Stem cell-derived double-compartment vesicles with synergistic anti-aging and osteogenesis functions and preparation and application of stem cell-derived double-compartment vesicles

Abstract

The invention belongs to the technical field of surgical configuration materials, and discloses a stem cell-derived double-compartment vesicle for synergic anti-aging and osteogenesis as well as preparation and application of the stem cell-derived double-compartment vesicle. The synergistic anti-aging and osteogenesis stem cell source double-compartment vesicle is of a concentric body structure and comprises an inner core and a shell covalently connected with the inner core. The core is a functionalized nano-vesicle of mesenchymal stem cells modified by azide groups, retains cell-derived proteins and signal components, and has natural biocompatibility and stem cell differentiation regulation ability. The shell is functionalized liposome entrapped with apparent regulation and control micromolecules, alkyne is introduced through lipid containing alkyne functional groups, the alkyne and azide groups on the surface of the inner core are subjected to a copper ion non-catalytic click chemical reaction, stable covalent linkage is achieved, and therefore the double-compartment vesicle with the concentric body structure is obtained. The stem cell-derived double-compartment vesicle can be used for preparing medicines for cell reprogramming, inflammatory microenvironment regulation and control and tissue regeneration, and is characterized in that the stem cell-derived double-compartment vesicle is applied to preparing medicines for bone aging diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical configurations, relates to a multi-functional collaborative targeted delivery system, and particularly relates to a stem cell-derived dual-compartment vesicle for synergistic anti-aging and osteogenesis, and its preparation and application. Background Art

[0002] Mesenchymal stem cell (MSC)-derived exosomes or nanovesicles have shown good prospects in the field of tissue regeneration. MSC-derived nanovesicles (MNVs) can partially replace the paracrine function of stem cells and have functions such as promoting osteogenic differentiation and regulating the inflammatory microenvironment, so they are widely used in bone regeneration research.

[0003] However, the aging microenvironment will significantly inhibit the function of stem cells, including their differentiation ability, immune regulation ability, and metabolic stability. Research shows that senescent cells are often accompanied by multiple pathological characteristics such as epigenetic dysregulation, heterochromatin loss, and mitochondrial dysfunction. Epigenetic regulation means are considered an important strategy for intervening in cell aging. Among them, S-adenosylmethionine (SAM), as the main methyl donor, can restore cellular epigenetic homeostasis and delay aging by promoting heterochromatin markers such as H3K9me3. However, SAM is a small molecule metabolite with a short half-life and poor targeting in vivo, and there are defects such as easy degradation and low efficiency when used directly.

[0004] Existing technologies have tried to use stem cell exosomes and epigenetic regulators in combination, but simple mixing or co-incubation methods are difficult to achieve progressive synergistic regulation, and there are problems such as uncontrollable release and chaotic action timing. Therefore, there is an urgent need for a new type of delivery system with controllable structure, functional synergy, and reasonable release timing to achieve efficient intervention in the aging microenvironment and activate the regenerative potential of stem cells. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a stem cell-derived dual-compartment vesicle for synergistic anti-aging and osteogenesis, and its preparation and application. The dual-compartment nanovesicle with a concentric structure constructed by click chemistry has the ability to precisely deliver multi-component bioactive substances, thereby achieving cascaded multi-functional regulation and being applicable to various biomedical scenarios such as cell fate regulation, epigenetic regulation, and tissue regeneration.

[0006] The stem cell-derived dual-compartment vesicle for synergistic anti-aging and osteogenesis, wherein the stem cell-derived dual-compartment vesicle has a concentric structure and includes a core and a shell covalently connected to the core.

[0007] The core is a functionalized nanovesicle of mesenchymal stem cells with azide groups modified on the surface, retaining proteins and signal components derived from mesenchymal stem cells, and having natural biocompatibility and the ability to regulate stem cell differentiation.

[0008] The outer shell is a functionalized liposome encapsulating epigenetic regulatory small molecules. An alkyne group is introduced through a lipid component containing a DBCO group, and a copper-free catalytic click chemical reaction occurs with the azide group on the surface of the inner core to achieve stable covalent connection, thereby forming a stable concentric structure double-compartment vesicle.

[0009] Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or dental pulp mesenchymal stem cells.

[0010] Preferably, the epigenetic regulatory small molecules have the functions of regulating the methylation / acetylation status of DNA or histones, improving heterochromatin formation and anti-aging, including S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), 5-azacytidine (5-Azacytidine), RG108, ascorbic acid (vitamin C), resveratrol (Resveratrol), nicotinamide mononucleotide (NMN) or butyrate (Butyrate).

[0011] A method for preparing a stem cell-derived double-compartment vesicle with synergistic anti-aging and osteogenic effects specifically includes the following steps: Step 1: Preparation of the functionalized inner core Mesenchymal stem cells are cultured in low-glucose DMEM medium supplemented with a metabolic precursor with an azide group, and the azide group is modified on the surface of mesenchymal stem cells by metabolic engineering. The cells are collected and the functionalized nanovesicle inner core retaining the proteins and signal components derived from mesenchymal stem cells is obtained by membrane extrusion.

[0012] Preferably, the azide group is conjugated to the surface protein of mesenchymal stem cells by a chemical reaction to modify the azide group on the surface of mesenchymal stem cells. Or the azide group is inserted into the surface protein of mesenchymal stem cells by an enzymatic reaction. Or by membrane fusion, phospholipids with azide modification are inserted into the membrane of mesenchymal stem cells.

[0013] Step 2: Preparation of the functionalized liposome outer shell The lipid containing an alkyne functional group is dissolved in an organic solvent. After the organic solvent is removed by rotary evaporation, PBS buffer containing 10 mM epigenetic regulatory small molecules is added for hydration, and the total lipid concentration is 2 mg / ml. After sonication, a functionalized liposome outer shell encapsulating epigenetic regulatory small molecules is formed.

[0014] Preferably, the lipid containing an alkyne functional group, neutral lipid, and cationic lipid are mixed and then dissolved in an organic solvent.

[0015] Step 3: Construction of the nano-concentric body The functionalized core is mixed and incubated with a functional liposome shell of equal concentration encapsulating small molecules for epigenetic regulation. The DBCO groups in the functional liposome shell undergo click reaction with the azide groups on the surface of the core to achieve stable covalent connection. Subsequently, a stem cell-derived nanovesicle with a two-compartment concentric structure is prepared by membrane extrusion.

[0016] The stem cell-derived two-compartment vesicles with synergistic anti-aging and osteogenic effects are applied in cell reprogramming, regulation of the inflammatory microenvironment, and tissue regeneration, especially in the preparation of drugs for bone aging diseases.

[0017] The present invention has the following beneficial effects: 1. A stable two-compartment structure is constructed through click chemical reaction, having good structural integrity and drug-loading stability; 2. Spatial separation and directional control of a multi-component delivery system can be achieved, having the potential to precisely regulate cell functions, and being particularly suitable for cutting-edge application scenarios such as cell reprogramming, regulation of the inflammatory microenvironment, and tissue regeneration.

[0018] 3. It combines the targeting property of natural biological membranes and the controllability of artificial liposomes, having good expandability and in vivo adaptability, and providing a new platform for the synergistic treatment of epigenetic modifiers (such as SAM) and biological membrane vesicles.

[0019] 4. SAM released from the outer layer can effectively enhance heterochromatin formation, maintain cell homeostasis, and improve the cell senescence phenotype, having a significant anti-aging function. Through the synergistic mechanism of epigenetic regulation and stem cell exosomes, the reversal and functional activation of aging stem cells are achieved, solving the technical problems of single action, poor targeting, and low differentiation efficiency in existing anti-aging strategies, and having broad application prospects. Description of the Drawings

[0020] Figure 1 The average particle size and Zeta potential of the stem cell-derived two-compartment vesicles measured in Example 3; Figure 2 The cryo-transmission electron microscopy image of the stem cell-derived two-compartment vesicles prepared in Example 3; Figure 3 The Western blot of the stem cell-derived two-compartment vesicles prepared in Example 3; Figure 4 The encapsulation efficiency of the stem cell-derived two-compartment vesicles prepared in Example 3 for SAM; Figure 5 The detection result of H3K9me3 expression in Example 7; Figure 6 The result of β-galactosidase staining in Example 7; Figure 7 The detection result of mitochondrial membrane potential in Example 7; Figure 8 Results of alkaline phosphatase staining and alizarin red staining in Example 7; Figure 9 Results of osteoblast gene expression in Example 7. Detailed implementation manners

[0021] The present invention will be further explained below with reference to the accompanying drawings.

[0022] Example 1 This example provides a method for preparing stem cell-derived double-compartment vesicles with synergistic anti-aging and osteogenic effects. The double-compartment vesicles are concentric structures, which are stably connected by click chemical reaction between a stem cell-derived nanovesicle core and a liposome shell loaded with SAM. The specific steps are as follows: Step 1: Preparation of functionalized core Human umbilical cord mesenchymal stem cells (OriCell, HUXUC-01001) were seeded in a 150 mm culture dish and cultured in low-glucose DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 50 µM Ac4ManNAz (azidoacetyl mannosamine) to modify azide groups on the surface of human umbilical cord mesenchymal stem cells through the sugar metabolism pathway. After the cell confluence reached 80%, the cells were digested with 0.25% trypsin-EDTA and centrifuged at 200g for 5 minutes to collect cell pellets.

[0023] The cell pellets were successively extruded mechanically through filters with pore sizes of 5 µm, 1 µm, 400 nm, and 200 nm to obtain nanovesicles rich in azide groups on the surface, and then centrifuged at 25000g for 60 minutes to obtain mesenchymal stem cell-derived nanovesicles (MNV), which were resuspended in PBS buffer for storage and standby.

[0024] Step 2: Preparation of functionalized liposome shell Lipid DOPE-PEG2000-DBCO containing alkyne functional groups was dissolved in chloroform, and chloroform was removed under reduced pressure by rotary evaporation to form a lipid film.

[0025] PBS buffer containing 10 mM SAM (New England Biolabs) was added for hydration, and the total lipid concentration was 2 mg / ml. Subsequently, it was placed in a 4°C ultrasonic water bath for 20 minutes to form liposomes loaded with SAM (SFL).

[0026] Step 3: Construction of nano-concentric bodies Mix the SFL prepared in step 1 with MNV in equal mass, let it stand and react at room temperature for 30 minutes to form a covalent connection through SPAAC reaction. Extrude successively through filter membranes with pore sizes of 1 µm, 400 nm and 200 nm, and further perform homogenization treatment to obtain a nano-concentric structure with uniform particle size. Recover after centrifugation at 25000g for 30 minutes.

[0027] Example 2 This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenic effects. On the basis of Example 1, use NHS-azide reagent to target the amino groups on the surface of bone marrow mesenchymal stem cells to prepare functionalized cores. And use the prepared lipid membrane to encapsulate nicotinamide mononucleotide to prepare a functionalized liposome shell.

[0028] Example 3 This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenic effects. On the basis of Example 1, dissolve DOPE, DOPE-PEG2000-DBCO, DOTAP and the tracer dye DiI in chloroform at a molar ratio of 0.5:0.5:1:0.1 to prepare a lipid membrane.

[0029] The hydrated particle size of the stem cell-derived nanovesicles with a dual-compartment concentric structure prepared was measured using a dynamic light scattering instrument (DLS). The results showed that the average particle size was about 120 nm and the Zeta potential was positive, as Figure 1 shown. Observation using a cryogenic transmission electron microscope (Cryo-TEM) showed a typical bilayer concentric structure, as Figure 2 shown. Western blot was as Figure 3 shown, and the specific membrane proteins of mesenchymal stem cells were retained. The encapsulation efficiency of SAM in the stem cell-derived dual-compartment vesicles was tested using an ELISA kit, as Figure 4 shown.

[0030] Example 4 This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenic effects. On the basis of Example 3, use maleimide-azide reagent to target the sulfhydryl groups on the surface of adipose mesenchymal stem cells to prepare functionalized cores. And use the prepared lipid membrane to encapsulate ascorbic acid to prepare a functionalized liposome shell.

[0031] Example 5 This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenic effects. On the basis of Example 3, mix and incubate phospholipids containing azide groups with dental pulp mesenchymal stem cells to prepare functionalized cores.

[0032] Example 6 This example provides a method for preparing stem cell-derived dual-compartment vesicles with collaborative anti-aging and osteogenic effects. On the basis of Example 5, the prepared lipid membrane is used to encapsulate 5-azacytidine to prepare a functionalized liposome shell.

[0033] Example 7 The stem cell-derived dual-compartment vesicles prepared in Example 3 are used in a bone aging disease model for cascade regulation at the cellular level. After the SAM encapsulated in the shell is released, it can promote heterochromatin formation by regulating the epigenetic mechanism and exert an anti-aging effect. The mesenchymal stem cell-derived functionalized nanovesicles in the inner core can enhance the osteogenic differentiation potential, thus synergistically promoting the differentiation of senescent stem cells into the osteogenic lineage.

[0034] To verify the cascade regulation function of the stem cell-derived dual-compartment vesicles at the cellular level, the following steps are used for testing: Step 1: Take mouse bone marrow mesenchymal stem cells (BMSCs) at passages 6-8 as model cells and maintain them in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin.

[0035] Step 2: Divide the cells into the following 4 groups: (1) Control group (Control): No treatment is performed on the cultured mouse bone marrow mesenchymal stem cells.

[0036] (2) Aging group (Doxo): The cultured mouse bone marrow mesenchymal stem cells are aged with 200 nM doxorubicin (Doxo) for 24 hours.

[0037] (3) Inner core group (Doxo+MNV): The mesenchymal stem cell-derived nanovesicles prepared in Step 1 of Example 3 are used for the mouse bone marrow mesenchymal stem cells after aging treatment, and the total protein amount is 100 µg / ml.

[0038] (4) Stem cell-derived dual-compartment vesicle group (Doxo+SMNV): The stem cell-derived dual-compartment vesicles prepared in Step 3 of Example 3 are used for the mouse bone marrow mesenchymal stem cells after aging treatment, where the concentration of SAM is 100 µM and the total protein amount is 100 µg / ml.

[0039] After acting for 48 hours, the cells in each group are tested.

[0040] Step 3: Verification of epigenetic anti-aging function S3.1: Detect the expression levels of histone H3K9me3, H3K9me2 and HP1α by Western blot, as Figure 5As shown, the level of H3K9me3 in the Doxo+SMNV group was significantly higher than that in the Control group and the Doxo+MNV group, suggesting that the SAM released by stem cell-derived double-compartment vesicles could effectively enhance heterochromatin formation.

[0041] s3.2. Perform β-galactosidase staining on the cells of each group. As Figure 6 shown, the proportion of positive cells in the Doxo+SMNV group decreased significantly, suggesting a decrease in the degree of cell senescence.

[0042] s3.3. Use JC-1 dye to detect the mitochondrial membrane potential of the cells in each group. As Figure 7 shown, the red / green fluorescence ratio in the Doxo+SMNV group increased significantly, indicating the recovery of the mitochondrial state of the cells, suggesting that stem cell-derived double-compartment vesicles have a strong anti-aging effect.

[0043] Step 4. Osteogenic differentiation function assessment s4.1. Transfer the cells of each group into osteogenic induction medium for culture. After 14 days, perform alkaline phosphatase (ALP) staining and alizarin red (ARS) staining. As Figure 8 shown, the staining area in the Doxo+SMNV group increased significantly, suggesting enhanced mineralization ability.

[0044] s4.2. Expression of osteogenesis-related genes Use qRT-PCR to detect the expression levels of RUNX2, Alpl, Opn, and Ocn genes in the cells of each group. As Figure 9 shown, the Doxo+SMNV group was significantly upregulated, higher than the Doxo+MNV group.

[0045] The above experimental results indicate that the stem cell-derived double-compartment vesicles can induce heterochromatin formation and inhibit senescence-related phenotypes through the outer SAM liposome, and at the same time enhance the osteogenic differentiation potential through the inner stem cell-derived MNV, realizing the "anti-aging - osteogenesis-promoting" cascade regulation at the cellular level, providing a new strategy for the treatment of bone aging diseases.

Claims

1. A stem cell-derived dual-compartment vesicle for synergistic anti-aging and osteogenesis, characterized in that: The stem cell-derived double-compartment vesicle has a concentric structure, including a core and a shell covalently linked to the core; The core is a functionalized nanovesicle of mesenchymal stem cells with azide groups modified on the surface, retaining proteins and signal components derived from mesenchymal stem cells; the shell is a lipid component containing alkyne functional groups and encapsulates epigenetic regulatory small molecules.

2. The stem cell-derived dual-compartment vesicles for collaborative anti-aging and osteogenesis according to claim 1, wherein: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or dental pulp mesenchymal stem cells.

3. The stem cell-derived dual-compartment vesicles for collaborative anti-aging and osteogenesis according to claim 1, characterized in that: The epigenetic regulatory small molecules have the functions of regulating the methylation / acetylation status of DNA or histones, improving heterochromatin formation and anti-aging, and include S-adenosylmethionine, S-adenosylhomocysteine, 5-azacytidine, RG108, ascorbic acid, resveratrol, nicotinamide mononucleotide or butyrate.

4. The stem cell-derived dual-compartment vesicles for collaborative anti-aging and osteogenesis according to claim 1, wherein: The shell is a combination of lipids containing alkyne functional groups, neutral lipids and cationic lipids.

5. The preparation method of the stem cell-derived dual-compartment vesicles for collaborative anti-aging and osteogenesis according to any one of claims 1 to 4, characterized in that: By means of metabolic engineering, azide groups are modified on the surface of mesenchymal stem cells, and then the mesenchymal stem cells are extruded through a membrane to prepare the core of the functionalized nanovesicle; a lipid membrane containing alkyne functional groups is prepared, hydrated with epigenetic regulatory small molecules in PBS buffer, and after ultrasonic treatment, a functional liposome shell encapsulating epigenetic regulatory small molecules is formed; the core of the functionalized nanovesicle is mixed and incubated with the functional liposome shell of the same concentration encapsulating epigenetic regulatory small molecules, and the stem cell-derived nanovesicle with a double-compartment concentric structure is obtained by membrane extrusion.

6. The preparation method of the stem cell-derived dual-compartment vesicles for collaborative anti-aging and osteogenesis according to claim 5, characterized in that: The azide groups are coupled to the surface proteins of mesenchymal stem cells through chemical reactions, or inserted into the surface proteins of mesenchymal stem cells through enzymatic reactions, or phospholipids with azide modifications are inserted into the mesenchymal stem cell membrane through membrane fusion.

7. The preparation method of the stem cell-derived dual-compartment vesicles for collaborative anti-aging and osteogenesis according to claim 5, characterized in that: The mesenchymal stem cells are inoculated in a culture dish and cultured in a low-glucose DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and 50 µM Ac4ManNAz; after the cell confluence reaches 80%, they are digested with 0.25% trypsin-EDTA, centrifuged at 200g for 5 minutes, and the cell pellet is collected; The cell pellet is mechanically extruded successively through filters with pore sizes of 5 µm, 1 µm, 400 nm and 200 nm to obtain nanovesicles rich in azide groups on the surface, and then centrifuged at 25000g for 60 minutes to obtain the core of the functionalized nanovesicle.

8. The preparation method of the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to claim 5, wherein: The lipid component containing alkyne functional groups is dissolved in an organic solvent, the organic solvent is removed by rotary evaporation, and then PBS buffer containing 10 mM epigenetic regulatory small molecules is added for hydration, and the total lipid concentration is 2 mg / ml; after ultrasonic treatment, a functional liposome shell encapsulating epigenetic regulatory small molecules is formed.

9. Use of the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to any one of claims 1 to 4, characterized in that: The application of the stem cell-derived double-compartment vesicle in the preparation of cell reprogramming, inflammation microenvironment regulation and tissue regeneration drugs.

10. Use of the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to claim 9, characterized in that: The application of the stem cell-derived double-compartment vesicle in the preparation of drugs for bone senile diseases.

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