Targeted nanovesicles for stage-specific tissue repair and their preparation and application methods

Abstract

The present invention discloses targeted nanovesicles for stage-specific tissue repair and methods for their preparation and application. The nanovesicles are formed by the fusion of the cell membranes and other masses of the dominant cells at different stages in the tissue repair process. They can be used for the precise delivery and regulation of key cells at different tissue repair stages, thereby promoting the dynamic remodeling of the regeneration environment and the coordinated advancement of tissue regeneration. The cell membrane is extracted using a low-temperature buffer system, and a fused cell membrane is formed after mixed incubation, and then the nanovesicles are prepared by a mechanical membrane extrusion method, which retains the membrane protein function and structural characteristics of the source cell membrane, has good stability in a physiological environment, and has good biosafety, and will not induce obvious immune response or toxicity. It has the advantages of low cost, mild process, and easy large-scale promotion. Through glucose functional modification, the active recognition and targeting capabilities are further enhanced, breaking through the limitations of traditional static targeting or timed release systems, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical preparations, and relates to nanomedicines for targeted delivery, in particular to targeted nanovesicles for stage-specific tissue repair, and methods for preparing and applying the same. Background Art

[0002] Tissue repair is a multi-stage, dynamic, and complex physiological process, primarily consisting of an inflammatory phase, a cell proliferation phase, and a tissue remodeling phase. During the inflammatory phase, immune cells such as macrophages and neutrophils dominate the repair process, clearing necrotic tissue and pathogens and initiating repair signals. During the cell proliferation and tissue remodeling phases, activation of mesenchymal stem cells, M2 macrophages, fibroblasts, and endothelial cells is the primary mechanism for promoting angiogenesis and matrix remodeling, thereby restoring tissue function. Because each stage of tissue repair has different dominant cells, and the repair process changes dynamically, precise regulation of the dominant cells at each stage is necessary.

[0003] In recent years, researchers have designed a variety of strategies to optimize cell-targeted therapy at different stages. For example, in the immune-inflammatory stage, pH- or ROS-responsive nanocarriers are used to deliver anti-inflammatory drugs to regulate the immune response in the local inflammatory environment. However, this method makes it difficult to distinguish between acute and chronic inflammatory signals, and the dynamic changes in the microenvironment have a significant impact on the response efficiency of drug delivery. In the cell proliferation and tissue remodeling stage, researchers use VEGF or integrin targeting peptides to modify nanocarriers to promote drug enrichment in new blood vessels and enhance vascular network reconstruction. However, the overlap of normal blood vessel and new blood vessel targets can easily lead to off-target effects. Gene editing technology can enhance 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] To adapt to the dynamic evolution of tissue repair, some studies have adopted a strategy of time-controlled drug release, triggering microenvironmental signals to release drugs in stages, thereby dynamically regulating cell behavior and improving therapeutic efficacy. However, drug-drug interactions can lead to side effects, and the complex release kinetics limit its clinical application.

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

[0006] To address the shortcomings of existing technologies, this paper proposes stage-specific tissue repair-targeted nanovesicles and methods for their preparation and application. By fusing the cell membranes of dominant cells at each stage of tissue repair, hybrid membrane materials are prepared to construct stage-specific cell-targeted nanovesicles. Furthermore, a glucose modification strategy is introduced to enhance their ability to recognize metabolically active cells. This addresses the existing difficulty in achieving precise targeting and dynamic adaptation of stage-dominant cells in tissue repair.

[0007] The targeted nanovesicles for stage-specific tissue repair are formed by the fusion of cell membranes of dominant cells at different stages in the tissue repair process, and the cell membranes of the dominant cells at different stages have equal mass.

[0008] Preferably, the nanovesicles are modified with glucose.

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

[0010] Preferably, the nanovesicles are formed by fusion of the cell membranes of bone marrow-derived macrophages and bone marrow-derived mesenchymal stem cells.

[0011] The preparation method of targeted nanovesicles for stage-specific tissue repair includes the following specific steps:

[0012] Step 1: Collect the leading cells at different stages of the tissue repair process and resuspend them in TM buffer.

[0013] Step 2: Repeatedly squeeze the cells to break them apart.

[0014] Step 3: Add 1 M sucrose solution to the lysate obtained in step 2 to adjust the sucrose concentration to 0.25 M.

[0015] Step 4: centrifuge the solution obtained in step 3 at low speed to remove impurities.

[0016] Step 5: Take the precipitate obtained after centrifugation in step 4, which is the membrane fraction, and wash the obtained membrane fraction with buffer to obtain purified cell membrane.

[0017] Step 6: Adjust the membrane protein concentration to 2 mg ml -1 .

[0018] Step 7: Take equal masses of purified cell membranes of dominant cells at different stages and mix them for incubation to form fusion cell membranes, which are then extruded to obtain nanovesicles.

[0019] Preferably, DSPE-PEG-Glucose is added to the fusion cell membrane solution, the mass ratio of DSPE-PEG-Glucose to membrane protein is 1:20, and the solution is shaken and incubated to modify the fusion cell membrane.

[0020] Preferably, after incubation with shaking, unbound free ligand is removed by ultrafiltration or spin column method.

[0021] The application method of targeted nanovesicles for stage-specific tissue repair, by constructing nanovesicles with stage-specific targeting functions, can achieve precise delivery and regulation of key cells in different tissue repair stages, thereby promoting the dynamic remodeling of the regenerative environment and the coordinated advancement of tissue regeneration.

[0022] Preferably, the nanovesicles are used for bone defect repair.

[0023] The present invention has the following beneficial effects:

[0024] 1. The targeted nanovesicles proposed in the present invention, by fusing stage-specific cell-derived membrane materials, can target and identify the dominant cells in different stages of tissue repair, adapt to the dynamic evolution of the dominant cell spectrum during the tissue repair process, and achieve precise delivery.

[0025] 2. The targeted nanovesicle preparation method proposed in the present invention does not require the use of high-precision equipment or chemical fusion agents. It only uses a low-temperature buffer system and mechanical membrane extrusion to achieve cell membrane fusion and nanovesicle construction. It has the advantages of low cost, mild process, and easy large-scale promotion.

[0026] 3. The targeted nanovesicles proposed and prepared by the present invention retain the functional and structural characteristics of the membrane proteins of the source cell membrane, have good stability in the physiological environment, and have good biosafety, and will not induce obvious immune response or toxicity.

[0027] 4. The present invention further enhances the active recognition and targeting capabilities of targeted nanovesicles through glucose functional modification, breaking through the limitations of traditional static targeting or timed release systems. It can achieve precise regulation of the entire 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The 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 in each independent experiment.

[0029] Figure 2 This is an electron micrograph of the targeted nanovesicles prepared in Example 2.

[0030] Figure 3 These are the results of protein composition determination of the targeted nanovesicles prepared in Example 2.

[0031] Figure 4 This is the membrane fusion verification result of the targeted nanovesicle prepared in Example 2.

[0032] Figure 5 This is the 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 indicates a statistically significant difference.

[0033] Figure 6 This is the 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.

[0034] Figure 7 This is the 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 indicates a statistically significant difference.

[0035] Figure 8 This is the 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 DESCRIPTION

[0036] The present invention will be further explained below with reference to the accompanying drawings.

[0037] Example 1

[0038] In this example, bone marrow mesenchymal stem cells (BMSCs) and bone marrow-derived macrophages (BMDMs) from C57BL / c mice were used to prepare stage-specific tissue repair targeted nanovesicles. The specific steps are as follows:

[0039] Step 1: Bone marrow mesenchymal stem cells from C57BL / c mice were collected and resuspended in TM buffer at 4°C. The TM buffer contained 30 mM Tris-HCl, 225 mM D-mannitol, 75 mM sucrose, and 0.2 mM EGTA.

[0040] Step 2: Use an Avanti mini-extruder to repeatedly extrude the cells collected in step 1 40 times to disrupt the cells.

[0041] Step 3: Add 1 M sucrose solution to the lysate from step 2 to adjust the final concentration to 0.25 M.

[0042] Step 4: Centrifuge the solution from 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.

[0043] Step 5: Take the precipitate from step 4 and wash the resulting membrane fraction with 0.25 M sucrose buffer to obtain purified cell membrane.

[0044] Step 6: Measure the protein concentration using the BCA method and adjust the membrane protein concentration to 2 mg ml -1 , and obtain mesenchymal stem cell membrane.

[0045] Step 7: Collect bone marrow-derived macrophages from C57BL / c mice and repeat steps 1 to 6 to obtain macrophage membranes.

[0046] 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, and then repeatedly extrude them through a 100 nm pore size polycarbonate membrane to form hybrid membrane targeted nanovesicles, which are then frozen and stored at –80°C until use.

[0047] Example 2

[0048] In this example, based on Example 1, glucose was modified to prepare hybrid membrane-targeted nanovesicles. Specifically, 100 µg of DSPE-PEG-Glucose was added to 1 ml of hybrid membrane vesicle solution. The solution was then incubated with gentle shaking at room temperature for 30 minutes. Unbound free ligand was removed by ultrafiltration or spin column methods to obtain targeted nanovesicles, which were then frozen and stored at -80°C until use.

[0049] Dynamic light scattering (DLS) was used to measure the average particle size and Zeta potential of the obtained targeted nanovesicles. Figure 1 As shown in Figure 2, the average particle size is about 250 nm, the Zeta potential is about –23 mV, and the stability is good. Figure 2 As shown in Figure 2, the structure of the targeted nanovesicles is similar to that of exosomes, which is evenly distributed and has clear edges. The protein composition of the targeted nanovesicles was determined by LC-MS / MS, and functional annotation was performed based on GO terms, such as Figure 3 As shown in the results, the obtained targeted nanovesicles retained the functional markers on both the mesenchymal stem cell membrane and the macrophage membrane, laying the molecular foundation for multi-stage targeting. Western blot analysis of the fusion of mesenchymal stem cell membrane and macrophage membrane was performed. Figure 4 As shown, it can be seen that the mesenchymal stem cell membrane and the macrophage membrane are successfully fused and retain the membrane protein characteristics specific to the source cells.

[0050] Test Example 1

[0051] This test example conducted an in vitro stage-specific cell targeting functional evaluation experiment on the stage-specific tissue repair targeted nanovesicles prepared in Example 2. Specifically:

[0052] The targeted nanovesicles prepared in Example 2 were fluorescently labeled with Cy5 and then incubated with three different cell types for 2 hours. Flow cytometry was then used to detect the uptake ratio of the targeted nanovesicles by each cell type. The different cell types were:

[0053] (1) Inflammatory bone marrow-derived macrophages stimulated by LPS are designated as M1 BMDMs.

[0054] (2) NIH3T3 fibroblasts, recorded as the negative control group.

[0055] (3) Bone marrow mesenchymal stem cells, denoted as BMSCs.

[0056] The results are as follows Figure 5 、 Figure 6 As shown, the uptake of targeted nanovesicles in M1 BMDMs and BMSCs was significantly higher than that in the negative control group, with M1 BMDMs having the highest uptake rate, demonstrating good targeting in the early stages of inflammation. This result suggests that the targeted nanovesicles have the ability to distinguish between different cell types and preferentially bind to dominant cells, providing a theoretical basis for phased delivery.

[0057] Test Example 2

[0058] This test example conducted an in vivo stage-specific cell targeting functional evaluation experiment on the stage-specific tissue repair targeted nanovesicles prepared in Example 2. Specifically:

[0059] The targeted nanovesicles prepared in Example 2 were fluorescently labeled with Cy5 and injected into the injured area via gelatin hydrogel in a mouse skull defect model. Single-cell suspensions were prepared from the skull defect area on days 3 and 14 after surgery, i.e., during the inflammatory and repair phases, respectively. Flow cytometry was used to analyze the uptake of the targeted nanovesicles by different cell types. Inflammatory macrophages were identified by Ly6C high expression, and mesenchymal stem cells were screened by CD105+ expression.

[0060] The results are as follows Figure 7 As shown in FIG, on the 3rd day after surgery, the targeted nanovesicles were mainly enriched in Ly6C^high inflammatory monocytes / macrophages; Figure 8As shown, on day 14 after surgery, the targeted nanovesicles were preferentially taken up by CD105+ mesenchymal stem cells, demonstrating their superior spatiotemporal and temporal stage-specific targeting capabilities. This demonstrates that the targeted nanovesicles can achieve a precise delivery strategy of "stage-based recognition and dynamic targeting" based on the microenvironmental characteristics and cellular composition changes at different stages of tissue repair.

Claims

1. Targeted nanovesicles for stage-specific tissue repair, characterized by: The nanocapsule is formed by the fusion of cell membranes of bone marrow-derived macrophages and bone marrow-derived mesenchymal stem cells, and its surface is modified with glucose.

2. The targeted nanovesicle for stage-specific tissue repair according to claim 1, characterized in that: The cell membrane masses of the bone marrow-derived macrophages and bone marrow-derived mesenchymal stem cells are equal.

3. The targeted nanovesicle for stage-specific tissue repair according to claim 1, characterized in that: DSPE-PEG-Glucose lipid molecules are inserted into the surface of the nanovesicles.

4. The method for preparing the targeted nanovesicles for stage-specific tissue repair according to any one of claims 1 to 3, characterized in that: Extract the cell membranes of the leading cells at different stages of tissue repair and adjust the membrane protein concentration to 2 mg ml -1 ; Take equal masses of cell membranes of dominant cells at different stages and mix them for incubation to form fusion cell membranes, and then obtain nanovesicles by extrusion.

5. The method for preparing the stage-specific tissue repair targeted nanovesicles according to claim 4, characterized in that: DSPE-PEG-Glucose was added to the fusion cell membrane solution and incubated with shaking. The mass ratio of DSPE-PEG-Glucose to membrane protein was 1:

20.

6. The method for preparing the stage-specific tissue repair targeted nanovesicles according to claim 5, characterized in that: Unbound free ligand is removed by ultrafiltration or spin column methods.

7. Use of the stage-specific tissue repair targeted nanovesicles according to any one of claims 1 to 3 in the preparation of a bone defect repair drug, characterized in that: The nanovesicles are used to prepare drugs to achieve precise delivery and regulation of key cells in different tissue repair stages, thereby promoting the dynamic remodeling of the regenerative environment and the coordinated advancement of tissue regeneration.

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