Synergistic anti-aging and osteogenesis-enhancing stem cell-derived dual-compartment vesicles and their preparation and application

Abstract

The present invention belongs to the technical field of surgical configuration materials, and discloses a stem cell-derived dual-compartment vesicle with synergistic anti-aging and osteogenesis, and its preparation and application. The stem cell-derived dual-compartment vesicle with synergistic anti-aging and osteogenesis is a concentric structure, comprising an inner core and an outer shell covalently connected to the inner core. The inner core is a functionalized nanovesicle of mesenchymal stem cells modified with an azide group, which retains cell-derived proteins and signal components, and has natural biological affinity and stem cell differentiation regulation capabilities. The outer shell is a functionalized liposome encapsulating epigenetic regulatory small molecules, and the alkyne group is introduced through a lipid containing an alkyne functional group, and a copper ion-free click chemistry reaction occurs with the azide group on the surface of the inner core to achieve a stable covalent connection, thereby obtaining a concentric structured dual-compartment vesicle. The stem cell-derived dual-compartment vesicle can be used for the preparation of drugs for cell reprogramming, inflammatory microenvironment regulation, and tissue regeneration, and is characterized by being used in the preparation of drugs for bone aging diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical preparations, and relates to a multifunctional synergistic targeted delivery system, in particular to a stem cell-derived dual-compartment vesicle with synergistic anti-aging and osteogenesis, and the preparation and application thereof. Background Art

[0002] Mesenchymal stem cell (MSC)-derived exosomes or nanovesicles have shown promising results in the field of tissue regeneration. MSC-derived nanovesicles (MNVs) can partially replace the paracrine function of stem cells, promoting osteogenic differentiation and modulating the inflammatory microenvironment, and are therefore widely used in bone regeneration research.

[0003] However, the aging microenvironment can significantly inhibit the function of stem cells, including their differentiation ability, immunoregulatory ability, and metabolic stability. Studies have shown that senescent cells are often accompanied by multiple pathological characteristics such as epigenetic dysregulation, loss of heterochromatin, and mitochondrial dysfunction. Epigenetic regulation is considered an important strategy for intervening in cellular aging. Among them, S-adenosylmethionine (SAM), as the main methyl donor, can restore cellular epigenetic homeostasis and delay aging by promoting heterochromatin marks such as H3K9me3. However, SAM is a small molecule metabolite with a short half-life in vivo and poor targeting. Direct use has the disadvantages of easy degradation and low efficiency.

[0004] Existing technologies attempt to combine stem cell exosomes with epigenetic regulators, but simple mixing or co-incubation approaches make it difficult to achieve progressive synergistic regulation and present issues such as uncontrollable release and chaotic action timing. Therefore, a novel delivery system with controllable structure, synergistic functions, and a rational release schedule is urgently needed to achieve efficient intervention in the aging microenvironment and activate the regenerative potential of stem cells. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention proposes stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenesis, as well as their preparation and application. The concentrically structured dual-compartment nanovesicles constructed by click chemistry have the ability to accurately deliver multi-component bioactive substances, thereby realizing cascade multifunctional regulation, which is suitable for various biomedical scenarios such as cell fate regulation, epigenetic regulation and tissue regeneration.

[0006] The invention relates to a stem cell-derived dual-compartment vesicle for synergistic anti-aging and osteogenesis. The stem cell-derived dual-compartment vesicle is a concentric structure, comprising an inner core and an outer shell covalently connected to the inner core.

[0007] The inner core is a functionalized nanocapsule of mesenchymal stem cells with azide groups modified on the surface, which retains proteins and signal components derived from mesenchymal stem cells and has natural biological affinity and stem cell differentiation regulation capabilities.

[0008] The shell is a functionalized liposome encapsulating epigenetic regulatory small molecules. Alkyl groups are introduced through lipid components containing DBCO groups, and copper ion-free click chemistry reactions occur with azide groups on the surface of the inner core to achieve stable covalent connection, thereby forming stable concentric structured dual-compartment vesicles.

[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, RG108, ascorbic acid (vitamin C), resveratrol, nicotinamide mononucleotide (NMN) or butyrate.

[0011] The preparation method of stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis specifically comprises the following steps:

[0012] Step 1: Preparation of functionalized core

[0013] Mesenchymal stem cells were cultured in low-glucose DMEM medium supplemented with metabolic precursors containing azide groups. Azide groups were then modified on the surface of the mesenchymal stem cells through metabolic engineering. The cells were harvested and extruded through membranes to create functionalized nanovesicle cores containing mesenchymal stem cell-derived proteins and signaling components.

[0014] Preferably, the azide groups are coupled to the surface proteins of the mesenchymal stem cell membrane via a chemical reaction to modify the surface of the mesenchymal stem cells with azide groups. Alternatively, the azide groups are inserted into the surface proteins of the mesenchymal stem cell membrane via an enzymatic reaction. Alternatively, azide-modified phospholipids are inserted into the mesenchymal stem cell membrane via membrane fusion.

[0015] Step 2: Preparation of functionalized liposome shell

[0016] Lipids containing alkyne functional groups were dissolved in an organic solvent. After rotary evaporation, the solvent was removed and hydrated with PBS buffer containing 10 mM epigenetic modulators, resulting in a total lipid concentration of 2 mg / ml. After sonication, the functionalized liposome shell containing the epigenetic modulators was formed.

[0017] Preferably, the lipid containing an alkyne functional group, the neutral lipid, and the cationic lipid are mixed and then dissolved in an organic solvent.

[0018] Step 3: Construction of Nanoconcentric Bodies

[0019] The functionalized core is mixed and incubated with an equal concentration of a functionalized liposome shell containing an epigenetic modulator. The DBCO groups in the functionalized liposome shell react with the azide groups on the core surface to achieve a stable covalent connection. Subsequently, membrane extrusion is used to prepare stem cell-derived nanovesicles with a dual-compartment concentric structure.

[0020] The application of stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenesis in cell reprogramming, inflammatory microenvironment regulation, and tissue regeneration, especially in the preparation of drugs for bone aging diseases.

[0021] The present invention has the following beneficial effects:

[0022] 1. A stable dual-compartment structure is constructed through click chemistry reaction, which has good structural integrity and drug loading stability;

[0023] 2. It can achieve spatial separation and directional control of multi-component delivery systems, and has the potential to precisely regulate cell functions. It is particularly suitable for cutting-edge application scenarios such as cell reprogramming, inflammatory microenvironment regulation, and tissue regeneration.

[0024] 3. It combines the targeting of natural biomembranes with the controllability of artificial liposomes, has good scalability and in vivo adaptability, and provides a new platform for the synergistic treatment of epigenetic modifiers (such as SAM) and biomembrane vesicles.

[0025] 4. The SAM released from the outer layer can effectively enhance heterochromatin formation, maintain cellular homeostasis, and improve cell aging phenotypes, demonstrating significant anti-aging properties. Through the synergistic mechanism of epigenetic regulation and stem cell exosomes, it achieves the reversal and functional activation of aging stem cells, resolving the technical challenges of existing anti-aging strategies, such as single action, poor targeting, and low differentiation efficiency, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The average particle size and zeta potential of the stem cell-derived dual-compartment vesicles measured in Example 3;

[0027] Figure 2 This is a cryo-transmission electron micrograph of the stem cell-derived dual-compartment vesicle prepared in Example 3;

[0028] Figure 3 Western blot of the stem cell-derived dual-compartment vesicles prepared in Example 3;

[0029] Figure 4 is the SAM encapsulation efficiency of the stem cell-derived dual-compartment vesicles prepared in Example 3;

[0030] Figure 5The results of H3K9me3 expression detection in Example 7;

[0031] Figure 6 This is the β-galactosidase staining result in Example 7;

[0032] Figure 7 The mitochondrial membrane potential detection results in Example 7;

[0033] Figure 8 The results of alkaline phosphatase staining and alizarin red staining in Example 7 are shown;

[0034] Figure 9 These are the osteoblast gene expression results in Example 7. DETAILED DESCRIPTION

[0035] The present invention will be further explained below with reference to the accompanying drawings.

[0036] Example 1

[0037] This embodiment provides a method for preparing stem cell-derived dual-compartment vesicles that synergistically resist aging and promote osteogenesis. The dual-compartment vesicles are concentric structures formed by stably connecting a stem cell-derived nanovesicle core and a SAM-loaded liposome shell via a click chemistry reaction. The method specifically comprises the following steps:

[0038] Step 1: Preparation of functionalized core

[0039] Human umbilical cord mesenchymal stem cells (OriCell, HUXUC-01001) were seeded in 150 mm culture dishes and cultured in low-glucose DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 50 µM Ac4ManNAz (azidoacetylmannosamine). Azide groups were modified on the surface of human umbilical cord mesenchymal stem cells via the glucose metabolism pathway. After reaching 80% confluency, the cells were digested with 0.25% trypsin-EDTA and centrifuged at 200g for 5 minutes to collect the cell pellets.

[0040] The cell clumps were mechanically extruded through filter membranes with pore sizes of 5 µm, 1 µm, 400 nm, and 200 nm in sequence to obtain nanovesicles with surface rich azide groups. The clumps were then centrifuged at 25,000 g for 60 minutes. The mesenchymal stem cell-derived nanovesicles (MNVs) were resuspended in PBS buffer and stored for later use.

[0041] Step 2: Preparation of functionalized liposome shell

[0042] The lipid DOPE-PEG2000-DBCO containing an alkyne functional group was dissolved in chloroform, and the chloroform was removed under reduced pressure by rotary evaporation to form a lipid film.

[0043] PBS buffer containing 10 mM SAM (New England Biolabs) was added for hydration, resulting in a total lipid concentration of 2 mg / ml. The liposomes were then sonicated at 4°C for 20 minutes to form SAM-entrapped liposomes (SFL).

[0044] Step 3: Construction of Nanoconcentric Bodies

[0045] Equal amounts of SFL prepared in step 1 and MNV were mixed and allowed to react at room temperature for 30 minutes. A covalent linkage was formed via SPAAC. The mixture was sequentially extruded through filters with pore sizes of 1 µm, 400 nm, and 200 nm and further homogenized to obtain a uniformly sized nanoconcentric structure. The mixture was centrifuged at 25,000 g for 30 minutes and recovered.

[0046] Example 2

[0047] This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenesis effects. Based on Example 1, an NHS-azide reagent was used to target amino groups on the surface of bone marrow mesenchymal stem cells to prepare a functionalized core. Nicotinamide mononucleotide was then encapsulated using the prepared lipid membrane to form a functionalized liposome shell.

[0048] Example 3

[0049] This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenesis. Based on Example 1, DOPE, DOPE-PEG2000-DBCO, DOTAP, and the tracer dye DiI were dissolved in chloroform at a molar ratio of 0.5:0.5:1:0.1 to prepare a lipid membrane.

[0050] Dynamic light scattering (DLS) was used to measure the hydrated particle size of the prepared dual-compartment concentric stem cell-derived nanovesicles. The results showed that the average particle size was about 120 nm and the Zeta potential was positive. Figure 1 As shown. Using cryo-transmission electron microscopy (Cryo-TEM) observation, it shows a typical double-layer concentric structure, as shown Figure 2 Protein blots were performed as shown. Figure 3 As shown in Figure 2, 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. Figure 4 shown.

[0051] Example 4

[0052] This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenesis effects. Based on Example 3, a maleimide-azide reagent was used to target sulfhydryl groups on the surface of adipose-derived mesenchymal stem cells to create a functionalized core. The prepared lipid membrane was then used to encapsulate ascorbic acid to create a functionalized liposome shell.

[0053] Example 5

[0054] This example provides a method for preparing stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis. Based on Example 3, a phospholipid containing an azide group is mixed and incubated with dental pulp mesenchymal stem cells to prepare a functionalized core.

[0055] Example 6

[0056] This example provides a method for preparing stem cell-derived dual-compartment vesicles with synergistic anti-aging and osteogenesis properties. Based on Example 5, the prepared lipid membrane is used to encapsulate 5-azacytidine to prepare a functionalized liposome shell.

[0057] Example 7

[0058] The stem cell-derived dual-compartment vesicles prepared in Example 3 were used in a bone aging disease model to conduct cascade regulation at the cellular level. The release of the SAM encapsulated in the outer shell promoted heterochromatin formation by regulating epigenetic mechanisms, exerting an anti-aging effect. The mesenchymal stem cell-derived functionalized nanovesicles within the inner core enhanced bone differentiation potential, thereby synergistically promoting the differentiation of aging stem cells into the osteogenic lineage.

[0059] In order to verify the cascade regulatory function of the stem cell-derived dual-compartment vesicles at the cellular level, the following steps were performed:

[0060] Step 1: Mouse bone marrow mesenchymal stem cells (BMSCs) of passage 6 to 8 were used as model cells and cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin.

[0061] Step 2: Divide the cells into the following 4 groups:

[0062] (1) Control group: No treatment was performed on the cultured mouse bone marrow mesenchymal stem cells.

[0063] (2) Aging group (Doxo): Cultured mouse bone marrow mesenchymal stem cells were aged with 200 nM doxorubicin (Doxo) for 24 hours.

[0064] (3) Core group (Doxo+MNV): The aged mouse bone marrow mesenchymal stem cells were treated with the mesenchymal stem cell-derived nanovesicles prepared in step 1 of Example 3, with a total protein content of 100 μg / ml.

[0065] (4) Stem cell-derived dual-compartment vesicle group (Doxo+SMNV): The aged mouse bone marrow mesenchymal stem cells were treated with the stem cell-derived dual-compartment vesicles prepared in step 3 of Example 3, wherein the SAM concentration was 100 μM and the total protein content was 100 μg / ml.

[0066] After 48 hours of treatment, the cells in each group were tested.

[0067] Step 3: Verification of epigenetic anti-aging function

[0068] s3.1. Detect the expression levels of histone H3K9me3, H3K9me2 and HP1α by Western blot. Figure 5 As shown in the figure, 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 SAM released by stem cell-derived bicompartmental vesicles can effectively enhance heterochromatin formation.

[0069] s3.2. Perform β-galactosidase staining on cells in each group. Figure 6 As shown in the figure, the proportion of positive cells in the Doxo+SMNV group decreased significantly, indicating that the degree of cell senescence decreased.

[0070] s3.3, JC-1 dye was used to detect the mitochondrial membrane potential of each group of cells, such as Figure 7 As shown, the red / green fluorescence ratio of the Doxo+SMNV group was significantly increased, indicating that the mitochondrial state of the cells was restored, indicating that the stem cell-derived dual-compartment vesicles have a strong anti-aging effect.

[0071] Step 4: Osteogenic differentiation function assessment

[0072] s4.1. Transfer cells from each group into osteogenic induction medium and culture them for 14 days. Alkaline phosphatase (ALP) staining and Alizarin red (ARS) staining were performed. Figure 8 As shown in the figure, the staining area of ​​the Doxo+SMNV group was significantly increased, indicating enhanced mineralization ability.

[0073] s4.2. Expression of Osteogenesis-Related Genes

[0074] qRT-PCR was used to detect the expression levels of RUNX2, Alpl, Opn and Ocn genes in each group of cells. Figure 9 As shown in the figure, the expression of mitochondria in the Doxo+SMNV group was significantly upregulated, higher than that in the Doxo+MNV group.

[0075] The above experimental results show that the stem cell-derived dual-compartment vesicles can induce heterochromatin formation and inhibit aging-related phenotypes through the outer layer SAM liposomes, while enhancing osteogenic differentiation potential through the inner layer stem cell-derived MNV, thereby achieving "anti-aging-promoting osteoblastic" cascade regulation at the cellular level, providing a new strategy for the treatment of bone aging diseases.

Claims

1. Synergistic anti-aging and osteogenesis stem cell-derived dual-compartment vesicles, characterized by: The stem cell-derived dual-compartment vesicle is a concentric structure, comprising an inner core and an outer shell covalently connected to the inner core; The inner core is a functionalized nanovesicle of mesenchymal stem cells with azide groups modified on the surface, which retains proteins and signal components derived from mesenchymal stem cells; the outer shell is a lipid component containing alkyne functional groups, which encapsulates an epigenetic regulatory small molecule; the epigenetic regulatory small molecule is S-adenosylmethionine.

2. The stem cell-derived dual-compartment vesicle for synergistic anti-aging and osteogenesis according to claim 1, characterized in that: 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 vesicle for synergistic anti-aging and osteogenesis according to claim 1, characterized in that: The shell is a combination of lipid containing alkyne functional groups, neutral lipid and cationic lipid.

4. The method for preparing the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenic effects according to any of claims 1 to 3, wherein: The surface of mesenchymal stem cells is modified with azide groups by metabolic engineering methods, and then the mesenchymal stem cells are extruded through a membrane to prepare a functionalized nanovesicle core; a lipid membrane containing alkyne functional groups is prepared, hydrated with epigenetic regulatory small molecules in PBS buffer, and ultrasonically treated to form a functional liposome shell encapsulating the epigenetic regulatory small molecules; the functionalized nanovesicle core is mixed and incubated with an equal concentration of a functional liposome shell encapsulating the epigenetic regulatory small molecules, and extruded through a membrane to obtain stem cell-derived nanovesicles with a dual-compartment concentric structure.

5. The method for preparing the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to claim 4, characterized in that: The azide group is coupled to the surface protein of the mesenchymal stem cell membrane through a chemical reaction, or the azide group is inserted into the surface protein of the mesenchymal stem cell membrane through an enzymatic reaction, or the phospholipid modified with azide is inserted into the mesenchymal stem cell membrane through membrane fusion.

6. The method for preparing the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to claim 4, characterized in that: Mesenchymal stem cells were seeded into culture dishes and cultured in low-glucose DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 50 μM Ac4ManNAz. After the cell confluence reached 80%, they were digested with 0.25% trypsin-EDTA and centrifuged at 200 g for 5 minutes to collect the cell clumps. The cell aggregates were mechanically extruded through filter membranes with pore sizes of 5 µm, 1 µm, 400 nm, and 200 nm in sequence to obtain nanovesicles with surface rich azide groups, and then centrifuged at 25,000 g for 60 minutes to obtain the functionalized nanovesicle cores.

7. The method for preparing the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to claim 4, characterized in that: The lipid component containing alkyne functional groups was dissolved in an organic solvent. After rotary evaporation to remove the organic solvent, PBS buffer containing 10 mM epigenetic regulatory small molecules was added for hydration, and the total lipid concentration was 2 mg / ml. After ultrasonic treatment, a functional liposome shell encapsulating the epigenetic regulatory small molecules was formed.

8. The use of the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to any one of claims 1 to 3, characterized in that: The stem cell-derived dual-compartment vesicles are used in the preparation of cell reprogramming, inflammatory microenvironment regulation, and tissue regeneration drugs.

9. The use of the stem cell-derived dual-compartment vesicles for synergistic anti-aging and osteogenesis according to claim 8, characterized in that: Application of the stem cell-derived dual-compartment vesicles in the preparation of drugs for bone aging diseases.

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