A silicon dioxide-engineered extracellular vesicle composite nano preparation for tendon-bone interface repair and a preparation method and application thereof

Abstract

The application discloses a kind of silicon dioxide-engineered extracellular vesicle composite nano-preparation for tendon-bone interface repair and its preparation method and application, the preparation includes carrier matrix and functional load component, functional load component is loaded in carrier matrix by the synergistic effect of physical adsorption and channel embedding;The carrier matrix is the bioactive silicon dioxide nanoparticle of core-shell structure, the mesoporous silicon dioxide with central radial channel is in the core, the shell is calcium phosphorus layer, and the nanoparticle surface is modified by bone targeting molecule;Functional load component is the engineered extracellular vesicle of bone marrow mesenchymal stem cell source preprocessed by the bioactive molecule with anti-inflammatory osteogenesis effect.The application is by the dual delivery strategy of " carrier + excretion component", on the one hand, active ion released by silicon dioxide carrier promotes osteogenesis, on the other hand, " high efficiency" exosome is stimulated, and local inflammatory microenvironment is adjusted, and the functional regeneration of tendon-bone interface is accelerated by synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of technology, and in particular to a silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair, its preparation method, and its application. Background Technology

[0002] The tendon-bone interface is a complex transitional structure connecting soft tissue (tendons / ligaments) and hard tissue (bone). In clinical practice, surgical reconstruction after injuries such as rotator cuff tears and anterior cruciate ligament ruptures often faces the problem of poor interface healing quality. The healing often manifests as fibrous scar tissue rather than a natural gradient hierarchical structure, resulting in weak biomechanical properties and a high postoperative recurrence rate.

[0003] In recent years, mesoporous silica (MSNs) and extracellular vesicles (EVs) have shown potential in tissue repair. However, existing technologies still have the following shortcomings: First, the pore size of ordinary mesoporous materials is small, resulting in low loading rates for larger extracellular vesicles; second, the release of exocrine components in the local microenvironment lacks spatiotemporal control and is difficult to precisely target the bone end of the insertion point; finally, the bioactivity of ordinary extracellular vesicles is limited, making it difficult to simultaneously meet the complex anti-inflammatory and osteogenic requirements of the tendon-bone interface. Therefore, there is an urgent need to develop a composite nanoparticle formulation that can precisely target, efficiently load, and possess multiple bioregulatory functions. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair, comprising a carrier matrix and a functional loading component. The functional loading component is loaded in the carrier matrix through a synergistic effect of physical adsorption and pore embedding. The carrier matrix consists of core-shell structured bioactive silica nanoparticles, with a core of mesoporous silica having centrally radial pores and a shell of calcium-phosphorus layer. The surface of the silica nanoparticles is modified with bone-targeting molecules. The functional loading component consists of engineered extracellular vesicles derived from bone marrow mesenchymal stem cells pretreated with bioactive molecules having anti-inflammatory and osteogenic effects.

[0006] Furthermore, the silica nanoparticles have a particle size of 180-320 nm and a specific surface area of ​​45-65 m². 2 / g, with a pore volume of 0.25-0.55cc / g.

[0007] Furthermore, the molar percentages of each component in the silica nanoparticles are: SiO2 92-98 mol%, CaO 1.5-4 mol%, and P2O5 0.5-2 mol.

[0008] Furthermore, the bone-targeting molecule is a D-Asp8 sequence polypeptide, delivered via MAL-PEG. 2000 -NHS coupling agent is grafted onto the surface of aminated silica nanoparticles; the amination modification uses 3-aminopropyltriethoxysilane as the coupling agent.

[0009] Furthermore, the extracellular vesicles have a diameter of 40-160 nm, express TSG101 and CD63 characteristic markers on their surface, do not express calnexin protein, and have a density of 1.13-1.19 g / mL.

[0010] Furthermore, the anti-inflammatory osteogenic bioactive molecule is selected from at least one of natural small molecule compounds, cytokines, or chemical inducers.

[0011] The second aspect is to provide a method for preparing the above-mentioned composite nano-formulation, including the following steps: Step 1, preparation of the carrier matrix: using hexadecyltrimethylammonium bromide as a template agent and tetraethyl orthosilicate as a silicon source, a mesoporous silica core with centrally radial channels is prepared by sol-gel method; then, calcium phosphate salts are deposited in situ on the surface of the core to form a core-shell structure, and after amination modification, bone-targeting molecule modification chains are grafted to obtain the targeting carrier matrix; Step 2, preparation of engineered extracellular vesicles: Bone marrow mesenchymal stem cells were cultured to 70-85% confluence, pretreated with induction medium containing anti-inflammatory osteogenic bioactive molecules, and then cultured in exosome-free serum medium for 12-24 hours; the cell culture supernatant was collected and engineered extracellular vesicles were obtained by separating and purifying them using a combination of gradient centrifugation and ultracentrifugation. Step 3, Composite Loading: The engineered extracellular vesicles and the targeting carrier matrix are mixed in a buffer system at a mass ratio of 1:(0.5-5) and incubated at 4°C in the dark for 6-24 hours to obtain the composite nano-formulation.

[0012] Further, in step one, the in-situ deposition process of calcium phosphate is carried out in an alkaline system with a pH of 8.5-9.5; in step two, the gradient centrifugation includes centrifugation at 300-500g for 10 min to remove live cells, centrifugation at 2000-3000g for 15 min to remove cell debris, and centrifugation at 10000-12000g for 30 min to remove organelles; the ultracentrifugation is centrifugation at 100,000g for 2 h.

[0013] Furthermore, in step two, the concentration of the anti-inflammatory osteogenic bioactive molecule is 0.5-5 μg / mL, and the pretreatment time is 20-28 h.

[0014] The third aspect is to provide the application of the above-mentioned composite nano-formulations in the preparation of rotator cuff injury repair materials.

[0015] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: The composite nano-formulation of the present invention uses a mesoporous silica carrier with centrally radial channels. Its special dendritic morphology provides a large exposed pore size, which significantly improves the loading efficiency and stability of extracellular vesicles. By introducing D-Asp8 bone-targeting peptide, the formulation can be precisely anchored at the bone end of the tendon-bone interface, achieving a high concentration of the drug at the lesion site.

[0016] This invention employs a dual delivery strategy of "carrier + exocrine component". On the one hand, it utilizes the active ions released by the silica carrier to promote osteoogenesis, and on the other hand, it utilizes the pre-stimulated "high-efficiency" exovesicles to regulate the local inflammatory microenvironment. Through synergistic effects, it accelerates the functional regeneration of the tendon-bone interface. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0018] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0019] Example 1 This embodiment 1 provides a silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair, comprising a carrier matrix and a functional loading component. The functional loading component is loaded in the carrier matrix through a synergistic effect of physical adsorption and pore embedding. The carrier matrix consists of core-shell structured bioactive silica nanoparticles, with a core of mesoporous silica having centrally radial pores and a shell of calcium-phosphorus layer. The surface of the silica nanoparticles is modified with bone-targeting molecules. The functional loading component consists of engineered extracellular vesicles derived from bone marrow mesenchymal stem cells pretreated with bioactive molecules that have anti-inflammatory and osteogenic effects.

[0020] The preparation method of the above-mentioned silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair is also provided, specifically including the following steps: Step 1: Preparation of the carrier matrix 0.3 g of cetyltrimethylammonium bromide (CTAB) was dissolved in 60 mL of deionized water, and 0.1 mL of triethanolamine was added. The mixture was stirred vigorously at 80 °C. 1.5 mL of tetraethyl orthosilicate (TEOS) was added dropwise, and the reaction was carried out for 2 h. The template agent was removed by high-temperature calcination (550 °C, 6 h) at a heating rate of 5 °C / min to obtain mesoporous silica cores. The obtained mesoporous silica cores were dispersed in a mixture of calcium nitrate and diammonium hydrogen phosphate, and the pH was controlled at 9.0. In-situ mineralization deposition was carried out for 4 h to obtain silica nanoparticles.

[0021] Characterization results showed that the obtained silica nanoparticles had a particle size of approximately 210 nm, a specific surface area of ​​52 m² / g, a pore volume of 0.40 cc / g, and a molar composition of 97.16 mol% SiO₂, 2.23 mol% CaO, and 0.61 mol% P₂O₅.

[0022] The aforementioned silica nanoparticles were first activated under vacuum at 110 °C for 2 h, dispersed in anhydrous ethanol, and then 3-aminopropyltriethoxysilane (APTES) was added. The mixture was then refluxed at 70 °C for 6 h under nitrogen protection to obtain aminated silica nanoparticles. Subsequently, MAL-PEG2000-NHS coupling agent was dissolved in PBS buffer (pH 7.4) and mixed with the aminated silica nanoparticles. The mixture was then reacted under nitrogen protection at room temperature in the dark for 1 h. After centrifugation and washing, a thiolized D-Asp8 sequence peptide was added, and the mixture was reacted at room temperature in the dark for 24 h to obtain the targeting carrier matrix.

[0023] Step 2: Preparation of engineered extracellular vesicles Bone marrow mesenchymal stem cells were cultured to 70-85% confluence and pretreated with induction medium containing 5 μg / mL fritillary acetate (an anti-inflammatory osteogenic bioactive molecule) for 24 h. Subsequently, the medium was replaced with exosome-free serum medium and cultured for another 18 h. The cell culture supernatant was collected, centrifuged at 300 g for 10 min to remove viable cells, centrifuged at 2000 g for 15 min to remove cell debris, and centrifuged at 10000 g for 30 min to remove organelles. Finally, the cells were ultracentrifuged at 100,000 g for 2 h to obtain engineered extracellular vesicles.

[0024] The resulting extracellular vesicles had a diameter of 90-120 nm, expressed TSG101 and CD63 characteristic markers on their surface, did not express calnexin protein, and had a density of 1.15 g / mL.

[0025] Step 3, Composite Load The engineered extracellular vesicles and the targeting carrier matrix were mixed at a mass ratio of 1:2 in a PBS buffer system (pH 7.4) and incubated at 4°C in the dark for 20 h. After incubation, the precipitate was collected by centrifugation (12,000 g, 30 min) to obtain the silica-engineered extracellular vesicle composite nanoparticle formulation.

[0026] The results showed that the diameter of the silica-engineered extracellular vesicle composite nanoparticles was 100-300 nm.

[0027] Example 2 This embodiment 2 provides a silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair, comprising a carrier matrix and a functional loading component. The functional loading component is loaded in the carrier matrix through a synergistic effect of physical adsorption and pore embedding. The carrier matrix consists of core-shell structured bioactive silica nanoparticles, with a core of mesoporous silica having centrally radial pores and a shell of calcium-phosphorus layer. The surface of the silica nanoparticles is modified with bone-targeting molecules. The functional loading component consists of engineered extracellular vesicles derived from bone marrow mesenchymal stem cells pretreated with bioactive molecules that have anti-inflammatory and osteogenic effects.

[0028] The preparation method of the above-mentioned silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair is also provided, specifically including the following steps: Step 1: Preparation of the carrier matrix (same as in Example 1) Step 2: Preparation of engineered extracellular vesicles Bone marrow mesenchymal stem cells were cultured to 70-85% confluence and pretreated with induction medium containing 0.5 μg / mL fritillary glycoside (an anti-inflammatory osteogenic bioactive molecule) for 24 h. Subsequently, the medium was replaced with exosome-free serum medium and cultured for another 18 h. The cell culture supernatant was collected, centrifuged at 300 g for 10 min to remove viable cells, centrifuged at 2000 g for 15 min to remove cell debris, and centrifuged at 10000 g for 30 min to remove organelles. Finally, the cells were ultracentrifuged at 100,000 g for 2 h to obtain engineered extracellular vesicles.

[0029] Step 3, Composite Load The engineered extracellular vesicles and the targeting carrier matrix were mixed at a mass ratio of 1:4 in a PBS buffer system (pH 7.4) and incubated at 4°C in the dark for 20 h. After incubation, the precipitate was collected by centrifugation (12,000 g, 30 min) to obtain the silica-engineered extracellular vesicle composite nanoformulation.

[0030] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content of the present invention specification should be included within the protection scope of the present invention.

Claims

1. A silica-engineered extracellular vesicle composite nanoparticle formulation for tendon-bone interface repair, characterized in that, The product comprises a carrier matrix and a functional loading component, wherein the functional loading component is loaded in the carrier matrix through a synergistic effect of physical adsorption and pore embedding; the carrier matrix consists of core-shell structured bioactive silica nanoparticles, with a core of mesoporous silica having centrally radial pores and a shell of calcium phosphate layer, and the surface of the silica nanoparticles is modified with bone-targeting molecules; the functional loading component consists of engineered extracellular vesicles derived from bone marrow mesenchymal stem cells pretreated with bioactive molecules having anti-inflammatory and osteogenic effects.

2. The composite nano-formulation according to claim 1, characterized in that, The silica nanoparticles have a particle size of 180-320 nm and a specific surface area of ​​45-65 m². 2 / g, with a pore volume of 0.25-0.55cc / g.

3. The composite nano-formulation according to claim 1, characterized in that, The molar percentages of each component in the silica nanoparticles are: SiO2 92-98 mol%, CaO 1.5-4 mol%, and P2O5 0.5-2 mol%.

4. The composite nano-formulation according to claim 1, characterized in that, The bone-targeting molecule is a D-Asp8 sequence polypeptide, delivered via MAL-PEG. 2000 -NHS coupling agent is grafted onto the surface of aminated silica nanoparticles; the amination modification uses 3-aminopropyltriethoxysilane as the coupling agent.

5. The composite nano-formulation according to claim 1, characterized in that, The extracellular vesicles have a diameter of 40-160 nm, express TSG101 and CD63 characteristic markers on their surface, do not express calnexin protein, and have a density of 1.13-1.19 g / mL.

6. The composite nano-formulation according to claim 1, characterized in that, The anti-inflammatory osteogenic bioactive molecule is selected from at least one of natural small molecule compounds, cytokines, or chemical inducers.

7. A method for preparing a composite nanoparticle formulation as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1, Preparation of the carrier matrix: Using hexadecyltrimethylammonium bromide as a template agent and tetraethyl orthosilicate as a silicon source, a mesoporous silica core with centrally radial channels was prepared by the sol-gel method. Subsequently, calcium phosphate salts were deposited in situ on the surface of the core to form a core-shell structure. After amination modification, bone-targeting molecule modification chains were grafted to obtain a targeting carrier matrix. Step 2, preparation of engineered extracellular vesicles: Bone marrow mesenchymal stem cells were cultured to 70-85% confluence, pretreated with induction medium containing anti-inflammatory osteogenic bioactive molecules, and then cultured in exosome-free serum medium for 12-24 hours; the cell culture supernatant was collected and engineered extracellular vesicles were obtained by separating and purifying them using a combination of gradient centrifugation and ultracentrifugation. Step 3, Composite Loading: The engineered extracellular vesicles and the targeting carrier matrix are mixed in a buffer system at a mass ratio of 1:(0.5-5) and incubated at 4°C in the dark for 6-24 hours to obtain the composite nano-formulation.

8. The preparation method according to claim 7, characterized in that, In step one, the in-situ deposition of calcium phosphate is carried out in an alkaline system with a pH of 8.5-9.5; in step two, the gradient centrifugation includes centrifugation at 300-500g for 10 min to remove live cells, centrifugation at 2000-3000g for 15 min to remove cell debris, and centrifugation at 10000-12000g for 30 min to remove organelles; the ultracentrifugation is centrifugation at 100,000g for 2 h.

9. The preparation method according to claim 7, characterized in that, In step two, the concentration of the anti-inflammatory osteogenic bioactive molecule is 0.5-5 μg / mL, and the pretreatment time is 20-28 h.

10. The use of the composite nanoformulation as described in any one of claims 1-6 in the preparation of rotator cuff injury repair materials.