Targeted nano-vesicles for stage-specific tissue repair and preparation and application methods of targeted nano-vesicles

Abstract

The invention discloses a targeted nano-vesicle for stage-specific tissue repair as well as a preparation method and an application method of the targeted nano-vesicle. The nano vesicles are formed by fusing cell membranes of dominant cells at different stages in the tissue repair process in an equal mass manner. And the system can be used for accurately delivering and regulating key cells in different tissue repair stages, so that the synergistic promotion of dynamic remodeling of a regeneration environment and tissue regeneration is promoted. The cell membrane is extracted by adopting a low-temperature buffer solution system, the fused cell membrane is formed after mixed incubation, and then the nano-vesicles are prepared by adopting a mechanical membrane extrusion method, so that the membrane protein function and structural characteristics of the source cell membrane are reserved, the stability in a physiological environment is good, the biological safety is good, and obvious immunoreaction or toxicity cannot be caused. And the method has the advantages of low cost, mild process and easiness in large-scale popularization. Through glucose functional modification, the active recognition and targeting capabilities are further enhanced, the limitation of a traditional static targeting or sequential release system is broken through, and the system has a wide application prospect.

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 targeted nanovesicles for stage-specific tissue repair and their preparation and application methods. Background Art

[0002] Tissue repair is a multi-stage, dynamic and complex physiological process, mainly including the inflammation stage, the cell proliferation stage and the tissue remodeling stage. In the inflammation stage, immune cells such as macrophages and neutrophils dominate the repair process, clear necrotic tissue and pathogens, and initiate repair signals. In the cell proliferation and tissue remodeling stages, the activation of mesenchymal stem cells, M2 macrophages, fibroblasts and endothelial cells, etc. is mainly relied on to promote angiogenesis and matrix remodeling and restore tissue function. Since each stage of tissue repair has different dominant cells and the repair process is dynamically changing, it is necessary to precisely regulate the dominant cells at different stages.

[0003] In recent years, researchers have designed a variety of strategies aimed at optimizing cell-targeted therapy at different stages. For example, for the immune inflammation stage, pH or ROS-responsive nanocarriers are used to deliver anti-inflammatory drugs to regulate the immune response in the local inflammatory environment. However, it is difficult to distinguish acute and chronic inflammation signals by this method, and the dynamic changes in the microenvironment will have a greater impact on the response efficiency of drug delivery. In the cell proliferation and tissue remodeling stages, researchers use VEGF or integrin-targeted peptides to modify nanocarriers to promote the enrichment of drugs at newly formed blood vessels and enhance the reconstruction of the vascular network. However, the overlap of normal and newly formed blood vessel targets easily leads to off-target effects. Gene editing technology can improve the repair function by editing stem cell genes, but there is also an off-target risk, which affects the homing efficiency of stem cells.

[0004] In order to adapt to the dynamic evolution process of tissue repair, some studies adopt the strategy of time-sequenced controlled drug release, trigger the release of drugs in stages through microenvironment signals, so as to dynamically regulate cell behavior and improve the therapeutic effect. However, the interaction between drugs may cause side effects, and the release kinetics is complex, which limits its clinical application.

[0005] Therefore, developing a strategy that can precisely target stage-specific cells and adapt to the dynamic changes in the repair process has become an important need in the field of tissue repair. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a targeted nanovesicle for stage-specific tissue repair, as well as its preparation method and application method. By fusing the cell membranes of the dominant cells at each stage of tissue repair, a hybrid membrane material (Hybrid Membranes) is prepared, a nanovesicle with stage-specific cell targeting is constructed, and a glucose modification strategy is introduced to enhance its recognition ability for actively metabolizing cells. The problem that the prior art is difficult to achieve precise targeting and dynamic adaptation to stage-dominant cells in tissue repair is solved.

[0007] The targeted nanovesicle for stage-specific tissue repair is formed by fusing the cell membranes of the dominant cells at different stages during the tissue repair process, and the masses of the cell membranes of the dominant cells at different stages are equal.

[0008] Preferably, the nanovesicle is modified with glucose.

[0009] Preferably, DSPE-PEG-Glucose lipid molecules are inserted on the surface of the nanovesicle.

[0010] Preferably, the nanovesicle is formed by fusing the cell membranes of bone marrow-derived macrophages and bone marrow-derived mesenchymal stem cells.

[0011] The preparation method of the targeted nanovesicle for stage-specific tissue repair is as follows: Step 1: Collect the dominant cells at different stages during the tissue repair process respectively and resuspend them in TM buffer.

[0012] Step 2: Repeatedly extrude the cells to rupture them.

[0013] Step 3: Add 1 M sucrose solution to the lysate obtained in Step 2 and adjust the sucrose concentration to 0.25 M.

[0014] Step 4: Centrifuge the solution obtained in Step 3 at a low speed to remove impurities.

[0015] Step 5: Take the precipitate obtained after centrifugation in Step 4, which is the membrane component, and wash the obtained membrane component with buffer to obtain purified cell membranes.

[0016] Step 6: Adjust the membrane protein concentration to 2 mg·ml -1 .

[0017] Step 7: Take equal masses of the purified cell membranes of the dominant cells at different stages, mix and incubate them to form a fused cell membrane, and then obtain nanovesicles by extrusion.

[0018] Preferably, DSPE-PEG-Glucose is added to the fused cell membrane solution, and the mass ratio of DSPE-PEG-Glucose to membrane protein is 1:20. After shaking and incubating, the fused cell membrane is modified.

[0019] Preferably, after shaking and incubating, unbound free ligands are removed by ultrafiltration or centrifugal column methods.

[0020] The application method of the stage-specific tissue repair targeted nanovesicles realizes the precise delivery and regulation of key cells at different tissue repair stages by constructing nanovesicles with stage-specific targeting functions, thereby promoting the coordinated advancement of the dynamic remodeling of the regeneration environment and tissue regeneration.

[0021] Preferably, the nanovesicles are used for bone defect repair.

[0022] The present invention has the following beneficial effects: 1. The targeted nanovesicles proposed by the present invention can target and recognize the dominant cells at different stages of tissue repair by fusing cell-derived membrane materials with stage specificity, can conform to the dynamic evolution of the dominant cell spectrum during tissue repair, and achieve precise delivery.

[0023] 2. The preparation method of the targeted nanovesicles proposed by the present invention does not require the aid of high-precision equipment or chemical fusion agents, and only uses a low-temperature buffer system and mechanical membrane extrusion method to achieve cell membrane fusion and nanovesicle construction, with the advantages of low cost, mild process, and easy large-scale promotion.

[0024] 3. The targeted nanovesicles proposed and prepared by the present invention retain the membrane protein functions and structural characteristics of the source cell membrane, have good stability in the physiological environment, and have good biosafety, and will not cause obvious immune reactions or toxicity.

[0025] 4. Through glucose functional modification, the present invention further enhances the active recognition and targeting ability of the targeted nanovesicles, breaks through the limitations of traditional static targeting or sequential release systems, and can achieve precise regulation of the whole process from inflammation control to tissue regeneration, and has broad application prospects in complex tissue repair fields such as chronic wound repair, bone tissue regeneration, and nerve regeneration. Description of the Drawings

[0026] Figure 1 Statistical results of the average particle size and Zeta potential of the targeted nanovesicles prepared in Example 2; the black dots represent the results obtained from each independent experiment.

[0027] Figure 2 Electron micrograph of the targeted nanovesicles prepared in Example 2.

[0028] Figure 3Results of the determination of the protein composition of the targeted nanovesicles prepared in Example 2.

[0029] Figure 4 Results of the verification of membrane fusion of the targeted nanovesicles prepared in Example 2.

[0030] Figure 5 Evaluation of the cell targeting function of the targeted nanovesicles in the early inflammatory stage in vitro in Test Example 1; each dot represents a biological replicate, and P < 0.05 represents a statistically significant difference.

[0031] Figure 6 Evaluation of the cell targeting function of the targeted nanovesicles in the late repair stage in vitro in Test Example 1; each dot represents a biological replicate, and P < 0.05 represents a statistically significant difference.

[0032] Figure 7 Evaluation of the cell targeting function of the targeted nanovesicles in the early inflammatory stage in vivo in Test Example 2; each dot represents a biological replicate, and P < 0.05 represents a statistically significant difference.

[0033] Figure 8 Evaluation of the cell targeting function of the targeted nanovesicles in the late repair stage in vivo in Test Example 2; each dot represents a biological replicate, and P < 0.05 represents a statistically significant difference. Detailed implementation manners

[0034] The present invention will be further explained and illustrated below with reference to the accompanying drawings.

[0035] Example 1

[0036] In this example, targeted nanovesicles for stage-specific tissue repair were prepared using bone marrow mesenchymal stem cells (BMSCs) and bone marrow-derived macrophages (BMDMs) from C57BL / c mice. The specific steps are as follows: Step 1: Collect bone marrow mesenchymal stem cells from C57BL / c mice and resuspend them in TM buffer at 4°C. The TM buffer includes 30 mM Tris-HCl, 225 mM D-mannitol, 75 mM sucrose, and 0.2 mM EGTA.

[0037] Step 2: Use an Avanti mini-extruder to extrude the cells collected in Step 1 repeatedly 40 times to rupture the cells.

[0038] Step 3: Add 1 M sucrose solution to the lysate in Step 2 to adjust the final concentration to 0.25 M.

[0039] Step 4: Centrifuge the solution in Step 3 at 2000 g for 10 min, take the supernatant and centrifuge it again at 3000 g for 30 min to remove impurities such as cell nuclei.

[0040] Step 5: Take the precipitate from Step 4, and wash the obtained membrane fraction with 0.25 M sucrose buffer to obtain purified cell membranes.

[0041] Step 6: Measure the protein concentration using the BCA method, and adjust the membrane protein concentration to 2 mg·ml -1 , to obtain mesenchymal stem cell membranes.

[0042] Step 7: Collect bone marrow-derived macrophages from C57BL / c mice, and repeat Steps 1 - 6 to obtain macrophage membranes.

[0043] Step 8: Take equal amounts of mesenchymal stem cell membranes and macrophage membranes, mix and incubate them in a 37°C water bath for 10 minutes, then repeatedly extrude them through a polycarbonate membrane with a pore size of 100 nm to form targeted nanovesicles of hybrid membranes, and freeze and store them at -80°C for later use.

[0044] Example 2

[0045] Based on Example 1, in this example, glucose modification was performed on the preparation of targeted nanovesicles of hybrid membranes. The specific method was as follows: Add 100 µg of DSPE-PEG-Glucose to 1 ml of the hybrid membrane vesicle solution. Then, gently shake and incubate at room temperature for 30 minutes, and remove the unbound free ligands by ultrafiltration or centrifugal column method to obtain targeted nanovesicles, which were frozen and stored at -80°C for later use.

[0046] The average particle size and Zeta potential of the obtained targeted nanovesicles were measured using a dynamic light scattering instrument (DLS). The results were as Figure 1 shown. Its average particle size was about 250 nm, and the Zeta potential was about -23 mV, with good stability. Observe the targeted nanovesicles through electron microscopy. As Figure 2 shown, they presented an exosome-like vesicle structure, were evenly distributed, and had clear edges. The protein composition of the targeted nanovesicles was determined by LC-MS / MS, and functional annotation was performed in combination with GO terms. As Figure 3 shown, the results showed that the obtained targeted nanovesicles retained the functional markers on both mesenchymal stem cell membranes and macrophage membranes, laying a molecular foundation for multi-stage targeting. Western blot was used to analyze the fusion of mesenchymal stem cell membranes and macrophage membranes. The results were as Figure 4 shown. It can be seen that the mesenchymal stem cell membranes and macrophage membranes were successfully fused, and the membrane protein characteristics specific to the source cells were retained.

[0047] Test Example 1 In this test example, an in vitro stage-specific cell targeting function evaluation experiment was carried out on the targeted nanovesicles for stage-specific tissue repair prepared in Example 2. Specifically: The targeted nanovesicles prepared in Example 2 were labeled with Cy5 fluorescence, and then co-incubated with three different types of cells for 2 hours. Then, flow cytometry was used to detect the uptake ratio of the targeted nanovesicles by various cells. The different types of cells were as follows: (1) Inflammatory bone marrow-derived macrophages stimulated by LPS, denoted as M1-type BMDMs.

[0048] (2) NIH3T3 fibroblasts, denoted as the negative control group.

[0049] (3) Bone marrow mesenchymal stem cells, denoted as BMSCs.

[0050] The results were as Figure 5 and Figure 6 shown. The uptake of the targeted nanovesicles in M1-type BMDMs and BMSCs was significantly higher than that in the negative control group. Among them, the uptake rate of M1-type BMDMs was the highest, indicating its good targeting ability in the early stage of inflammation. This result suggests that the targeted nanovesicles have the ability to distinguish different cell types and preferentially bind to the dominant cells, providing a theoretical basis for stage-specific delivery.

[0051] Test Example 2 In this test example, an in vivo stage-specific cell targeting function evaluation experiment was conducted on the stage-specific tissue repair targeted nanovesicles prepared in Example 2. Specifically: The targeted nanovesicles prepared in Example 2 were labeled with Cy5 fluorescence. In a mouse skull defect model, the labeled targeted nanovesicles were locally loaded and injected into the injury area through a gelatin hydrogel. Samples were taken on the 3rd and 14th days after surgery, namely the inflammatory phase and the repair phase, to prepare single-cell suspensions of the skull defect area, and flow cytometry was used to analyze the uptake of the targeted nanovesicles by different types of cells. Among them, inflammatory macrophages were identified by Ly6C^high labeling, and mesenchymal stem cells were screened by CD105^+.

[0052] The results were as Figure 7 shown. On the 3rd day after surgery, the targeted nanovesicles were mainly enriched in Ly6C^high inflammatory monocytes / macrophages; as Figure 8 shown, on the 14th day after surgery, the targeted nanovesicles were more likely to be taken up by CD105^+ mesenchymal stem cells, indicating that the targeted nanovesicles have good spatio-temporal stage-specific targeting ability. It was proved that the targeted nanovesicles can achieve a precise delivery strategy of "recognizing by stage and dynamically targeting" according to the microenvironmental characteristics and cell composition changes at different stages of tissue repair.

Claims

1. Targeted nanovesicles for stage-specific tissue repair, characterized in that: The nanovesicles are formed by the fusion of cell membranes of dominant cells at different stages during tissue repair.

2. The targeted nanovesicles for stage-specific tissue repair according to claim 1, characterized in that: The cell membranes of dominant cells at different stages have equal mass.

3. The targeted nanovesicles for stage-specific tissue repair according to claim 2, wherein: The nanovesicles are formed by the fusion of cell membranes of bone marrow-derived macrophages and bone marrow-derived mesenchymal stem cells.

4. The targeted nanovesicles for stage-specific tissue repair according to claim 1, characterized in that: Glucose modification is performed on the nanovesicles.

5. The targeted nanovesicles for stage-specific tissue repair according to claim 4, wherein: DSPE-PEG-Glucose lipid molecules are inserted on the surface of the nanovesicles.

6. The preparation method of the stage-specific tissue repair-targeted nanovesicles according to any one of claims 1 to 5, characterized in that: Extract the cell membranes of the dominant cells at different stages during tissue repair, and adjust the membrane protein concentration to 2 mg·ml -1 ; Mix and incubate the cell membranes of the dominant cells at different stages with equal mass to form fused cell membranes, and then obtain nanovesicles by extrusion.

7. The preparation method of the stage-specific tissue repair-targeted nanovesicles according to claim 6, characterized in that: DSPE-PEG-Glucose is added to the fused cell membrane solution and incubated with shaking. The mass ratio of DSPE-PEG-Glucose to membrane protein is 1:

20.

8. The preparation method of the stage-specific tissue repair-targeted nanovesicles according to claim 6, characterized in that: Unbound free ligands are removed by ultrafiltration or centrifugal column methods.

9. The application method of the stage-specific tissue repair-targeted nanovesicles according to any one of claims 1 to 5, characterized in that: The nanovesicles are used to prepare drugs to achieve precise delivery and regulation of key cells at different tissue repair stages, thereby promoting the coordinated advancement of the dynamic remodeling of the regenerative environment and tissue regeneration.

10. The application method of the stage-specific tissue repair-targeted nanovesicles according to any one of claims 1 to 5, characterized in that: The nanovesicles are used to prepare drugs for bone defect repair.

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