solution was freely drunk by C57BL/6 mice, and the mice were continuously fed for 10 days to establish a model of ulcerative colitis.

Example 15: effect of inflammation in ulcerative colitis model

To further investigate the effect of chimeric apoptotic bodies on inflammation in vivo, the site-specific ability of inflammation of chimeric apoptotic bodies was assessed by an in vivo imaging system. Injecting the DiR-marked mesoporous silica nanoparticles, the apoptotic small body membrane, the chimeric apoptotic small body and PBS into a mouse body of the ulcerative colitis model through a tail vein, detecting the biological distribution of the DiR-marked mesoporous silica nanoparticles at the damaged part of the mouse after 24 hours, and carrying out quantitative statistics on the DiR-marked mesoporous silica nanoparticles, the apoptotic small body membrane, the chimeric apoptotic small body and the PBS according to the fluorescence intensity of an imaging system. As shown in fig. 10, the in vivo imaging results show that the chimeric apoptotic bodies or apoptotic body membranes have enhanced targeting ability to inflammatory wound sites compared to the bare mesoporous silica nanoparticles. The results indicate the targeting ability of chimeric apoptotic bodies in vivo, while mesoporous silica nanoparticles accumulate little around the inflammatory region.

Example 16: treatment of ulcerative colitis models

To further evaluate the effect of chimeric apoptotic bodies on wound healing, we treated mice model for ulcerative colitis. Mice, a skin inflammation injury model, were randomly divided into four groups (n = 6 per group), and injected into caudal vein after surgery, 100. Mu.l PBS + 100. Mu.g cABs, respectively ^MicroRNA-21 And 100. Mu.l PBS + 100. Mu.g cABs ^Cur . Mouse body weights were measured and counted on

days

2, 4, 6, 8 and 10 after treatment. At the end point, all mice were euthanized and injury site tissues were collected for colon length and histology studies.

The results are shown in FIGS. 11A to 12E, and FIG. 11A shows the results of monitoring the body weight of mice, and the results show that the control group shows more significant weight loss than the PBS group with time, and that cABs are formed ^MicroRNA-21 Group and cABs ^Cur Group treatment can inhibit the rapid decrease of body weight and the worsening of disease; FIG. 11B is a colon length result with a significant shortening of the colon due to dextran sodium sulfate administration, accompanied by severe congestion and ulceration. While using cABs ^MicroRNA-21 And cABs ^Cur The therapeutic effect exerted can partially restore the length of the colon and improve the lesion; FIGS. 12A-12E are HE staining results of injury sites in ulcerative colitis models in different dosing groups, showing massive transmural inflammatory cell infiltration, crypt injury and loss of mucosal epithelial integrity with the PBS group colon, cABs ^MicroRNA-21 And cABs ^Cur The colon tissue of the treated group retained normal crypt and intestinal gland structures and reduced inflammatory infiltration.

The reagents used in the above embodiments of the present invention are, unless otherwise specified, commercially available reagents or reagents that can be manufactured according to the prior art, and the related detection devices or experimental devices, if not specifically specified, refer to conventional instruments that can meet the requirements of related detection or experiment, and are all commercially available.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments and application fields, and the above-described embodiments are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the claims appended hereto.

Claims

1. The functional chimeric apoptotic body is characterized by comprising an apoptotic body membrane and mesoporous silica nanoparticles wrapped in the apoptotic body membrane, wherein the apoptotic body membrane is derived from immune cells, and the mesoporous silica nanoparticles are loaded with curcumin; after the curcumin is loaded in the pores of the mesoporous silica nanoparticles through physical adsorption, the mesoporous silica nanoparticles are encapsulated by the polyglycerol dimethacrylate.

2. The functional chimeric apoptotic body of claim 1, wherein said immune cell is selected from lymphocytes selected from T cells.

3. The functional chimeric apoptotic body of claim 1, wherein when said functional chimeric apoptotic body is targeted to a site of inflammation and phagocytosed by macrophages, said polyglycerol dimethacrylate is degraded by esterase within the target cell, thereby responsively releasing curcumin within the target cell.

4. A method of producing functional chimeric apoptotic bodies of any one of claims 1-3, comprising the steps of:

3) Mixing the mesoporous silica nano particles co-loaded with the polyglycerol dimethacrylate and the curcumin with the apoptotic body membrane, then intermittently ultrasonically treating the mixed solution for 2min in an ultrasonic instrument, centrifuging the treated suspension for 10min under the condition of 5000g, taking the precipitate to obtain the functional chimeric apoptotic body, and re-suspending the functional chimeric apoptotic body in water at 4 ℃ for storage.

5. The preparation method according to claim 4, wherein in the step 3), the mass ratio of the mesoporous silica nanoparticles co-loaded with the polyglycerol dimethacrylate and the curcumin to the apoptotic body membrane is 1.

6. Use of a functional chimeric apoptotic body of any one of claims 1 to 3 in the manufacture of a product for the treatment of an inflammatory disease.

7. Use according to claim 6, characterized in that said inflammatory diseases comprise skin inflammation, colitis, pneumonia, hepatitis or nephritis.

8. A medicament for the treatment of an inflammatory disease, comprising a functional chimeric apoptotic body of any one of claims 1-2 and a pharmaceutically acceptable carrier.

9. The pharmaceutical of claim 8, wherein the pharmaceutically acceptable carrier is selected from water, PBS buffer, and physiological saline.